Expression of Dmrt1 in a turtle with temperature-dependent sex determination.

There is a variety of sex determining mechanisms among vertebrates. Many reptiles possess temperature-dependent sex determination (TSD), in which the incubation temperature of the egg determines the sex of the hatchling. The red-eared slider turtle, Trachemys scripta has often been used as a model system for examining the physiology of TSD. In the current study, the expression of Dmrt1 was examined during TSD in this turtle. Dmrt1 is a putative regulator of sex determination/differentiation and has been identified in a variety of vertebrates, including fishes, amphibians, reptiles, birds, and mammals. Specifically, Dmrt1 has been shown to be up-regulated in a male-specific pattern during embryonic development in many vertebrates. In the current study, the expression patterns of Dmrt1 were examined in the developing adrenal-kidney-gonad complexes of T. scripta during embryonic development. Using a quantitative competitive RT-PCR, Dmrt1 was shown to be up-regulated during the thermosensitive period of sex determination in males. In contrast, levels of Dmrt1 remained low in females throughout the thermosensitive period. These data suggest that the up-regulation of Dmrt1 may play a role in male sex determination/sex differentiation during TSD in T. scripta.

Developmental expression of steroidogenic factor 1 in a turtle with temperature-dependent sex determination.

A variety of reptiles possess temperature-dependent sex determination (TSD) in which the incubation temperature of a developing egg determines the gonadal sex. Current evidence suggests that temperature signals may be transduced into steroid hormone signals with estrogens directing ovarian differentiation. Steroidogenic factor 1 (SF-1) is one component of interest because it regulates the expression of steroidogenic enzymes in mammals and is differentially expressed during development of testis and ovary. Northern blot analysis of SF-1 in developing tissues of the red-eared slider turtle (Trachemys scripta), a TSD species, detected a single primary SF-1 transcript of approximately 5.8 kb across all stages of development examined. Analysis by in situ hybridization indicated nearly equivalent SF-1 expression in early, bipotential gonads at male (26 degrees C)- and female (31 degrees C)-producing incubation temperatures. In subsequent stages, as gonadal sex first becomes histologically distinguishable during the temperature-sensitive period, SF-1 expression increased in gonads at a male-producing temperature and decreased at a female-producing temperature, suggesting a role for SF-1 in the sex differentiation pathway. SF-1 message was also found in adrenal and in the periventricular region of the preoptic area and diencephalon, but there was no apparent sex bias in these tissues at any stage examined. The overall developmental pattern of SF-1 mRNA expression in T. scripta appears to parallel that found in mammals, indicating possible homologous functions.

Steroid levels and steroid metabolism in relation to early gonadal development in the tilapia Oreochromis niloticus (Teleostei: cyprinoidei).

Sex steroid levels and steroid metabolism were investigated in relation to early gonadal development in a mixed sex population of the tilapia Oreochromis niloticus. Androstenedione (AD), testosterone (T), 11-ketotestosterone (KT), and estradiol (E2) were quantified by radioimmunoassay (RIA) of whole body extracts. Androstenedione metabolism was assessed by incubations in vitro with 3H-AD and metabolites were identified by thin-layer chromatography coupled with radioisotope image analysis. Histology revealed the presence of gonadal structures at 15 days postfertilization (dpf) and ovaries at 36 dpf, with other individuals exhibiting undifferentiated gonads containing germinal cells, presumably eventual testes. Androgen levels were initially high in eggs then decreased severalfold prior to the emergence of gonads. A transient increase in the levels of T and KT occurred at 22 dpf. Levels of E2 were either low or undetectable except for a transient increase (43 dpf) after ovaries were present. Levels of T approached bimodality from 57 to 64 dpf. Steroid metabolism generally increased throughout development. Metabolites were generally similar, consisting of T predominantly as well as 5beta-reduced androgen derivatives and 11-oyxgenated derivatives. Estriol was tentatively identified. Conjugated steroids were not formed. Two types of steroid metabolic profiles occurred at 50 dpf. These results demonstrate that changes in the steroidogenic profile occur during early transitions of gonadal development. Notably, (1) steroid biosynthetic capacity preceeds gonadal differentiation, (2) evidence for estrogens occurs after ovarian development has begun, and (3) bimodality of levels of T and differential steroid metabolism later in development may reflect the onset of sexual divergence.

Temperature-dependent sex determination in the red-eared slider turtle, Trachemys scripta.

Temperature-dependent sex determination (TSD) in the red-eared slider turtle, Trachemys scripta, has been the subject of a variety of past studies. Incubation temperature appears to affect sex determination in a dose-dependent fashion. This suggests that temperature could be affecting a dosage-sensitive element in the sex-determination cascade. Sex determination in T. scripta is sensitive to estrogen, and data from many studies support the hypothesis that endogenous estrogen production may be involved in female sex determination. However, this hypothesis has not yet been evaluated through aromatase expression studies in this species. Several recent studies have cloned cDNAs for genes that could be involved in sex determination and/or sex differentiation. The cDNAs for SF-1 and MIS have been cloned in T. scripta, indicating that these may represent conserved elements in the sex-determination/sex-differentiation cascade of reptiles. The SOX9 cDNA also has been cloned in T. scripta (Spotila et al., '98), and it shows a sex-specific expression pattern. Future studies targeted at aromatase expression as well as the expression of factors such as SOX9, SF-1, and MIS will begin to provide a more comprehensive picture of the events involved in TSD in T. scripta. Further, such studies could help pinpoint the temperature-sensitive element(s).

Cloning and in situ hybridization analysis of estrogen receptor in the developing gonad of the red-eared slider turtle, a species with temperature-dependent sex determination.

Many reptiles exhibit temperature-dependent sex determination where the incubation temperature of the egg determines the gonadal sex of the individual. If exogenous estrogen is administered during the temperature-sensitive period to embryos incubating at a male-producing temperature, the temperature effects can be overridden and females will be produced. Inhibiting production of endogenous estrogens at female-biased incubation temperatures results in embryos developing as males rather than females. Thus, estrogen-estrogen receptor-dependent mechanisms appear to play a key role in female sex determination. The present study characterized the expression of the estrogen receptor during the critical period of temperature sensitivity in the red-eared slider turtle, Trachemys scripta. Polymerase chain reaction was used to amplify estrogen receptor cDNA. A portion of the estrogen receptor cDNA was used to produce probes for in situ hybridization analyses to localize and quantitate levels of estrogen receptor mRNA at different stages of development in embryos from different incubation temperatures. Estrogen receptor mRNA is expressed in the gonadal tissues of both putative males and putative females even before the gonads begin to resolve as ovaries or testes. There is a greater abundance of estrogen receptor mRNA in putative females at the beginning of the temperature-sensitive period as compared to putative males. In embryos from a female-producing incubation temperature, levels of estrogen receptor mRNA are higher in the beginning of the temperature-sensitive window compared to levels after the ovary is differentiated. These results support the hypothesis that estrogen-estrogen receptor dependent processes are important during sex determination and gonadal differentiation in temperature-dependent sex determination.

Steroid-induced sex determination at incubation temperatures producing mixed sex ratios in a turtle with TSD.

Previous studies have shown that exogenous steroid hormones can affect sex determination in reptiles with temperature-dependent sex determination (TSD). These studies have also suggested that the sensitivity of TSD to exogenous steroids may vary with incubation temperature. The majority of these studies, however, have utilized incubation temperatures producing all males or all females in the control groups, rather than temperatures which produced mixed sex ratios in control groups. The goals of the current study were to examine the effects of steroids on sex determination in a turtle (Trachemys scripta) at temperatures which produced mixed sex ratios in the control groups. Collectively, the results of single-treatment experiments indicate that at incubation temperatures producing mixed sex ratios in control groups, (1) estradiol-17 beta, tamoxifen, norethindrone, and testosterone all showed a similar "type" of effect (i.e., feminizing) as in previous studies utilizing male-producing temperatures, (2) sex determination has significantly increased sensitivity to estradiol-17 beta in comparison to its effect at temperatures producing all males, and (3) sex determination is sensitive to the masculinizing effects of dihydrotestosterone (DHT) (in previous studies utilizing female-producing temperature DHT did not affect sex determination). Last, a set of double-treatment experiments was performed in which eggs received both estradiol-17 beta and DHT treatments. No significant increases in the production of males were detected. Significant increases in the production of females were detected, but only in the groups receiving the highest dosage of estradiol-17 beta (1.0 micrograms).(ABSTRACT TRUNCATED AT 250 WORDS)

Putative aromatase inhibitor induces male sex determination in a female unisexual lizard and in a turtle with temperature-dependent sex determination.

Treatment of developing embryos of two diverse species of reptiles with fadrozole (a potent and specific nonsteroidal inhibitor of aromatase activity in mammals) resulted in the induction of male sex determination. In the first experiment, males were produced in an all-female parthenogenic species of lizard (Cnemidophorus uniparens). In the second experiment, male sex determination was induced in a turtle (Trachemys scripta) with temperature-dependent sex determination. The results support the hypothesis that the endogenous production of oestrogen may represent a pivotal step in the sex determination cascade of reptiles. Further, the production of male C uniparens indicates that the genes required for male sexual differentiation have not been lost in this parthenogenic lizard.

Temperature-dependent sex determination in reptiles: proximate mechanisms, ultimate outcomes, and practical applications.

In many egg-laying reptiles, the incubation temperature of the egg determines the sex of the offspring, a process known as temperature-dependent sex determination (TSD). In TSD sex determination is an "all or none" process and intersexes are rarely formed. How is the external signal of temperature transduced into a genetic signal that determines gonadal sex and channels sexual development? Studies with the red-eared slider turtle have focused on the physiological, biochemical, and molecular cascades initiated by the temperature signal. Both male and female development are active processes--rather than the organized/default system characteristic of vertebrates with genotypic sex determination--that require simultaneous activation and suppression of testis- and ovary-determining cascades for normal sex determination. It appears that temperature accomplishes this end by acting on genes encoding for steroidogenic enzymes and steroid hormone receptors and modifying the endocrine microenvironment in the embryo. The temperature experienced in development also has long-term functional outcomes in addition to sex determination. Research with the leopard gecko indicates that incubation temperature as well as steroid hormones serve as organizers in shaping the adult phenotype, with temperature modulating sex hormone action in sexual differentiation. Finally, practical applications of this research have emerged for the conservation and restoration of endangered egg-laying reptiles as well as the embryonic development of reptiles as biomarkers to monitor the estrogenic effects of common environmental contaminants.

Estrogen- and temperature-induced medullary cord regression during gonadal differentiation in a turtle.

Gonadal differentiation associated with estrogen-induced female sex determination was examined in a turtle with temperature-dependent sex determination, and was compared to ovarian differentiation at a female-producing temperature. Freshly ladi eggs of the red-eared slider, Trachemys scripta, were incubated at a male-producing temperature (26 degrees C) and were experimentally manipulated at one of three embryonic stages: stage 15, 17, or 20 (i.e. early, midway, or late in the temperature-sensitive and estrogen-sensitive periods). At those developmental stages, groups of eggs were either: (1) treated with a control solution (95% ethanol) and placed back at the male-producing temperature, (2) treated with 10 micrograms of estradiol-17 beta and placed back at the male-producing temperature, or (3) shifted to a female-producing temperature (31 degrees C). Additionally, a control group of freshly laid eggs was continually incubated at 31 degrees C throughout embryonic development. To examine morphological events occurring after the treatments, a subset of embryos from each group was examined at the time of the treatment and at 1-2 stage intervals following the treatments. The results indicate that estradiol-17 beta as well as female-producing temperature may ensure female sex determination by facilitating medullary cord regression. Further, the results reveal a chronology of differentiation in which medullary cord regression temporally precedes cortical proliferation.

Steroid hormone-induced male sex determination in an amniotic vertebrate.

In many reptiles, sex is determined by the temperature at which the eggs are incubated (i.e., temperature-dependent sex determination, or TSD). Past studies have shown that exogenous steroid hormones can override the effects of temperature and induce female sex determination. However, past attempts to induce male sex determination have consistently failed. In the present study, sex determination was studied in a turtle with TSD. By utilizing an incubation temperature regimen that resulted in approximately a 1:1 sex ratio in the control group, sex determination was shown to be sensitive to both exogenous androgen and estrogen treatments: androgen induced the production of male hatchlings, whereas estrogen induced the production of female hatchlings. This is the first report of an amniotic vertebrate in which an exogenous steroid hormone induces male sex determination.

Serum gonadotropins and gonadal steroids associated with ovulation and egg production in sea turtles.

Changes in serum concentrations of gonadotropins and gonadal steroids during the periovulatory period were monitored in green, Chelonia mydas, and loggerhead, Caretta caretta, sea turtles. Turtles were from natural populations that nest on a coral island on the Great Barrier Reef. After nesting, each turtle was transferred to a holding tank and held for a maximum of 8 days. A time series of blood samples was obtained from each of five sea turtles (three C. mydas and two C. caretta) starting immediately after nesting and then at approximately 12-hr intervals until the time of release. Prior to release back into the ocean, each turtle was examined by laparoscopy to verify that ovulation had occurred. Serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (PRO), and testosterone (T) in both species exhibited significant changes during this period. Surges of FSH, LH, and PRO were evident within approximately 20 to 50 hr after each turtle had nested. The significant change in FSH concentration during the periovulatory period is the first such report for a reptile. Coincident with maximal concentrations of FSH, LH, and PRO was a decline in T concentrations in both species. Estradiol-17 beta concentrations were near or below assay sensitivity in the C. mydas, whereas those in the C. caretta were detectable but exhibited no significant changes. The dynamic changes in FSH, LH, PRO, and T concentrations are consistent with the hypothesis that these hormones facilitate specific physiological events during ovulation and egg production.

Specificity of steroid hormone-induced sex determination in a turtle.

The specificity of steroid hormone-induced sex determination was investigated in the red-eared slider, Trachemys scripta, a turtle with temperature-dependent sex determination. All eggs were incubated at either a female-producing temperature (31 degrees C) or a male-producing temperature (26 degrees C) and received control or experimental treatments at stage 17-18 of embryonic development. A variety of treatments induced female sex determination at the male-producing temperature. Oestradiol-17 beta, diethylstilboestrol (DES) (an oestrogen agonist) and norethindrone (NET) (a progestin with reputed oestrogenic as well as anti-oestrogenic properties) were the most effective in inducing female sex determination. Other reputed oestrogen antagonists/partial agonists (i.e. tamoxifen, nafoxidine and clomiphene citrate) were also capable of inducing female sex determination, but to a lesser extent. A high dosage of testosterone resulted in the production of some females (7 of 15 hatchlings) whereas dihydrotestosterone had no detectable effect on sex determination. This latter finding suggests that testosterone could be acting via aromatization to oestradiol-17 beta. A few females resulted from eggs that had been treated with aromatase inhibitor, 1,4,6-androstatrien-3,17-dione (ATD) (3 of 97), the antiandrogen hydroxyflutamide (1 of 55) and progesterone (3 of 36), suggesting the possibilities of non-specific effects of these compounds when used in large dosages. Alternatively, metabolites of these compounds may be oestrogenic. Collectively, the results at the male-producing temperature are consistent with the hypothesis that steroid-induced female sex determination is mediated via an oestrogen-specific receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Sites of estrogen uptake in embryonic Trachemys scripta, a turtle with temperature-dependent sex determination.

Female sex determination can be induced in embryonic red-eared slider turtles (Trachemys scripta) by exogenous estrogen, as well as by incubation at warm temperature. In the present study, estrogen target areas were identified in embryos before (stage 15), during (stage 18), and after (stage 22) the critical period for sex determination. Both hyperfilm and emulsion autoradiography were used to localize tritium accumulation after the injection of radiolabeled 17 beta-estradiol. Site-specific tritium-labelling was found at all stages, notably in the mesonephros at stage 15, in the mesonephros and oviduct at stage 18, and in the mesonephros, oviduct, and the interrenal gland at stage 22. Few if any cells in the gonad were tritium-labeled at any stage. The large number of estrogen-concentrating cells in the mesonephros and interrenal and the lack of binding to gonadal tissues indicates that estrogen action on gonadal differentiation during the period of sex determination may be indirect.

Hydroxysteroid dehydrogenase activity associated with sexual differentiation in embryos of the turtle Trachemys scripta.

Many turtles exhibit temperature-dependent sex determination. We have examined the hypothesis that incubation temperature causes a differential expression of steroidogenic enzymes in embryonic turtles. The activities of three steroidogenic enzymes were studied histochemically in turtle (Trachemys scripta) embryos at different developmental stages: sexually undifferentiated (Stage 15), differentiating (Stage 17), and differentiated (Stage 26; i.e., hatchling). Steroidogenic enzymes were detected in several tissues prior to, during, and after gonadal differentiation in embryos incubated at both male-producing and female-producing temperatures. In all embryos, ene-5-3 beta-hydroxysteroid dehydrogenase (HSDH) was detected only in adrenal tissue. 3 alpha-HSDH was localized in adrenal tissue, as well as in the mesonephros and liver. 17 beta-HSDH was evident in mesonephric, hepatic, and gut tissues. In hatchlings, ene-5-3 beta-HSDH and 3 alpha-HSDH were evident in the adrenal gland, whereas 3 alpha-HSDH and 17 beta-HSDH were present in the mesonephros and liver. While there was some variation in the activities of these enzymes during development, no temperature-specific pattern was apparent. At no stage were the enzymes observed in genital ridge/gonad. Our results show that T. scripta embryos possess enzymes necessary for steroid hormone synthesis. The segregated distributions of the enzymes suggest that a multi-organ regulatory system may mediate embryonic steroidogenesis. Our results do not indicate the genital ridge/gonad the principle site of steroid synthesis, although it may possess other enzymes that influence steroidogenesis.

Chronology and morphology of temperature-dependent sex determination.

Temperature sensitivity and gonadal differentiation were studied in a turtle, Trachemys scripta, with temperature-dependent sex determination. Sex determination was sensitive to both the duration and magnitude of incubation temperature. Temperature exerted an "all or none" effect on the ovarian or testicular nature of most gonads, but affected the length of ovaries in a graded fashion. Collectively, the results indicate that sex determination is controlled by the quantitative effect of temperature during a period beginning prior to (histologically detectable) sexual differentiation and extending to a time when initial sex specific changes are evident in the gonads.

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.

Estrogen and sex reversal in turtles: a dose-dependent phenomenon.

Exogenous estradiol benzoate (EB) or estradiol-17 beta (E2) caused dose-dependent gonadal feminization of slider turtle (Trachemys scripta) embryos incubated at a male-producing temperature (26 degrees), suggesting that sex reversal requires a threshold dosage of these hormones. Even at dosages resulting in mixtures of males and females, nearly all hatchlings had normal-appearing ovaries or testes. Only 7 of 241 hatchlings had gonads that had not differentiated fully into ovaries or testes. Thus, with rare exception, estrogens exerted an "all or none" effect. The transport of hormone into the embryo varied with mode of administration and E2 was more effective than was EB at the lowest dosage used. These studies suggest either that exogenous estrogen is capable of coordinating cortical/medullary development in the gonad so that intersexes are prevented or that estrogen acts "upstream" of the developmental processes responsible for coordinating gonadal development.

Sex-determining potencies vary among female incubation temperatures in a turtle.

Reptiles whose sex is determined by incubation temperature typically exhibit all-male or all-female sex ratios over a wide range of incubation temperatures. The question arises as to whether the various all-female temperatures (or the various all-male temperatures) are equivalent in their "potency", or capacity to effect female determination. In map turtles, warm incubation temperatures produce all females and cool ones produce all males. We compared sex determining potencies of two all-female temperatures, 31 degrees C and 32.5 degrees C, by incubating eggs first at a male-producing temperature (26 degrees C) and then shifting them to the warm temperatures. The resulting sex ratio was significantly more male biased in the 26 degrees C----31 degrees C shift than in the 26 degrees C----32.5 degrees C shift, indicating that 32.5 degrees C has the greater female potency. These results point to the possibility that sex determination depends on a quantitative rather than qualitative level of gene expression.

Seasonal changes in serum gonadal steroids associated with migration, mating, and nesting in the loggerhead sea turtle (Caretta caretta).

Adult male loggerhead sea turtles, Caretta caretta, exhibited a "prenuptial" spermatogenic cycle that was coincident with increased concentrations of serum testosterone (T). Serum T was high during the months when migration and mating have been recorded for males. In contrast to females, males appear to be annual breeders. Nine reproductively active female C. caretta (as verified through laparoscopy) were tagged with sonic transmitters and were repeatedly bled prior to migration. Four months prior to the nesting season, the ovaries of reproductively active females had hundreds of vitellogenic follicles of approximately 1.5 cm in diameter (i.e., half the size of ovulatory follicles). Approximately 4-6 weeks prior to migration from feeding grounds to mating and nesting areas, serum estradiol-17 beta (E2) concentrations increased significantly and remained high for approximately 4 weeks, suggesting a period of increased vitellogenesis. During a 1- to 2-week period prior to migration, serum E2 decreased significantly, while serum T concentrations increased (at least) until the time of migration. Serum T, E2, and progesterone (PRO) were elevated during nesting if a turtle was going to nest again during that nesting season. During the last nesting of a season, turtles had low serum concentrations of T, E2, and Pro. The prenuptial pattern of gonadal recrudescence and gonadal steroid production in both male and female C. caretta contrasts with those of many temperate freshwater turtles, and this type of reproductive pattern may have been facilitated by adaptation to a tropical marine environment.