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Biology of Reproduction 61, 1275-1280 (1999)
© 1999 Society for the Study of Reproduction, Inc.

Articles

Incubation Temperature Influences Sex-Steroid Levels in Juvenile Red-Eared Slider Turtles, Trachemys scripta, a Species with Temperature-Dependent Sex Determination1

Turk Rhen2,a, Emily Willinghama, Jon T. Sakataa, and David Crewsa

a Section of Integrative Biology, University of Texas, Austin, Texas 78712


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Incubation temperature determines gonadal sex in the red-eared slider turtle, Trachemys scripta. However, little is known about the long-term effects of incubation temperature on traits other than gonadal sex in this species. To investigate the hypothesis that incubation temperature (independent of gonadal sex) influences sex steroid levels after hatching, we incubated eggs of the red-eared slider turtle at three temperatures (26, 28.6, and 31°C). We then measured plasma levels of dihydrotestosterone, estradiol, progesterone, and testosterone in 6-wk-old males from 26°C and 28.6°C eggs, and in 6-wk-old females from 28.6°C and 31°C eggs. We found that dihydrotestosterone levels were not influenced by incubation temperature or gonadal sex. However, progesterone levels were significantly higher in males from 26°C eggs than in males from 28.6°C eggs. In contrast, testosterone levels did not differ between males from 26°C versus males from 28.6°C eggs, but they were significantly higher in females from 28.6°C than in females from 31°C eggs. Progesterone and testosterone levels did not differ between males and females from 28.6°C eggs. Temperature also influenced estradiol levels in both sexes, but the effects were enigmatic. We conclude that incubation temperature has lasting effects on sex steroid levels even after hatching.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Temperature during embryonic development determines gonadal sex in some lizards, many turtles, and all crocodilians studied to date (reviewed in [1–3]). Although advances have been made toward understanding the developmental mechanism of temperature-dependent sex determination (TSD; reviewed in [4–6]), most in-depth research on this topic has been conducted on a small number of species. Likewise, incubation temperature effects on traits other than gonadal sex have been examined in just a few TSD species. Unfortunately, the vast majority of studies on any given TSD species have focused on either 1) the mechanism of sex determination during embryogenesis or 2) the effects of incubation temperature on other traits after embryonic development is completed. As a result, it is difficult to integrate these studies of different species in order to obtain a coherent picture of how incubation temperature simultaneously determines gonadal sex and influences post-hatching traits like body size, energy reserves, metabolism and growth, behavioral thermoregulation, and adult sociosexual behaviors [7–14].

For example, there is a relatively large data base on TSD in the red-eared slider turtle, Trachemys scripta, but virtually no information concerning incubation temperature effects after hatching (but see [15]). In this species, low temperatures (i.e., < 28.4°C) produce all males, intermediate temperatures (i.e., 28.4–29.4°C) produce mixed sex ratios, while high temperatures (i.e., > 29.4°C) produce all females [16]. Various experiments suggest that temperature determines gonadal sex by influencing sex steroid metabolism during embryonic development. First, exogenous natural and man-made estrogens induce ovarian differentiation at normally male-producing temperatures and only have their effect during the temperature-sensitive period of gonadal development [17–19]. Second, these estrogens act synergistically with increasing incubation temperatures to produce more females than would be expected if estrogens and temperature act via separate developmental pathways [20, 21]. Finally, aromatase inhibitors can block ovarian differentiation at temperatures that normally produce females [22, 23]. Consequently, it is thought that temperature directly (or indirectly) regulates the expression of aromatase enzyme, which in turn affects estrogen production and ovarian differentiation [4].

Because sex determination is a threshold trait (i.e., sex ratio varies, but no hermaphrodites are produced), individuals with estrogen levels below a certain threshold develop as males and individuals with estrogen levels above the threshold develop as females. This model predicts that individuals of the same sex from different incubation temperatures are exposed to different hormonal milieus during embryonic development. Consequently, temperature-induced variation in hormone production could affect numerous physiological and behavioral traits later in life. To investigate the hypothesis that embryonic incubation temperature (independent of gonadal sex) influences sex steroid levels after hatching, we incubated eggs of the red-eared slider turtle at three different temperatures (26, 28.6, and 31°C). Whereas 26°C produces only males and 31°C produces only females, a temperature of 28.6°C produces ~4:1 ratio of males to females. We then raised turtles to 6 wk of age and determined basal levels of sex steroids and steroid levels in response to FSH. In another turtle species, FSH treatment stimulates male-specific production of testosterone [24], and we were anticipating a similar sex-specific (and possibly temperature-influenced) steroid response in the red-eared slider turtle. By choosing these incubation temperatures, we were able to compare temperature effects within each sex and test for sex differences in turtles from the same temperature.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Egg Incubation and Turtle Husbandry

Animals were treated according to a research protocol approved by the University of Texas institutional animal care and use committee. Turtle eggs were purchased from a commercial supplier (Robert Kliebert, Hammond, LA) and transported to the lab for candling, used to establish embryo viability. The eggs used in this experiment were collected and brought to the lab within 24 h of oviposition. To avoid clutch effects, eggs were randomized among treatment groups before incubation. Viable eggs were placed in groups of 35 into trays containing 1:1 vermiculite:water and were incubated at 26°C, 28.6°C, or 31°C in computer-controlled incubators. Temperature was also monitored by HOBO temperature loggers (Onset Computer Corporation, Pocassett, MA), and by daily readings of in-incubator thermometers. After hatching, turtles were maintained at room temperature (~25°C) in plastic shoeboxes filled with tap water approximately 3 cm deep and were fed lettuce and commercial turtle food on a daily basis.

Gonadotropin Challenge and Blood Sampling

A blood sample was drawn from 112 turtles (37 males from 26°C, 43 males and 16 females from 28.6°C, and 16 females from 31°C) at 6 wk of age: red-eared slider turtles are sexually immature at this age. Turtles from each temperature were randomly assigned to one of three groups: 1) a nontreated control, 2) a vehicle-treated control (0.5 ml of nanopure water), and 3) an FSH group (50 µg ovine FSH dissolved in 0.5 ml of nanopure water; 1 U/mg; Sigma, St. Louis, MO). Nontreated turtles were anesthetized on ice and then pithed while vehicle-treated and FSH-treated turtles were first injected i.p. with either water vehicle (0.5 ml H2O) or FSH, maintained at room temperature for 3 h, anesthetized on ice, and then pithed. Blood was then collected directly from the heart with a heparinized 1-cc syringe, transferred to 0.5-ml microfuge tubes, and centrifuged at 3000 rpm for 10 min at 4°C. Plasma was drawn off and frozen at -80°C. When killed, each turtle was diagnosed for gonadal sex by examination of the structure of the gonads with a dissecting microscope. This procedure is very accurate and has been validated a number of times using histological methods [17–23].

RIA

Plasma was pooled from 2–3 individuals to make 600-µl samples for RIA because a pilot study indicated that hormone levels were very low. Samples were then assayed for dihydrotestosterone (DHT), estradiol-17ß (E2), progesterone (P4), and testosterone (T) using the methods of Tousignant and Crews [10]. The antibodies used for RIA were DT3–351 for DHT, E26–47 for E2, P11–192 for P4, and T3–125 for T (Endocrine Sciences, Calabasas Hills, CA). Briefly, plasma samples were mixed with approximately 500 cpm each of tritiated DHT, E2, and T. After a 1-h equilibration time at 25°C, steroids were extracted with 3 ml of ether. Extracts were then dried under a stream of nitrogen in a dry bath at 37°C. Steroids were resuspended in 500 µl of isooctane saturated with ethylene glycol and separated on celite columns. The eluted steroids were dried under a stream of nitrogen in a dry bath at 37°C and resuspended overnight at 4°C in 330 µl of PBS with gelatin.

A single 100-µl aliquot of each resuspended sample was used to determine individual recoveries. Duplicate aliquots of each resuspended sample (100 µl) were used for RIA with approximately 2000 cpm (50 µl) of the appropriate labeled steroid and the corresponding antibody (100 µl). Assay sensitivity was 13 pg DHT/ml plasma, 13 pg E2/ml plasma, 106 pg P4/ml plasma, and 10 pg T/ml plasma. Intraassay coefficients of variation for a pooled sample of leopard gecko plasma were 16% for DHT, 18% for E2, 12% for P4, and 17% for T. Interassay coefficients of variation for the same sample were 18% for DHT, 17% for E2, 20% for P4, and 13% for T. We also ran quality control standards of known concentration in the low, medium, and high ranges of the standard curve for each steroid, with the following results, respectively. For DHT, intraassay coefficients of variation were 12%, 6%, and 6%, and interassay coefficients of variation were 18%, 9%, and 11%. For E2, intraassay coefficients of variation were 11%, 4%, and 6%, and interassay coefficients of variation were 10%, 8%, and 9%. For P4, intraassay coefficients of variation were 17%, 7%, and 8%, and interassay coefficients of variation were 14%, 1%, and 5%. For T, intraassay coefficients of variation were 9%, 4%, and 5%, and interassay coefficients of variation were 14%, 9%, and 10%.

Statistical Analysis

Plasma steroid levels were log-transformed to eliminate heteroscedasticity. Transformed steroid levels were compared using separate two-way ANOVAs for males and females for each steroid. We used two-way analyses with incubation temperature and FSH treatment as main effects because a three-way, fully factorial analysis was not possible (no females are produced at 26°C and no males are produced at 31°C). Since untreated and vehicle-treated groups did not differ within any of the experimental groups, they were combined into a single control group for the following analyses. Thus, independent variables in the two-way ANOVAs were incubation temperature, juvenile FSH administration 3 h before turtles were killed at 6 wk of age (combined controls vs. FSH treated), and their interaction. The following hierarchical set of comparisons among treatment groups was planned a priori. We first determined whether the interaction between incubation temperature and juvenile FSH administration was significant. Since this interaction was not significant for any sex steroid for males or for females, we examined the main effects of incubation temperature and FSH treatment. Because there were only two temperature and two gonadotropin groups, there was no need to make any correction for multiple comparisons. An analogous procedure was used to analyze sex effects, FSH effects, and sex-by-FSH effects for males and females from 28.6°C eggs. Recoveries for a number of P4 samples were low, which prevented their inclusion in our statistical analysis. An outlier for T in the 26°C male group was also thrown out. All statistics were done using Version 3.1 of JMP [25] for Apple Macintosh (Cupertino, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Incubation Temperature Effects in Males

Levels of DHT in 6-wk-old males were not influenced by incubation temperature, FSH challenge, or the interaction between incubation temperature and FSH challenge (P > 0.05 in all cases; 26°C males = 40 ± 6 pg/ml plasma; 28.6°C males = 33 ± 5 pg/ml plasma).

P4 levels in 6-wk-old males were influenced by incubation temperature during embryonic development: males from 26°C eggs had significantly higher P4 levels than males from 28.6°C (ANOVA; F ratio = 5.45; df = 1; P = 0.04; Fig. 1). However, there was no significant effect of FSH challenge nor a significant interaction between incubation temperature and FSH challenge (P > 0.05).



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  		FIG. 1. Plasma P4 levels of male and female juvenile red-eared slider turtles as a function of incubation temperature. P4 levels are least-squares means (± 1 SE) from the ANOVA described in the text. These means represent the means for each group while controlling for all other independent variables in the experiment. Sample sizes for each group are indicated at the base of the histogram bars

Like DHT, T levels in 6-wk-old males were not influenced by incubation temperature (Fig. 2), FSH challenge, or the interaction between incubation temperature and FSH challenge (P > 0.05 in all cases).



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  		FIG. 2. Plasma T levels of male and female juvenile red-eared slider turtles as a function of incubation temperature. T levels are least-squares means (± 1 SE) as noted in Figure 1. Sample sizes for each group are indicated at the base of the histogram bars

Six-wk-old male turtles from 26°C eggs had E2 levels well above the level of assay sensitivity (t-test = 8.088, P < 0.0001), while males from 28.6°C had undetectable levels of E2 (i.e., < 13 pg E2/ml plasma), indicating a highly significant incubation temperature effect (Fig. 3). There was no effect of FSH challenge on E2 levels in males from 26°C (t-test = 1.13, P = 0.28). Since E2 levels were undetectable in males from 28.6°C, we were not able to test for FSH effects.



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  		FIG. 3. Plasma E2 levels of male and female juvenile red-eared slider turtles as a function of incubation temperature. E2 levels in males and females from 28.6 were below assay sensitivity (i.e., < 13 pg E2/ml plasma). E2 levels are least-squares means (± 1 SE) as noted in Figure 1. Sample sizes for each group are indicated at the base of the histogram bars

Incubation Temperature Effects in Females

Levels of DHT in 6-wk-old females were not influenced by incubation temperature, FSH challenge, or the interaction between incubation temperature and FSH challenge (P > 0.05 in all cases; 28.6°C females = 39 ± 5 pg/ml plasma; 31°C females = 40 ± 4 pg/ml plasma).

Six-wk-old females from 28.6°C eggs tended to have higher P4 levels than females from 31°C (Fig. 1). However, P4 levels did not differ significantly between incubation temperatures (ANOVA; F ratio = 3.63; df = 1; P = 0.09). There was no significant effect of FSH challenge nor a significant interaction between incubation temperature and FSH challenge for P4 levels (P > 0.05).

Incubation temperature influenced the level of T in females: 6-wk-old females from 28.6°C eggs had significantly higher T levels than females from 31°C (ANOVA; F ratio = 5.32; df = 1; P = 0.047; Fig. 2). There was no significant effect of FSH challenge nor a significant interaction between incubation temperature and FSH challenge for T levels (P > 0.05).

Incubation temperature also significantly influenced plasma E2 levels in females (Fig. 3). Six-wk-old female turtles from 31°C eggs had E2 levels above the level of assay sensitivity (t-test = 8.69, P < 0.0001), while E2 levels were undetectable in females from 28.6°C (i.e., < 13 pg E2/ml plasma). FSH challenge did not influence E2 levels in females from 31°C (t-test = 0.97, P = 0.38). Since E2 levels were undetectable in females from 28.6°C, we were not able to test for FSH effects at this temperature.

Sex Differences

There was no detectable sex difference in the levels of DHT, P4, T, or E2 in 6-wk-old turtles from an incubation temperature of 28.6°C (P > 0.05 in all cases; Figs. 1, 2, and 3). In addition, there were no FSH effects nor an interaction between gonadal sex and FSH challenge in turtles from this temperature (P > 0.05 in all cases).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we found that embryonic incubation temperature had lasting effects on post-embryonic sex steroid levels in both male and female red-eared slider turtles, a species with TSD. Levels of P4 were inversely related to incubation temperature in both sexes whereas T levels only varied with temperature in females. However, there were no detectable differences in P4 or T levels between males and females from the same incubation temperature. The latter result indicates that differences in hormone levels between males from 26°C eggs (i.e., higher P4 and T) and females from 31°C eggs (i.e., lower P4 and T) are primarily due to incubation temperature and not gonadal sex. Interestingly, the pattern of incubation temperature effects (i.e., 26 vs. 31°C) on 6-wk-old turtles is the same as previously described for whole-body P4 and T concentrations during the temperature-sensitive period of embryonic development [26]. Conferring additional veracity to the current data, both the concentrations of hormones and the magnitude of the temperature differences were very close to those observed in the earlier study of embryos. In addition to these in vivo effects, incubation temperature similarly influenced in vitro steroidogenesis in adrenal-kidney-gonad complexes taken from embryos incubated at 26 and 31°C [27, 28]. In our study, incubation temperature also influenced E2 levels in both sexes. Although it is unclear why E2 levels were significantly higher in males from 26°C and females from 31°C and undetectable in both sexes from 28.6°C, similar U-shaped responses have been reported for various physiological traits. Despite this enigma, plasma E2 levels in males from 26°C and females from 31°C at 6 wk of age were generally similar to those reported during the temperature-sensitive period of embryonic development [26]. In contrast to P4, T, and E2 levels, we did not detect any incubation temperature or gonadal sex effects on DHT levels. However, we cannot judge the validity of these results, as DHT levels have not been measured previously in embryos or young red-eared slider turtles. Finally, we did not detect any change in sex steroid levels in response to FSH challenge. Considering results from in vitro studies, it is possible that the ambient temperature of the turtles in our experiment (i.e., ~25°C) reduced their responsiveness to gonadotropin treatment. Cool ambient temperatures (i.e., 10 and 20°C), relative to a warm ambient temperature (i.e., 28°C), eliminated or blunted T secretion in response to ovine FSH treatment of testes taken from sexually mature males [29, 30].

However, the lack of response for any steroid in either sex in the juvenile turtles in our experiment could also be due to developmental differences in sensitivity to mammalian gonadotropins. In fact, in vitro studies of testicular steroid secretion reveal that ovine FSH stimulated testosterone production about 10-fold in testes from adults but did not increase testosterone production in adrenal-kidney-gonad complexes from putative male (or female) embryos [28–30]. Although adrenal-kidney-gonad complexes from embryos did respond to FSH treatment with significant increases in P4 and E2, the responses were dependent upon developmental stage and incubation temperature and were relatively weak [28]. Unfortunately, we cannot make many other comparisons among developmental stages for the hormones that we measured because there are few published studies on sex steroid levels in sexually mature red-eared sliders. Nevertheless, plasma E2 levels in our 6-wk-old turtles were generally similar to those reported in ovariectomized adult females [31]. While this suggests that the gonads may be quiescent with respect to E2 production in immature turtles, we do not know what the normal circulating levels of E2 are like in intact, cycling females. In another study of female red-eared sliders [32], plasma levels of P4 were similar to, but slightly lower than, those we found in juvenile females from 31°C eggs. Yet samples in that study were taken after females were administered P4: plasma P4 levels were presumably at basal levels immediately after hormone administration and increased thereafter. Another caveat is that the authors did not report the age, the size, nor the reproductive status of the females used in their experiment so it is difficult to make any inferences about developmental (or any other) changes in P4 levels. In the only study that has reported circulating levels of sex steroids in mature males, P4 levels were one to two orders of magnitude higher than those we found for immature males, whereas T and DHT levels were generally undetectable [33]. Although the level of detectability for each hormone was not explicitly reported, it is interesting that P4 levels are substantially higher than both T and DHT levels in both immature and mature male red-eared sliders. Another fascinating finding from this study was that melanistic and nonmelanistic males differed with respect to their serum and testicular levels of progesterone. Consequently, it will be important to determine whether incubation temperature effects on pigmentation in hatchlings [15] are mediated by temperature-induced differences in sex steroid levels and whether such differences are correlated with color polymorphism in adulthood.

Overall, the results from our experiment and these earlier studies suggest that embryonic incubation temperature may have lasting effects on steroid-dependent physiological and behavioral traits much later in life. Indeed, in many vertebrates, variation in early sex steroid exposure of this kind has permanent developmental effects on adult sexual and aggressive behavior. This finding is significant because one would have to wait years to examine incubation temperature effects in adult red-eared sliders: males attain sexual maturity at 3–5 yr of age while females reach maturity at approximately 8 yr of age [34]. In fact, it would be difficult to study the persistence of incubation temperature effects in the majority of TSD reptiles because of their longevity. Nevertheless, previous work on an early-maturing TSD lizard, the leopard gecko, Eublepharis macularius, indicates that such temperature-induced variation in sex steroid levels, and consequently behavior, persist into adulthood (reviewed in [12]).

Perhaps the most intriguing question is how, at a developmental level, incubation temperature maintains its persistent effects on sex steroid levels in TSD reptiles. Although we have no clear answer, lessons may be drawn from intrauterine position effects in mammals. The intrauterine position of a mammalian fetus relative to same- or opposite-sex siblings affects its exposure to estradiol and testosterone [35, 36], thus influencing subsequent morphology, physiology, and behavior [37–40]. For example, adult male rats that were located between male siblings in utero, and thereby exposed to higher testosterone levels, have significantly higher testosterone and lower estradiol levels than adult male rats that were located between female siblings in utero [38]. In rats and mice, the effects of this early steroid exposure on adult steroid physiology are mediated by changes in steroidogenic enzyme activities in the reproductive tract [37, 41]. Thus, it would be interesting to determine whether similar changes in the activity (or expression) of steroidogenic enzymes occur in the reproductive system of TSD reptiles.

Considering recent evidence that much of the molecular machinery for gonadogenesis is evolutionarily conserved, an analogy between incubation temperature and intrauterine position effects on subsequent gonadal physiology may reflect common developmental mechanisms. Indeed, the gonadal anlagen is initially bipotential and consists of a cortical region that gives rise to the ovary and a medullary region that gives rise to the testis in all amniotic vertebrates [42]. Moreover, genes clearly involved in mammalian sex determination (e.g., AMH, SF-1, WT1, and SOX9) [43–45] have also been identified and implicated in avian sex determination [43, 46–48] and temperature-dependent sex determination (TSD) in the red-eared slider turtle [43, 49, 50] (A. Fleming and D. Crews, personal communication). Some of these genes (i.e., AMH and SF-1) are known to regulate the expression of steroidogenic enzymes during gonadal differentiation in mammals and may also do so in reptiles with TSD. In conclusion, our results with the red-eared slider turtle show that incubation temperature has lasting effects on sex steroid levels even 6 wk after embryonic development has been completed. It remains to be determined, however, whether this variation in steroid levels is related to temperature-induced differences in metabolism, growth, and behavior that have been demonstrated in other TSD reptiles [7–14] or to variation in pigmentation in the red-eared slider turtle [15, 33].


   ACKNOWLEDGMENTS
 
We thank Dr. Andrea Gore and three anonymous reviewers for helpful comments on the manuscript. We also thank Sarah Woolley for assistance with blood sampling.


   FOOTNOTES
 
1 This work was supported by NSF Grant IBN-9723617. Back

2 Correspondence: Turk Rhen, Section of Integrative Biology, University of Texas, Austin, TX 78712. FAX: 512 471 9651; turkrhen.uta{at}mail.utexas.edu Back

Accepted: June 22, 1999.

Received: March 22, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ewert MA, Jackson D, Nelson C. Patterns of temperature-dependent sex determination in turtles. J Exp Zool 1994; 270:3–15.[CrossRef]
  2. Lang JW, Andrews HV. Temperature-dependent sex determination in crocodilians. J Exp Zool 1994; 270:28–44.[CrossRef]
  3. Viets B, Ewert MA, Talent LG, Nelson CE. Sex determining mechanisms in squamate reptiles. J Exp Zool 1994; 270:45–56.[CrossRef]
  4. Crews D. Temperature-dependent sex determination: the interplay of steroid hormones and temperature. Zool Sci 1996; 13:1–13.[Medline]
  5. Pieau C. Temperature variation and sex determination in reptiles. Bioessays 1996; 18:19–26.
  6. Lance VA. Sex determination in reptiles: an update. Am Zool 1997; 37:504–513.
  7. Gutzke WHN, Crews D. Embryonic temperature determines adult sexuality in a reptile. Nature 1988; 332:832–834.[CrossRef][Medline]
  8. Allsteadt J, Lang JW. Incubation temperature affects body size and energy reserves of hatchling alligators. Physiol Zool 1994; 68:76–97.
  9. Rhen T, Lang JW. Phenotypic plasticity for growth in the common snapping turtle: effects of incubation temperature, clutch, and their interaction. Am Nat 1995; 146:726–747.[CrossRef]
  10. Tousignant A, Crews D. Incubation temperature and gonadal sex affect growth and physiology in the leopard gecko (Eublepharis macularius), a lizard with temperature-dependent sex determination. J Morphol 1995; 224:1–12.[CrossRef]
  11. O'Steen S. Embryonic temperature influences juvenile temperature choice and growth rate in snapping turtles Chelydra serpentina. J Exp Biol 1998; 201:439–449.
  12. Crews D, Sakata J, Rhen T. Developmental effects on intersexual and intrasexual variation in growth and reproduction in a lizard with temperature-dependent sex determination. Comp Biochem Physiol C 1998; 119:229–241.
  13. Rhen T, Lang JW. Embryonic and juvenile temperature independently influence growth in hatchling snapping turtles, Chelydra serpentina. J Therm Biol 1999; 24:33–41.
  14. Rhen T, Lang JW. Incubation temperature and sex affect mass and energy reserves of hatchling snapping turtles (Chelydra serpentina). Oikos 1999; 86:311–319.
  15. Etchberger CR, Ewert MA, Phillips JB, Nelson CE. Environmental and maternal influences on embryonic pigmentation in a turtle (Trachemys scripta elegans). J Zool (Lond) 1993; 230:529–539.
  16. Crews D, Bergeron JM, Flores D, Bull JJ, Skipper JK, Tousignant A, Wibbels T. Temperature-dependent sex determination in reptiles: proximate mechanisms, ultimate outcomes, and practical applications. Dev Genet 1994; 15:297–312.[CrossRef][Medline]
  17. Crews D, Bull JJ, Wibbels T. Estrogen and sex reversal in turtles: a dose-dependent phenomenon. Gen Comp Endocrinol 1991; 81:357–364.[CrossRef][Medline]
  18. Wibbels T, Bull JJ, Crews D. Chronology and morphology of temperature-dependent sex determination. J Exp Zool 1991; 260:371–381.[CrossRef][Medline]
  19. Bergeron JM, Crews D, McLachlan JA. PCBs as environmental estrogens: turtle sex determination as a biomarker of environmental contamination. Environ Health Perspect 1994; 102:780–781.[Medline]
  20. Wibbels T, Bull JJ, Crews D. Synergism between temperature and estradiol: a common pathway in turtle sex determination? J Exp Zool 1991; 260:130–134.
  21. Crews D, Cantu AR, Rhen T, Vohra R. The relative effectiveness of estrone, estradiol-17ß, and estriol in sex reversal in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination. Gen Comp Endocrinol 1996; 102:317–326.[CrossRef][Medline]
  22. Crews D, Bergeron JM. Role of reductase and aromatase in sex determination in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination. J Endocrinol 1994; 143:279–289.[Abstract/Free Full Text]
  23. Wibbels T, Crews D. Putative aromatase inhibitor induces male sex determination in a female unisexual lizard and in a turtle with TSD. J Endocrinol 1994; 141:295–299.[Abstract/Free Full Text]
  24. Rhen T, Elf PK, Fivizzani AJ, Lang JW. Sex-reversed and normal turtles display similar sex steroid profiles. J Exp Zool 1996; 274:221–226.[CrossRef]
  25. SAS. JMP User's Guide, Version 3.1 of JMP. Cary, NC: Statistical Analysis Systems Institute., Inc.; 1995.
  26. White RB, Thomas P. Whole-body and plasma concentrations of steroids in the turtle, Trachemys scripta, before, during, and after the temperature-sensitive period for sex determination. J Exp Zool 1992; 264:159–166.[CrossRef]
  27. White RB, Thomas P. Adrenal-kidney and gonadal steroidogenesis during sexual differentiation of a reptile with temperature-dependent sex determination. Gen Comp Endocrinol 1992; 88:10–19.[CrossRef][Medline]
  28. White RB, Thomas P. Stimulation of in vitro steroidogenesis by pituitary hormones in a turtle (Trachemys scripta) within the temperature sensitive period for sex determination. Biol Reprod 1992; 47:952–959.[Abstract]
  29. Licht P, Denver RJ, Pavgi S. Temperature dependence of in vitro pituitary, testis, and thyroid secretion in a turtle, Pseudemys scripta. Gen Comp Endocrinol 1989; 76:274–285.[CrossRef][Medline]
  30. Licht P, Pavgi S, Denver RJ. The role of hormone binding in the cold suppression of hormone stimulation of the pituitary, thyroid, and testis of the turtle. Gen Comp Endocrinol 1990; 80:381–392.[CrossRef][Medline]
  31. Selcer KW, Palmer BD. Estrogen downregulation of albumin and a 170-kDa serum protein in the turtle, Trachemys scripta. Gen Comp Endocrinol 1995; 97:340–352.[CrossRef][Medline]
  32. Selcer KW, Leavitt WW. Progesterone downregulates progesterone receptor, but not estrogen receptor, in the estrogen-primed oviduct of a turtle (Trachemys scripta). Gen Comp Endocrinol 1991; 83:316–323.[CrossRef][Medline]
  33. Gartska WR, Cooper WE Jr, Wasmund KW, Lovich JE. Male sex steroids and hormonal control of male courtship behavior in the yellow-bellied slider turtle, Trachemys scripta. Comp Biochem Physiol 1991; 98A:271–280.
  34. Gibbons JW, Semlitsch RD, Greene JL, Schubauer JP. Variation in size and age at maturity of the slider turtle (Pseudemys scripta). Am Nat 1981; 117:841–845.[CrossRef]
  35. vom Saal FS, Bronson F. Sexual characteristics of adult female mice are correlated with their blood testosterone levels during prenatal development. Science 1980; 208:597–599.[Abstract/Free Full Text]
  36. Even MD, Dhar MG, vom Saal FS. Transport of steroids between fetuses via amniotic fluid in relation to the intrauterine position phenomenon in rats. J Reprod Fertil 1992; 96:709–716.[Abstract/Free Full Text]
  37. Nonneman DJ, Ganjam VK, Welshons WV, vom Saal FS. Intrauterine position effects on steroid metabolism and steroid receptors of reproductive organs in male mice. Biol Reprod 1992; 47:723–729.[Abstract]
  38. van der Hoeven TH, Lefevre R, Mankes R. Effects of intrauterine position on the hepatic microsomal polysubstrate monooxygenase and cytosolic glutathione s-transferase activity, plasma sex steroids and relative organ weights in adult male and female Long-Evans rats. J Pharmacol Exp Ther 1992; 263:32–39.[Abstract/Free Full Text]
  39. Palanza P, Parmigiani S, vom Saal FS. Urine marking and maternal aggression of wild female mice in relation to anogenital distance at birth. Physiol Behav 1995; 58:827–835.[CrossRef][Medline]
  40. Clark MM, Vonk JM, Galef BG. Reproductive profiles of adult mongolian gerbils gestated as the sole fetus in a uterine horn. Physiol Behav 1997; 61:77–81.[CrossRef][Medline]
  41. Cooke GM, Price CA, Oko RJ. Effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin TCDD on serum androgens and steroidogenic enzyme activities in the male rat reproductive tract. J Steroid Biochem Mol Biol 1998; 674:347–354.
  42. Witshi E. Age of sex determining mechanisms in vertebrates. Science 1959; 130:829–846.[Free Full Text]
  43. Di Clemente N, Ghaffari S, Pepinsky RB, Pieau C, Josso N, Cate RL, Vigier B. A quantitative and interspecific test for biological activity of anti-Müllerian hormone: the fetal aromatase assay. Development 1992; 114:721–727.[Abstract]
  44. Lou X, Ikeda Y, Parker KL. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 1994; 77:481–490.[CrossRef][Medline]
  45. Nachtigal MA, Hirokawa Y, Enyeart-VanHouten DL, Flanagan JN, Hammer GD, Ingraham HA. Wilms' tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression. Cell 1998; 93:445–454.[CrossRef][Medline]
  46. Da Silva SM, Hacker A, Harley V, Goodfellow P, Swain A, Lovell Badge R. Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. Nat Genet 1996; 14:62–68.[CrossRef][Medline]
  47. Kent J, Wheatley SC, Andrews JE, Sinclair AH, Koopman P. A male-specific role for SOX9 in vertebrate sex determination. Development 1996; 122:2813–2822.[Abstract]
  48. Smith CA, Smith MJ, Sinclair AH. Expression of chicken steroidogenic factor-1 during gonadal sex differentiation. Gen Comp Endocrinol 1999; 113:187–196.[CrossRef][Medline]
  49. Spotila LD, Spotila JR, Hall SE. Sequence and expression analysis of WT1 and Sox9 in the red-eared slider turtle, Trachemys scripta. J Exp Zool 1998; 281:417–427.[CrossRef][Medline]
  50. Wibbels T, Cowan J, LeBoeuf R. Temperature-dependent sex determination in the red-eared slider turtle, Trachemys scripta. J Exp Zool 1998; 281:409–416.[CrossRef][Medline]



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