Sex Determination In Amphibians Research Articles

Male heterogametic sex determination in Rana dybowskii based on sex-linked molecular markers.

Identifying the mechanism for sex determination in amphibians is challenging. Very little is known about sex determination mechanisms of Rana dybowskii, a species of importance to evolutionary and conservation biology. We screened for sex‐linked molecular markers in R. dybowskii in China using target region amplification polymorphism with 2 fixed primers against the sequences of Dmrt1. We found 2 male‐linked molecular markers in R. dybowskii, which were 222 bp and 261 bp long. The detection rates of 222 bp marker in males form Xinglong, Huadian, and Dandong were 93.79%, 69.64%, and 13.64%, respectively, while the rate in females from Huadian was 27.50%. Besides, the detection rates of 261 bp marker in the above 3 regions were only observed in males at the rate of 93.79%, 87.50%, and 32.73%, respectively. The inheritance patterns of sex‐linked molecular markers showed that the 2 sex‐linked molecular markers were heterozygous. Compared to the XY‐male parent, progeny from XX‐pseudo‐male parent possessed lower sex reversal ratio at the same rearing temperature, and the proportion of female froglets from an XX‐pseudo‐male parent was more than 95% at low rearing temperature (15°C). Our findings suggest that R. dybowskii displays male heterogamety, and the 2 sex‐linked molecular markers may have a guiding significance for the protection and utilization of R. dybowskii.

Identification of critical sex-biased genes in Andrias davidianus by de novo transcriptome.

The Chinese giant salamander Andrias davidianus is a protected amphibian with high nutritional and economic value. Understanding its sex determination mechanism is important for improving culture techniques and sex control in breeding. However, little information on the characterization of critical genes involved in sex is available. Herein, sequencing of ovary and test produced 40,783,222 and 46,128,902 raw reads, respectively, which were jointly assembled into 80,497 unigenes. Of these, 36,609 unigenes were annotated, of which 8907 were female-biased and 10,385 were male-biased. Several sex-related pathways were observed, including the Wnt signaling pathway. After elevated temperature and estrogen exposure, neomale and neofemale specimens were identified by a female-specific marker for the first time. RT-qPCR analysis showed the expression profile of ten selected sex-biased genes to be exhibited consistently in male and neomale and in female and neofemale, with the exception of the Amh and TfIIIa genes. Results suggested that these genes may play important roles in A. davidianus sex determination and gonad development. This provides a basis for further investigation of the molecular mechanisms of sex determination in amphibians.

High temperatures influence sexual development differentially in male and female tadpoles of the Indian skipper frog, Euphlyctis cyanophlyctis.

Although sex determination in amphibians is believed to be a genetic process, environmental factors such as temperature are known to influence the sex differentiation and development. Extremely low and high temperatures influence gonadal development and sex ratio in amphibians but the mechanism of action is not known. In the present study, effect of different temperatures on gonadal development, sex ratio and metamorphosis was studied in the Indian skipper frog, Euphlyctis cyanophlyctis. The embryos of Gosner stage 7 were exposed to 20, 22, 24, 26, 28, 30 and 32 degrees C up to tadpole stage 42. The embryos (stage 7) were also exposed to 20 and 32 degrees C up to tadpole stage 25 (non-feeding stages). Tadpoles of stage 25 were reared at 20 and 32 degrees C up to stage 42 (feeding stages). The results show that exposure to higher temperatures (28, 30 and 32 degrees C) during stages 7-42 produced male-biased sex ratio. Rearing of tadpoles at 32 degrees C during stages 25-42 produced malebiased sex ratio, while exposure during stages 7-25 did not affect sex ratio. Embryos and tadpoles exposed to lower temperatures (20 and 22 degrees C) died during the early stages. High temperatures stimulated testis development, and disturbed ovary development. Exposure to high temperatures resulted in the early metamorphosis of tadpoles with reduced body size. These results demonstrated that high temperatures influence gonadal development differently in male and female tadpoles, leading to male-biased sex ratio. These results suggest that high temperature probably acts through stress hormones and favours the small-sized sex.

Sex-linked markers in the North American green frog (Rana clamitans) developed using DArTseq provide early insight into sex chromosome evolution.

BackgroundThe extent to which sex reversal is associated with transitions in sex determining systems (XX-XY, ZZ-ZW, etc.) or abnormal sexual differentiation is predominantly unexplored in amphibians. This is in large part because most amphibian taxa have homomorphic sex chromosomes, which has traditionally made it challenging to identify discordance between phenotypic and genetic sex in amphibians, despite all amphibians having a genetic component to sex determination. Recent advances in molecular techniques such as genome complexity reduction and high throughput sequencing present a valuable avenue for furthering our understanding of sex determination in amphibians and other taxa with homomorphic sex chromosomes like many fish and reptiles.ResultsWe use DArTseq as a novel approach to identify sex-linked markers in the North American green frog (Rana clamitans melanota) using lab-reared tadpoles as well as wild-caught adults from seven ponds either in undeveloped, forested habitats or suburban ponds known to be subject to contamination by anthropogenic chemicals. The DArTseq methodology identified 13 sex-linked SNP loci and eight presence-absence loci associated with males, indicating an XX-XY system. Both alleles from a single locus show partial high sequence homology to Dmrt1, a gene linked to sex determination and differentiation throughout Metazoa. Two other loci have sequence similarities to regions of the chimpanzee and human X-chromosome as well as the chicken Z-chromosome. Several loci also show geographic variation in sex-linkage, possibly indicating sex chromosome recombination. While all loci are statistically sex-linked, they show varying degrees of female heterozygosity and male homozygosity, providing further evidence that some markers are on regions of the sex chromosomes undergoing higher rates of recombination and therefore further apart from the putative sex determining locus.ConclusionThe ease of the DArTseq platform provides a useful avenue for future research on sex reversal and sex chromosome evolution in vertebrates, particularly for non-model species with homomorphic or cryptic or nascent sex chromosomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3209-x) contains supplementary material, which is available to authorized users.

Is a Sex-Determining Gene(s) Necessary for Sex-Determination in Amphibians? Steroid Hormones May Be the Key Factor

Amphibians have 2 genetic sex-determining systems, one with male (XX/XY) and one with female (ZZ/ZW) heterogamety. While the ancestral state of sex-determination is thought to be female heterogamety, male and female heterogametic types were probably once interchangeable. The Japanese frog Rana rugosa has both XX/XY and ZZ/ZW systems within a single species in certain local populations. However, steroid hormones can alter the phenotypic sex epigenetically. In R. rugosa, steroidogenic enzyme expression starts before sex-determination in the indifferent gonad, and these enzymes become active in both male and female tadpoles. Androgens are produced in the indifferent gonad of male tadpoles at high levels, whereas estrogens are synthesized in females. In this regard, the observed enhanced expression of the hormone-metabolizing genes, CYP19 in the female gonad and CYP17 in males, may be crucial for sex-determination. Moreover, with FSH known to increase estrogen synthesis in the vertebrate ovary, observed upregulation of FSH receptor (FSHR) expression in the indifferent gonad of female tadpoles is intriguing. These data suggest that steroid hormones could be crucial for sex-determination in R. rugosa, with the consequence that upregulation of CYP19 and FSHR expression is necessary for female and CYP17 for male sex-determination.

Does the speciation clock tick more slowly in the absence of heteromorphic sex chromosomes?

Squamates may be an attractive group in which to study the influence of sex chromosomes on speciation rates because of the repeated evolution of heterogamety (both XY and ZW), as well as an apparently large number of taxa with environmental sex-determination.

Diversity in the origins of sex chromosomes in anurans inferred from comparative mapping of sexual differentiation genes for three species of the Raninae and Xenopodinae

Amphibians employ genetic sex determination systems with male and female heterogamety. The ancestral state of sex determination in amphibians has been suggested to be female heterogamety; however, the origins of the sex chromosomes and the sex-determining genes are still unknown. In Xenopus laevis, chromosome 3 with a candidate for the sex- (ovary-) determining gene (DM-W) was recently identified as the W sex chromosome. This study conducted comparative genomic hybridization for X. laevis and Xenopus tropicalis and FISH mapping of eight sexual differentiation genes for X. laevis, X. tropicalis, and Rana rugosa. Three sex-linked genes of R. rugosa--AR, SF-1/Ad4BP, and Sox3--are all localized to chromosome 10 of X. tropicalis, whereas AR and SF-1/Ad4BP are mapped to chromosome 14 and Sox3 to chromosome 11 in X. laevis. These results suggest that the W sex chromosome was independently acquired in the lineage of X. laevis, and the origins of the ZW sex chromosomes are different between X. laevis and R. rugosa. Cyp17, Cyp19, Dmrt1, Sox9, and WT1 were localized to autosomes in X. laevis and R. rugosa, suggesting that these five genes probably are not candidates for the sex-determining genes in the two anuran species.

A Sex-Specific Marker Reveals Male Heterogamety in European Tree Frogs

Most amphibians examined so far show undifferentiated sex chromosomes. The heterogametic sex's identity, usually revealed through indirect means, often varies among closely related species or even populations (as do sex-linkage groups), suggesting great evolutionary instability of the sex-determining genes. Here we take advantage of a sex-specific marker that amplifies in several related species of European tree frogs (Hyla arborea group) to disclose a homogeneous pattern of male heterogamety. Besides relevance for evolutionary studies of sex determination in amphibians, our results have potential for addressing practical issues in conservation biology because sex reversal by anthropogenic endocrine disruptors is considered one possible cause of amphibian decline.

Sex determination: the amphibian models

We review and discuss current knowledge about sex determination in amphibians. The astonishing wide variety of mechanisms of genotypic sex determination is presented and discussed in an evolutionary context. We recall the natural occurrence of transitory juvenile hermaphroditism in some species. Our present knowledge of the mechanisms of sex determination in amphibians is compared to that in mammals. The influence of epigenetic factors, and especially temperature is highlighted. In amphibians, the influence of temperature on sexual differentiation, that can prevail over genotypic sex determination, remains poorly considered in publications. We suggest that studies on genetic and epigenetic factors of gonadal sex differentiation in amphibians could provide substantial information on the evolutionary process of sex determination mechanisms in current living vertebrates.

Sex determination and primary sex differentiation in amphibians: Genetic and developmental mechanisms

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.

Evolutionary changes of heterogametic sex in the phylogenetic history of amphibians

AbstractA survey of sex‐determining systems in amphibians revealed widespread variation in the heterogametic sex and the extent of sex chromosome differentiation. Systems of sex determination have now been resolved in 63 species of salamanders and frogs. Species with ZW female heterogamety, OW female heterogamety, and XY male heterogamety have all been reported. A phylogenetic analysis suggests that the ancestral state for amphibians was female heterogamety. XY/XX sex determination has evolved independently at least seven times, whereas there is only one case in which the data suggest that male heterogamety has subsequently reversed to female heterogamety. Differentiation of heteromorphic sex chromosomes in Amphibia has occured repeatedly. A model of an ancestral two‐locus sex‐determination mechanism, based on the findings of the phylogenic analysis and the condition found in the primitive frog Leiopelma hochstetteri, allows a recessive mutation to result in the eventual acquisition of male heterogamety. Reversal to female heterogamety requires de novo evolution of a functional control locus under the model. The model is consistent with all information on sex determination in amphibians, and explains the observed bias in evolution from ZW/ZZ to XY/XX systems.

The Mechanism of Temperature Dependent Sex Determination in Crocodilians: A Hypothesis

Incubation of alligator eggs at 30°C produces 100% females, at 33°C 100% males; temperatures in between produce varying sex ratios. Wild nests of Alligator mississippiensis show similar effects and the populations are biased towards females. The incidence and patterns of temperature dependent sex determination (TSD) in other crocodilians are reviewed. Temperature also affects hatchling size and pigmentation patterns, post-hatching growth rates and thermoregulation by juvenile crocodilians. The significance of temperature sensitive periods defined by temperature shift experiments is questioned in relation to a hypothesis to explain the mechanism of TSD in crocodilians. It is postulated that there is an initial sex differentiation mechanism which involves a quantum period of time and a threshold for a dose of a male determining factor. The conditions for induction of males are precise but exhibit variation between individuals within the population. Females develop by default. The hypothalamus may have an important role in a later sex differentiation mechanism. The hypothesis is used to explain the late temperature sensitive periods defined by high to low temperature shift experiments, why cooler temperatures are more effective at determining sex, how intermediate temperatures can produce both sexes, the differences in the pattern between turtles and crocodilians and geographical similarities in the pattern of TSD within crocodilians despite differing climates. The phylogenetic advantages of TSD in crocodilians are concerned with the overall reproductive strategy of the animals. Those crocodilians which are incubated and grow to maturity under optimal environmental conditions will be both large and male. Larger males are more likely to produce more offspring. A review of the effects of the environment on sex determination in amphibians and fish suggests that there is a general relationship between size and sex in vertebrates

Studies on sex differentiation and sex determination in amphibians. IX. Quantitative relationships in the induction of sex differentiation, and the problem of sex reversal in parabiotic salamanders

Studies on sex differentiation and sex determination in amphibians. IX. Quantitative relationships in the induction of sex differentiation, and the problem of sex reversal in parabiotic salamanders

Studies on sex differentiation and sex determination in amphibians. VIII. Experiments on inductive inhibition of sex differentiation in parabiotic twins of salamanders

Studies on sex differentiation and sex determination in amphibians. VIII. Experiments on inductive inhibition of sex differentiation in parabiotic twins of salamanders

Studies on sex differentiation and sex determination in amphibians. VII. Sex in two local races of the spotted salamander, Amblystoma maculatum Shaw

Studies on sex differentiation and sex determination in amphibians. VII. Sex in two local races of the spotted salamander, Amblystoma maculatum Shaw

Studies in sex differentiation and sex determination in amphibians. VI. The nature of Bidder's organ in the toad

Studies in sex differentiation and sex determination in amphibians. VI. The nature of Bidder's organ in the toad

Studies on sex differentiation and sex determination in amphibians. V. Range of the cortex‐medulla antagonism in parabiotic twins of Ranidae and Hylidae

Studies on sex differentiation and sex determination in amphibians. V. Range of the cortex‐medulla antagonism in parabiotic twins of Ranidae and Hylidae

Studies on sex differentiation and sex determination in amphibians. IV. The geographical distribution of the sex races of the European grass frog (Rana temporaria, L.). A contribution to the problem of the evolution of sex

Studies on sex differentiation and sex determination in amphibians. IV. The geographical distribution of the sex races of the European grass frog (Rana temporaria, L.). A contribution to the problem of the evolution of sex

Studies on sex differentiation and sex determination in amphibians. III. rudimentary hermaphroditism and Y chromosome in Rana temporaria

Studies on sex differentiation and sex determination in amphibians. III. rudimentary hermaphroditism and Y chromosome in Rana temporaria

Studies on sex differentiation and sex determination in amphibians. I. Development and sexual differentiation of the gonads of Rana sylvatica

Studies on sex differentiation and sex determination in amphibians. I. Development and sexual differentiation of the gonads of Rana sylvatica