Yolk steroid hormones and sex determination in reptiles with TSD

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1 General and Comparative Endocrinology 132 (2003) Review Yolk steroid hormones and sex determination in reptiles with TSD P.K. Elf * University of Minnesota Crookston, 2900 University Avenue, Crookston, MN , USA University of North Dakota, GrandForks, ND 58202, USA Accepted 24February 2003 GENERAL AND COMPARATIVE ENDOCRINOLOGY Abstract In reptiles with temperature-dependent sex determination (TSD), the temperature at which the eggs are incubated determines the sex of the offspring. The molecular switch responsible for determining sex in these species has not yet been elucidated. We have examined the dynamics of yolk steroid hormones during embryonic development in the snapping turtle, Chelydra serpentina, and the alligator, Alligator mississippiensis, and have found that yolk estradiol (E 2 ) responds differentially to incubation temperature in both of these reptiles. Based upon recently reported roles for E 2 in modulation of steroidogenic factor 1, a transcription factor known to be significant in the sex differentiation process, we hypothesize that yolk E 2 is a link between temperature and the gene expression pathway responsible for sex determination and differentiation in at least some of these species. Here we review the evidence that supports our hypothesis. Ó 2003 Elsevier Science (USA). All rights reserved. 1. Temperature-dependent sex determination Sex determination is thought to occur in two basically different modes. There is genetic sex determination (GSD), in which sex chromosomes determine the sex of the individual and environmental sex determination (ESD), where environmental factors determine sex. In one form of ESD, temperature-dependent sex determination (TSD), the temperature at which the eggs are incubated determines the sex of the hatchlings. There are three different patterns or temperature profiles that have been described for TSD species, male female (MF), female male (FM), and female male female (FMF). In the MF pattern, low temperatures produce a majority of males, high temperatures produce mostly females, and intermediate temperatures produce a ratio of males to females. The intermediate temperature that produces a 1:1 ratio of males to females is referred to as the pivotal temperature for the species. Several turtle species have been reported to show this profile, including the painted turtle, Chrysemys picta and the red-eared slider turtle, Trachemys scripta (Ewert et al., 1994). In the FM pattern, the temperature regimen is reversed, with high * Fax: address: pelf@mail.crk.umn.edu. temperatures producing mainly males, low temperatures producing primarily females, and again, intermediate temperatures producing ratios of males to females. This pattern has been reported for some lizards (Viets et al., 1994), including the skink, Eulamprus tympanum, the only viviparous TSD lizard reported to date (Robert and Thompson, 2001). In the third TSD pattern, FMF, females are produced at low temperatures, a majority of males are produced at an intermediate temperature, and predominantly females are produced again at high temperatures. In this system there are two pivotal temperatures at which ratios of males to females are produced. This pattern is displayed in all the crocodilians studied to date, including the American alligator, Alligator mississippiensis (Lang and Andrews, 1994). In the snapping turtle, Chelydra serpentina, the usual TSD pattern is FMF (Ewert et al., 1994), however, the TSD pattern in some populations of snapping turtles varies slightly from that described, being MF, with males predominating at lower temperatures, females at higher temperatures, and a single pivotal temperature range. The period of development during which sex is determined, the thermosensitive period (TSP), falls within the middle one-third to one half of the total incubation time (Wibbels et al., 1991a), and temperature influences the rate of development as well as the sex of the hatchling /03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi: /s (03)

2 350 P.K. Elf / General andcomparative Endocrinology 132 (2003) Fig. 1. Percentages of males produced in 24clutches of eggs from Chelydra serpentina incubated at male-producing, female-producing, and intermediate temperatures through-out development (adapted from Rhen and Lang, 1998). Temperature is apparently not the only factor influencing sex determination, at least in some of these species. There are reports of large variations in the ratios of males to females produced among clutches of eggs laid by different females at the pivotal temperature where one would expect to see a 1:1 ratio (Rhen and Lang, 1998, Fig. 1). This would indicate that other factors, perhaps some maternal contribution could influence the outcome of the sex determining process. Clutch identity or clutch effects have also been reported to influence other aspects of offspring fitness, including residual yolk mass, fat body mass and total mass of hatchling snapping turtles (Rhen and Lang, 1999). Moreover, studies of post-hatch growth of snapping turtles showed significant clutch effects in growth rates that were independent of egg mass (Rhen and Lang, 1995). These differences could also be due to differential hormone deposition in yolk, as has been reported in some avian species (Frank et al., 1991; Schwabl, 1996; Schwabl et al., 1997). 2. Gene expression patterns during sex differentiation of TSD reptiles What is known about the sex differentiation process in reptiles with TSD? The gene expression pattern that leads to sex determination and subsequent testis or ovary differentiation, has been defined best in mammalian species, which utilize GSD. SRY (Sex-determining region of the Y chromosome) is thought to be the primary determinant of testis differentiation in mouse and human systems (reviewed by Koopman et al., 2001), but there is no known homologue of SRY in TSD reptiles. There are a number of candidate genes that are present in mammalian, avian and reptilian systems whose roles in sex determination and differentiation are currently being investigated. The SOX9 gene, which is expressed downstream of SRY in mammals, is essential for stimulating production of anti-mullerian hormone (AMH) in the human and mouse systems, and is male-specific in its expression (reviewed in Koopman et al., 2001). AMH plays duel roles, in that it is necessary for regression of the Mullerian ducts, which would differentiate into female reproductive structures (Arango et al., 1999; de- Santa Barbera et al., 1998), and also is expressed early in Sertoli cell differentiation, at least in the mouse (Munsterberg and Lovell-Badge, 1991). Differential SOX9 expression has been documented in all the TSD reptile species studied to date, including the alligator (Western et al., 1999), the sea turtle (Moreno-Mendoza et al., 1999), the red-eared slider turtle (Spotila et al., 1998), and the leopard gecko (Valleley et al., 2001). However, unlike the mouse system, AMH expression precedes that of SOX9 in the alligator (reviewed by Western and Sinclair, 2001), so there are apparently earlier genes involved in AMH stimulation in at least this TSD species. Besides SOX9, SF-1 (steroidogenic factor one) is necessary for activation of AMH in mammalian species (reviewed by Koopman et al., 2001). SF-1, a transcription factor which regulates the expression of steroidogenic enzymes, is expressed very early in sex differentiation and is thought to be necessary for germinal ridge formation. It is also expressed in gonadotropes of the developing pituitary of mice and influences expression of genes required for gonadotropin production (Ingraham et al., 1994). SF-1 expression patterns in developing gonads have been investigated in two TSD reptiles, the alligator (Western et al., 2000), and the redeared slider turtle (Fleming et al., 1999). Results of these two reports both demonstrate sexually dimorphic expression of SF-1 early in the TSP, during gonadal differentiation, but they document opposite patterns of expression. SF-1 expression is increased in the female gonad of the alligator and decreased in the male gonad. This is similar to the pattern reported in the chicken. Contrarily, SF-1 appears to be up-regulated in the developing male gonad in the red-eared slider turtle, and down-regulated in the female, as in mammals. Further, in the red-eared slider turtle, addition of exogenous estradiol (E 2 ) suppresses SF-1 expression (Fleming and Crews, 2001). Murdock and Wibbels (2002) recently reported opposite results with respect to SF-1 expression in the red-eared slider turtle. They performed quantitative competitive RT-PCR for SF-1 on total RNA isolated from gonad adrenal mesenephros complexes (GAMs) taken at male and female producing temperatures during development. They documented greater levels of SF-1 expression in GAMs from female producing temperatures compared to male producing temperatures,

3 P.K. Elf / General andcomparative Endocrinology 132 (2003) but since the embryonic adrenal gland is extremely active, these results do not accurately reflect activity of the gonad alone (T. Wibbels, personal communication). Since in mammalian species SF-1 works in conjunction with SOX9 to up-regulate AMH for male differentiation, SF-1 must participate in completely different interactions in chickens and alligators, where it is upregulated in females. Recent reports indicate that DAX1, an orphan nuclear receptor, inhibits the expression of genes in the male differentiation pathway possibly by modulating the activity of SF-1 (reviewed by Parker and Schimmer, 2002). DAX1 also has reported interactions with estrogen receptors and is thought to act as a corepressor, so could play a role in estrogen signaling pathways (Zhang et al., 2000). Cytochrome P450 aromatase expression, a gene in the estrogen synthesis pathway, has also been documented in TSD species. Several investigations have examined aromatase expression in the alligator and in some turtle species. In the alligator, aromatase expression in developing gonadal tissue is not measurable until after the TSP, at which time it is more highly expressed in ovary than testis (Gabriel et al., 2001; Smith et al., 1995). Differential expression of aromatase has been reported in developing gonads of the European pond turtle toward the end of the TSP (Desvages and Pieau, 1992), but the timing would make this appear to be a result of sex determination, rather than the cause of it. Further, Murdock and Wibbels (2003) reported aromatase expression in the red-eared slider turtle similar to that reported in the alligator, i.e., not measurable until after the TSP, near the end of development. It has also been hypothesized that aromatase activity in the brain might be responsible for determining gonadal sex in TSD reptiles through the production of E 2 (Jeyasuria and Place, 1998; Place et al., 2001), but again, evidence from different species is contradictory (Gabriel et al., 2001; Jeyasuria and Place, 1998; Merchant-Larios and Villalpando, 1990). Another gene whose expression has recently been described in the red-eared slider turtle is Dmrt1 (Kettlewell et al., 2000). Dmrt1 is related to DMRT1 in humans, which is required in two copies for testicular differentiation to occur (Raymond et al., 1999). Differential expression of this gene in T. scripta has been reported as early as stage 15 during development, preceding Sox9 expression. These findings were corroborated by Murdock and Wibbels (2003) who reported similar Dmrt1 expression measured using a competitive PCR assay. There appears to be extensive variation in the interactions of transcription factors that work in conjunction with each other to modulate the expression of the genes directly involved in the processes of sex determination and differentiation in mammalian, avian, and reptilian species. 3. Yolk steroid hormone dynamics in TSD species The importance of estradiol (E 2 ) in the sexual differentiation process in turtles has been well documented. Studies addressing sex reversal in the red-eared slider turtle have shown estrogens to be potent agents for the production of a greater number of female offspring at male-producing and pivotal temperatures (Crews et al., 1995; Crews et al., 1996), a phenomenon that has been demonstrated to be dose-dependent (Crews et al., 1991). In the snapping turtle, E 2 can increase the number of females produced at male temperatures and aromatase inhibitors are capable of producing greater percentages of males at temperatures that typically produce a majority of females. In contrast, testosterone treatment does not increase the number of males produced at female temperatures (Rhen and Lang, 1994). These findings provide support for an essential role for estrogens in the sex determination process in these species. Further support for E 2 involvement in the sex determination process was provided in a report by Bowden et al. (2000). They found a seasonal variation in yolk steroid hormone content in eggs from the painted turtle, C. picta, with elevated E 2 levels in second clutches correlated with a female sex bias in the offspring. Wibbels et al. (1991b) have documented a synergism between E 2 and incubation temperature in the red-eared slider turtle. They reported that as incubation temperature was increased toward that which produces 100% females, less exogenous E 2 was needed to produce larger female/male ratios than would naturally occur. Based on these results, they hypothesized that E 2 and temperature are both components of the same sex-determining pathway. We have investigated initial levels of yolk steroid hormones, T and E 2, in several species of turtles (Elf et al., 2002a; Gerum, 1999), the leopard gecko (Elf, 2003), and also androstenedione (A) in the alligator (Conley et al., 1997; Elf et al., 2001). In all the species examined, initial hormone levels were in the nanogram per gram of yolk range. We have also documented significant differences in the levels of the hormones between clutches of eggs from individual females, or clutch effects (Conley et al., 1997; Elf et al., 2002a; Elf, 2003). These clutch effects could be responsible for the differences reported in several parameters of sex determination and growth in turtles (Rhen and Lang, 1995, 1998, 1999), similar to the variations attributed to yolk T differences in finches and canaries (Schwabl, 1996). We have monitored the dynamics of developmental changes in yolk steroid hormones in the eggs of snapping turtles, (Elf et al., 2002b), and alligators (Conley et al., 1997; Elf et al., 2001). Both systems demonstrated a decline in E 2 and T from initial levels to hatching. In the snapping turtle, we documented a temperature-de-

4 352 P.K. Elf / General andcomparative Endocrinology 132 (2003) pendent decline in E 2 levels, with eggs incubated at female producing temperatures (29.5 C) maintaining the greatest amount, eggs incubated at male producing temperatures (26 C) maintaining the least E 2, and eggs incubated at the pivotal temperature (28 C) having an intermediate amount of E 2. This temperature-dependent decline was apparent prior to the TSP, and continued through development (Fig. 2). There was also a decline in T, but no differential temperature effect was observed. Further examination of E 2 levels in eggs incubated at the pivotal temperature revealed the possibility of two separate populations, one with slightly greater and one with slightly less E 2 (Elf et al., 2002b). A recent publication (Bowden et al., 2002) reported decreases in yolk steroid hormones in the red-eared slider turtle during development, also. However, their assay system was not sensitive enough to measure E 2 levels after oviposition, so their concentrations represent only one egg from each of their subsequent sampling periods. Further, the three samples they were able to quantify came from different clutches, so individual female effects could also be a factor in the amounts reported. More accurate analysis of E 2 changes during embryogenesis need to be done before the true dynamics of the hormone are understood in this species. In the alligator we documented differences, though not significant, in E 2 levels at stage 21 (during the sex determining period). E 2 was greatest at 34 C (high temperature females produced), least at 33 C (male producing), and intermediate at 31 C (low temperature females produced) (Conley et al., 1997). In residual yolk material from alligator hatchlings incubated at male (33 C), female (29, 31 C), and an intermediate temperature (32 C), we did document a significant difference in E 2 that demonstrated the same pattern as that seen in snapping turtles (Elf et al., 2001). The hatchlings incubated at the two female producing temperatures had similar E 2 levels that were significantly greater than those from male hatchlings, and hatchlings from the intermediate temperature had intermediate levels of E 2, regardless of hatchling sex. Temperature appears to produce a different E 2 profile in this system, and yet, an elevated E 2 environment is established for female embryos, regardless of whether they are produced at higher or lower temperatures. This conservation of elevated E 2 levels at female producing temperatures suggests that a similar mechanism is operating in both of these TSD species, even though E 2 is influenced in different ways. There are other differences in the sex determination process between the turtle system and the alligator system, as demonstrated by the reversed pattern of SF-1 expression in the alligator (Western et al., 2000). 4. Gene/yolk hormone interactions in TSD reptiles Based upon the temperature-dependent dynamics of E 2 we have documented in alligators and snapping turtles, along with the evidence that SF-1 lies very early in the sex-determination pathway and is influenced by E 2 levels, we hypothesize that temperature is influencing E 2 levels within the yolk, E 2 levels are impacting the expression of SF-1, and SF-1 is then up-regulated or down-regulated, depending upon species, resulting in either female or male development (Fig. 3). And though Fig. 2. Incubation temperature effects on E 2 in yolks of snapping turtle eggs through development (adapted from Elf et al., 2002b). Data were log transformed and analyzed using a mixed model two-way ANOVA for temperature, time and temperature-time interactions. P < 0:001; F ¼ 31:534; df ¼ 2, 306 for temperature. P < 0:001; F ¼ 178:643; df ¼ 2, 306 for time. P ¼ 0:600; F ¼ 0:689; df ¼ 4, 306 for time-temperature interactions. Fig. 3. Predicted sex determination pathways in TSD species. In turtles, an increase in incubation temperature causes maintenance of greater E 2 levels, which suppresses SF-1 expression and leads to differentiation of ovaries, whereas at low incubation temperatures E 2 is not maintained, SF-1 is expressed and testes result. In the alligator, high or low incubation temperatures maintain greater E 2 levels which increase SF-1 expression and lead to ovarian differentiation. At the intermediate temperature, E 2 is not maintained, SF-1 is suppressed and testes form.

5 P.K. Elf / General andcomparative Endocrinology 132 (2003) Nomura et al. (1995) found no E 2 regulatory region in the SF-1 gene in the rat system, that doesnõt preclude its existence in reptilian systems, and perhaps more importantly it doesnõt preclude the possibility that E 2 acts in conjunction with other factors to impact SF-1 expression. What we are hypothesizing is that E 2 and SF-1 are part of the same pathway which is involved in the process of sex determination. Further, we hypothesize that there exists a threshold level of E 2 that is required to stimulate or suppress SF-1 expression, and that some temperature-dependent factor is responsible for either maintaining E 2 at female producing temperatures, or for metabolizing it at male producing temperatures. At intermediate temperatures, clutches with initially greater levels of yolk E 2 would have sufficient hormone present during the TSP to exceed the necessary threshold, and females would be produced. Contrarily, in eggs from clutches with lesser initial yolk E 2 levels, threshold would not be maintained, and males will be produced. So, maternal differences in initial egg hormone levels, or clutch effects, would influence the ratios of males to females produced, consistent with results observed in the snapping turtle (Rhen and Lang, 1998). This hypothesis is also consistent with reported synergies between E 2 and temperature with regards to production of female hatchlings (Wibbels et al., 1991b) and would explain why addition of exogenous E 2 can reverse sex in clutches incubated at male producing temperatures (Crews et al., 1991, 1995, 1996; Rhen and Lang, 1994). Aromatase inhibitors, which are not as potent at reversing sex, probably influence the system in later stages of sex differentiation, interfering with the embryonic production of E 2 which is necessary for development of the ovary (Belaid et al., 2001). In support of this hypothesis, we have evidence that low concentrations of exogenous 3 H-estradiol administered to the shell of snapping turtle eggs exhibits the same temperature dependent effects as endogenous yolk E 2. We find that at the female producing temperature, equilibrium is established between the 3 H-E 2 concentration in the albumin and the 3 H-E 2 levels in the yolk material as it is absorbed. However, at the male producing temperature, 3 H-E 2 appears to be sequestered in the albumin, and levels remain lower in the yolk throughout the thermo-sensitive period of development. Additions of higher concentrations of exogenous E 2 would proportionally increase the amount incorporated into the yolk at male temperatures, and so cause sex reversal. Other differences include significantly greater uptake of 3 H-E 2 by gonads at the female producing temperature, and temporal differences in absorption by brain tissue with greater uptake of 3 H-E 2 in the brain at the male producing temperature at the later stages of development (Elf et al., 2002c). 5. Female variability in clutchsteroid hormone levels What determines the amount of steroid hormone deposited in reptilian eggs? We have documented differences in yolk hormone levels among eggs laid by different females (clutch effects) in the alligator (Conley et al., 1997), several species of turtles (Elf et al., 2002a; Gerum, 1999) and the leopard gecko (Elf, 2003), while within clutch variation in these species is almost nonexistent. There are several factors that could affect the concentrations of these hormones. Among them are female fitness, seasonal effects, female age/size, and possible innate factors specific to individual females. When females are stressed due to environmental factors, they could respond to that stress by producing a normal number of smaller eggs, which would then contain less yolk material, including hormones; they could produce fewer regular sized eggs with normal provisioning of hormone; or, they could completely forego reproduction for the season. Bowden et al. (2000) documented an increase in E 2 in clutches of C. picta eggs in second clutches laid in a season, but since females were not marked, there was no way to determine if females with greater levels of hormones in first clutches maintained greater levels in second clutches also (R. Bowden, personal communication). This study does, however, demonstrate a seasonal variation in yolk hormone levels. In some reptilian species, as females grow and age, the mass of their clutches also increases (Congdon and Gibbons, 1985). With increased clutch mass, is there also an increase in hormones contributed to yolk, or put simply, does female age affect the amount of yolk hormones in eggs? Analyses done in our lab of several snapping turtle and painted turtle clutches indicate that there is no correlation between total clutch mass, i.e., female age or size, and yolk hormone content of the eggs (Elf et al., 2003). This would indicate that at least in these turtle species, there are other factors responsible for determining individual female hormone contributions to eggs. Do environmental stresses affect levels of hormones maternally contributed to the yolk? We have demonstrated female differences in steroid hormone concentrations in yolks of eggs from chickens, Gallus domesticus (Elf and Fivizzani, 2002d), and leopard geckos (Elf, 2003) raised in controlled environments, so these clutch effects exist even when environmental stress is not a factor. Is there, then, some innate predisposition in individual females to contribute more or less hormone to their eggs, and if so, what is responsible for establishing that predisposition? In conclusion, there are a number of factors, among them, yolk steroid/gene interactions during development and possible environmental impacts or inherited factors that may play roles in determining maternal contributions to yolk. These factors need further investigation in

6 354 P.K. Elf / General andcomparative Endocrinology 132 (2003) order to understand the dynamics of yolk steroid hormones and their subsequent influences on growth, behavior, and sex determination and differentiation of embryos with TSD. Future studies need to be designed to address some of these questions. References Arango, N.A., Lovell-Badge, R., Behringer, R.R., Targeted mutagenesis of the endogenous mouse Mis gene promoter: in vivo definition of genetic pathways of vertebrate sexual development. Cell 99, Belaid, B., Richard-Mercier, N., Pieau, C., Dorizzi, M., Sex reversal and aromatase in the European pond turtle: Treatment with letrozole after the thermosensitive period for sex determination. J. Exp. Zool. 290, Bowden, R.M., Ewert, M.A., Nelson, C.E., Environmental sex determination in a reptile varies seasonally and with yolk hormones. Proc. R. Soc. Lond. 267, Bowden, R.M., Ewert, M.A., Nelson, C.E., Hormone levels in yolk decline throughout development in the red-eared slider turtle (Trachemys scripta elegans). Gen. Comp. Endocrinol. 129, Congdon, K.A., Gibbons, J.W., Egg components and reproductive characteristics of turtles: relationship to body size. Herpetologia 41, Conley, A.J., Elf, P., Corbin, C.J., Dubowsky, S., Fivizzani, A., Lang, J.W., Yolk steroids decline during sexual differentiation in the alligator. Gen. Comp. Endocrinol. 107, Crews, D., Bull, J.J., Wibbels, T., Estrogen and sex reversal in turtles: a dose-dependent phenomenon. Gen. Comp. Endocrinol. 81, Crews, D., Cantu, A., Bergeron, J.M., Rhen, T., The relative effectiveness of androstenedione, testosterone, and estrone, precursors to estradiol, in sex reversal in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination. Gen. Comp. Endocrinol. 100, Crews, D., Cantu, A., Rhen, T., Vohra, R., The relative effectiveness of estrone, estradiol-17beta, and estriol in sex reversal in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination. Gen. Comp. Endocrinol. 102, desanta Barbera, P., Bonneaud, N., Boizet, B., Desclozeaux, M., Moniot, B., Sudbeck, P., Scherer, G., Poulat, F., Berta, P., Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-mullerian hormone gene. Mol. Cell Biol. 18, Desvages, G., Pieau, C., Aromatase activity in gonads of turtle embryos as a function of the incubation temperature of eggs. J. Steroid Biochem. Mol. Biol. 41, Elf, P.K., Allsteadt, J., Lang, J.W., Fivizzani, A.J., The role of yolk steroid hormones in reptile sex determination. Proceedings of 14th International Endocrine Congress. pp Elf, P.K., Lang, J.W., Fivizzani, A.J., 2002a. Yolk hormone levels in the eggs of snapping turtles and painted turtles. Gen. Comp. Endocrinol. 127, Elf, P.K., Lang, J.W., Fivizzani, A.J., 2002b. Dynamics of yolk steroid hormones during development in a reptile with temperature-dependent sex determination. Gen. Comp. Endocrinol. 127, Elf, P.K., Lang, J.W., Fivizzani, A.J., 2002c. Dynamics of exogenous 3 H-estradiol applied to snapping turtle eggs during embryonic development. SICB Annual Meeting. Anaheim, CA Final Program and Abstracts. 24.1, p Elf, P.K., Fivizzani, A.J., 2002d. Changes in sex steroid levels in yolks of the leghorn chicken, Gallus domesticus, during embryonic development. J. Exp. Zool. 293, Elf, P.K., Lang, J.W., Fivizzani, A.J., Correlations between female age/body size and yolk steroid hormone levels in two species of TSD turtles. SICB Annual Meeting. Toronto, Canada Final Program and Abstracts. Ewert, M.A., Jackson, D.R., Nelson, C.E., Patterns of temperature-dependent sex determination in turtles. J. Exp. Zool. 270, Fleming, A., Wibbels, T., Skipper, J.K., Crews, D., Developmental expression of steroidogenic factor 1 in a turtle with temperature-dependent sex determination. Gen. Comp. Endocrinol. 116, Fleming, A., Crews, D., Estradiol and incubation temperature modulate SF1 expression in the embryonic red-eared slider turtle. Endocrinology 142 (4), Frank, L.G., Glickman, S.E., Licht, P., Fatal sibling aggression, precocial development, and androgens in neonatal spotted hyenas. Science 252, Gabriel, W.N., Blumberg, B., Sutton, S., Place, A.R., Lance, V., Alligator aromatase cdna sequence and its expression in embryos at male and female incubation temperatures. J. Exp. Zool. 290, Gerum, S.V., Endogenous yolk steroids in five species of turtles: a comparison between sex determination mechanisms. M.Sc. thesis. University of North Dakota, Grand Forks. Ingraham, H.A., Lala, D.S., Ikeda, Y., Luo, X., Nachtigal, M.W., Abbud, R., Nilson, J.H., Parker, K.L., The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev. 8, Jeyasuria, P., Place, A.R., Embryonic brain-gonadal axis in temperature-dependent sex determination of reptiles: a role for P450 aromatase (CYP19). J. Exp. Zool. 281, Kettlewell, J.R., Raymond, C.S., Zarkower, D., Temperaturedependent expression of turtle Dmrt1 prior to sexual differentiation. Genesis 26, Koopman, P., Bullejos, M., Bowles, J., Regulation of male sexual development by Sry and Sox9. J. Exp. Zool. 290, Lang, J.W., Andrews, H.V., Temperature-dependent sex determination in crocodilians. J. Exp. Zool. 270, Merchant-Larios, H., Villalpando, I., Effect of temperature on gonadal sex differentiation in the sea turtle it Lepidochelys olivacea: an organ culture study. J. Exp. Zool. 254, Moreno-Mendoza, N., Harley, V.R., Merchant-Larios, H., Differential expression of SOX9 in gonads of the sea turtle Lepidochelys olivacea at male- or female-promoting temperatures. J. Exp. Zool. 284, Munsterberg, A., Lovell-Badge, R., Expression of the mouse anti-mullerian hormone gene suggests a role in both male and female sexual differentiation. Development 113, Murdock, C., Wibbels, T., Steroidogenic factor-1 mrna levels in the embryonic adrenal/kidney/gonadal complexes of Trachemys scripta. SICB Annual Meeting. Anaheim, CA. Final Program and Abstracts. P3.64, p Murdock, C., Wibbels, T., Dmrt1 mrna levels in the embryonic adrenal/kidney/gonadal complexes of Trachemys scripta. SICB Annual Meeting. Toronto, Canada. Final Program and Abstracts. 62.4, p Nomura, M., Bartsch, S., Nawata, H., Omura, T., Morohashi, K., An E box element is required for the expression of the ad4bp gene, a mammalian homologue of ftz-f1 gene, which is essential for adrenal and gonadal development. J. Biol. Chem. 270 (13), Parker, K.L., Schimmer, B.P., Genes essential for early events in gonadal development. Ann. Med. 34,

7 P.K. Elf / General andcomparative Endocrinology 132 (2003) Place, A.R., Lang, J., Gavasso, S., Jeyasuria, P., Expression of P450arom in Malaclemys terrapin and Chelydra serpentina: a tale of two sites. J. Exp. Zool. 290, Raymond, C.S., Parker, E.D., Kettlewell, J.R., Brown, I.G., Page, D.C., Kusz, K., Jaruzelska, J., Reinberg, Y., Flejter, W.L., Bardwell, V., Hirsch, B., Zarkower, D., A region of human chromosome 9p required for testis development contains two genes related to known sexual regulators. Human Mol. Genet. 8, Rhen, T., Lang, J.W., Temperature-dependent sex determination in the snapping turtle: Manipulation of the embryonic sex steroid environment. Gen. Comp. Endocrinol. 96, Rhen, T., Lang, J.W., Phenotypic plasticity for growth in the common snapping turtle: Effects of incubation temperature, clutch, and their interaction. Am. Nat. 146, Rhen, T., Lang, J.W., Among-family variation for environmental sex determination in reptiles. Evolution 52 (5), Rhen, T., Lang, J.W., Incubation temperature and sex affect mass and energy reserves of hatchling snapping turtles, Chelydra serpentina. OIKOS 86, Robert, K.A., Thompson, M.B., Viviparous lizard selects sex of embryos. Nature 412, Schwabl, H., Maternal testosterone in the avian egg enhances postnatal growth. Comp. Biochem. Physiol. 114A, Schwabl, H., Mock, D.W., Gieg, J.A., A hormonal mechanism for parental favouritism. Nature 386, 231. Smith, C.A., Elf, P.K., Lang, J.W., Joss, J.M.P., Aromatase enzyme activity during gonadal sex differentiation in alligator embryos. Differentiation 58, Spotila, L.D., Spotila, J.R., Hall, S.E., Sequence and expression analysis of WT1 and Sox9 in the red-eared slider turtle, Trachemys scripta. J. Exp. Zool. 281, Elf, P.K., Yolk steroids and their possible roles in TSD species. In: Valenzuela, N., Lance, V.(Eds.), Temperature-dependent Sex Determination. Smithsonian Institution Press, Washington, D.C., in press. Valleley, M.A., Cartwright, N.J., Croft, N.J., Markham, A.F., Coletta, P.L., Characterisation and expression of Sox9 in the leopard gecko, Eublepharis macularius. J. Exp. Zool. 291, Viets, B.E., Ewert, M.A., Talent, L.G., Nelson, C.E., Sexdetermining mechanisms in squamate reptiles. J. Exp. Zool. 270, Western, P.S., Harry, J.L., Marshall-Graves, J.A., Sinclair, A.H., Temperature-dependent sex determination in the American alligator: AMH precedes SOX9 expression. Dev. Dyn. 216, Western, P.S., Hary, J.L., Marshall Graves, J.A., Sinclair, A.H., Temperature-dependent sex determination in the American alligator: expression of SF1, Wti, and DAX1 during gonadogenesis. Gene 241, Western, P.S., Sinclair, A.H., Sex, genes, and heat: triggers of diversity. J. Exp. Zool. 290, Wibbels, T., Bull, J.J., Crews, D., 1991a. Chronology and morphology of temperature-dependent sex determination. J. Exp. Zool. 260, Wibbels, T., Bull, J.J., Crews, D., 1991b. Synergism between temperature and estradiol: a common pathway in turtle sex determination? J. Exp. Zool. 260, Zhang, H., Thomsen, J.S., Johansson, L., Gustafsson, J., Treuter, E., DAX-1 functions as an LXXLL-containing corepressor for activated estrogen receptors. J. Biol. Chem. 275,