THE JOURNAL OF EXPERIMENTAL ZOOLOGY 281:373–399 (1998)

Sex Determination and Primary Sex Differentiation

in Amphibians: Genetic and Developmental
Mechanisms
TYRONE B. HAYES*
Laboratory for Integrative Studies in Amphibian Biology, Group in
Endocrinology, Museum of Vertebrate Zoology, and Department of Integrative
Biology, University of California, Berkeley, California 94720

ABSTRACT Most amphibians lack morphologically distinguishable sex chromosomes, but a

number of experimental techniques have shown that amphibian sex determination is controlled
genetically. The few studies suggesting that environment influences sex determination in amphib-
ians have all been conducted at temperatures outside of the range normally experienced by the
species under study, and these effects probably do not occur under natural conditions. No sex-
determining genes have been described in amphibians, and sex differentiation can be altered by
treatment with exogenous steroid hormones. The effects of sex steroids vary extensively between
species, and a variety of steroids can alter the sex ratios of treated larvae. The role of endogenous
sex steroids in gonadal differentiation has not been fully explored; thus the natural role of ste-
roids in amphibian gonadal differentiation is unknown. Sex steroid receptors have not been exam-
ined in amphibian gonads, and the mechanism of steroid action on the gonad is unclear. In addition
to steroids, the thyroid hormones may play a role in gonadal differentiation. Pituitary gonado-
trop(h)ins affect gonadal growth, but not differentiation or maturation of gonads.
In addition to the issue of resolving the mechanisms underlying hormone action in gonadal differ-
entiation, other debates concerning interactions between the developing gonads and the invading
germ cells, and even the origin of the medullary and cortical portions of the developing gonads,
remain unresolved. Studies examining links between sex determination and gonadal differentiation
are needed. In addition, examinations of variation in steroidal effects on gonadal development in a
phylogenetic context are lacking. J. Exp. Zool. 281:373–399, 1998. © 1998 Wiley-Liss, Inc.

Amphibians are excellent models to study sex
differentiation, for a number of reasons. Genetic
sex-determining systems and sex chromosomes
have evolved a number of times in this class, and
gonadal differentiation (although under genetic
control) is responsive to experimental manipula-
tions. In addition, as laboratory animals, amphib-
ians are ideal; because females produce up to
10,000 eggs per clutch in some species, large
sample sizes can be obtained. Also, large num-
bers of individuals can be treated easily in ex-
perimental manipulations because larvae are
typically aquatic and readily absorb hormones and
other chemicals added to rearing water.
Despite their accessibility as study animals,
relatively few recent studies have addressed sex
determination and differentiation in amphibians.1
Also, there are no recent reviews that address both
sex determination and sex differentiation in am-
phibians, although a few reviews addressed each
topic separately: Dodd (’60) and Dournon et al.
(’90) reviewed genetic and environmental mecha-
nisms of sex determination (addressing amphib-
ians and reptiles), and Schmid (’83) and Hillis and
Green (’90) examined genetic mechanisms of sex
determination with special reference to the evo-
lution of sex chromosomes in amphibians. Gonadal
differentiation in amphibians was reviewed by
Gallien (’65), and at least four reviews (Burns, ’49,
’61; Adkins-Regan, ’81, ’87) addressed the effects
of hormones on sex differentiation in vertebrates
in general, with extensive discussions of amphib-
1
Although often used interchangeably, there is a fundamental dif-
ference between sex determination and sex differentiation. Here, “sex
determination” will refer to mechanisms that direct sex (gonadal)
differentiation, and “sex differentiation” will refer specifically to the
development of testes or ovaries from the undifferentiated or
bipotential gonad. In other words, sex determination refers to the
switch (or the decision), whereas sex differentiation refers to the ac-
tual developmental process.
Grant sponsor: NSF; Grant numbers: IBN-9513362 and IBN-
9508996.
*Correspondence to: Tyrone B. Hayes, Ph.D., Assistant Professor,
Department of Integrative Biology, 3060 Valley Life Sciences Build-
ing, University of California, Berkeley, CA 94720-3140. E-mail:
tyrone@socrates.berkeley.edu
Received 17 February 1998; Accepted 18 February 1998

© 1998 WILEY-LISS, INC.

374 T.B. HAYES
ians. A single review addressed both sex determi-
nation and differentiation in amphibians (Witschi,
’50), but this work was published prior to many
primary studies on the topics. A second review
(Adkins-Regan, ’87) addressed both topics in
nonmammalian vetebrates in general with refer-
ence to amphibians. Thus, with the exception of
the recent reviews addressing sex chromosome evo-
lution (Schmid, ’83; Hillis and Green, ’90), reviews
on sex determination and sex differentiation (and,
in particular, reviews addressing both sex deter-
mination and differentiation) are lacking. The goal
of the current review is to synthesize work on sex
determination and sex differentiation in amphib-
ians. This simultaneous review of sex determina-
tion and differentiation will attempt to place the
available information into an evolutionary frame-
work and explore interactions between mechanisms
of sex determination and sex differentiation.
SEX DETERMINATION
Genetic sex determination versus
environmental sex determination
There are at least two mechanisms of sex de-
termination recognized in vertebrates: genetic sex
determination (GSD) and environmental sex de-
termination (ESD). Genetic sex determination is
a mechanism by which genes directly determine
whether the gonads differentiate into testes or
ovaries without external (environmental) influ-
ences. In ESD, the sex of individuals is determined
by environmental factors: For example, in tem-
perature-dependent sex determination (recognized
in some fish, many turtles, some lizards, and all
crocodilians), sex is determined by the incubation
temperature of the eggs. Thus each individual has
the full capacity to develop into males (testicular
differentiation) or females (ovarian differentia-
tion). In fact, sex differentiation is likely controlled
by genetic mechanisms in animals with ESD and
only the switch “sex determination” may be influ-
enced by environmental factors.
Environmental sex determination
Several studies have suggested that some am-
phibian species display ESD (for review, see Dodd,
’60; Gallien, ’74; Dournon and Houillon, ’84;
Dournon et al., ’90). In frogs, one species of Bufo
(Piquet, ’30) and four species of Rana (Witschi,
’14; ’29a; Piquet, ’30; Yoshikura, ’59b; Hsü et al.,
’71) were examined, in addition to three species
of salamanders from two genera Pleurodeles
(Dournon and Houillon, ’84; Dournon et al., ’84;
Chardard et al., ’95) and Hynobius (Uchida,
’37a,b). These studies all showed that the tem-
perature of the rearing water can alter the sex
ratios of larvae. In all of these studies, however,
effects were obtained by exposure to temperatures
that are not normally experienced by the species
under study. In fact, with one exception, these ef-
fects were produced only at high temperatures (27
to 36°C). In all of the studies on frogs, 100% males
were produced at high temperatures, whereas ei-
ther 100% females (Pleurodeles poireti; Dournon
et al., ’84) or 100% males (Hynobius retardatus:
Uchida, ’37b; Pleurodeles waltl: Dournon and
Houillon, ’84) were produced by high-temperature
treatments in salamanders. A single study (Uchida,
’37b) showed that low temperatures (10°C) can pro-
duce 100% females also.
When reared at temperatures within the ranges
experienced naturally by the species under study,
a 50:50 sex ratio was obtained in all of the stud-
ies cited above. For example, in Witschi’s (’29a)
study on Rana sylvatica, larvae were reared at
32°C (which Witschi reported as a temperature
near the lethal limit and a temperature that can
be tolerated only for short periods). Rana sylvatica
lives in northern North America where eggs are
typically laid in early spring when water tempera-
tures are as low as 4°C (personal observation; and
Wright and Wright, ’49; Herreid and Kinney, ’66;
Seale, ’82; Waldman, ’82). Larvae begin metamor-
phosing within 8 weeks, and water temperatures
do not typically exceed 20°C during this time.
When larvae were reared at 20°C in Witschi’s
(’29a) study, or reared at a range of temperatures
naturally experienced by this species (Hayes and
Waldman, unpublished), the sex ratio was not sig-
nificantly different from 50:50. Similarly, we
(Hayes et al., unpublished) reared other species
(Hyperolius viridiflavus, Bufo boreas, and Pyxi-
cephalus adspersus) over a range of temperatures
appropriate for each species and did not observe
any effects on sex ratios.
The natural role of temperature in the sex de-
termination in reptiles and fish is much more con-
vincing, because effects have been observed at
temperatures within ranges experienced by the
species in the wild (fish: Conover and Heins, ’87;
turtles: Bull et al., ’82), and effects have been
shown in natural nests in turtles (Bull, ’85; Rhodes
and Lang, ’95). Thus, given the lack of effects on
sex ratio at temperatures appropriate for the spe-
cies in the studies on amphibians, it is not likely
that temperature is important in normal sex de-
termination in amphibians. The reported environ-
 SEX DETERMINATION IN AMPHIBIANS
mental influences are probably artifacts of the ab-
normally high temperatures at which the animals
were reared. In addition, reptiles and fish display-
ing ESD typically lack sex chromosomes (morpho-
logically distinguishable or otherwise), which is
not the case in amphibians. Thus the available
evidence for amphibians suggests that sex deter-
mination is controlled exclusively by genetics un-
der natural conditions (see below).
Genetic sex determination
In genetic sex determination, the direction of
sex differentiation is controlled genetically, such
that one sex has a gene (or more likely a set of
genes) that the other lacks. The sex-determining
genes likely control only the “decision,” and are
not likely involved in sex differentiation, but
rather initiate a sequence of events that results
in gonadal differentiation. Normally, the different
sexes develop because individuals are directed
through different developmental pathways as a
result of the activation of the sex-determining
gene(s). This last point is important, because all
individuals may have the capacity to develop into
either sex. This capacity is evident from the abil-
ity to experimentally sex-reverse amphibians: De-
pending on the species, genetic males can be
experimentally induced to develop ovaries and vice
versa with steroid hormone treatments.
The presence of sex chromosomes is the clearest
indication that a species possesses GSD. In these
cases, the sex-determining genes are on a chro-
mosome that has evolved such that it is morpho-
logically distinguishable from its homologue upon
cytological examination (the X and Y in mammals,
for example). The evolution of sex chromosomes
often involves the loss of genetic material: For ex-
ample, in mammals, the Y chromosome is smaller
than the X as a result of limited chromatid ex-
change between the X and Y chromosome and sub-
sequent loss of genes from the Y chromosome (see
J. Graves, this issue).
Sex chromosomes can be distinguished morpho-
logically by a number of techniques. Examining
preparations of metaphase chromosomes from
males and females for chromosomes in which the
homologues differ in size between the sexes is the
simplest technique. Some sex chromosomes differ
in the centromeric index as a result of pericentric
inversions, which can be observed cytologically. In
addition, as chromosomes lose genetic material
(such as in the mammalian Y chromosome), they
accumulate repeats, resulting in heterochromatism.
Heterochromatism can be detected by a variety of
375
techniques that produce different banding patterns
on the chromosomes. Finally, some accumulated
repeats result in differences in replication time
between chromosomes. A technique using the in-
corporation of bromodesoxyuridine can detect dif-
ferences in replication time to identify otherwise
homomorphic sex chromosomes (Schmid, ’83).
More than 1500 amphibian species have been
examined cytologically using the techniques de-
scribed above. As few as two species of frogs
(Pyxicephalus adspersus: Schmid, ’80; Schmid and
Bachman, ’81; Gastrotheca riobambae: Schmid, ’83)
and 11 salamanders (all Plethodontidae: Mancino,
’65; Kezer and MacGregor, ’71; León and Kezer, ’78;
Sessions, ’84; Nardi et al., ’86) have sex chromo-
somes that differ in size. Sex chromosomes are dis-
tinguishable in at least another 30 species of
salamanders and frogs by examining banding pat-
terns or differences in replication time. So, less than
4% of the amphibians examined cytologically pos-
sess morphologically distinguishable sex chromo-
somes (no data are available for caecelians).
Despite the absence of morphological distin-
guishable sex chromosomes in the majority of am-
phibians, sex is probably under genetic control in
all amphibians. The inability to distinguish sex
chromosomes in amphibians may indicate only
that the sex chromosomes have not evolved to
where they are morphologically distinguishable:
Although there may be chromosomes possessing
sex-specific genes, the differentiated regions are
small and not distinguishable cytologically. Hillis
and Greene (’90) suggested that the lack of differ-
entiation in amphibian sex chromosomes indicates
that a much smaller region may be involved in
sex determination in amphibian sex chromosomes
relative to other vertebrates with morphologically
distinct sex chromosomes.
Furthermore, other experimental techniques that
do not require morphological identification of sex
chromosomes have been used to identify the sex-
determining systems in amphibians. In most cases,
experimental manipulations were used to obtain
information regarding heterogamety. These stud-
ies involved back-crossing experimentally “sex-re-
versed” individuals to normal individuals, and
using the resulting sex ratio to infer the genetic
constitution (male heterogamety: XX/XY versus fe-
male heterogamety: ZZ/ZW). For example, 100%
females were produced when larvae of the African
clawed frog (Xenopus laevis) were exposed to exog-
enous estrogens (Witschi and Allison, ’50). When
raised to sexual maturity, approximately 50% of
these females produced 100% male offspring when
376 T.B. HAYES
bred to normal males. Thus 50% of the estrogen-
treated animals were assumed to be sex-reversed
males (ZZ females) that produced only male (ZZ)
offspring, when crossed back to normal (ZZ) males
(Chang and Witschi, ’55b; Hayes, ’98; Figure 1).
These types of studies have been used to show fe-
male heterogamety in other species (Ambystoma
spp.; Humphrey, ’45; ’48). In similar studies with
Rana spp., androgen treatment produced 100%
males (Kawamura and Yokota, ’59; Richards and
Fig. 1. Experimental techniques used by Mikamo and
Witschi (’64) to determine the genetic sex-determining sys-
tem of the African clawed frog (Xenopus laevis). Normal males
and females were bred to produce larvae. The resulting lar-
vae were treated with estrogen and produced 100% females.
Fifty percent of the treated larvae were genetic females, and
the remaining 50% were genetic males, “sex-reversed” by the
hormone treatment so that they developed ovaries. When bred
back to normal males, the estrogen-treated females produced
a normal sex ratio of 50% male/50% female offspring. The
sex-reversed males, however, produced only male offspring
when bred back to normal males, indicating that males are
homogametic (ZZ) in this species.
Nace, ’78). Approximately 50% of these males pro-
duced 100% female offspring when crossed back to
normal females, showing that the species under
study were male heterogametic (XX/XY). Other
studies used breeding experiments to track sex-
linked (but not sex-determining) genes in frogs
(Wright and Richards, ’83; Sumida and Nishioka,
’94; Rudolf and Dournon, ’96).
Expression of the HY antigen (see Bennet et
al., ’75; Wachtel et al., ’75; Zaborski et al, ’88) has
been used to identify the heterogametic sex in
frogs (Engle and Schmid, ’81) also. In this study,
species in which the heterogametic sex was iden-
tifiable by independent methods were used to test
the use of HY antigen as a tool for identifying the
heterogametic sex. HY antigen was always ex-
pressed in the heterogametic sex in this and sub-
sequent studies, suggesting that the test was valid
(Wachtel et al., ’75; Zaborski, ’79). Later studies
showed that the HY antigen was not the testis
inducer, although it was sex-linked (McLaren et
al., ’84, ’88; Goldberg, ’88). Furthermore, in
nonmammalian vertebrates, the gene for HY an-
tigen is present in both sexes (not sex-linked) and
can be induced by hormone treatment. For ex-
ample, in birds, only females (ZW) normally ex-
press HY antigen. When males were treated with
estrogens, however, they expressed HY antigen
(Müller et al., ’79, ’80; Zaborski et al., ’81; Eben-
sperger et al., ’88). Given the presence (but not
necessarily expression) of the HY antigen gene in
both sexes in lower vertebrates, its use as a tool
to identify the heterogametic sex in amphibians
is limited, and results must be confirmed by other
methods. Nevertheless, experimental techniques,
such as the ones described above, identified the
sex-determining systems in another 17 species of
frogs (in addition to those identified by cytologi-
cal techniques) and two more salamanders.
One other method for assessing the sex-deter-
mining mechanism in amphibians has provided
confusing results. Several workers showed that
amphibian eggs can be induced to divide without
fertilization by pricking the eggs with a glass needle
(Parmenter, ’25; Kawamura, ’39, ’49; Moriwaki, ’54,
’57, ’59, ’60a,b). Pricking induced chromosome
doubling in some eggs (diploidy) and presumably
normal development in these “parthenogenetic”
embryos. All of these studies were conducted in
Rana spp., which are male heterogametic (XX/XY:
Schmid, ’83; Hillis and Green, ’90), so all parthe-
nogenetically produced animals should be females
(the eggs contain only an X chromosome). In sev-
eral studies, however, males resulted from pricked
 SEX DETERMINATION IN AMPHIBIANS
(unfertilized) eggs. For example, Parmenter (’25)
reported 18 male and three female diploid animals
produced by pricking in Rana pipiens; Kawamura
(’49) reported 32 normal females, seven females
with degenerating ovaries, three males with rudi-
mentary testes, and two normal males in Rana
nigromaculata and R. japonica; and Moriwaki (’54)
reported seven males and nine females in R.
japonica. One interpretation is that the sexes were
misidentified, but when a parthenogenetically pro-
duced male was used to fertilized a normal female
(Moriwaki, ’54), 100% females (72 animals) were
produced, compared to 56% females (out of 86 ani-
mals) in controls. These data suggest that the par-
thenogenetically produced male was indeed a
homogametic (XX) individual. It is difficult to rec-
oncile these data with other evidence for male het-
erogamety in this genus, and no recent studies have
followed up on these studies.
Evolution of sex chromosomes
Using cytological data and data from published
studies such as the ones described above, Hillis
and Greene (’90) examined the evolution of sex-
determining mechanisms in amphibians. Unlike
many other major vertebrate groups (all mammals
are male heterogametic, all birds are female het-
erogametic, etc.), sex chromosomes have evolved
several times in amphibians. The ancestral state
appears to be female heterogamety, and although
male heterogamety has evolved several times (Fig-
ure 2), a reversion to female heterogamety in a
male heterogametic lineage has occurred only once
(Chiropterotriton dimidatus in the bolitoglossin
plethodontids).
Schmid (’83) pointed out that the ancestral Z
chromosome in amphibians does not share a com-
mon ancestor with the Z chromosome of snakes
and birds. Similarly, it is not likely that the sex
chromosomes are related between amphibian
families: The X and Y chromosomes in derived
Ranids are not evolved from the Z or W chromo-
somes of the more basal Pyxicephalus adspersus
and Tomopterna spp. For example, the nucleolus
organizing region (NOR) is on the Z chromosome
in Leiopelma (Green, ’88), on the X chromosome
in H. femoralis (Anderson, ’91) and Gastrotheca
(Schmid et al., ’86, ’88, ’90), on the Z chromosome
in Buergeria buergeria (Ohta, ’86; Schmid et al.,
’93), but is not on either of the sex chromosomes in
Pyxicephalus adspersus (Schmid, ’80; Schmid and
Bachman, ’81). Each change in heterogamety likely
involved the cooption of a different set of autosomes
to produce the sex chromosomes, just as the Z and
377
W chromosomes in birds and the X and Y chromo-
somes in mammals are independently derived from
different autosomes.
The number of times that sex chromosomes
evolved and the number of transitions between
heterogametic types are not known, because the
genetic sex-determining system is known for only
nine of the 21 extant families of frogs and five of
the ten urodele families. (There are no data avail-
able for caecelians.) In most cases, the type of het-
erogamety for each family is inferred from one or
two species and, usually, a single genus. Further
analysis may reveal even more transitions (such
as the two types of heterogamety in the Ranidae:
female heterogamety in two genera, and male het-
erogamety in at least one). In addition, Nishioka
et al. (’94) and Nishioka and Hanada (’94) sug-
gested that the sex-determining system can
vary even between populations of a single spe-
cies: They reported that Rana rugosa showed
both male (XX/XY) and female (ZZ/ZW) hetero-
gamety, depending on the population under
study. So sex-determining systems may be com-
plex even within a species.
Sex-determining genes and
genetic mechanisms
To date, no sex-determining genes have been
identified in amphibians. A single study (Chardard
et al., ’93) reported identification of an SrY Box
(SOX) gene in a salamander (Pleurodeles waltl).
This gene is related to the SRY gene identified in
mammals, but is not sex-linked in P. waltl. Also,
unlike in mammals, gene-dosage compensation
does not occur in amphibians. Bar bodies or other
mechanisms of deactivation of the homogametic
sex chromosome have not been observed. For ex-
ample, in Buergeria buergeri the NOR is on the Z
chromosome (Ohta, ’86; Schmid et al., ’93). As a
result, males have two nucleoli per nucleus and
females only one. Males express twice as much
ribosomal RNA as females, further suggesting that
there is no dosage compensation.
SEX (GONADAL) DIFFERENTIATION
Developmental mechanisms
Gonadal development
Once sex determination mechanisms are acti-
vated, primary sex differentiation ensues. The ma-
jority of amphibians are gonochoristic, and gonadal
differentiation follows a similar pattern in frogs and
salamanders (little is known about caecilians). The
gonads consist of the somatic gonad and germ cells.
378 T.B. HAYES
Fig. 2. Evolution of genetic sex-determining mechanisms
in amphibians. Sex-determining systems are shown for anu-
ran (a) and urodele (b) families. Data for families are based
on studies from the literature reviewed by Hillis and Green
(’90). Heavy lines show female heterogametic families, normal
lines show male heterogametic families, and dashed lines show
families where the sex-determining system is not known. Fe-
male heterogamety appears to be the ancestral state for am-
phibians, and male heterogamety has evolved several times.
In most cases, however, the sex-determining system assumed
for each family is based on studies from a single genus and in
many cases a single species from each genus. In addition, the
sex-determining system has been examined in approximately
only half of the extant anuran (nine families, for which nei-
ther the genetic sex-determining system nor the effects of ste-
roids are known, are not shown here) and urodele families,
with no studies in caecelians. Also, within the Ranids, the basal
genus Pyxicephalus has a different sex-determining mechanism
from the other Ranids and is treated separately.
The effect of exogenous androgens (A) and estrogens (E) is
shown, when data are available for anurans: MF indicates
The somatic gonadal tissues in males and females
likely have different origins, and the primordial
germ cells arise from yet a third origin.
The gonads originate from an outpocket of cells
on the ventral surface of the kidney. Initially, there
are no histologically observable differences be-
tween males and females. The undifferentiated (or
bipotential) gonad is a solid structure with an in-
tact cortex and medulla. In females, the cortex
develops and grows, whereas the medulla re-
gresses, leaving a hole (ovarian vesicle) which is
that normal males and females occur at normal frequencies
following the treatments (no effect); F indicates that 100%
females resulted from the treatment (sex reversal of genetic
males); “F or M” indicates that 100% males or 100% females
can result (in this case, depending on the dose of estrogen); I
indicates that intersexes result from the treatment (hermaph-
roditic or ambiguous gonads in all individuals); IF indicates
that the males show partial sex reversal (hermaphroditic go-
nads) and females are unaffected; M indicates that 100%
males result (sex reversal of genetic females); MI indicates
that normal males result, but that females show partial (her-
maphroditic gonads) sex reversal. A “?” indicates that these
data were not reported. It should be noted that in most cases
the effects of steroids for each family are based on a limited
number of studies in usually one species, and in many cases
the species used to determine the sex-determining mecha-
nism is not the same one used in experimental manipula-
tions with exogenous hormones. The literature base used to
evaluate the effects of steroids in this figure is presented in
Table 1. In salamanders (b), data for the effects of steroids
are not shown because of the limited available data; see text.
observable upon histological analysis. The cells in
the cortex become large follicles, and oocytes can
be observed during fairly early stages of differen-
tiation. Male development is characterized by
medullary development and cortical regression.
The gonads in males have smaller densely packed
cells (Figure 3).
Origin of the somatic gonadal tissue
There is still debate regarding the origin of the
cells that form the gonadal ridge (which becomes
 SEX DETERMINATION IN AMPHIBIANS
Fig. 3. Transverse histological cross sections through
bipotential gonads (top panel), ovaries (bottom left), and tes-
tes (bottom right) from the African clawed frog (Xenopus
laevis). Bipotential gonads were dissected from early larvae,
and differentiated gonads were dissected from newly meta-
morphosed animals. Ovarian differentiation is characterized
by the regression of the medullary portion of the bipotential
the undifferentiated gonad) and whether differ-
ent tissues contribute to the cortex and medulla.
In Witschi’s studies on Rana sylvatica (Witschi,
’27) and R. sylvatica, R. arora, and Hyla regilla
(Witschi, ’31), he reported that the cortex (the pri-
mordium of the ovary) is derived from the coelomic
epithelium and that the medulla (the testicular pri-
mordium) originated from the mesonephric blas-
tema, with no contributions from the coelomic
epithelium. Several later studies and reviews
agreed with Witschi’s findings (Witschi, ’67;
Mittwoch, ’73; Deuchar, ’75), but Vannini (’41, ’42)
suggested that the medulla was derived from the
interrenal blastema and not the mesonephric
blastema in Rana agilis. Studies in Bufo bufo and
Bufo viridis (Vannini and Busetto, ’45), in Bom-
bina pachypus (Vannini, ’47), and in two sala-
379
gonad (resulting in a hole, or ovarian vesicle, in the center),
and testicular differentiation is characterized by the regres-
sion of the cortical portion. All sections were cut from paraf-
fin-embedded tissues at 10µ and stained with hematoxylin/
eosin. Arrows show gonads in each panel; bar = 250µ; K =
kidney; OV = ovarian vesicle.
manders (Triton spp.; Papi, ’47, ’49) produced find-
ings similar to Vannini (’41, ’42). Later studies in
Rana esculenta (Sabbadin, ’51a; Vannini and
Sabbadin, ’54) and Rana dalmatina (Sabbadin,
’51b) suggested that the interrenal blastema con-
tributed cells to the cortex (which is derived pri-
marily from cells from the coelomic epithelium)
and to the medulla of the developing gonads. In
these early stages of gonadal development, the cor-
tex and medulla are reportedly not distinct and
migrations from the interrenal blastema to the
cortex do not cease after the two layers separate.
Vannini and Sabbadin (’54) suggested that the dif-
ferences in their findings and those findings of
Witschi were, in part, due to what they interpreted
as Witschi’s unclear understandings of the origins
of the mesonephric and interrenal blastema. The
380 T.B. HAYES
significance of the difference between Witschi’s and
Vannini’s model is that the gonadal primordia and
the interrenal primordia (both steroid-producing tis-
sues) share a common origin in Vannini’s model,
whereas the gonads share a common origin with
the kidney primordia (non steroid-producing) in
Witchi’s model.
Recent studies examining the origins of the go-
nadal ridge created more confusion: Studies in Xe-
nopus laevis and R. pipiens (Merchant-Larios and
Villalpando, ’78, ’81) suggested that the meso-
nephric blastema may not contribute to the gonadal
medulla (in agreement with Vannini’s proposal),
but further suggested that the coelomic epithelium
was the origin of the gonadal medulla, giving the
cortex and medulla a common origin. Lopez (’89)
suggested that there was no medulla in Bombina
orientalis initially, and that the germ cell–filled cor-
tex was invaded by the medulla, which originated
from cells of the mesonephric blastema. Further-
more, Tanimura and Iwasawa (’87, ’89) suggested
that the entire gonad was formed from the coelo-
mic epithelium in Rhacophorus arboreus and that
the medulla is formed by cells segregating within
the primordial gonad. Furthermore, at least two
studies suggested that cells migrate from the cor-
tex into the medulla during male development
(Vannini and Sabbadin, ’54; Amanuma, ’63). Thus
an examination of several studies reveals conflict-
ing results. Although species variation is possible,
more comparative ultrastructural studies may re-
solve this problem.
Origin of the primordial germ cells
Like the origins of the cortex and the medulla,
the origin of the primordial germ cells in amphib-
ians has been heavily debated. The early history
of these debates was documented by Witschi (’29c):
The origin of the primordial germ cells was first
described by Goette (1875), who suggested that they
arose from yolk-laden embryonic cells. Allen (’07)
was the first to report that the primordial germ
cells had an endodermal origin. Other authors de-
scribed secondary germ cells, and some claimed
that these secondary germ cells had a mesodermal
origin (Bouin, ’01; Dustin, ’07). Humphrey (’25) even
reported that germ cells in salamanders were de-
rived from mesoderm, whereas germ cells in frogs
were endoderm-derived. Humphrey suggested also
that germ cells did not migrate or move indepen-
dently, but rather shifted their position through-
out development as a result of “growth of shifting
of related parts” (Humphrey, ’25; page 1). Other
authors had previously made similar claims that
germ cells were mesoderm-derived in salamanders
(Hall, ’04; Dustin, ’07; Allen, ’11; Schapitz, ’12;
Beccari, ’22). Kuschakewitsch (’10) reported second-
ary germ cells that arose from a variety of tissues;
and Gatenby (’16) claimed that oogonia arose de
novo in the ovary. King (’08), Witschi (’14), Beccari
(’24, ’26), Bounoure (’25) did not find any evidence
for secondary germ cell differentiation, and it is
not likely that such occurs, based on recent stud-
ies cited below.
Most workers currently agree that the germ cells
originate in the endoderm from the dorsal mesent-
ary of the gut. The formation and migration of the
primordial germ cells have been well studied in
Xenopus laevis (Blackler, ’58; Al-Mukhtar and
Webb, ’71; Kerr and Dixon, ’74; Ijiri and Egami,
’75; Ikenishi and Kotani, ’75; Whitington and
Dixon, ’75; Wylie and Heasman, ’76; Kamimura et
al., ’76; Wylie et al., ’76; Züst and Dixon, ’77;
Yamaguchi and Iwasawa, ’81), including light mi-
croscopic and ultrastructural electron microscopi-
cal studies. These studies found that germ cells
originated solely from primordial germ cells that
differentiated and migrated as early as the gas-
trula stage. Thus no secondary or spontaneous dif-
ferentiation of germ cells in the gonads are likely.
Blackler (’62) provided additional evidence
against secondary formation of germ cells: Germ
cells were reciprocally transferred between two sub-
species of Xenopus laevis (one that produced light
brown eggs and one that produced dark brown eggs).
In some crosses, the eggs produced by adult females
were solely of the donor type: If any secondary pro-
duction from a source other than the grafted pri-
mordial germ cells occurred, then all individuals
would have produced eggs of mixed types.
Although all of the studies cited above agree in
regards to the origin of primordial germ cells, very
little comparative work has been conducted. Two
recent studies (Tanimura and Iwasawa, ’87;
Nomura and Iwasawa, ’92) examined germ cell
kinetics in a toad (Bufo japonicus formosus) and
a salamander (Onychodactylus japonicus), respec-
tively, but no studies have examined the ultra-
structure and migration of primordial germ cells
to the extent of the studies in Xenopus laevis.
Germ cell–somatic gonadal tissue interactions
Several studies examined the potential role of
the germ cells in gonadal differentiation, with
mixed results. Witschi (’29c) and Cheng (’32) sug-
gested that the histological changes associated
with masculine and feminine differentiation oc-
curred before the germ cells migrated into the go-
 SEX DETERMINATION IN AMPHIBIANS
nad, but Dantchakoff (’32, ’33) proposed that the
germ cells secreted a substance that induced go-
nadal differentiation.
The most convincing experimental evidence sug-
gesting that the gonads differentiate independent
of germ cells was presented by Humphrey (’38),
who conducted reciprocal unilateral transplants
of the intermediate segment and adjacent meso-
derm in Rana sylvatica during early tail bud
stages (prior to migration of the primordial germ
cells). Thus the mesonephros and gonad on the
operated side differentiated from the donor’s me-
soderm, whereas the primordial germ cells that
migrated into the developing gonad were from the
host’s endoderm (because no endoderm was trans-
planted). In cases where the sex of the donor and
host differed, the gonad on the operated side fol-
lowed the sex of the donor, as did the germ cells.
For example, when male mesoderm was trans-
planted to a female embryo, an ovary developed
on the unoperated side, whereas a testes devel-
oped on the operated side. The primordial germ
cells that entered this testes developed into sperm,
showing that the developing testes induced the
invading germ cells to develop into sperm rather
than oocytes. Similar studies were conducted in
salamanders (Humphrey, ’27), and showed that
gonads developed in the absence of primordial
germ cells, but only testicular development was
described in detail. Humphrey’s studies showed,
however, that gonads develop in the absence of
germ cells and that germ cells do not stimulate
differentiation of the gonads.
Later ultrastructural studies suggested that
germ cells may even differentiate independent of
the gonads (Kalt, ’73). Primordial germ cells were
more numerous (Ijiri and Egami, ’75), and the
germ cell division rate was faster in females than
in males (Züst and Dixon, ’77), even before the
germ cells migrated into the gonads. These data
suggested that germ cells may sexually differen-
tiate independent of the gonads, although Hum-
phrey’s findings (’27, ’38) suggested that the
gonads can redirect germ cell differentiation.
Despite evidence that germ cells are not re-
quired for gonadal differentiation, more recent
studies suggested, on the contrary, that germ cells
were inductive and directed differentiation of the
gonads. Shirane pressed eggs of Rana japonica
and Rana brevipoda (’82) and Rana nigromaculata
(’84) against quartz and exposed the vegetal pole
to UV irradiation to destroy primordial germ cells
before they differentiated and migrated to the go-
nadal ridge. In the earlier (’82) study, germ cell–
381
free embryos were produced, and although the go-
nads were sometimes smaller in size, fertile males
and females were produced. Shirane (’82) reported
a skewed ratio at sexual maturity in Rana brevi-
poda, but the sample size (33 total: 8 females/25
males) was low, and mortality between metamor-
phosis and the stage of examination was greater
than 30%. The sex ratio determined before mor-
tality (at metamorphosis) was normal (54% fe-
male), and although the author claimed that the
skewed sex ratio at sexual maturity was due to
sex reversal (female to male), it is equally likely
that the change was due to differential mortality.
In Shirane’s later study (’84), he reported female-
to-male sex reversal (but again sample sizes were
low). He hypothesized that UV irradiation reduced
the number of germ cells that migrated into the
gonadal ridge. He claimed that ovarian differentia-
tion failed and that medullary cells surrounded the
remaining germ cells and induced testicular devel-
opment (female-male sex reversal). With this evi-
dence, he concluded that germ cells were necessary
for ovarian differentiation.
Shirane (’87) further tested the hypothesis that
germ cells affected gonadal differentiation by us-
ing the peanut lectin (PNA), which binds to the
surface of migrating primordial germ cells. Shi-
rane (’87) suggested that the observation that the
primordial germ cells lost their affinity for PNA
coincident with gonadal differentiation indicated
that the germ cells induced gonadal differentia-
tion. He further suggested that the primordial
germ cells secreted substances that induced gene
expression in the developing gonad, resulting in
differentiation (Shirane, ’87). To test these hypoth-
eses, Shirane injected embryos with PNA or N-
acetylgalactosamine (galNA; which is recognized
by PNA). He proposed that the sex-inducing ac-
tivity of the migrating primordial germ cells would
be decreased by treatment with lectins (which
would presumably occupy the binding sites of the
lectins and prevent binding and activation of the
secreted substances in the gonadal ridge) and that
treatment with galNA would enhance the activ-
ity. Shirane (’87) suggested that PNA inhibited fe-
male development in Rana japonica and inhibited
both male and female development in R. nigro-
maculata based on the results of this study.
Furthermore, treatment with galNA reportedly in-
hibited male development in both species.
There are several problems with Shirane’s (’87)
study and the interpretation of results, however.
The sample sizes were low (30 or fewer in the
experimental groups discussed) and not adequate
382 T.B. HAYES

for assessing effects on the sex ratio. In addition,

Shirane examined gonadal development only in
early developmental stages (TK V; Taylor and
Kollros, ’46) and admits that “the results at TK V
were sometimes ambiguous” and “perhaps discrep-
ancies in these results may be caused by the fact
that the decision about gonadal sex was histologi-
cally more difficult at TK V” (Shirane, ’87; page
500). Attempts to reanalyze data at TK X failed
because treatments with galNA were lethal by this
stage, and the experiment was discontinued. Fur-
thermore, the author discussed the effects of his
treatment on male and female genes, when there
is no evidence that any observed histological ef-
fects resulted from changes in gene expression.
Shirane’s (’82, ’86, ’87) studies are thus inconclu-
sive, and earlier studies (discussed previously) pro-
vided stronger evidence for the lack of a role for
primordial germ cells in gonadal differentiation.
Cortical-medullary interactions
Witschi (’27, ’31, ’34, ’57) proposed that gonadal
differentiation was the result of antagonistic in-
teractions between the developing cortex and me-
dulla (Figure 4). He proposed that the cortex of
genetic females produced a compound (cortexin)
that induced regression of the medulla (or alter-
natively accelerated cortical growth) and/or that
a substance from the medulla (medullarin) in-
duced cortical regression (or medullary growth)
in males. Furthermore, Witschi proposed that
these compounds did not behave like typical hor-
mones, but traveled in tissue rather than being Fig. 4. Potential mechanism controlling gonadal differen-
tiation (proposed by Witschi, ’47). On the left, activation of
secreted into the blood (Witschi, ’27). female-producing genes results in the secretion of putative
Several studies showed that sex reversals oc- compounds that inhibit (cause regression) of the medulla and/
curred when males and females of the same spe- or promote development of the cortex. On the right, male-
cies were surgically connected as parabiotic twins, determining genes induce the secretion of similar putative
which Witschi viewed as evidence supporting his compounds that cause regression of the cortex and/or pro-
mote development and growth of the medulla which forms
theory: Witschi and McCurdy (’29) showed that the testes.
ovaries transformed into testes when female lar-
vae were paired with male larvae in Taricha torosa
(Triturus torosus) with only minor effects on the testes. Witschi viewed the above data as evidence
testes by the female. Burns’s (’30) data supported for his inductor substances (Witschi, ’31), but
these findings and showed that male Ambystoma these data suggested that the putative inductors
tigrinum induced sex reversal when parabiosed traveled in the blood shared by the parabiotic
with females. In frogs, similar findings were re- twins (not only through tissues as suggested by
ported: Parabiosis between male and female Xe- Witschi [’27]).
nopus laevis resulted in sex reversal of females Other studies examined interactions between
(degeneration of the cortex and oocytes, and hy- ovaries and testes by transplanting testes into fe-
pertrophy of the medulla; Chang, ’53). Similarly, males and ovaries into males, for comparisons with
in parabiotic twins of Rana sylvatica (Witschi, same-sex reciprocal transplants as controls. Such
’29c), Rana aurora, and Hyla regilla (Witschi, ’31) studies in salamanders (Ambystoma maculatum
females initially developed ovaries when para- and A. tigrinum: Humphrey, ’29; ’31a; Cynops
biosed with males, but subsequently developed (Triturus) pyrrhogaster: Ichikawa, ’37; A. mexi-
 SEX DETERMINATION IN AMPHIBIANS
canum: Humphrey, ’42) showed that transplanted
donor testes developed normally, but caused sex
reversal (transformation of ovaries into testes) in
female hosts and transformation of donor ovaries
when placed in males (provided that the donor and
host were of similar age and developmental stage).
Humphrey (’31b) later showed that when the
grafted testes were removed from the female hosts,
the sex-reversed females (neomales) were sterile
(Humphrey, ’42). Furthermore, the rudimentary
ovary underwent reversal again (after testes re-
moval) and produced functioning ovaries, showing
that the effects were not permanent.
Humphrey (’35) showed that heterosexual para-
biosis between two different species produced dif-
ferent results: Testes donated from A. maculatum
were transformed into ovaries by the female A.
jeffersonium. Also, A. tigrinum female hosts trans-
formed testes grafted from A. maculatum into ova-
ries (Humphrey, ’35). In these cases, the effects of
the ovary on the testes were believed to be due to
the intrinsic faster development rate of the female
host species.
In frogs, similar examinations revealed signifi-
cant variation between studies. In Rana tem-
poraria (Witschi, ’27), testes implanted into
sexually undifferentiated tadpoles had no effect
on developing ovaries. These studies (Witschi, ’27)
were conducted in a race described as protogynous,
so this peculiar sex differentiation in this species
may have affected results. Witschi (’27) viewed
these data as evidence that did not support his
inductor theory of sex differentiation, as he pre-
dicted that the testes should interfere with corti-
cal development. A number of studies in Xenopus
laevis, however, yielded positive results, but cre-
ated some confusion: When implanted with tes-
tes, genetic females developed testes (Chang, ’53;
Mikamo and Witschi, ’63, ’64). In addition, Chang
(’53) found that testis grafts induced sex reversal
(development of testes from ovaries in females)
only when the grafts were made during the lar-
val phase. Also, testes from only immature post-
metamorphic males induced complete sex reversal:
The testes of adult sexually mature males lost
their inductive capabilities.
The ability of implanted testes from immature
males (but not adult males) in Xenopus laevis is
especially interesting, in light of experiments ex-
amining the effects of crystalline sex steroids on
gonadal development in amphibians. Testosterone
was incapable of inducing male development in
Xenopus laevis larvae; early studies suggested
that testosterone produced 100% males, but
383
proved incorrect. The gonads were not affected
by androgen treatment, although androgens in-
duced secondary sex characters such as thumb
pad development (Chang and Witschi, ’55a). Thus
sex steroids were likely candidates for Witschi’s
cortexin and medullarin, except that testosterone
had no effect on gonads, whereas whole testes
were effective. In fact, if androgens were the in-
ductive substances, then the testes of adult males
should be more potent, but Chang’s (’53) studies
showed that only immature testes were capable
of inducing testicular development in females.
Furthermore, estrogens were 100% effective at
inducing complete sex reversal of genetic males,
but in parabiosis experiments, the ovary was af-
fected by the male (or the testis graft) and not
the other way around. Even more confusing, ova-
rian extracts were capable of inducing sex inver-
sion in adult male Triturus pyrrhogaster, whereas
estrogen treatment could not produce this effect
(Hanaoka, ’61). Chang and Witschi (’55a) turned
away from the idea that steroids were the puta-
tive inductors, speculating that the cortexin and
medullarin were proteins, and perhaps that the
cortex even produced antibodies (antimedullarin)
against the medulla and vise versa. In the same
report (Chang and Witschi, ’55a), the authors also
reexamined Witschi’s earlier thesis (Witschi, ’27)
that the inductor substances traveled through tis-
sues rather than through blood, and suggested
that the putative inducers were blood-bound.
Role of steroid hormones
Padoa (’36) was the first study to examine the
effects of exogenous steroids on gonadal differen-
tiation in amphibians. In Padoa’s (’36) study on
Rana esculenta, exogenous estrogen treatment re-
sulted in 100% male offspring. Later studies
showed that a variety of steroids “sex-reverse” frog
and salamander larvae when added to the water
or injected into larvae (Gallien, ’59), although
Swingle (’22) suggested that the true transforma-
tions do not occur. Paradoxically, a number of
chemically unrelated steroids have similar effects:
For example, progestins, corticoids, androgens,
and estrogens can all induce testicular develop-
ment producing 100% male progeny (Table 1).
Equally puzzling, a single steroid can have a va-
riety of effects, depending on the species: Estra-
diol can produce 100% females, have no effect at
all, or produce 100% males (see Table 1). A single
steroid can have drastically different effects even
in the same species, depending on dose. Moreover,
in ranids estradiol induces 100% females at low
384 T.B. HAYES

TABLE 1. Steroidal effects on primary sex differentiation in anurans

Steroid1 Conc2 Species3 Effect4 References

Progestins
Pregnandiol .03–3.9 Rana sylvatica MF Witschi (’48)
Pregnenalone .06–.08 Rana sylvatica MF Witschi and Allison (’50)
Pregnenalone .3–1.6 Rana sylvatica M Witschi and Allison (’50)
Pregnenalone .32 Rana sylvatica M Witschi and Chang (’50)
Pregnenalone ? Rana pipiens M Eversole and D’Angelo (’42)
Pregnenalone 3.95 Rana temporaria M Jost (’43)
Progesterone ? Rana agilis M Vannini (’46)
Progesterone .16–.95 Rana esculenta M Padoa (’42)
Progesterone ? Rana esculenta M Vannini (’45)
Progesterone .06–.32 Rana sylvatica MF Witschi (’48)
Progesterone .63–1.6 Rana sylvatica M Witschi (’48)
Progesterone .79 Rana sylvatica M Witschi and Chang (’50)
Corticoids
Corticosterone 1.1 Bufo boreas MF Hayes, Chan, and Licht, unpubl.
Cortisone 2.78 Rana sylvatica M Witschi and Chang (’50)
Cortisol 2.76 Rana esculenta MF Padoa (’57)
DOC ? Rana agilis M Vannini (’46)
DOC 1.5–3.0 Hyla arborea MF Uchida and Takahashi (’59)
DOC .06–.12 Rana esculenta F Padoa (’42)
DOC ? Rana esculenta F Vannini (’45)
DOC 1.5–3.0 Rana ? IF Uchida and Takahashi (’59)
DOCA 1 Rana catesbeiana M Witschi and Chang (’50)
DOCA 0.4 Rana dalmatina F Vannini (’47)
DOCA .53–.67 Rana sylvatica MF Witschi (’48)
DOCA 1.3–2.6 Rana sylvatica M Witschi (’48)
Androgens
Androsterone .017 Rana sylvatica M Witschi (’48)
Androsterone .07–.09 Rana sylvatica MIF Witschi (’48)
Androsterone .34–1.7 Rana sylvatica MI Witschi (’48)
Androsterone .3–3 Xenopus laevis MF Chang and Witschi (’55b)
DHEA .07–.35 Rana sylvatica MF Witschi (’48)
DHEA .69–.87 Rana sylvatica MI Witschi (’48)
DHEA 3.5–4.3 Rana sylvatica M Witschi (’48)
DHT .34 Pyxicephalus adspersus MF Hayes and Licht, unpubl.
Testosterone ? Alytes obstetricians M Witschi and Allison (’50)
Testosterone .87 Buergeria buergeri MIF Ohta (’87)
Testosterone 1.4 Bufo boreas MF Hayes, Chan, and Licht, unpubl.
Testosterone ? Bufo japonica MF Chang (’55)
Testosterone 1.5 Bombina varieagata MF Gallien (’65)
Testosterone .034–0.34 Hyperolius argus M Hayes et al., unpubl.
Testosterone .17 Hyperolius viridiflavus M Richards (’82)
Testosterone 0.34 Hyperolius viridiflavus M Hayes (’97b)
Testosterone .35 Pyxicephalus adspersus MF Hayes and Licht, unpubl.
Testosterone .17–1.7 Pseudacris nigrita M Witschi et al. (’58)
Testosterone ? Rana agilis M Vannini (’46)
Testosterone .02–.14 Rana dalmatina MI Padoa (’47)
Testosterone .28–1.1 Rana dalmatina M Padoa (’47)
Testosterone .17 Rana pipiens M Richards and Nace (’78)
Testosterone ? Rana sylvatica M Mintz et al. (’45)
Testosterone Inj Rana temporaria M Gallien (’37, ’43)
Testosterone .037–.074 Rhacophorus arboreus M Iwasawa et al. (’79)
ET .06–60 Discoglossis picta F Gallien (’50)
ET .01–1 Rana sylvatica MF Witschi and Allison (’50)
ET 1–250 Rana sylvatica M Witschi and Allison (’50)
MT .07–1.7 Bufo americanus MF Chang (’55)
MT 2.88 Bufo vulgaris MF Takahashi (’56)
MT .002–.13 Rana sylvatica M Witschi (’48)
MT .07 Rana spp. M Chang and Witschi (’55c)
MT .032–.064 Rhacophorus arboreus M Iwasawa et al. (’79)
MT .064–.160 Rhacophorus schlegelii MIF Amanuma (’63a,b)
(continued)
 SEX DETERMINATION IN AMPHIBIANS 385

TABLE 1. Steroidal effects on primary sex differentiation in anurans (continued)

Steroid1 Conc2 Species3 Effect4 References

TP .006–5.8 Discoglossis picta MF Gallien (’50)

TP ? Pelodytes punctatus MF Gallien and Collenot (’58)
TP .6 Bufo vulgaris MF Takahashi (’56)
TP Inj (500) Rana catesbeiana MF Pucket (’39, ’40), Witschi (’48)
TP Inj (500) Rana clamitans M Mintz et al. (’45), Foote and Witschi (’39)
TP .001–.003 Rana sylvatica IF Witschi (’48)
TP .006–14 Rana sylvatica M Witschi (’48)
TP .03–2.9 Rana sylvatica MIF Mintz (’48)
TP 6–2,900 Rana sylvatica M Mintz (’48)
TP .03–29.2 Xenopus laevis MF Chang and Witschi (’55b)
TP 1.7 Xenopus laevis MI Gallien (’56)
TP 1.7 Xenopus laevis MIF Gallien (’62)
Estrogens
Estrone 3.7 Bufo vulgaris MF Takahashi (’56)
Estrone UI Rana esculenta MIF Padoa (’38)
Estrone UI Rana esculenta M Padoa (’38)
Estrone UI Rana esculenta M Padoa (’36)
Estradiol 3.7 Bufo vulgaris MF Takahashi (’56)
Estradiol .07–.14 Buergeria buergeria MIF Ohta (’87)
Estradiol .46–1.85 Buergeria buergeria M Ohta (’87)
Estradiol ? Alytes obstetricians F Witschi and Allison (’50)
Estradiol .37–3.7 Bufo americanus IF Chang (’55)
Estradiol 1.0 Bufo boreas MF Hayes et al., unpubl.
Estradiol 0.037–0.37 Hyperolius argus IF Hayes et al., unpubl.
Estradiol 0.37 Hyperolius viridiflavus IF Hayes et al., unpubl.
Estradiol .18 Hyperolius viridiflavus MIF Richards (’82)
Estradiol 0.37 Pyxicephalus adspersus MF Hayes and Licht, unpubl.
Estradiol ? Rana agilis F Vannini (’46)
Estradiol Inj Rana clamitans MF Foote and Witschi (’39), Mintz et al. (’45)
Estradiol Inj Rana catesbeiana MF Pucket (’39, ’40)
Estradiol UI Rana esculenta M Padoa (’38)
Estradiol .1–.21 Rana esculenta F Padoa (’42)
Estradiol .42 Rana esculenta MIF Padoa (’42)
Estradiol .92–3.7 Rana esculenta M Padoa (’42)
Estradiol ? Rana esculenta M Padoa (’36)
Estradiol 3.68 Rana japonica MF Yoshikura (’62)
Estradiol .07–.18 Rana pipiens F Richards and Nace (’78)
Estradiol 3.68 Rana pipiens M Richards and Nace (’78)
Estradiol .03–.92 Rana sylvatica F Witschi (’48)
Estradiol .7 Rana sylvatica M Dale (’62)
Estradiol 3.7–4.6 Rana sylvatica IF Witschi (’48)
Estradiol ? Rana temporaria M Gallien (’40, ’41)
Estradiol .07–.37 Rana spp. F Chang and Witschi (’55c)
Estradiol 1.85 Rana spp. MIF Chang and Witschi (’55c)
Estradiol 3.7–18.5 Rana spp. M Chang and Witschi (’55c)
Estradiol .04 Xenopus laevis MF Chang and Witschi (’55a,b)
Estradiol .18–.36 Xenopus laevis F Chang and Witschi (’55a,b)
Estradiol .185–2.2 Xenopus laevis F Gallien (’53)
Estradiol .185–2.2 Xenopus laevis F Gallien (’57)
EB ? Xenopus laevis IF Gallien (’54)
EB .13–5.2 Discoglossis picta IF Gallien (’50)
EB Inj (150) Pelobates cultripes F Gallien and Collenot (’58)
EB Inj (150) Pelobytes punctatus IF Gallien and Collenot (’58)
EB .21 Rana esculenta M Padoa (’42)
EB .11–.66 Rana sylvatica MF Witschi (’48)
EB 1.6 Rana sylvatica F Witschi (’48)
EB .13 Xenopus laevis F Gallien (’56)
EB .001–1.6 Xenopus laevis F Gallien (’55, ’62)
EBB Inj Rana clamitans MF Mintz et al. (’45)
EDP Inj Rana clamitans MF Mintz et al. (’45)
(continued)
386 T.B. HAYES

TABLE 1. Steroidal effects on primary sex differentiation in anurans (concluded)

Steroid1 Conc2 Species3 Effect4 References

EE .03 Rana sylvatica MIF Witschi and Allison (’50)

EE .3–4.2 Rana sylvatica F Witschi and Allison (’50)
EQ .02–.04 Rana sylvatica MIF Witschi and Allison (’50)
EQ .37–4.7 Rana sylvatica F Witschi and Allison (’50)
1
Steroids are listed by class. Within classes, steorids are listed by alphabetical order starting with natural followed by synthetic compounds.
DHEA = dehydroepiandrosterone; DOC = deoxycorticosterone; DOCA = deoxycorticosterone acetate; DHT = dihydrosterosterone; EB = estra-
diol benzoate; EBB = estradiol benzyl buterate; EDP = estradiol dipropionate; EQ = equilenin; ET = ethynyl estradiol; MT = methyl testoster-
one; TP = testosterone propionate.
2
Concentrations reflect ranges of concentrations of steroids added to water except where indicated. Inj indicates that the indicated concentra-
tion of hormone was injected into larvae. A ‘‘?’’ indicates that the information was not provided. UI indicates that the concentration was
reported in international units and cannot be converted into µM quantitites.
3
For each steroid, data are grouped by species tested and in alphabetical order. In one case multiple species were used and not distinguished
in the report and are listed as Rana spp. In one other case, the species name was incorrectly reported and is reported as Rana?
4
F = female; I = intersex; M = male. MF indicates that normal males and females occur at normal frequencies following the treatments (no
effect); F indicates that 100% females resulted from the treatment (sex reversal of genetic males); ‘‘F or M’’ indicates that 100% males or
100% females can result (in this case, depending on the dose of estrogen); I indicates that interesexes result from the treatment (hermaphro-
ditic or ambiguous gonads in all individuals); IF indicates that the males show partial sex reversal (hermaphroditic gonads) and females are
unaffected; M indicates that 100% males result (sex reversal of genetic females); MI indicates that normal males result, but that females
show partial (hermaphroditic gonads) sex reversal; MIF indicates that normal males, normal females, and intersexes are all produced.

doses, but treatment at higher doses results in 100%
males (see Table 1). Furthermore, examining these
data in a phylogenetic context, taking into account
the genetic sex-determining system, reveals no pat-
terns or trends (see Figure 2) in anuran amphib-
ians: There are no similar patterns of effects within
clades or within clades of anurans with a similar
genetic sex-determining system.
An extensive data set for the effects of exog-
enous hormones is not available for salamanders,
but estrogen treatments produced all female
offspring in Ambystoma punctatum (Burns, ’38,
’39), A. tigrinum (Ackart and Leavy, ’39), A. macu-
latum (Foote, ’40, ’41), Hynobius retardatus
(Hanaoki, ’41a), H. nebulosus (Asayama and
Miyamori, ’57), Triturus helviticus (Gallien and
Collenot, ’60), and Pleurodeles waltl (Gallien, ’50,
’54). Androgens reportedly had no effect or inhib-
ited gonadogenesis in some salamander species
(Burns, ’39; Hanaoka, ’41b; Asayama and Mat-
suzaki, ’58; Gallien, ’65), but produced 100% fe-
males in other species (Gallien, ’54; Asayama and
Amanuma, ’57; Amanuma, ’61, ’63). Thus species
from only three salamander families have been
examined in this regard, and no data are avail-
able for caecilians.
Mechanisms of steroid action
Mechanism of steroid action on the gonads are
not known. No steroid receptors have been iden-
tified in amphibian gonads. Furthermore, gonads
respond to steroids at times when other tissues
lack sensitivity to steroids. For example, estro-
gens induce ovarian development in Xenopus
laevis larvae during stages when this same hor-
mone is unable to induce vitellogenesis in the liver,
oviductal growth, or cloacal labial growth (Hayes,
’97b). In the case of the liver, estradiol is not ca-
pable of inducing vitellogenin because the liver
lacks estrogen receptors until metamorphosis
(Rabelo et al., ’94), and this mechanism likely ex-
plains why other tissues do not respond. Similarly,
testosterone induced testicular differentiation in
Hyperolius argus and Hyperolius viridiflavus dur-
ing stages in which this hormone is unable to in-
duce secondary sex characters such as gular pouch
development (Hayes, ’97a). So, either sex steroids
do not act through conventional steroid receptors
in their actions on the gonads, or gonadal expres-
sion of steroid receptors is regulated differently
when compared with other tissues.
Tadpoles metabolize steroids administered into
the water or injected into the body cavity (Dale,
’62; Rao et al., ’68, ’69; Hayes and Licht, ’93, ’95),
and this metabolism may play a role in the var-
ied effects of steroids on sex differentiation. Spe-
cies that are unaffected by steroids may deactivate
these hormones faster by metabolizing them to
inactive compounds, or alternatively, species that
are affected may activate the hormones by me-
tabolizing them to more potent compounds. Data
from one study (Hayes et al., submitted) suggested
that the metabolites of estradiol produced by Xe-
nopus laevis larvae are active compounds capable
of inducing ovarian differentiation. Rao et al. (’68)
also claimed that female tadpoles metabolized ste-
roids to active compounds, whereas male tadpoles
converted them to inactive compounds.
 SEX DETERMINATION IN AMPHIBIANS
Incomplete reporting and inconsistent designs
between published studies create a problem when
interpreting data on the effects of steroids in dif-
ferent species. For example, with a few exceptions,
none of the studies used to estimate the effects of
steroids in amphibians (see Figure 2) reported the
total amount of hormone added to the water, the
number of tadpoles per treatment tank, the de-
velopmental stages during which the tadpoles
were treated, the total amount of time over which
animals were treated, or the temperature main-
tained during treatments (Hayes and Licht, ’95).
Variation in effects between species may be an
artifact of different experimental designs and
treatment regimes: The total amount of hormone
and number of tadpoles are important in deter-
mining dose (in addition to reporting hormone con-
centrations). A given amount of hormone likely
decreases in effectiveness as more tadpoles are
added to a tank (50 tadpoles sequester and use
more hormone than 10 tadpoles). Also, the devel-
opmental stage during the treatment is important:
Estrogens induce ovarian differentiation in Xeno-
pus laevis only during specific stages of develop-
ment (Chang and Witschi, ’55a; Gallien and
Foulgoc, ’60; Witschi, ’71; Iwasawa et al., ’79;
Villalpando and Merchant-Larios, ’90; Hayes et
al., ’97b). Thus some studies may fail to show ef-
fects of steroids on gonadal development because
the treatments were administered during incor-
rect stages. In addition, these critical periods
likely vary between species. Likewise, the total
amount of time that animals are exposed and the
temperature during exposure likely affect the po-
tency of steroids. In addition, all of these factors
(concentration, total mass of steroid, developmen-
tal stage, temperature, and duration of exposure)
alter steroid metabolic rates (which also vary be-
tween species; Hayes and Licht, ’93, ’95). Thus, if
steroid metabolism by tadpoles represents acti-
vation and/or deactivation of the steroids (and ste-
roid metabolism likely affects potency), then the
interaction between tadpoles and treatment con-
ditions and effects on steroid metabolism provide
another mechanism by which artifacts may be pro-
duced when comparing effects between species from
reports in the literature. These problems may be
solved by careful studies of multiple species under
similar controlled conditions in parallel studies.
Endogenous steroids
It is not likely that steroid hormones play a role
in sex determination, although they may be in-
volved in normal gonadal differentiation in am-
387
phibians. In order for males and females to pro-
duce different sex steroids, they have to express
different steroidogenic enzymes. If males and fe-
males express different functional enzymes, then
they are already differentiated (even if this dif-
ferentiation cannot be detected ultrastructurally).
In fact, the steroids may be responsible for induc-
ing this detectable (structural and ultrastructural)
differentiation, but the hormones themselves must
be the product of prior differentiation.
In addition to the lack of understanding of the
mechanisms of exogenous steroid action on go-
nadal differentiation, little is known about the role
of endogenous steroids in normal gonadal differ-
entiation. Amphibian larvae possess steroidogenic
enzymes in their gonads, and steroid metabolism
and production have been measured, but only af-
ter gonadal differentiation had occurred (Hsü et
al., ’77, ’78, ’79, ’85) or when the sexes were not
identifiable (Kang et al., ’95). Also, several stud-
ies have shown that amphibian larvae metabo-
lize exogenous steroids (Rana sylvatica and Rana
pipiens: Dale, ’62; Pleurodeles waltl: Ozon, ’63;
Xenopus laevis: Rao et al., ’68, ’69; nine species,
representing eight families: Hayes and Licht, ’93,
’95), and one study even suggested that male and
female larvae metabolize estrogens differently
(Rao et al., ’68). Although steroid metabolism was
not localized to the gonads, these data showed that
larvae are capable of metabolizing and possibly
producing sex steroids. In salamanders, Chardard
et al. (’95) showed that Pleurodeles waltl had ac-
tive aromatase in its gonads that increased in
females during larval development. Rearing fe-
male larvae at high temperatures (which sex-
reverses female P. waltl) caused a decrease in
aromatase activity, suggesting that estrogens may
be involved in sex differentiation of females in this
species (Chardard et al., ’95).
Despite the paucity of studies examining endog-
enous hormone production, a few studies have ad-
dressed the role of endogenous hormones in
amphibian gonadal differentiation by treating am-
phibian larvae with reagents that block endog-
enous steroid production. Zaccanti et al. (’94)
treated larval Bufo bufo and Rana dalmatina with
androsten-3 one 17 β-carbossilic acid, which in-
hibits 5 α reductase (prevents the production of
5α reduced androgens). This study showed accel-
erated ovarian development with larger and more
numerous oocytes in the Bidder’s organ of Bufo
bufo and in the ovary of Rana dalmatina. Al-
though sex reversal did not occur, these data sug-
gested that endogenous androgens might be
388 T.B. HAYES
involved in gonadal differentiation. Hsü et al. (’79,
’81a) also showed that blocking estrogen synthe-
sis resulted in transformation of ovaries into tes-
tes and that blocking both androgen and estrogen
synthesis with 17 β-ureide (Hsü et al., ’81b) re-
sulted in sterile gonads. Similarly Yu et al. (’93)
showed that endogenous steroids may be involved
in sex differentiation in Rana catesbeiana. Im-
planting tadpoles with capsules containing 4-OH-
androstenedione (an aromatase inhibitor) resulted
in testicular development. The authors suggested
that the sex reversal was due to a buildup of an-
drogen that acted on the gonad to induce sex re-
versal. These studies all suggest that aromatase
(and low androgens) may be required for ovarian
differentiation. An earlier study suggested that
endogenous steroids may not be involved in nor-
mal sex differentiation, however. Treatments with
an anti-estrogen (tamoxifen) or an anti-androgen
(cyproterone acetate) were unable to block the ef-
fect of exogenous estrogen or testosterone treat-
ment, respectively. Also, neither anti-steroid
affected normal sex differentiation (Rastogi and
Chieffi, ’75).
One difficulty in understanding the potential
role of steroids in normal gonadal differentiation
is that steroid production must be analyzed prior
to sex differentiation to discern whether sex ste-
roids organize the gonad or whether differences
in sex steroid production are the result of organi-
zation (sex differentiation) that has occurred al-
ready. This task is difficult, because identification
of sexes in most species is based on gonadal mor-
phology, since only a few species have easily dis-
tinguishable sex chromosomes. Although steroid
levels were measured in eggs of two species (Rana
sylvatica and Xenopus laevis; Hayes, unpublished),
the effects of steroids during these early stages
are not known to influence sex differentiation.
Also, hormones found in eggs are of maternal ori-
gin, because the steroidogenic tissue has not de-
veloped in early embryos at these stages.
Other hormones
Thyroid hormones
Thyroid hormones are best known for their role
in amphibian metamorphosis, but have been im-
plicated in gonadal development in amphibians.
Their role in gonadal differentiation is debated,
and some workers claim that gonadal differentia-
tion is one of the few aspects of development that
is not under the control of thyroid hormones
(Hayes, ’97b; for review).
The order and coordination of developmental
events are relatively consistent in amphibians:
Forelimbs emerge before the tail reabsorbs (in
frogs), the lungs mature before the gills reabsorb
(in frogs and metamorphosing salamanders), etc.
The timing of gonadal development varies signifi-
cantly, however. For example, Xenopus laevis have
differentiated gonads (based on histological analy-
sis) by NF (Nieuwkoop and Faber, ’56) Stage 52,
whereas Hyperolius viridiflavus are differentiated
as early as NF Stage 48 (even by morphological
examination). Other species, such as Bufo boreas,
are not sexually differentiated until after meta-
morphosis (Gosner [’60] Stage 48 = NF Stage 66),
and gonads are not distinguishable by histologi-
cal examination even 1 week after metamorpho-
sis in Pyxicephalus adpsersus (Hayes and Licht,
’92). Even within a species, there may be varia-
tion in the timing of gonadal differentiation
(Iwasawa, ’69; Chang and Hsu, ’87), especially in
the sexually undifferentiated races of Rana
temporaria (Witschi, ’30) and Rana catesbeiana
(Pucket, ’40). In addition, Ohta (’87) reported that
Buergeria buergeri from Hiroshima had differ-
entiated gonads by Gosner Stage 40, but our ex-
perience raising the same species from Yamanashi
under similar conditions showed that gonads were
not differentiated even at metamorphosis (Hayes
and Wong, unpublished). One interpretation is
that this variation is an indication that thyroid
hormones do not affect gonadal differentiation.
On the other hand, thyroid hormones may regu-
late gonadal differentiation, but different species
may show different sensitivities: some respond to
very low doses of thyroid hormones and differenti-
ate early in development, whereas others require
high titers of thyroid hormone and differentiate
at metamorphic climax, etc. Also, thyroid hormones
may regulate gonadal differentiation in some spe-
cies, but not others. This possibility is certainly
realized in other tissues, such as development of
the hind limbs, which are thyroid hormone–depen-
dent in Xenopus laevis but not in Bufo boreas
(Hayes, ’95, ’97a).
Several studies suggested that thyroid hor-
mones regulate gonadal development. In recent
studies, we (Hayes, ’97b) showed that treatment
with thiourea (a goitrogen) resulted in 100% fe-
males in Xenopus laevis (a species in which
estradiol induced ovarian differentiation but tes-
tosterone had no effect) and 100% males in
Hyperolius viridiflavus (an unrelated species in
which estradiol had no effect but testosterone in-
duced testicular development). In fact, the ste-
 SEX DETERMINATION IN AMPHIBIANS
roids effective at inducing sex reversal also in-
hibited larval development and the thyroid axis,
and it is possible that sex steroids alter gonad
differentiation simply by inhibiting the thyroid
axis. The alteration of gonadal differentiation by
blocking thyroid hormones in two unrelated spe-
cies suggests a generalized role for thyroid hor-
mones in gonadal development across anurans.
Also, other studies have suggested that thyroid
hormones inhibit ovarian development (Hoskins
and Hoskins, ’19; Iwasawa, ’55a,b, ’61; Yoshikura,
’60, ’61, ’62b,c; Yü et al., ’72; Rengel et al., ’93).
On the contrary, several studies suggested that
thyroid hormones are not involved in gonadal dif-
ferentiation (Allen, ’18; Yoshikura, ’60; Hsü et al.,
’74; Robertson and Kelley, ’96). Thus a definite
role for thyroid hormones and their mechanism
of action remains to be shown.
Pituitary hormones
Several studies addressed the role of pituitary
hormones and the pituitary gonadotrop(h)ins in sex
differentiation. Gonads developed normally in both
males and females when larvae were hypophysec-
tomized early in development prior to gonadal dif-
ferentiation in Rana japonica (Yoshikura, ’59a,b;
Iwasawa, ’61). The gonads from hypophysectomized
animals were smaller than controls, however, sug-
gesting that pituitary hormones stimulated growth
of the gonads, but that differentiation was inde-
pendent of these hormones. Gonads grown in cul-
ture differentiated into testes and ovaries, but
growth was impaired (especially testes) in Rana
catesbeiana (Foote and Foote, ’58). Schreiber and
Rugh (’45), Asayama (’55), and Iwasawa (’60)
showed that pituitary injections accelerated growth
of testes and ovaries in Rana pipiens, but did not
affect differentiation or maturation. In addition, the
pituitary did not affect sex steroid effects on gonad
differentiation (Mintz and Gallien, ’54; Yoshikura,
’60a) and gonads respond to steroids in vitro (Foote
and Foote, ’59). These studies all agreed that the
pituitary is not required for early gonadal differ-
entiation, but may be involved in gonadal growth
after differentiation occurs.
Atypical sex gonadal differentiation
Most of the data discussed so far, were based
on studies of gonochoristic species. Enough varia-
tion in sex developmental patterns exists in am-
phibians to warrant discussion of these exceptions,
however. These exceptions include reported un-
differentiated races, hermaphrodites, and parthe-
nogenetic species.
389
Undifferentiated races
So called undifferentiated races have been re-
ported in a number of ranid species, including
Rana temporaria (Pflüger, 1882; Ponse, ’30;
Witschi, ’30), R. catesbeiana (Pucket, ’40), and R.
esculenta, R. ornativentris (Iwasawa, ’69) and in
a single bufonid (Bufo bufo: Ponse, ’49; Foote, ’64)
and in Rhacophorus arboreus (Iwasawa, ’69a,b).
Similar reports were made in salamanders (Hyno-
bius retardatus: Uchida, ’37b; and Ambystoma
maculatum: Witschi, ’33b). In R. catesbeiana
(Pucket, ’40), the animals were not truly undif-
ferentiated, but ovarian differentiation was ob-
served in larval stages. The testes remained a
“prototestis” (as described by Swingle, ’26), how-
ever, until metamorphosis, when they developed
into recognizable testes. Development in undiffer-
entiated races of R. temporaria is quite distinct,
however. In some populations, all of the animals
metamorphose with ovaries. In the following 6 to
9 months after metamorphosis, some of the indi-
viduals reportedly transform into males going
through an intersexual stage (Witschi, ’21, ’30).
Similar reports were made for Rhacophorus
schlegelii (Amanuma, ’63a,b).
Sequential hermaphroditism and
spontaneous sex reversal
Although Witschi (’29b, page 236) stated, “Some
of the most common anurans are rudimentary her-
maphrodites which eventually may ripen fertile
eggs and sperms either in consecutive years, as
in the toad Bufo vulgaris, or simultaneously, as
in the frog Rana temporaria” and even claimed
that artificial self-fertilization was possible in
some of these rudimentary hermaphrodites, he
does not cite any reference where data and obser-
vations were reported. Crew (’21) also reported a
number of incidences of abnormal sex reversals
and intersexual animals, but not much informa-
tion was provided for review, and the report is
simply a categorical listing of cases.
At least two published studies since Witschi
(’29b) and Crew (’21) addressed the possibility of
sequential hermaphroditism (natural sex rever-
sal without any hormone treatments, etc.). Grafe
and Linsenmair (’89) suggested that seven out of
24 captive female Hyperolius viridiflavus om-
matostictus (which reportedly laid at least one
clutch of eggs) began calling, developed gular
pouches, and apparently fertilized eggs. No histo-
logical evidence or further examinations were pub-
lished in this case, however. In another study
390 T.B. HAYES
(Collenot et al., ’94), the salamander (Pleurodeles
waltl) reportedly underwent spontaneous sex re-
versal in captivity. The authors reported that
some animals (all apparently of the ZZ type) de-
veloped into females, and intersexes. This study
used a supposed sex-linked peptidase in which
one type is supposedly linked to the W chromo-
some (Ferrier et al., ’80; Dournon et al., ’88) to
identify genetic types of individuals. Animals
lacking the proposed W-linked peptidase were as-
sumed to be ZZ, and any animals possessing an
ovary but lacking the W-linked form of the en-
zyme were assumed to be sex-reversed. An al-
ternative explanation is that the peptidase may
not be as tightly linked as proposed and thus
may not be a reliable sex marker. Their results
may be explained by exchange between the Z and
W sex chromosomes. Evidence for such exchange
is found in the X and Y chromosome in mam-
mals, for example (Kent et al., ’88).
Rudimentary hermaphroditism in Bufonids
The natural condition in Bufonids is the most
compelling case for hermaphroditism in amphib-
ians (although not functional). In all bufonids,
both males and females possess Bidder’s organs.
Bidder’s organs are a pair of rudimentary ovaries
that develops at the anterior portion of the gonad
in both males and females. Witschi (’33a) provided
an excellent review of the early descriptions of the
Bidder’s organ: The Bidder’s organ was first de-
scribed as fat (Roesel, 1758) and then as the pri-
mordium of the testes (Rathke, 1825). Ironically,
Bidder (1846), for whom the organ was named,
argued that the Bidder’s organ was an accessory
structure involved in male gametogenesis. Later,
Von Wittich (1853) suggested that the Bidder’s or-
gan was a rudimentary ovary developing in both
males and females, and further histological data
from several workers supported Von Wittich’s
claim (see references in Witschi, ’33a). Even more
convincing, Harms (’21, ’23) and Guyénot and
Ponse (’23a,b,c, ’27) showed that the Bidder’s or-
gan developed into a fully functional ovary that
can produce fertilizable eggs after the testes were
surgically removed.
Since its discovery, a number of papers have de-
scribed the development of the Bidder’s organ
(Witschi, ’33a; Horié, ’38; Vannini, ’56; Zaccanti et
al., ’71; Tanimura and Iwasawa, ’86), and more re-
cent studies have examined its contribution to ste-
roid hormones (Colombo and Colombo-Belvedere,
’80; Ghosh et al., ’84; Pancak-Roessler and Norris,
’91). The Bidder’s organs always develop at the
cephalic end of the gonad and may differentiate
at the same time as the gonads. Meiotic activity
in the Bidder’s organ is greater in females than in
males (Tanimura and Iwasawa, ’86), and these
data, combined with the observation that removal
of the testes results in the transformation of the
Bidder’s organ into fully functional ovaries (ref-
erences above), suggested that the testicular se-
cretions may antagonize the development and
function of this rudimentary ovary in males.
Despite the presence of the Bidder’s organ, how-
ever, bufonids are not truly hermaphroditic (nei-
ther simultaneous or sequential): Under normal
conditions, the Bidder’s organs do not function as
ovaries in males. Although Witschi suggested that
sequential hermaphroditism occurred in Bufo vul-
garis, the primary description of this observation
and details of studies are not cited and the litera-
ture is not available. Thus the function (if any)
and the mechanisms underlying development of
this structure remain unclear. The realization that
the Bidder’s organ is a rudimentary ovary stimu-
lated much discussion about the ancestral state
of amphibians, however. Several authors proposed
that the rudimentary ovary in bufonids is evidence
of a hermaphroditic ancestry of the bufonids, if
not all amphibians (Witschi, ’33a). Interestingly,
the cephalic portion of the gonad in Xenopus laevis
can be induced to develop an ovarian-like struc-
ture (with male characteristics developing cau-
dally) if male larvae are treated with estrogens
for a short time near the critical period (Chang
and Witschi, ’55a; Hayes et al., unpublished). The
fate of such intersex animals is unknown since
none have been followed to metamorphosis and
adulthood. These data suggest that the cephalic
portion of the gonad is more sensitive to sex ste-
roids and transforms before the caudal end. Alter-
natively, gonads normally differentiate posterior to
anterior, so that short-term hormone treatments
transform the cephalic end, which remains bi-
potential, but are unable to transform the caudal
end, which may differentiate earlier. Whatever the
mechanism, such studies may provide interesting
models for examining mechanisms underlying
Bidder’s organ development in bufonids.
Parthenogenesis
Natural parthenogenesis has been reported in
Ambystomatid salamanders and is well studied.
The Jeffersonian salamander complex consists of
two diploid species (Ambystoma jeffersonium and
Ambystoma laterale), which reproduce normally.
There are at least two triploid species (Ambystoma
 SEX DETERMINATION IN AMPHIBIANS
platineum and Ambystoma tremblayi) whose
ranges overlap with the ranges of the diploid spe-
cies (Clanton, ’34; Minton, ’54; Uzzell, ’64; Mac-
Gregor and Uzzell, ’64). The two triploid species
primarily consist of females. These triploids de-
pend on diploid males because sperm is required
to stimulate development of the eggs of the
parthenogens, although genetic material from the
males is not incorporated (Licht, ’89).
Uzzell (’64) showed that triploid females con-
sistently produced only female (and triploid) off-
spring, and suggested that the triploid species
resulted from hybridization between the two dip-
loid species (Uzzell, ’63). Later electrophoretic
studies supported this hypothesis (Uzzell and
Goldblatt, ’67; Downs, ’78; Bogart et al., ’85;
Lowcock and Bogart, ’89; Kraus, ’89; Lowcock, ’89)
and showed that some island species may have
arisen from hybridizations between Ambystoma
laterale and Ambystoma texanum as well. Uzzell
and Goldblatt (’67) went further to hypothesize
that a tetraploid hybrid, producing diploid ga-
metes, may have served as an intermediate that,
when back-crossed to one of the diploid parental
species (producing haploid gametes), produced
triploid offspring.
Triploid salamanders have been produced in the
laboratory also. Ambystoma tigrinum and Am-
bystoma mexicanum eggs can be induced to de-
velop as triploids by cold treatment and by
pricking with a needle. These animals undergo
normal ovarian development during early stages,
but oocytes degenerate later in development. This
degeneration may be related to the ploidy of the
animals and not some physiological deficiency, since
grafted ovaries from normal diploid females develop
normally (Humphrey and Frankhauser, ’46).
Studies in natural triploid Notophthalamus
(Triturus) viridescens (Salamandridae) showed
that, unlike Ambystomatids, both male and female
triploids developed (Frankhauser, ’40). In addition,
not only did males develop normally, but females
suffered a loss of germ cells, and only rudimen-
tary ovaries developed. However, this study
(Frankhauser, ’40) was based on only four ani-
mals (one male and three females) that were cap-
tured from the wild and raised in the laboratory.
One possible explanation for the potential differ-
ences in the effects of triploidy between the Am-
bystoma spp. and Notophthalamus may be related
to the different sex-determining systems (Am-
bystoma are ZZ/ZW whereas Notophthalamus spp.
are XX/XY). Studies on these animals may con-
tribute to our understanding of the relationship
391
between mechanisms of sex determination and sex
differentiation.
The existence of parthenogenesis has not been
reported in frogs, although natural polyploids ex-
ist in frogs (Wasserman, ’70; Bogart and Tandy,
’76; Bogart, ’80). Frogs can be induced to develop
polyploidy (Parmenter, ’33; Rugh and Marsland,
’43; Dasgupta, ’62), and a single study showed that
both males and females resulted from triploidy
induced in Rana pipiens by cold shock, but by
metamorphosis all females were sex-reversed and
developed testes (Humphrey et al., ’50).
CONCLUSIONS
All amphibians probably display genetic sex de-
termination, but external factors can influence sex
(gonadal) differentiation. The external factors in-
clude temperature, physical manipulations, and ex-
ogenous hormones. Only endogenous hormones may
naturally affect gonadal differentiation, however.
A lack of work in the field over the last two
decades has left many questions concerning sex
differentiation unanswered. Mechanisms regulat-
ing germ cell differentiation are unclear. The con-
tributions of coelomic epithelium and kidney and
interrenal primordia to the cortex and medulla of
the gonads remain undetermined. The role of en-
dogenous steroids and thyroid hormones and their
mechanisms of action on the development of the
gonad are also unclear. Other future studies
should address the relationship between mecha-
nisms of GSD (female versus male heterogamety)
and mechanisms of sex differentiation and how
GSD may be related to the susceptibility to sex
reversal by some steroids but not by others
(known to be active in other species).
ACKNOWLEDGMENTS
I thank V. Lance for the invitation to partici-
pate in this symposium. I also thank R. Liu, S.
Wong, T. Gill, J. Yeh, and K. Menendez who have
participated in various studies on this topic and
an anonymous reviewer whose comments im-
proved the manuscript. I also thank Katherine
Kim for her support.
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