Methyltestosterone alters sex determination in the American alligator (Alligator mississippiensis).

Effects of xenobiotics can be organizational, permanently affecting anatomy during embryonic development, and/or activational, influencing transitory actions during adulthood. The organizational influence of endocrine-disrupting contaminants (EDC's) produces a wide variety of reproductive abnormalities among vertebrates that exhibit temperature-dependent sex determination (TSD). Typically, such influences result in subsequent activational malfunction, some of which are beneficial in aquaculture. For example, 17-αmethyltestosterone (MT), a synthetic androgen, is utilized in tilapia farming to bias sex ratio towards males because they are more profitable. A heavily male-biased hatchling sex ratio is reported from a crocodile population near one such tilapia operation in Guanacaste, Costa Rica. In this study we test the effects of MT on sexual differentiation in American alligators, which we used as a surrogate for all crocodilians. Experimentally, alligators were exposed to MT in ovo at standard ecotoxicological concentrations. Sexual differentiation was determined by examination of primary and secondary sex organs post hatching. We find that MT is capable of producing male embryos at temperatures known to produce females and demonstrate a dose-dependent gradient of masculinization. Embryonic exposure to MT results in hermaphroditic primary sex organs, delayed renal development and masculinization of the clitero-penis (CTP).

Chronology, magnitude and duration of expression of putative sex-determining/differentiation genes in a turtle with temperature-dependent sex determination.

The red-eared slider turtle (Trachemys scripta) possesses temperature-dependent sex determination (TSD) in which the incubation temperature determines gonadal sex. Although a number of mammalian gene homologues have been identified in reptiles with TSD, the exact sex-determining trigger(s) is not known. To date, the current study represents the most comprehensive simultaneous evaluation of the chronology of mRNA expression profiles of putative sex-determining/differentiation genes (Dmrt1, Sox9, Amh, Lhx9, and Foxl2) from gonads incubated at male- and female-producing temperatures in T. scripta. Additionally, sex-reversing treatments with 17ß-estradiol and letrozole were examined. At a male-producing temperature, Dmrt1 expression was sexually dimorphic by stage 17, Sox9 by 19 and Amh by 21. In contrast, Foxl2 did not significantly increase until after the thermosensitive period at a female-producing temperature. Treatment with 17ß-estradiol resulted in reduced gonad size and/or inhibited gonadal development and differentiation. Gene expression was subsequently low in this group. Sex reversal utilizing letrozole failed to produce testes at a female-producing temperature and as such, gene expression was comparable to ovary. These results indicate that Dmrt1 and Sox9 are potential triggers for testis differentiation and Amh, Lhx9 and Foxl2 represent a conserved core set of genes in the sex-determining/differentiation pathway of TSD species.

Exogenous application of estradiol to eggs unexpectedly induces male development in two turtle species with temperature-dependent sex determination.

Steroid hormones affect sex determination in a variety of vertebrates. The feminizing effects of exposure to estradiol and the masculinizing effects of aromatase inhibition during development are well established in a broad range of vertebrate taxa, but paradoxical findings are occasionally reported. Four independent experiments were conducted on two turtle species with temperature-dependent sex determination (Chrysemys picta and Chelydra serpentina) to quantify the effects of egg incubation temperature, estradiol, and an aromatase inhibitor on offspring sex ratios. As expected, the warmer incubation temperatures induced female development and the cooler temperatures produced primarily males. However, application of an aromatase inhibitor had no effect on offspring sex ratios, and exogenous applications of estradiol to eggs produced male offspring across all incubation temperatures. These unexpected results were remarkably consistent across all four experiments and both study species. Elevated concentrations of estradiol could interact with androgen receptors or inhibit aromatase expression, which might result in relatively high testosterone concentrations that lead to testis development. These findings add to a short list of studies that report paradoxical effects of steroid hormones, which addresses the need for a more comprehensive understanding of the role of sex steroids in sexual development.

The cloning and expression analysis of Lhx9 during gonadal sex differentiation in the red-eared slider turtle, Trachemys scripta, a species with temperature-dependent sex determination.

Many reptiles, including the red-eared slider turtle (Trachemys scripta), possess a temperature-dependent sex determination (TSD) mechanism where the temperature at which the developing embryos are incubated dictates the gonadal sex of the animal. A number of mammalian gene orthologues have been identified in the sex determination/differentiation cascade of reptiles with TSD although the exact trigger(s) is not well understood. A potential early regulator of gonadal differentiation, Lhx9, controls the proliferation of gonadal cells in mice and its absence prevents gonadal development and drastically reduces the expression of Sf-1, a gene that regulates the expression of steroidogenic enzymes in the bipotential gonad. In the current study, we cloned Lhx9 from T. scripta and analyzed its expression throughout the thermosensitive period of gonad development using quantitative PCR. We examined the expression profiles of Lhx9 in embryos incubated under control conditions at male- and female-producing temperatures and with the application of exogenous 17ß-estradiol or an aromatase inhibitor, Letrozole, to induce sex reversal. The T. scripta Lhx9 cDNA and predicted amino acid sequence showed high homology to those of chicken, anole, and mouse. Lhx9 was expressed at both male- and female-producing temperatures with expression levels increasing throughout the thermosensitive period. Letrozole induced sex-reversal did not alter Lhx9 expression levels. 17ß-estradiol treatments appeared to inhibit or delay gonadal differentiation and resulted in lower Lhx9 expression from the presumptive gonadal ridge region. The structural homology and temporal expression pattern of Lhx9 suggests that this represents a conserved element in the gonadal differentiation cascade of T. scripta.

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.

Estrogen inhibits caudal progression but stimulates proliferation of developing müllerian ducts in a turtle with temperature-dependent sex determination.

Müllerian duct development appears to be similar in many vertebrate groups, and previous studies have shown that this development is estrogen sensitive. For example, embryonic exposure to diethylstilbestrol (DES) in humans and mice, and estrogen exposure in chickens, can have multiple, usually adverse, effects on müllerian duct differentiation and growth. The current study investigates 17beta-estradiol's effects on müllerian duct development in a reptile, the turtle Trachemys scripta. In T. scripta, normal müllerian duct development proceeds cranially to caudally over developmental stages 15 to 21. To ascertain 17beta-estradiol's effect on this process, four groups of eggs were incubated at a female-producing temperature. Each group was treated with 50 mug of 17beta-estradiol or a vehicle control at one of four stages (15, 17, 19, 21). The degree of müllerian duct development was assessed by examining gross morphology and histology. Results showed that estradiol-17beta blocked development of the müllerian duct if applied before differentiation began. If applied afterwards, 17beta-estradiol caused hypertrophy in the differentiated portion, but prevented further differentiation of the müllerian duct in more caudal regions. Therefore, reptilian müllerian ducts in T. scripta are estrogen sensitive and estrogen's effects may be similar to those reported for birds.

Dmrt1 expression in response to estrogen treatment in a reptile with temperature-dependent sex determination.

Dmrt1 has been implicated as an important factor in sex determination in all classes of vertebrates, including reptiles with temperature-dependent sex determination (TSD). Specifically, early embryonic expression of Dmrt1 appears to be an integral part of normal testicular development in vertebrates. Recently, a number of TSD studies have placed Dmrt1 expression at the top of a short list of putative temperature-sensitive events for TSD. Dmrt1 expression has been shown to be up-regulated at male-producing temperatures during the thermosensitive period (TSP) of several TSD reptile species. An interesting finding in Dmrt1 studies of fish and amphibians has been that in species where exogenous steroids can stimulate sex reversal, Dmrt1 expression can also be manipulated by these steroid treatments. In the current study, we examine the effects of exogenous 17beta-estradiol treatment on Dmrt1 expression at male-producing temperature (26 degrees C) in a reptile with TSD, the red-eared slider turtle, Trachemys scripta. Previous studies have demonstrated that exogenous estrogens can stimulate sex reversal (at male-producing temperatures) in this species of turtle. In the current study, T. scripta embryos that received estradiol treatment displayed a significant decrease in Dmrt1 expression during the TSP, while embryos in the control group (no estradiol treatment) showed significant increases in Dmrt1 expression during the TSP. Furthermore, the pattern of Dmrt1 expression for the estradiol-treated group was similar to the pattern we previously reported for Dmrt1 at female-producing temperature (31 degrees C). These findings indicate that exogenous estrogen is acting at or before the Dmrt1 event in the sex determination/sex differentiation cascade of T. scripta. Further, the results are consistent with the hypothesis that exogenous estrogen could be exerting its feminizing effect by down-regulating the expression of Dmrt1.

Cloning and expression of aromatase in a turtle with temperature-dependent sex determination.

It has been hypothesized that estrogen production may play a pivotal role in the sex determination of reptiles with temperature-dependent sex determination (TSD). This hypothesis has been furthered by studies that have shown higher aromatase activity in the developing ovaries in some reptiles. However, other studies have not consistently supported this hypothesis. In the current study we addressed this issue by cloning P450 aromatase cDNA in the turtle, Trachemys scripta, and developing a quantitative competitive RT-PCR for aromatase. This assay was then used to quantify aromatase mRNA levels in adrenal-kidney-gonad complexes (AKG) during TSD. Aromatase mRNA was detected in the AKGs at both male- and female-producing temperatures from the earliest stage of development sampled (stage 15), through hatching (stage 26). However, levels remained relatively constant during the thermosensitive period of TSD. Further, no significant difference was detected between male- and female-producing temperatures during the thermosensitive period. After the thermosensitive period, aromatase mRNA levels increased in females (this coincides with the period during which the ovaries are differentiating). These results are consistent with those of several previous studies of certain reptiles with TSD. The current results suggest that the expression of aromatase may not be a pivotal regulatory step in the sex determination cascade of this turtle.

Androgen and estrogen metabolism during sex differentiation in mono-sex populations of the Nile tilapia, Oreochromis niloticus.

Androgen and estrogen metabolism were examined in the period of steroid sensitivity during sex differentiation in mono-sex populations of Oreochromis niloticus. Fry (XX, XY, and YY genotypes) were maintained at 28 degrees and were sampled at 8, 10, 11, and 13 days postfertilization. Subsamples (n = 2-4) of pooled fry from each maternally distinct family were homogenized and incubated with either [(3)H]androstenedione or [(3)H]estradiol. Metabolites present in organic extracts were identified by thin-layer chromatography, microchemical reactions, and recrystallization to constant specific activity. Androstenedione was metabolized into at least seven readily identifiable compounds by all genotypes. In the XY genotype, 5beta-androstane-3alpha,17beta-diol synthesis decreased rapidly from 8 to 13 days postfertilization, with a concomitant increase in testosterone synthesis. Testosterone synthesis did not increase in the XX genotype. Testosterone synthesis in the YY genotype was intermediate to that of the XY and XX genotypes. Estrogens were not synthesized by any genotype. We hypothesize that 5beta-reduction (or further hydroxylation) is a mechanism important in regulating testosterone production and subsequent sex differentiation. Results of incubations with estradiol show an age-dependent increase in metabolism which did not vary among genotypes. Metabolites synthesized included estrone and up to five unidentified compounds.