Sex Determination In Turtles Research Articles

Screening of temperature-responsive signalling molecules during sex differentiation in Asian yellow pond turtle (Mauremys mutica)

BackgroundThe Asian yellow pond turtle (Mauremys mutica) is an important commercial freshwater aquaculture species in China. This species is a highly sexually dimorphic species, with males growing at a faster rate than females and exhibits temperature-dependent sex determination (TSD), in which the incubation temperature during embryonic development determines the sexual fate. However, the mechanisms of the sex determination or sex differentiation in the Asian yellow pond turtle are remain a mystery.ResultsTemperature-specific gonadal transcriptomics of the Asian yellow pond turtle were performed during the thermosensitive period (stage 15) using RNA-seq technology to identify candidate genes that initiate gonadal differentiation. We uncovered candidates that were the first to respond to temperature. These candidates were sexually dimorphic in expression, reflecting differences in gonadal (Cirbp, Runx1) and germline differentiation (Vasa, Nanos1, Piwil2), gametogenesis (Hmgb3, Zar1, Ovoinhibitor-like, Kif4), steroid hormone biosynthesis (Hsd17b5, Hsd17b6), heat shock (Dnajb6, Hsp90b1, Hsp90aa1) and transient receptor potential channel genes (Trpm1, Trpm4, Trpm6, Trpv1).ConclusionsOur work will provide important genetic information to elucidate the mechanisms of sex control in the Asian yellow pond turtles, and will contribute important genetic resources for further studies of temperature-dependent sex determination in turtles.

Pengaruh Suhu Inkubasi Terhadap Profil Ekspresi Gen Determinasi Seks Tukik Lepidochelys olivacea

Sex determination in turtles is not only based on genotype, but also depends on the incubation temperature. In addition, sex differentiation takes place during the thermosensitive period (TSP). This review article aims to examine the effect of incubation temperature on the expression profile of the sex determination gene for tortoises. Fertile eggs are incubated at two temperatures, namely the feminine temperature (30-33 ° C), and the masculine temperature (26-27 ° C). The results of bioreproduction and phenotype measurements of hatchlings, namely, embryo growth, incubation time, morphometrics, and locomotor performance of hatchlings were analyzed by regression analysis and Student's t test. The results of analysis from several literatures showed that the expression of aromatase and Rspond 1 genes (which play a role in ovarian differentiation) after TSP from incubated embryos at a higher feminine temperature and different from the masculine temperature. Regarding bioreproduction, the incubation period at the feminine temperature is shorter than the masculine temperature. Likewise embryos grow more rapidly at feminine temperatures. Incubation at feminine temperature had a significant effect on the phenotype of hatchlings, namely carapace width, plastron length and width, long flipper and hind arms, long neck, and flipper swing frequency.

Some like it hot - sex determination in turtles.

A recent study in Science reports the mechanism by which temperature affects sex determination in the red-eared slider turtle, Trachemys scripta elegans.

A new approach for measuring temperature inside turtle eggs.

For turtles, the thermal environment experienced during development plays critical roles in many biological processes. While the temperature inside an egg is assumed to match the substrate temperature, many factors such as evaporative cooling, metabolic heating and the insulating properties of extra-embryonic components can lead to thermal differences. However, no method developed to date has allowed for measurement of the embryonic temperature in live chelonian eggs. We designed a thermocouple-based technique to measure embryonic temperature, achieving 94% survival in Trachemys scripta This methodology may be applicable to other reptile species. We found that, while the temperature in the substrate adjacent to the eggshell accurately reflects the internal egg temperature, it differs from air temperature (∼2°C) in a moisture-dependent manner. Our results demonstrate that external egg temperature, but not air temperature, is suitable for assessing the effects of temperature on biological processes, which could be critical when considering that processes such as temperature-dependent sex determination in turtles occurs within a 4°C window.

Embryonic PCB exposure alters phenotypic, genetic, and epigenetic profiles in turtle sex determination, a biomarker of environmental contamination.

In species with temperature-dependent sex determination, embryonic gonadal differentiation can be modified by exposure to exogenous chemicals such as environmental contaminants. Although phenotypic outcomes of such events are well documented, the underlying molecular mechanisms are rarely described. Here we examine the genetic and epigenetic effect of the embryonic exposure to polychlorinated biphenyls (PCBs) on gonad differentiation in red-eared slider turtles (Trachemys scripta). Some PCB congeners are without effect whereas others synergize to alter sex determination in this species. Application of two potent PCB congeners alter the physiological processes of gonad development normally dictated by the male-producing temperature (MPT), resulting sex ratios significantly biased toward female hatchlings. Of these PCB-induced females, oviduct formation is prominently distorted regardless of ovary development. Further, gonadal expression of ovarian markers, aromatase, FoxL2, and Rspo1, is activated whereas testicular markers, Dmrt1 and Sox9, are suppressed compared with typical expression patterns observed at MPT. DNA methylation profiles of the aromatase promoter in PCB-treated gonads do not follow the typical methylation pattern observed in embryos incubating at female-producing temperature. Rather, the MPT-typical methylation profiles is retained despite the induced ovarian formation. Overall, our studies demonstrate that PCB exposure alters the transcriptional profiles of genes responsible for gonadal differentiation but does not re-establish the epigenetic marks of the aromatase promoter normally set by incubation temperatures in embryonic gonads.

Effect of daily water treatment on hatchling sex ratios in a turtle with temperature‐dependent sex determination

Some previous studies indicate that the local hydric environment may influence sex determination in turtles with temperature-dependent sex determination. In this study, the effect of a daily application of 0.77 mL of ddH(2)0 per egg using an incubation temperature of 29.1 degrees C was examined during the temperature-sensitive period for two consecutive nesting seasons. This regimen yielded sex ratios of 11.8 and 11.1% male in control groups not receiving water supplementation, whereas daily water treatments resulted in sex ratios of 86.7 and 45.7% male during the 2006 and 2007 nesting seasons, respectively. The results indicate that daily water treatments significantly influenced sex ratios (P<0.001). In addition to providing insight on the physiology of sex determination, these results could have implications for studies predicting sex ratios from nests on natural nesting beaches that are periodically exposed to rain.

Gonadal expression of Sf1 and aromatase during sex determination in the red-eared slider turtle ( Trachemys scripta), a reptile with temperature-dependent sex determination

Many egg-laying reptiles have temperature-dependent sex determination (TSD), where the offspring sex is determined by incubation temperature during a temperature-sensitive period (TSP) in the middle third of development. The underlying mechanism transducing a temperature cue into an ovary or testis is unknown, but it is known that steroid hormones play an important role. During the TSP, exogenous application of estrogen can override a temperature cue and produce females, while blocking the activity of aromatase ( Cyp19a1), the enzyme that converts testosterone to estradiol, produces males from a female-biased temperature. The production of estrogen is a key step in ovarian differentiation for many vertebrates, including TSD reptiles, and temperature-based differences in aromatase expression during the TSP may be a critical step in ovarian determination. Steroidogenic factor-1 ( Sf1) is a key gene in vertebrate sex determination and regulates many steroidogenic enzymes, including aromatase. We find that Sf1 and aromatase are differentially expressed during sex determination in the red-eared slider turtle, Trachemys scripta elegans. Sf1 is expressed at higher levels during testis development while aromatase expression increases during ovary determination. We also assayed Sf1 and aromatase response to sex-reversing treatments via temperature or the modulation of estrogen availability. Sf1 expression was redirected to low-level female-specific patterns with feminizing temperature shift or exogenous estradiol application and redirected to more intense male-specific patterns with male-producing temperature shift or inhibition of aromatase activity. Conversely, aromatase expression was redirected to more intense female-specific patterns with female-producing treatment and redirected toward diffuse low-level male-specific patterns with masculinizing sex reversal. Our data do not lend support to a role for Sf1 in the regulation of aromatase expression during slider turtle sex determination, but do support a critical role for estrogen in ovarian development.

Adrenal–kidney–gonad complex measurements may not predict gonad‐specific changes in gene expression patterns during temperature‐dependent sex determination in the red‐eared slider turtle (Trachemys scripta elegans)

Many turtles, including the red-eared slider turtle (Trachemys scripta elegans) have temperature-dependent sex determination in which gonadal sex is determined by temperature during the middle third of incubation. The gonad develops as part of a heterogenous tissue complex that comprises the developing adrenal, kidney, and gonad (AKG complex). Owing to the difficulty in excising the gonad from the adjacent tissues, the AKG complex is often used as tissue source in assays examining gene expression in the developing gonad. However, the gonad is a relatively small component of the AKG, and gene expression in the adrenal-kidney (AK) compartment may interfere with the detection of gonad-specific changes in gene expression, particularly during early key phases of gonadal development and sex determination. In this study, we examine transcript levels as measured by quantitative real-time polymerase chain reaction for five genes important in slider turtle sex determination and differentiation (AR, ERalpha, ERbeta, aromatase, and Sf1) in AKG, AK, and isolated gonad tissues. In all cases, gonad-specific gene expression patterns were attenuated in AKG versus gonad tissue. All five genes were expressed in the AK in addition to the gonad at all stages/temperatures. Inclusion of the AK compartment masked important changes in gonadal gene expression. In addition, AK and gonad expression patterns are not additive, and gonadal gene expression cannot be predicted from intact AKG measurements.

Sox8 is expressed at similar levels in gonads of both sexes during the sex determining period in turtles.

A critical gene involved in mammalian sex determination and differentiation is the Sry-related gene Sox9. In reptiles, Sox9 resembles that of mammals in both structure and expression pattern in the developing gonad, but a causal role in male sex determination has not been established. A closely related gene, Sox8, is conserved in human, mouse, and trout and is expressed in developing testes and not developing ovaries in mouse. In this study, we tested the possibility of Sox8 being important for sex determination or sex differentiation in the red-eared slider turtle Trachemys scripta, in which sex is determined by egg incubation temperature between stages 15 and 20. We cloned partial turtle Sox8 and anti-Müllerian hormone (Amh) cDNAs, and analyzed the expression patterns of these genes in developing gonads by reverse transcriptase-polymerase chain reaction and whole-mount in situ hybridization. While Amh is expressed more strongly in males than in females at stage 17, Sox8 is expressed at similar levels in males and females throughout the sex-determining period. These observations suggest that differential transcription of Sox8 is not responsible for regulation of Amh, nor responsible for sex determination in turtle.

Turtle sex determination assay: Mass balance and responses to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin and 3,3′,4,4′,5‐pentachlorobiphenyl

Polyhalogenated hydrocarbons have been implicated in the anomalous sexual differentiation of mammals and reptiles. Here, a temperature-sensitive turtle sex determination assay using the red-eared slider (Trachemys scripta elegans) was used to determine the estrogenic or antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (PCB-126). Neither TCDD nor PCB-126 showed a statistically significant difference in the resulting sex ratios (Fisher's exact test, p < 0.45). As a consequence of the dosing technique (eggshell spotting), the shell retained 90 and 96% of the dose for PCB- 126 and TCDD, respectively, similar to retention of estradiol- 17beta. However, the dosing allowed transfer of sufficient chemical to achieve tissue concentrations that were greater than most concentrations reported for environmentally incurred residues. Similar relative mass distributions of PCB-126 and TCDD were observed in albumin (14-20%), yolk (55-70%), and embryo (16-25%). Relative concentration distributions in the embryo approached those in the yolk, 37 to 40% and 40 to 52%, respectively, while relative concentrations in the albumin remained at 11 to 20%. Lipid-normalized TCDD and PCB-126 concentrations were 30- to 40-fold greater in the embryo than in the yolk. It is hypothesized that nonpassive partitioning processes may have occurred in the embryo.

Expression of P450(arom) in Malaclemys terrapin and Chelydra serpentina: a tale of two sites.

The formation of estrogens from androgens in all vertebrates is catalyzed by the "aromatase" complex, which consists of a membrane bound P(450) enzyme, P(450) aromatase (which binds the androgen substrate and inserts an oxygen into the molecule), and a flavoprotein (NADPH-cytochrome P450 reductase). Among vertebrates, the two major sites of aromatase expression are the brain and gonads. Given the importance of estrogen in reptile sex determination, we set out to examine whether P450arom was involved in the initiation and/or stabilization of sex determination in turtles. We examined the expression of aromatase activity in the brain and gonads of two turtle species exhibiting temperature dependent sex determination (TSD), the diamondback terrapin (Malaclemys terrapin), and the common snapping turtle (Chelydra serpentina). Estradiol when applied at stage 14 of the terrapin induces expression of aromatase in the gonad of embryos incubated at male temperatures (26.5 degrees C). The level of expression is similar to that of a normal embryonic ovary. When applied at stage 22, estradiol does not induce aromatase expression in the terrapin. The xenoestrogen, nonylphenol, sex reverses terrapin embryos at 26.5 degrees C. Letrazole, a nonsteroidal aromatase inhibitor, suppresses aromatase activity in the brain at either incubation temperature. Ovotestes are produced by letrazole administration in the terrapin when incubated at 30.5 degrees C. In the snapping turtle at stage 23, gonadal and brain aromatase activity in embryos incubated at female temperatures (30.5 degrees C) is nearly half that exhibited in terrapin embryos at the same temperature. Moreover, letrazole administration suppresses aromatase expression to nearly basal levels. At male incubation temperatures (26.5 degrees ), brain aromatase expression is nearly three times higher than at female temperatures, while gonadal expression levels are nearly one third lower. However, the gonadal expression levels at male temperatures in the snapping turtle are nearly 25 times higher than that found in the terrapin. Estradiol administration elevates this level nearly three fold. These data suggest that is not merely the expression of aromatase that is important for ovarian development, but that the level of expression may be more important.

Temperature-dependent sex determination: the interplay of steroid hormones and temperature.

Sex determination is the product of coordinated gene expression. Mutational analyses have yielded great progress in our understanding of mammalian sex determination, and insight into the evolution of this sex chromosome system would be valuable. Mammals arose from turtle-like reptiles, and in many turtles the incubation temperature of the egg determines the sex of the offspring, a process known as temperature-dependent sex determination. There is mounting evidence that sex steroid hormones are the physiological equivalent of incubation temperature and serve as the proximate trigger for male and female sex determination. Temperature appears to accomplish this end by acting on genes coding for steroidogenic enzymes and sex steroid hormone receptors. The ability to manipulate sex determination in turtles both by temperature and by sex steroid hormones extends our understanding of the evolution as well as the physiology and molecular biology of sex determination.

The role of estrogen in turtle sex determination and the effect of PCBs.

In the current model of vertebrate sex determination and sexual differentiation, gonadal sex is fixed at fertilization by specific chromosomes, a process known as genotypic sex determination (GSD). Only after the gonad is formed do hormones begin to exert an influence that modifies specific structures that eventually will differ the sexes. Many egg-laying reptiles do not exhibit GSD but rather depend on the temperature of the incubating egg to determine the gonadal sex of the offspring, a process termed temperature-dependent sex determination (TSD). Research on TSD indicates that sex determination in these species is fundamentally different in at least one way. Gonadal sex is not irrevocably set by the genetic composition inherited at fertilization but depends ultimately on which genes encoding for steroidogenic enzymes and hormone receptors are activated during the midtrimester of embryonic development by temperature. Incubation temperature modifies the activity as well as the temporal and spatial sequence of enzymes and hormone receptors such that sex-specific hormone milieus, created in the urogenital system of the developing embryo, determine gonad type. Estrogen is the physiologic equivalent of incubation temperature and the proximate cue that initiates female sex determination. There is increasing evidence that some polychlorinated biphenyl (PCB) compounds are capable of disrupting reproductive and endocrine function in fish, birds, and mammals, including humans. Reproductive disorders resulting from exposure to these xenobiotic compounds may include reductions in fertility, hatch rate in fish and birds, and viability of offspring, as well as alterations in hormone levels or adult sexual behaviors, all of which have further implications, particularly in wildlife population dynamics. Research on the mechanism through which these compounds may be acting to alter reproductive function indicates estrogenic activity, by which the compounds may be altering sexual differentiation. In TSD turtles, the estrogenic effect of some PCBs reverses gonadal sex in individuals incubating at an otherwise male-producing temperature. Furthermore, certain PCBs are synergistic in their effect at very low concentrations.

The Role of Estrogen in Turtle Sex Determination and the Effect of PCBs

The Role of Estrogen in Turtle Sex Determination and the Effect of PCBs

Patterns of temperature‐dependent sex determination in turtles

AbstractAmong reptiles that show temperature‐dependent sex determination, sex ratios vary across constant incubation temperatures in ways sufficiently predictable to allow classification into patterns. One common pattern shows low temperatures yielding only males and high temperatures yielding only females. Another common pattern has low as well as high temperatures yielding only or mostly females and some intermediate temperatures yielding mostly males. Patterns tend to be associated with the direction of sexual dimorphism in adult size, especially for species with strong dimorphism.Pivotal temperatures (those yielding 1:1 sex ratios) within the best‐documented species and genera tend to increase with both latitude and longitude across central and southern North America. These geographic trends probably reflect factors that affect nest temperatures (duration of growing season, insolation, and prevailing amounts of shading by vegetation).Data from a population of the alligator snapping turtle (Macroclemys temminckii) suggest that some embryos are temperature‐independent females because these individuals become females even when they are shifted among male‐producing temperatures during development. These individuals are also more frequent in clutches of small eggs. In this and several other species, no constant incubation temperatures yield more than 75% males. © 1994 Wiley‐Liss, Inc.

PCBs as Environmental Estrogens: Turtle Sex Determination as a Biomarker of Environmental Contamination

PCBs as Environmental Estrogens: Turtle Sex Determination as a Biomarker of Environmental Contamination

Genetic contribution to sex determination in turtles with environmental sex determination

SummaryIn many reptiles, sex determination is temperature-sensitive. This phenomenon has been shown to take place in the laboratory as well as in nature, but its effect on natural populations remains questionable. In the turtleEmys orbicularis, the effects of temperature override a weak mechanism of genetic sex determination which is revealed in incubation at pivotal temperature. At this temperature, the sexual phenotype is concordant with the expression of the serologically defined H-Y antigen (H-Ys) in non-gonadal tissues; males are H-Ys negative (H-Y−) whereas females are H-Ys positive (H-Y+). To estimate the importance of sexual inversion (sexual phenotype and H-Ys expression discordant) in populations of Brenne (France), the frequencies of male and female sexual phenotypes among H-Ys phenotypes were determined. The frequencies of sex reversed individuals are low, only 6 % of phenotypic females being H-Y−and 11 % of phenotypic males being H-Y+. According to these data, two theoretical models have been constructed to estimate the contribution to sex determination of individuals in relation to their genotype. The first model excludes any influence of incubation temperature and sexual phenotype on the fitness of individuals. The second one considers that these parameters influence fitness because this model has been previously shown to favour environmental sex determination. In both models, it appears that sex determination can be viewed as genotypic and monogenic with some individuals sexually inverted by theaction of temperature. One category of homozygous animals differentiates mainly into one sex, and the heterozygous animals differentiate mainly into the other sex. The second category of homozygotes has a low frequency in the populations and can differentiate as male or female without high constraint. Then it is estimated that in Brenne approximately 83% of the eggs are incubated in conditions allowing the genetic component to influence sex determination.

Synergism between temperature and estradiol: A common pathway in turtle sex determination?

In many reptiles, the temperature at which the eggs are incubated determines the sex of the hatchlings. Administration of estradiol will counteract the masculinizing effects of a male-producing temperature, resulting in female hatchlings. To address whether temperature and estrogen are biologically equivalent, two experiments were conducted with the red-eared slider turtle, Trachemys scripta. In the first experiment, varying dosages of estrogen were administered at Stage 17 (the middle of the temperature-sensitive window) to eggs maintained at two temperatures, 26 degrees C (which normally produces all males) and 28.2 degrees C (which produces mostly males but lies at the threshold of the transition from male- to female-producing temperatures). Results indicate that estrogen and temperature exert a synergistic effect on sex determination. In the second experiment, estrogen was administered at different stages of embryonic development. The results indicate an estrogen-sensitive period ranging from Stage 14 through Stage 21, a period similar to the temperature-sensitive period for this species. The results of these experiments are consistent with the hypothesis that temperature and estradiol act in a common pathway in temperature-dependent sex determination.

Sensitive periods during embryogeny for hormonally induced sex determination in turtles

Administration of exogenous estradiol between embryonic stages 10 and 19 inclusive caused embryos of snapping turtles (a species which exhibits temperature-dependent sex determination) to develop ovaries rather than testes, while administration of hormone either prior to stage 10 or after stage 22 did not affect the predicted sex ratio of offspring. Thus the sensitive period for hormonal effects on gonadal development apparently coincides with the period of normal gonadal differentiation. In light of these and prior results use of the term “sex reversal” when discussing the effects of hormones on gonadal differentiation may be a misnomer. We suggest the alternative phrase “hormonally induced gonadal differentiation” (HGD) as a substitute. Results also indicate that variations in the timing of injections among studies may account fur at least some of the interspecific differences previously reported for effects of exogenous hormones on gonad differentiation.

H-Y Antigen and Sex-Determination in Turtles

H-Y antigen was investigated in 14 turtle species belonging to five different families. In 13 species the female was typed as H-Y antigen positive, only in Chinemys reevesi was the male H-Y antigen positive. Since in all vertebrate species studied till now, the expression of H-Y antigen is strictly correlated with the heterogametic sex, it can be assumed that in turtles both types of sex determining mechanisms are realized, namely the ZZ/ZW-and the XX/XY mechanism; both mechanisms are realized in species belonging to one and the same turtle family. However, most turtle species seem to be endowed with a ZZ/ZW sex determining mechanism.