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Developmental Expression of Steroidogenic Factor 1 in a Turtle with Temperature-Dependent Sex Determination

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Developmental Expression of Steroidogenic Factor 1 in a Turtle with Temperature-Dependent Sex Determination General and Comparative Endocrinology 116, 336–346 (1999) Article ID gcen.1999.7360, available online at http://www.idealibrary.com on Developmental Expression of Steroidogenic Factor 1 in a Turtle with Temperature-Dependent Sex Determination Alice Fleming,* Thane Wibbels,† James K. Skipper,* and David Crews*,1 *Section of Integrative Biology, University of Texas at Austin, Austin, Texas 78712; and †Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-1170 Accepted July 24, 1999 A variety of reptiles possess temperature-dependent sex indicating possible homologous functions. ௠ 1999 Academic determination (TSD) in which the incubation tempera- Press ture of a developing egg determines the gonadal sex. Key Words: steroidogenic factor 1, SF-1; Ad4BP; Current evidence suggests that temperature signals may FTZ-F1; reptile; turtle; Trachemys scripta; temperature- be transduced into steroid hormone signals with estro- dependent sex determination. gens directing ovarian differentiation. Steroidogenic fac- tor 1 (SF-1) is one component of interest because it regulates the expression of steroidogenic enzymes in INTRODUCTION mammals and is differentially expressed during develop- ment of testis and ovary. Northern blot analysis of SF-1 in developing tissues of the red-eared slider turtle Gonadal sex of species with temperature-dependent (Trachemys scripta), a TSD species, detected a single sex determination (TSD) is determined by the tempera- primary SF-1 transcript of approximately 5.8 kb across ture at which their eggs are incubated. In the red-eared all stages of development examined. Analysis by in situ slider turtle (Trachemys scripta), only males are pro- hybridization indicated nearly equivalent SF-1 expres- duced when eggs are incubated at 26°C, and only sion in early, bipotential gonads at male (26ЊC)- and females are produced at 31°C (Bull et al., 1982). The female (31ЊC)-producing incubation temperatures. In temperature-sensitive period (TSP) for sex determina- subsequent stages, as gonadal sex first becomes histologi- tion occurs between Yntema (1968) stages 15 and 21, cally distinguishable during the temperature-sensitive the middle third of incubation (Wibbels et al., 1991). period, SF-1 expression increased in gonads at a male- Commitment to gonadal sex occurs within that period. producing temperature and decreased at a female- Sex steroid hormones are implicated in the process producing temperature, suggesting a role for SF-1 in the of TSD, and estrogen, in particular, appears essential in sex differentiation pathway. SF-1 message was also found female sex determination (Crews, 1996; Crews et al., in adrenal and in the periventricular region of the 1994; Wibbels et al., 1998; Lance, 1997). Estrogens preoptic area and diencephalon, but there was no appar- applied exogenously to T. scripta eggs incubating at a ent sex bias in these tissues at any stage examined. The male-producing temperature override the temperature overall developmental pattern of SF-1 mRNA expression effect, and female hatchlings result (Crews et al., 1991; in T. scripta appears to parallel that found in mammals, Wibbels and Crews, 1992). Exogenously applied inhibi- tors of aromatase—the enzyme that converts testoster- 1 To whom correspondence should be addressed. Fax: (512) 471- one to estrogen (Simpson et al., 1994)—override a 6078. E-mail: [email protected]. female-producing incubation temperature, and male 336 0016-6480/99 $30.00 Copyright ௠ 1999 by Academic Press All rights of reproduction in any form reserved. SF-1 Expression in Embryonic Turtle 337 hatchlings result (Crews and Bergeron, 1994; Wibbels message and possible alternate transcripts. A single and Crews, 1994). transcript was found in all stages and tissues exam- Work with other turtle species has shown a correla- ined. Adrenal, kidney, and gonad cannot be effectively tion between female incubation temperatures and separated at early developmental stages in T. scripta, increased levels of endogenous aromatase transcript precluding traditional quantitative measures of SF-1 in and activity in the putative ovary during the TSP gonad alone. For this reason, in situ hybridization was (Desvages and Pieau, 1992; Jeyasuria and Place, 1997, selected as the most appropriate technique to both 1998). Other researchers have found aromatase activity localize and quantify SF-1 in the embryonic turtle. SF-1 in the turtle brain prior to that found in the gonad at message was found in similar amounts and distribu- tion in the bipotential gonad of males and females. female-producing temperatures and have proposed During stages when the sex of gonads is becoming that the brain, rather than the gonad, is the sex- distinct and committed, SF-1 message increased at a determining source of estrogen (Merchant-Larios, 1998; male-producing temperature and decreased at a female- Jeyasuria and Place, 1998). Whatever the endogenous producing temperature. SF-1 message was also de- source of estrogen, gonads of putative females and tected in developing adrenal and the periventricular males are receptive to its effect as both express estro- region of the preoptic area and diencephalon in similar gen receptor, albeit differentially, throughout the TSP amounts and distribution at male- and female-produc- (Bergeron et al., 1998). ing temperatures. The sex-based differential expres- Male sex determination can be manipulated by sion of SF-1 in the turtle gonad during a critical period exogenously applied dihydrotestosterone, a nonaroma- of gonadal sex development mirrors that found in tizable androgen, and by inhibitors of its endogenous mammals, suggesting homologous functions and pos- synthesis (Crews and Bergeron, 1994; Wibbels and sible involvement in temperature-sensitive sex determi- Crews, 1992, 1995; Wibbels et al., 1992). This effect is nation and differentiation. less striking than that of estrogen in female sex determina- tion and is only seen at intermediate, or less potent, incubation temperatures. Nevertheless, steroid hormones are undoubtedly a part of TSD in both males and females. MATERIALS AND METHODS To begin exploring the underlying molecular mecha- nisms of steroid hormones in TSD, we examined the Tissue Collection distribution of steroidogenic factor 1 (SF-1) (Lala et al., 1992), also called Ad4BP (Morohashi et al., 1992), in T. T. scripta eggs were purchased within 2 days of laying from Robert Kliebert (Kliebert Turtle Farms, scripta. SF-1, encoded by the FTZ-F1 gene and a Hammond, LA), brought to the laboratory, and kept at member of the nuclear receptor superfamily, is known room temperature until viability was established by in mammals to regulate transcription of many genes candling. Viable eggs were placed in containers with within the reproductive axis (reviewed in Morohashi moistened vermiculite (1:1 vermiculite to water) and and Omura, 1996; Parker and Schimmer, 1997). In randomized across containers to eliminate clutch ef- steroidogenic tissue, it regulates the gene activity of fects. The containers were placed in incubators (Preci- many proteins involved in the synthesis of testoster- sion, Chicago) at either 26 or 31°C. Continuous incuba- one and estrogen, including steroidogenic acute regu- tion of T. scripta eggs at 26°C produces all male ␤ latory protein, P450scc, P45017␣,3 -HSD, and aromatase hatchlings whereas incubation at 31°C produces all (reviewed in Morohashi, 1999; Parker et al., 1999). female hatchlings (Bull et al., 1982). During mammalian development, SF-1 is differentially Temperature of the incubators was continuously expressed in testes and ovaries (Ikeda et al., 1994; monitored with HOBO data loggers (Onset Computer Hatano et al., 1994). Corp.), supplemented by daily checks of in-incubator In this study we examined the pattern of SF-1 thermometers. Temperature fluctuations were less than mRNA expression in T. scripta. Northern blot analysis 0.1°C. Egg boxes were rotated within the incubators was performed to determine presence and size of each day, and eggs were checked periodically for Copyright ௠ 1999 by Academic Press All rights of reproduction in any form reserved. 338 Fleming et al. developmental stage according to Yntema’s staging (1989). Probes were synthesized to a specific activity of guidelines (1968). By these guidelines, the temperature- 9 ϫ 108 cpm/µg and were used at a concentration of sensitive period in T. scripta is from approximately 0.3 µg probe ϫ length (kb)/ml hybridization solution. stage 15 through 21, and eggs hatch at stage 26. For in situ hybridization, embryos were taken at Northern Blot Analysis stages 13 through 19 and at stage 23 from each incuba- tion temperature, quickly frozen on dry ice, and stored Total RNA was isolated according to Sambrook et al. at Ϫ80°C until sectioning. For all other molecular (1989) or RNAgents Total RNA Isolation System. T. work, embryos were decapitated; the adrenal–kidney– scripta tissues were AKG complexes from early, middle, gonad (AKG) complex and brain were then quickly and post TSP (stages 15, 18/19, and 23, respectively) at dissected out, frozen in liquid nitrogen or isopentane, both male- and female-producing temperatures (26 and stored at Ϫ80°C until use. and 31°C); whole brain from the middle of the TSP (stages 18/19) at both temperatures; and adult ovary. Probe Preparation Twenty-five micrograms of total RNA from each tissue and RNA Millenium Markers (Ambion) were loaded. The open reading frame of T. scripta SF-1 cDNA has The blot was prepared using Ambion’s Northern
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