5

5
HORMONAL
HORMONAL S EX C
SEX ONTROL AND
CONTROL AND ITS
ITS
APPLICATION
APPLICATION TO FISH
FISH C ULTURE
CULTURE
A. HUNTER AND EDWARD M
GEORGE A. M.. DONALDSON
West Vancouver
Vancouver Laboratory,
Laboratory, Fisheries
Fisheries Research
Research Branch
Dept.
Dept. of
of Fisheries
Fisheries and
and Oceans
Oceans
West Vancouver, British Columbia,
Vancouver, British Columbia, Canada
Canada

Introduction.. ....................................................
I. Introduction .................................................. 223
223
II. Determination and Differentiation ..............................
11. Sex Determination ............................ 225
225
A. Genotypic
Genotypic Sex Sex and and Sex Sex Chromosomes
Chromosomes.. . . .........................
....................... 225
B. Models
B. Determination ..................................
Models of Sex Determination ................................ 226
226
C.
C. Models Differentiation ..................................
Models of Sex Differentiation ................................ 229
229
D. Summary ....................................................
D. Summary .................................................. 241
241
III. Hormonal Sex Control
111. Control.. . . .. .. .. .. .. .. ....... .. .. .. .. .. .. .. . . . . . . . .. .. .. .. .. .. ..... .. .. .. .. . . . . . 242
A. Management .................................................
A. Management ............................................... 243
243
B. Treatment ...................................................
B. Treatment ................................................. 250
250
C. Evaluation
C. Evaluation.. . . .. .. .. .. .. .. .. .. .. . . . ...........................
................................... 268
268
IV. Economically Important Species. Species. ...................................
................................. 268
268
A. Cichlids . . . . . . . . . . . .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . .. .. .. .. .. ....... .. .. .. .. . 269
269
B. Salmonids . . . . . . . . . ..........................................
........................................ 276
C. Cyprinids .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Cyprinids 288
288
Conclusions .....................................................
V. Conclusions ................................................... 290
290
References .. .. .. . . . . . . . . . .. .. .. .. .. ......... .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References 291
291

I. INTRODUCTION
INTRODUCTION

Studies involving experimental sex manipulation by hormones in fish

have become increasingly popular. This is attributable largely to the tremen­
tremen-
dous economic potential that sex-control techniques have for the culture of
economically important species of fish. For the purposes of this discussion,
hormonal sex control refers only to the general control of sexual processes
which
which can
can be achieved
achieved byby manipulating
manipulating gonadal
gonadal sex.
sex. The
The hormones
hormones pri­
pri-
marily involved in experimental studies of the manipulation of gonadal sex
have
have been
been the sex
sex steroids.
steroids.
The first
first major
major proliferation
proliferation of
of studies
studies directed
directed toward
toward the
the influence
influence of
of
hormones on the gonads of fish occurred in the late 1930s
1930s and early 1940s.
1940s.
223
223
FISH PHYSIOLOGY. VOL. IXB
PHYSIOLOGY, VOL. Copyrig
Copyrightht ©
0 1983
1983 by Academic Press.
Press, Inc.
Inc.
All rights
rights of reproduction in
in any form reserved.
reserved.
ISBN 0·12-350429-5
0-12-350429-5
224 GEORGE A.
GEORGE A. HUNTER
HUNTER AND
A N D EDWARD M.. DONALDSON
EDWARD M DONALDSON

Although
Although most most ofof these
these studies
studies examined
examined the the effects
effects ofof the
the sex
sex steroids
steroids on
on
secondary sexual characteristics, several suggested the possibility that func­ func-
tional
tional sex
sex inversion
inversion could
could bebe achieved
achieved in fish. In
in fish. In the
the following
following 22 decades
decades the
the
efficacy
efficacy of hormonal sex control was demonstrated in several gonochorist
species.
species. Within
Within this
this period
period anan extensive
extensive series
series of
of studies
studies were
were conducted on on
the
the medaka,
medaka, Oryzias Zutipes (Yamamoto,
Oryzius latipes (Yamamoto, 1953,1953, 1955,
1955, 1958,
1958, 1959a,b, 1961,
1959a,b, 1961,
1962,
1962, 1964a,b,
1964a,b, 1965,
1965, 1967,
1967, 1968;
1968; Yamamoto et al. al.,, 1968;
1968; Yamamoto
Yamamoto and
Matsuda,
Matsuda, 1963; 1963; Yamamoto
Yamamoto and and Suzuki,
Suzuki, 1955),
1955), which
which formed
formed the basis of
the basis of aa
comprehensive
comprehensive review review of of the
the subject
subject (Yamamoto,
(Yamamoto, 1969).
1969). Synthesizing
Synthesizing the
results of these numerous studies of the effect of gonadal steroids on sex
differentiation
differentiation in in fish
fish with
with the concept
concept of of aa dual
dual inductor
inductor system
system ofof sex
sex
differentiation
differentiation proposed
proposed for for the
the amphibians
amphibians by by Witschi
Witschi (1929),
(1929), Yamamoto
Yamamoto
concluded that the sex steroids, androgens and estrogens, were in fact the
respective male male and
and female
female sex
sex inducers
inducers responsible
responsible for for gonadogenesis
gonadogenesis in in
fish.
fish. Further, he laid down specific criteria for successful successful treatment
application.
The establishment of an effective protocol for sex manipulation in fish
marked a divergence between those studies concerned with the fundamental
aspects of hormonal sex control and those directed toward the optimization
of treatment, usually in economically important species. The former studies,
which were primarily concerned concerned with the elucidation of the role of sex
steroids with respect to sex determination and sex differentiation, were also
reviewed by Vanyakina (1969) (1969) and later by Harrington
Harrington (1974).
(1974). Of additional
value are several publications dealing with the role of sex steroids in induced
or natural sex inversion in hermaphroditic
hermaphroditic species (Chan,(Chan, 1970,
1970, 1977;
1977; Chan
et al. 1975; Reinboth, 1970,
al.,, 1975; 1970, 1972).
1972).
Schreck (1974)
(1974) provided the first review of the literature in the light of of
the newly established economic objectives. Several recent articles have
elaborated the specific
specific genetic and hormonal techniques applied in general
(Yamazaki,
(Yamazaki, 1983) 1983) or specifically
specifically to the economically important cichlids (Shel­ (Shel-
ton
ton et al.al.,, 1978;
1978; Guerrero,
Guerrero, 1979)
1979) and
and salmonids
salmonids (Donaldson
(Donaldson and and Hunter,
Hunter,
1982a).
1982a).
A discussion of hormonal sex control inherently involves consideration of
the
the assumptions on on which
which the
the action
action of
of the
the hormone
hormone is is based,
based, ultimately
ultimately the
mechanisms of sex determination and sex differentiation.
differentiation. Although pursued
intensively during the 1900s, 19OOs, a unifying model of sex determination and sex
differentiation has proven illusive. This is, perhaps, not surprising in light of of
the bewildering diversity of sexual expression found within the vertebrates,
particularly the largest group the Pisces. Therefore, the discussion first con­ con-
centrates
centrates on on the
the theoretical
theoretical context
context within
within which
which hormonal
hormonal sex sex control
control stud­
stud-
ies are conducted. Second,Second, the various components of hormonal sex control
studies are examined with particular reference to those factors which influ-
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL
CONTROL AND
AND ITS
ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 225

ence treatment success.

success. Finally, studies that have involved several eco­
eco-
nomically important species
species are discussed in detail.
It is notable that the majority of studies on hormonal sex control have
involved gonochorist species. Therefore, these species are given emphasis.
Hermaphroditic species are considered where applicable;
applicable; however, these
species are discussed in greater detail in Chapter 4, this volume.

II.
11. SEX DETERMINATION AND
DIFFERENTIATION
DIFFERENTIATION

Within the Pisces, a wide range of reproductive strategies

strategies are encoun­
encoun-
tered.
tered. The great majority of fish are either differentiated or undifferentiated
undifferentiated
gonochorists. However, numerous species exhibit synchronous, pro­ pro-
togynous, or protandrous hermaphroditism. Further, a number of species
are capable of gynogenetic reproduction
reproduction.. The diversity of physiological re­
re-
productive systems and ethological sexes
sexes has been the topic of numerous
reviews including Atz (1964),
(1964), Yamamoto (1969),
(1969), Chan (1970,
(1970, 1977),
1977), and
Reinboth (1970).
(1970). Although it is
is generally accepted that in fish, as in other
vertebrates, sex has a genetic basis, the specific mechanisms by which genet­
genet-
ic sex is determined and expressed are unclear.

A. Genotypic
Genotypic Sex
Sex and
and Sex
Sex Chromosomes
Chromosomes

Among the vertebrates the evolutionary tendency has been to aggregate

all those genes responsible for sexual development onto a single pair of
heteromorphic sex chromosomes, thereby providing a vehicle for the inheri­inheri-
tance of sex as a Mendelian trait. The two two basic sex chromosomal systems
which
which have
have thus
thus evolved
evolved are
are either
either male
male heterogametic-female
heterogametic-female homogame­
homogame-
tic (XY:XX)
(XY:XX) as in the case of the mammals or male homogametic-female
homogametic-female
heterogametic (ZZ:WZ)
(ZZ:WZ) as in the birds (Ohno,
(Ohno, 1967).
1967).
Approximately 10001000 species of fish have been examined cytologically.
cytologically.
The results of these investigations indicate that very few of these species
contain
contain easily
easily discernable
discernable heteromorphic
heteromorphic sex sex chromosomes
chromosomes (Voronstov,
(Voronstov,
1973; Yamazaki,
1973; Yamazaki, 1983).
1983). However, recent advances in cyotogical
cyotogical techniques
have permitted the identification of heterosomes in species such as the
rainbow trout, Salmo gairdneri, which are barely discernable from auto­
S a l m gairdneri, auto-
somes and are presumably in the early stages of heteromorphic evolution
(Thorgaard, 1977). Both male and female digamety systems
(Thorgaard, 1977). systems have been dem­
dem-
onstrated as well as several variations of these basic models including species
with multiple sex chromosomes.
chromosomes. Overall, the XY:XX
XY:XX system appears to be
most prevalent in fish (Yamazaki,
(Yamazaki, 1983).
1983).
226 GEORGE
GEORGE A.
A. HUNTER
HUNTER AND
A N D EDWARD
EDWARD M.
M. DONALDSON
DONALDSON

Although
Although in in the majority
majority ofof species
species heterosomes
heterosomes are are not
not distinguishable
distinguishable
by
by cytological
cytological techniques,
techniques, the presence
presence ofof heterosomal
heterosomal systems
systems have
have been
been
demonstrated
demonstrated by by genetic
genetic techniques.
techniques. Aida
Aida (1921,
(1921, 1936)
1936) first
first demonstrated
demonstrated
sex linked color genes and the male heterogametic-female
heterogametic-female homogametic sex sex
chromosomal system in the medaka. Vanyakina Vanyakina (1969)
(1969)and Yamamoto
Yamamoto (1969)
(1969)
have
have reviewed
reviewed the the use
use of
of similar
similar techniques
techniques to to describe
describe the heterosomal
heterosomal
systems
systems inin several
several tropical
tropical fish,
fish, notably
notably in
in the
the family
family Poeciliidae.
Poeciliidae. Yamamoto
Yamamoto
(1969)
(1969)also
also provides a brief,
brief, but interesting,
interesting, review of the early work involv­involv-
ing inter- and intraspecific matings which has provided much of our current
knowledge
knowledge of of these systems. Of
these systems. Of particular
particular interest
interest is is the
the demonstration
demonstration of of
both XX:XY
XX:XY and ZZ:WZ ZZ:WZ (YY:WY) systems in the M exican and British Hon­
Mexican Hon-
duran races of platyfish Xiphophorus (Platypoecilus) maculatus (Gordon,
(Platypoecilus) maculatus
1947;
1947; Kallman,
Kallman, 1965).
1965). Similar
Similar systems
systems have
have been
been reported
reported forfor Oreochromis
Oreochromis
mossambicus
mossambicus (Tilapia rnossambica) (Hickling,
(Tilapia mossambica) (Hickling, 1960).
1960). Bull
Bull and
and Charnov
Charnov
(1977)
(1977) have explored the possible transition from male to female hetero­ hetero-
gamety or vice versa through a intermediate polygenic system of sex deter­ deter-
mination. More recently, analysis of the sex of progeny following gynogen­ gynogen-
esis has proven useful for the determination of of female homogamety in
several species including grass carp, Ctenopharyngodon
Ctenopharyngodon idella (Stanley, (Stanley,
1976),
1976), common carp, Cyprinus carpio (Nagy (Nagy et al.al.,, 1978, 1981), and coho
1978, 1981),
Oncorhynchus kisutch (Refstie et al.
salmon, Oncorhynchus al.,, 1982).
1982). A detailed examination
of this technique
technique is presented in Chapter 8, this volume. Further, the analy­ analy-
sis of the progeny produced by the matings of hormonally sex-inverted and
sis
untreated individuals, first described in the medaka by Yamamoto Yamamoto (1953),
(1953),
has been used effectively for the identification of heterosomal systems
(Table
(Table I).
I).
The control
control over
over sex
sex determination
determination exerted
exerted by by mechanisms
mechanisms associated
associated
with the sex chromosomes is relatively strict within the higher vertebrates.
This also appears to be the case in a number of fish species examined.
However,
However, in in several
several species
species that
that have an
an apparent
apparent heterosomal system, sex sex
determination does not appear to be strictly bound to the sex chromosomes.

B. Models of
of Sex Determination
Several models of sex determination have been proposed since the dis­
dis-
covery early in the century of
of the sex chromosomes. The models proposed
for the vertebrates, developed primarily from studies on the amniotes have
been based either on chromosomal or genic inheritance.

l. CHROMOSOMAL
1. CHROMOSOMAL INHERITANCE
INHERITANCE
Sex determination based on chromosomal inheritance was proposed
proposed by
Mittwoch (1971)
(1971) for the higher
higher vertebrates. The model stated that the pres-
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND ITS
CONTROL AND ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 227

Table
Table I
Demonstration
Demonstration of
of Heterosomal Systems by tbe
the Mating of Untreated
Untreated and Sex-Inverted
Sex-Inverted Individuals

Species
Species Chromosomal
Chromosomal mechanism
mechanism Reference
Reference

Oryzias
0y z i a s latipes XX:XY
xx:XY Yamamoto
Yamamoto (1953)
(1953)
Oreochromis mossambicus
mossambicus XX:XY
xx:XY Clemens and
Clemens and Inslee
Inslee (1968)
(1968)
Carassius
Carassius auratus XX:XY
xx:XY Yamamoto
Yamamoto and and Kajishima
Kajishima (1969)
(1969)
Oreochromis niloticus
niloticus XX:XY
xx:XY Jalabert al. ((1974)
Jalabert et al. 1974)
H emihaplochromis multicolor
Hemihaplochromis XX:XY
xx:XY Hackmann
Hackmann and and Reinboth
Reinboth (1974)
(1974)
Oreochromis aureus
aureus wz:zz
WZ:ZZ Guerrero (1975),
(1975), LiuLiu (1977)
(1977)
Poecilia reticulata XX:XY
xx:XY Takahashi
Takahashi (19715
(197%) a)
Salmo gairdneri XX:XY
xx:XY Okada al. (1979),
Okada et al. (1979),
Johnstone
Johnstone et al.al. (1979a)
(1979a)
Oncorhynchus kisutch
Oncorhynchus kisutch XX:XY
xx:XY Hunter al. (1982a)
Hunter et al. (1982a)
Oncorhynchus
Oncorhynchus tsawytscha XX:XY
xx:XY Hunter al. (1983)
Hunter et al. (1983)

ence or absence of the whole Y or W chromosome determines

determines the respective
dominant male or female sex. sex. The resulting differential chromosome vol­ vol-
umes along with Y- Y- or W-linked RNA synthesis were presumed to induce
gonadogenesis
gonadogenesis mediated by a higher mitotic rate in the heterogametic sex. sex.
In mammals and birds heterogamety had been previously previously correlated with
early sex differentiation (Hamilton,
(Hamilton, 1965).
1965).
This model probably does not apply to the teleosts. First, as previously
mentioned, the majority of fish species do not have heteromorphic chromo­ chromo-
somes. Second, early differentiation of germ cells apparently does not corre­
somes. Second, corre-
late with the heterogametic sex. (1965) reported early
sex. Eckstein and Spira (1965)
differentiation in the female cichlid, Oreochromis aureus (Tilapia
(Tilapia aurea),
aurea),
which has been demonstrated
demonstrated to be heterogametic (Guerrero, 1975). 1975). How­
How-
ever,
ever, differentiation occurs first in the homogametic female Oryzias
O y z i a s latipes
(Satoh and Egami, 1972;1972; Onitake, 1972;
1972; Quirk and Hamilton, 1973),
1973), the
cichlid, Oreochromis mossambicus (Nakamura and Takahashi, 1973)
cichlid, 1973),, and
'
the goldfish, Carassius auratus (Nakamura, 1978). Early differentiation of
(Nakamura, '1978).
the female has also
also been reported in the cichlid, Hemihaplochromis multi­ multi-
color (Muller,
(Miiller, 1969),
1969), Ti tapia zillii (Yoshikawa
Tilapia (Yoshikawa and Oguri, 1978),
1978), the trout,
Salmo trutta (Ashby, 1957), Ctenopharyngodon idella, (Shelton and Jensen,
(Ashby, 1957),
1979), and the salmonids, Oncorhynchus masou,
1979), masou, Oncorhynchus keta, and
leucomaenis (Nakamura, 1978),
Salvelinus leucomuenis
Salvelinus 1978), and the threespined stickleback,
Gasterosteus aculeatus (Shimizu
(Shimizu and Takahashi, 1980). Early differentiation
Takahashi, 1980).
of the male in a differentiated gonochorist teleost has not been reported.reported.
Although a correlation between hetero- or homogamety is not evident, evident, de­ de-
veloping germ cells of many species examined do demonstrate a differential
(1972) observed that following the oral administration
mitotic rate. Onitake (1972)
228 GEORGE A. HUNTER
GEORGE A. HUNTER AND
A N D EDWARD M. DONALDSON
EDWARD M. DONALDSON

of estrone to genetically male medaka the germ cells began an an atypical rapid
proliferation which preceded sex differentiation. Onitake suggested that the
rapid mitotic increase was necessary to to the process of differentiation. In an
intensive examination of factors affecting sex inversion in the cyprinodont,
mumoratus, Harrington
Rivulus marmoratus, Harrington (1975)
(1975) reported
reported that
that rearing
rearing temperatures
temperatures
of
of 19°C
19°C or
or 26°C
26°C resulted
resulted in
in higher
higher proportions
proportions of
of primary
primary males
males and
and her­
her-
maphrodites, respectively,
respectively, and these results were correlated with mitotic
activity in the developing gonad. The role of differential mitotic growth in
the
the process
process of
of sex
sex differentiation
differentiation remains
remains to
to be determined.
determined.

2. GENIC INHERITANCE
2. GENIC INHERITANCE AND POLYGENIC
AND POLYGENIC SEX
SEX
DETERMINATION
DETERMINATION
The concept
concept ofof genic
genic balance
balance established
established byby Bridges
Bridges (1925,
(1925, 1936)
1936) was
was
initially modified by Winge (1934)(1934) to apply to sex determination in Poecilia
(Lebistes).Winge proposed that the X and Y chromosomes contained superi­
(Lebistes). superi-
or
or male
male and
and female
female sex-determining
sex-determining genes.
genes. Minor
Minor male
male and
and female
female sex­
sex-
determining factors were held in the autosomes.autosomes. Normally the autosomal
genes are maintained in balance allowing sex to be determined by the het- het­
erosomal mechanism. However, in exceptional individuals, autosomal com­ com-
binations or recombinations may occur that result in an excess excess of autosomal
factors
factors of one sex capable of overriding the heterosomal mechanism. The
outcome is an individual with a phenotypic sex differing from its heterosomal
sex. Similar polygenic sex-determining systems have been hypothesized for
sex.
several
several xiphophorin
xiphophorin fishes
fishes (Kosswig
(Kosswig and
and Oktay,
Oktay, 1955;
1955; Kosswig,
Kosswig, 1964;
1964; Anders
and
and Anders, 1963;1963; Dzwillo
Dzwillo and
and Zander,
Zander, 1967), lutipes (Yamamoto,
1967), Oryzias latipes (Yamamoto,
1963, 1969), and Betta
1963, 1969), Bettu splendens (Lowe and Larkin, 1975). 1975). Exhaustive re­ re-
views
views of of the
the early
early research
research onon the development
development of of the
the polygenic
polygenic concept
have been provided by Kosswig (1964) (1964) and Yamamoto (1969).(1969). Yamamoto
(1969)
(1969)summarized
summarized the results obtained
obtained byby asserting that
that "sex
“sex determination
determination
in gonochorists is polyfactorial
polyfactorial with or without epistatic sex genes in sex
chromosomes.
chromosomes.”" When the total strength of the male factors exceeds that of of
the female factors, the zygote will be male and vice versa. Therefore, de­ de-
pending on the control exerted by epistatic genes on the sex chromosomes
the
the stability
stability of
of sexual
sexual determination
determination will vary
vary from
from species to to species.
species.
Within the tilapia, interspecific crosses of several types result in non­ non-
Mendelian sex ratios (Pruginin
(Pruginin et al. 1975). Avtalion and Hammerman
al.,, 1975).
(1978)
(1978)examined an extensive series of hybrid crosses between homozygous
(Tilapiu) hornorum males and Oreochromis mossambicus
Oreochromis (Tilapia)
females and between homozygous Oreochromis macrochir mucrochir males and
Oreochromis niloticu8 (Tilapia niliotica) females conducted by Chen (1969)
niloticus (Tilapia (1969)
and
and Jalabert et al.al. (1971),
(1971), respectively. Assuming that the Y and Z chromo-
 HORMONAL SEX
5. HORMONAL SEX CONTROL
CONTROL AND
AND ITS
ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 229

somes were identical and that an autosomal influence exists, Avtalion and
Hammerman proposed that the simplest sex-determining mechanism in
Hammerman
tilapia would consist of three gonosomes (X, (X, W, Y) in pairs of of two (XX, XY,
XW, WY, WW, and YY) YY) similar to the system proposed proposed for Xiphophorus
Xiphophorus
macuiatus by Gordon (1947)
maculatus (1947) but with the addition of of a pair ofof autosomes.
(AA,
(AA, Aa, and aa). The resulting 18 possible possible combinations of of autosomes and
gonosomes could be used to predict predict the results obtained by Chen, but not of of
Jalabert et al. (1971). Hammerman and Avtalion (1979)
ai. (1971). (1979) suggested that the
results of of Jalabert et al. (1971) could be explained by assigning different
ai. (1971)
strengths to each of of the chromosomes involved. involved. Analysis of of the sex-deter-
sex-deter­
mining mechanism has been hampered because of of the fact that in tilapia, sex
chromosomes cannot be identified by karyotypic karyotypic analysis and no sex-linked
color markers are present. Avtalion et al. ai. (1975)
(1975) and Hardin (1976) (1976) have
identified a male-specific electrophoretic marker in adult male Oreochromis
(Sarotherodon)
(Sarotherodon)aureus. aureus. However, it has only been found in sexually mature
individuals and may, therefore, be hormonally induced. The usefulness of
individuals
these
these markers
markers is is dependent
dependent on on whether
whether theythey are
are sex
sex linked
linked asas opposed
opposed to to sex
sex
limited. For further review, the reader is referred to Wolhfarth and Hulata
(1981)
(1981) and Avtalion (1982). (1982).
The
The concept
concept of of aa polygenic
polygenic mechanism
mechanism in in which
which autosomal
autosomal genesgenes may may
play
play aa decisive
decisive role
role inin the
the process
process of of sex
sex determination
determination has has dominated
dominated the the
research
research on on fish.
fish. However,
However, no no single
single polygenic
polygenic system
system has
has been
been capable
capable of of
being
being reconciled
reconciled withwith allall empirical
empirical datadata (Harrington,
(Harrington, 1974).
1974). Despite
Despite excep­
excep-
tions,
tions, the
the concept
concept of of aa polygenic
polygenic system
system remains
remains suited
suited to
to the
the majority
majority of of
data
data from
from studies
studies of of fish.
fish. An
An interesting
interesting example
example is is the
the recent
recent report
report by by
Streisinger
Streisinger et al. (1981).
et al. (1981).In In this
this study,
study, clones
clones ofof homozygous
homozygous diploid
diploid female
female
zebrafish,
zebrafish, Brachydanio rerio, rerio, were
were produced
produced by by hydrostatic
hydrostatic pressure
pressure or or
temperature
temperature shocksshocks administered
administered to to ova,
ova, activated
activated byby ultraviolet
ultraviolet (UV)-treat­
(UV)-treat-
ed
ed sperm.
sperm. SomeSome of of the
the clones
clones were
were predominately
predominately male male and
and produced
produced high high
proportions
proportions of of males
males in in subsequent
subsequent generations.
generations. Such Such results
results are
are difficult
difficult toto
reconcile
reconcile with
with any
any ofof the
the current
current models
models of of sex
sex determination
determination otherother thanthan aa
polygenic
polygenic system.
system. FromFrom the the analysis
analysis of of single
single gene
gene mutations
mutations it it is
is clear
clear that
that
even
even inin the
the mammals,
mammals, genetic genetic sex
sex cannot
cannot be be explained
explained by by the
the constitution
constitution of of
the
the sex
sex chromosomes
chromosomes alone. alone. Autosomal
Autosomal genes genes which
which may
may play
play an
an important
important
role
role in
in gonadal
gonadal differentiation
differentiation have have been
been reported
reported in in pigs
pigs Gohnston
(Johnston et et ai.
al.,,
1958),
1958), goats
goats (Hamerton
(Hamerton et al.,, 1969),
et ai. 1969), and
and mice
mice (Cattanach
(Cattanach et al.,, 1971).
et al. 1971).

C.
C. Models
Models of
of Sex
Sex Differentiation
Differentiation

Several
Several models
models of
of sex
sex differentiation
differentiation have
have been
been proposed
proposed which
which are
are com­
com-
patible
patible with
with single
single or
or multiple
multiple gene
gene action.
action.
230
230 GEORGE A. HUNTER
GEORGE A. AND
HUNTER A N D EDWARD M.. DONALDSON
EDWARD M DONALDSON

1.
1. It-Y
H-Y ANTIGEN
ANTIGEN

Sex differentiation
differentiation based on the action of an individual gene or genes has
recently been
been given considerable
considerable support
support in in the aves
aves and
and mammalia
mammalia with with the
the
discovery ofof the male specific histocompatibility-Y
male specific histocompatibility-Y chromosome
chromosome (H-Y) (H -Y) anti-
anti­
gen.
gen. The antigen,
antigen, first
first discovered
discovered inin mice by by Eichwald and and Silmser
Silmser (1955)
(1955)isis
ubiquitously
ubiquitously associated
associated with with the heterogametic
heterogametic sex sex in
in mammalian
mammalian and and some
some
nonmammalian
nonmammalian vertebrates.
vertebrates. TheseThese observations
observations havehave ledled toto the
the hypothesis
hypothesis
that
that the
the antigen
antigen plays
plays aa major
major role
role in
in gonadal
gonadal sexsex differentiation.
differentiation. The The current
current
hypothesis is that a gene or genes on the Y or W chromosome code for the H- H­
Y antigen
antigen and
and the presence
presence of of the antigen
antigen on on the
the surface
surface of of somatic
somatic cells
cells of
of
the indifferent gonad results in the development of the heterogametic gonad
(Wachtel et al. 1975; Zenzes et al.
aZ.,, 1975; 1978). However, the mechanics of the
al.,, 1978).
action or regulation of H-Y antigen with respect to gonadal differentiation
have yet to be discovered.
discovered. Ohno et al. al. (1978)
(1978) have demonstrated that the
membrane H-Y antigen receptor is present only on gonadal cells in both
sexes. Ohno
sexes. Ohno andand co-workers
co-workers usedused H-Y antibody
antibody to to strip
strip H-Y
H-Y antigen
antigen from
from
mice
mice gonadal cells of known genetic constitution. Culture of the cells re­ re-
vealed that removal of of the H-Y antigen resulted in the development of of
spherical aggregates that resembled ovarian follicles. follicles. Un stripped cells
Unstripped
formed cylindrical tubular structures morphologicallymorphologically similar to semi­ semi-
niferous tubules. Ohno and co-workers concluded that there was a causal
relationship between the presence of the H-Y antigen on the surface of
gonadal cells and the development of the testes testes.. They further suggested that
the H-Y antigen could act as a short-range hormone-inducing specific specific gene
expression.
al. (1979)
Miiller et al. (1979) inverted the sex of of chicken embryo testes (ZZ) with
estrogen. He found that the normally H-Y (W) (W) antigen-negative testes were
positive after sex inversion, which indicated that the gene for the antigen
was expressed in the absence of of the W chromosome and, therefore, must be
present in both sexes, supporting the report by Ohno et al. al. (1978).
(1978).Assuming
the the presence of the H-Y (W) (W) antigen was responsible for the formation of
the ovary, M iiller et al.
Muller al. (1979)
(1979) suggested that the hormone-induced sex
inversion was an indirect effect mediated by H-Y (W) (W) antigen. They also
indicated that there was a correlation between morphogenetic changes and
H-Y (W)
(W)titer in the gonad.
gonad. Therefore, Miiller
Muller and co-workers suggested that
the induction of the H H-Y-Y (W)
(W) antigen by estrogen did not operate as a strict
on-off
on-off switch mechanism, again similar to the conclusion arrived at by Ohno
al. (1978).
et al. (1978). Similarily,
Similarily, induced H-Y antigen-positive ovarian tissue in the
ovaries of sex-reversed (ZZ) (ZZ) individuals has been demonstrated in Zenopus
laeuis (Wachtel et al.
laevis al.,, 1980)
1980) and Pleurodeles waltlii (Zaborski
(Zaborski and Andrieux,
1980).
1980). In a series of experiments reviewed by Zaborski (1982) (1982) involving
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND ITS
CONTROL AND ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 231
231

amphibians,
amphibians, H H-Y-Y expression was repressed in Pleurodeles waltlii ovaries ovaries
(ZW)
(ZW) or Rana ridibunda testes (XY) (XY) by the administration
administration of dihydrotesto­
dihydrotesto-
sterone or estradiol,
estradiol, respectively.
respectively. Therefore;
Therefore; although
although in the lower verte­ verte-
brates the sex hormones
hormones appear to be inducers of H H-Y-Y antigen,
antigen, an inhibitory
control of its expression
expression is also suggested.
suggested. Because
Because both androgens
androgens and
estrogens
estrogens are produced in each sex, sex, the nature of hormonal controlcontrol of the
antigen system is presumed to be quantitative.
antigen system However, recent research
quantitative. However,
with rainbow trout gonadal
gonadal homogenates
homogenates suggests
suggests that during the period of
sex differentiation
differentiation the gonads
gonads may not be capable of estrogen production
(van
(van den Hurk et al. al.,, 1982).
1982).
Sex-specific
Sex-specific antigens have been detected in nonmammalian vertebrates
including
including birds (Bacon,
(Bacon, 1970;
1970; Wachtel
Wachtel et al.al.,, 1975;
1975; Muller et al. al.,, 1980),
1980),
amphibians pipiens, Xenopus laevis (Wachtel
amphibians such as Rana pipiens, (Wachtelet al. 1975), and
al.,, 1975),
reptiles such as Emys orbicularis (Zabroski
(Zabroski et al. 1979). Recently,
al.,, 1979). Recently, Shalev
Shalev
and Huebner (1980),
(1980), citing unpublished work, have reported the presence
of the antigen in invertebrates.
The presence of the antigen system has also been demonstrated in sever­ sever-
al species
species of fish.
fish. Muller and Wolf Wolf (1979)
(1979) tested absorption of mammalian
mammalian
anti-H-Y
anti-H-Y antiserum in the teleosts teleosts Salvelinus alpinus,
alpinus, Salmo
Salmo gairdneri,
Rutilus rutilus,
rutilus, Carassius auratus, Barbus tetrazona, Poecilia reticulata
(Lebistes reticulatus), and Xiphophorus helleri.
(Lebistes reticulatus), helleri. The gonads
gonads of the more
primitive orders Ostariphysi, represented by Rutilus, Carassius,
Carassius, and Bar­
Bar-
bus, and Isospondyli,
Zsospondyli, represented by Salvelinus and Salmo,Salmo, absorbed anti­
anti-
H-Y antiserum; however,
however, a clear sexsex difference
difference was not observed.
observed. In the
more advanced poeciliids Poecilia and Xiphophorus, the anti-H-Y
advanced poeciliids anti-H-Y antiserum
was absorbed almost exclusively
exclusively by the gonadal
gonadal tissues of the male but not
the female.
female. AsAs previously mentioned, Poecilia, Salmo, and Carassius all
Poecilia, Salmo,
have male heterogametic-female homogametic systems. systems. The sex-determin­
sex-determin-
ing mechanism in Xiphophorus
Xiphophorw does not appear to be strictly heterosomal
(Kosswig,
(Kosswig, 1964).
1964). The presence of the antigen systemsystem in Poecilia reticulata
has been recently confirmed by Shalev Shalev and Heubner (1980).
(1980). Further sup­
sup-
port for
for a sex-specific
sex-specific antigen expression in advanced species
species comes
comes from
research by Pechan et al. al. (1979).
(1979). In this study, anti-H-Y antiserum absorp­
absorp-
tion was found exclusively
exclusively in male cells of Xiphophorus maculatus,
maculatus, Haplo­
Haplo-
chromis burtoni, Oryzias latipes, and several
several tilapia hybrids.
hybrids. The H-Y anti­
anti-
gen was detected most readily in Xiphophorus maculatus males (YY). Male
heterogamety has not yet been determined for the cichlid Haplochromis
Haplochromis
burtoni.
burtoni.
The presence of of the sex-specific
sex-specific antigen system in fi sh is certainly
fish certainly in­
in-
teresting from an evolutionary
evolutionary perspective. Further examination
examination of the anti­
anti-
gen system
system in the lower vertebrates may provideprovide an answer to the basic
question
question of when in the course
course of evolution
evolution the antigen system
system assumed a
232
232 GEORGE A. HUNTER
GEORGE A. AND EDWARD
HUNTER AND M.. DONALDSON
EDWARD M DONALDSON

primary
primary role
role in
in the
the process
process ofof sex
sex differentiation.
differentiation. The
The demonstration
demonstration H-YH-Y
antigen
antigen expression
expression inin the
the ovaries
ovaries of
of homozygous
homozygous chickschicks and
and amphibians
amphibians sex
sex
inverted
inverted with
with estradiol
estradiol suggests
suggests aa major
major rolerole of
of the
the antigen
antigen in
in the
the process
process of
of
hormonal
hormonal sex
sex inversion
inversion inin these
these groups.
groups. Examination
Examination of of the
the role
role of
of the
the
antigen
antigen system
system inin the natural or
the natural or hormonally
hormonally induced sex inversion
induced sex inversion of
of both
both
hermaphroditic
hermaphroditic andand gonochorist
gonochorist fish
fish is
is aa promising
promising area
area of
of study.
study.

2. THE
2. THECORTICOMEDULLARY
CORTICOMEDULLARYINDUCTOR
INDUCTORMODELS
MODELS
The corticomedullary inductor model was proposed by Witschi (1929) (1929)to
explain sex differentiation in amphibians. Witschi observed that the primor­
primor-
dium of the amphibian gonad, like those of of most vertebrates, is comprised of of
both
both an
an outer
outer cortex
cortex and
and inner medulla
medulla ultimately
ultimately derived
derived from
from the
the germinal
germinal
epithelium. During differentiation, either the cortex or the medulla devel­ devel-
ops at the expense of the other resulting in the development of an ovary or
testis, respectively. Witschi theorized
theorized that the genetic male and female
factors
factors embodied
embodied byby the balance
balance theory
theory of of sex
sex determination
determination were
were phe­
phe-
notypically manifested in the dualistic character of the primordial gonad.
The action of these genetic factors results in the production of an embryonic
cortexin or medullarin, which in turn initiated ovarian and testicular differ­
differ-
entiation, respectively. The existence of of these hypothetical inductors re­ re-
mains to be demonstrated. As a result, consideration of the suitability of of
Wistchi's
Wistchi’s model has remained a conceptual debate.
In later publications, Witschi (1965,
(1965, 1967)
1967) modifi ed his theory to include
modified
an
an antagonist action of the inductors. The theory of antagonism has been
illustrated in the amphibians by ablation of the dominant component of the
gonad which results in differentiation of the remaining component (Haffen,(Haffen,
1977).
1977). Witschi (1967)
(1967) has suggested that the interaction ofof the inductors is
similar to an immune reaction. Therefore, each has the capability of inhibit­
inhibit-
ing or destroying the other.
other. Reinboth (1982)
(1982) has recognized the similarity
between Witschi's
Witschi’s (1967)
(1967)model and the model of mammalian sex differentia­
differentia-
tion involving the H-Y antigen system and a presumptive ovarian factor. The
existence of the ovarian factor and the nature of its interaction with the H-Y
antigen system remain a subject of study and debate (Wachtel and Koo,
1981).
1981).
With regard to fish, the debate surrounding the dual-inductor concept
has centered on reconciliation of the model with the proposed unitary origin
of the teleost gonad and the common occurrence within the teleosts of
various forms of hermaphroditism.
various
The discrete topographical division of of the primoridal amphibian gonad
provided Witschi with strong support for his dual-inducer concept. This dual
embryonic nature appeared ideally suited to providing the separate chemical