Sex Determination and Primary Sex Differentiation in Amphibians: Genetic and Developmental Mechanisms TYRONE B

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 amphibians 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 steroids in amphibian gonadal differentiation is unknown. Sex steroid receptors have not been examined 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 gonadotrop(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 differentiation, 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. 281:373399, 1998. © 1998 Wiley-Liss, Inc.

Amphibians are excellent models to study sex nisms of sex determination (addressing amphib- differentiation, for a number of reasons. Genetic ians and reptiles), and Schmid (’83) and Hillis and sex-determining systems and sex chromosomes Green (’90) examined genetic mechanisms of sex have evolved a number of times in this class, and determination with special reference to the evo- gonadal differentiation (although under genetic lution of sex chromosomes in amphibians. Gonadal control) is responsive to experimental manipula- differentiation in amphibians was reviewed by tions. In addition, as laboratory animals, amphib- Gallien (’65), and at least four reviews (Burns, ’49, ians are ideal; because females produce up to ’61; Adkins-Regan, ’81, ’87) addressed the effects 10,000 eggs per clutch in some species, large of hormones on sex differentiation in vertebrates sample sizes can be obtained. Also, large num- in general, with extensive discussions of amphib- bers of individuals can be treated easily in experimental manipulations because larvae are 1Although often used interchangeably, there is a fundamental dif- typically aquatic and readily absorb hormones and ference between sex determination and sex differentiation. Here, “sex other chemicals added to rearing water. determination” will refer to mechanisms that direct sex (gonadal) differentiation, and “sex differentiation” will refer specifically to the Despite their accessibility as study animals, development of testes or ovaries from the undifferentiated or relatively few recent studies have addressed sex bipotential gonad. In other words, sex determination refers to the 1 switch (or the decision), whereas sex differentiation refers to the ac- determination and differentiation in amphibians. tual developmental process. Also, there are no recent reviews that address both Grant sponsor: NSF; Grant numbers: IBN-9513362 and IBN- 9508996. sex determination and sex differentiation in am- *Correspondence to: Tyrone B. Hayes, Ph.D., Assistant Professor, phibians, although a few reviews addressed each Department of Integrative Biology, 3060 Valley Life Sciences Build- ing, University of California, Berkeley, CA 94720-3140. E-mail: topic separately: Dodd (’60) and Dournon et al. [email protected] (’90) reviewed genetic and environmental mecha- Received 17 February 1998; Accepted 18 February 1998 © 1998 WILEY-LISS, INC. 374 T.B. HAYES ians. A single review addressed both sex determi- Chardard et al., ’95) and Hynobius (Uchida, nation and differentiation in amphibians (Witschi, ’37a,b). These studies all showed that the tem- ’50), but this work was published prior to many perature of the rearing water can alter the sex primary studies on the topics. A second review ratios of larvae. In all of these studies, however, (Adkins-Regan, ’87) addressed both topics in effects were obtained by exposure to temperatures nonmammalian vetebrates in general with refer- that are not normally experienced by the species ence to amphibians. Thus, with the exception of under study. In fact, with one exception, these ef- the recent reviews addressing sex chromosome evo- fects were produced only at high temperatures (27 lution (Schmid, ’83; Hillis and Green, ’90), reviews to 36°C). In all of the studies on frogs, 100% males on sex determination and sex differentiation (and, were produced at high temperatures, whereas ei- in particular, reviews addressing both sex deter- ther 100% females (Pleurodeles poireti; Dournon mination and differentiation) are lacking. The goal et al., ’84) or 100% males (Hynobius retardatus: of the current review is to synthesize work on sex Uchida, ’37b; Pleurodeles waltl: Dournon and determination and sex differentiation in amphib- Houillon, ’84) were produced by high-temperature ians. This simultaneous review of sex determina- treatments in salamanders. A single study (Uchida, tion and differentiation will attempt to place the ’37b) showed that low temperatures (10°C) can pro- available information into an evolutionary frame- duce 100% females also. work and explore interactions between mechanisms When reared at temperatures within the ranges of sex determination and sex differentiation. experienced naturally by the species under study, a 50:50 sex ratio was obtained in all of the stud- SEX DETERMINATION ies cited above. For example, in Witschi’s (’29a) Rana sylvatica, Genetic sex determination versus study on larvae were reared at environmental sex determination 32°C (which Witschi reported as a temperature near the lethal limit and a temperature that can There are at least two mechanisms of sex de- be tolerated only for short periods). Rana sylvatica termination recognized in vertebrates: genetic sex lives in northern North America where eggs are determination (GSD) and environmental sex de- typically laid in early spring when water tempera- termination (ESD). Genetic sex determination is tures are as low as 4°C (personal observation; and a mechanism by which genes directly determine Wright and Wright, ’49; Herreid and Kinney, ’66; whether the gonads differentiate into testes or Seale, ’82; Waldman, ’82). Larvae begin metamor- ovaries without external (environmental) influ- phosing within 8 weeks, and water temperatures ences. In ESD, the sex of individuals is determined do not typically exceed 20°C during this time. by environmental factors: For example, in tem- When larvae were reared at 20°C in Witschi’s perature-dependent sex determination (recognized (’29a) study, or reared at a range of temperatures in some fish, many turtles, some lizards, and all naturally experienced by this species (Hayes and crocodilians), sex is determined by the incubation Waldman, unpublished), the sex ratio was not sig- temperature of the eggs. Thus each individual has nificantly different from 50:50. Similarly, we the full capacity to develop into males (testicular (Hayes et al., unpublished) reared other species differentiation) or females (ovarian differentia- (Hyperolius viridiflavus, Bufo boreas, and Pyxi- tion). In fact, sex differentiation is likely controlled cephalus adspersus) over a range of temperatures by genetic mechanisms in animals with ESD and appropriate for each species and did not observe only the switch “sex determination” may be influ- any effects on sex ratios. enced by environmental factors. The natural role of temperature in the sex determination in reptiles and fish is much more con- Environmental sex determination vincing, because effects have been observed at Several studies have suggested that some am- temperatures within ranges experienced by the phibian species display ESD (for review, see Dodd, species in the wild (fish: Conover and Heins, ’87; ’60; Gallien, ’74; Dournon and Houillon, ’84; turtles: Bull et al., ’82), and effects have been Dournon et al., ’90). In frogs, one species of Bufo shown in natural nests in turtles (Bull, ’85; Rhodes (Piquet, ’30) and four species of Rana (Witschi, and Lang, ’95). Thus, given the lack of effects on ’14; ’29a; Piquet, ’30; Yoshikura, ’59b; Hsü et al., sex ratio at temperatures appropriate for the spe- ’71) were examined, in addition to three species cies in the studies on amphibians, it is not likely of salamanders from two genera Pleurodeles that temperature is important in normal sex de- (Dournon and Houillon, ’84; Dournon et al., ’84; termination in amphibians. The reported environ- SEX DETERMINATION IN AMPHIBIANS 375 mental influences are probably artifacts of the ab- techniques that produce different banding patterns normally high temperatures at which the animals on the chromosomes. Finally, some accumulated were reared. In addition, reptiles and fish display- repeats result in differences in replication time ing ESD typically lack sex chromosomes (morpho- between chromosomes. A technique using the in- logically distinguishable or otherwise), which is corporation of bromodesoxyuridine can detect dif- not the case in amphibians. Thus the available ferences in replication time to identify otherwise evidence for amphibians suggests that sex deter- homomorphic sex chromosomes (Schmid, ’83). mination is controlled exclusively by genetics un- More than 1500 amphibian species have been der natural conditions (see below). examined cytologically using the techniques described above. As few as two species of frogs Genetic sex determination (Pyxicephalus adspersus: Schmid, ’80; Schmid and In genetic sex determination, the direction of Bachman, ’81; Gastrotheca riobambae: Schmid, ’83) sex differentiation is controlled genetically, such and 11 salamanders (all Plethodontidae: Mancino, that one sex has a gene (or more likely a set of ’65; Kezer and MacGregor, ’71; León and Kezer, ’78; genes) that the other lacks. The sex-determining Sessions, ’84; Nardi et al., ’86) have sex chromo- genes likely control only the “decision,” and are somes that differ in size. Sex chromosomes are dis- not likely involved in sex differentiation, but tinguishable in at least another 30 species of rather initiate a sequence of events that results salamanders and frogs by examining banding pat- in gonadal differentiation. Normally, the different terns or differences in replication time. So, less than sexes develop because individuals are directed 4% of the amphibians examined cytologically pos- through different developmental pathways as a sess morphologically distinguishable sex chromo- result of the activation of the sex-determining somes (no data are available for caecelians). gene(s). This last point is important, because all Despite the absence of morphological distin- individuals may have the capacity to develop into guishable sex chromosomes in the majority of am- either sex. This capacity is evident from the abil- phibians, sex is probably under genetic control in ity to experimentally sex-reverse amphibians: De- all amphibians. The inability to distinguish sex pending on the species, genetic males can be chromosomes in amphibians may indicate only experimentally induced to develop ovaries and vice that the sex chromosomes have not evolved to versa with steroid hormone treatments. where they are morphologically distinguishable: The presence of sex chromosomes is the clearest Although there may be chromosomes possessing indication that a species possesses GSD. In these sex-specific genes, the differentiated regions are cases, the sex-determining genes are on a chro- small and not distinguishable cytologically. Hillis mosome that has evolved such that it is morpho- and Greene (’90) suggested that the lack of differ- logically distinguishable from its homologue upon entiation in amphibian sex chromosomes indicates cytological examination (the X and Y in mammals, that a much smaller region may be involved in for example). The evolution of sex chromosomes sex determination in amphibian sex chromosomes often involves the loss of genetic material: For ex- relative to other vertebrates with morphologically ample, in mammals, the Y chromosome is smaller distinct sex chromosomes. than the X as a result of limited chromatid ex- Furthermore, other experimental techniques that change between the X and Y chromosome and sub- do not require morphological identification of sex sequent loss of genes from the Y chromosome (see chromosomes have been used to identify the sex- J. Graves, this issue). determining systems in amphibians. In most cases, Sex chromosomes can be distinguished morpho- experimental manipulations were used to obtain logically by a number of techniques. Examining information regarding heterogamety. These stud- preparations of metaphase chromosomes from ies involved back-crossing experimentally “sex-re- males and females for chromosomes in which the versed” individuals to normal individuals, and homologues differ in size between the sexes is the using the resulting sex ratio to infer the genetic simplest technique. Some sex chromosomes differ constitution (male heterogamety: XX/XY versus fe- in the centromeric index as a result of pericentric male heterogamety: ZZ/ZW). For example, 100% inversions, which can be observed cytologically. In females were produced when larvae of the African addition, as chromosomes lose genetic material clawed frog (Xenopus laevis) were exposed to exog- (such as in the mammalian Y chromosome), they enous estrogens (Witschi and Allison, ’50). When accumulate repeats, resulting in heterochromatism. raised to sexual maturity, approximately 50% of Heterochromatism can be detected by a variety of these females produced 100% male offspring when 376 T.B. HAYES bred to normal males. Thus 50% of the estrogen- Nace, ’78). Approximately 50% of these males pro- treated animals were assumed to be sex-reversed duced 100% female offspring when crossed back to males (ZZ females) that produced only male (ZZ) normal females, showing that the species under offspring, when crossed back to normal (ZZ) males study were male heterogametic (XX/XY). Other (Chang and Witschi, ’55b; Hayes, ’98; Figure 1). studies used breeding experiments to track sex- These types of studies have been used to show fe- linked (but not sex-determining) genes in frogs male heterogamety in other species (Ambystoma (Wright and Richards, ’83; Sumida and Nishioka, spp.; Humphrey, ’45; ’48). In similar studies with ’94; Rudolf and Dournon, ’96). Rana spp., androgen treatment produced 100% Expression of the HY antigen (see Bennet et males (Kawamura and Yokota, ’59; Richards and 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 identifiable by independent methods were used to test the use of HY antigen as a tool for identifying the heterogametic sex. HY antigen was always expressed 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 antigen is present in both sexes (not sex-linked) and can be induced by hormone treatment. For example, in birds, only females (ZW) normally express 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 cytological techniques) and two more salamanders. One other method for assessing the sex-determining mechanism in amphibians has provided confusing results. Several workers showed that amphibian eggs can be induced to divide without Fig. 1. Experimental techniques used by Mikamo and fertilization by pricking the eggs with a glass needle Witschi (’64) to determine the genetic sex-determining sys- (Parmenter, ’25; Kawamura, ’39, ’49; Moriwaki, ’54, tem of the African clawed frog (Xenopus laevis). Normal males ’57, ’59, ’60a,b). Pricking induced chromosome and females were bred to produce larvae. The resulting larvae were treated with estrogen and produced 100% females. doubling in some eggs (diploidy) and presumably Fifty percent of the treated larvae were genetic females, and normal development in these “parthenogenetic” the remaining 50% were genetic males, “sex-reversed” by the embryos. All of these studies were conducted in hormone treatment so that they developed ovaries. When bred Rana spp., which are male heterogametic (XX/XY: back to normal males, the estrogen-treated females produced Schmid, ’83; Hillis and Green, ’90), so all parthe- a normal sex ratio of 50% male/50% female offspring. The sex-reversed males, however, produced only male offspring nogenetically produced animals should be females when bred back to normal males, indicating that males are (the eggs contain only an X chromosome). In sev- homogametic (ZZ) in this species. eral studies, however, males resulted from pricked SEX DETERMINATION IN AMPHIBIANS 377 (unfertilized) eggs. For example, Parmenter (’25) W chromosomes in birds and the X and Y chromo- reported 18 male and three female diploid animals somes in mammals are independently derived from produced by pricking in Rana pipiens; Kawamura different autosomes. (’49) reported 32 normal females, seven females The number of times that sex chromosomes with degenerating ovaries, three males with rudi- evolved and the number of transitions between mentary testes, and two normal males in Rana heterogametic types are not known, because the nigromaculata and R. japonica; and Moriwaki (’54) genetic sex-determining system is known for only reported seven males and nine females in R. nine of the 21 extant families of frogs and five of japonica. One interpretation is that the sexes were the ten urodele families. (There are no data avail- misidentified, but when a parthenogenetically pro- able for caecelians.) In most cases, the type of het- duced male was used to fertilized a normal female erogamety for each family is inferred from one or (Moriwaki, ’54), 100% females (72 animals) were two species and, usually, a single genus. Further produced, compared to 56% females (out of 86 ani- analysis may reveal even more transitions (such mals) in controls. These data suggest that the par- as the two types of heterogamety in the Ranidae: thenogenetically produced male was indeed a female heterogamety in two genera, and male het- homogametic (XX) individual. It is difficult to rec- erogamety in at least one). In addition, Nishioka oncile these data with other evidence for male het- et al. (’94) and Nishioka and Hanada (’94) sug- erogamety in this genus, and no recent studies have gested that the sex-determining system can followed up on these studies. vary even between populations of a single species: They reported that Rana rugosa showed Evolution of sex chromosomes both male (XX/XY) and female (ZZ/ZW) hetero- Using cytological data and data from published gamety, depending on the population under studies such as the ones described above, Hillis study. So sex-determining systems may be com- and Greene (’90) examined the evolution of sex- plex even within a species. determining mechanisms in amphibians. Unlike many other major vertebrate groups (all mammals Sex-determining genes and are male heterogametic, all birds are female het- genetic mechanisms erogametic, etc.), sex chromosomes have evolved To date, no sex-determining genes have been several times in amphibians. The ancestral state identified in amphibians. A single study (Chardard appears to be female heterogamety, and although et al., ’93) reported identification of an SrY Box male heterogamety has evolved several times (Fig- (SOX) gene in a salamander (Pleurodeles waltl). ure 2), a reversion to female heterogamety in a This gene is related to the SRY gene identified in male heterogametic lineage has occurred only once mammals, but is not sex-linked in P. waltl. Also, Chiropterotriton dimidatus ( in the bolitoglossin unlike in mammals, gene-dosage compensation plethodontids). does not occur in amphibians. Bar bodies or other Schmid (’83) pointed out that the ancestral Z mechanisms of deactivation of the homogametic chromosome in amphibians does not share a com- sex chromosome have not been observed. For ex- mon ancestor with the Z chromosome of snakes ample, in Buergeria buergeri the NOR is on the Z and birds. Similarly, it is not likely that the sex chromosome (Ohta, ’86; Schmid et al., ’93). As a chromosomes are related between amphibian result, males have two nucleoli per nucleus and families: The X and Y chromosomes in derived females only one. Males express twice as much Ranids are not evolved from the Z or W chromo- ribosomal RNA as females, further suggesting that Pyxicephalus adspersus somes of the more basal there is no dosage compensation. and Tomopterna spp. For example, the nucleolus organizing region (NOR) is on the Z chromosome SEX (GONADAL) DIFFERENTIATION Leiopelma in (Green, ’88), on the X chromosome Developmental mechanisms in H. femoralis (Anderson, ’91) and Gastrotheca (Schmid et al., ’86, ’88, ’90), on the Z chromosome Gonadal development in Buergeria buergeria (Ohta, ’86; Schmid et al., Once sex determination mechanisms are acti- ’93), but is not on either of the sex chromosomes in vated, primary sex differentiation ensues. The ma- Pyxicephalus adspersus (Schmid, ’80; Schmid and jority of amphibians are gonochoristic, and gonadal Bachman, ’81). Each change in heterogamety likely differentiation follows a similar pattern in frogs and involved the cooption of a different set of autosomes salamanders (little is known about caecilians). The to produce the sex chromosomes, just as the Z and gonads consist of the somatic gonad and germ cells. 378 T.B. HAYES

Fig. 2. Evolution of genetic sex-determining mechanisms that normal males and females occur at normal frequencies in amphibians. Sex-determining systems are shown for anu- following the treatments (no effect); F indicates that 100% ran (a) and urodele (b) families. Data for families are based females resulted from the treatment (sex reversal of genetic on studies from the literature reviewed by Hillis and Green males); “F or M” indicates that 100% males or 100% females (’90). Heavy lines show female heterogametic families, normal can result (in this case, depending on the dose of estrogen); I lines show male heterogametic families, and dashed lines show indicates that intersexes result from the treatment (hermaph- families where the sex-determining system is not known. Fe- roditic or ambiguous gonads in all individuals); IF indicates male heterogamety appears to be the ancestral state for am- that the males show partial sex reversal (hermaphroditic go- phibians, and male heterogamety has evolved several times. nads) and females are unaffected; M indicates that 100% In most cases, however, the sex-determining system assumed males result (sex reversal of genetic females); MI indicates for each family is based on studies from a single genus and in that normal males result, but that females show partial (her- many cases a single species from each genus. In addition, the maphroditic gonads) sex reversal. A “?” indicates that these sex-determining system has been examined in approximately data were not reported. It should be noted that in most cases only half of the extant anuran (nine families, for which nei- the effects of steroids for each family are based on a limited ther the genetic sex-determining system nor the effects of ste- number of studies in usually one species, and in many cases roids are known, are not shown here) and urodele families, the species used to determine the sex-determining mecha- with no studies in caecelians. Also, within the Ranids, the basal nism is not the same one used in experimental manipula- genus Pyxicephalus has a different sex-determining mechanism tions with exogenous hormones. The literature base used to from the other Ranids and is treated separately. evaluate the effects of steroids in this figure is presented in The effect of exogenous androgens (A) and estrogens (E) is Table 1. In salamanders (b), data for the effects of steroids shown, when data are available for anurans: MF indicates are not shown because of the limited available data; see text.

The somatic gonadal tissues in males and females observable upon histological analysis. The cells in likely have different origins, and the primordial the cortex become large follicles, and oocytes can germ cells arise from yet a third origin. be observed during fairly early stages of differen- The gonads originate from an outpocket of cells tiation. Male development is characterized by on the ventral surface of the kidney. Initially, there medullary development and cortical regression. are no histologically observable differences be- The gonads in males have smaller densely packed tween males and females. The undifferentiated (or cells (Figure 3). bipotential) gonad is a solid structure with an in- tact cortex and medulla. In females, the cortex Origin of the somatic gonadal tissue develops and grows, whereas the medulla re- There is still debate regarding the origin of the gresses, leaving a hole (ovarian vesicle) which is cells that form the gonadal ridge (which becomes SEX DETERMINATION IN AMPHIBIANS 379

Fig. 3. Transverse histological cross sections through gonad (resulting in a hole, or ovarian vesicle, in the center), bipotential gonads (top panel), ovaries (bottom left), and tes- and testicular differentiation is characterized by the regres- tes (bottom right) from the African clawed frog (Xenopus sion of the cortical portion. All sections were cut from paraf- laevis). Bipotential gonads were dissected from early larvae, fin-embedded tissues at 10µ and stained with hematoxylin/ and differentiated gonads were dissected from newly meta- eosin. Arrows show gonads in each panel; bar = 250µ; K = morphosed animals. Ovarian differentiation is characterized kidney; OV = ovarian vesicle. by the regression of the medullary portion of the bipotential the undifferentiated gonad) and whether differ- manders (Triton spp.; Papi, ’47, ’49) produced find- ent tissues contribute to the cortex and medulla. ings similar to Vannini (’41, ’42). Later studies in In Witschi’s studies on Rana sylvatica (Witschi, Rana esculenta (Sabbadin, ’51a; Vannini and ’27) and R. sylvatica, R. arora, and Hyla regilla Sabbadin, ’54) and Rana dalmatina (Sabbadin, (Witschi, ’31), he reported that the cortex (the pri- ’51b) suggested that the interrenal blastema con- mordium of the ovary) is derived from the coelomic tributed cells to the cortex (which is derived pri- epithelium and that the medulla (the testicular primarily from cells from the coelomic epithelium) mordium) originated from the mesonephric blas- and to the medulla of the developing gonads. In tema, with no contributions from the coelomic these early stages of gonadal development, the cor- epithelium. Several later studies and reviews tex and medulla are reportedly not distinct and agreed with Witschi’s findings (Witschi, ’67; migrations from the interrenal blastema to the Mittwoch, ’73; Deuchar, ’75), but Vannini (’41, ’42) cortex do not cease after the two layers separate. suggested that the medulla was derived from the Vannini and Sabbadin (’54) suggested that the dif- interrenal blastema and not the mesonephric ferences in their findings and those findings of blastema in Rana agilis. Studies in Bufo bufo and Witschi were, in part, due to what they interpreted Bufo viridis (Vannini and Busetto, ’45), in Bom- as Witschi’s unclear understandings of the origins bina pachypus (Vannini, ’47), and in two sala- of the mesonephric and interrenal blastema. The 380 T.B. HAYES significance of the difference between Witschi’s and germ cells were mesoderm-derived in salamanders Vannini’s model is that the gonadal primordia and (Hall, ’04; Dustin, ’07; Allen, ’11; Schapitz, ’12; the interrenal primordia (both steroid-producing tis- Beccari, ’22). Kuschakewitsch (’10) reported second- sues) share a common origin in Vannini’s model, ary germ cells that arose from a variety of tissues; whereas the gonads share a common origin with and Gatenby (’16) claimed that oogonia arose de the kidney primordia (non steroid-producing) in novo in the ovary. King (’08), Witschi (’14), Beccari Witchi’s model. (’24, ’26), Bounoure (’25) did not find any evidence Recent studies examining the origins of the go- for secondary germ cell differentiation, and it is nadal ridge created more confusion: Studies in Xe- not likely that such occurs, based on recent stud- nopus laevis and R. pipiens (Merchant-Larios and ies cited below. Villalpando, ’78, ’81) suggested that the meso- Most workers currently agree that the germ cells nephric blastema may not contribute to the gonadal originate in the endoderm from the dorsal mesent- medulla (in agreement with Vannini’s proposal), ary of the gut. The formation and migration of the but further suggested that the coelomic epithelium primordial germ cells have been well studied in was the origin of the gonadal medulla, giving the Xenopus laevis (Blackler, ’58; Al-Mukhtar and cortex and medulla a common origin. Lopez (’89) Webb, ’71; Kerr and Dixon, ’74; Ijiri and Egami, suggested that there was no medulla in Bombina ’75; Ikenishi and Kotani, ’75; Whitington and orientalis initially, and that the germ cell–filled cor- Dixon, ’75; Wylie and Heasman, ’76; Kamimura et tex was invaded by the medulla, which originated al., ’76; Wylie et al., ’76; Züst and Dixon, ’77; from cells of the mesonephric blastema. Further- Yamaguchi and Iwasawa, ’81), including light mi- more, Tanimura and Iwasawa (’87, ’89) suggested croscopic and ultrastructural electron microscopi- that the entire gonad was formed from the coelo- cal studies. These studies found that germ cells mic epithelium in Rhacophorus arboreus and that originated solely from primordial germ cells that the medulla is formed by cells segregating within differentiated and migrated as early as the gas- the primordial gonad. Furthermore, at least two trula stage. Thus no secondary or spontaneous dif- studies suggested that cells migrate from the cor- ferentiation of germ cells in the gonads are likely. tex into the medulla during male development Blackler (’62) provided additional evidence (Vannini and Sabbadin, ’54; Amanuma, ’63). Thus against secondary formation of germ cells: Germ an examination of several studies reveals conflict- cells were reciprocally transferred between two sub- ing results. Although species variation is possible, species of Xenopus laevis (one that produced light more comparative ultrastructural studies may re- brown eggs and one that produced dark brown eggs). solve this problem. In some crosses, the eggs produced by adult females were solely of the donor type: If any secondary pro- Origin of the primordial germ cells duction from a source other than the grafted pri- Like the origins of the cortex and the medulla, mordial germ cells occurred, then all individuals the origin of the primordial germ cells in amphib- would have produced eggs of mixed types. ians has been heavily debated. The early history Although all of the studies cited above agree in of these debates was documented by Witschi (’29c): regards to the origin of primordial germ cells, very The origin of the primordial germ cells was first little comparative work has been conducted. Two described by Goette (1875), who suggested that they recent studies (Tanimura and Iwasawa, ’87; arose from yolk-laden embryonic cells. Allen (’07) Nomura and Iwasawa, ’92) examined germ cell was the first to report that the primordial germ kinetics in a toad (Bufo japonicus formosus) and cells had an endodermal origin. Other authors de- a salamander (Onychodactylus japonicus), respec- scribed secondary germ cells, and some claimed tively, but no studies have examined the ultra- that these secondary germ cells had a mesodermal structure and migration of primordial germ cells origin (Bouin, ’01; Dustin, ’07). Humphrey (’25) even to the extent of the studies in Xenopus laevis. reported that germ cells in salamanders were derived from mesoderm, whereas germ cells in frogs Germ cell–somatic gonadal tissue interactions were endoderm-derived. Humphrey suggested also Several studies examined the potential role of that germ cells did not migrate or move indepen- the germ cells in gonadal differentiation, with dently, but rather shifted their position through- mixed results. Witschi (’29c) and Cheng (’32) sug- out development as a result of “growth of shifting gested that the histological changes associated of related parts” (Humphrey, ’25; page 1). Other with masculine and feminine differentiation oc- authors had previously made similar claims that curred before the germ cells migrated into the go- SEX DETERMINATION IN AMPHIBIANS 381 nad, but Dantchakoff (’32, ’33) proposed that the free embryos were produced, and although the go- germ cells secreted a substance that induced gonads were sometimes smaller in size, fertile males nadal differentiation. and females were produced. Shirane (’82) reported The most convincing experimental evidence sug- a skewed ratio at sexual maturity in Rana brevi- gesting that the gonads differentiate independent poda, but the sample size (33 total: 8 females/25 of germ cells was presented by Humphrey (’38), males) was low, and mortality between metamor- who conducted reciprocal unilateral transplants phosis and the stage of examination was greater of the intermediate segment and adjacent meso- than 30%. The sex ratio determined before mor- derm in Rana sylvatica during early tail bud tality (at metamorphosis) was normal (54% fe- stages (prior to migration of the primordial germ male), and although the author claimed that the cells). Thus the mesonephros and gonad on the skewed sex ratio at sexual maturity was due to operated side differentiated from the donor’s me- sex reversal (female to male), it is equally likely soderm, whereas the primordial germ cells that that the change was due to differential mortality. migrated into the developing gonad were from the In Shirane’s later study (’84), he reported female- host’s endoderm (because no endoderm was trans- to-male sex reversal (but again sample sizes were planted). In cases where the sex of the donor and low). He hypothesized that UV irradiation reduced host differed, the gonad on the operated side fol- the number of germ cells that migrated into the lowed the sex of the donor, as did the germ cells. gonadal ridge. He claimed that ovarian differentia- For example, when male mesoderm was trans- tion failed and that medullary cells surrounded the planted to a female embryo, an ovary developed remaining germ cells and induced testicular devel- on the unoperated side, whereas a testes development (female-male sex reversal). With this evi- oped on the operated side. The primordial germ dence, he concluded that germ cells were necessary cells that entered this testes developed into sperm, for ovarian differentiation. showing that the developing testes induced the Shirane (’87) further tested the hypothesis that invading germ cells to develop into sperm rather germ cells affected gonadal differentiation by us- than oocytes. Similar studies were conducted in ing the peanut lectin (PNA), which binds to the salamanders (Humphrey, ’27), and showed that surface of migrating primordial germ cells. Shi- gonads developed in the absence of primordial rane (’87) suggested that the observation that the germ cells, but only testicular development was primordial germ cells lost their affinity for PNA described in detail. Humphrey’s studies showed, coincident with gonadal differentiation indicated however, that gonads develop in the absence of that the germ cells induced gonadal differentia- germ cells and that germ cells do not stimulate tion. He further suggested that the primordial differentiation of the gonads. germ cells secreted substances that induced gene Later ultrastructural studies suggested that expression in the developing gonad, resulting in germ cells may even differentiate independent of differentiation (Shirane, ’87). To test these hypoth- the gonads (Kalt, ’73). Primordial germ cells were eses, Shirane injected embryos with PNA or N- more numerous (Ijiri and Egami, ’75), and the acetylgalactosamine (galNA; which is recognized germ cell division rate was faster in females than by PNA). He proposed that the sex-inducing ac- in males (Züst and Dixon, ’77), even before the tivity of the migrating primordial germ cells would germ cells migrated into the gonads. These data be decreased by treatment with lectins (which suggested that germ cells may sexually differen- would presumably occupy the binding sites of the tiate independent of the gonads, although Hum- lectins and prevent binding and activation of the phrey’s findings (’27, ’38) suggested that the secreted substances in the gonadal ridge) and that gonads can redirect germ cell differentiation. treatment with galNA would enhance the activ- Despite evidence that germ cells are not re- ity. Shirane (’87) suggested that PNA inhibited fe- quired for gonadal differentiation, more recent male development in Rana japonica and inhibited studies suggested, on the contrary, that germ cells both male and female development in R. nigro- were inductive and directed differentiation of the maculata based on the results of this study. gonads. Shirane pressed eggs of Rana japonica Furthermore, treatment with galNA reportedly in- and Rana brevipoda (’82) and Rana nigromaculata hibited male development in both species. (’84) against quartz and exposed the vegetal pole There are several problems with Shirane’s (’87) to UV irradiation to destroy primordial germ cells study and the interpretation of results, however. before they differentiated and migrated to the go- The sample sizes were low (30 or fewer in the nadal ridge. In the earlier (’82) study, germ cell– 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 histologically 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 effects resulted from changes in gene expression. Shirane’s (’82, ’86, ’87) studies are thus inconclu- sive, and earlier studies (discussed previously) provided 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 interactions between the developing cortex and medulla (Figure 4). He proposed that the cortex of genetic females produced a compound (cortexin) that induced regression of the medulla (or alternatively accelerated cortical growth) and/or that a substance from the medulla (medullarin) induced cortical regression (or medullary growth) in males. Furthermore, Witschi proposed that these compounds did not behave like typical hormones, but traveled in tissue rather than being Fig. 4. Potential mechanism controlling gonadal differentiation (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 promote development and growth of the medulla which forms theory: Witschi and McCurdy (’29) showed that the testes. ovaries transformed into testes when female larvae 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 383 canum: Humphrey, ’42) showed that transplanted proved incorrect. The gonads were not affected donor testes developed normally, but caused sex by androgen treatment, although androgens in- reversal (transformation of ovaries into testes) in duced secondary sex characters such as thumb female hosts and transformation of donor ovaries pad development (Chang and Witschi, ’55a). Thus when placed in males (provided that the donor and sex steroids were likely candidates for Witschi’s host were of similar age and developmental stage). cortexin and medullarin, except that testosterone Humphrey (’31b) later showed that when the had no effect on gonads, whereas whole testes grafted testes were removed from the female hosts, were effective. In fact, if androgens were the in- the sex-reversed females (neomales) were sterile ductive substances, then the testes of adult males (Humphrey, ’42). Furthermore, the rudimentary should be more potent, but Chang’s (’53) studies ovary underwent reversal again (after testes re- showed that only immature testes were capable moval) and produced functioning ovaries, showing of inducing testicular development in females. that the effects were not permanent. Furthermore, estrogens were 100% effective at Humphrey (’35) showed that heterosexual para- inducing complete sex reversal of genetic males, biosis between two different species produced dif- but in parabiosis experiments, the ovary was af- ferent results: Testes donated from A. maculatum fected by the male (or the testis graft) and not were transformed into ovaries by the female A. the other way around. Even more confusing, ova- jeffersonium. Also, A. tigrinum female hosts trans- rian extracts were capable of inducing sex inver- formed testes grafted from A. maculatum into ova- sion in adult male Triturus pyrrhogaster, whereas ries (Humphrey, ’35). In these cases, the effects of estrogen treatment could not produce this effect the ovary on the testes were believed to be due to (Hanaoka, ’61). Chang and Witschi (’55a) turned the intrinsic faster development rate of the female away from the idea that steroids were the puta- host species. tive inductors, speculating that the cortexin and In frogs, similar examinations revealed signifi- medullarin were proteins, and perhaps that the cant variation between studies. In Rana tem- cortex even produced antibodies (antimedullarin) poraria (Witschi, ’27), testes implanted into against the medulla and vise versa. In the same sexually undifferentiated tadpoles had no effect report (Chang and Witschi, ’55a), the authors also on developing ovaries. These studies (Witschi, ’27) reexamined Witschi’s earlier thesis (Witschi, ’27) were conducted in a race described as protogynous, that the inductor substances traveled through tis- so this peculiar sex differentiation in this species sues rather than through blood, and suggested may have affected results. Witschi (’27) viewed that the putative inducers were blood-bound. these data as evidence that did not support his inductor theory of sex differentiation, as he pre- Role of steroid hormones dicted that the testes should interfere with corti- Padoa (’36) was the first study to examine the cal development. A number of studies in Xenopus effects of exogenous steroids on gonadal differen- laevis, however, yielded positive results, but cre- tiation in amphibians. In Padoa’s (’36) study on ated some confusion: When implanted with tes- Rana esculenta, exogenous estrogen treatment re- tes, genetic females developed testes (Chang, ’53; sulted in 100% male offspring. Later studies Mikamo and Witschi, ’63, ’64). In addition, Chang showed that a variety of steroids “sex-reverse” frog (’53) found that testis grafts induced sex reversal and salamander larvae when added to the water (development of testes from ovaries in females) or injected into larvae (Gallien, ’59), although only when the grafts were made during the lar- Swingle (’22) suggested that the true transforma- val phase. Also, testes from only immature post- tions do not occur. Paradoxically, a number of metamorphic males induced complete sex reversal: chemically unrelated steroids have similar effects: The testes of adult sexually mature males lost For example, progestins, corticoids, androgens, their inductive capabilities. and estrogens can all induce testicular develop- The ability of implanted testes from immature ment producing 100% male progeny (Table 1). males (but not adult males) in Xenopus laevis is Equally puzzling, a single steroid can have a va- especially interesting, in light of experiments ex- riety of effects, depending on the species: Estra- amining the effects of crystalline sex steroids on diol can produce 100% females, have no effect at gonadal development in amphibians. Testosterone all, or produce 100% males (see Table 1). A single was incapable of inducing male development in steroid can have drastically different effects even Xenopus laevis larvae; early studies suggested in the same species, depending on dose. Moreover, that testosterone produced 100% males, but 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) 1Steroids 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 = estradiol benzoate; EBB = estradiol benzyl buterate; EDP = estradiol dipropionate; EQ = equilenin; ET = ethynyl estradiol; MT = methyl testosterone; TP = testosterone propionate. 2Concentrations reflect ranges of concentrations of steroids added to water except where indicated. Inj indicates that the indicated concentration 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. 3For 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? 4F = 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 (hermaphroditic 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% laevis larvae during stages when this same hor- males (see Table 1). Furthermore, examining these mone is unable to induce vitellogenesis in the liver, data in a phylogenetic context, taking into account oviductal growth, or cloacal labial growth (Hayes, the genetic sex-determining system, reveals no pat- ’97b). In the case of the liver, estradiol is not ca- terns or trends (see Figure 2) in anuran amphib- pable of inducing vitellogenin because the liver ians: There are no similar patterns of effects within lacks estrogen receptors until metamorphosis clades or within clades of anurans with a similar (Rabelo et al., ’94), and this mechanism likely ex- genetic sex-determining system. plains why other tissues do not respond. Similarly, An extensive data set for the effects of exog- testosterone induced testicular differentiation in enous hormones is not available for salamanders, Hyperolius argus and Hyperolius viridiflavus dur- but estrogen treatments produced all female ing stages in which this hormone is unable to in- offspring in Ambystoma punctatum (Burns, ’38, duce secondary sex characters such as gular pouch ’39), A. tigrinum (Ackart and Leavy, ’39), A. macu- development (Hayes, ’97a). So, either sex steroids latum (Foote, ’40, ’41), Hynobius retardatus do not act through conventional steroid receptors (Hanaoki, ’41a), H. nebulosus (Asayama and in their actions on the gonads, or gonadal expres- Miyamori, ’57), Triturus helviticus (Gallien and sion of steroid receptors is regulated differently Collenot, ’60), and Pleurodeles waltl (Gallien, ’50, when compared with other tissues. ’54). Androgens reportedly had no effect or inhib- Tadpoles metabolize steroids administered into ited gonadogenesis in some salamander species the water or injected into the body cavity (Dale, (Burns, ’39; Hanaoka, ’41b; Asayama and Mat- ’62; Rao et al., ’68, ’69; Hayes and Licht, ’93, ’95), suzaki, ’58; Gallien, ’65), but produced 100% fe- and this metabolism may play a role in the var- males in other species (Gallien, ’54; Asayama and ied effects of steroids on sex differentiation. Spe- Amanuma, ’57; Amanuma, ’61, ’63). Thus species cies that are unaffected by steroids may deactivate from only three salamander families have been these hormones faster by metabolizing them to examined in this regard, and no data are avail- inactive compounds, or alternatively, species that able for caecilians. are affected may activate the hormones by metabolizing them to more potent compounds. Data Mechanisms of steroid action from one study (Hayes et al., submitted) suggested Mechanism of steroid action on the gonads are that the metabolites of estradiol produced by Xe- not known. No steroid receptors have been iden- nopus laevis larvae are active compounds capable tified in amphibian gonads. Furthermore, gonads of inducing ovarian differentiation. Rao et al. (’68) respond to steroids at times when other tissues also claimed that female tadpoles metabolized ste- lack sensitivity to steroids. For example, estro- roids to active compounds, whereas male tadpoles gens induce ovarian development in Xenopus converted them to inactive compounds. SEX DETERMINATION IN AMPHIBIANS 387 Incomplete reporting and inconsistent designs phibians. In order for males and females to pro- between published studies create a problem when duce different sex steroids, they have to express interpreting data on the effects of steroids in dif- different steroidogenic enzymes. If males and fe- ferent species. For example, with a few exceptions, males express different functional enzymes, then none of the studies used to estimate the effects of they are already differentiated (even if this dif- steroids in amphibians (see Figure 2) reported the ferentiation cannot be detected ultrastructurally). total amount of hormone added to the water, the In fact, the steroids may be responsible for induc- number of tadpoles per treatment tank, the de- ing this detectable (structural and ultrastructural) velopmental stages during which the tadpoles differentiation, but the hormones themselves must were treated, the total amount of time over which be the product of prior differentiation. animals were treated, or the temperature main- In addition to the lack of understanding of the tained during treatments (Hayes and Licht, ’95). mechanisms of exogenous steroid action on go- Variation in effects between species may be an nadal differentiation, little is known about the role artifact of different experimental designs and of endogenous steroids in normal gonadal differ- treatment regimes: The total amount of hormone entiation. Amphibian larvae possess steroidogenic and number of tadpoles are important in deter- enzymes in their gonads, and steroid metabolism mining dose (in addition to reporting hormone con- and production have been measured, but only af- centrations). A given amount of hormone likely ter gonadal differentiation had occurred (Hsü et decreases in effectiveness as more tadpoles are al., ’77, ’78, ’79, ’85) or when the sexes were not added to a tank (50 tadpoles sequester and use identifiable (Kang et al., ’95). Also, several stud- more hormone than 10 tadpoles). Also, the devel- ies have shown that amphibian larvae metabo- opmental stage during the treatment is important: lize exogenous steroids (Rana sylvatica and Rana Estrogens induce ovarian differentiation in Xeno- pipiens: Dale, ’62; Pleurodeles waltl: Ozon, ’63; pus laevis only during specific stages of develop- Xenopus laevis: Rao et al., ’68, ’69; nine species, ment (Chang and Witschi, ’55a; Gallien and representing eight families: Hayes and Licht, ’93, Foulgoc, ’60; Witschi, ’71; Iwasawa et al., ’79; ’95), and one study even suggested that male and Villalpando and Merchant-Larios, ’90; Hayes et female larvae metabolize estrogens differently al., ’97b). Thus some studies may fail to show ef- (Rao et al., ’68). Although steroid metabolism was fects of steroids on gonadal development because not localized to the gonads, these data showed that the treatments were administered during incor- larvae are capable of metabolizing and possibly rect stages. In addition, these critical periods producing sex steroids. In salamanders, Chardard likely vary between species. Likewise, the total et al. (’95) showed that Pleurodeles waltl had ac- amount of time that animals are exposed and the tive aromatase in its gonads that increased in temperature during exposure likely affect the po- females during larval development. Rearing fe- tency of steroids. In addition, all of these factors male larvae at high temperatures (which sex- (concentration, total mass of steroid, developmen- reverses female P. waltl) caused a decrease in tal stage, temperature, and duration of exposure) aromatase activity, suggesting that estrogens may alter steroid metabolic rates (which also vary be- be involved in sex differentiation of females in this tween species; Hayes and Licht, ’93, ’95). Thus, if species (Chardard et al., ’95). steroid metabolism by tadpoles represents acti- Despite the paucity of studies examining endog- vation and/or deactivation of the steroids (and ste- enous hormone production, a few studies have ad- roid metabolism likely affects potency), then the dressed the role of endogenous hormones in interaction between tadpoles and treatment con- amphibian gonadal differentiation by treating am- ditions and effects on steroid metabolism provide phibian larvae with reagents that block endog- another mechanism by which artifacts may be pro- enous steroid production. Zaccanti et al. (’94) duced when comparing effects between species from treated larval Bufo bufo and Rana dalmatina with reports in the literature. These problems may be androsten-3 one 17 β-carbossilic acid, which in- solved by careful studies of multiple species under hibits 5 α reductase (prevents the production of similar controlled conditions in parallel studies. 5α reduced androgens). This study showed accelerated ovarian development with larger and more Endogenous steroids numerous oocytes in the Bidder’s organ of Bufo It is not likely that steroid hormones play a role bufo and in the ovary of Rana dalmatina. Al- in sex determination, although they may be in- though sex reversal did not occur, these data sug- volved in normal gonadal differentiation in am- gested that endogenous androgens might be 388 T.B. HAYES involved in gonadal differentiation. Hsü et al. (’79, The order and coordination of developmental ’81a) also showed that blocking estrogen synthe- events are relatively consistent in amphibians: sis resulted in transformation of ovaries into tes- Forelimbs emerge before the tail reabsorbs (in tes and that blocking both androgen and estrogen frogs), the lungs mature before the gills reabsorb synthesis with 17 β-ureide (Hsü et al., ’81b) re- (in frogs and metamorphosing salamanders), etc. sulted in sterile gonads. Similarly Yu et al. (’93) The timing of gonadal development varies signifi- showed that endogenous steroids may be involved cantly, however. For example, Xenopus laevis have in sex differentiation in Rana catesbeiana. Im- differentiated gonads (based on histological analy- planting tadpoles with capsules containing 4-OH- sis) by NF (Nieuwkoop and Faber, ’56) Stage 52, androstenedione (an aromatase inhibitor) resulted whereas Hyperolius viridiflavus are differentiated in testicular development. The authors suggested as early as NF Stage 48 (even by morphological that the sex reversal was due to a buildup of an- examination). Other species, such as Bufo boreas, drogen that acted on the gonad to induce sex re- are not sexually differentiated until after meta- versal. These studies all suggest that aromatase morphosis (Gosner [’60] Stage 48 = NF Stage 66), (and low androgens) may be required for ovarian and gonads are not distinguishable by histologi- differentiation. An earlier study suggested that cal examination even 1 week after metamorpho- endogenous steroids may not be involved in nor- sis in Pyxicephalus adpsersus (Hayes and Licht, mal sex differentiation, however. Treatments with ’92). Even within a species, there may be varia- an anti-estrogen (tamoxifen) or an anti-androgen tion in the timing of gonadal differentiation (cyproterone acetate) were unable to block the ef- (Iwasawa, ’69; Chang and Hsu, ’87), especially in fect of exogenous estrogen or testosterone treat- the sexually undifferentiated races of Rana ment, respectively. Also, neither anti-steroid temporaria (Witschi, ’30) and Rana catesbeiana affected normal sex differentiation (Rastogi and (Pucket, ’40). In addition, Ohta (’87) reported that Chieffi, ’75). Buergeria buergeri from Hiroshima had differ- One difficulty in understanding the potential entiated gonads by Gosner Stage 40, but our ex- role of steroids in normal gonadal differentiation perience raising the same species from Yamanashi is that steroid production must be analyzed prior under similar conditions showed that gonads were to sex differentiation to discern whether sex ste- not differentiated even at metamorphosis (Hayes roids organize the gonad or whether differences and Wong, unpublished). One interpretation is in sex steroid production are the result of organi- that this variation is an indication that thyroid zation (sex differentiation) that has occurred al- hormones do not affect gonadal differentiation. ready. This task is difficult, because identification On the other hand, thyroid hormones may regu- of sexes in most species is based on gonadal mor- late gonadal differentiation, but different species phology, since only a few species have easily dis- may show different sensitivities: some respond to tinguishable sex chromosomes. Although steroid very low doses of thyroid hormones and differenti- levels were measured in eggs of two species (Rana ate early in development, whereas others require sylvatica and Xenopus laevis; Hayes, unpublished), high titers of thyroid hormone and differentiate the effects of steroids during these early stages at metamorphic climax, etc. Also, thyroid hormones are not known to influence sex differentiation. may regulate gonadal differentiation in some spe- Also, hormones found in eggs are of maternal ori- cies, but not others. This possibility is certainly gin, because the steroidogenic tissue has not de- realized in other tissues, such as development of veloped in early embryos at these stages. the hind limbs, which are thyroid hormone–dependent in Xenopus laevis but not in Bufo boreas Other hormones (Hayes, ’95, ’97a). Several studies suggested that thyroid hor- Thyroid hormones mones regulate gonadal development. In recent Thyroid hormones are best known for their role studies, we (Hayes, ’97b) showed that treatment in amphibian metamorphosis, but have been im- with thiourea (a goitrogen) resulted in 100% fe- plicated in gonadal development in amphibians. males in Xenopus laevis (a species in which Their role in gonadal differentiation is debated, estradiol induced ovarian differentiation but tes- and some workers claim that gonadal differentia- tosterone had no effect) and 100% males in tion is one of the few aspects of development that Hyperolius viridiflavus (an unrelated species in is not under the control of thyroid hormones which estradiol had no effect but testosterone in- (Hayes, ’97b; for review). duced testicular development). In fact, the ste- SEX DETERMINATION IN AMPHIBIANS 389 roids effective at inducing sex reversal also in- Undifferentiated races hibited larval development and the thyroid axis, So called undifferentiated races have been re- and it is possible that sex steroids alter gonad ported in a number of ranid species, including differentiation simply by inhibiting the thyroid Rana temporaria (Pflüger, 1882; Ponse, ’30; axis. The alteration of gonadal differentiation by Witschi, ’30), R. catesbeiana (Pucket, ’40), and R. blocking thyroid hormones in two unrelated spe- esculenta, R. ornativentris (Iwasawa, ’69) and in cies suggests a generalized role for thyroid hor- a single bufonid (Bufo bufo: Ponse, ’49; Foote, ’64) mones in gonadal development across anurans. and in Rhacophorus arboreus (Iwasawa, ’69a,b). Also, other studies have suggested that thyroid Similar reports were made in salamanders (Hyno- hormones inhibit ovarian development (Hoskins bius retardatus: Uchida, ’37b; and Ambystoma and Hoskins, ’19; Iwasawa, ’55a,b, ’61; Yoshikura, maculatum: Witschi, ’33b). In R. catesbeiana ’60, ’61, ’62b,c; Yü et al., ’72; Rengel et al., ’93). (Pucket, ’40), the animals were not truly undif- On the contrary, several studies suggested that ferentiated, but ovarian differentiation was ob- thyroid hormones are not involved in gonadal dif- served in larval stages. The testes remained a ferentiation (Allen, ’18; Yoshikura, ’60; Hsü et al., “prototestis” (as described by Swingle, ’26), how- ’74; Robertson and Kelley, ’96). Thus a definite ever, until metamorphosis, when they developed role for thyroid hormones and their mechanism into recognizable testes. Development in undiffer- of action remains to be shown. entiated races of R. temporaria is quite distinct, Pituitary hormones however. In some populations, all of the animals metamorphose with ovaries. In the following 6 to Several studies addressed the role of pituitary 9 months after metamorphosis, some of the indi- hormones and the pituitary gonadotrop(h)ins in sex viduals reportedly transform into males going differentiation. Gonads developed normally in both through an intersexual stage (Witschi, ’21, ’30). males and females when larvae were hypophysec- Similar reports were made for Rhacophorus tomized early in development prior to gonadal dif- schlegelii (Amanuma, ’63a,b). ferentiation in Rana japonica (Yoshikura, ’59a,b; Iwasawa, ’61). The gonads from hypophysectomized Sequential hermaphroditism and animals were smaller than controls, however, sug- spontaneous sex reversal gesting that pituitary hormones stimulated growth of the gonads, but that differentiation was inde- Although Witschi (’29b, page 236) stated, “Some pendent of these hormones. Gonads grown in cul- of the most common anurans are rudimentary her- ture differentiated into testes and ovaries, but maphrodites which eventually may ripen fertile growth was impaired (especially testes) in Rana eggs and sperms either in consecutive years, as catesbeiana (Foote and Foote, ’58). Schreiber and in the toad Bufo vulgaris, or simultaneously, as Rugh (’45), Asayama (’55), and Iwasawa (’60) in the frog Rana temporaria” and even claimed showed that pituitary injections accelerated growth that artificial self-fertilization was possible in of testes and ovaries in Rana pipiens, but did not some of these rudimentary hermaphrodites, he affect differentiation or maturation. In addition, the does not cite any reference where data and obser- pituitary did not affect sex steroid effects on gonad vations were reported. Crew (’21) also reported a differentiation (Mintz and Gallien, ’54; Yoshikura, number of incidences of abnormal sex reversals ’60a) and gonads respond to steroids in vitro (Foote and intersexual animals, but not much informa- and Foote, ’59). These studies all agreed that the tion was provided for review, and the report is pituitary is not required for early gonadal differ- simply a categorical listing of cases. entiation, but may be involved in gonadal growth At least two published studies since Witschi after differentiation occurs. (’29b) and Crew (’21) addressed the possibility of sequential hermaphroditism (natural sex rever- Atypical sex gonadal differentiation sal without any hormone treatments, etc.). Grafe Most of the data discussed so far, were based and Linsenmair (’89) suggested that seven out of on studies of gonochoristic species. Enough varia- 24 captive female Hyperolius viridiflavus om- tion in sex developmental patterns exists in am- matostictus (which reportedly laid at least one phibians to warrant discussion of these exceptions, clutch of eggs) began calling, developed gular however. These exceptions include reported un- pouches, and apparently fertilized eggs. No histo- differentiated races, hermaphrodites, and parthe- logical evidence or further examinations were pub- nogenetic species. lished in this case, however. In another study 390 T.B. HAYES (Collenot et al., ’94), the salamander (Pleurodeles cephalic end of the gonad and may differentiate waltl) reportedly underwent spontaneous sex re- at the same time as the gonads. Meiotic activity versal in captivity. The authors reported that in the Bidder’s organ is greater in females than in some animals (all apparently of the ZZ type) de- males (Tanimura and Iwasawa, ’86), and these veloped into females, and intersexes. This study data, combined with the observation that removal used a supposed sex-linked peptidase in which of the testes results in the transformation of the one type is supposedly linked to the W chromo- Bidder’s organ into fully functional ovaries (ref- some (Ferrier et al., ’80; Dournon et al., ’88) to erences above), suggested that the testicular se- identify genetic types of individuals. Animals cretions may antagonize the development and lacking the proposed W-linked peptidase were as- function of this rudimentary ovary in males. sumed to be ZZ, and any animals possessing an Despite the presence of the Bidder’s organ, how- ovary but lacking the W-linked form of the en- ever, bufonids are not truly hermaphroditic (nei- zyme were assumed to be sex-reversed. An al- ther simultaneous or sequential): Under normal ternative explanation is that the peptidase may conditions, the Bidder’s organs do not function as not be as tightly linked as proposed and thus ovaries in males. Although Witschi suggested that may not be a reliable sex marker. Their results sequential hermaphroditism occurred in Bufo vul- may be explained by exchange between the Z and garis, the primary description of this observation W sex chromosomes. Evidence for such exchange and details of studies are not cited and the litera- is found in the X and Y chromosome in mam- ture is not available. Thus the function (if any) mals, for example (Kent et al., ’88). and the mechanisms underlying development of this structure remain unclear. The realization that Rudimentary hermaphroditism in Bufonids the Bidder’s organ is a rudimentary ovary stimu- The natural condition in Bufonids is the most lated much discussion about the ancestral state compelling case for hermaphroditism in amphib- of amphibians, however. Several authors proposed ians (although not functional). In all bufonids, that the rudimentary ovary in bufonids is evidence both males and females possess Bidder’s organs. of a hermaphroditic ancestry of the bufonids, if Bidder’s organs are a pair of rudimentary ovaries not all amphibians (Witschi, ’33a). Interestingly, that develops at the anterior portion of the gonad the cephalic portion of the gonad in Xenopus laevis in both males and females. Witschi (’33a) provided can be induced to develop an ovarian-like struc- an excellent review of the early descriptions of the ture (with male characteristics developing cau- Bidder’s organ: The Bidder’s organ was first de- dally) if male larvae are treated with estrogens scribed as fat (Roesel, 1758) and then as the pri- for a short time near the critical period (Chang mordium of the testes (Rathke, 1825). Ironically, and Witschi, ’55a; Hayes et al., unpublished). The Bidder (1846), for whom the organ was named, fate of such intersex animals is unknown since argued that the Bidder’s organ was an accessory none have been followed to metamorphosis and structure involved in male gametogenesis. Later, adulthood. These data suggest that the cephalic Von Wittich (1853) suggested that the Bidder’s or- portion of the gonad is more sensitive to sex ste- gan was a rudimentary ovary developing in both roids and transforms before the caudal end. Alter- males and females, and further histological data natively, gonads normally differentiate posterior to from several workers supported Von Wittich’s anterior, so that short-term hormone treatments claim (see references in Witschi, ’33a). Even more transform the cephalic end, which remains bi- convincing, Harms (’21, ’23) and Guyénot and potential, but are unable to transform the caudal Ponse (’23a,b,c, ’27) showed that the Bidder’s or- end, which may differentiate earlier. Whatever the gan developed into a fully functional ovary that mechanism, such studies may provide interesting can produce fertilizable eggs after the testes were models for examining mechanisms underlying surgically removed. Bidder’s organ development in bufonids. Since its discovery, a number of papers have described the development of the Bidder’s organ Parthenogenesis (Witschi, ’33a; Horié, ’38; Vannini, ’56; Zaccanti et Natural parthenogenesis has been reported in al., ’71; Tanimura and Iwasawa, ’86), and more re- Ambystomatid salamanders and is well studied. cent studies have examined its contribution to ste- The Jeffersonian salamander complex consists of roid hormones (Colombo and Colombo-Belvedere, two diploid species (Ambystoma jeffersonium and ’80; Ghosh et al., ’84; Pancak-Roessler and Norris, Ambystoma laterale), which reproduce normally. ’91). The Bidder’s organs always develop at the There are at least two triploid species (Ambystoma SEX DETERMINATION IN AMPHIBIANS 391 platineum and Ambystoma tremblayi) whose between mechanisms of sex determination and sex ranges overlap with the ranges of the diploid spe- differentiation. cies (Clanton, ’34; Minton, ’54; Uzzell, ’64; Mac- The existence of parthenogenesis has not been Gregor and Uzzell, ’64). The two triploid species reported in frogs, although natural polyploids ex- primarily consist of females. These triploids de- ist in frogs (Wasserman, ’70; Bogart and Tandy, pend on diploid males because sperm is required ’76; Bogart, ’80). Frogs can be induced to develop to stimulate development of the eggs of the polyploidy (Parmenter, ’33; Rugh and Marsland, parthenogens, although genetic material from the ’43; Dasgupta, ’62), and a single study showed that males is not incorporated (Licht, ’89). both males and females resulted from triploidy Uzzell (’64) showed that triploid females con- induced in Rana pipiens by cold shock, but by sistently produced only female (and triploid) off- metamorphosis all females were sex-reversed and spring, and suggested that the triploid species developed testes (Humphrey et al., ’50). resulted from hybridization between the two diploid species (Uzzell, ’63). Later electrophoretic CONCLUSIONS studies supported this hypothesis (Uzzell and All amphibians probably display genetic sex de- Goldblatt, ’67; Downs, ’78; Bogart et al., ’85; termination, but external factors can influence sex Lowcock and Bogart, ’89; Kraus, ’89; Lowcock, ’89) (gonadal) differentiation. The external factors in- and showed that some island species may have clude temperature, physical manipulations, and ex- arisen from hybridizations between Ambystoma ogenous hormones. Only endogenous hormones may laterale and Ambystoma texanum as well. Uzzell naturally affect gonadal differentiation, however. and Goldblatt (’67) went further to hypothesize A lack of work in the field over the last two that a tetraploid hybrid, producing diploid ga- decades has left many questions concerning sex metes, may have served as an intermediate that, differentiation unanswered. Mechanisms regulat- when back-crossed to one of the diploid parental ing germ cell differentiation are unclear. The con- species (producing haploid gametes), produced tributions of coelomic epithelium and kidney and triploid offspring. interrenal primordia to the cortex and medulla of Triploid salamanders have been produced in the the gonads remain undetermined. The role of en- laboratory also. Ambystoma tigrinum and Am- dogenous steroids and thyroid hormones and their bystoma mexicanum eggs can be induced to de- mechanisms of action on the development of the velop as triploids by cold treatment and by gonad are also unclear. Other future studies pricking with a needle. These animals undergo should address the relationship between mecha- normal ovarian development during early stages, nisms of GSD (female versus male heterogamety) but oocytes degenerate later in development. This and mechanisms of sex differentiation and how degeneration may be related to the ploidy of the GSD may be related to the susceptibility to sex animals and not some physiological deficiency, since reversal by some steroids but not by others grafted ovaries from normal diploid females develop (known to be active in other species). normally (Humphrey and Frankhauser, ’46). Studies in natural triploid Notophthalamus ACKNOWLEDGMENTS (Triturus) viridescens (Salamandridae) showed I thank V. Lance for the invitation to partici- that, unlike Ambystomatids, both male and female pate in this symposium. I also thank R. Liu, S. triploids developed (Frankhauser, ’40). In addition, Wong, T. Gill, J. Yeh, and K. Menendez who have not only did males develop normally, but females participated in various studies on this topic and suffered a loss of germ cells, and only rudimen- an anonymous reviewer whose comments im- tary ovaries developed. However, this study proved the manuscript. I also thank Katherine (Frankhauser, ’40) was based on only four ani- Kim for her support. mals (one male and three females) that were cap- tured from the wild and raised in the laboratory. LITERATURE CITED One possible explanation for the potential differ- Ackart, R.J., and S. Leavy (1939) Experimental reversal in ences in the effects of triploidy between the Am- salamanders by injection of oestrone. Proc. Soc. Exp. 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