Effect of feed restriction during calfhood on serum concentrations of metabolic hormones, gonadotropins, testosterone, and on sexual development in bulls.

The objective of the present study was to evaluate the effects offeed restriction during calfhood on serum concentrations of metabolic hormones, gonadotropins, and testosterone, and on sexual development in bulls. Eight beef bull calves received a control diet from 10 to 70 weeks of age. An additional 16 calves had restricted feed (75% of control) from 10 to 26 weeks of age (calfhood), followed by either control or high nutrition (n=8/group) during the peripubertal period until 70 weeks of age. Restricted feed during calfhood inhibited the hypothalamic GnRH pulse generator, reduced the pituitary response to GnRH, impaired testicular steroidogenesis, delayed puberty, and reduced testicular weight at 70 weeks of age, regardless of the nutrition during the peripubertal period. Restricted feed reduced serum IGF-I concentrations, but concentrations of leptin, insulin, and GH were not affected. In conclusion, restricted feed during calfhood impaired sexual development in bulls due to adverse effects on every level of the hypothalamus-pituitary-gonad axis and these effects were not overcome by supplemental feeding during the peripubertal period. Furthermore, based on temporal associations, the effects of restricted feed on the hypothalamus-pituitary-gonad axis might be mediated by serum IGF-I concentrations. These results supported the hypotheses that the pattern of LH secretion during the early gonadotropin rise during calfhood is the main determinant of age of puberty in bulls and that gonadotropin-independent mechanisms involved in testicular growth during the peripubertal period are affected by previous LH exposure.

Steroid signaling system responds differently to temperature and hormone manipulation in the red-eared slider turtle (Trachemys scripta elegans), a reptile with temperature-dependent sex determination.

Many reptiles, including the red-eared slider turtle (Trachemys scripta elegans), exhibit temperature-dependent sex determination (TSD). Temperature determines gonadal sex during the middle of embryogenesis, or the temperature-sensitive period (TSP), when gonadal sex is labile to both temperature and hormones--particularly estrogen. The biological actions of steroid hormones are mediated by their receptors as defined here as the classic transcriptional regulation of target genes. To elucidate estrogen action during sex determination, we examined estrogen receptor alpha (Esr1, hereafter referred to as ERalpha), estrogen receptor beta (Esr2, hereafter referred to as ERbeta), and androgen receptor (Ar, hereafter referred to as AR) expression in slider turtle gonads before, during and after the TSP, as well as following sex reversal via temperature or steroid hormone manipulation. ERalpha and AR levels spike at the female-producing temperature while ovarian sex is determined, but none of the receptors exhibited sexually dimorphic localization within the gonad prior to morphological differentiation. All three receptors respond differentially to sex-reversing treatments. When shifted to female-producing temperatures, embryos maintain ERalpha and AR expression while ERbeta is reduced. When shifted to male-producing temperatures, medullary expression of all three receptors is reduced. Feminization via estradiol (E(2)) treatment at a male-producing temperature profoundly changed the expression patterns for all three receptors. ERalpha and ERbeta redirected to the cortex in E(2)-created ovaries, while AR medullary expression was transiently reduced. Although warmer incubation temperature and estrogen result in the same endpoint (ovarian development), our results indicate different steroid signaling patterns between temperature- and estrogen-induced feminization.

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).

The distributions of the duplicate oestrogen receptors ER-beta a and ER-beta b in the forebrain of the Atlantic croaker (Micropogonias undulatus): evidence for subfunctionalization after gene duplication.

Teleost fishes have three distinct oestrogen receptor (ER) subtypes: ER-alpha, ER-beta a (or ER-gamma) and ER-beta b. ER-beta a and ER-beta b arose from a duplication of an ancestral ER-beta gene early in the teleost lineage. Here, we describe the distribution of the three ER mRNAs in the hypothalamus and cerebellum of the Atlantic croaker to address two issues: the specific functions of multiple ERs in the neuroendocrine system and the evolution and fate of duplicated genes. ER-alpha was detected in nuclei of the preoptic area (POA) and hypothalamus previously shown to possess ER-alphas in teleosts. AcER-beta b, but not ER-beta a, labelling was detected in the magnocellular neurons of the POA, nucleus posterior tuberis, the nucleus recessus posterior and cerebellum. By contrast, acER-beta a, but not ER-beta b, was detected in the dorsal anterior parvocellular POA and suprachiasmatic nucleus. Both ER-betas were found in posterior parvocellular and ventral anterior POA nuclei, the ventral hypothalamus, and periventricular dorsal hypothalamus. The differences we observed in ER subtype mRNA distribution within well-characterized brain nuclei suggest that ER-beta a and ER-beta b have distinct functions in the neuroendocrine control of reproduction and behaviour, and provide evidence that the teleost ER-beta paralogues have partitioned functions of the ancestral ER-beta gene they shared with tetrapods.

Vasoactive intestinal polypeptide contacts on gonadotropin-releasing hormone neurones increase following puberty in female rats.

Successful reproduction requires precise temporal coordination among various endocrine and behavioural events. The circadian system regulates daily temporal organization in behaviour and physiology, including neuroendocrine rhythms. The main circadian pacemaker in mammals is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The SCN sends direct efferents to the reproductive axis via monosynaptic projections to gonadotropin-releasing hormone (GnRH) neurones. This communication generates circadian endocrine rhythms as well as the preovulatory luteinizing hormone (LH) surge necessary for successful ovulation. One SCN peptide thought to be important for the regulation of oestrous cycles is vasoactive intestinal polypeptide (VIP). VIP neurones from the SCN contact GnRH cells, and these cells are preferentially activated during an LH surge in rats. Unlike adult rats, prepubertal females do not exhibit oestrous cycles, nor do they exhibit an LH surge in response to oestradiol positive-feedback. The present study was undertaken to determine the extent to which the development of a 'mature' reproductive axis in female rats is associated with modifications in VIP contacts on GnRH neurones. The brains of diestrus adult (approximately 60 days of age) and prepubertal (21 days of age) female rats were examined using double-label fluorescence immunohistochemistry for VIP and GnRH, with light and confocal microscopy. Although the total number of GnRH-immunoreactive neurones did not differ between adult and prepubertal females, adults had a significant increase in the percentage of GnRH cells receiving VIP contacts compared to juveniles. These data suggest that the development of reproductive hormone rhythms and oestrous cyclicity may be, in part, due to modifications of VIP input to the GnRH system.

Role of steroidogenic factor 1 and aromatase in temperature-dependent sex determination in the red-eared slider turtle.

Red-eared slider turtles are genetically bipotential for sex determination. In this species, as in many other reptiles, incubation temperature of the egg determines gonadal sex. At higher incubation temperatures females are produced and increasing temperature appears to increase estrogen production in the embryonic brain. Treatment of eggs incubating at a male-producing temperature with exogenous estrogen causes ovaries to form. At a female-biased incubation temperature, prevention of estrogen biosynthesis or administration of nonaromatizable androgens results in the development of testes. In mammals, steroidogenic factor 1 (SF-1) regulates most genes required for estrogen biosynthesis, including aromatase. In both mammals and red-eared sliders, SF-1 is differentially expressed in males and females during gonadogenesis. We have examined both SF-1 gene expression and aromatase activity in embryos incubating at different temperatures and after manipulation to change the course of gonadal development. Our findings indicate a central role for SF-1 in enacting the effect of estrogen. Estrogen treatment directly or indirectly downregulates SF-1 and, ultimately, causes development of females. The inhibition of estrogen results in upregulation of SF-1 and male hatchlings. Thus, SF-1 may lie at the center of one molecular crossroad in male versus female differentiation of the red-eared slider.

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.

Control of attractivity and receptivity in female red-sided garter snakes.

Female red-sided garter snakes emerge from their hibernacula in the spring attractive and receptive to males. Attractivity is communicated by a pheromone released through the female's skin and is a consequence of ovarian recrudescence the previous summer. Receptivity, on the other hand, is stimulated by ovarian estrogen secretion during emergence itself. Mating renders females both unattractive and unreceptive. Another "mating" pheromone of male origin is important in making females unattractive after mating. To investigate the role of cloacal stimulation in the loss of attractivity and receptivity we injected a local anesthetic (lidocaine or tetracaine) in the cloacal region of females before mating. This does not prevent mating, although it blocks neural transmission of copulatory sensory stimuli. The time course of transition from attractive and receptive states was then observed. Females treated with local anesthetic as well as control females were unattractive within 15 min of mating. However, when retested 2-3 and 24 h after mating, a significantly higher proportion of treated females regained their attractivity, while mated control females remained unattractive. This restorative effect was transient, though, as treated females retested 48 h after mating were as unattractive as the controls. Both anesthetized and control females were unreceptive when tested following mating and did not regain receptivity with time. Last, the mating-induced surge in circulating concentrations of prostaglandin was diminished in females that received a local anesthetic prior to mating. Taken together these results indicate that the loss of attractivity and receptivity following mating in the red-sided garter snake is due to combined effects of a mating pheromone and a physiological, neurally mediated response to the sensation of stimuli associated with the act of mating.

Estradiol and incubation temperature modulate regulation of steroidogenic factor 1 in the developing gonad of the red-eared slider turtle.

Red-eared slider turtles are genetically bipotential for sex determination, with incubation temperature of the egg determining gonadal sex. At higher incubation temperatures, females are produced, possibly due to increased biosynthesis of estrogen. Exogenous estrogen causes the formation of ovaries, and prevention of estrogen biosynthesis results in the development of testes. In mammals, steroidogenic factor 1 (SF-1) regulates most genes required for estrogen biosynthesis from cholesterol. In mammals as well as red-eared slider turtles, SF-1 is differentially expressed in males and females during gonadogenesis. To examine a possible role for SF-1 in temperature-dependent sex determination, we assayed its expression in red-eared slider turtles after treatments that alter sex development during gonadogenesis of the wild-type organism. We examined gonadal SF-1 expression in embryos 1) incubating at three different temperatures, 2) after treating eggs with estrogen at a male-producing temperature, and 3) after inhibition of estrogen biosynthesis at a female-producing temperature. Our findings suggest that both temperature and estrogen lie upstream of SF-1 in a sex-determining regulatory hierarchy in red-eared slider turtles and that estrogen directly or indirectly modulates the regulation of SF-1 expression.

Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts.

This paper describes three distinct estrogen receptor (ER) subtypes: ERalpha, ERbeta, and a unique type, ERgamma, cloned from a teleost fish, the Atlantic croaker Micropogonias undulatus; the first identification of a third type of classical ER in vertebrate species. Phylogenetic analysis shows that ERgamma arose through gene duplication from ERbeta early in the teleost lineage and indicates that ERgamma is present in other teleosts, although it has not been recognized as such. The Atlantic croaker ERgamma shows amino acid differences in regions important for ligand binding and receptor activation that are conserved in all other ERgammas. The three ER subtypes are genetically distinct and have different distribution patterns in Atlantic croaker tissues. In addition, ERbeta and ERgamma fusion proteins can each bind estradiol-17beta with high affinity. The presence of three functional ERs in one species expands the role of ER multiplicity in estrogen signaling systems and provides a unique opportunity to investigate the dynamics and mechanisms of ER evolution.

Endocrine disruptors: present issues, future directions.

A variety of natural products and synthetic chemicals, known collectively as endocrine-disrupting compounds (EDCs), mimic or interfere with the mechanisms that govern vertebrate reproductive development and function. At present, research has focused on (i) the morphological and functional consequences of EDCs; (ii) identifying and determining the relative potencies of synthetic and steroidal compounds that have endocrine-disrupting effects; (iii) the mechanism of action of EDCs at the molecular level; and (iv) the recognition that in "real life," contamination usually reflects mixtures of EDCs. Future research must examine (i) the interactive nature of EDCs, particularly whether the threshold concept as developed in traditional toxicological research applies to these chemicals; (ii) when and how EDCs act at the physiological level, particularly how they may organize the neural substrates of reproductive physiology and behavior; (iii) the various effects these compounds have on different species, individuals, and even tissues; and (iv) how adaptations may evolve in natural populations with continued exposure to EDCs. Several predictions are offered that reflect these new perspectives. Specifically, (i) the threshold assumption will be found not to apply to EDCs because they mimic the actions of endogenous molecules (e.g., estrogen) critical to development; hence, the threshold is automatically exceeded with exposure. (ii) Behavior can compound and magnify the effects of EDCs over successive generations; that is, bioaccumulated EDCs inherited from the mother not only influence the morphological and physiological development of the offspring but also the offsprings' reproductive behavior as adults. This adult behavior, in turn, can have further consequences on the sexual development of their own young. (iii) The sensitivity of a species or an individual to a compound is related to species (individual)-typical concentrations of circulating gonadal steroid hormones. Related to this is the recent finding that alternate forms of the putative receptors are differentially distributed, thereby contributing to the different effects that have been observed. (iv) Except in extraordinary situations, populations often continue to exist in contaminated sites. One possible explanation for this observation that needs to be considered is that animals can rapidly adapt to the nature and level of contamination in their environment. It is unlikely that successive generations coincidentally become insensitive to gonadal steroid hormones fundamentally important as biological regulators of development and reproduction. Rather, adaptive alterations in the genes that encode steroid receptors may occur with chronic exposure to EDCs, allowing the sex hormone receptor to discriminate natural steroids from EDCs.

Aromatase activity during embryogenesis in the brain and adrenal-kidney-gonad of the red-eared slider turtle, a species with temperature-dependent sex determination.

Gonadal sex in the red-eared slider turtle is determined by the incubation temperature that the embryo experiences during the mid-trimester of development. High temperatures result in female-biased sex ratios, and low temperatures produce male-biased sex ratios. The physiological equivalent of temperature appears to be a combination of the nature and abundance of steroidogenic enzymes and their products-including estradiol and its precursor, testosterone-and aromatase, the enzyme that converts testosterone to estradiol. Aromatase has been hypothesized to play a major role in the female developmental pathway in this species, and research in other species with temperature-dependent sex determination points to the brain as an organ that transduces the temperature signal into an aromatase response. In this study, we used a tritiated water assay to compare the pattern of estradiol biosynthesis at male- and female-producing temperatures in the brain and adrenal-kidney-gonad (AKG) through development. The pattern for both sexes in the AKG was one of increased activity after the temperature-sensitive period (TSP), but with no significant difference between sexes. In the brain, however, putative females exhibited a significantly higher level of aromatase activity than putative males at the beginning of the TSP, after which activity in both male and female brains decreased, dropping below detection in females before hatch. These results point to the brain as a site of aromatase response to temperature in this species, and they suggest that the product of aromatase activity, estradiol, may induce alterations in the neuroendocrine axis controlling gonadal sex steroid hormone production.

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.

Androgenic regulation of steroid hormone receptor mRNAs in the brain of whiptail lizards.

Sex and species differences in androgenic regulation of steroid hormone receptor mRNAs were examined in the diencephalon of two species of whiptail lizards: Cnemidophorus inornatus is a sexual species and the direct evolutionary ancestor to Cnemidophorus uniparens, an all-female parthenogenetic species. Lizards were gonadectomized and treated with different doses of either aromatizable testosterone or nonaromatizable dihydrotestosterone. The relative abundances of androgen-, oestrogen-, and progesterone-receptor mRNAs were compared in various nuclei following in situ hybridization with homologous riboprobes. A diversity of patterns in androgenic regulation was observed, with effects differing according to brain region, the steroid-receptor mRNA being considered and, in some cases, between androgens. In the ancestral sexual species, intact males had lower androgen-receptor mRNA abundances than castrated, blank-implanted males in the medial preoptic area. Testosterone significantly decreased androgen-receptor mRNA abundance in the medial preoptic area of castrated males. Males had higher androgen-receptor mRNA levels in the preoptic area than females generally and neither the sexual or parthenogenetic females showed a decrease in androgen-receptor mRNA with androgen treatment. Both testosterone and dihydrotestosterone increased oestrogen-receptor mRNA abundance in the ventromedial hypothalamus of C. inornatus, but no sex differences in this effect were observed. Gonadectomy decreased, whereas androgen treatment increased, progesterone-receptor mRNA abundance in the ventromedial hypothalamus. There was a sex difference in this response to androgen in the sexual species, with males having greater amounts than females in this brain area. The parthenogenetic species exhibited a similar pattern to females of the sexual species, but the levels were higher overall, possibly because Cnemidophorus uniparens is triploid. The periventricular preoptic area showed a different pattern, with testosterone treatment increasing progesterone-receptor mRNA abundance in both sexes of the sexual species and in the parthenogenetic species, while dihydrotestosterone did not. The diversity of patterns in androgen effects indicates that gonadal sex, aromatization of androgen, and perhaps gene dosage all influence the expression of steroid-receptor mRNAs in the lizard brain.

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.

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.

Hormonal regulation of progesterone receptor mRNA expression in the hypothalamus of whiptail lizards: regional and species differences.

The effects of gonadal steroid hormones on steroid receptor mRNA expression vary across nuclei within the brain, between the sexes, and between species. We report that exogenous estrogen increases progesterone receptor (PR) mRNA levels in the periventricular preoptic area in an ancestor and descendant species pair of whiptail lizards, and also that this effect of estrogen is significantly stronger in females of the descendant species. Second, while progesterone strongly decreases PR mRNA in the ventromedial hypothalamus of whiptail lizards and rodents, we find that there is no discernible effect of progesterone on PR mRNA levels in the periventricular preoptic area in females of the ancestral member of this species pair. These findings are a further demonstration of the variability of steroid effects on steroid receptor mRNA levels across brain nuclei. This variability may be important both in behavioral transitions over the course of the ovarian cycle in this ancestor-descendant species pair of lizards and in the evolution of pseudosexual behavior in the descendant parthenogen species.

Sex reversal effects of environmentally relevant xenobiotic concentrations on the red-eared slider turtle, a species with temperature-dependent sex determination.

Xenobiotics suspected of being estrogenic-the PCB aroclor 1242 and the pesticides toxaphene, dieldrin, p,p'-DDD, cis-Nonachlor, trans-Nonachlor, p,p'-DDE, and chlordane-were examined for their ability to override a male-producing incubation temperature and result in female hatchlings in the red-eared slider, a turtle with temperature-dependent sex determination. Compounds were assayed in the environmentally relevant concentrations detected in alligator eggs from Lake Apopka, Florida, singly, in concert with one another, and with estradiol. Compounds assayed alone and resulting in significant sex reversal were trans-Nonachlor, cis-Nonachlor, aroclor 1242, p,p'-DDE, and chlordane. When administered with estradiol, only one of the compounds, chlordane, caused sex reversal at significant levels. When applied together, however, the eight compounds assayed resulted in significant sex reversal.

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.

No threshold dose for estradiol-induced sex reversal of turtle embryos: how little is too much?

Risk assessments for nongenotoxic chemicals assume a threshold below which no adverse outcomes are seen. However, when an endogenous chemical, such as 17ss-estradiol (E2), occurs at a concentration sufficient to cause an effect, the threshold is already exceeded. Under these circumstances, exogenous estradiol is not expected to provide a threshold dose. This principle is demonstrated for E2 in the red-eared slider, a turtle with temperature-dependent sex determination. In this species, gonadal sex is determined by egg incubation temperature; female development requires endogenous estrogen produced by elevated temperature. While normal production of females by endogenous estrogens is not an adverse effect, exogenous estrogens can sex reverse presumptive males, which can be an adverse effect. A large dose-response study was conducted using seven doses and a vehicle control (starting n = 300/group); a single E2 dose was applied to the eggshell of recently laid eggs. Animals were sexed after hatching. The incubation temperature chosen, 28.6 degrees C, generates a minority of females. Thus, the criteria for testing the threshold hypothesis were met, i.e., there is evidence that there is endogenous estrogen and that it generates an irreversible response. The lowest E2 dose tested, 400 pg/egg (40 ng/kg), sex reversed 14.4% of the animals, demonstrating very low dose sensitivity. The data were fit with a modified Michaelis-Menten equation, which provided an estimate of 1.7 ng/egg for endogenous estradiol. The median effective dose (ED50) was 5.0 +/- 2.0 ng/egg (95% confidence limits), of which 1.7 ng/egg was endogenous estradiol and 3.3 ng/egg came from the applied estradiol. There was no apparent threshold dose for E2. A smaller replication confirmed these results. These results provide a simple biologically based dose-response model and suggest that chemicals which act mechanistically like E2 may also show no threshold dose. If so, even low environmental concentrations of such chemicals may carry risk for sex reversal.