Primer Effects by Murine Pheromone Signaling - Pheromonal Influences On Reproductive Conditions (PDFDrive)

SPRINGER BRIEFS IN ANIMAL SCIENCES
Sachiko Koyama
Primer Effects by
Murine Pheromone
Signaling
Pheromonal
Influences on
Reproductive
Conditions
SpringerBriefs in Animal Sciences
More information about this series at http://www.springer.com/series/10153
Sachiko Koyama
Primer Effects by Murine

Pheromone Signaling
Pheromonal Influences on Reproductive
Conditions
123
Sachiko Koyama
School of Medicine, Medical Science
Program
Indiana University
Bloomington, IN
USA
ISSN 2211-7504 ISSN 2211-7512 (electronic)

SpringerBriefs in Animal Sciences
ISBN 978-3-319-13932-6 ISBN 978-3-319-13933-3 (eBook)
DOI 10.1007/978-3-319-13933-3
Library of Congress Control Number: 2016933470
© The Author(s) 2016

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt from
the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the
authors or the editors give a warranty, express or implied, with respect to the material contained herein or
for any errors or omissions that may have been made.
Printed on acid-free paper
This Springer imprint is published by Springer Nature

The registered company is Springer International Publishing AG Switzerland
Preface
Nowadays the word “pheromone” is well known. However, the popular image of
pheromones usually involves some mysterious, invisible chemicals that make
people attracted to the opposite sex perhaps because of the use of the word in the
names of some perfumes. Pheromones have many functions. Indeed some of them
function as attractants between the sexes, and some stimulate aggression between
members of the same sex, especially in males. The function that perhaps is not yet
well known is their affect on the reproductive status of others. In this context,
males’ pheromones stimulate females’ reproductive status and females’ pher-
omones stimulate males’ reproductive status. Although studies in mice have pro-
gressed substantially, mechanistic clarification of these phenomena may provide
valuable applicability to humans as well.
The study of olfactory communication in mice advanced dramatically during the
last half of the twentieth century, beginning with the first observation findings on
physiological changes in females in the 1950s to the chemical identification of the
responsible pheromones that followed in the 1980s. The first observations were
of the influences of conspecific odors on the estrous cycles and establishment of
pregnancy in female mice, effects that came to be called Lee-Boot effect, Whitten
effect, Bruce effect, and Vandenbergh effect (Chap. 3). These influences of con-
specific odors on physiological conditions are called “primer effects,” whereas the
influences of conspecific odors on behaviors are called “releaser effects.” In this
book, I will focus on the primer effects, other than in Chap. 4 Identification of
Pheromones, where I described about some releaser effects in explaining the
pheromones that had been identified so far.
In the 1990s, olfactory neuroscience research produced a transition of the
investigation of olfactory communication from classic behavioral biology studies to
studies using the techniques in molecular biology and/or neuroscience, studies that
included the use of transgenic mice. My own studies at college included animal
psychology and I became interested in the field of ethology, the evolution of animal
behaviors and their adaptive functions. I subsequently obtained my Ph.D. in
Ethology and I later studied cell biology and the reproductive physiology of sperm
v
vi Preface
cells as a postdoctoral fellow at the University of Tokyo in the laboratory of Prof.

Shinji Kamimura (currently at Chuo University, Japan). I found that sperm density
in the males exposed to female-soiled bedding is enhanced and that sperm motility
in the subordinate males is suppressed compared to dominant males (see Chap. 3).
My training in cell biology opened my eyes to microscopic studies of cells, a
major transition from my earlier macroscopic studies of social behaviors in mice
using mazes or encounter boxes. Following that, I expanded my professional
training to neurogenesis and studies of mammary glands. That led to two additional
new findings, an enhanced development of mammary glands in female mice by
exposure to a male murine pheromone, and an enhanced cognitive function in the
next generation of these females (both discussed in Chap. 5). I also identified the
pheromones that stimulate adult neurogenesis (Chap. 5). In summary, my studies
and collaborations with the people working in these diverse fields led me to dis-
cover four new primer effects that will be the main topics of this book: effects on
sperm motility, sperm density, mammary glands, and enhanced cognitive function
in the next generation.
I have intentionally included in this book the information that is difficult to write
in academic papers. Examples include the details of mouse biology that may affect
odors of mice and their responses to odors of other mice, as well as the key steps in
the scientific endeavor that led to exciting results in successful experiments, or that
led to negative results, which sometimes give us important information as well.
This information may be important for students and for junior scientists. I believe
that the ability to include such information is one of the benefits in writing a book.
Acknowledgments
There are many people to whom I would like to express my thanks. First of all, I
would like to express my sincere thanks to Shinji Kamimura of the University of
Tokyo (currently at Chuo University, Japan), who expanded my knowledge and
techniques from animal psychology and ethology to cell biology of sperm cells. If I
did not have the chance to collaborate with him, we could never find the primer
effects in males that we found. He was generous enough to combine the knowledge
from different study fields, which I now know that it is extremely rare, and, besides,
he enthusiastically used the metaphor of “a fusion of atoms gives off enormous
energy” in explaining our interdisciplinary project to his colleagues at the
University of Tokyo. Nothing is more encouraging than this in conducting a totally
new interdisciplinary scientific activity. The pheromone projects on neurogenesis,
mammary glands, and trans-generational influences of pheromones were conducted
in collaboration with many people, but especially with Milos V. Novotny and
Helena A. Soini of the Department of Chemistry, and John Foley of the School of
Medicine of Indiana University. Utilization of the synthetic analogues of pher-
omones, which Milos Novotny generated, enabled precise control of exposure to
pheromones, and collaboration with John Foley whose profession is molecular
biology of mammary glands enabled expansion of the projects to include mammary
glands and molecular level analyses. I would also like to express my sincere
gratitude to John Watkins, III of the School of Medicine of Indiana University for
all his support. Another special sincere gratitude goes to Robert Karn of University
of Arizona, who thoroughly edited my English in the Preface and Chap. 1, and
provided many comments on mouse biology from his profession of wild mouse
genetics and geographical distribution of wild house mouse subspecies. I appreciate
Silvia Herold and Claus-Dieter Bachem of SpringerBrief for providing me this
exciting chance to write a book on murine pheromone signaling. And last but not
least, I appreciate my family in Japan for understanding that I am a scientist who
would never stop studying and pursuing my science. It is always exciting to think
about a new hypothesis.
vii
Contents
1 Introduction: The Laboratory Mice . . . . . . . . . . . . . . . . . . . ..... 1

1.1 Ethology and the Study Field of Olfactory Communication
in Laboratory Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 1
1.2 The Social System and Breeding System . . . . . . . . . . . . . ..... 3
1.3 Pregnancy, Delivery, and the Growth and Development
of the Offspring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 6
1.4 Mouse Biology, Hormones, Pheromones, and Pheromone
Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 6
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 7
2 The Olfactory Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... 11
2.1 The Olfactory Systems . . . . . . . . . . . . . . . . . . . . . . . . ....... 11
2.2 The Main Olfactory System and Its Pathway in Relation
to Pheromone Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.3 Accessory Olfactory System (Vomeronasal System) . . . . . . . . . . . 15
2.4 Responses to Pheromone Are not Always the Same . . . . . . . . . . . 17
2.5 Sex Differences in the Responses to Pheromones . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3 The Primer Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.1 The Lee–Boot Effect, Whitten Effect, and Vandenbergh Effect . . . 23
3.2 Bruce Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.3 Primer Effect in Males . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.3.1 Social Dominance and Sperm Motility . . . . . . . . . . . . . . . 29
3.3.2 Female Odors and Sperm Density . . . . . . . . . . . . . . . . . . 31
3.3.3 Influence of Adult Males’ Odor on Sperm Morphology . . . 35
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
ix
x Contents
4 Identification of Pheromones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.1 Pheromones to Induce Primer Effects . . . . . . . . . . . . . . . . . . . . . 39
4.2 Pheromones to Induce Releaser Effects . . . . . . . . . . . . . . . . . . . . 43
4.2.1 Aggression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.2.2 Sexual Behaviors and Pheromones in Tears: The Esps . . . . 44
4.2.3 Kin Recognition, Subspecies Recognition,
and Mate Preference . . . . . . . . . . . . . . . . . . . . . . . . ... 45
4.2.4 Rooting Behaviors by Neonates: Are There
Pheromones in Mouse Milk? . . . . . . . . . . . . . . . . . . ... 50
4.2.5 Parental Behaviors. . . . . . . . . . . . . . . . . . . . . . . . . . ... 52
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 55
5 New Primer Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5.1 From Early Studies to New Studies . . . . . . . . . . . . . . . . . . . . . . 59
5.2 Adult Neurogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.3 Odors of Opposite Sex Stimulate Neurogenesis . . . . . . . . . . . . . . 60
5.4 Identification of Pheromones that Stimulate Cell Proliferation
in the Brain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.4.1 Female Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.4.2 Male Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
5.5 Mammary Gland Expansion by Exposure to Male Pheromone. . . . 68
5.6 Trans-generational Influence of Exposure to Pheromones . . . . . . . 70
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
6 Ontogeny. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
6.1 Ontogeny of the Olfactory System . . . . . . . . . . . . . . . . . . . . . . . 75
6.2 Developmental Changes in the Responses to Pheromones . . . . . . . 78
6.3 Epigenetic Changes on the Sensitivity to Smell . . . . . . . . . . . . . . 79
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
7 Adaptive Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 85
7.1 What is their Function in Reproductive Success? . . . . . . . . ..... 85
7.2 Function of Enhanced Adult Neurogenesis by Exposure to
Pheromones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
7.3 Possible Pheromonal Signaling in Humans and its Function . . . . . 87
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Chapter 1
Introduction: The Laboratory Mice
Abstract The biology of mice has been studied for centuries with the main
purpose of pest control. With the advent of scientific approaches to human medical
care, mice have become the most popular animal model system because of the
many similarities between their physiology and that of humans. More recently, they
have been used in an even broader range of scientific studies aimed at manipulating
their genome. With the development of olfactory neuroscience from 1990s and
sequencing the whole mouse genome at the beginning of 21st century, olfactory
communication is now studied at cell and molecular levels. This work requires
using various transgenic mice to visualize neuronal connections, cellular responses
at the single sensory neuron level to exposure of the animal to odors and/or
pheromones, and their neural circuits in the brain. Some of this work has involved
genetically altering laboratory strains of mice to produce and secrete less chemical
components in their urine compared to wild house mice. Nonetheless mice are still
mice, with all, or many of their behavioral characteristics intact and still possessing
the communication system typical to mice. The knowledge of mouse biology helps
in optimally designing mouse behavioral experiments but also mouse housing
conditions and helps to avoid contamination of olfactory signaling factors that may
affect the results significantly.
Keywords Mouse biology Social system Breeding system
1.1 Ethology and the Study Field of Olfactory

Communication in Laboratory Mice
In a paper published in 1963, Niko Tinbergen, who was one of the founders of the
field of ethology, listed four issues that ethologists focus on, i.e., ontogeny, cau-
sation, survival value, and evolution (Tinbergen 1963). These four words/phrases
can explain (i) how behaviors develop, (ii) how behaviors are controlled, (iii) what
the adaptive function(s) are, and (iv) how the behaviors evolved, which, taken
together, constitute the main theme in ethology. The ultimate goal for ethologists is
© The Author(s) 2016 1

S. Koyama, Primer Effects by Murine Pheromone Signaling,
SpringerBriefs in Animal Sciences, DOI 10.1007/978-3-319-13933-3_1
2 1 Introduction: The Laboratory Mice
to “reconstruct the phylogeny of motor patterns” (Eibl-Eibesfeldt 1970), in a way

similar to comparative anatomists try to reconstruct the phylogeny of morphological
characteristics. “They explore the processes underlying the ontogenetic develop-
ment and finally search for its causes, investigating the releasing stimuli and the
underlying physiological processes” (Eibl-Eibesfeldt 1970). The rapid growth of
the field of ethology during the over 50 years since the paper by Tinbergen was
published shows his foresight in predicting how the field would grow. Ethologists
study how behaviors develop (ontogeny) and what controls the behaviors (causa-
tion), and attempt to understand the adaptive function of the behaviors of animals.
Ultimately, ethologists endeavor to understand the adaptive functions represented
by the evolution of behaviors. This requires developing knowledge about the social
system of mice, their natural habitats, their mating systems, and the way they rear
their offsprings as prerequisites for further research. A thing that is also necessary to
be noted is that, although the laboratory mouse strains are a descendant of a single
species of house mouse, Mus musculus, they are also “produced” through a mixture
of several subspecies. Although most of the genome of laboratory mice strains are
derived from one of the house mouse subspecies, Mus musculus domesticus (M.m.
domesticus) (94.3 %), the rest comes from Mus musculus musculus (M.m. mus-
culus) and then from Mus musculus castaneus (M.m. castaneus) (Yang et al. 2011).
Although the percentage is small, the mixture of multiple subspecies in the labo-
ratory strains might have produced some changes in their behaviors. For example,
in a strain generated by breeding mice strains DBA and C3H, a gene region Abp
had entirely the M.m. musculus genome characteristics although other regions were
almost entirely those of M.m. domesticus (R. Karn personal communication;
Laukaitis et al. 1997, 2012). This suggests the possibility that the specific strain
would show behavioral characteristics typical to the M.m. musculus in the mating
preference behaviors that Abp regulates or vague in their behaviors compared to the
pure subspecies’ behaviors (Laukaitis et al. 1997). It is always necessary to be
cautious in taking it into consideration the possible existence of other regions with
“genomic contamination” that might be influencing the responses to social stimuli
in their physiological conditions and behaviors. Another factor that is necessary to
consider is that the long-term history of breeding under laboratory conditions.
Decades of mating determined by humans without opportunities to show mating
preference through chemical signaling could have affected their chemical and
behavioral characteristics. This can affect in a way that the laboratory mice strains
lose secretions of pheromones that are used in mating preferences. For example,
major urinary protein (MUP) of laboratory mouse strains shows less variation than
wild-type variation (Cheetham et al. 2009). Yet, the knowledge on what is known
as the social system of mice, their natural habitats, their mating systems, and the
way they rear their offspring is still extremely important in order to be able to
handle the factors that can affect the results on primer effects. This is because such
knowledge includes information on the housing conditions that affect physiological
conditions. The knowledge will enable avoiding housing mice in ways that affect
1.1 Ethology and the Study Field of Olfactory Communication in Laboratory Mice 3
their physiological conditions unexpectedly. This might induce contamination of

multiple conditions without knowing, leading to vague results, and even possibly
misleading to wrong conclusions.
1.2 The Social System and Breeding System
Mice live in groups in the wild. As a mammalian species with very small body size,
it is considered that living in groups and huddling together in their nests help them
to avoid losing their body temperature. The necessity of huddling would be stronger
in the area with colder temperature and also during the seasonally harsh period.
Eisenberg has classified the social system in animals into 3 different types; (i) dis-
persed (solitary), (ii) communal (each individual does not have separate space and
protects a territory as a group), and (iii) colonial (each individual possesses their
space and protects a territory around the colony) (Eisenberg 1981). Depending on
the species, it is not simple to define the type of their social system, because some
species will have different social system depending on the sex and some species
show seasonal changes as well. In case of house mice, males and females establish
communal groups and the groups usually consist of one dominant male, which sires
most of the offspring, with several adult females, and young adults and infants of
both sex (Berry 1981; Berry and Bronson 1992). These males and females share the
same nest and huddle together even when the females are breeding (communal
breeders) (Solomon and French 1997). The females nurse offspring of their own as
well as those of other females sharing the same nest.
When animals live in groups, often there is social dominance among the group
members. In house mice, males typically establish social dominance. Nonbreeding
females are less aggressive and there is no obvious social dominance to be deter-
mined by the amount of aggression directed to group-mates especially in laboratory
cage housing conditions. Younger females around postpuberty are sometimes
aggressive to non-cagemates (Ropartz and Haug 1975; personal observation by
author as well). Breeding females show maternal aggression to unfamiliar female
intruders. There are also reports on social “classification” of females depending on
breeding status and accessibility to resources of food, water, and nest box to rep-
resent dominance in female house mice kept in large enclosures (Lloyd and
Christian 1969; DeLong 1978; Hurst 1987). The social dominance in male mice was
studied well in early 20th century. Uhlich (1938) showed that “despotic” type is the
most typical type of social dominance in male mice. In this type of social domi-
nance, one male is dominant, showing most or all of the aggressive behaviors to
other group-mates and to intruders in the territory, and other males do not show
aggressive behaviors. When aggression takes place, typically the subordinates
would be in the corners without movements, with their face directed to the dominant
male when he is attacking others, or showing submissive upright posture without
movement, with their face up avoiding direct eye contact with the dominant male
when they are attacked by him (Grant and Mackintosh 1963; Mackintosh 1981).
When the dominant male is removed, usually another male becomes aggressive and
becomes the new dominant male, which shows that the lack of aggressive behaviors
among subordinates does not necessarily indicate the lack of aggressiveness but it is
often because their aggressiveness is suppressed by the dominant male. The next
frequently observed social dominance type in mice is the so-called “pecking order”
type, in which there is hierarchical order of social status determined by the amount
of aggressive behaviors each male shows. The name of this type of social domi-
nance originates from studies in cocks by Schjelderup-Ebbe (Ito 1959).
Stability of social dominance alters depending on the number of mice in the
cages. When more than three males are kept in cages, the change in social status is
observed as often as once in a month (Koyama, personal observation). The most
stable dominant—subordinate relationships in male mice without changes in social
status are observed when two males are kept in one cage (Koyama personal
observation). When mice are singly housed, they often show abnormally high
aggressiveness (King 1957; Baer 1971; Cairns and Nakelski 1971; Cairns et al.
1985; Koyama 1993a, b). The behavior patterns also become abnormal especially if
the singly housed condition is in long term from immediately after weaning
(Valzelli 1973; Koyama 1993a, b, 1995a). For example, when male mice are singly
housed starting from immediately after weaning for long term, they rarely show
“genital sniffing” behaviors (Koyama 1985), the sequence of behavior is abnormal
(Koyama 1993b), and the way they respond to urine of other males lack the
characteristics seen in the males that grew in social environment (Koyama 1995a).
These studies suggest that, although mice use odor as major means of social
communication (Brown 1979, 1985), learning is required in the olfactory com-
munication and the social interaction around puberty is critical for the learning
(Koyama 1985).
The use of odor covers not only the recognition of individuals (Brown 1985),
group members (Hurst 1989), kinship (Yamazaki et al. 1979), but also the territorial
boundary (Harrington 1976), physiological state, emotional state, and social rela-
tionship (Brown 1985). Jones and Nowell (1973) suggested that male mice dis-
criminate the odor of dominant and subordinate mice, and that the odor of dominant
males had aversive property compared to that of subordinate males. Koyama
(1995a, b) showed that the way to respond to the urine of dominant and subordinate
males depends on the social status of the responder and that it depends also on the
context (familiar male versus. unfamiliar male, or familiar male that the subordinate
male observed being beaten by his cagemate dominant male).
The information on social dominance and its stability as well as the influences of
singly housing is important because the odor of mice is affected by the social status
(see Chap. 3). If males are to be used as odor donors in the experiments on olfactory
communication, the housing conditions need to be strictly controlled and domi-
nance of males may need to be determined. Behavioral responses to the odor of
other males also change by their own social status other than by the social status of
the odor donor (Koyama 1995a, b). Whether the response to odor is different at
1.2 The Social System and Breeding System 5
signaling level is not known yet, but it is better to consider possibilities of

differences.
Dominant and subordinate males show scent marking behaviors as well. In
studies examining the pattern of urination by dominant and subordinate males, it
was found that dominant males urinate within the broader area and also at the
border area, making more spots of urination, whereas subordinate males urinated at
less location, typically at one corner. These differences suggest the function of urine
spots as scent marking or territorial marking and that the dominant males are
involved in these territorial marking.
Singly housing is very stressful for females. When females were kept either
singly, four in one cage, or twelve in a cage, the singly housed females showed the
highest serum corticosterone concentration and the females kept in four per cage
showed the lowest level of it (Koyama unpublished data). These differences by the
number of females per cage affect their estrous cycles as well (see Chap. 3). The
chemical components included in the urine of females change depending on the
estrous stage and stress makes the odor change as well. The secretion of female
specific pheromones also changes by the number of females kept per cage (see
Chap. 3). This indicates that the housing conditions of females need to be strictly
controlled in the experiments on olfactory communication because the responses to
odor can be different depending on these factors. It is always better to keep records
of the housing conditions and estrous cycles/statuses of females to be precisely
following the possible factors that might have affected the results.
The estrous cycle of females is usually 4–5 days (see Chap. 3 for the changes by
housing conditions) with 4 stages, i.e., proestrus, estrus, postestrus, and di-estrus.
The changes in the estrous cycle by housing conditions also indicate that the cyclic
changes in hormone secretion are affected by the housing conditions. Studies have
shown that secretion of estrogen (Wood et al. 2007; Christensen et al. 2011) and
prolactin (DeLeon et al. 1990; DeMaria et al. 1998; Christensen et al. 2011)
increases at proestrus stage to estrous stage and the concentration of dopamine
decreases when prolactin shows peak (DeMaria et al. 1998; Christensen et al.
2011). There are studies showing that estrogen stimulates prolactin secretion by
up-regulating dopamine-activated K+ channels in pituitary lactotrophs and thus the
increase of prolactin follows that of estrogen (DeLeon et al. 1990; Christensen et al.
2011). The levels of concentrations at the peak vary among the studies and species.
For example, the level of peak in prolactin concentration ranges between about
60 ng/mL in plasma (mice, DeLeon et al. 1990), about 300 ng/mL in serum (rat,
DeMaria et al. 1998) and above 800 ng/mL (hamster, Reburn and Wynne-Edwards
1996). The results of studies on progesterone are showing some differences, i.e.,
some showing its increase at diestrous stage (in mice, Wood et al. 2007), some at
estrous stage (in mice, DeLeon et al. 1990), and some showing two peaks, a small
peak at diestrous and a higher peak at estrous stage (in hamster, Reburn and
Wynne-Edwards 1996), from unknown reason. There are also evidences showing
that cellular and stromal remodeling in uterus (Wood et al. 2007) and mammary
glands (Brisken and O’Malley 2011) take place along the stages of estrous cycle.
These studies indicate that how females are housed would affect not only the length
of estrous cycles but it can also affect the morphology or functional activity of the
tissues and organs, which are under the control of the hormones that change their
secretion along estrous cycles.
1.3 Pregnancy, Delivery, and the Growth

and Development of the Offspring
The duration of pregnancy is 19 days in house mice. People who use embryos in
their studies often determine embryonic day by the days after females are mated (for
example, E14 if the embryo is collected 14 days after females were mated). This is
not precise. Copulation may take place immediately on the day the mice were
mated or it may take place several days after the day they were housed together
depending on the estrous stage the female was on the day they were mated.
Utilization of Theiler’s embryonic table (Theiler 1989) is necessary to precisely
determine the embryonic stage when the embryos are collected.
In case of continuous breeding without removing the males after the pregnancy
is obvious, copulation will take place several hours after the first delivery because
of the increase of estrogen in females at the time of delivery. In these cases, females
will nurse the first offspring and the process of pregnancy of the next offspring will
start in parallel. Pups can be weaned at 21 days of age and the next offspring are
often born around that time. The second litter in case of continuous breeding often
shows delay in implantation, and the duration until the next delivery is not always
19 days but often much longer. This is due to the day that implantation of the
fertilized eggs on uterus becomes often delayed in the continuous breeding. The
vagina opening of the female pups takes place around 26–28 days in age, and, in
males, sperm start reaching to cauda epididymis around 40 days in age with several
days’ differences depending on the individuals and the strains. Mice have 5 pairs of
nipples and the size of mammary glands vary depending on the location. #4
mammary glands, which are located between the hind legs, are the largest mam-
mary glands, and #1 mammary glands, which are located at the neck, are the
smallest. It is expected that the amount of milk secreted would be different
depending on these locations and, depending on experimental conditions, it is better
to control the litter size to control the growth of the pups.
1.4 Mouse Biology, Hormones, Pheromones,

and Pheromone Signaling
In this chapter, what I tried to write is that the physiological conditions of mice can
be affected by various factors. Hormone secretions will be affected by their social
status, number of mice kept per cages, estrous status, breeding status, and age. The
1.4 Mouse Biology, Hormones, Pheromones, and Pheromone Signaling 7
chemical compounds included in urine vary by these changes, and hence cause
differences in the response to the urine, if urine is used as stimulus to test responses
of mice. The response to the same pheromone compound can also be affected by
these conditions of the mice exposed to the urine. That is, the response to odor
stimuli can be altered by the factors on the odor donor side and on the odor receiver
side. Important thing is that it is necessary to know that these various factors can
affect the results and it is necessary to take them into consideration in order to
control the conditions when we design the experiments. As social status and iso-
lation can affect results, it would be hard or almost impossible to completely control
the conditions and prepare every single mouse in the same conditions. However, if
we know the factors that can affect results, we can analyze results taking these
factors in consideration and/or design experiments in a way that we can avoid
mixture of factors that may affect the results. In short, it is better to keep mice with
same number of mice per cage to avoid influences of density on hormone secretion,
avoid isolation housing from weaning if you expect mice to obtain normal social
behaviors, and check the social status of males if you need to use males as odor
donors and also when you use them to test the respond to odors.
References
Baer H (1971) Long-term isolation stress and its effects on drug response in rodents. Lab Anim Sci
21:341–349
Berry RJ (1981) Population dynamics of the house mouse. Symp Zool Soc Lond 47:395–425
Berry RJ, Bronson FH (1992) Life history and bioeconomy of the house mouse. Biol Rev
67:519–550
Brisken C, O’Malley B (2011) Hormone action in the mammary gland. In: Bissell MJ, Polyak K,
Rosen JM (eds) The mammary gland as an experimental model. Cold Spring Harb. Perspect.
Biol. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 71–85. doi:10.1101/
cshperspect.a003178
Brown RE (1979) Mammalian social odors: a critical review. In: Rosenblatt JS, Hinde RA, Baer C,
Busnel M-C (eds) Advances in the study of behavior, vol 10, Academic Press, NY, pp 103–162
Braown RE (1985) The rodents II: suborder Myomorpha. In: Brown RE, MacDonald DW
(eds) Social odours in mammals, vol 1, Clarendon Press, Oxford, pp 345–457
Cairns RB, Nakelski JS (1971) On fighting in mice: ontogenetic and experiential determinants.
J Comp Physio Psychol 74(3):354–364
Cairns RB, Hood KE, Midlam J (1985) On fighting in mice: is there a sensitive period for isolation
effects? Anim Behav 33:166–180
Cheetham SA, Smith AL, Armstrong SD, Beynon RJ, Hurst JL (2009) Limited variation in the
major urinary proteins of laboratory mice. Physiol Behav 96(2):253–261
Christensen HR, Zeng Q, Murawsky MK, Gregerson KA (2011) Estrogen regulation of the
dopamine-activated GIRK channel in pituitary lactotrophs: implications for regulation of
prolactin release during the estrous cycle. Am J Physiol Regul Integr Comp Physiol 301:
R746–R756
DeLong KT (1978) The effect of the manipulation of social structure on reproduction in house
mice. Ecology 59:922–933
DeLeon DD, Zelinski-Wooten MB, Barkley MS (1990) Hormonal basis of variation in oestrous
cyclicity in selected strains of mice. J Reprod Fert 89:117–126
DeMaria JE, Livingstone JD, Freeman ME (1998) Characterization of the dopaminergic input to
the pituitary gland throughout the estrous cycle of the rat. Neuroendocrinology 67:377–383
Eibl-Eibesfeldt I (1970) Ethology. The biology of behavior. Holt, Rinehart and Winston, Inc., New
York
Eisenberg JF (1981) The mammalian radiations: an analysis of trends in evolution, adaptation, and
behavior. Univ Chicago Press, Chicago, London
Grant EC, Mackintosh JH (1963) A comparison of the social posture of some common laboratory
rodents. Behaviour 21:246–259
Harrington JE (1976) Recognition of territorial boundaries by olfactory cues in mice (Mus
musculus L.). Z Tierpsychol 41(3):295–306
Hurst JL (1987) Behavioural variation in wild house mice Mus domesticus Rutty: a quantitative
assessment of female social organization. Anim Behav 35:1846–1857
Hurst JL (1989) The complex network of olfactory communication in populations of wild house
mice Mus domesticus Rutty: urine marking and investigation within family groups. Anim
Behav 37:705–725
Ito Y (1959) Comparative Ecology. Iwanami Shoten, Tokyo
Jones RB, Nowell NW (1973) Aversive and aggression-promoting properties of urine from
dominant and subordinate male mice. Anim Learn Behav 1:207–210
King JA (1957) Relationships between early social experience and adult aggressive behavior in
inbred mice. J Genet Psychol 90(2):151–166
Koyama S (1985) The effects of social deprivation on the social behavior of mice (Mus musculus).
Ann Anim Psychol 35:79–90
Koyama S (1993a) Isolation effect in mice (Mus musculus): (i) does it really induce aggression? J
Ethol 11:117–130
Koyama S (1993b) Isolation effect in mice (Mus musculus): (ii) variance in aggression. J Ethol
11:131–140
Koyama S (1995a) The differential responses to the odour of unfamiliar male conspecifics in
dominant, subordinate, and isolated mice. J Ethol 13:165–173
Koyama S (1995b) Differential response to the odour of familiar intruder mice in male mice (Mus
musculus). J Ethol 13:47–56
Laukaitis CM, Critser ES, Karn RC (1997) Salivary androgen-binding protein (ABP) mediates
sexual isolation in Mus musculus. Evolution 51(6):2000–2005
Laukaitis CM, Mauss C, Karn RC (2012) Congenic strain analysis reveals genes that are rapidly
evolving components of a prezygotic isolation mechanism mediating incipient reinforcement.
PLoS ONE 7(4):e35898
Lloyd JA, Christian JJ (1969) Reproductive activity of individual females: three experimental
freely-growing populations of house mouse (Mus musculus L.). J Mammal 50:49–59
Mackintosh JH (1981) Behaviour of the house mouse. Symp Zool Soc Lond. 47:337–365
Reburn CJ, Wynne-Edwards KE (1996) Novel patterns of progesterone and prolactin in plasma
during the estrous cycle in the Djungarian hamster (Phodopus campbelli) as determined by
repeated sampling of individual females. Biol Reprod 54:819–825
Ropartz P, Haug M (1975) Olfaction and aggressive behavior in female mice. In: Denton DA,
Coghlan JP (eds) Olfaction and taste: 5th symposium. Academic Press, New York, pp 411–412
Solomon NG, French JA (1997) Cooperative breeding in mammals. Cambridge Univ Press,
Cambridge
Theiler K (1989) The house mouse: atlas of embryonic development. Springer-Verlag, New York
Tnbergen N (1963) On aims and methods of ethology. Z Tierpsychol 20:410–433
Uhrich L (1938) The social hierarchy in albino mice. J Comp Physio Psychol 25:373–413
Valzelli L (1973) The “isolation syndrome” in mice. Psychopharmakologia (Berl.) 31:79–94
Wood GA, Fata JE, Watson KLM, Khokha R (2007) Circulating hormones and estrous stage
predict cellular and stromal remodeling in murine uterus. Reproduction 133:1035–1044
Wu Z, Autry AE, Bergan JF, Watabe-Uchida M, Dulac C (2014) Galanin neurons in the medial
preoptic area govern parental behavior. Nature 509:325–330
References 9
Yamazaki K, Yamaguchi M, Baranoski L, Bard J, Boyse EA, Thomas L (1979) Recognition

among mice. Evidence from the use of a Y-maze. Differentially scented by congenic mice of
different major histocompatibility types. J Exp Med 150:755–760
Yang H, Wang JR, Didion JP, Buus RJ, Bell TA et al (2011) Subspecific origin and haplotype
diversity in the laboratory mouse. Nat Genet 43:648–655
Chapter 2
The Olfactory Systems
Abstract The 1990s became the decade that olfactory neuroscience showed
extraordinary development. Now we have much better understanding of how we
distinguish various odors and how the signaling pathways are in the brain. We also
know that the olfactory system is not a single system but it is a group of systems in
the nasal cavity that respond to chemical compounds. It has been believed for
decades that pheromones are received at the vomeronasal organ and the signaling
pathway from there reaches to hypothalamus and activates GnRH neurons, how-
ever, recent studies have determined that the pathway that reaches to GnRH neu-
rons starts from the main olfactory epithelium. There are pheromones that have
receptors in the accessory olfactory system releasing behaviors. The responses to
odors/pheromones are not always the same but they change at the sensory neuron
level and these changes are regulated by hormones. It is a chemical signaling
process.
Keywords Main olfactory system

Accessory olfactory system Olfactory

receptors Signaling pathways Hormones
2.1 The Olfactory Systems
The most well-known olfactory system is the main olfactory system and the
accessory olfactory system (vomeronasal system) (Brennan and Zufall 2006; Spehr
et al. 2006) (Fig. 2.1). There are several other systems (Breer et al. 2006; Fleischer
et al. 2009), for example Grüneberg ganglion and septal organ (the organ of
Masera), which are located in different regions in the nasal cavity. The Grüneberg
ganglion is located at the tip of the nose and recent studies have suggested its role in
detecting life threatening toxic chemicals or alarm pheromones. The septal organ is
located in the central area or middle between the main olfactory system and
accessory olfactory system in the nasal cavity. There are some others classified to
be different in the type of receptors they carry but located in the main olfactory
system area as well, for example, trace amine-associated receptors (TAARs) and

12 2 The Olfactory Systems
Fig. 2.1 The olfactory

system in mice (from Brennan
and Zufall 2006). AOB
accessory olfactory bulb,
GCD guanylylcyclase type D
system, GG Grüneberg
ganglion, MOB main
olfactory bulb, MOE main
olfactory epithelium, NC
nasal cavity, NG necklace
glomeruli, SOM septal organ
of Masera, VNO vomeronasal
organ
olfactory-specific guanylyl cyclase type D receptor (GC-D). Much is not known yet
about the function of olfactory systems other than the main and accessory olfactory
system. There are many review articles and books on the olfactory neuroscience
that here I will just briefly list and summarize the main and accessory olfactory
system.
2.2 The Main Olfactory System and Its Pathway

in Relation to Pheromone Signaling
What we usually call “the nose” in humans is precisely saying the main olfactory
system. The olfactory epithelium of the main olfactory system is located at the
posterior end of the nasal cavity (top of nasal cavity in case of humans) on the
surface of cartilage of labyrinth like shape protuberance. Structurally it is composed
of olfactory sensory neurons, supporting cells, and basal cells. The olfactory sen-
sory neurons are bipolar neurons with ciliated dendrite, which extend into the
mucosal surface and G protein–coupled receptors for the chemical signals are
located on these cilia. Olfactory receptor genes started to be identified from early
1990s (Buck and Axel 1991). Over 1000 different types of olfactory receptors are
identified in mouse (compared to less than 400 in humans) (Sullivan 2002; Zhang
and Firestein 2002; Tirindelli et al. 2009). The axon terminals from olfactory
sensory neurons with the same olfactory receptor create a single (or a couple of)
glomerulus in the olfactory bulb. Thus, activation of many olfactory sensory neu-
rons with the same olfactory receptor will be converted into an activation of a single
glomerulus in the olfactory bulb, which enables the discrimination of odors in the
environment. Higher concentration of an odor will activate larger number of
olfactory sensory neurons with the receptors for the specific odor and thus stronger
2.2 The Main Olfactory System and Its Pathway … 13
signaling will be conveyed to induce the cognitive outcome of the “notice of strong
smell”.
In other words, it is possible to say that the main function of the main olfactory
system has been traditionally considered to be the detection, discrimination, and
recognition of odorants, i.e., involved in cognitive function, whereas, the accessory
olfactory system was considered to be an unconscious pathway, detecting pher-
omones and altering hormone secretion. However, recent studies have shown that
the main olfactory system is the one that transfers signals toward gonadotropin
releasing hormone (GnRH) neurons, i.e., the neurons that synthesize and secrete
gonadotropin releasing hormone (Yoon et al. 2005) (Fig. 2.2). In relation to social
contexts, the main olfactory system seems to be involved in mate preference, mate
recognition (Baum 2012), and onset of maternal behaviors (Baum and Cherry 2014)
and recent studies have shown that pheromones are detected at the main olfactory
system as well (Xu et al. 2005; reviewed in Baum and Cherry 2014). It is highly
possible that the main olfactory system is involved in inducing primer effects.
These findings on the pathway from main olfactory system to GnRH neurons
had strong impact to the studies of species that are known to lack functional
accessory olfactory system, like in the higher primates including humans. Studies
have been reporting the phenomenon that suggest the existence of pheromonal
signaling in these species, although the accessory olfactory system had been found
to be residual without functional activity or without axon terminals from the
vomeronasal organ reaching to accessory olfactory bulb. Whether the signaling
from main olfactory system to the GnRH neurons is causing the primer effects in
higher primates is necessary to be clarified hopefully in near future. Conversion of
main and accessory pathway was also found in the accessory olfactory system, i.e.,
some portion of the signaling goes to the main olfactory pathway and reaches to the
cortex (Von Campenhausen and Mori 2000; Boehm et al. 2005). This suggests that
the accessory olfactory pathway may also not be an “unconscious” pathway. It is
maybe more possible that the system used in detecting specific pheromones
depends on the characteristics of the pheromones as chemical compounds (for
example, chemical structure or molecular weight which is related to volatility) or
the adaptive function of the pheromones (in what social context it is related; mating,
parenting, affecting reproductive condition, or informing life threatening danger
nearby).
The main signaling pathway from the olfactory epithelium reaches to main
olfactory bulb (MOB), which then projects to the anterior cortical nucleus of
amygdala (ACN), posterolateral cortical amygdaloid nucleus (PLCN), and to the
ventrolateral surface of the brain, the anterior olfactory nucleus (AON), the olfac-
tory tubercle (OT), and the olfactory cortex (piriform cortex (Pir), and entorhinal
cortex (EC)) (Figs. 2.2 and 2.3). Thus the signaling from the main olfactory bulb
projects to hypothalamus and amygdala (Boehm et al. 2005; Yoon et al. 2005;
Kang et al. 2009), which suggest that the alterations of hormone secretion can be
mediated by the main olfactory system. Studies have shown that, when female mice
were exposed to volatile odor of male urine (wind of air blown over urine without
direct contact to the urine), immediate early gene Fos was expressed in their
Fig. 2.2 The pathway to GnRH neurons was determined to be the main olfactory pathway (right
in blue) using infection to Ba2001 virus, which carries tau-GFP, infects neurons selectively
noninvasively, and spread in a retrograde way from postsynaptic to presynaptic (from Yoon et al.
2005). This was against the common knowledge that the pathway from accessory olfactory system
stimulates GnRH neurons. Transgenic mice with CRE recombinase expressed in GNRH1 (LHRH)
gene were infected with Ba2001 to let gfp expressed in the GnRH neurons and spread backwards.
Photos on the right show immunofluorescence staining against gfp. Scale bars = 50 um. AON
anterior olfactory nucleus, EC entorhinal cortex, MOB main olfactory blb, MOE main olfactory
epithelium, OT olfactory tubercle, PIR piriform cortex, PLCN posterolateral cortical amygdaloid
nucleus
amygdala but female urine and cat urine did not make significant changes (Kang
et al. 2009), which suggested that the main olfactory system transfers pheromonal
information, although it may not be all types of them. However, in other studies
using a single type of female pheromone, 2-Heptanone, activation of both main and
accessory olfactory bulb of female mice was observed in high-resolution functional
magnetic resonance imaging (fMRI) (Xu et al. 2005). In some studies both males
2.2 The Main Olfactory System and Its Pathway … 15
and females showed increased Fos immunoreactivity at the vomeronasal neurons,

bulbs, and in the brain, when they were exposed to male-soiled bedding directly
(and not to wind containing volatile odor) compared to clean bedding (Halem et al.
1999). These studies suggest that the differences in the influences may depend on
the direct/indirect access to the source and also by the way the activation was
measured.
2.3 Accessory Olfactory System (Vomeronasal System)
Accessory olfactory system, or the vomeronasal organ, is located in the bottom of

the nasal cavity. It is a blind alley hole in the bone with epithelium on one side of
the surface of the hole. The amazing mechanical system to bring chemicals into this
blind alley is the blood vessel running along the vomeronasal organ. This
mechanical system is called vomeronasal pump. The changes in the size opening of
the hole caused by the pulsation serve to mechanically pull the chemicals inside the
hole from the entrance of vomeronasal organ and to reach to the area where sensory
neurons are located. The faster and stronger the heart beats, the more the chemicals
will be mechanically pulled into the vomeronasal organ. The sensory neurons, i.e.,
the vomeronasal neurons, are bipolar neurons like the olfactory sensory neurons but
have villi instead of cilia, with receptors on the villi. Axon terminal integrates into
glomeruli at the backside of the olfactory bulb (accessory olfactory bulb) and,
interestingly, the axons with different receptor types construct a glomerulus (in the
main olfactory system, axons with the same receptor type construct a glomerulus).
These studies suggest that multiple chemical compounds can induce the same
phenomenon by activating different types of vomeronasal neurons and they pre-
sumably reach to the same vomeronasal amygdala region in the brain. These
hypotheses need to be further tested in future. The signaling pathway was tradi-
tionally considered to be the one that reaches to hypothalamus and amygdala and
produces changes in the hormone secretion by stimulating GnRH neurons, and thus
stimulating the secretion of luteinizing hormone (LH). However, as written above,
recent studies have shown that the signaling pathway that reaches to GnRH neurons
and thus most likely related to primer effects is the main olfactory system (Yoon
et al. 2005). Instead, there are some recent studies showing that pheromones
involved in releasing effect are detected by the vomeronasal system (Kimoto et al.
2005; Chamero et al. 2007; Haga et al. 2010; Papes et al. 2010).
Pheromone receptor gene family was first reported in 1995 (Dulac and Axel
1995). So far there are two groups of vomeronasal receptor genes classified, V1R
and V2R, from the type of G protein expressed, i.e., Gαi2 and Gαo (Matsunami and
Buck 1997; Buck 2000), respectively, which are both different G protein alpha
subunit from those expressed in the main olfactory system (Sullivan 2002).
Vomeronasal neurons with V1R receptors are located in the apical layer of the
vomeronasal organ and that with V2R receptors are located in its bottom layer. The
region in the accessory olfactory bulb that the axon terminals reach is also different.
Those from V1R type vomeronasal neurons reach to the rostral area of the acces-
sory olfactory bulb and those from V2R type vomeronasal neurons reach to its
posterior area. From the accessory olfactory bulb, the signaling reaches to four
nuclei of the limbic system, the medial (MeA/MeP) and posteromedial amygdaloid
cortical nucleus (PMCN), and bed nucleus of the accessory olfactory tract (NAOT)
and posteromedial bed nucleus of the stria terminalis (BNST) (Yoon et al. 2005;
Dulac and Wagner 2006; Rodriguez and Boehm 2008; Baum and Kelliher 2009)
(Fig. 2.3). From these areas, the neurons relay to hypothalamic nuclei (medial
preoptic area (MPOA), and ventromedial hypothalamus (VMH), and premammilary
and supraoptic nuclei) (Dulac and Wagner 2006). It is known that most of the
volatile and semi-volatile pheromones identified to induce or suppress estrus in
female mice stimulate V1R neurons and nonvolatile major urinary proteins
Fig. 2.3 Signaling pathway of the main olfactory system (right) and accessory olfactory system
(left) (from Yoon et al. 2005). ACN anterior cortical nucleus of amygdala, AOB accessory olfactory
bulb, BNSTp posterior division of bed nucleus of the stria terminalis, EC entorhinal cortex, MeA
medial amygdaloid nucleus, MOE main olfactory epithelium, MOB main olfactory bulb, OT
olfactory tubercle, PLCN posterolateral cortical amygdaloid nucleus, PMCN posteromedial
cortical amygdaloid nucleus, TT tenia tecta, VNO vomeronasal organ, scale bars = 50 um
2.3 Accessory Olfactory System (Vomeronasal System) 17
(MUP) and MHC class 1 peptides stimulate V2R neurons (see Chap. 3 for the
details of these pheromones) (Tirindelli et al. 2009). In addition, MUP is known to
have strong affinity with a male murine pheromone 2-sec-butyl-4,5-dihydrothiazole
(SBT) (Zidek et al. 1999; Sharrow et al. 2002), which is one of the volatile male
pheromone that stimulates V1R (Tirindelli et al. 2009) and induce estrus in adult
females (Jemiolo et al. 1986). Binding of SBT with the large nonvolatile MUP
delays the loss of function of SBT as a pheromone due to evaporation and dis-
persion (Hurst and Beynon 2004).
2.4 Responses to Pheromone Are not Always the Same
In the history of studies on olfactory sense, it has been generally considered that
olfactory sense is stronger in females and also generally known that people will lose
sensitivity to smell by ageing. Changes in the olfactory sense in the same person
have been considered to happen also by menstrual cycles but mechanistic details
had not been known yet. A new study using mice led by Lisa Stowers has shown
recently (Dey et al. 2015) that the sensitivity to detect male pheromone changes in
female mice along their estrous cycles, i.e., they become more sensitive during
estrous stage and less sensitive during post- and di-estrous stages.
The study first showed that, when female mice were in estrus, they showed
preference to one of the two rooms of the test apparatus where there was a male
pheromone MUP (see Chap. 4) absorbed in a blotting paper on a wall, but they did
not show the preference when they were in di-estrus (Fig. 2.4a). Such differences in
the behavior could be explained in a way that they detected the smell of male
pheromone but they were ignoring it because they were not in reproductively active
state. However, the study showed that the differences were at the sensory system
level, and the number of vomeronasal neurons that became activated indicated by
calcium influx following exposure to recombinant MUP (rMUP), was significantly
higher in the neurons obtained from estrous females than those from di-estrous
females (Fig. 2.4b).
As these changes took place along the estrous cycles, Stowers group hypothe-
sized that maybe the sex hormones were involved in modifying the responses. They
isolated vomeronasal neurons from ovariectomized mice, added either estrogen or
progesterone in the culture buffer, and exposed them to rMUP (Fig. 2.5a). When
there was nothing added in the culture buffer, the level of responses to rMUP was
not different from the neurons obtained from females in estrus state. When estrogen
was added, the level of response was again not different. When progesterone was
added at the level of females in estrus, the level of responses was similar, however,
when progesterone was added at the high level of progesterone in the females in
di-estrous status, the level of responses dropped.
The Stowers group then conducted a thorough investigation of the gene
expression in the vomeronasal organ and found that progesterone receptor
membrane-component 1 protein (PGRMC1) was expressed. To determine if
Fig. 2.4 Responses to rMUP (modified from Dey et al. 2015). a Preference to the space with the
odor of rMUP was high in females in estrus compared to those in di-estrus. b Percentage of
vomeronasal neurons (VN) activated by exposure to rMUP was high in the VNs from females in
estrus than in di-estrus. Blank bar estrous female or VN from estrous female, black bar: di-estrous
females or VN from di-estrous females, **P < 0.01. ****P < 0.00001
Fig. 2.5 Responses to rMUP in the estrogen or progesterone treated VNs from ovariectomized
female mice (modified from Dey et al. 2015). a VNs from ovariectomized females treated with
estrogen (E2) or treated with progesterone (P4) at basal level or at the level of females in di-estrous
stage. b VNs from ovariectomized females of transgenic mice that lack progesterone receptors
(PGRMC1−/−) or its wild-type (+/+) treated with/without P4 at di-estrous level. ****P < 0.00001,
ns not significant
2.4 Responses to Pheromone Are not Always the Same 19
PGRMC1 is involved in the suppression of the responses of vomeronasal neurons

to rMUP, they added PGRMC1 antagonist, A205, in the culture buffer for
vomeronasal neurons, and then added progesterone at a high concentration as the
females in di-estrous status and exposed the neurons to rMUP. They found that the
suppressive impact of progesterone was avoided, i.e., it was determined that pro-
gesterone is involved in the suppression of the responses of vomeronasal neurons to
rMUP at di-estrous status. They also used transgenic mice, which lack PGRMC1
and found that these mice showed preference to the smell of rMUP at any stage of
estrous cycle. The vomeronasal neurons from these knockout mice also did not
show suppressed responses of activation when they were exposed to rMUP
(Fig. 2.5b). These results indicate that the change of responses of the vomeronasal
neurons to rMUP is regulated by progesterone and these changes are affecting the
preference behaviors to the odor.
Other than the responses to pheromones, recent studies by a group led by Jörg
Strotmann of Hohenheim University have also found that the sensitivity of olfaction
becomes stronger when mice are hungry (Loch et al. 2015). When we feel hungry,
the smell of food always seem to be strong and it is easy to think that it is because
you are looking for food and you are thinking of food, i.e., not a change at the
sensory system but a change in the cognitive system. However, these studies
showed that it was a change in the olfactory system itself and that these changes
were regulated by a hormone, ghrelin. Ghrelin was discovered in 1999 as a ligand
for a receptor called growth hormone secretagogues (GHS). It is a 28 amino acid
peptide that is secreted in the stomach by neuroendocrine cells into the circulation
system (Inui et al. 2004). The secretion increases in hunger condition and it is
known to be involved in feeding behaviors, memory, and antidepressant effects
(Kojima et al. 1999; Sakata and Sakai 2010). The concentration of ghrelin is
considered to be high in the hypothalamus but the receptors are activated most at
the stomach (Inui et al. 2004). Strotmann group found that ghrelin receptor gene,
ghsr1, was expressed in the olfactory system. They also tested if ghrelin was
secreted locally at the olfactory system or if it reached there through circulating
system by testing the gene expression of preproghrelin, which produces ghrelin by
posttranslational process of prohormone convertase (PC)1/3 (Walia et al. 2009), and
found that ghrelin was not produced locally at the olfactory system. Then they
found that, when mice were exposed to odorants (benzaldehyde, 2,3-hexanedione,
or 1-heptanal) for one hour after ghrelin (1 uL of ghrelin diluted in water to 1 nM)
was applied directly to the nasal epithelium of a mouse, the expression of imme-
diate early gene, Egr1, was higher (Loch et al. 2015). Studies using clawed frog
(Xenopus) larvae also showed that the calcium influx in the olfactory sensory
neurons in response to exposure to odorants (mixture of 19 amino acids, which has
been known to be food odorants for aquatic animals) was higher when they were
kept on hunger condition for 6 h or 12 h than when they were fed ad libitum
(Breunig et al. 2010). They also found that one of the endocannabinoids,
2-arachidonoylglycerol (2-AG), and one of the enzymes that is involved in the
synthesis of 2-AG, i.e., diacylglycerol lipase alpha (DAGlα), was expressed more in
the olfactory epithelium when the frog larvae is on 6 or 12 h’ hunger conditions.
When the antagonist to DAGlα, RHC80267, was added to the cultured olfactory
epithelium tissue and it was exposed to the food odorant, the calcium influx of the
olfactory sensory neurons was suppressed, suggesting the role of 2-AG in the
changes of responses. These studies show that the sensitivity of olfactory sense
becomes modified, becoming more sensitive in a situation where there are needs in
the things that the odor source represents, whether it is food or it is a male to mate,
and that, in case of mice, hormone has significant role in regulating the sensitivity.
2.5 Sex Differences in the Responses to Pheromones
The gender differences in the responses to pheromones have been reported in many
studies. The most thorough studies have been conducted by collaborative studies by
James Cherry and Michael Baum groups of Psychology Department and Biology
Department, respectively, both from Boston University. Studies so far have shown
that there are sex differences in the immediate gene c-fos expression in the
vomeronasal organ and medial amygdala when mice were exposed to male-soiled
bedding, i.e., females showed more expression than males (Baum 2012). They also
showed that the expression of c-fos was not different between males and females
when they were exposed to the volatile components of urine from males and
females (Martel and Baum 2007). The results of these studies suggest that the odor
of the same sex may not have impact on vomeronasal pathway but they may have
through the main olfactory system.
In our studies on the influences of exposure to synthetic analogs of murine
pheromones on cell proliferation in the brain, we found that the exposure to the
pheromones of the opposite sex enhanced cell proliferation both in males and
females but it did not affect it when mice were exposed to the pheromones of the
same sex (Koyama et al. 2013, 2014). However, earlier studies have shown that
when female mice were kept in groups their estrous cycles become suppressed
(Lee-Boot effect) (Lee and Boot 1955, 1956) and this is caused by the female
pheromones (Ma et al. 1998), which is an example that the physiological conditions
of females are under the control of the pheromones from the same sex and that
indicates that the pheromones of the same sex are detected and processed to cause
changes. I have also shown in my early studies that when male mice were housed
and established social status, the sperm activity was lower in the subordinate mice
(Koyama and Kamimura 1999). Such differences diminished when the vomeronasal
organ was removed (Koyama and Kamimura 2003), suggesting that the differences
were mediated by pheromones. These studies suggest that pheromones of the same
sex do have influences. It is possible that the concentration to affect the same sex
need to be higher or also that the reproductive organs are more sensitive than the
brain to cause changes.
References 21
References
Baum MJ (2012) Contribution of pheromones processed by the main olfactory system to mate
recognition in female mammals. Front Neuroanat 6. doi:10.3389/fnana.2012.00020
Baum MJ, Kelliher KR (2009) Complementary roles of the main and accessory olfactory systems
in mammalian mate recognition. Annu Rev Phys 71:141–160. doi:10.1146/annurev.physiol.
010908.163137
Baum MJ, Cherry JA (2014) Processing by the main olfactory system of chemosignals that
facilitate mammalian reproduction. Horm Behav 68:53–64
Boehm U, Zou Z, Buck LB (2005) Feedback loops link odor and pheromone signaling with
reproduction. Cell 123:683–695
Breer H, Fleischer J, Strotmann J (2006) The sense of smell: multiple olfactory subsystems. Cell
Mol Life Sci 63:1465–1475
Brennan PA, Zufall F (2006) Pheromonal communication in vertebrates. Nature 444:308–315
Breunig E, Manzini I, Pischitelli F, Gutermann B, Di Marzo V, Schild D, Szesnik D (2010) The
endocannabinoid 2-arachidonoyl-glycerol controls odor sensitivity in larvae of enopus laevis.
J Neurosci 30(26):8965–8973
Buck L, Axel RA (1991) A novel multigene family may encode odorant receptors: a molecular
basis for odor recognition. Cell 65:175–187
Buck L (2000) The molecular architecture of odor and pheromone sensing in mammals. Cell
100:611–618
Chamero P, Marton TF, Logan DW, Flanagan K, Cruz JR, Saghatelian A, Cravatt BF, Stowers L
(2007) Identification of protein pheromones that promote aggressive behaviour. Nature
450:899–902
Dey S, Chamero P, Pru JK, Chien MS, Ibarra-Soria X, Spencer KR, Logan DW, Matsunami H,
Peluso JJ, Stowers L (2015) Cyclic regulation of sensory perception by a female hormone
alters behavior. Cell 16(6):1334–1344
Dulac C, Axel R (1995) A novel family of genes encoding putative pheromone receptors in
mammals. Cell 83:195–206
Dulac C, Wagner S (2006) Genetic analysis of brain circuits underlying pheromone signaling.
Annu Rev Genet 40:449–467
Fleischer J, Breer H, Strotmann J (2009) Mammalian olfactory receptors. Front Cell Neuroci 3
(article 9)
Haga S, Hattori T, Sato T, Sato K, Matsuda S, Kobayakawa R, Sakano H, Yoshihara Y, Kikusui T,
Touhara K (2010) The male mouse pheromone ESP1 enhances female sexual receptive
behavior through a specific vomeronasal receptor. Nature 466:118–122
Halem A, Cherry JA, Baum MJ (1999) Vomeronasal neuroepithelium ad forebrain fos responses
to male pheromones in male and female mice. J Neurobiol 39(2):249–263
Hurst JL, Beynon RJ (2004) Scent wars: the chemobiology of competitive signaling in mice.
BioEssays 26(12):1288–1298
Inui A, Asakawa A, Bowers CY, Mantovani G, Laviano A, Meguid MM, Fujimiya M (2004)
Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ.
The FASEB J. 18:439–456
Jemiolo B, Harvey S, Novotny M (1986) Promotion of the Whitten effect in female mice by
synthetic analogues of male urinary constituents. Proc Natl Acad Sci USA 83:4576–4579
Kang N, Baum MJ, Cherry JA (2009) A direct main olfactory bulb projection to the ‘vomeronasal’
amygdala in female mice selectively responds to volatile pheromones from males. Eur J
Neurosci 29:624–634
Kimoto H, Haga S, Sato K, Touhara K (2005) Sex-specific peptides from exocrine glands
stimulate mouse vomeronasal sensory neurons. Nature 437:898–901
Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K (1999) Ghrelin is a
growth-hormone-releasing acylated peptide from stomach. Nature (London) 402:656–660
Koyama S, Kamimura S (1999) Lowered sperm motility in mice of subordinate social status. Phys
Behav 65:665–669
Koyama S, Kamimura S (2003) Effects of vomeronasal organ removal on the sperm motility in
male mice. Zool Sci 20:1355–1358
Koyama S, Soini HA, Foley J, Novotny MV, Lai C (2013) Stimulation of cell proliferation in the
subventricular zone by synthetic murine pheromones. Front Behav Neurosci. doi:10.3389/
fnbeh.2013.00101eCollection
Koyama S, Soini HA, Foley J, Novotny MV, Lai C (2014) Pheromone-induced cell proliferation
in the murine subventricular zone. Biochem Soc Trans 42(4):882–885
Lee S, van der Boot LM (1955) Spontaneous pseudopregnancy in mice. Acta Phys Pharmacol
Neerl 4:442–443
Lee S, van der Boot LM (1956) Spontaneous pseudopregnancy in mice II. Acta Phys Pharmacol
Neerl 5:213–214
Loch D, Breer H, Strotmann J (2015) Endocrine modulation of olfactory responsiveness: effects of
the orexigenic hormone ghrelin. Senses, Chem. doi:10.1093/chemse/bjv028
Ma W, Miao Z, Novotny MV (1998) Role of the adrenal gland and adrenal—mediated
chemosignals in suppression of estrus in the house mouse: the Lee-Boot effect revisited. Biol
Reprod 59:1317–1320
Martel KL, Baum MJ (2007) Sexually dimorphic activation of the accessory, but not the main,
olfactory bulb in mice by urinary volatiles. Eur J Neurosci 26(2):463–475
Matsunami H, Buck LB (1997) A multigene family encoding a diverse array of putative
pheromone receptors in mammals. Cell 90:775–784
Papes F, Logan DW, Stowers L (2010) The vomeronasal organ mediates interspecies defensive
behaviors through detection of protein pheromone homologs. Cell 141(4):692–701
Rodriguez I, Boehm U (2008) Pheromone sensing in mice. Results Probl Cell Differ 77–96.
doi:10.1007/400_2008_8
Sakata I, Sakai T (2010) Ghrelin cells in the gastrointestinal tract. Int J Pept 2010 (Article ID
945056)
Sharrow SD, Vaughn JL, Zidek L, Novotny MV, Stone MJ (2002) Pheromone binding by
polymorphic mouse major urinary proteins. Protein Sci 11(9):2247–2256
Spehr M, Spehr J, Ukhanov K, Kelliher KR, Leinders-Zufall T, Zufall F (2006) Parallel processing
of social signals by the mammalian main and accessory olfactory systems. Cell Mol Life Sci
63:1476–1484
Sullivan SL (2002) Mammalian chemosensory receptors. NeuroReport 13:A9–A17
Tirindelli R, Dibattista M, Pifferi S, Menni A (2009) From pheromones to behavior. Physiol Rev
80:921–956
Von Campenhausen H, Mori K (2000) Convergence of segregated pheromonal pathways from the
accessory olfactory bulb to the cortex in the mouse. Eur J Neurosci 12:33–46
Walia P, Asaki A, Kieffer TJ, Johnson JD, Chanoine J-D (2009) Ontogeny of ghrelin, obestatin,
preproghrelin, and prohormone convertases in rat pancreas and stomach. Ped Res 65:39–44
Xu F, Schaefer M, Kida I, Liu N, Rothman DL, Hyder F, Restrepo D, Shepherd GM (2005)
Simultaneous activation of mouse main and accessory olfactory bulbs by odors or pheromones.
J Comp Neurol 489(4):491–500
Yoon H, Enquist LW, Dulac C (2005) Olfactory inputs to hypothalamic neurons controlling
reproduction and fertility. Cell 123:669–682
Zhang X, Firestein S (2002) The olfactory receptor gene superfamily of the mouse. Nat Neurosci 5
(2):124–133
Zidek L, Stone MJ, Lato SM, Pagel MD, Miao Z, Ellington AD, Novotny MV (1999) NMR
mapping of the recombinant mouse major urinary protein I binding site occupied by the
pheromone 2-sec-butyl-4,5-dihydrothiazole. Biochemistry 38(31):9850–9861
Chapter 3
The Primer Effects
Abstract The discovery of primer effects in mice began in the 1950s. The findings
at that time were all made with female mice. When female mice were housed in
groups without the odor of males, their estrous cycles were extended (Lee–Boot
effect) resulting in less frequently coming into estrus. Exposure to male odor
reversed this, i.e., the females with longer estrous cycles returned to short estrous
cycle length (Whitten effect). This influence of male odor was stronger when the
females were kept in larger group sizes. Influences of male odors on females were
also observed to accelerate the timing of vaginal opening in female pups
(Vandenbergh effect), such that their vaginal opening occurred earlier following
exposure to male odor. These influences of male odors on the estrous cycles of
females and timing of puberty suggested that sex hormone secretion is stimulated
by the odor of opposite sex. Another primer effect found in the 1950s was the
disruption of the establishment of pregnancy by exposure to the odor of an unfa-
miliar male (Bruce effect). Similar observations in male mice were not to occur until
much later. For example, the sperm density in males exposed to female-soiled
bedding was higher and sperm motility of subordinate males was low. This sug-
gested that the influence was mediated by the odor of the dominant male. Primer
effects are found in both males and females affecting reproductive status.

Keywords Lee–boot effect Whitten effect Vandenbergh effect Bruce effect

Sperm density Sperm motility in males
3.1 The Lee–Boot Effect, Whitten Effect,

and Vandenbergh Effect
The early findings have been called by the name of the people who found the
phenomena. Lee and van der Boot were the first to find a primer effect. They
noticed that when females are kept in groups without access to males’ odor, the
estrous cycle became longer and this was called Lee–Boot effect (Lee and van der
Boot 1955, 1956). Estrous cycle has four stages, i.e., proestrus, estrus, post-estrus,

24 3 The Primer Effects
and diestrus. The shortest duration of estrous cycle is 4 days and females can come
into estrus every 4 days. When females are kept singly housed or 2 females per
cage, they will show this short estrous cycle. However, when the group size become
larger, the estrous cycle becomes longer and females come into estrus less fre-
quently. The larger the group size is, the longer the estrous cycle becomes. In my
previous experiments, when females were kept either singly, 4 females per cage, or
12 females in a cage, the estrous cycle length showed positive correlation with the
group size. Besides, of the 12 females kept in one cage, 25 % even lost the estrous
cycle and stayed in diestrous stage (see Sect. 3.3.2). Later it was found that there
are some chemical compounds that are secreted significantly more in the females
kept in larger group size which was responsible for the longer estrous cycle in
group-housed females.
After the finding by Lee and van der Boot, Wesley Kingston Whitten found that
when these females are exposed to males’ odor, the estrous cycle becomes short and
“reset” making most of the females come into estrus 2–3 days later, and showing
synchronization of the cycle (Whitten 1956, 1958). This was called Whitten effect.
Later, the chemical compounds responsible to Whitten effect were identified in
male murine urine. What these studies suggested is that the odor of males stimulates
hormone secretion in females and turns them into a sexually receptive physiological
condition. Following the finding by Whitten, John Vandenbergh found that the
timing of puberty also become affected by the odor of males. The males’ presence
and odor were found to accelerate puberty of female pups, which was called
Vandenbergh effect (Vandenbergh 1967, 1969). Studies have shown that
Vandenbergh effect does not occur in the females when the vomeronasal organ was
removed (Lomas and Keverne 1982) indicating the critical involvement of
vomeronasal system in Vandenbergh effect. The studies on Lee–Boot effect,
Whitten effect, and Vandenbergh effect suggest that males’ odor stimulates secre-
tion of sex hormone in females, which produces induction of estrus in adult females
and acceleration of puberty in juvenile females. They also show that females’ body
condition related to reproduction becomes suppressed or decelerated without males’
odor, which is an adaptive change when there are no males to mate in the
environment.
3.2 Bruce Effect
Another well-known phenomenon in primer effects found in the early days is the
Bruce effect. Hilda Bruce found that when female mouse was placed with an
unfamiliar male mouse immediately after she mated, the female could not establish
the pregnancy (Bruce effect) (Bruce 1959; Parkes and Bruce 1961). If the
replacement of males took place after the pregnancy was established around 4 days
after mating, the pregnancy continued. The influence of unfamiliar male on the
disturbance of establishing pregnancy did not require the existence of the male itself
but only the soiled bedding worked similarly, which indicated that it was mediated
3.2 Bruce Effect 25
by the odor of the male. Studies later have determined the most sophisticated
mechanisms involved in this phenomenon. As written in Whitten effect, males’
odor stimulates females to come into estrus indicating that it enhances the secretion
of estrogen, which increases at the proestrous stage in the estrous cycle. When
pregnancy is established, increase in the secretion of prolactin and progesterone
takes place, and, if estrogen secretion is increased by the exposure to males’ odor,
this disturbs the establishment of pregnancy, which means that mice cannot have
offspring unless the stimulation of estrogen secretion is somehow suppressed when
females mated.
Studies from the 1980s by the research group led by Barry Keverne of
Cambridge University have determined that, in mice, a temporary olfactory
memory of the stud that lasts between 30–50 days mediates the temporary sup-
pression of estrogen secretion to the odor of males that females mated, and that if
the female gives birth the memory resets even if the days between is less than 30–
50 days. Figure 3.1 shows the results of experiments showing the occurrence of
Bruce effect in several different experimental conditions (Kaba et al. 1988). Bruce
effect was found to more likely occur when the odors of the male mice are different
Fig. 3.1 Recognition of stud measured by failure to induce Bruce effect (from Kaba et al. 1988).
If females were mated with Balb/c male and immediately disrupted, the establishment of
pregnancy by an exposure to F1 male bedding, and then remate with F1 male and exposed to
Balb/c male, Bruce effect due to exposure to the Balb/c male did not happen if the exposure to the
Balb/c male after remating with F1 male took place within 30 days after the first mating with
the Balb/c. However, Bruce effect happened if the remating was after 50 days. This indicates the
memory of the Balb/c continued for 30 days but not 50 days. When the female was mated with the
Balb/c male and gave birth, remated with F1 male and exposed to Balb/c male, the exposure to
Balb/c male after the remating caused Bruce effect. This suggests the role of estrogen at
post-parturition in destroying the memory
and, in case of inbred mice, males from different strains were more effective in
inducing Bruce effect. In the experiments in Fig. 3.1, two different strains of mice,
Balb/c and the F1 of cross between CBA/2 and C57BL/6 were used. When females
were mated with Balb/c male and exposed to F1 male’s bedding, the female will not
establish pregnancy (Bruce effect). When this female is mated with a F1 male later
and then reexposed to Balb/c, Bruce effect does not happen if the reexposure to
Balb/c male is 10, or 20, or 30 days after the first mating with the Balb/c male.
However, if the reexposure to Balb/c male is 50 days after the first mating with the
male, Bruce effect occurs showing that the memory is lost during the 50 days. If the
female is mated with Balb/c male and the pregnancy is established and delivery
takes place, and then she is remated with F1 male and then reexposed to Balb/c
male, Bruce effect occurs even if the period is less than 30 days. This indicates that
parturition makes a reset of the memory of the male earlier. In case of mice, it is
well known that post-parturition estrus takes place and females mate on the day of
their delivery a few hours after the delivery of the first litter. This is caused by the
surge of estrogen at the time of parturition. The study group of Keverne (Kaba et al.
1988) conducted silastic implantation of beta estradiol and found that the increased
estrogen level resets the memory of the mated male and it will make the male
capable of inducing Bruce effect within 30 days. These results indicate that estrogen
surge around the time of parturition can be involved in the reset of the memory of
odor of the stud male.
The results above show that female mice are using a sophisticated method to
enable pregnancy using the memory of the odor of males they mated. Changes in
the secretion of estrogen by exposure to unfamiliar male were considered to be the
key factor inducing Bruce effect, whereas the secretion of prolactin was found to be
the key factor in avoiding Bruce effect. When females recently mated were exposed
to bedding soiled by unfamiliar male twice per day for 4 h each time coinciding
with the nocturnal and diurnal surges of prolactin, 80 % of these females showed
Bruce effect. Moreover, when these females were injected with dopamine agonist
(bromocriptine; 2 mg/kg body weight), which suppress prolactin secretion; it
caused Bruce effect in 90 % of recently mated females without the exposure to
unfamiliar males (Rosser et al. 1989). In the establishment of pregnancy, the surge
of prolactin and progesterone is known to be inevitable. If prolactin secretion drops,
the secretion of progesterone decrease and estrogen secretion increase, and the
female will obtain the cyclic changes of estrus again losing the possibility of
establishing pregnancy. This was considered to be the process of Bruce effect.
A question here is how the memory system works to avoid the Bruce effect.
Keverne group has found the role of interneurons in the accessory olfactory bulb
in avoiding Bruce effect. They first found that it is necessary for the females to be
with the stud for 4 to 5 h postcoitus during which she established the memory of the
odor of the males and vaginocervical stimulation during mating stimulated the
secretion of noradrenaline (NA) in their accessory olfactory bulbs (Rosser and
Keverne 1985). NA is secreted mostly in the granule cell layer in the olfactory bulb
and functions inhibitory to the mitral cells, which relays the signaling from the axon
terminals of olfactory/vomeronasal neurons to brain (Halasz and Shepherd 1983).
3.2 Bruce Effect 27
The experimental suppression of NA by infusion of NA antagonist in the accessory

olfactory bulb was found to disturb the establishment of memory of the stud (Kaba
and Keverne 1988). NA secretion is considered to stimulate release of
gamma-aminobutyric acid (GABA) in the granule cells, which have synapses on
the mitral cells in the olfactory bulb. It was found that microinfusion of GABA
itself, without exposure to unfamiliar male after mating induces Bruce effect (Kaba
and Keverne 1988) showing the key role of GABA in the effect. These studies show
that the increase in NA following vaginocervical stimulation during mating sup-
presses the secretion of GABA, which inhibits the signal transduction from the
vomeronasal neurons to reach to amygdala/hypothalamus by being exposed to the
odors of the stud. And the lack of signal transduction avoids the increase in estrogen
secretion by the exposure to the odors of the stud male, thus avoiding induction of
Bruce effect. This decrease in the response was confirmed with immunohisto-
chemistry in the percentage of neurons showing c-fos expression in the arcuate
dopaminergic neurons response to the stud’s soiled bedding. When females were
exposed to the soiled bedding of males they mated, the expression of c-fos was
similar to the control level of virgin females, whereas when they were exposed to
unfamiliar males’ soiled bedding it was as high as the level that virgin females
showed when they were exposed to male-soiled bedding (Matthews et al. 2013).
The Bruce effect is thus a block of establishment of pregnancy by the increase of
estrogen and decrease of prolactin and progesterone. After the pregnancy is
established, Bruce effect does not take place. If the stud is replaced with unfamiliar
male after 4 days, Bruce effect does not take place and the process of pregnancy
continues. However, at this time period, still early in pregnancy, progesterone still
has critical role in maintenance of the progress of pregnancy. A recent study has
shown that the lack of Bruce effect after the establishment of pregnancy may be due
to the less functional olfaction in the main olfactory system because of the increased
dopamine (Serguera et al. 2008). This study was the first to show that main
olfactory system can be involved in memorizing the odor of the stud to avoid
abortion. They found that the number of tyrosine hydroxylase positive cells
involved in dopamine synthesis increased in the juxtaglomerular dopaminergic
interneurons after mating, reached to plateau of increase 4.5 days after mating, and
returned to original level at 12.5 days after mating. Level of dopamine in mated
females became higher than virgin females from 2.5 days after mating. The time
length of sniffing was less in the females 6.5 days after mating compared to 0.5 days
after mating and compared to virgin females in estrus. The expression of immediate
early gene c-fos in the glomerular layer, mitral layer, and granular layer in the main
olfactory bulb after exposure to male-soiled bedding was high in virgin females in
estrus compared to virgin females in estrus without exposure to male-soiled bed-
ding. Pregnant females, 4.5 days after mating, exposed to male-soiled bedding
showed significantly less c-fos expression in these layers compared to virgin female
in estrus, and there was no significant difference between them and virgin females
in estrus without exposure to male-soiled bedding (Serguera et al. 2008). Whether
this difference is due to the less sniffing time or due to the less functional olfactory
system is not determined yet.
The avoidance of Bruce effect is mediated by memorizing the odor of the stud
male. This indicates that there is no specific pheromone compound that can be
universally used to avoid Bruce effect. Instead, it suggests the role of chemical
compounds that specify individualities, like the odor that are regulated by genes of
major histocompatibility complex (MHC), which is known to determine the odor
specific to each individual (see Chap. 4). In 2004, it was reported that MHC class I
peptides could function as the ligands to activate the signaling that induced Bruce
effect (Leinders-Zufall et al. 2004). When female mice mated with males of
C57BL/6 strain were exposed to MHC peptide specific to different strain, BALB/c
(H-2d, namely, SYFPEITHI for H-2d haplotype), it caused Bruce effect at high
percentage, but when they were exposed to MHC peptide of their own strain (H-2b,
namely, AAPDNRETF for H-2b haplotype), it did not cause Bruce effect. These
peptides are detected by the vomeronasal neurons in the basal layer of vomeronasal
organ. The vomeronasal neurons are classified into two types by the gene families
of receptors they carry, V1R and V2R. Vomeronasal neurons with V1R type
receptor are located in the surface layer in the vomeronasal organ, whereas those
with V2R are located in the basal layer. They extend their axons in the apical area
of accessory olfactory bulb (AOB) and posterior area of AOB, respectively, as well.
The V1R type vomeronasal neurons are also known to detect volatile pheromones
which are involved in Lee–Boot effect, Whitten effect, and Vandenbergh effect,
whereas V2R type neurons respond to nonvolatile proteins and peptides like MHC
class I peptides, ESP1 (male-specific peptide in tears), and major urinary proteins
(MUP) (Tirindelli et al. 2009). Some of the V2R neurons, specifically, these with
their cell body in the basal subdivision of the basal layer of the vomeronasal organ
coexpress a family of nine nonclassical class I Mhc genes, the H2-Mv genes (Ishii
and Mombaerts 2008). These neurons, V2rf2 neurons, were also found to extend
their axons to the posterior region of the posterior part of the AOB as well (Ishii and
Mombaerts 2008). Recent studies have shown that vomeronasal neurons detect
pheromones at extremely low concentration, i.e., “ultrasensitive chemodetection”
(Leinders-Zufall et al. 2000). However, without functional H2-Mv gene expressed
in the V2rf2 neurons, the sensitivity of the neurons becomes low, and requires
higher concentration to detect chemicals, although they do not totally lose their
function to detect (Leinders-Zufall et al. 2014). It is possible that these neurons that
carry H2-Mv genes are involved in the detection and discrimination of the odor of
the stud and may be involved in memorizing their odor. However, it is still possible
that there are some other mechanisms to discriminate the odor of stud male without
using the vomeronasal neurons as well. Studies have shown that mice with
genetically ablated vomeronasal system (TRPC-/-) still showed Bruce effect,
whereas, when the vomeronasal system was mechanically destroyed (vomeronasal
ectomy), Bruce effect did not take place (Kelliher et al. 2006). Electrophysiological
experiments found that the microvillous layer of sensory epithelia of the vomer-
onasal organs from TRPC2-/- mice can still show action potential responses to
MHC peptide ligands (Kelliher et al. 2006). This suggests that there are some
undetermined signal transduction mechanisms to detect MHC in the vomeronasal
organ, which is independent of the vomeronasal neurons.
3.3 Primer Effect in Males 29
3.3 Primer Effect in Males
Ideas from mouse biology and cell biology produced a series of new findings, just like a
fusion of atoms giving off an enormous amount of energy
—Shinji Kamimura
The University of Tokyo has been a research institute with long history of extensive
scientific activities in the study field of spermatology with a number of world-class
scientists working on biophysics, biochemistry, or cell biology studies of sperm
cells of various animal species, from invertebrates to vertebrates. It has been well
known by the spermatologists that the motility of sperm (% of motile sperm) is
homogenously high in invertebrate sperm, whereas mammalian sperm cells often
showed large variance in motility depending on individuals, for unknown reason
(Kamimura, pers. comm.). In the study field of mouse biology, it has been known
that the serum testosterone concentration of the dominant males is higher than that
of the subordinate males. As testosterone is involved in spermatogenesis and sperm
maturation, it is possible that there are differences in the sperm density and sperm
activity (velocity and motility) between dominant and subordinate males. Our study
on the sperm activity and sperm density of male mice depending on their social
status started from the observations of large variance among individuals in mam-
malian sperm and the studies on testosterone concentration depending on social
status.
3.3.1 Social Dominance and Sperm Motility
Males were kept in male-male pairs from weaning because this produced stable
dominant–subordinate relationships (see Chap. 1). The social status of males in
each cage was determined by intruder tests. In mice and rats, it has been known that
the dominant male attacks the intruders in their territories (home cages are the
territories in case of laboratory mice and rats) (Blanchard et al. 1977). We used
several males of the same age as intruder mice and the intruder mice were used only
as intruders and not used in tests to examine sperm activity and sperm density. The
intruder tests were conducted with both of the two resident males present in their
home cages. The procedure was as follows: one intruder male mouse was placed in
the cage for 10 min or until any attack/fight was observed. The mouse (or mice) that
showed attack/fight and the latency until the attack started were recorded. We
conducted intruder tests multiple times to determine the dominant male in each cage
and only the pairs that show clear social dominance were used at the end to examine
sperm activity and sperm density. The criteria of clear social dominance were:
(i) one male attacks the intruder and (ii) no change in the male that attacks the
intruder during the intruder test period. These mean that, when both resident males
attacked the intruder male mouse, or when the existence of intruder stimulated both
resident male to start fighting, the social dominance of these male pairs was
determined to be “not clear/unestablished”. Also, when neither of the resident males
showed attack to the intruder male mouse, the social dominance was determined to
be “not clear/nonaggressive”.
Another key point in these experiments was that the bedding was not changed
during the experimental period. This was strictly kept unchanged to keep the odor
environment stable in the cages. In order to enable this, we used a type of bedding
which had extremely high absorbability (Petline Co., Gifu, Japan). Using this
bedding, inside the cage was usually very dry for 10 weeks and it was possible to
replace only small portion of the bedding that became soaked and too wet with
urine or with water that accidentally dripped from water bottle. In our preliminary
experiments, we found that changing of bedding had a large influence on the results
of sperm. When bedding was changed weekly we did not see differences in the
sperm density or sperm motility between the dominant and subordinate males,
which suggested the significant role of the odor environment in the cage. The time
length of keeping the males before examining the sperm activity and density was
determined considering the duration for spermatogenesis. In case of mice, sper-
matogenesis takes about 50 days and sperm maturation, which is a process that
takes place when the sperm migrates through epididymis and acquires swimming
ability by establishing protein localization on the sperm surface, takes about
12 days.
After the time period of determining social dominance under these strictly
controlled housing conditions, we collected small volume of sperm from cauda
epididymis. Collected sperm was released into Biggers, Whitten and Whittingham’s
buffer (BWW buffer) and observed under phase-contrast microscope with
thermo-plate (Tokai-Hit Co. LtD., Shizuoka, Japan) on its stage, which maintains
the slide glass with sperm at a controlled temperature (37 °C). We video recorded
the images and analyzed the motility, density, velocity, and morphology of the
sperm. Motility was analyzed by placing transparent sheet (overhead projector
sheet) on the monitor screen and moving the video tape frame by frame to precisely
determine the number of sperm cells that showed change in the shape of sperm
flagella between frames, which indicated it was swimming, and recorded them as
“motile” sperm (Manual counting is the most precise method in analyzing sperm
activities—S. Kamimura). The sperm cells that did not show movement in the
flagella were counted as “nonmotile” even if the location itself changed. Some
sperm moved between frames without movements in the flagella and these were
considered to be “floating” and not motile. The velocity of sperm was calculated by
counting the number of frames to swim through a certain distance or measuring the
distance moved during one frame, which is 1/30 s, and converted it to velocity. The
morphology was determined by classifying the sperm cells into “normal” and
“abnormal”, and abnormality was determined to be “lack of head”, “bent head and
flagella”, “abnormal shape of head”, and “existence of cytoplasmic droplet”. Sperm
density was determined by obtaining the whole amount of sperm from cauda epi-
didymis, homogenously dissolving them in a determined volume of saline, counting
the number of cells in a blood cell counter, and converting it into density. As a
Fig. 3.2 Sperm motility

(percentage of motile sperm)
depending on social status
(from Koyama and Kamimura
1999). *0.05 < P < 0.10,
*P < 0.05, ***P < 0.001
result, we found that the sperm motility, but not sperm density or sperm velocity,
was significantly higher in the dominant males (Koyama and Kamimura 1999)
(Fig. 3.2). The differences diminished when the vomeronasal organ of the subor-
dinate males was removed (Koyama and Kamimura 2003a). The variance of sperm
motility in the sperm obtained from males around puberty was small and sperm
motility was generally very high. Sperm starts to reach cauda epididymis from 35 to
40 days old (Koyama and Kamimura 2003b), with some differences depending on
the individual and the strain, and the sperm motility was high with less variance at
this time. Variance in sperm motility became large in full adult males in a way that
some males kept showing high sperm motility as males did around puberty and
some showing drop in it (Koyama and Kamimura 2003b).
3.3.2 Female Odors and Sperm Density
We then examined the influence of exposure to female bedding on sperm density

and sperm activities. For the exposure, shredded filter paper was used as the bed-
ding for females and the female-soiled bedding (control group males received same
amount of clean shredded filter paper) was put in males’ home cages every other
day from 5 weeks in age to 10 weeks in age. Females were kept singly housed and
males were exposed to the soiled bedding of a randomly changed female in order to
avoid any possible influence of specific female’s odor. Other parts in the methods
(keeping two males per cage, intruder tests to examine the social dominance, and
using the same bedding without change) were the same as the first experiment. In
this study, we found that males exposed to female-soiled bedding showed higher
sperm density especially in the dominant males and not in the subordinate males but
sperm motility was not affected by the exposure to female-soiled bedding (Koyama
and Kamimura 2000). Similar phenomenon was found in rats as well, i.e., when
male rats were housed with females, the sperm density was higher (Taylor et al.
1987). Also, the puberty was earlier in male mice when they were housed with adult
females (Vandenbergh 1971).
As housing conditions affect females’ estrous cycle length, and the influence of
females’ odor to males may be different depending on the specific odor profile of
females, we thought that females of different housing conditions might have dif-
ferential influences on males’ sperm density. We kept females in three different
housing conditions, i.e., singly housed, 4 females per cage, and 12 females per
cage. We also mixed the bedding of 12 females per condition and provided same
amount in weight of the bedding to each males’ cage (12 singly housed females’
bedding mixed together or 3 cage of females kept in four mixed together, or one
cage of 12 females’ bedding mixed thoroughly). In this way, all the males received
same amount of female-soiled bedding that contained information of 12 females but
of one of the 3 types of housing conditions. The estrous cycle of females was
determined and we found that singly housed females had the shortest estrous cycle
coming into estrus most frequently, whereas the females that were kept in the
largest group size showed the longest estrous cycle coming into estrus least fre-
quently (Fig. 3.3a). The length of estrous cycle of females kept in four came in the
Fig. 3.3 a Estrous cycle length of females kept isolated, 4 females per cage, and 12 females per
cage. Sizes of cages were adjusted in a way that area size per female in cage is roughly the same.
25 % of the females kept as 12 females per cage did not show changes in estrous cycle and stayed
in diestrous status, and these females were not included in obtaining average estrous cycle length.
Estrous cycle length was significantly longer in the grouped females (F (2,40) = 27.676,
P < 0.001). b Percentage of females in estrus on the day the soiled bedding was collected to be
exposed to males. Percentage of estrous females was significantly higher in the isolated females
(Chi-square = 22.614, df = 2, p < 0.001). ***P < 0.001
middle (Fig. 3.3a). Twenty-five percentage of the 12 females kept in one cage even
did not show estrous cycle at all staying at the diestrus stage. From these differences
in the estrous cycle length, the males exposed to 12 singly housed females’ soiled
bedding were exposed to the odor of females in estrus most frequently, and the
males exposed to the soiled bedding of 12 females in one cage were exposed to the
odor of females in estrus least frequently (Fig. 3.3b shows the estrous status of each
female on the day their soiled bedding was collected). We first expected that odor of
females in estrus might have the strongest impact on males’ sperm density.
However, the results were opposite. The sperm density was lowest in the males
exposed to singly housed females’ soiled bedding and the influences of 12 females
kept in 4 and kept in 12 were both significantly higher than the sperm density of
males exposed to singly housed females (Fig. 3.4). These results suggested that
some pheromone(s) included in the soiled bedding of females kept in group housing
conditions, like 2,5-dimethylpyrazine, strongly enhanced spermatogenesis in males.
It is possible that the odors that inform stressful condition had negative impacts
on males and the females kept in isolation housing might have had some negative
impact on males because of isolation stress. We measured corticosterone level of
the females at each housing condition and found that serum corticosterone level was
highest in the females kept in isolation (Fig. 3.5) suggesting the higher stress in
these females. Females kept in large group size also showed rather high corticos-
terone level, although it was not as high as the isolated females showing no sig-
nificant difference with either the isolated females or with the females kept in small
group size. We also compared the aggressiveness at the intruder tests in the males
exposed to these female-soiled bedding and found that the dominant males exposed
to mixture of 12 isolated females showed low aggressiveness whereas those
Fig. 3.4 Sperm density of

dominant (D) and subordinate
(S) males exposed to the
soiled bedding of 12 females
kept in one cage (hgf),
mixture of soiled bedding of 3
cages of 4 females kept in one
cage (lgf), mixture of soiled
bedding of 12 isolated
females (mix) and soiled
bedding of one isolated
female (if). Group-housed
females had stronger
influence than the isolated
females (Fgroup
(3,63) = 5.811, P = 0.001)
Fig. 3.5 Serum

corticosterone level in the
females of various housing
conditions. There was a
significant difference among
conditions (F2,21 = 3.619,
P = 0.045) and isolated
females showed significantly
higher corticosterone level
than the females kept in 4 per
cage. **P < 0.01
exposed to only one isolated female’s soiled bedding showed high aggressiveness
(Fig. 3.6). We then examined if there are differences in the preferences to the odor
of these different types of soiled bedding by measuring the time length that males
approach and stay close to the soiled bedding. We used naïve males that lack
experiences of exposure to the female-soiled bedding in these tests. The males were
released in an open field where there were 5 containers, each with a small petri dish
inside. The petri dishes had one of the 4 types of soiled bedding (of 12 females kept
Fig. 3.6 Percentage of

aggressive pairs at intruder
tests. The aggressiveness of
males was significantly
different among groups
(Chi-square 8.792, df = 3,
P < 0.05). Males exposed to
soiled bedding of females kept
in larger group and soiled
bedding of only one isolated
female showed significantly
high percentage of
aggressiveness
Fig. 3.7 a Open field test apparatus to examine the preference to female-soiled bedding and clean
bedding and b the time naive males spent close to each type of soiled bedding and clean bedding
during 10 min’ preference tests. Sniffing time: the time length males entered and stayed in the
container. Time with their head and front legs in the container was measured. *P < 0.05,
**P < 0.01
in one group, of 12 females kept in 3 groups (4 females per cage), mixture of 12

isolated female, and soiled bedding of only one isolated female) or clean bedding in
it (Fig. 3.7a). The location of the containers was randomly changed among tests and
the time length and frequency that males entered the container during 10 min’ tests
were measured. We found that males stayed significantly close to the soiled bed-
ding of 4 females per cage (F5,147 = 3.989, P = 0.002) and that of the mixture of 12
isolated females was also preferred than clean bedding (Fig. 3.7b). Males showed
less preference to the soiled bedding of females kept in a large group (12 females
per cage) and to that of only one isolated female (Fig. 3.7b). These results suggest
that the isolation housing could be a stressful housing condition for female mice but
isolation stress did not have negative influences on the behaviors of males. As the
influence of the soiled bedding of females kept in large group was as strong as that
of females kept in small group, and as their stress level was close to isolated
females, it is possible to say that the stress of females did not have negative
influences on sperm density. It is more likely that some female pheromones that are
secreted in group-housed conditions have positive influences.
3.3.3 Influence of Adult Males’ Odor on Sperm Morphology
Although, the studies on the influence of odors (pheromones) in male mice have
been few, there have been studies showing that exposure of juvenile males to the
soiled bedding of adult males has negative influence on their behaviors and sperm
morphology (Novikov et al. 1981, 1984; Aref’ev et al. 1985). When juvenile male
mice (sexual maturation takes place from around 40 days old, although depending
on individual and on strains of mice) were exposed to soiled bedding of adult male
mice (3–4 months old) for only 2 h/day for 10 from 21 days old, the mice showed
less movements in an open field at postnatal day 31 compared to juvenile males
either exposed to clean bedding or no treatment (Novikov et al. 1981). When
juvenile males of postnatal day 31 was exposed to adult male bedding for 2 h for
only one time and killed 2, 4, 6, or 8 h after the exposure, spermatocyte cells at
diakinesis/metaphase I stage from males killed 8 h after the exposure showed higher
percentage of cells with meiotic defects compared to control group males exposed
to clean bedding or no treatment (Novikov et al. 1981). Using olfactometer, 30 days
old juvenile male mice were exposed to volatile odorants from freshly collected
urine of adult males (5–8 month old) for 2 h for one time (Novikov et al. 1984;
Aref’ev et al. 1985). When spermatocytes of diakinesis/metaphase I stage was
collected 8 h after the start of exposure, they found that juvenile males exposed to
odorants from the urine of adult males showed significantly more meiotic distur-
bances (Novikov et al. 1984). When sperm was collected from cauda epididymis 8–
35 days later, the percentage of anomalous sperm was significantly higher in the
mice exposed to volatile odorants from adult males but the differences were
observed after 17 days and not in the males sacrificed 8 days after the exposure to
the odor (Aref’ev et al. 1985). These studies show that the reproductive system of
juvenile males can be negatively affected by the odor of adult males’ odor. They
also suggested that the influences of exposure to adult male on spermatocytes could
be observed 8 h after the exposure and in the sperm 17 days after the exposure.
References
Aref’ev AA, Daev EV, Kaidanov LZ, Lopatina NG, Novikov SN (1985) Anomalous
spermatogenesis in laboratory mice after the influence of volatile compounds contained in
urine of sexually mature males. Dokl Akad Nauk SSSR 291(5):1257–1259
Blanchard RJ, Takahashi LK, Blanchard DC (1977) The development of intruder attack in
colonies of laboratory rats. Anim Learn Behav 5(4):365–369
Bruce HM (1959) An exteroceptive block to pregnancy in the mouse. Nature 184:105
Halasz N, Shepherd GM (1983) Neurochemistry of the vertebrate olfactory bulb. Neuroscience
10(3):579–619
Ishii T, Mombaerts P (2008) Expression of nonclassical class I major histocompatibility genes
defines a tripartite organization of the mouse vomeronasal system. J Neurosci 28(10):
2332–2341
Kaba H, Keverne EB (1988) The effect of microinfusions of drugs into the accessory olfactory
bulb on the olfactory block to pregnancy. Neuroscience 25(3):1007–1011
Kaba H, Rosser AE, Keverne EB (1988) Hormonal enhancement of neurogenesis and its
relationship to the duration of olfactory memory. Neuroscience 24(1):93–98
Kelliher KR, Spehr M, Li X-H, Zufall F, Leinders-Zufall T (2006) Pheromonal recognition
memory induced by TRPC2-independent vomeronasal sensing. Eur J Neurosci 23:3385–3390
Koyama S, Kamimura S (1999) Lowered sperm motility in mice of subordinate social status. Phys
Behav 65:665–669
Koyama S, Kamimura S (2000) Influence of social dominance and female odour on the sperm
activity of male mice. Phys Behav 71:415–422
References 37
Koyama S, Kamimura S (2003a) Effects of vomeronasal organ removal on the sperm motility in
male mice. Zool Sci 20:1355–1358
Koyama S, Kamimura S (2003b) Study on the developmental changes, variance of sperm motility,
and social dominance of male mice. Phys Behav 80:267–272
Leinders-Zufall T, Lane AP, Puche AC, Ma W, Novotny MV, Shipley MT, Zufall F (2000)
Ultrasensitive pheromone detection by mammalian vomeronasal neurons. Nature 405:792–796
Leinders-Zufall T, Brennan P, Widmayer P, ChandramaniP Maul-Pavicic A, Jager M, Li X-H,
Breer H, Zufall F, Boehm T (2004) MHC class I peptides as chemosensory signals in the
vomeronasal organ. Science 306:1033–1037
Leinders-Zufall T, Ishii T, Chamero P, Hendrix P, Oboti L, Schmid A, Kircher S, Pyrski M,
Akiyoshi S, Khan M, Vaes E, Zufall F, Mombaerts P (2014) A family of nonclassical class I
MHC genes contributes to ultrasensitive chemodetection by mouse vomeronasal sensory
neurons. J Neurosci 34(15):5121–5133
Lee S, van der Boot LM (1955) Spontaneous pseudopregnancy in mice. Acta Phys Pharmacol
Neerl 4:442–443
Lee S, van der Boot LM (1956) Spontaneous pseudopregnancy in mice II. Acta Phys Pharmacol
Neerl 5:213–214
Lomas DE, Keverne EB (1982) Role of the vomeronasal organ and prolactin in the acceleration of
puberty in female mice. J Reprod Fert 66:101–107
Matthews GA, Patel R, Walsh A, Davies O, Martinez-Ricos J, Brennan PA (2013) Mating
increases neuronal tyrosine hydroxylase expression and selectively gates transmission of male
chemosensory information in female mice. PLoS ONE 8(7):e69943
Novikov SN, Tsapygina RI, Daev EV, Togo EF (1981) Influenc of natural compounds of biogenic
origin on behavior and reproductive function in the male house mouse Mus musculus. Dokl
Akad Nauk SSSR 262(3):746–748
Novikov SN, Daev EV, Tsapygina RI (1984) Action of volatile urine components on the
generative function of non-sexually mature males of the house mouse Mus musculus L. Dokl
Akad Nauk SSSR 281(6):1506–1508
Parkes AS, Bruce HM (1961) Olfactory stimuli in mammalian reproduction. Science 134:
1049–1054
Rosser AE, Keverne EB (1985) The importance of central noradrenergic neurones in the formation
of an olfactory memory in the prevention of pregnancy block. Neuroscience 15(4):1141–1147
Rosser AE, Remfry CJ, Keverne EB (1989) Restricted exposure of mice to primer pheromones
coincident with prolactin surges blocks pregnancy by changing hypothalamic dopamine
release. J Reprod Fert 87:553–559
Serguera C, Triaca V, Kelly-Barrett J, Al Banchaabouchi M, Minichiello L (2008) Increased
dopamine after mating impairs olfaction and prevents odor interference with pregnancy. Nat
Neurosci 11(8):949–956
Taylor GT, Weiss J, Rupich R (1987) Male rat behavior, endocrinology and reproductive
physiology in a mixed-sex, socially stressful colony. Phys Behav 39:429–433
Tirindelli R, Dibattista M, Pifferi S, Menini A (2009) From pheromones to behavior. Phys Rev
89:921–956
Vandenbergh JG (1967) Effect of the presence of a male on the sexual maturation of female mice.
Endocrinology 81(2):345–349
Vandenbergh JG (1969) Male odor accelerates female sexual maturation in mice. Endocrinology
84(3):658–660
Vandenbergh JG (1971) The influence of social environment on sexual maturation in male mice.
J Reprod Fertil 24:383–390
Whitten WK (1956) Modification of the oestrus cycle of the mouse by external stimuli associated
with the male. J Endocrinol 13:399–404
Whitten WK (1958) Modification of the oestrus cycle of the mouse by external stimuli associated
with the male: changes in the oestrus cycle determined by vaginal smears. J Endocrinol
17:307–313
Chapter 4
Identification of Pheromones
Abstract Identification of the pheromones responsible for these primer effects,

which were started to be found in the 1950s, began in the 1980s. 3,4-Dehydro-
exo-brevicomin (DHB), 2-sec-butyl-4,5-dihydrothiazole (SBT), E,E-α-farnesene,
and E-β-farnesene present in male mouse urine were found to induce the Whitten
effect. The Lee–Boot effect was found to be induced by 2,5-dimethylpyrazine in
female urine. Major urinary proteins (MUPs) were found to bind to SBT and delay
its evaporation. It was also found that MUP attracts females, whereas SBT itself did
not attract females. Recent studies have also found that there are small molecular
weight peptides in the tears that stimulate females to be receptive to males. In the
rabbits there are pheromones from nipples that stimulate rabbit pups to show
rooting behaviors, but studies so far have shown that, in mice, there may be no
pheromones secreted from nipples to induce rooting behaviors. Learning of smells
start from prenatal stage and newly born mouse pups could react to the specific odor
they learned in uterus. It is a process of chemical signaling.
Keywords Pheromones in urine, tears, saliva Rabbit pheromone Embryonic

learning of odors in amniotic fluid
4.1 Pheromones to Induce Primer Effects
Whether it is possible to call a specific chemical compound as a pheromone has

been argued for long time especially in case of chemical communication in
mammals. The word “pheromone” was coined by Karlson and Lüscher in the
middle of twentieth century (Karlson and Lüscher 1959). It was defined as “sub-
stances secreted to the outside by an individual and received by a second individual
of the same species in which they release a specific reaction, for example a definite
behavior or a developmental process.” The studies were based on insects at these
days and the first pheromone identified was called bombykol from silk moth
(Bombyx mori). Debates started when it was found that mammals use chemical
communication and that it is more complicated in case of mammals in several

40 4 Identification of Pheromones
aspects, i.e., other than species-specific chemical compounds, (i) there are chemi-
cals that are “learned” and obtained the function to affect the physiological con-
ditions of others like in Bruce effects and rooting behaviors of rabbit pups and mice
pups, (ii) a study in the 1970s showed that the menstrual cycles in women who
spent their time together became synchronized. Studies from the 1950s have shown
in mice that estrous cycles of female mice become suppressed when they are group
housed without the odors of male mice, and the cycle become shortened and
synchronized when they are exposed to males’ odors. The possibility itself that
humans may be using odors in communication might have been unacceptable at
first, suggesting unexpected continuance to lower creatures. The following sen-
tences clearly address this; “What are we going to do if it turns out that we have
pheromones? What on earth would we be doing with such things? With the richness
of speech, and all our new devices for communication, why would we want to
release odors into air to convey information about anything? We can send notes,
telephone, whisper cryptic invitations, announce the giving of parties, even bounce
words off the moon, and make them carom around the planets. Why a gas, or
droplets of moisture made to be deposited on fence posts?” (Thomas 1974). It was
maybe at first dishonorable to consider the possibility that humans might use odors
in communications, imagining the continuances to dogs which disgustingly urinate
on the fences to send messages to other dogs or further continuances to even lower
creatures like insects leaving smell in the air. There are still debates on whether
humans have functional vomeronasal system (Meredith 2001). As vomeronasal
system was at first considered to be the pathway to cause primer effects, the lack of
accessory olfactory bulb and thus the lack of connectivity to the brain area involved
in changes of hormone secretions became a “positive” proof for people who were
against chemical communication in humans. As written in Chap. 2, it is now
determined that the main olfactory system also conveys pheromone signaling, and
thus the pathway and chemical communication in humans can not be declined by
the lack of functional vomeronasal system. In an article recently published (Wyatt
2009), we see a more positive way of thinking on the possibility of chemical
communication in humans; “As we’re mammals, we are likely to use pheromones.
Our armpits are prime candidates as sources, as their smells develop along with
other changes at puberty” and “A strong contender for the first real human pher-
omone is some compound in women’s armpit extract that apparently causes
menstrual synchrony in females living in close quarters. Its identification is keenly
awaited, not least as it could potentially open the door to sniffable contraceptives.
There may never be a magic potion to make us irresistible, but I’m sure human
pheromones will surprise us yet” (Wyatt 2009).
Studies using mice made an extraordinary progress since the first phenomenon
was found in the 1950s. The development of olfactory neuroscience from the 1990s
enabled us to understand better the brain neural circuitry of olfactory information
other than the signaling process at molecular level at each step of the pathway, i.e.,
the sensory system, the olfactory bulbs, and further at the regions in the brain
involved in the odorant and pheromone signaling. The whole genome project
determined the whole genome in mice, which, with the development of genetic
4.1 Pheromones to Induce Primer Effects 41
engineering, enabled production of various transgenic mice possible to use to

determine the role of various genes involved in olfactory and pheromone signaling.
The development of analytical instruments of chemical compounds enabled us to
further understand the nature of the chemicals involved in chemical communica-
tion. The starting of this, i.e., the identification of murine pheromones, goes back to
the 1980s, when the first murine pheromones were identified. The first pheromones
identified were male murine pheromones, the farnesenes, 3,4-dehydro-exo-
brevicomin (DHB) and 2-sec-butyl-4,5-dihydrothiazole (SBT) to induce estrus in
female mice (Jemiolo et al. 1986; Ma et al. 1999) (Fig. 4.1; Table 4.1). These
pheromones as well as major urinary protein (MUP) (see below) were found to
accelerate puberty in females as well (Novotny et al. 1999). In these studies, direct
exposure to pheromones diluted with water is used as methods and, for puberty
studies, the weights of uterine are used to determine the influences (Novotny et al.
1999). The exposure to pheromones started from postnatal day 21, twice a day of
direct deposit of 30 uL of pheromone solutions (concentration depends on the
pheromone types), or urine from castrated or normal male, or MUP (see below), or
water. Juvenile females were sacrificed at postnatal day 27 and the weights of uterus
were measured. The results showed that the juvenile females exposed to MUP, the
farnesenes, SBT, and DHB had heavier uterus at postnatal day 27 (Novotny et al.
1999).
It was also found that female origin pheromone 2,5-dimethylpyrazine affects the
females to delay their puberty (Jemiolo and Novotny 1994). In these studies, the
pups were exposed to 2,5-dimethylpyrazine from the day of their births until
postnatal day 30. The method of exposing to 2,5-dimethylpyrazine was not direct
deposit on their nostril like the studies written above, but they added 25 uL of
2,5-dimethylpyrazine (2000 ppm) on a cotton ball and placed the cotton ball in a
plastic capsule with small holes, and the capsule was attached to the lid of the home
cages. The mice were exposed to freshly prepared pheromone daily. There were
other groups exposed to either water, male urine, or urine of group housed females.
With this method, they found that the weights of uterus of female pups exposed to
Fig. 4.1 Male murine

pheromones identified to
induce Whitten effect and
female murine pheromone
identified to induce
Vandenbergh effect (from
Dr. Soini and Dr. Novotny by
courtesy)
Table 4.1 Pheromones, their origins, and their detection thresholds in in vitro experiments (from
Leinders-Zufall et al. 2000)
urine of group housed females and 2,5-dimethylpyrazine was less than half of the
weights of female pups exposed to water or male urine. Body weights of these
females were not different.
Studies using synthetic analogos of these pheromones have shown that each of
these pheromones can affect the physiological conditions of mice, respectively.
However, it is also found that MUP binds to some of the male pheromones, for
example SBT and DHB (Robertson et al. 1993; Sharrow et al. 2002; Armstrong
et al. 2005). It is considered that the binding of MUP with small pheromone
molecules may serve to protect these ligands from chemical degradation and also
delay the release and disperse of these small volatile pheromones from the urine
scent marks and thus let them stay in the scent mark site longer (Hurst et al. 1998;
Armstrong et al. 2005). Scent marking is a behavior typical to dominant male mice
with the function to advertise territory ownership (Hurst and Beynon 2004).
Dominant males urinate in a broader area as spots, rather than using one area, and
especially along the borders of their territories, which serves better in advertising
the boundaries. And for this purpose, it is more functional if the smell stays at the
site longer. The male pheromones SBT, DHB, the farnesenes are secreted more by
the dominant males (Apps et al. 1988; Harvey et al. 1989; Novotny et al. 1990).
The more territory marking by dominant males and the larger amount of pher-
omones secreted by the dominant males indicate that these pheromones detected in
4.1 Pheromones to Induce Primer Effects 43
the scent marked areas will be information for other mice about the social status of
the males that marked the area. And this will explain the reason that it stimulates
aggression from other males (see Sect. 4.2.1).
4.2 Pheromones to Induce Releaser Effects
4.2.1 Aggression
The two major roles of pheromones in communication are priming changes in

physiological conditions (primer effects) and stimulating specific behaviors (re-
leaser effect). Studies have found that some pheromones that induced primer effects
induced releaser effect as well and some pheromones are considered to have their
functions on either primer effect or releaser effect, at least so far. For example, SBT
and DHB were found to stimulate females to come into estrus (primer effect) and
also stimulate males to show aggression to other males (Novotny et al. 1985). In
this study, castrated male mice were introduced twice in home cages of singly
housed males (intruder test) for 4 min each time (Novotny et al. 1985). The first
intruder test was conducted without any addition of odor. At the second intruder
tests, the castrated intruder mice were painted on their back with urine from
non-castrated male, or castrated male, or castrated male with SBT and/or DHB
added. Records were made on the differences of the aggression from the resident
male to the intruder (Novotny et al. 1985). The results clearly showed that castrated
males painted with urine from castrated males without addition of SBT or DHB
received much less aggression than castrated males painted with urine from cas-
trated males with SBT and/or DHB added at about 1.3 ppm. These pheromones
were found to be secreted more by socially dominant males (Harvey et al. 1989),
which suggest the role of these pheromones to inform other mice their social status.
It is possible that the odor that inform dominant males stimulate more aggression
from other males.
Recent studies have determined that MUP stimulates aggression in males as well
and that it is through stimulating Vmn2r putative pheromone receptors (V2Rs)
(Chamero et al. 2007). They first showed that transgenic mice, which lack func-
tional vomeronasal system, do not show aggression to the castrated males with
urine from intact male, indicating that the stimulus that induce aggression is
detected by the accessory olfactory system. They eventually determined that there
were five fractions (F15–19) in the male urine with rather high molecular mass that
stimulates aggression and that these fractions were MUPs. An interesting note that
their study added was that there seemed to be some unidentified different pher-
omone of low molecular weight that is received by the main olfactory system and
inducing aggression and both main and accessory olfactory need to be intact for
aggressive behaviors.
4.2.2 Sexual Behaviors and Pheromones in Tears: The Esps
One of the most recently found pheromone group is a pheromone in tears, which
induces sexually receptive behaviors in females. It is a 7 kDa peptide called exo-
crine gland-secreting peptide 1 (ESP1) (Kimoto et al. 2005, 2007; Haga et al.
2010). ESP1 is secreted only in the males, and not in the females, from the
extraorbital lacrimal gland into tears (Kimoto et al. 2005). ESP1 is a multigene
family with 38 members in mice and 10 in rats but absent in humans (Kimoto et al.
2007). It stimulates vomeronasal neurons that express vomeronasal receptor gene
V2Rp5, one of the 7 genes of V2Rp family (Haga et al. 2010) and it enhances
female sexual receptive behaviors (Haga et al. 2010).
These studies started from the finding that the basal layer of vomeronasal
epithelium, i.e., vomeronasal neurons with V2R receptors, becomes activated
(expressed immediate early genes, c-fos and egr-1) by some nonvolatile male
specific compounds that seemed to be secreted from a gland rather than in urine
(Kimoto et al. 2005). The study group led by Kazushige Touhara of the University
of Tokyo tested exocrine glands and found that secretions from the extraorbital
lacrimal gland (ELG) cause activation of V2R neurons (Fig. 4.2a). They then found
using in situ hybridication that 87 % of the V2R neurons of female mice that
became active by exposure to male-soiled bedding or ELG expressed a receptor
Fig. 4.2 a ESP1 secreted from extraorbital lacrimal gland (ELG) of male mice stimulates
vomeronasal neurons in the VNO of female mice (from Kimoto et al. 2005). b ESP1 is a ligand of
V2Rp5 of vomeronasal sensory neurons (VSN). The signal is tranferred to AOB and from there to
BSTN, MeA, PMCN, VMH (see Chap. 2 for the original names of the locations), and cause
receptive behaviors in female mice (from Haga et al. 2010)
4.2 Pheromones to Induce Releaser Effects 45
type called V2Rp. These results suggested that the nonvolatile compound that
activated V2R neurons are secreted from the ELG.
They then purified and identified compounds in the ELG extract and found that
recombinant ESP1 (rESP1) induced c-fos expression in females but not in males
and the neurons showing c-fos expression were V2Rp neurons (Kimoto et al. 2005).
An interesting part of their results is that although males did not show c-fos
expression when they were exposed to rESP1, they did show electrical responses
there. Their interpretation is that, as males are always exposed to rESP1, maybe that
is hindering the c-fos expression to the exposure to rESP1.
Following the first finding that male specific compound from ELG, i.e., ESP1,
stimulates vomeronasal neurons with V2Rp receptor in female mice, Touhara group
found that among the seven types of V2Rp genes (V2Rp1 to V2Rp7), V2Rp5 was the
one that responded to ESP1 (Haga et al. 2010). They then generated a transgenic
mice strain that express IRES-tWGA/DsRed reporter gene in V2Rp5 gene region.
The DsRed positive vomeronasal neurons projected to 3–5 glomeruli in the caudal
part of accessory olfactory bulb. Interestingly, the C57BL/6 strain males did not
secrete ESP1 as much as Balb/c strain males, and using this fact, they conducted
intruder test with female C57BL/6 mouse placing the mouse into the home cage of
Balb/c male or C57BL/6 male. They found that Balb/c males induce twice more
lordosis behaviors in females than C57BL/6 males, which suggested that ESP1
works as a releaser pheromone, inducing sexually receptive behaviors in females
(Haga et al. 2010) (Fig. 4.2b). After these series of experiments on the role of ESP1
secreted by young and full adult males (ESP1 starts to be secreted in males after
4 weeks in age), it was found that another type of ESP, ESP22, is secreted from the
lacrimal gland into tears by 2–3 week old mice (Ferrero et al. 2013). When ESP22
was painted on adult female mice, adult males showed much less mounting
behaviors and much longer latency to show mounting behaviors to the female,
indicating that ESP22 inhibits sexual behaviors from males.
ESP1 and ESP22 are secreted in the tears and mice often show self-grooming
behaviors, which would function to spread the ESP1 and 22 to cover their bodies.
When mice meet, they show sniffing behaviors and detect the pheromones, ESP1
and 22. These studies show that the ESP1 from males serves to stimulate sexual
behaviors in females and ESP22 from juvenile mice serves to avoid the sexual
behavior from males to juvenile mice that are not ready for breeding.
4.2.3 Kin Recognition, Subspecies Recognition, and Mate

Preference
4.2.3.1 Individual Recognition: Major Histocompatibility Complex

(MHC)
The use of dogs to search for missing people makes people understand generally
that every individual has their own odor profile that represents the person.
The scientific studies on the genes that determine individual odors were conducted
in the twentieth century and it was found that genes related to major histocom-
patibility complex (MHC), the system involved in immune system, were in charge
of determining the odor profile of individuals as well. This major finding started
from the innovative thought by Lewis Thomas (1913–1993) of Memorial Sloan
Kettering Cancer Center at that time. He thought that the MHC of the immune
system, which is involved in distinguishing self and nonself, might be involved in
determining the smell of individuals as well. And this turned out to be right.
In mice, the genes of MHC are located in a region called H-2 on chromosome
17, and, in case of human, they are called HLA and located on chromosome 6.
Kunio Yamazaki conducted studies using congenic mice with the only difference in
genes at the H-2 region. They tested the preference of male mice to female mice
with the same or different H-2 type. What they found was that males had the
tendency to mate with females of different H-2 type (Yamazaki et al. 1976; Boyse
et al. 1987). There were also debates on whether “males” were more choosy or
“females” were more choosy, and the results showed differences by studies (Penn
and Potts 1999). Such differences can be due to the differences in experimental
procedures and/or the types of data the conclusions were based on. Studies also
showed that when the litters were cross-fostered and brought up by a mouse with
different H-2 type, the preference was found to become opposite, i.e., in preference
tests they preferred the H-2 type of their own compared to the H-2 type of the foster
parents, showing that the preference was mediated by learning process during early
post-natal days (Yamazaki et al. 1988; Eklund 1997; Arcaro and Eklund 1999).
These studies indicate that the results can become affected by various factors and
the experimental conditions as well as housing conditions could affect the results,
causing diversities in the results of studies. However, what is possible to say
through these studies is that mice show mate preferences based on the MHC odor
type and the preference is obtained by learning during early postnatal stage odor
environment. This mate preference is considered to function as the system to avoid
incest and enhance genetic diversity. Considering that the function of ABP (see
below) is to avoid mating with individuals with subspecies’ level differences, it is
possible to say that the abilities to distinguish kin relationship and subspecies’ level
differences are synergistically working in the mating through chemical signaling.
There are also hypotheses from behavioral ecology view points on its function that
it may enhance immunocompetence by producing MHC-hetorozygous offspring,
and that the diversity will avoid a specific type of MHC alleles to become too
common (Penn and Potts 1999). “As MHC alleles have different susceptibilities to a
particular parasite, then the most resistant allele will be favored and spread through
the population” (Penn and Potts 1999). However, this might cause the increase of
parasites that do not recognize common MHC allele, and there will be an indefinite
race between the host and parasites. The mating preference in a direction to enhance
diversity will shift the target preferable alleles for parasites along time (moving
target or Red Queen hypothesis, Penn and Potts 1999).
The chemical compounds that determined individual odor profile depending on
the MHC type has been another topic. If MHC type determines individual odor
profile, this can be called a “fingerprint” by odor, and just like the fingerprints, the
odor profile can be used as an ID of each individual. Some day, the “odor profile
ID” may start to be used at the airport customs in addition to (or instead of?)
fingerprints.
4.2.3.2 Major Urinary Protein (MUP) and Its Various Functions
Urine is a source of various species-specific proteins that have the function to

provide information to other conspecifics. One group of these proteins is the major
urinary protein (MUP), which is a group of proteins that are small in molecular size
(18–19 kDa) and belong to lipocalin family (Hurst and Beynon 2013). Over 30 of
MUP genes were encoded in mouse chromosome 4 (Beynon and Hurst 2004).
MUPs are produced in the liver, secreted into plasma, pass the filtration by glo-
meruli in the kidney, and thus excreted out by urine of mice and rats. First, they
were considered to be produced only by sexually mature males (Cavaggioni and
Mucignat-Caretta 2000; Beynon and Hurst 2004), but it is now known that females
also produce about 30 % less than males (Hurst and Beynon 2013). These changes
in the results were due to the fact that early studies have used laboratory strains of
mice and that the females of laboratory strains do not produce MUPS. Later studies
using wild caught female mice or wild origin female mice bred under captivity
produced MUPs, although much less than males (Hurst and Beynon 2013).
MUPs play multiple roles in chemical communication. They are known to bind
with other pheromones, for example, SBT (Zidek et al. 1999; Sharrow et al. 2002;
Armstrong et al. 2005), which stimulates females to come into estrus (Jemiolo et al.
1986) and MHC-related peptides. This is considered to delay the evaporation of
volatile pheromones and thus lengthen the time that pheromones can function to
transfer their information to other conspecifics (Beynon and Hurst 2003, 2004).
Male mice scent mark their territories repetitively with their urine and these scent
marks will provide information about the territorial ownership and the frequency of
scent marking (or the time passed after they were deposited). Slower release of
pheromones bound to MUP will provide possibilities of pheromones to stay longer
in the area where they were deposited and the ratio of release itself can be the
information about the freshness or oldness. The role of MUP to control the pher-
omone longevity in the area and territorial ownership suggests the possibility of
another role, i.e., the information on individuality by MUP, just like MHC has been
found to function. This was proved to be true. When urine of his own was placed, a
male mouse did not countermark on the urine more than water control, however,
when urine of other male was placed; the male spent longer time around the urine
and countermarked on it (Rich and Hurst 1999; Beynon and Hurst 2004; Kaur et al.
2014). Mass spectrometry of outbred mice showed that the urinary MUP pattern of
unrelated mice are highly diverse compared to genetically closely related mice
(Hurst and Beynon 2013). The fact that mice can distinguish their odor from others
using MUP and that the MUP pattern of related mice are similar compared to
unrelated mice indicate that MUP can be a signal to tell kinship as well, just like
MHC does.
Another feature that needs to be addressed concerning MUP is that one of the
MUPs itself turned out to function as a pheromone. A urinary MUP named darcin
was found to attract female mice and make them stay close to the smell long, thus
named after Darcy in Pride and Prejudice by Jane Austen (Roberts et al. 2010). It
binds to male pheromone SBT (Zidek et al. 1999; Sharrow et al. 2002; Armstrong
et al. 2005) and it was found that darcin, but not SBT, was attractant to female mice.
These responses of females to MUP have also been found to be different depending
on the estrous stage of the females (Dey et al. 2015) (Fig. 2.2). The responses to
recombinant MUP20 (rMUP), which was produced in bacteria and purified, were
higher when females were in estrus and less when they were in diestrous status. In
addition, such differences were found to exist at the sensory neuron levels. The rate
of vomeronasal neurons (VN) that showed calcium influx when they were exposed
to rMUP was higher (*5 %) in the VNs dissociated from estrous females compared
to these dissociated from diestrous females (1 %). The VNs of ovariectomized
females showed as high response as those from the females in estrus and, when
progesterone was added in the culture media for VNs at the level of diestrous
females, the responses to rMUP dropped. Eventually, it was found that proges-
terone receptor membrane-component 1 protein, PGRMC1, identified on total VNO
cDNA (Ibarra-Soria et al. 2014) is responsible to the changes in the responses of
VNs toward rMUPs (Dey et al. 2015).
Chemicals, which possess positive reproductive factor for females, sometimes
have negative meaning for males, because attractive feature for females means
higher competitive ability in reproductive situation for males. It was found that
MUP stimulates aggression in males, when castrated males had MUP swabbed on
their body (Chamero et al. 2007). Thus, studies have found that MUP has various
significant roles in chemical communication in mice, indicating their ID, their
sexual competitiveness to females and to males, thus affecting behaviors of females
and males.
4.2.3.3 Subspecies Recognition: Androgen-Binding Protein

(ABP) in Male Mice
Robert Karn and his group were originally interested in salivary protein poly-
morphism in mice. They noticed that the mobility of a certain protein in the saliva
collected from wild mice showed variable migration in electrophoresis gels. They
also noticed that the mobility changes along age and they are expressed only in
males. Eventually they determined that a gene, sex-limited salivary protein gene
(Ssp) (now renamed as Androgen-binding protein, Abp), is involved in causing the
differences of mobility, which was due to the differences in molecular weights, and
the expression of the gene depended on the level of testosterone and androgen
receptors. This protein, named androgen-binding protein (ABP), was secreted in the
saliva of male mice (Dlouhy and Karn 1983). ABP was a dimer with an alpha
subunit common to all forms of ABP, encoded by Abpa located on chromosome 7

(Dlouhy et al. 1987), and had differences in the other subunits, which were encoded
by Abpb or Abpg (Dlouhy et al. 1987). Full sequences of the genes were also
determined later (Laukaitis et al. 2003) and Abp genes were also found to be more
diverse than the three types originally found, with 30 Abpa genes and 34 Abpbg
genes, which encodes the beta and gamma subunits (Emes et al. 2004; Laukaitis
et al. 2008).
Following the findings of the androgen-binding protein and the polymorphism of
the protein, Karn group conducted a test on the diversity of ABP type in wild mice
from extensively broad areas, expanding from various locations in Europe to Asia,
and they found that there were specificities in the types of ABP and the locations
where the mice were from (Fig. 4.3). Abpaa, one of the variants of alpha subunit
gene, was found in Mus musculus domesticus in Western Europe and
Mediterranean basin. Another one variant of alpha subunit gene, Abpab, was found
in M. m. musculus, which had their habitat in Eastern Europe to northern China. In
Southeast Asia and Malaysia, a different variant of alpha subunit gene, Abpac, was
observed in M. m. castaneus (Karn and Dlouhy 1991; Karn et al. 2002). There was
also a hybrid zone running through Danish Jutland, Baltic, to the Black Sea, where
both Abpaa and Abpab type mice were observed and the mechanisms of repro-
ductive isolation were unknown.
One of the questions that comes out by the existence of hybrid zone is whether
mice mate between the different ABP types and some post-zygotic mechanisms
mediate reproductive isolation, or whether there are some pre-zygotic mechanisms
that establish reproductive isolation among subspecies. As a method of pre-zygotic
Fig. 4.3 Distribution of Abpaa,b,c type genes. Dotted line indicates the hybrid zone of Abpaa and
Abpab type mice (from Dr. Karn by courtesy)
mechanisms, it is possible to consider mate preferences and these mate preferences

are based on chemical signaling (odor), visual stimuli, and behavioral sequences.
Karn group considered that the ABP types could be used as chemical
signaling-based mate preferences. Female mice of Abpaa type (inbred laboratory
mice strain C3H was used to represent M. m. domesticus) and Abpab type (an inbred
strain, CZECH II, with origin of Central Europe to represent M. m. musculus) was
allowed to explore a test box where males of the same ABP type and different ABP
type spent 5 days. The first choice of the side of the test apparatus and the time they
spent in each area was analyzed and they found that female mice prefer the smell of
the male with the same ABP type. In a test that males were in the test apparatus with
tether, they found that females preferred to mate with males of the same ABP type
(Laukaitis et al. 1997). ABP is not found in urine (Dlouhy et al. 1986) and, when
females were exposed to urine, females did not show significant differences in their
preferences to urine depending on the ABP types of the urine donor (Talley et al.
2001).
4.2.4 Rooting Behaviors by Neonates: Are There

Pheromones in Mouse Milk?
The European rabbits (Oryctolagus cuniculus) are known to nurse their offspring
only 4–5 min/day during the first couple of weeks. It is critical for the offspring to
reach to the nipples as quickly as possible and suckle as much as possible imme-
diately. Benoit Schaal group in France first used a gas chromatograph
(GC) equipped with a sniffing device and identified 21 compounds that GC peaks
overlap with the peaks in behavioral responses by newborn rabbit pups, i.e., turning
their heads toward the sniffing device or trying to grasp it with their mouths (Schaal
et al. 2003). When these 21 compounds were tested by presenting them on a glass
rod at 1 ug/ml−1, one volatile in milk, 2-methylbut-2-enal (2MB2), induced sig-
nificantly stronger response than other compounds in the rabbit pups (Fig. 4.4). The
response was strong at certain range of concentration and not only lower but also
higher concentration induced less response from rabbit pups. The most effective
range of concentration was between 10 ng/mL−1 and 1 ug/mL−1. The rabbit pups
newly born and deprived of contact with their mothers’ belly or milk also showed
responses indicating that learning is not necessary to show proper responses. Pups
delivered by cesarean section one day prior to the delivery date also showed proper
response to 2MB2, although 2MB2 was not detected by GC-MS in amniotic fluid,
showing that the response was not due to learning in uterus.
The ability to respond to 2MB2 without learning does not mean that rabbit pups
do not memorize other odors and utilize them as cues. Pups of females fed with
food of certain flavors during pregnancy showed preferences to these smell, indi-
cating embryonic capability of olfaction and learning of the smells (Coureaud et al.
2006, 2010). When pups of dams fed with certain smell was cross-fostered to a dam
Fig. 4.4 a A 2-day old rabbit pup resting (left) and grasping a glass rod with 2MB2 (right) (from
Schaal et al. 2003). b Responses of rabbit pups to the 21 compounds identified in rabbit milk.
Blank bars indicate searching behaviors and black bars indicate grasping of the glass rod. Numbers
in the parenthesis indicate the number of pups used
fed with a different smell, the pups showed difficulties in locating the nipples and
also ingested less milk (Coureaud et al. 2010), which shows that embryonic
learning of smell has significant influences on the olfactory guidance toward milk.
Learning takes place postnatally as well and it was found that 2MB2 works as a
reinforcer in olfactory learning. When newborn rabbit pups were exposed to a
neutral odorant mixed with 2MB2 for only 5 min, they showed searching and
grasping responses when they were exposed to the odorant alone 24 h later. This
learning took place between the day of birth until 4 days later (sensitive period) and
not after postnatal day 5 (critical period). As 2MB2 is species specific and it will not
specify who their mother is, it might be that the additional odorants learned function
to establish odor component profile that enables pups to distinguish the mother. As
another possibility, it is also possible that the additional odorants enable pups to
notice the smell faster and make the odor guidance to the nipple stronger.
Whether a pheromone(s) is secreted from mouse nipples has been one of the
unsolved questions. The answer to this question is so far being negative. A research
group led by Lisa Stowers of Scripps Institute has shown that there is no pheromone
in mouse milk. Darren Logan, a post-doctoral fellow of Stowers group at that time
(currently at Wellcome Trust Sanger Institute), first showed that, when nipples were
washed, it made the newly born mouse pups slow to grasp and suckle a nipple
compared to unwashed nipples, suggesting that olfactory cues are helping the pups
to find the nipples (Logan et al. 2012). Pups, which lacked functional vomeronasal
system (TRPC2-/- mice), did not have problem in finding unwashed nipples,
however, pups without functional main olfactory system (Cnga2-/-) showed as slow
reach to the nipples as the wild-type pups did to washed nipples. This showed that
main olfactory system but not the accessory olfactory system is involved in the
odor-guided nipple searching behaviors of the mouse pups. To determine the
olfactory cues, nipples were washed and various types of olfactory cues were
applied to the washed nipples. When amniotic fluid or maternal saliva or milk was
applied on the washed nipple, it made the pups reach to the nipple faster, indicating
that all these stimuli can function as the olfactory cues. However, when mouse were
delivered by cesarean section, maternal saliva, milk, and colostrum were found to
lack the function as olfactory cues to guide to the nipples and only amniotic fluid
did. These results showed that there is no pheromone included in mouse milk,
which will function as a guide to the nipples for mouse pups.
The authors of these studies also showed that the olfactory guidance by amniotic
fluid was not mediated by a single molecule but a combination of small molecular
weight components that were less than 3 kDa and 3–10 kDa were involved. They
also showed that, similar to European rabbit pups, when the mother mice were fed
with food of specific smell during pregnancy (vanillin or garlic), the pups showed
faster reach to the washed nipples added with the specific smell mixed with
amniotic fluid. This showed that pups could learn the smell of the food their
mothers have been eating during embryonic stages and obtain strong preference
toward these smells.
4.2.5 Parental Behaviors
The stimulus that triggers parental behaviors from male and female mice or rats has
been studied extensively in the study field of animal psychology and psychoneu-
roendocrinology. Recent development of genetic engineering enabled determining
the neurons involved in parental behaviors. A group led by Catherine Dulac of
Harvard University first tested whether parental behaviors were triggered by the
stimuli through the main or accessory olfactory system (Wu et al. 2014). They used
TRPC2 (transient receptor potential cation channel, subfamily C, member 2)
knockout mice, which lack functional vomeronasal system, and wild type of these
mice as control, they conducted intruder tests placing 4 unrelated neonatal pups
(postnatal day 1–3 pups of C57BL/6 strain) in the home cage of male or female
adult mice. The behaviors were observed from the moment the mice showed
sniffing behavior to a pup until the occurrence of attack or during the 30 min after
the mouse first sniffed the pups. As parental behaviors, retrieving behavior,
crouching over, and nest building behaviors were recorded. They found that, in case
of female mice, both TRPC2 knockout and wild type showed retrieving behaviors,
which indicate that parental behaviors are not triggered by the stimuli through
vomeronasal organ. The results of male mice were largely different from those of
females. TRPC2 knockout mice showed parental behaviors like female mice,
however, almost 80 % of the wild-type mice attacked the neonatal mice. This
indicates that the aggressive behaviors in male mice are triggered by the stimuli
through accessory olfactory system.
Dulac group also found that such high aggressiveness to the “intruder” pup
decreases when the males are mated. When the males were tested 1–2 or
10–12 days after mating, the aggressiveness did not change and males attacked the
pups. However, when they were tested 17–20 days after mating, the males did not
show attack and half of them showed parental behaviors. When the males were
tested 25–27 days after mating, all the males showed parental behaviors (Wu et al.
2014). Studies utilizing the expression of immediate early genes determined that
when father males were exposed to pups, immediate early gene, c-fos, was not
observed in the vomeronasal neurons, indicating that the changes are at the main
sensory system (Tachikawa et al. 2013).
In order to determine the differences in the brain activities between the mice that
showed parental behaviors and those that did not, Dulac group compared the
expression of c-fos in the brain, especially in the hypothalamus, amygdala and other
areas in the brain related to social behaviors, after the exposure to pups. They found
there are several areas in the brain of mice that show expression of c-fos common in
mice that showed parental behaviors and especially the medial preoptic area,
MPOA, showed strongest activation (Fig. 4.5a–d). As these areas are related to
various behaviors other than parental behaviors (Fig. 4.5e) and also in the regu-
lation of body temperature, Dulac group examined if the neurons that become
activated are different depending on the differences in the behaviors that mice
showed. As c-fos first becomes expressed in the nucleus and then in the cytoplasm,
it is possible to utilize the change in the location of expression along time course.
For example, when mice were exposed to pups first and then mated, it is possible to
examine where the expressions are, whether they are expressed in the same neurons
or in different ones by comparing with the results of the expression after mice were
exposed to pups twice with time between. In this way, they found that when mice
are exposed to pups two times, c-fos was expressed in the same neurons at the rate
of 70 %, whereas, when the mice were exposed to pups and then mated, only
20–30 % of the cells expressed c-fos was the same cells (Fig. 4.5f). They then tested
Fig. 4.5 Expression of c-fos in virgin males (a), fathers (b), virgin females (c) at MPOA (d) (from
Wu et al. 2014). e Expression of c-fos depending on the types of situations (aggression, parenting,
mating, and control). NS: not significant, ***P < 0.001. f Percentage of overlap in the cells
expressing c-fos in the MPOA for the first situation (parentin or mating) and second situation in
males. **P < 0.01. g Influences of the ablation of MPOA galanin neurons of fathers on their
responses to pups. Fishers’ exact test, P < 0.01. h Influences of optogenetic activation of MPOA
galanin neurons in the virgin males on attack behaviors to pups. NS: not significant, ***P < 0.001,
Stim: optogenetically stimulated, No stim: not stimulated
various markers to determine what is expressed in the neurons that are responsive to
parental behaviors and found it is a neuropeptide galanin. In case of virgin females
38.3 % of the cells that expressed c-fos showed expression of galanin gene as well,
which was 43.9 % in case of dams and 33.4 % in case of father male mice. These
neurons were also mostly inhibitory GABA neurons. Galanin was found in 1970s
and considered to be involved in various situations, for example, inhibition of
learning and memory, activation of feeding behavior, and inhibition of male sexual
behaviors (Lang et al. 2007).
To confirm the role of galanin neurons in parental behaviors, Dulac group used a
transgenic mouse strain that lack functional galanin neurons and showed that
female mice without functional galanin neurons show attack when they are exposed
to pups (Wu et al. 2014) (Fig. 4.5g). They also used a transgenic mice strain with
galanin specific Cre system expressed and injected in the area of MPOA with virus
that has Cre dependent channelrhodopsin-2 gene fused with enhanced yellow
fluorescence (eYFP) (AAV-ChR2:EYFP), and this will make the galanin neurons
express channelrhodopsin-2 gene and its expression can be confirmed by the
expression of eYFP. They also implanted optical fiber in the MPOA area in the
brain to stimulate only the galanin neurons in the MPOA by exposure to blue light.
Channelrhodopsin-2 is originally a light-gate ion channel, which serves as pho-
toreceptors in the chlamydomonas, known to be involved in phototaxis. The
channel opens with exposure to blue light and the signaling starts. By exposing the
galanin neurons to blue light using the optical fiber, it is possible to activate galanin
neurons. When male mice without mating experiences had their galanin neurons
activated by blue light, they did not show aggressive behaviors in 16 out of 18
trials, whereas, when the same mice were not exposed to blue light, they attacked
the pups (Fig. 4.5h). These experiments showed that galanin neurons have sup-
pressive influences on aggression.
This study showed that galanin neurons suppress the aggression toward pups,
which disturbs mice from showing parental behaviors, however, it does not directly
show what stimuli are involved in releasing parental behaviors. Studies in the past
have shown that the sensory cues involved in releasing parental behaviors are not
single but multiple. Parental behaviors of lactating dams were not suppressed by
removing either the olfactory sense (anosmia), or visual sense (blind), or touch
(anaptic), but it was suppressed when anosmia and tactile sense were both deprived
(Dulac et al. 2014).
The birth of pups, which is a sudden emergence of nonself in the nest, requires
some mechanisms to suppress aggression so that the pups will not be killed and to
stimulate parental behaviors so that the pups will not be ignored. Galanin serves to
suppress aggression and the multiplicity of sensory cues that serves to release
parental behaviors will ensure that pups will survive and grow. How mating
stimulates secretion of galanin and enhance parenting would be the question to be
answered in near future.
References
Apps PJ, Rasa A, Viljoen HW (1988) Quantitative chromatographic profiling of odours associated
with dominance in male laboratory mice. Aggressive Behav 14:451–461
Arcaro KF, Eklund A (1999) A review of MHC-based mate preferences and fostering experiments
in two congenic strains of mice. Genetica 104:241–244
Armstrong SD, Robertson DH, Cheetham SA, Hurst JL, Beynon RJ (2005) Structural and
functional differences in isoforms of mouse major urinary proteins: a male-specific protein that
preferentially binds a male pheromone. Biochem J 391(Pt2):343–350
Beynon RJ, Hurst JL (2003) Multiple roles of major urinary proteins in the house mouse. Mus
domesticus. Biochem Soc Trans 31(Pt1):142–146
Beynon RJ, Hurst JL (2004) Urinary proteins and the modulation of chemical scents in mice and
rats. Peptides 25(9):1553–1663
Boyse EA, Beauchamp GK, Yamazaki K (1987) The genetics of body scent. Trends Genet
3:97–102
Cavaggioni A, Mucignat-Caretta C (2000) Major urinary proteins, alpha (2U)-globulins and

aphrodisin. Biochim Biophys Acta 1482(1–2):218–228
(2007) Identification of protein pheromones that promote aggressive behaviour. Nature 450
(7171):899–902
Coureaud G, Moncomble A-S, Montigny D, Dewas M, Perrier G, Schaal B (2006) A pheromone
that rapidly promotes learning in the newborn. Curr Biol 16:1956–1961
Coureaud G, Charra R, Datiche F, Sinding C, Thomas-Danguin T, Languille S, Hars B, Schaal B
(2010) A pheromone to behave, a pheromone to learn: the rabbit mammary pheromone.
J Comp Phys A 196:779–790
Dey S, Chamero P, Pru JK, Chien M-S, Ibarra-Soria X, Spencer KR, Logan DW, Matsunami H,
Peluso JJ, Stowers L (2015) Cyclic regulation of sensory perception by a female hormone
alters behavior. Cell 161:1334–1344
Dlouhy SR, Karn RC (1983) Multiple gene action determining a mouse salivary protein
phenotype: identification of the structural gene for androgen binding protein (Abp). Biochem
Genet 22:657–667
Dlouhy SR, Nichols WC, Karn RC (1986) Production of an antibody to mouse salivary androgen
binding protein (abp) and its use in identifying a prostate protein produced by a gene distinct
from abp. Biochem Genet 24:743–763
Dlouhy SR, Taylor BA, Karn RC (1987) The genes for mouse salivary androgen-binding protein
(ABP) subunits alpha and gamma are located on chromosome 7. Genetics 115:535–543
Dulac C, O’Connell LA, Wu Z (2014) Neural control of maternal and paternal behaviors. Science
345(6198):756–770
Eklund A (1997) The effect of early experience on MHC-based mate preferences in two B10.W
strains of mice (Mus domesticus). Behav Genet 27:223–229
Emes RD, Riley MC, Laukaitis CM, Goodstadt L, Karn RC, Ponting CP (2004) Comparative
evolutionary genomics of androgen-binding protein genes. Genome Res 14:1516–1529
Ferrero DM, Moeller LM, Osakada T, Horio N, Li Q, Roy DS, Cichy A, Spehr M, Touhara K,
Liberles SD (2013) A juvenile mouse pheromone inhibits sexual behavour through the
vomeronasal system. Nature 502:368–371
Harvey S, Jemiolo B, Novotny M (1989) Pattern of volatile compounds in dominant and
subordinate male mouse urine. J Chem Ecol 15(7):2061–2072
Hurst JL, Robertson DHL, Tolladay U, Beynon RJ (1998) Proteins in urine scent marks of male
house mice extend the longevity of olfactory signals. Anim Behav 55:1289–1297
Hurst JL, Beynon RJ (2004) Scent wars: the chemobiology of competitive signaling in mice.
BioEssays 26:1288–1298
Hurst JL, Beynon R (2013) Rodent urinary proteins used in scent communications. In: East ML,
Dehnhard M (eds) Chemical signals in vertebrates. Springer, New York, pp 117–133
Iberra-Soria X, Levitin MO, Saraiva LR, Logan DW (2014) The olfactory transcriptomes of mice.
PLoS Genet 10(9):e1004593
Jemiolo B, Novotny M (1994) Inhibition of sexual maturation in juvenile female and male mice by
a chemosignal of female origin. Physiol Behav 55(3):519–522
Karlson P, Luscher M (1959) Pheromones’: a new term for a class of biologically active
substances. Nature 183:55–56
Karn RC, Dlouhy SR (1991) Salivary androgen-binding protein variation in Mus and other
rodents. J Heredity 82:453–458
Karn RC, Orth A, Bonhomme F, Boursot P (2002) The complex history of a gene proposed to
participate in a sexual isolation mechanism in house mice. Mol Biol Evol 19:462–471
References 57
Kaur AW, Ackels T, Kuo TH, Cichy A, Dey S, Hays C, Kateri M, Logan DW, Marton TF,
Spehr M, Stowers L (2014) Murine pheromone proteins constitute a context-dependent
combinatorial code governing multiple social behaviors. Cell 157(3):676–688
Kimoto H, Sato K, Nodari F, Haga S, Holy TE, Touhara K (2007) Sex- and strain-specific
expression and vomeronasal activity of mouse ESP family peptides. Curr Biol 17:1879–1884
Lang R, Gundlach AL, Kofler B (2007) The galanin peptide family: receptor pharmacology,
pleiotropic biological actions, and implications in health and disease. Pharmcol Ther
115:177–207
Laukaitis CM, Critser ES, Karn RC (1997) Salivary androgen-binding protein (ABP) mediates
sexual isolation in Mus musculus. Evolution 51(6):2000–2005
Laukaitis CM, Dlouhy SR, Karn RC (2003) The mouse salivary androgen-binding protein
(ABP) gene cluster on chromosomes 7: characterization and evolutionary relationships. Mamm
Genome 14(10):679–691
Laukaitis CM, Heger A, Blakley TD, Munclinger P, Ponting CP, Karn RC (2008) Rapid bursts of
androgen-binding protein (Abp) gene duplication occurred independently in diverse mammals.
BMC Evol Biol 8:46–63
Ultrasensitive pheromone detection by mammalian vomeronasal neurons. Nature 405:792–796
Logan DW, Brunet LJ, Webb WR, Cutforth T, Ngai J, Stowers L (2012) Learned recognition of
maternal signature odors mediates the first suckling episode in mice. Curr Biol 22:1998–2007
Ma W, Miao Z, Novotny MV (1999) Induction of estrus in grouped female mice (Mus domesticus)
by synthetic analogues of preputial gland constituents. Chem Senses 24:289–293
Meredith M (2001) Human vomeronasal organ function: a critical review of best and worst cases.
Chem Senses 26:433–445
Novotny M, Harvey S, Jemiolo B, Alberts J (1985) Synthetic pheromones that promote inter-male
aggression in mice. Proc Natl Acad Sci USA 82:2059–2061
Novotny M, Harvey S, Jemiolo B (1990) Chemistry of male dominance in the house mouse, Mus
domesticus. Experientia 46:109–113
Novotny MV, Ma W, Wiesler D, Zidek I (1999) Positive identification of the puberty-accelerating
pheromone of house mouse: the volatile ligands associating with the major urinary protein.
Proc R Soc Lond B 266:2017–2022
Penn DJ, Potts WK (1999) The evolution of mating preferences and major histocompatibility
complex genes. Am Nat 153(2):145–164
Rich TJ, Hurst JL (1999) The competing countermarks hypothesis: reliable assessment of
competitive ability by potential mates. Anim Behav 58(5):1027–1037
Roberts SA, Simpson DM, Armstrong SD, Davidson AJ, Robertson DH, McLean L, Beynon RJ,
Hurst JL (2010) Darcin: a male pheromone that stimulates female memory and sexual
attraction to an individual male’s odour. BMC Biol Jun 3(8):75. doi:10.1186/1741-7007-8-75
Robertson DHL, Beynon RJ, Evershed RP (1993) Extraction, characterization, and binding
analysis of two pheromonally active ligands associated with major urinary protein of house
mouse (Mus musculus). J Chem Ecol 19(7):1405–1416
Schaal B, Coureaud G, Langlois D, Ginies C, Semon E, Perrier G (2003) Chemical and
behavioural characterization of the rabbit mammary pheromone. Nature 424:68–72
Sharrow SD, Vaughn JL, Zidek L, Novotny MV, Stone MJ (2002) Pheromone binding by
polymorphic mouse major urinary proteins. Protein Sci 11(9):2247–2256
Tachikawa KS, Yoshihara Y, Kuroda KO (2013) Behavioral transition from attack to parenting in
male mice: a crucial role of the vomeronasal system. J Neurosci 33(12):5120–5126
Talley HM, Laukaitis CM, Karn RC (2001) Female preference for male saliva: implications for
sexual isolation of Mus musculus subspecies. Evolution 55(3):631–634
Thomas L (1974) The lives of a cell. Notes of a biology watcher. The Viking Press, New York
Wu Z, Autry AE, Bergen JF, Watabe-Uchida M, Dulac CG (2014) Galanin neurons in the medial
preoptic area govern parental behavior. Nature 509(7500):325–330
Wyatt TD (2009) Fifty years of pheromones. Nature 457:262–263

Yamazaki K, Boyse EA, Mike V, Thaler HT, Mathieson BJ, Abbott J, Boyse J, Zayas ZA (1976)
Control of mating preferences in mice by genes in the major histocompatibility complex. J Exp
Med 144:1324–1335
Yamazaki K, Beauchamp GK, Kupniewski D, Bard J, Thomas L, Boyse EA (1988) Familial
imprinting determines H-2 selective mating preferences. Science 240:1331–1332
Zidek L, Stone MJ, Lato SM, Pagel MD, Miao Z, Ellington AD, Novotny MV (1999) NMR
mapping of the recombinant mouse major urinary protein I binding site occupied by the
pheromone 2-sec-butyl-4,5-dihydrothiazole. Biochemistry 38(31):9850–9861
Chapter 5
New Primer Effects
Abstract From the end of twentieth century to the beginning of twenty-first

century, several new primer effects were found, i.e., enhanced adult neurogenesis
by the exposure to pheromone of opposite sex, enhanced expansion of mammary
gland in females by exposure to male pheromone, enhanced sperm density by
exposure to female-soiled bedding, suppressed sperm activity of subordinate males,
and transgenerational influences of exposure to pheromones. It is not only the
reproductive system. It enhances the whole reproductive success.
Keywords Adult neurogenesis Social status Sperm motility Sperm density
5.1 From Early Studies to New Studies
The earliest findings of primer effects started in the 1950s, mostly focusing on the
reproductive conditions in female mice, i.e., influences of conspecific odors on the
estrous cycles of females (Lee–Boot effect and Whitten effect), puberty acceleration/
delay (Vandenbergh effect), and disruption of establishment of pregnancy (Bruce
effect). There were almost no studies focusing on male mice during these early years.
This could have been because of the lack of estrous cycles, which is easy to examine
in measuring the influences, in males. Studies on males started from the end of
twentieth century and it was found that the reproductive conditions of males are
under the control of odor environment as in females. Newer studies also found that
not only the reproductive system but also the rate of neurogenesis in the neurogenic
regions in the brain, subventricular zone and dentate gyrus, is also regulated by
pheromones. Furthermore, recent studies have found that the expansion of mammary
glands is affected by exposure to pheromones as well and such influences on the
mammary glands have extended influences on the next generation, most likely
through the influences on milk synthesis. These recent studies typically employed
state-of-the-art techniques in the fields of cell biology, molecular biology, genetic
engineering, and interdisciplinary approaches, which must have contributed in the
new findings and in providing mechanistic insights into these findings.

60 5 New Primer Effects
5.2 Adult Neurogenesis
It was generally believed that neurons do not regenerate or turn over in the adult
systems. However, this turned out to be wrong. Not only the sensory neurons of the
main and accessory olfactory system, which turn over periodically, but also the
neurons of central nervous system were found to regenerate continuously all
through the life. This is called adult neurogenesis. Several areas in the brain were
found to show adult neurogenesis, the subventricular zone (SVZ), dentate gyrus
(DG), and hypothalamus. Cells proliferate at the subventricular zone migrate to
olfactory bulb and become integrated as interneurons. When neurogenesis was
suppressed using genetically engineered mice, which had tamoxifen inducible
system that disturbed neurogenesis, the olfactory bulb shrank to half of the normal
size (Imayoshi et al. 2008), which suggested that adult neurogenesis may have a
critical role in maintaining the function of transferring information from the
olfactory system to the brain at the olfactory bulb. The newly born cells at the
dentate gyrus and hypothalamus are integrated in the hippocampus and hypotha-
lamus, respectively, suggesting their roles in organizing and/or maintaining the
memory system and controlling hormone secretions.
Historically, the reports on cell proliferation in the adult brain were actually
already seen in the beginning of twentieth century (Allen 1912). However, it took
over 50 more years until a series of papers on adult neurogenesis started to be
published (Altman 1962, 1963) and it had to wait until 1980s to be officially
recognized. This delay was caused because of the generally strongly believed
concept that neurons do not regenerate after the central neural system is developed
and also because the studies in these early years did not contain fully convincing
results. The first person who proved the existence of adult neurogenesis was
Fernando Nottebohm using canary birds (Paton and Nottebohm 1984). He showed
using tritiated thymidine where new neurons are added in adult brains and this
seemed to facilitate their song learning in spring season. The development of
molecular biology and bioengineering in the late twentieth century enabled the
enormous progress in the science of adult neurogenesis and details of the process,
molecular environment at the niche where adult neurogenesis takes place, and the
factors that affect it started to be determined.
5.3 Odors of Opposite Sex Stimulate Neurogenesis
Various factors are known to affect the rate of adult neurogenesis. One of the factors
newly found to affect adult neurogenesis was the odor of the opposite sex. There
was a paper published in 2007 showing that one week of exposure to male-soiled
bedding enhanced the neurogenesis in the SVZ and DG of female mice (Mak et al.
2007). They also found that the bedding soiled by dominant male has significant
influence in enhancing neurogenesis, whereas that of subordinate male did not
5.3 Odors of Opposite Sex Stimulate Neurogenesis 61
affect neurogenesis. The method they used to expose females to male odors was
placing female mice in the cages that were used by males for certain time length.
Using transgenic mice that lack estrogen receptor or prolactin receptor, they showed
that the enhanced neurogenesis by exposure to male-soiled bedding was mediated
by estrogen in the DG and by prolactin in the SVZ.
Following this study, there was another paper showing the same enhanced
neurogenesis in female mice by the exposure to males with wire mesh between
(Larsen et al. 2008). This group, led by David Grattan, showed the change in serum
concentration of prolactin after the exposure to males started. Prolactin concen-
tration showed a steep rise from one day after the exposure started and showed a
peak on the second and third days, and then started to decrease from 4 days after the
exposure to males started. The result on neurogenesis in their group showed a peak
at one week after the exposure, similarly to the first study by Samuel Weiss group in
Canada (Mak et al. 2007).
There was another group in Italy, which was published showed similar result in
the next year (Oboti et al. 2009). In their study, they showed that if the soiled
bedding was placed in a glass bottle with wire mesh on it to avoid direct contact, the
soiled bedding did not affect the rate of neurogenesis. The group claimed that the
compound(s) involved in the enhanced neurogenesis in the females exposed to
male-soiled bedding is nonvolatile.
5.4 Identification of Pheromones that Stimulate Cell

Proliferation in the Brain
5.4.1 Female Studies
5.4.1.1 Volatility and Influences
As written in Chap. 4, there are male murine pheromones that are secreted sig-
nificantly more by dominant males. In the adult neurogenesis studies, Samuel Weiss
group has shown that the influence of exposure to male-soiled bedding was sig-
nificantly stronger when the bedding was soiled by dominant males (Mak et al.
2007). These studies suggested that the male murine pheromones of dominant
males could be the pheromones that stimulated neurogenesis in female mice.
A question is the results shown by Oboti et al. (2009) with the conclusion that the
chemical compounds were nonvolatile, because the dominant male specific pher-
omones are volatile (Harvey et al. 1989). So, a hypothesis to explain this was the
differences in the method of exposure to male odor in these studies. In the study by
Mak et al. (2007), they placed female mice in the cages used by males for certain
time period. In the study by Oboti et al. (2009), they placed a bottle, which contains
soiled bedding of males, in females’ cages. A big difference in these situations is the
time length that females are exposed to males’ odors. How often the females go
close to the bottle with male-soiled bedding will make a large difference in the
amount that females are exposed to males’ odors, whereas if females are placed in
cages used by males, the cages are covered with males’ odors everywhere. So, these
studies and hypotheses suggested that the pheromones specific to dominant males
could be the pheromones that stimulate adult neurogenesis in female mice.
We tested this hypothesis, exposing females to 2-sec-butyl-4,5-dihydrothiazole
(SBT), 3,4-dehydro-exo-brevicomin (DHB), and the farnesenes (E,E-α-farnesene
and E-β-farnesene) (Koyama et al. 2013, 2014). At first, we tested if exposure
without direct access will still enhance neurogenesis in mice, by putting a cotton tip
with pheromone solution (50 uL) in an Eppendorf tube with holes and putting the
tube in a wire mesh container so that the mice cannot chew the tube. The wire mesh
containers were either hung on the wall of mice cages or left on top of the bedding.
When female mice were exposed to male pheromone using this method, there was
no difference between the pheromone exposure group and control group. These
results suggested that direct contact or very close contact to pheromones was
necessary to induce the enhanced neurogenesis, or the pheromones we used were
not the ones that affect female mice.
Next, we exposed females to male pheromones by grasping the mice and directly
depositing the same 50 uL of pheromone buffer (all 250 ppm) on their nostril. Mice
were exposed to pheromone twice daily for one week. Using this method, we found
that SBT and the farnesenes stimulate cell proliferation in the SVZ of female mice,
but DHB, which is also one of the dominant male pheromones, did not significantly
affect cell proliferation (Fig. 5.1).
These results provide interesting conclusions and further questions. First, it
explains that the method in Oboti et al. (2009) was negative maybe because the
extent of exposure of females to the odors of males was not enough to enhance adult
neurogenesis, or maybe the odor donors were subordinate males. In the former case,
it suggests that females are not “attracted” to the pheromones, which are specific to
dominant males. There are studies actually showing that this is right.
5.4.1.2 Exposure to Male-Soiled Bedding
We also exposed females to male-soiled bedding to compare with the results of

exposure to the synthetic analogs of pheromones. Odor donor males were kept in
groups of 5 males, which will ensure establishment of social dominance among the
males (see Chap. 1). We first measured the amount of SBT, the farnesenes, and
DHB included in male-soiled bedding depending on the time length males used the
bedding. To collect the volatile chemical compounds in the bedding, magnetic stir
bars with silicone (polydimethylsiloxane, PDMS) coating were put in Eppendorf
tubes with holes, which enables entering of volatile chemical compounds, and the
Eppendorf tubes were covered by wire mesh to avoid the mice to chew the tubes.
Several of these coated stir bars were placed on top of the clean bedding. Males
were introduced in the cages with the clean bedding with coated stir bars and then
the stir bars were retrieved 2 days later, 4 days later, and 7 days later. Collected stir
5.4 Identification of Pheromones that Stimulate Cell Proliferation in the Brain 63
Fig. 5.1 Cell proliferation (BrdU+) in the SVZ of female mice after exposure to bedding and
synthetic analogs of murine pheromones (modified from Koyama et al. 2013). ***P < 0.001, CB
clean bedding, FB female-soiled bedding, MB male-soiled bedding, WT water, BT benzothiazole,
FN farnesene, 25D 2,5-dimethylpyrazine. Error bars show standard error
bars were used to determine the concentration of male pheromones in the bedding
using mass spectrometry–gas chromatography (MS–GC). We found that SBT and
the farnesenes showed increase in their concentration along time, whereas DHB did
not show significant increase (Fig. 5.2). In the studies by Mak et al. (2007), they
showed that male-soiled bedding, which was used by males for 2 days did not have
significant influence on enhancing adult neurogenesis in females, whereas
7-day-old soiled bedding enhanced it. Their results and our results using synthetic
analogs of male pheromones, and the results on changes in concentration of
pheromones suggest that the concentration of SBT and the farnesenes were not
enough in the 2-days-old male-soiled bedding in the study by Mak et al. (2007).
Based on our results on the changes in concentration of pheromones along time
course and also on the results by Mak et al. (2007), we exposed female mice to
7-day-old male-soiled bedding to compare with the results of exposure to synthetic
analogs of pheromones. As a procedure, females were placed in the cages, which
were used by males for 7 days, every other day, after moving the males to new
cages. Control group females were placed in new cages with clean bedding every
other day. On the eighth day, all the females were cardiac perfused after injections
Fig. 5.2 Change in the concentration of male murine pheromones included in male-soiled
bedding depending on the length of days the bedding was used. Error bars show standard error
of bromodeoxyuridine (BrdU) (300 mg/kgbw) with 4 % paraformaldehyde and the

brains were dissected out. We found that cell proliferation in the brain of females
exposed to male-soiled bedding was significantly enhanced than females exposed to
new soiled bedding and it was as high as the cell proliferation in the brain of
females exposed to synthetic analogs of SBT and the farnesenes (Fig. 5.1). We also
exposed females to male-soiled bedding, which was used by males for different
time length, and we found that the bedding, which was used by males for longer
time period, had stronger influence on females.
5.4.1.3 Age and Influence of Male Pheromones
In early studies, John Vandenbergh has shown that females kept without the odors
of males show delay in their puberty, in other words, male odors accelerate puberty
(Vandenbergh effect). The male pheromones that were found to enhance cell pro-
liferation in the brain of females are the pheromones that stimulate Vandenbergh
effect. This suggests that they may enhance neurogenesis in the prepubertal females
as well. But it is also possible that as neurogenesis is high during early stages in life,
male pheromones may not enhance the already high neurogenesis at younger ages.
We tested these hypotheses by exposing females of various ages to the male
pheromones that enhanced cell proliferation in the SVZ of adult females. Females
of postnatal day 20 (prepuberty), 30 (postpuberty), 40 (young), and 70 (adult) were
exposed to SBT and the farnesenes, both 50 uL of 250 ppm solution, twice per day
Fig. 5.3 Cell proliferation

(BrdU+) in the SVZ of female
mice after exposure to SBT
(filled circle) or water (empty
circle) (from Koyama et al.
2013). Circles connected with
line show the average at each
age
for 7 days and we found that exposure to male pheromones enhances cell prolif-
eration only in the adult females and not in pre-, postpubertal females nor young
females (Fig. 5.3).
5.4.1.4 Threshold Concentration to Produce Influences
We chose the concentration of the male pheromones by our earlier studies, in which
they successfully induced estrus in females (Whitten effect). Recent studies have
shown that, at the peripheral system, the sensory neurons (vomeronasal neurons)
are ultrasensitive and detect pheromones at extremely low concentration
(Leinders-Zufall et al. 2000). In our previous study, in which we examined the dose
dependent differences in the influences of pheromones on cell proliferation in the
brain, we found that enhanced proliferation occurred at much higher concentration
than the threshold concentration at the sensory system (Koyama et al. 2013, 2014;
Leinders-Zufall et al. 2000) (Fig. 5.4). These differences may due to experimental
differences of in vitro and in vivo. However, it is also possible that activation of
more than certain amount of sensory neurons is necessary to enhance neurogenesis
in the brain by pheromone exposure. This can be determined by conducting dose
dependent exposure and examining the changes in molecular environment at the
peripheral system and in the brain depending on the concentration of the pher-
omone used.
5.4.1.5 Pheromones from the Same Sex versus from the Opposite Sex
In earlier studies it was found that, when there are no males’ odor in the envi-
ronment and when females are kept in groups, the estrous cycle becomes extended
and the frequency that females come into estrus decreases (Lee–Boot effect). Later,
Fig. 5.4 Cell proliferation

(BrdU+) in the SVZ of female
mice after exposure to SBT
(black circle) or farnesene
(lighter circle) of various
concentrations. X and Y
indicate the threshold
concentration that SBT
(black) and farnesene (lighter
color) activated vomeronasal
neurons in in vitro studies
(from Koyama et al. 2013).
Control conditions show
results of mice exposed to
water
2,5-dimethylpyrazine was found to be responsible to this phenomenon (Ma et al.

1998). We thought that, if 2,5-dimethylpyrazine suppresses estrous cycle sup-
pressing secretion of estrogen and prolactin, it may suppress adult neurogenesis in
females as well. Adult females were exposed to 2,5-dimethylpyrazine, 250 ppm,
twice per day for 7 days and we found that there was no suppressive influence by
exposure to 2,5-dimethylpyrazine (Fig. 5.1). There could be some other chemical
compounds in female-soiled bedding that affect adult neurogenesis in females. To
test this hypothesis, we exposed females to female-soiled bedding and there was no
suppressive influence, suggesting that, although the estrous cycles are suppressed
by female pheromones, they do not affect the rate of neurogenesis (Fig. 5.1).
5.4.2 Male Studies
5.4.2.1 Exposure to Female Pheromone 2,5-Dimethylpyrazine
In my studies using males, we found that, when males were exposed to female-soiled
bedding, their sperm density increased. In our studies using females, we found that,
when females were exposed to male-soiled bedding and synthetic analogs of male
pheromones, the cell proliferation in the brain became enhanced. These results sug-
gested that odors of the opposite sex stimulate cell proliferation in the reproductive
system and in the brain, which suggest that exposure to female pheromones may
stimulate cell proliferation in the brain of male mice other than in their reproductive
system. 2,5-Dimethylpyrazine is a female pheromone identified to suppress estrus in
females (Lee–Boot effect). The secretion of 2,5-dimethylpyrazine increases when
females are kept in larger groups than in small groups or in isolated condition. In our
studies using males exposing them to the soiled bedding of females kept in various
group sizes, the influence of enhancing sperm density was larger when males were
exposed to females kept in groups (see Chap. 4). These studies suggested that
2,5-dimethylpyrazine can be a female pheromone that stimulate cell proliferation in
males and also that cell proliferation may not be limited to the reproductive system but
also in the brain. We tested if exposure to 2,5-dimethylpyrazine will enhance adult
neurogenesis in male mice. We exposed males to 2,5-dimethylpyrazine for 7 days, in a
same way as to experiments using females (see 5.4.1.1), and cardiac perfused them on
the 8th day after injecting BrdU (300 mg/kgbw) and dissected out their brains. Control
group males were exposed to water. We also exposed males to the bedding used by
females kept in groups for 7 days. Group sizes were 4 to 5 females per cage. Method
of exposure was similar to the method used in exposing females to male-soiled
bedding (see 5.4.1.2), i.e., placing males in the cages used by females for 7 days and
moving the males to another soiled bedding every other day for 7 days (4 times) and
cardiac perfused on the 8th day.
Figure 5.5 shows the results. Male mice exposed to 2,5-dimethylpyrazine
showed significantly high cell proliferation in the SVZ compared to control males
exposed to water. Males exposed to female-soiled bedding also showed high cell
proliferation in the SVZ compared to control males exposed to clean bedding. This
Fig. 5.5 Cell proliferation (BrdU+) in the SVZ of male mice after exposure to bedding or murine
pheromones (modified from Koyama et al. 2013). *P < 0.05, ***P < 0.001, CB clean bedding, FB
female-soiled bedding, MB male-soiled bedding, WT water, FN farnesene, 25D
2,5-dimethylpyrazine. Error bars show standard error
was a new finding of a primer effect found in male mice. These studies showed that
the pheromones of the opposite sex stimulate cell proliferation in the brain other
than in the reproductive system.
5.4.2.2 Exposure to Male Pheromones, SBT and the Farnesenes
In the early studies in 1950s, it was shown that female pheromones suppress estrous
cycle of females (Lee–Boot effect). In my study using males, sperm motility of
subordinate male was suppressed presumably by the dominant male pheromones as
well (Koyama and Kamimura 1999). These studies suggested that the exposure to
the pheromones of the same sex might have some suppressive influences on the
adult neurogenesis as well. We tested this hypothesis by exposing males to male
pheromones, SBT and found that, as similar to the lack of suppressive influence of
female pheromone to females, male mouse pheromones did not suppress cell
proliferation in the brain of male mice (Fig. 5.5). These results suggested that
pheromones of the same sex suppress the reproductive system of other same sex
individuals, but they do not suppress neurogenesis of others.
5.5 Mammary Gland Expansion by Exposure to Male

Pheromone
Starting from the 1950s, studies have shown that exposure to the odors of other
conspecific animals can affect the physiological conditions, suppressing estrus,
inducing estrus, disturbing establishment of pregnancy, enhancing sperm density,
suppressing sperm motility, and stimulating adult neurogenesis. Studies on adult
neurogenesis have shown that the changes are mediated by estrogen and prolactin
in case of females’ adult neurogenesis enhanced by the exposure to male-soiled
bedding. Inducing estrus in females by exposure to male-soiled bedding also
suggests that it stimulates secretion of estrogen and prolactin, which increase their
secretion in pro-estrous to estrous status. Recent studies using transneuronal
markers have shown that exposure to pheromone stimulates vomeronasal neurons,
which activate gonadotropin-releasing hormone (GnRH). These studies suggest that
the activation of GnRH neurons by exposure to pheromones stimulated the secre-
tion of sex hormones and prolactin. And, if so, it is possible that there are other
organs and body systems, which are under the control of these hormones, affected
by the exposure to pheromones because of the changes in the secretion of these
hormones.
One of such organs is the mammary gland. Changes in the secretion of hormones
affect the morphology of mammary glands and the system is known to show
changes along the estrous cycles. If the secretion of sex hormones is affected by
5.5 Mammary Gland Expansion by Exposure to Male Pheromone 69
exposure to male pheromones, it is possible that the mammary glands also go

through some structural changes following the exposure to the pheromones.
We exposed adult female mice to male pheromone, SBT (50 uL of 250 ppm
solution; control group was exposed to 50 uL of water), twice per day for one week,
as we did in adult neurogenesis studies. After one week of exposure, mammary
glands were collected, fixed with Tellyesnizky fixative (70 % EtOH, gracial acetic
acid, formaldehyde, 20:1:1), stained with carmine red staining solution, went
through dehydration process, and observed in toluene buffer (Tiran and Elson
2003). The toluene buffer clears the fat pad and makes the stained mammary gland
visible (Fig. 5.6). The mammary glands have tree-like structure with branches and
the end of branches have the developing point called terminal end buds (TEB). We
counted the number of branches and TEBs and compared between the groups and
found that the mammary glands of the females exposed to SBT had significantly
more branches and TEBs (Fig. 5.6) (Koyama et al. 2015). The mammary glands
structure changes along the estrous cycles but the changes by the exposure to SBT
were larger than the changes by estrous cycle.
Fig. 5.6 Influence of exposure of female mice to SBT on the number of terminal end buds
(TEB) and branches in the mammary tree (from Koyama et al. 2015). ***P < 0.001, Error bars
show standard error. #4 mammary gland of a female exposed to SBT (a) and water (b). Number of
terminal end buds (c) and branches (d) of females exposed to SBT, farnesene and control group.
Fig. 5.7 Influence of exposure of female mice of various ages to SBT on the number of terminal
end buds (TEB) and branches in the mammary tree (modified from Koyama et al. 2015).
***P < 0.001, error bars show standard error
As we observed differences in the influence of exposure to male pheromone on

neurogenesis in females by females’ age, we questioned if there are any differences
in the influence to mammary gland as well. The influence to expand mammary
gland was strong in adult females and, although the influence to molecular envi-
ronment in the mammary gland still needs to be determined, it did not affect the
mammary gland tree-like structure in young females around puberty and early after
puberty (Fig. 5.7).
We then mated the females for 2 weeks after they were exposed to a male
pheromone, SBT (50 uL of 250 ppm solution twice daily for one week). Control
group females were exposed to water (50 uL, twice daily for one week) and then
mated for one week. We hypothesized that mating and exposure to male mouse will
stimulate the mammary gland of the control group females to expand and the
difference between them and the females exposed to pheromone may disappear. Or,
the mating will stimulate the mammary glands of both the control group and
pheromone group females to further expand and differentiate and the difference
between the two groups remain. We weighed the mammary glands on postdelivery
day 10 and found that the mammary glands of the females exposed to SBT were
still heavier than the control group females (Fig. 5.8) (Koyama et al. 2015).
5.6 Trans-generational Influence of Exposure

to Pheromones
It has been thought for decades that experiences in the life of an individual will not
be inherited and affect the next generation. However, recent studies have shown that
this is not true. A recent study has shown that the offspring of male mice, which
5.6 Trans-generational Influence of Exposure to Pheromones 71
Fig. 5.8 Exposure to SBT to

virgin females had long-term
influence through pregnancy
until lactating stage (modified
from Koyama et al. 2015).
The mammary gland was
heavier in the dams exposed
to SBT before mated.
**P < 0.01, NT females
without treatment of exposure
to SBT or water
experienced fear conditioning with a non-pheromone odorant, acetophenone, were

more sensitive to the smell of acetophenone. These offspring showed fear response
to acetophenone from the first exposure to it and showed avoidance at lower
concentration than the first generation (Dias and Ressler 2014). When Dias and
Ressler (2014) used transgenic mice with tauLacZ gene tagged to the gene of
olfactory receptors for acetophenone, M71, they found that the offspring of males
which went through fear conditioning with acetophenone had more olfactory sen-
sory neurons with receptors for acetophenone expressed in their main olfactory
system (Dias and Ressler 2014), which is considered to have contributed to the
higher sensitivity, i.e., showing responses from lower concentration, to the smell of
acetophenone.
Acetophenone is not a pheromone. In insects, there are studies showing that
when aphids are exposed to alarm pheromones, the ratio of aphids with wings in the
next generation becomes higher (Podjasek et al. 2005). Interestingly, there was
positive correlation between the concentration of pheromone and the ratio of aphids
with wings, i.e., the exposure to higher concentration of the pheromone was fol-
lowed by higher ratio of aphids with wings in the next generation (Podjasek et al.
2005). This suggests that the offspring of aphids exposed to alarm pheromone might
be faster in escaping at emergency situations. Review papers on the influence of the
environmental conditions on the next generation have shown that about 75 % of the
factors that had influence on the next generation was negative factors (cold tem-
perature, malnutrition, low oxygen, and handling stress) (Burton and Metcalfe
2014). One of the factors that has positive influences in the next generation is
maternal behavior. There are studies showing that, in rats, the female offspring of
mothers, which showed more maternal behaviors (grooming and licking), became
good mothers showing more maternal behaviors (Champagne 2011). These studies
have shown that such changes in the maternal behaviors of the offspring are due to
differences in the expression of genes through epigenetic modifications.
In our studies on the influences of exposure to male pheromone on mammary
gland expansion, we observed the enhanced expansion of mammary gland by male
pheromone continuing until the postdelivery stage. We hypothesized that this might
have impact on the growth of their offspring. The body weights of the offspring
were not different from the offspring of control group dams. However, at adult, they
showed better performances in spatial memory tests using Morris water maze
(Fig. 5.9). Cross-fostered offspring of control group dams raised by pheromone-
exposed dams also showed good performance in Morris water maze indicating that
the differences are caused by some postnatal factor.
Possible differences to cause postnatal differences in the offspring are the milk or
the maternal behaviors. From our results on the differences in mammary glands
between the groups, we suspected that milk might have caused differences in the
offspring. Studies have also shown that sialic acid in the milk can serve as an
exogenous source of polysialic acid (PSA) and polysialylated neural cell adhesion
molecule (PSA-NCAM) is involved in the development of the brain neural system
(Wang 2009; Hiratsuka et al. 2013). PSA-NCAM is widely expressed on the sur-
face of cells in the central nervous system. PSA is found at high level in the early
developmental stages, i.e., embryonic and early postnatal stages, and in adults it is
maintained at a high level in the hippocampus and olfactory system, where adult
neurogenesis takes place (Bonfanti et al. 1992; Seki and Arai 1993a, b; Bonfanti
2006). NCAM regulates cell migration, neurite outgrowth, axon elongation and
synaptic formation, and plasticity (Wang 2009). It is possible that the offspring of
females exposed to SBT received more sialic acid containing oligosaccharides,
which can serve as exogenous source of sialic acid for the offspring. It is also
possible that polysialyltransferases, which polymerize sialic acid to PSA, are more
expressed in the offspring of dams exposed to SBT that they can synthesize more
Fig. 5.9 Offspring of females

exposed to SBT showed faster
learning and higher plateau
level in finding the location of
hidden platform in the Morris
water maze as adults.
*P < 0.05, **P < 0.01, NT
offspring of females without
treatment before mated, SBT
offspring of females exposed
to SBT before mated, Water
offspring of females exposed
to water before mated
5.6 Trans-generational Influence of Exposure to Pheromones 73
PSA. We measured the mRNA of polysialyltransferasee, which polymerize sialic

acids, in the brains of postnatal day 10 pups and found that one of the types of
polysialyltransferase, ST8SiaIV, was expressed significantly more in the brain of
the offspring of pheromone-exposed dams. These results suggest that the enhanced
amount of sialic acid in the milk that the offspring of pheromone-exposed dams had
received might have caused enhanced development of the brain in them and led to
better cognitive function as adults. This also suggests that studies on the milk
components and brain development may provide significant information on the
influences of nutrition during the early postnatal stages and the cognitive function
as adults.
References
Allen E (1912) The cessation of mitosis in the central nervous system of the albino rat. J Comp
Neurol 22:547–568
Altman J (1962) Are new neurons formed in the brains of adult mammals? Science 135:1128–1129
Altman J (1963) Autoradiographic investigatin of cell proliferation in the brains of rats and cats.
Anat Rec 145:573–591
Bonfanti L, Olive S, Poulain DA, Theodosis DT (1992) Mapping of the distribution of
polysialylated neural cell adhesion molecule throughout the central nervous system of the adult
rat: an immunohistochemical study. Neuroscience 49(2):419–436
Bonfanti L (2006) PSA-NCAM in mammalian structural plasticity and neurogenesis. Prog
Neurobiol 80:129–164
Burton T, Metcalfe NB (2014) Can environmental conditions experienced in early life influence
future generations? Proc R Soc B 281:20140311
Champagne FA (2011) Maternal imprints and the origins of variation. Horm Behav 60(1):4–11
Dias BG, Ressler KJ (2014) Parental olfactory experience influences behavior and neural structure
in subsequent generations. Nat Neurosci 17(1):89–96
Harvey S, Jemiolo B, Novotny M (1989) Pattern of volatile compounds in dominant and
subordinate male mouse urine. J Chem Ecol 15:2061–2071. doi:10.1007/BF01207438
Hiratsuka S, Honma H, Saitoh Y, Yasuda Y, Yokogoshi H (2013) Effects of dietary sialic acid in
n-3 fatty acid-deficient dams during pregnancy and lactation on the learning abilities of their
pups after weaning. J Nutr Sci Vitaminol (Tokyo) 59(2):136–143
Imayoshi I, Sakamoto M, Ohtsuka T, Takao K, Miyakawa T, Yamaguchi M, Mori K, Ikeda T,
Itohara S, Kageyama R (2008) Roles of continuous neurogenesis in the structural and
functional integrity of the adult forebrain. Nat Neurosci 11:1153–1161
Koyama S, Kamimura S (1999) Lowered sperm motility in subordinate social status of mice.
Physiol Behav 65:665–669
fnbeh.2013.00101 eCollection 2013
Koyama S, Soini HA, Wager-Miller J, Alley WR, Pizzo MJ, Rodda C, Alberts J, Crystal JD,
Lai C, Foley J, Novotny MV (2015) Cross-generational impact of a male murine Pheromone
2-sec-butyl-4,5-dihydrothiazole in female mice. Proc Biol Sci 282(1811) doi:10.1098/rspb.
2015.1074 PMID: 26136453 http://dx.doi.org/10.1098/rspb.2015.1074
Larsen CM, Kokay IC, Grattan DR (2008) Male pheromones initiate prolactin-induced
neurogenesis and advance maternal behavior in female mice. Horm Behav 53(4):509–517
Ultrasensitive pheromone detection by mammalian vomeronasal neurons. Nature
405(6788):792–796
chemosignals in suppression of estrus in the house mouse: the Lee-Boot effect revisited. Biol
Reprod 59:1317–1320
Mak GK, Enwere EK, Gregg C, Pakarainen T, Poutanen M, Huhtaniemi I, Weiss S (2007) Male
pheromone–stimulated neurogenesis in the adult female brain: possible role in mating
behavior. Nat Neurosci 10:1003–1011
Oboti L, Savalli G, Giachino C, De Marchis S, Panzica GC, Fasolo A, Peretto P (2009) Integration
and sensory experience-dependent survival of newly-generated neurons in the accessory
olfactory bulb of female mice. Eur J Neurosci 29:679–692
Paton JA, Nottebohm FN (1984) Neurons generated in the adult brain are recruited into functional
circuits. Science 225:1046–1048
Podjasek JO, Bosnjak LM, Brooker DJ, Mondor EB (2005) Alarm pheromone induces a
transgenerational wing polyphenism in the pea aphid, Acyrthosiphon pisum. Can J Zool
83:1138–1141
Seki T, Arai Y (1993a) Distribution and possible roles of the highly polysialylated neural cell
adhesion molecule (NCAM-H) in the developing and adult central nervous system. Neurosci
Res 17(4):265–290
Seki T, Arai Y (1993b) Highly polysialylated neural cell adhesion molecule (NCAM-H) is
expressed by newly generated granule cells in the dentate gyrus of the adult rat. J Neurosci
13(6):2351–2358
Tiran Z, Elson A (2003) Whole mount analysis of mammary gland structure in PTP epsilon
transgenic mice. Methods Enzymol 366:124–132. doi:10.1016/S0076-6879(03)66011-8
Wang B (2009) Sialic acid is an essential nutrient for brain development and cognition. Annu Rev
Nutr 29:177–222
Chapter 6
Ontogeny
Abstract When do we start to respond to pheromones? Olfactory sensory neurons

and glomeruli in the olfactory bulbs develop mostly in postnatal stages, but studies
have shown that learning and establishment of preferences to odors start from late
embryonic stages. The mechanisms of olfactory learning during embryonic stages
need further investigations to determine the mechanisms. Recent studies have found
that experience in one generation affects the olfactory sense in the next generation.
The exposure to some odorants combined with fear experience caused epigenetic
changes in the expression to the receptors to these odorants and enhanced the
sensitivity to these odors in the next generation. These studies show that not only
the preference/avoidance to odors are acquired from early stages in life but also
show that olfactory sense itself becomes modified, affecting the behaviors and such
changes will most likely affect how strongly the primer effects will take place as
well. It is a long-term influence!
Keywords Development of olfactory sense Epigenetic modification
6.1 Ontogeny of the Olfactory System
There is a primer effect called Vandenbergh effect, in which female pups reach
puberty earlier when there is male odor in the environment. In other words, female
pups reach puberty late if there are no odors of males in the environment. This
indicates that females can detect and respond to males’ odors from very early stage
in their lives. As, without males in the environment, female pups will not breed
early, the delay of puberty is an adaptive change produced by the lack of male odors
and it is also adaptive to be sensitive to the odors of males from early develop-
mental stages. In case of male pups, however, it can be more beneficial to be less
responsive to odors of the opposite sex at their early developmental stages, because
the odors of their own mothers may stimulate male pups. When mice start to
respond the odors/pheromones of the opposite sex and whether there are gender
differences in it, are critical questions in primer effects.

76 6 Ontogeny
Studies have shown that olfactory system starts to develop during the
middle-to-late embryonic stages (Fig. 6.1). The duration of pregnancy is 19 days in
mice and the dendrites of olfactory sensory neurons extend to the surface as early as
embryonic stage (E) 11 and the cilia extends from the dendritic knobs around E14
(Cuschieri and Bannister 1975; Lam and Mombaerts 2013). The expression of
olfactory receptor genes starts from E9 to E16, the day depending on the type of
receptors. The olfactory receptor protein first start to accumulate in the dendrite
knobs before the cilia develops, and once the cilia extends the olfactory receptor
protein diminishes from the dendrite knobs, presumably migrating along with the
extension of the cilia from the knobs (Schwarzenbacher et al. 2005). The percentage
of olfactory sensory neurons that show responses to odor is still low (about 8 % of
the olfactory sensory neurons) during E15.5 to E18.5 and almost no responses are
observed at E14.5 (Lam and Mombaerts 2013). The development of glomeruli at
the olfactory bulb with the axons from olfactory sensory neurons is still later, and at
E18.5 they are only starting to show web-like structure (Potter et al. 2001). The
structure typical to glomeruli develops during postnatal stages. The number of
olfactory sensory neurons increases extensively during the early postnatal period
before postnatal day (P) 24 (Fig. 6.1) with peak at P14, and the diameter of
glomeruli becomes about 4 times larger at P24 compared to that at P1 (Lam and
Mombaerts 2013) (Fig. 6.2). Neurogenesis of olfactory sensory neurons decreases
after the peak but continues at certain level through life (Ma et al. 2014).
One of the amazing mysteries in the development of the olfactory system is how
axons produce the glomeruli with other axons that carry the same type of olfactory
receptor. The development of glomeruli with the same olfactory receptor is the key
finding that explains how olfactory messages are converted to the discrimination of
odor types. Recent study by Ron Yu group of Stowers Institute showed that
activities of olfactory sensory neurons during the first 21 postnatal days have
critical impact on the formation of glomeruli (Ma et al. 2014). They used a
Fig. 6.1 Changes in the

number of olfactory sensory
neurons along the
development (from Lam and
Mombaerts 2013). With
average and SEM
6.1 Ontogeny of the Olfactory System 77
Fig. 6.2 Change in the size of glomeruli along development (from Lam and Mombaerts 2013).
Gfp expression in the glomeruli along development for olfactory receptor gene S1 (also called
Olfr749 and MOR106-1) (a) and MOR23 (also called Olfr16 and MOR267-13) (b). c Diameter of
the glomeruli along development. Scale bars = 100 um, shown with average and SEM
transgenic mice strain that has overexpression of K+ ion channels and the over-
expression of K+ ion channels could be suppressed by feeding food that contains
doxycycline. K+ has suppressive influences on the activities of olfactory sensory
neurons. By feeding food with doxycycline to the dams with pups at different
developmental stages, it is possible to test the influences of suppression/activation
of the activities of olfactory sensory neurons on the development of glomeruli in the
olfactory bulb. The mice they used also had green fluorescence protein
(GFP) tagged to one of the olfactory receptors M72 so they could examine the
growth of the glomeruli for olfactory receptor M72. The number of glomeruli for a
same type of olfactory receptor is known to be 1 or some cases 2 and, at early
postnatal stages, it is higher (average about 1.7) but decreases as they become adult
(average about 1.2 at P60 and over P90) (Zou et al. 2004). Yu group first found that
when the dams are not fed with doxycycline food, which will activate the olfactory
system, and the activity of olfactory sensory neurons were thus suppressed, the
number of glomeruli for M72 becomes multiple, up to about 5 (Ma et al. 2014).
They also found that once the glomeruli become multiple, it does not reduce the
number to the normal one glomerulus later even if the pups are fed doxycycline
food after P21. When they started to feed the dams from various time points, they
found that if the starting day is later than when the pups are P5, the glomeruli
become multiple. As it takes 2 days for the doxycycline to show influences, they
concluded that P7 is the critical period for the normal development of glomeruli.
This time point matches to the timing of glomeruli development as it is considered
that the glomeruli are developed and become connected with mitral cells around
78 6 Ontogeny
this age. Such results resembled the results obtained in another study, which did
naris closure at various postnatal days and examined its influence on the number of
glomeruli (Zou et al. 2004). In that study the naris closure treatment made the
number of glomeruli to become multiple with the average around 2, which was
much less than in Ron Yu’s study from unknown reason, and the influence of naris
closure was stronger when the treatment was conducted before P10 (Zou et al.
2004).
These studies show that the olfactory system of mice show development rather
late, starting from late embryonic stage and completed postnatally. For the neonatal
mice pups, the activation of olfactory sensory neurons by exposure to olfactory cues
is considered to have significant roles in this completion of olfactory system
development. A very interesting thing in relation to the completion of olfactory
system and learning of odors and showing preferences/avoidance to odors is that
there are studies from decades ago showing that the olfactory learning starts from
embryonic stages and these learning affects the preferences to the odor in the
neonates. For example, studies using rats have shown that rat pups exposed to apple
juice with small amount of lithium chloride (LiCl) at embryonic stage 20 showed
avoidance to the smell of apple juice at postnatal day 10 (Smotherman 1982). As
glomeruli are not developed yet at the embryonic stages, it is possible that detec-
tion, discrimination, and learning of odors takes place through direct connection of
the olfactory sensory neurons and mitral cells at the olfactory bulb, and then the
information goes to the brain. This will need to be determined in future.
6.2 Developmental Changes in the Responses

to Pheromones
As written above, earlier studies have found that exposure to male-soiled bedding
stimulates female pups to accelerate puberty (Vandenbergh effect) (Vandenbergh
1967, 1969). This indicates that female pups can detect male pheromones and their
hormone secretion becomes affected by the exposure. However, sex differences
were found in this detectability at early postnatal stages. Studies have shown that
the microvillar membranes from male mice pups (20 to 21 days old, prepubertal
males) did not show inositol (1,4,5)-triphosphate (IP3) production in the microvillar
membrane of vomeronasal neurons (VN), which are used to measure activation of
VNs, when they were exposed to urinary chemicals (Thompson et al. 2004). There
was also no significant increase in the IP3 when it was exposed to 2-heptanone or
2,5-dimethylpyrazine, which are female specific pheromones (Thompson et al.
2004). When the microvillar membranes from the vomeronasal organ of the same
age female pups were exposed to urine from adult females or adult males, there was
a significant increase in the IP3 level (Thompson et al. 2004). The responses were
also high when it was exposed to single chemical compounds, 2-heptanone or
2,5-dimethylpyrazine. These results show that at early postnatal stages, VNs from
6.2 Developmental Changes in the Responses to Pheromones 79
males are not sensitive to pheromones of opposite sex. In our previous study,
exposure of adult males to 250 ppm 2,5-dimethylpyrazine directly on their nostril
induced a strong influence on enhancing cell proliferation in the brain (Koyama
et al. 2013, 2014). When males start to show responses to female pheromone is not
determined and whether it is related to the changes at puberty is necessary to be
elucidated.
In our previous study (Koyama et al. 2013, 2014), the exposure to male pher-
omones enhanced cell proliferation in the brain in adult females but it did not in
prepubertal, postpubertal, and young adult females. As females show responses to
pheromones in in vitro cell level studies, these differences along age are possibly
not peripheral level differences but might be due to some differences in the
molecular environment in the brain that hinders the influences of pheromones to
affect cell proliferation in the brain. The changes before and after puberty seems to
be not making the changes as the postpubertal and young adult females also did not
show enhanced cell proliferation in the brain by exposure to male pheromones.
What exactly is causing these changes along age is an important topic in under-
standing the mechanisms of adult neurogenesis.
6.3 Epigenetic Changes on the Sensitivity to Smell
As written in Chap. 5, recent studies have found that when mouse experience fear
accompanied by a specific odor, their offspring express more olfactory sensory
neurons with the olfactory receptor specific to the odor, which makes the offspring
more sensitive to the odor. In this study acetophenone was used as the olfactory
receptor gene is known (Olfr151) for the receptor (M71), and it is possible to
visualize the expression by using a transgenic mice with LacZ gene expressed to
M71 (M71-IRES-tauLacZ) (Dias and Ressler 2014). They did 5 trials per day for
3 days of fear conditioning using 8 weeks old male mice (F0). The fear conditioning
was conducted with 10 s of odor exposure (either acetophenone or propanol) and
slight electric shock (0.4 mA) during the last 0.25 s of the 10 s of odor exposure.
Ten days later, these males were mated for 10 days and then separated from the
females, which meant that their offspring (F1) never had the chance to meet their
father. The olfactory bulbs of the offspring were stained with 5-bromo-4-
chloro-3-indolyl-β-d-galactoside (X-gal) to visualize the number of olfactory sen-
sory neurons that carry the tauLacZ tagged to Olfr151. They found that the glo-
meruli of M71 receptor were significantly larger in the F1 of males exposed to
acetophenone beforehand (Fig. 6.3). These F1 mice also showed startle behaviors to
the odor than control and avoidance to the odor (stay in a chamber without odor) at
lower concentration than the offspring of male mice that experienced fear condi-
tioning with propanol (Fig. 6.4). Further surprising results of them were that these
80 6 Ontogeny
Fig. 6.3 Sizes of acetophenone-responding glomeruli and number of olfactory sensory neurons
(OSN) in the F1 males of males experienced fear conditioning with the odor of acetophenone
(F1-Ace-M71) (from Dias and Ressler 2014). Control groups were F1 males of males exposed to
propanol (F1-Prop-M71) and F1 males of males left in home cages without fear conditioning with
some odor (F1-Home-M71). (a–f) X-gal staining of tauLacZ expressed in M71 gene, which is the
olfactory receptor gene for acetophenone. F1 males of males exposed to acetophenone while fear
conditioning are showing larger volume of staining in the dorsal (a–c) and medial (d–f) olfactory
bulb. Size of glomeruli measured as pixel size at the dorsal area (g) and medial area (h). Number of
olfactory sensory neurons with M71 gene (i). With average and SEM, *P < 0.05, **P < 0.01,
***P < 0.01, ****P < 0.0001. Scale bar = 1 mm
differences between the groups were still observed at the F2 stage (Fig. 6.5). The F2
of the male that experienced fear conditioning with acetophenone had larger glo-
meruli for M71 and they showed more fear response to the odor than the F2 of
control group mice. Studies using insects have also shown that when mother
generation of aphids were exposed to alarm pheromone, (E)-ß-farnesene, which is
identified as a male murine pheromone as well, the next generation had higher
percentage of aphids with wings (Podjasek et al. 2005).
One of the questions we encounter from these studies is whether only “fearful or
negative experiences” have transgenerational influences in the descending genera-
tions. In a review paper on transgenerational influences of environmental conditions
(nutrition level, low temperature, hypoxia, etc.) about 75 % of the cases listed were
some negative conditions (Burton and Metcalfe 2014). Although it is possible that
negative influences of severe conditions are more noticeable, it is still possible that
6.3 Epigenetic Changes on the Sensitivity to Smell 81
Fig. 6.4 Responses to the odor of acetophenone and propanol in the F1 males of males
experienced fear conditioning with acetophenone or propanol (from Dias and Ressler 2014).
Odor-potentiated startle (OPS) responses to acetophenone (a) and propanol (b). OPS was
measured as follows: habituation stage (3 separate days of experience in the startle box for 5 to
10 min), followed by 15 trials of “noise alone” (105 dB noise burst), and then 10 trials of “noise
presented with 10 types of odor” (odor was presented for 10 s with a noise for 50 ms at the end).
Each “odor + noise” trial had “noise-alone” trials between. F1 males also showed aversion to the
odor their fathers experienced fear conditioning from lower concentration. Odor aversion to
acetophenone (c) and propanol (d) was measured by the time spent in a chamber with and without
the odor. Aversion index was calculated by subtracting the time in the odorless chamber from the
time in the odor chamber. Shown with average and SEM. *P < 0.05, **P < 0.01
negative conditions have larger impact on the following generations. The severe
nutritious conditions at a part of Netherland occupied by German during WWII
(Dutch hunger or Dutch famine) is known to have produced tendencies in the
children of women pregnant during that time to be smaller and also to show
increased incidences of some diseases. The studies on the mechanisms of changes
in the sensitivity of olfactory sense may have possibilities to become an animal
model system to determine the mechanisms that transgenerational modifications of
environmental conditions.
82 6 Ontogeny
Fig. 6.5 Sizes of glomeruli and OPS responses in the F2 males of males experienced fear
conditioning with acetophenone (from Dias and Ressler 2014). The sizes were larger (a and b) and
the OPS responses in these males were stronger (e). Scale bar = 200 um, *P < 0.05. With average
and SEM
References
Burton T, Metcalfe NB (2014) Can environmental conditions experienced in early life influence
future generations? Proc R Soc B 281:20140311
Cuschieri A, Bannister LH (1975) The development of the olfactory mucosa in the mouse: electron
microscopy. J Anat 119:471–498
Dias BG, Ressler KJ (2014) Parental olfactory experience influences behavior and neural structure
in subsequent generations. Nat Neurosci 17(1):89–96
subventricular zone by synthetic murine pheromones. Front Behav Neurosci. DOI:10.3389/
in the murine subventricular zone. Biochem Soc Trans 42(4): 882–885
Lam RS, Mombaerts P (2013) Odorant responsiveness of embryonic mouse olfactory sensory
neurons expressing the odorant receptors S1 or MOR23. Eur J Neurosci 38:2210–2217
Ma L, Wu Y, Qiu Q, Scheerer H, Moran A, Yu CR (2014) A developmental switch of axon
targeting in the continuously regenerating mouse olfactory system. Science 344(6180):194–197
Podjasek JO, Bosnjak LM, Brooker DJ, Mondor EB (2005) Alarm pheromone induces a
transgenerational wing polyphenism in the pea aphid, Acyrthosiphon pisum. Can J Zool
83:1138–1141
Potter SM, Zheng C, Koos DS, Feinstein P, Fraser SE, Mombaerts P (2001) Structure and
emergence of specific olfactory glomeruli in the mouse. J Neurosci 21(24):9713–9723
Schwarzenbacher K, Fleischer J, Breer H (2005) Formation and maturation of olfactory cilia
monitored by odorant receptor-specific antibodies. Hisochem Cell Biol 123:419–428
Smotherman WP (1982) Odor aversion learning by the rat fetus. Physiol Behav 29:769–771
Thompson RN, Robertson BK, Napier A, Wekesa KS (2004) Sex-specific responses to urinary
chemicals by the mouse vomeronasal organ. Chem Senses 29:749–754
Vandenbergh JG (1967) Effect of the presence of a male on the sexual maturation of female mice.
Endocrinology 81(2):345–349
References 83
Vandenbergh JG (1969) Male odor accelerates female sexual maturation in mice. Endocrinology
84(3):658–660
Zou DJ, Feinstein P, Rivers AL, Mathews GA, Kim A, Greer CA, Mombaerts P, Firestein S
(2004) Postnatal refinement of peripheral olfactory projections. Science 304(5679):1976–1979
Chapter 7
Adaptive Functions
Abstract What is the function of primer effects? Suppression of estrous cycle when
males are not around (Lee–Boot effect) and coming into estrus when females are
exposed to males’ odors (Whitten effect) would be highly adaptive in reproduction.
Also, being unable to establish pregnancy (Bruce effect) by exposure to unfamiliar
males’ odor is also adaptive considering that there is higher risk of infanticide by the
male. Enhanced neurogenesis in virgin female mice by exposure to male pher-
omones enabled them to distinguish and prefer dominant males than subordinate
males, which would enhance reproductive success with dominant males. These
studies suggest that primer effects can have high adaptive function. Studies in
humans have shown possibilities that primer effects are possible in humans as well.
Studies using mice as model animal may provide further insights into the possible
primer effects in humans. How can we enhance our survival using odors?
Keywords Primer effects Reproductive success
7.1 What is their Function in Reproductive Success?
In our daily life, people are surrounded by other individuals of the same and
opposite sex unless they are in some unusual extreme situation from some reasons.
Whether daily life situation is a multi-sex environment or not depends on the social
system of animal species. In case of house mice, this environmental situation is the
same and both in house mice and in humans, primer effects are found. This suggests
that primer effects may have some positive functions especially developed in
socially living animal species. So, what is the function of primer effects? Is there
any benefit for mice to adjust or modify their reproductive system and neurogenesis
using pheromones? Studies on the evolution of social system have postulated the
benefits of living in groups as (1) enhancing productivity by collaborations in
working, (2) enhancing protection against predators/enemies by counterattacking
together, or (3) enhancing survival against negative environmental factors like cold
temperature by huddling. In case of house mice with small body size, the advantage

86 7 Adaptive Functions
of living in groups is considered to be the third reason, i.e., to enhance survival by

huddling together.
In wild animals, mating season is the time period that protective activities by the
group members against enemies become least effective because of the increased
attention to the possible mating partners, fighting to obtain the mating partners,
protecting the mating partners from competitors, and engaging themselves in mating
behaviors. These situations decrease the attention to possible enemies, i.e., the
predators and non-group member individuals trying to invade in the territory. Frank
Fraser Darling hypothesized that group living animals show shortened
mating/breeding season and such synchronization of breeding enhances the repro-
ductive success (Fraser Darling effect). This hypothesis was postulated from his
observations in herring gulls and became expanded to other animal species. The
behaviors and other sensory stimuli emitted by the individuals in the group that came
into estrus early stimulate others, pulling the whole group into the mating/breeding
status. Such influences eventually shorten the period of mating/breeding season,
which is beneficial for the group by minimizing the season that group members are
distracted from the activities to protect the group. Studies of primer effects show that
the odors of each other, other than behaviors, visual stimuli (like some color change
specific to estrous condition) and vocal stimuli (like songs of birds or some vocal
expression that inform that the individual is in estrus), can stimulate other individ-
uals in and outside the group to enhance the reproductive conditions.
For a mammalian species with small body size like mice, it will be beneficial to
live in groups to help maintaining the body temperature. For small mammalian
species that live in groups, it will be beneficial to give births simultaneously so that
dams can share the nursing efforts of pups with other dams in the same group
(called “communal breeding”). Synchronized estrous cycles enable simultaneous
deliveries. So, the primer effects can be adaptive if they influence the females in the
same groups to synchronize the reproduction. Then, why should females suppress
each other’s estrous cycle (Lee-Boot effect)? Lee–Boot effect becomes stronger
when the group size of females become larger (see Fig. 3.3 in Chap. 3), which made
people think in early days that it is a negative influence due to overcrowded
situation. However, this may not be necessarily the case. Even when the group size
of females is large, Lee–Boot effect does not happen if there are males’ odors. It
happens when there are many females but no male in the environment, and in this
condition, there is no need to come into estrus as the lack of males’ odors indicates
there is no chance of breeding. This suggests that it evolved with a positive function
of avoiding unnecessary changes in the physiological conditions and staying in a
“dormant” condition. This hypothesis can be tested by, for example, measuring
energy expenditure in the females in Lee–Boot effect condition compared to
females with estrous cycles.
The function of Bruce effect (the pregnancy block by unfamiliar males’ odor)
was puzzling and it was often compared with the infanticide behaviors by males
that took over breeding colonies in hanuman langur monkeys (Semnopithecus
entellus) and lions (Panthera leo). The typical characteristics of the breeding
colonies of these animal species are that they establish one male breeding colonies
7.1 What is their Function in Reproductive Success? 87
with multiple females and bachelor males make their own groups around the
breeding colonies. These bachelor males occasionally get rid of the breeder males
and take over the breeding colonies. This breeding system induces a high com-
petition among the males and limited time period for them to reproduce. Thus,
when a bachelor male takes over a breeding colony, the first thing he does is killing
the unweaned offspring in the colony. When females are nursing, they do not come
into estrus. When infanticide happens, it will stimulate the females to come into
estrus immediately (Sugiyama 1993) and thus the new breeder male can immedi-
ately mate with these females.
In case of mice, studies have shown that males will show infanticide at about
30–50 % of the cases when breeder male was replaced after the pregnancy is
already established and Bruce effect did not take place. If Bruce effect happened to
the females, the female will come into estrus in about a week. If it did not happen,
female will come into estrus on the day of delivery (post parturition estrus), and
Bruce effect will make the timing of mating for the new male about 10 days earlier.
This difference does not sound large difference for humans, but for house mice with
short longevity and high vulnerability to predators, it may be a worthwhile dif-
ference. The Bruce effect functions for males to obtain offspring earlier and enables
females to avoid infanticide of their offspring after all the process of pregnancy.
7.2 Function of Enhanced Adult Neurogenesis

by Exposure to Pheromones
The function of adult neurogenesis was a question asked by multiple scientists.

Recent studies have shown that after the exposure to male-soiled bedding and the
following enhanced adult neurogenesis, female mice distinguished and showed
preference to dominant males (Mak et al. 2007). Considering the possibilities that,
in the field, stronger males will have better food resources, this may enhance the
reproductive success. Females with enhanced neurogenesis by exposure to male
odors showed better memory of the males they mated (Oboti et al. 2011). Studies
on the function of enhanced adult neurogenesis by exposure to pheromones are still
few and there may be more functions to be found in future.
7.3 Possible Pheromonal Signaling in Humans

and its Function
We humans are one of the species that mostly live in social groups, often working
in groups to enhance productivities and living in family groups. Our modern
technology and civilized lifestyle may make us hard to believe that we may be
“still” under the control of odors of each other. There are still discussions on
whether there are pheromones in humans that affect mate selection and physio-
logical conditions in humans. There are chemical compounds that have been known
as “pheromone candidates” in humans, but they are still not totally determined to be
pheromones. The less developed olfactory system is an encouraging fact for people
who are against the influences of odors on physiological conditions and behaviors.
Besides, because traditionally it was considered that the accessory olfactory system
is responsible to the pheromone-induced changes, the results of anatomical studies
showing that the vomeronasal system in the nasal cavity of humans is only a vestige
that lack functional activity especially in postnatal stages became a strong evidence
that pheromonal signaling is not expectable in humans. The strong scientific
arguments against the pheromonal influences in humans were mostly based on this
lack of functional activity in the vomeronasal organs in humans. However, the
recent studies showing the main olfactory system are the pathway that the signaling
pathway reaches to GnRH neurons (Yoon et al. 2005) have made the situation
totally changed. The understanding of the mechanisms of primer effects in mice
may be of help for the people who are hoping to enhance their reproductive
conditions.
There was a paper published in Nature by Martha McClintock reporting on
menstrual cycle synchronization among women who live in the same dormitory of a
women’s college (McClintock 1971). The onset of menstrual periods in the 135
females, aged 17–22 years, showed smaller differences (closer onset) in March,
later in the academic year, compared to October, soon after the new semester
started. This tendency was especially clear in the close friend groups, suggesting
that the more time they spend together the closer their onset becomes. It was even
reported as an example that one woman had a cycle of 6 months, which shortened
to 4.5 weeks when she began to date, but returned to longer cycle when she stopped
dating. These results on menstrual onset and the length of cycles resemble the Lee–
Boot effect and Whitten effect in mice (see Chap. 3). Much later it was also found
that, when axillary compounds were collected from odor donor women who were at
follicular phase of menstrual cycle and exposed them to women, their menstrual
cycles became shorter (−1.7 ± 0.9 days), whereas, when these were collected from
the same odor donor women at ovulatory phase and exposed them to women, their
menstrual cycles became longer (+1.4 ± 0.5 days) (Stern and McClintock 1998).
Odor of men also activates the hypothalamus in women (Savic and Berglund 2010)
supporting the possible primer effects in humans.
Even though the pheromones of humans are not determined precisely yet, if we
can understand the mechanisms of the changes in the physiological conditions
induced by exposure to pheromones in mice, we might be able to utilize the
knowledge in clinical trials or improving health conditions of humans. The basic
studies on (1) how pheromones are detected at the peripheral sensory systems,
(2) how the detection is different by sex and changes along age, (3) how the
detection is affected by hormone secretions, (4) how activation of the sensory
neurons affect the brain, and (5) how hormone secretion is in turn affected by
pheromones will have important implications on maintaining and improving human
7.3 Possible Pheromonal Signaling in Humans and its Function 89
health, in the young to elderlies. The utilization of mice for these basic studies will
certainly provide many of the answers or mechanistic insights to the answers that
we need.
Appendix
Table A.1 List of mouse releaser effects and primer effects

Pheromones Receptor or References Chapter
olfactory
system
Releaser effects
Male-male MUP VNO Chamero et al. 4
aggression (2007)
? MOE
3,4-dehydro-exo-brevicomin Novotny et al. 4
(DHB) (1985)
2-sec-butyl-4,
5-dihydrothiazole (SBT)
E,E-α-farnesene and
E-β-farnesene
Preference/attraction Darcin (MUP20) Roberts et al. 4
to male (2010)
Parental behavior Females: Wu et al. (2014) 4
multiple types
of sensory
stimuli induce
parental
behavior
Males:
accessory
olfactory
system (could
be through
altering
aggressiveness)
Receptive behavior ESP1 V2R (V2Rp5) Kimoto et al. 4
for mating (2005)
Haga et al. 4
(2010)
Suppression of ESP22 Ferrero et al. 4
mating behavior (2013)
Primer effects
Lee-Boot effect 2,5-dimethylpyrazine Ma et al. (1998) 2 and 5
Whitten effect 3,4-dehydro-exo-brevicomin Jemiolo et al. 2 and 4
(DHB), (1986)
2-sec-butyl-4,5-dihydrothiazole
(SBT)
E,E-α-farnesene and Ma et al. (1999) 4
E-β-farnesene
(continued)
Table A.1 (continued)

Pheromones Receptor or References Chapter
olfactory
system
Bruce effect MHC: SYFPEITHI for H-2d V2R (V2rf2 Leinders-Zufall 3
haplotype with H2-Mv) et al. (2004)
MHC: AAPDNRETF for H-2b Ishii and 3
haplotype Mombaerts
(2008)
Kelliher et al.
(2006)
Vandenbergh effect 3,4-dehydro-exo-brevicomin Novotny et al. 4
(DHB), (1999)
2-sec-butyl-4,5-dihydrothiazole
(SBT)
E-β-farnesene, MUP (facilitate)
2,5-dimethylpyrazine (delay) Jemiolo and 4
Novotny (1994)
Sperm motility (male odors) Koyama and 3
Kamimura
(1999)
Sperm density (female odors) Koyama and 3
Kamimura
(2000)
Sperm morphology Novikov et al. 3
(1981)
Novikov et al. 3
(1984)
Aref’ev et al. 3
(1985)
Cell proliferation in 2-sec-butyl-4,5-dihydrothiazole Koyama et al. 5
female brain (SBT) (2013)
E-β-farnesene
Cell proliferation in 2,5-dimethylpyrazine Koyama et al. 5
male brain (2013)
Mammary gland SBT Koyama et al. 5
expansion (2015)
References
Aref’ev AA, Daev EV, Kaidanov LZ, Lopatina NG, Novikov SN (1985) Anomalous
spermatogenesis in laboratory mice after the influence of volatile compounds contained in
urine of sexually mature males. Doklady Akademii Nauk SSSR 291(5):1257–1259
(2007) Identification of protein pheromones that promote aggressive behaviour. Nature 450
(7171):899–902
References 91
Ferrero DM, Moeller LM, Osakada T, Horio N, Li Q, Roy, DS, Cichy A, Spehr M, Touhara K,
Liberles SD (2013) A juvenile mouse pheromone inhibits sexual behaviour through the
vomeronasal system. Nature 502:368–371
Ishii T, Mombaerts P (2008) Expression of nonclassical class I major histocompatibility genes defines
a tripartite organization of the mouse vomeronasal system. J Neurosci 28(10):2332–2341
Jemiolo B, Novotny M (1994) Inhibition of sexual maturation in juvenile female and male mice by
a chemosignal of female origin. Physiol Behav 55(3):519–522
Kelliher KR, Spehr M, Li X-H, Zufall F, Leinders-Zufall T (2006) Pheromonal recognition
memory induced by TRPC2-independent vomeronasal sensing. Eur J Neurosci 23:3385–3390
Koyama S, Kamimura S (1999) Lowered sperm motility in mice of subordinate social status.
Physiol Behav 65:665–669
Koyama S, Kamimura S (2000) Influence of social dominance and female odour on the sperm
activity of male mice. Physiol Behav 71:415–422
Koyama S, Soini HA, Wager-Miller J, Alley WR, Pizzo MJ, Rodda C, Alberts J, Crystal JD,
Lai C, Foley J, Novotny MV (2015) Cross-generational Impact of a Male Murine Pheromone
2-sec-butyl-4,5-dihydrothiazole in Female Mice. Proc Biol Sci. 282(1811) doi:10.1098/rspb.
2015.1074 PMID: 26136453 http://dx.doi.org/10.1098/rspb.2015.1074
Leinders-Zufall T, Brennan P, Widmayer P, Chandramani P, Maul-Pavicic A, Jager M, Li X-H,
Breer, H, Zufall F, Boehm T (2004) MHC class I peptides as chemosensory signals in the
vomeronasal organ. Science 306:1033–1037
chemosignals in suppression of estrus in the house mouse: the Lee–Boot effect revisited. Biol
Reprod 59:1317–1320
Ma W, Miao Z, Novotny MV (1999) Induction of estrus in grouped female mice (Mus domesticus)
by synthetic analogues of preputial gland constituents. Chem Senses 24:289–293
Mak GK, Enwere EK, Gregg C, Pakarainen T, Poutanen M, Huhtaniemi I, Weiss S (2007) Male
pheromone—stimulated neurogenesis in the adult female brain: possible role in mating
behavior. Nat Neurosci 10:1003–1011
McClintock MK (1971) Menstrual Synchrony and Suppression. Nature 229:244–245
Novikov SN, Tsapygina RI, Daev EV, Togo EF (1981) Influenc of natural compounds of biogenic
origin on behavior and reproductive function in the male house mouse Mus musculus. Doklady
Akademii Nauk SSSR 262(3):746–748
Novikov SN, Daev EV, Tsapygina RI (1984) Action of volatile urine components on the
generative function of non-sexually mature males of the house mouse Mus musculus L.
Doklady Akademii Nauk SSSR 281(6):1506–1508
Novotny M, Harvey S, Jemiolo B, Alberts J (1985) Synthetic pheromones that promote inter-male
aggression in mice. Proc Natl Acad Sci USA 82:2059–2061
Novotny MV, Ma W, Wiesler D, Zidek I (1999) Positive identification of the puberty-accelerating
pheromone of house mouse: the volatile ligands associating with the major urinary protein.
Proc R Soc Lond B 266:2017–2022
Oboti L, Savalli G, Giachino C, De Marchis S, Panzica GC, Fasolo A, Peretto P (2009) Integration
and sensory experience-dependent survival of newly-generated neurons in the accessory
olfactory bulb of female mice. Eur J Neurosci 29:679–692
Oboti L, Schellino R, Giachino C, Chamero P, Pyrski M, Leinders-Zufall T, Zufall F, Fasolo A,
Peretto P (2011) Newborn interneurons in the accessory olfactory bulb promote mate
recognition in female mice. Front Neurosci 5:113. doi:10.3389/fnins.2011.00113
Roberts SA, Simpson DM, Armstrong SD, Davidson AJ, Robertson DH, McLean L, Beynon RJ,
Hurst JL (2010) Darcin: a male pheromone that stimulates female memory and sexual
attraction to an individual male’s odour. BMC Biol Jun 3;8:75. doi:10.1186/1741-7007-8-75
Savic I, Berglund H (2010) Androstenol—a steroid derived odor activates the hypothalamus in
women. PLoS ONE 5(2):e8651. doi:10.1371/journal.pone.0008651
Smith TD, Laitman JT, Bhatnagar KP (2014) The shrinking anthropoid nose, the human
vomeronasal organ, and the language of anatomical reduction. Anat Rec 297:2196–2204
Stern K, McClintock MK (1998) Regulation of ovulation by human pheromones. Nature 392:177–179
Sugiyama Y. (1993) 子殺しの行動学講談社学術文庫1057. 講談社, 東京
Wu Z, Autry AE, Bergen JF, Watabe-Uchida M, Dulac CG (2014) Galanin neurons in the medial
preoptic area govern parental behavior. Nature 509(7500):325–330
Yoon H, Enquist LW, Dulac C (2005) Olfactory inputs to hypothalamic neurons controlling
reproduction and fertility. Cell 123:669–682

Primer Effects by Murine Pheromone Signaling - Pheromonal Influences On Reproductive Conditions (PDFDrive)

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Primer Effects by Murine Pheromone Signaling - Pheromonal Influences On Reproductive Conditions (PDFDrive)

Uploaded by

Copyright:

Available Formats

SPRINGER BRIEFS IN ANIMAL SCIENCES

Primer Effects by Murine

ISSN 2211-7504 ISSN 2211-7512 (electronic)

Library of Congress Control Number: 2016933470

© The Author(s) 2016

Printed on acid-free paper

This Springer imprint is published by Springer Nature

cells as a postdoctoral fellow at the University of Tokyo in the laboratory of Prof.

1 Introduction: The Laboratory Mice . . . . . . . . . . . . . . . . . . . ..... 1

Keywords Mouse biology Social system Breeding system

1.1 Ethology and the Study Field of Olfactory

© The Author(s) 2016 1

to “reconstruct the phylogeny of motor patterns” (Eibl-Eibesfeldt 1970), in a way

their physiological conditions unexpectedly. This might induce contamination of

1.2 The Social System and Breeding System

signaling level is not known yet, but it is better to consider possibilities of

1.3 Pregnancy, Delivery, and the Growth

1.4 Mouse Biology, Hormones, Pheromones,

Yamazaki K, Yamaguchi M, Baranoski L, Bard J, Boyse EA, Thomas L (1979) Recognition

Keywords Main olfactory system

2.1 The Olfactory Systems

© The Author(s) 2016 11

Fig. 2.1 The olfactory

2.2 The Main Olfactory System and Its Pathway

and females showed increased Fos immunoreactivity at the vomeronasal neurons,

2.3 Accessory Olfactory System (Vomeronasal System)

Accessory olfactory system, or the vomeronasal organ, is located in the bottom of

2.4 Responses to Pheromone Are not Always the Same

PGRMC1 is involved in the suppression of the responses of vomeronasal neurons

2.5 Sex Differences in the Responses to Pheromones

3.1 The Lee–Boot Effect, Whitten Effect,

© The Author(s) 2016 23

3.2 Bruce Effect

The experimental suppression of NA by infusion of NA antagonist in the accessory

3.3 Primer Effect in Males

3.3.1 Social Dominance and Sperm Motility

Fig. 3.2 Sperm motility

3.3.2 Female Odors and Sperm Density

We then examined the influence of exposure to female bedding on sperm density

Fig. 3.4 Sperm density of

Fig. 3.5 Serum

Fig. 3.6 Percentage of

in one group, of 12 females kept in 3 groups (4 females per cage), mixture of 12

3.3.3 Influence of Adult Males’ Odor on Sperm Morphology

Abstract Identiﬁcation of the pheromones responsible for these primer effects,

Keywords Pheromones in urine, tears, saliva Rabbit pheromone Embryonic

4.1 Pheromones to Induce Primer Effects

Whether it is possible to call a speciﬁc chemical compound as a pheromone has

© The Author(s) 2016 39

engineering, enabled production of various transgenic mice possible to use to

Fig. 4.1 Male murine

4.2 Pheromones to Induce Releaser Effects

The two major roles of pheromones in communication are priming changes in

4.2.2 Sexual Behaviors and Pheromones in Tears: The Esps

4.2.3 Kin Recognition, Subspecies Recognition, and Mate

4.2.3.1 Individual Recognition: Major Histocompatibility Complex

4.2.3.2 Major Urinary Protein (MUP) and Its Various Functions

Urine is a source of various species-speciﬁc proteins that have the function to

4.2.3.3 Subspecies Recognition: Androgen-Binding Protein

subunit common to all forms of ABP, encoded by Abpa located on chromosome 7

mechanisms, it is possible to consider mate preferences and these mate preferences

4.2.4 Rooting Behaviors by Neonates: Are There

4.2.5 Parental Behaviors