Segregating variation for temperature-dependent sex determination in a lizard.

Temperature-dependent sex determination (TSD) was first reported in 1966 in an African lizard. It has since been shown that TSD occurs in some fish, several lizards, tuataras, numerous turtles and all crocodilians. Extreme temperatures can also cause sex reversal in several amphibians and lizards with genotypic sex determination. Research in TSD species indicates that estrogen signaling is important for ovary development and that orthologs of mammalian genes have a function in gonad differentiation. Nevertheless, the mechanism that actually transduces temperature into a biological signal for ovary versus testis development is not known in any species. Classical genetics could be used to identify the loci underlying TSD, but only if there is segregating variation for TSD. Here, we use the 'animal model' to analyze inheritance of sexual phenotype in a 13-generation pedigree of captive leopard geckos, Eublepharis macularius, a TSD reptile. We directly show genetic variance and genotype-by-temperature interactions for sex determination. Additive genetic variation was significant at a temperature that produces a female-biased sex ratio (30°C), but not at a temperature that produces a male-biased sex ratio (32.5°C). Conversely, dominance variance was significant at the male-biased temperature (32.5°C), but not at the female-biased temperature (30°C). Non-genetic maternal effects on sex determination were negligible in comparison with additive genetic variance, dominance variance and the primary effect of temperature. These data show for the first time that there is segregating variation for TSD in a reptile and consequently that a quantitative trait locus analysis would be practicable for identifying the genes underlying TSD.

Molecular mechanisms of sex determination in reptiles.

Charles Darwin first provided a lucid explanation of how gender differences evolve nearly 140 years ago. Yet, a disconnect remains between his theory of sexual selection and the mechanisms that underlie the development of males and females. In particular, comparisons between representatives of different phyla (i.e., flies and mice) reveal distinct genetic mechanisms for sexual differentiation. Such differences are hard to comprehend unless we study organisms that bridge the phylogenetic gap. Analysis of variation within monophyletic groups (i.e., amniotes) is just as important if we hope to elucidate the evolution of mechanisms underlying sexual differentiation. Here we review the molecular, cellular, morphological, and physiological changes associated with sex determination in reptiles. Most research on the molecular biology of sex determination in reptiles describes expression patterns for orthologs of mammalian sex-determining genes. Many of these genes have evolutionarily conserved expression profiles (i.e., DMRT1 and SOX9 are expressed at a higher level in developing testes vs. developing ovaries in all species), which suggests functional conservation. However, expression profiling alone does not test gene function and will not identify novel sex-determining genes or gene interactions. For that reason, we provide a prospectus on various techniques that promise to reveal new sex-determining genes and regulatory interactions among these genes. We offer specific examples of novel candidate genes and a new signaling pathway in support of these techniques.

Expression of putative sex-determining genes during the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina.

Modes of sex determination are quite variable in vertebrates. The developmental decision to form a testis or an ovary can be influenced by one gene, several genes, environmental variables, or a combination of these factors. Nevertheless, certain morphogenetic aspects of sex determination appear to be conserved in amniotes. Here we clone fragments of nine candidate sex-determining genes from the snapping turtle Chelydra serpentina, a species with temperature-dependent sex determination (TSD). We then analyze expression of these genes during the thermosensitive period of gonad development. In particular, we compare gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature. Expression of Dmrt1 and Sox9 mRNA increased gradually at the male-producing temperature, but was suppressed at the female-producing temperature. This finding suggests that Dmrt1 and Sox9 play a role in testis development. In contrast, expression of aromatase, androgen receptor (Ar), and Foxl2 mRNA was constant at the male-producing temperature, but increased several-fold in embryos at the female-producing temperature. Aromatase, Ar, and Foxl2 may therefore play a role in ovary development. In addition, there was a small temperature effect on ER alpha expression with lower mRNA levels found in embryos at the female-producing temperature. Finally, Dax1, Fgf9, and SF-1 were not differentially expressed during the sex-determining period, suggesting these genes are not involved in sex determination in the snapping turtle. Comparison of gene expression profiles among amniotes indicates that Dmrt1 and Sox9 are part of a core testis-determining pathway and that Ar, aromatase, ER alpha, and Foxl2 are part of a core ovary-determining pathway.

Reproductive tradeoffs and yolk steroids in female leopard geckos, Eublepharis macularius.

Life history theory predicts tradeoffs among reproductive traits, but the physiological mechanisms underlying such tradeoffs remain unclear. Here we examine reproductive tradeoffs and their association with yolk steroids in an oviparous lizard. Female leopard geckos lay two eggs in a clutch, produce multiple clutches in a breeding season, and reproduce for several years. We detected a significant tradeoff between egg size and the number of clutches laid by females during their first two breeding seasons. Total reproductive effort was strongly condition-dependent in the first season, but much less so in the second season. Although these and other tradeoffs were unmistakable, they were not associated with levels of androstenedione, oestradiol, or testosterone in egg yolk. Female condition and egg size, however, were inversely related to dihydrotestosterone (DHT) levels in egg yolk. Finally, steroid levels in egg yolk were not directly related to steroid levels in the maternal circulation when follicles were developing, indicating that steroid transfer to eggs is regulated. These findings suggest that maternal allocation of DHT could mitigate tradeoffs that lead to poor offspring quality (i.e. poor female condition) and small offspring size (i.e. small egg size).

Variation in reproductive behaviour within a sex: neural systems and endocrine activation.

Intrasexual variation in reproductive behaviour, morphology and physiology is taxonomically widespread in vertebrates, and is as biologically and ecologically significant as the differences between the sexes. In this review, we examine the diverse patterns of intrasexual variation in reproductive behaviours within vertebrates. By illustrating the genetic, cellular, hormonal and/or neural mechanisms underlying behavioural variation in a number of species, another level of complexity is added to studies of brain organization and function. Such information increases our understanding of the unique and conserved mechanisms underlying sex and individual differences in behaviour in vertebrates as a whole. Here, we show that intrasexual variation in behaviour may be discrete or continuous in nature. Moreover, this variation may be due to polymorphism at a single genetic locus or many loci, or may even be the result of phenotypic plasticity. Phenotypic plasticity simply refers to cases where a single genotype (or individual) can produce (or display) different phenotypes. Defined in this way, plasticity subsumes many different types of behavioural variation. For example, some behavioural phenotypes are established by environmental factors during early ontogeny, others are the result of developmental transitions from one phenotype early in life to another later in life, and still other strategies are facultative with different behaviours displayed in different social contexts.

Distribution of androgen and estrogen receptor mRNA in the brain and reproductive tissues of the leopard gecko, Eublepharis macularius.

Incubation temperature during embryonic development determines gonadal sex in the leopard gecko, Eublepharis macularius. In addition, both incubation temperature and gonadal sex influence behavioral responses to androgen and estrogen treatments in adulthood. Although these findings suggest that temperature and sex steroids act upon a common neural substrate to influence behavior, it is unclear where temperature and hormone effects are integrated. To begin to address this question, we identified areas of the leopard gecko brain that express androgen receptor (AR) and estrogen receptor (ER) mRNA. We gonadectomized adult female and male geckos from an incubation temperature that produces a female-biased sex ratio and another temperature that produces a male-biased sex ratio. Females and males from both temperatures were then treated with equivalent levels of various sex steroids. Region-specific patterns of AR mRNA expression and ER mRNA expression were observed upon hybridization of radiolabeled (35S) cRNA probes to thin sections of reproductive tissues (male hemipenes and female oviduct) and brain. Labeling for AR mRNA was very intense in the epithelium, but not within the body, of the male hemipenes. In contrast, expression of ER mRNA was prominent in most of the oviduct but not in the luminal epithelium. Within the brain, labeling for AR mRNA was conspicuous in the anterior olfactory nucleus, the lateral septum, the medial preoptic area, the periventricular preoptic area, the external nucleus of the amygdala, the anterior hypothalamus, the ventromedial hypothalamus, the premammillary nucleus, and the caudal portion of the periventricular nucleus of the hypothalamus. Expression of ER mRNA was sparse in the septum and was prominent in the ventromedial hypothalamus, the caudal portion of the periventricular nucleus of the hypothalamus, and a group of cells near the torus semicircularis. Many of these brain regions have been implicated in the regulation of hormone-dependent, sex-typical reproductive and agonistic behaviors in other vertebrates. Consequently, these nuclei are likely to control such behaviors in the leopard gecko and also are candidate neural substrates for mediating temperature effects on behavior.

Sex-limited mutations and the evolution of sexual dimorphism.

Although the developmental and genetic mechanisms underlying sex differences are being elucidated in great detail in a number of species, there remains a breach between proximate and evolutionary studies of sexual dimorphism. More precisely, the evolution of sex-limited gene expression at autosomal loci has not been well reasoned using either theoretical or empirical methods. Here, I show that a Mendelian genetic model including elementary details of sexual differentiation provides novel insight into the evolution of sex differences via sex limitation. This model indicates that the nature of allelic effects and the pattern of selection must be known in both sexes to predict the evolution of sex differences. That is, selection interacts with genetic variation for sexual dimorphism to produce unanticipated patterns of trait divergence or convergence between the sexes. Ultimately, this model may explain why previous models for the evolution of sexual dimorphism do not predict the erratic behavior of the sex difference during artificial selection experiments.

Sex steroid levels across the reproductive cycle of female leopard geckos, Eublepharis macularius, from different incubation temperatures.

Incubation temperature during embryonic development determines gonadal sex in many reptiles, including the leopard gecko (Eublepharis macularius). In this study, we examined the hormonal and behavioral changes that occur during the reproductive cycle of female leopard geckos from four (i.e., 26, 30, 32.5, and 34 degrees C) incubation temperatures. Controlling for reproductive status, plasma levels of dihydrotestosterone (DHT), testosterone (T), and progesterone (P) varied with incubation temperature but estradiol 17-beta (E2) levels did not. Controlling for the effects of incubation temperature, DHT and T levels were low when females were previtellogenic, increased slightly during early vitellogenesis, increased dramatically during late vitellogenesis (i.e., prior to ovulation), and then decreased to previtellogenic levels after ovulation. In contrast, E2 levels increased gradually from the previtellogenic stage to the early vitellogenic stage, peaked during late vitellogenesis, and decreased to previtellogenic levels after ovulation. Levels of P increased from the previtellogenic stage to the early vitellogenic stage, remained elevated during late vitellogenesis, and then decreased after ovulation. Moreover, we determined that females were not sexually receptive when previtellogenic, were somewhat receptive during early vitellogenesis (approximately 20% receptive), were most receptive during late vitellogenesis (approximately 80% receptive), and were again unreceptive after ovulation. Incubation temperature did not influence receptivity. Overall, these data show that hormone levels and behavior change coordinately during the reproductive cycle. Although incubation temperature has persistent effects on endocrine physiology in adult female leopard geckos, these effects are modest compared to hormonal changes across the reproductive cycle.

Organization and activation of sexual and agonistic behavior in the leopard gecko, Eublepharis macularius.

Gonadal sex is determined by the temperature experienced during incubation in the leopard gecko (Eublepharis macularius). Furthermore, both factors, incubation temperature and gonadal sex, influence adult sexual and agonistic behavior in this species. Yet it is unclear whether such differences in behavior are irreversibly organized during development or are mediated by differences in hormone levels in adulthood. To address this question, we gonadectomized adult females and males generated from a female-biased (30 degrees C) and a male-biased (32.5 degrees C) incubation temperature and treated them with equivalent levels of various sex steroids. We found that 17beta-estradiol (E(2)) activated sexual receptivity in females but not males, suggesting an organized sex difference in behavioral sensitivity to E(2). There were also organized and activated sex differences in attractivity to stimulus males. Although females were more attractive than males when treated with E(2), both sexes were equally unattractive when treated with dihydrotestosterone (DHT) or testosterone (T). Likewise, sex differences in aggressive and submissive behavior were organized and activated. Attacks on stimulus males were activated by T in males but not in females. In contrast, hormones did not influence flight behavior in males but did affect female submissiveness. Overall, males also evoked more attacks by stimulus males than did females. Nevertheless, females and males treated with androgens evoked more attacks than animals of the same sex that were treated with cholesterol or E(2). Incubation temperature had some weak effects on certain behaviors and no effect on others. This suggests that temperature effects in gonadally intact geckos may be due primarily to differences in circulating levels of hormones in adulthood. We conclude that gonadal sex has both organizational and activational effects on various behaviors in the leopard gecko.

Embryonic treatment with xenobiotics disrupts steroid hormone profiles in hatchling red-eared slider turtles (Trachemys scripta elegans).

Many compounds in the environment capable of acting as endocrine disruptors have been assayed for their developmental effects on morphogenesis; however, few studies have addressed how such xenobiotics affect physiology. In the current study we examine the effects of three endocrine-disrupting compounds, chlordane, trans-nonachlor, and the polychlorinated biphenyl (PCB) mixture Aroclor 1242, on the steroid hormone concentrations of red-eared slider turtle (Trachemys scripta elegans) hatchlings treated in ovo. Basal steroid concentrations and steroid concentrations in response to follicle-stimulating hormone were examined in both male and female turtles treated with each of the three compounds. Treated male turtles exposed to Aroclor 1242 or chlordane exhibited significantly lower testosterone concentrations than controls, whereas chlordane-treated females had significantly lower progesterone, testosterone, and 5[alpha]-dihydrotestosterone concentrations relative to controls. The effects of these endocrine disruptors extend beyond embryonic development, altering sex-steroid physiology in exposed animals.

Effects of testosterone on sexual behavior and morphology in adult female leopard geckos, Eublepharis macularius.

The leopard gecko, Eublepharis macularius, is a species in which testosterone (T) is the primary circulating sex hormone in adults of both sexes. There are, however, sex differences in T physiology. Whereas males have prolonged periods with high T levels, T levels cycle in accord with follicular development in females. Specifically, T concentration increases during vitellogenesis, drops after ovulation, and then remains at previtellogenic levels until eggs are laid and the next follicular cycle begins. To determine the function of T in females, we manipulated both the level and the duration of T elevation using Silastic implants in intact, adult female leopard geckos. Females had low ( approximately 1 ng/ml), medium ( approximately 100 ng/ml), or high ( approximately 200 ng/ml) T levels for either a short (8 days) or a long (35 days) duration. Behavior tests with males were conducted on days 1-5 in the short-duration group or on days 29-33 in the long-duration group. For both short- and long-duration groups, T treatment decreased attractivity in females with medium and high T levels compared to females with low T levels. In contrast, females with a medium T level were more receptive than females with a low T level in the short-duration group. Females in the long-duration group were unreceptive regardless of T level. Females treated for a long duration also displayed more aggression toward and evoked more aggression from males than short duration females. Short-duration T treatment had no masculinizing effect on female morphology, whereas medium and high T levels for a long duration induced development of hemipenes. Overall, these results suggest that T can both increase and decrease sexual behaviors in the female leopard gecko.

Incubation temperature influences sex-steroid levels in juvenile red-eared slider turtles, Trachemys scripta, a species with temperature-dependent sex determination.

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 degrees C). We then measured plasma levels of dihydrotestosterone, estradiol, progesterone, and testosterone in 6-wk-old males from 26 degrees C and 28.6 degrees C eggs, and in 6-wk-old females from 28.6 degrees C and 31 degrees 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 degrees C eggs than in males from 28.6 degrees C eggs. In contrast, testosterone levels did not differ between males from 26 degrees C versus males from 28.6 degrees C eggs, but they were significantly higher in females from 28.6 degrees C than in females from 31 degrees C eggs. Progesterone and testosterone levels did not differ between males and females from 28.6 degrees 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.

Embryonic temperature and gonadal sex organize male-typical sexual and aggressive behavior in a lizard with temperature-dependent sex determination.

Temperature during embryonic development determines gonadal sex in the leopard gecko, Eublepharis macularius. Moreover, both embryonic temperature and gonadal sex influence adult behavior. Yet it remains unclear whether the effects of embryonic temperature and gonadal sex on behavior are irreversibly organized during development. To address this question, we gonadectomized adult females and males generated from a temperature that produces mostly females (30 C) and a temperature that produces mostly males (32.5 C). Females and males from both temperatures were then treated with equivalent levels of various sex steroids. We found that both embryonic temperature and gonadal sex had persistent effects on the expression of male-typical sexual and aggressive behaviors. For example, adult females do not scent mark and display very little courtship and mounting behavior even when treated with levels of hormones (primarily androgens) that activate these behaviors in males. In contrast, species-typical aggressive displays were less sex specific and were activated by both dihydrotestosterone and testosterone (T) in males and by T in females. Nevertheless, the average duration of aggressive displays was significantly shorter in T-treated females than that in T-treated males. With regard to submissive behavior, androgens decreased flight behavior in males, but had no effect in females. Embryonic temperature had enduring effects on certain behaviors in males. For instance, males from a male-biased embryonic temperature scent-marked more than males from a female-biased embryonic temperature when treated with dihydrotestosterone or T. Conversely, and across hormone treatments, males from a female-biased embryonic temperature mounted more than males from a male-biased embryonic temperature. Finally, treatment with 17beta-estradiol decreased submissive behavior in males from a male-biased embryonic temperature compared with that in males from a female-biased embryonic temperature. Courtship and aggressive behavior were not influenced by temperature. These results strongly suggest that male-typical behaviors in the adult leopard gecko are permanently organized by both embryonic temperature and gonadal sex during development.

Developmental synergism of steroidal estrogens in sex determination.

Gonadal sex in the red-eared slider turtle, Trachemys scripta, is determined by incubation temperature during embryonic development. Evidence suggests that temperature determines sex by influencing steroid hormone metabolism and/or sensitivity: steroidogenic enzyme inhibitors or exogenous sex steroid hormones and their man-made analogs override (or enhance) temperature effects on sex determination. Specifically, nonaromatizable androgens and aromatase inhibitors induce testis differentiation at female-producing temperatures, whereas aromatizable androgens and estrogens induce ovary differentiation at male-producing temperatures. Moreover, natural estrogens and temperature synergize to produce more females than would be expected if estrogens and temperature had purely additive effects on sex determination. In this study, we use sex reversal of turtle embryos incubated at a male-producing temperature to examine synergism among steroidal estrogens: estrone, 17ss-estradiol, and estriol. A low dose of 17ss-estradiol (200 ng) showed significant synergism when administered with a single low dose of estriol (10 ng). Likewise, a single low dose of estrone (250 ng) had a synergistic effect when combined with the same low dose of estriol (10 ng). We conclude that the weak natural estrogens estrone and 17ss-estradiol synergize with a low dose of the more potent estriol to reverse gonadal sex during the critical period of sexual differentiation. These results suggest that weak environmental estrogens may also synergize with stronger natural estrogens.

Developmental effects on intersexual and intrasexual variation in growth and reproduction in a lizard with temperature-dependent sex determination.

The mechanisms that control growth and reproduction have received considerable attention by molecular and cellular endocrinologists, yet there has been relatively little effort to link these two aspects of physiology. On the other hand, evolutionary biologists have long commented on the relationship between growth and reproduction in many species, yet have generally neglected the mechanisms underlying such complex traits. An approach that integrates the multiple proximate levels promises to provide significant insight into the evolution of neuroendocrine control mechanisms. In this chapter, we take this approach in reviewing environmental influences on growth and reproduction in the leopard gecko, Eublepharis macularius. In this species, incubation temperature during embryonic development not only determines gonadal sex, but also underlies within-sex differences in growth, adult morphology, aggressiveness, reproductive physiology and behaviour, and brain organization. Thus, the leopard gecko is an excellent model to elucidate the developmental interactions among the environment and the endocrine and nervous systems that control growth and reproduction.

The relative effectiveness of estrone, estradiol-17 beta, and estriol in sex reversal in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination.

In many turtles the temperature during the middle of incubation determines the gonadal sex of the hatchling. Sex steroid hormones have been implicated in temperature-dependent sex determination in the red-eared slider turtle, Trachemys scripta; nonaromatizable androgens are involved in male sex determination and estrogens and aromatizable androgens in female sex determination. Administration of exogenous estradiol-17 beta to eggs incubating at a temperature that normally produces only males can overcome the effect of temperature and result in all offspring being female. Further, estradiol-17 beta and incubation temperature synergize to produce a greater feminizing effect at intermediate incubation temperatures that produce mixed sex ratios. This study demonstrates that, in the red-eared slider, there is a complex interaction between incubation temperature, different estrogens, and the dosage effect of each hormone. There are changes in potency of different estrogens with incubation temperature such that estriol is more potent than estrone and estradiol-17 beta at 26 degrees (an all-male producing incubation temperature), estrone and estriol are equipotent to each other and more potent than estradiol-17 beta at 28.8 degrees (an incubation temperature that produced a male-biased sex ratio), and estradiol-17 beta is more potent than estrone and estriol at 29 degrees (an incubation temperature that produced equal numbers of males and females). These changes may be due to differences in synergism between the hormones and incubation temperature. Estriol treatment also resulted in cranially hypertrophied oviducts at all incubation temperatures in a dose-dependent manner, whereas animals treated with estradiol-17 beta and estrone had normal oviducts. These results support the hypothesis that estrogens are involved in the final common pathway of female sex determination in this species.

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.

In many turtles the temperature during the middle of incubation determines the gonadal sex of the hatchling. Sex steroid hormones have been implicated in temperature-dependent sex determination in the red-eared slider turtle, Trachemys scripta; androgen is involved in male sex determination and estradiol in female sex determination. Administration of exogenous estradiol and its agonists to eggs incubating at a male-producing temperature can overcome the effect of temperature and result in all-female offspring. Exogenous testosterone will also result in some female hatchlings if administered to eggs incubated at a male-producing temperature, an effect due to the aromatization of testosterone to estradiol. This study demonstrates that in the red-eared slider, androstenedione, the precursor to both testosterone and estradiol, has a similar effect. In addition, both testosterone and androstenedione synergize with incubation temperature to exert a greater effect at intermediate incubation temperatures that normally produce mixed sex ratios, indicating that as with estradiol, androstenedione and testosterone are involved in the final common pathway of sex determination in this species. At the single dosage administered, estrone and estradiol produced all females at a male-producing incubation temperature.

Temperature-dependent sex determination in the snapping turtle: manipulation of the embryonic sex steroid environment.

In certain reptiles with temperature-dependent sex determination (TSD), estrogens act as a signal for female differentiation. Because aromatase produces estrogens from androgens, this enzyme plays a pivotal role in TSD. Whether androgens act as the signal for male differentiation in TSD species in not yet clear. We manipulated the hormonal environment in eggs of the common snapping turtle (Chelydra serpentina) to determine the effects of an estrogen (estradiol 17-beta), an aromatase inhibitor (fadrozole; CGS 16949A), and androgens (testosterone and dihydrotestosterone) on sex determination in this TSD species. Test solutions were applied topically to representative eggs (total tested = 1054 from 27 clutches) and incubated at two male-producing temperatures (24 and 26.5 degrees) and at a predominantly female-producing temperature (29 degrees). In this species, application of an estrogen induced female development at all temperatures tested. In contrast, the aromatase inhibitor had no effect at the male-producing temperatures, but induced male development at the predominantly female-producing temperature. At this temperature, aromatase inhibitor plus testosterone had a similar male-producing effect, but when applied alone, testosterone failed to augment male production. Dihydrotestosterone had a similar effect, in contrast to its reported androgenic effects in other TSD species. In the snapping turtle, male differentiation may not be androgen dependent; rather, it may proceed in the absence of female differentiation. In this species, female development is clearly estrogen-dependent and is altered by aromatase inhibition at female-producing temperatures. Our results not only provide additional evidence that sex steroids mediate gonadal differentiation in TSD species, but also suggest caution with respect to generalizations about the proximal mechanisms of TSD in reptiles.