Temperature fluctuations and estrone sulfate affect gene expression via different mechanisms to promote female development in a species with temperature-dependent sex determination.

Variation in developmental conditions can affect a variety of embryonic processes and shape a number of phenotypic characteristics that can affect offspring throughout their lives. This is particularly true of oviparous species where development typically occurs outside of the female, and studies have shown that traits such as survival and behavior can be altered by both temperature and exposure to steroid hormones during development. In species with temperature-dependent sex determination (TSD), the fate of gonadal development can be affected by temperature and by maternal estrogens present in the egg at oviposition, and there is evidence that these factors can affect gene expression patterns. Here, we explored how thermal fluctuations and exposure to an estrogen metabolite, estrone sulfate, affect the expression of several genes known to be involved in sexual differentiation: Kdm6b, Dmrt1, Sox9, FoxL2 and Cyp19A1. We found that most of the genes responded to both temperature and estrone sulfate exposure, but that the responses to these factors were not identical, in that estrone sulfate effects occur downstream of temperature effects. Our findings demonstrate that conjugated hormones such as estrone sulfate are capable of influencing temperature-dependent pathways to potentially alter how embryos respond to temperature, and highlight the importance of studying the interaction of maternal hormone and temperature effects.

Is Thermal Responsiveness Affected by Maternal Estrogens in Species with Temperature-Dependent Sex Determination?

In species with temperature-dependent sex determination (TSD), incubation temperatures regulate the expression of genes involved in gonadal differentiation and determine whether the gonads develop into ovaries or testes. For most species, natural incubation conditions result in transient exposure to thermal cues for both ovarian and testis development, but how individuals respond to this transient exposure varies and can drive variation in the resulting sex ratios. Here, we argue that variation in the timing to respond to temperature cues, or thermal responsiveness, is a trait needing further study. Recent work in the red-eared slider turtle (Trachemys scripta) has found that when embryos experience transient exposure to warm conditions (i.e., heatwaves), some embryos show high responsiveness, requiring only short exposures to commit to ovarian development, while others show low responsiveness, developing testes even after more extended exposures to warm conditions. We discuss how maternal estrogens might influence thermal responsiveness for organisms that develop under thermal fluctuations. Examining the interplay of molecular responses to more subtle thermal and endocrine environments may reveal significant insights into the process of sex determination in species with TSD.

Red-eared slider hatchlings (Trachemys scripta) show a seasonal shift in behavioral types.

Correlated and repeatable patterns of behavior, termed behavioral types, can affect individual fitness. The most advantageous behavioral type may differ across predictable environments (e.g., seasonally), and maternally mediated effects may match hatchling behavior to the environment. We measured righting response, an indicator of behavioral type, of juvenile red-eared slider turtles (Trachemys scripta) emerging from early and late season clutches to understand if the production of behavioral types differs across the nesting season. There was a significant effect of season, with early season hatchlings righting more quickly than late season hatchlings, and we explored two potential underlying mechanisms, maternal estrogens and maternal investment (e.g., yolk allocation). We dosed early season eggs with an estrogen mixture to mimic late season eggs and assayed hatchling righting response, but found no significant effect of this maternal effect. We assessed maternal investment by measuring egg, hatchling, and residual yolk masses. We found a seasonal pattern in yolk allocation, where early season eggs have more yolk than late season eggs. Early season hatchlings used more yolk for growth rather than maintenance of existing tissues, resulting in larger hatchlings. Interestingly, across both seasons, hatchlings that received less maternal yolk appeared to be more efficient at converting yolk to tissue, but we found no direct correlation with righting behavior. We demonstrate that the prevalence of behavioral types varies across the nesting season, creating correlated suites of seasonal phenotypes in turtle hatchlings, but it appears that neither maternal estrogens or investment in yolk directly underlie this shift in behavior.

Year-round plasma steroid hormone profiles and the reproductive ecology of gopher tortoises (Gopherus polyphemus) at the southernmost edge of their range.

Populations of wide ranging ectotherms often exhibit variation in traits that are influenced by local environmental conditions. Although the gopher tortoise, Gopherus polyphemus, is well studied in pine flatwoods habitats across their range, little attention has been given to coastal populations existing in the southern extreme portion of the range. We examined the reproductive physiology of a coastal dune population in southwest Florida to determine if reproductive cycles vary across populations. Here we present the first year-round sex hormone profiles for a wild population of gopher tortoises. Male testosterone concentrations varied across the year (F11,54 = 2.52, P = 0.015) with elevated values from September to December and minimal levels from April to July, with the exception of a secondary peak during the month of June. Female testosterone and estradiol concentrations varied across the sampling period (T: F11,66 = 8.54, P < 0.001, E: F11,66 = 4.57, P < 0.001) with highest values from August to February, and lowest levels from May to July. Female progesterone concentrations varied over the year (F11,64 = 3.29, P = 0.002) and increased in late fall with a peak in March. These data suggest this population has an extended breeding season from fall through spring with mating likely occurring from September through March, and nesting in winter through spring. This pattern is similar to reproductive patterns described for tropical and sub-tropical chelonians but differs from that of gopher tortoise populations in northern portions of the range where hibernation may last for five months and a single clutch of eggs are deposited in late spring.

In ovo inhibition of steroid metabolism by bisphenol-A as a potential mechanism of endocrine disruption.

During embryonic development, endogenous signals, for example steroid hormones, and exogenous signals, for example endocrine disrupting chemicals (EDCs), have the capacity to produce phenotypic effects that persist into adulthood. As the actions of steroids are mediated through the binding of steroid receptors, most studies of EDCs have assumed that they too elicit their effects by binding steroid receptors. We tested an alternative hypothesis, namely that EDCs elicit their effects during embryonic development by disrupting the metabolism of maternally derived steroids, thereby allowing maternally derived steroids to bind steroid receptors and elicit effects. Specifically, we examined the ability of the EDC, bisphenol-A (BPA) to inhibit the normal metabolism of oestradiol during the first nine days of embryonic development in the red-eared slider turtle (Trachemys scripta). We found that, when BPA was present, oestrogen metabolism was inhibited when compared to control eggs. In particular, the formation of oestrone sulfate was blocked in BPA-treated eggs. We postulate that the oestrogenic effects of EDCs may be driven, at least in part, by inappropriate oestrogen signalling. The retention of oestrogens at points of development when they would normally be metabolized to inactive forms might also help explain low-dose effects frequently reported for EDCs.

Sulfonation of maternal steroids is a conserved metabolic pathway in vertebrates.

All vertebrate embryos develop in the presence of maternally derived steroids, and maternal steroids have been hypothesized to link phenotype of the offspring to maternal physiology. In placental vertebrates, it is known that maternally derived steroids are metabolized during development via the sulfonation pathway. We used eggs from the red-eared slider turtle (Trachemys scripta) to determine whether the same metabolic pathway is used to metabolize maternally derived steroids in an oviparous vertebrate. To examine the relationship between estradiol and estrogen sulfates during development, levels of maternally derived estradiol were compared with levels of estradiol sulfate, estrone sulfate, and estriol sulfate at oviposition and after 20 days of embryonic development. Estrone sulfate was the only detectable estrogen sulfate. At oviposition, levels of both estradiol and estrone sulfate varied seasonally with clutches from later in the nesting season having significantly higher concentrations of both steroids. Levels of estrone sulfate increased during development, demonstrating that the sulfonation of maternally derived steroids occurs in oviparous vertebrates as well as in placental vertebrates. We also found that exogenous estrone sulfate increases the production of female hatchlings, thereby demonstrating the ability of this metabolite to influence embryonic development. To examine the role of sulfonation in the metabolism of maternal progesterone and testosterone, we characterized the metabolic fate of both steroids by applying tritiated forms of each steroid at oviposition and characterizing metabolites after 20 days of incubation. Similar to what was demonstrated for estradiol, both progesterone and testosterone are converted to sulfonated metabolites during embryonic development. These data suggest that steroid sulfates, both those that are maternally derived and those resulting from the metabolism of maternal steroids, are a key component of the mechanism underlying steroid-mediated maternal effects.

No evidence that estrogens affect the development of the immune system in the red-eared slider turtle, Trachemys scripta.

Exposure to maternally derived substances during development can affect offspring phenotype. In ovo exposure to maternally derived steroids has been shown to influence traits such as growth and behavior in the offspring. The development of the immune system also can be altered by exposure to both androgens and glucocorticoids in a variety of species, but much less is known about the potential for estrogens to influence the development of this system. We examined the effect of estradiol on the development of both innate and adaptive immune components in the red-eared slider turtle (Trachemys scripta). A bacterial killing assay was used to assess innate immunity, a delayed-type hypersensitivity test for cellular immunity, and total immunoglobulin levels to measure the humoral immune response. We found no effect of in ovo estradiol treatment on any of our immune measures despite using doses that are known to influence other phenotypic parameters during development and varying the timing of dosing across development. Our results suggest that maternally derived estradiol does not affect the development of the immune system in T. scripta.

Characterizing the metabolism and movement of yolk estradiol during embryonic development in the red-eared slider (Trachemys scripta).

Eggs of oviparous amniotes can contain substantial quantities of several steroids at the time of oviposition. These maternally derived steroids appear to affect the phenotype of developing offspring, but not all steroid sensitive traits are affected by maternal steroids, and little is known about how these effects may arise. In this study, we applied tritiated estradiol to the eggs of red-eared sliders (Trachemys scripta) at the time of oviposition and characterized the subsequent metabolism and movement throughout embryonic development. Results indicate that very early in development, estradiol is converted to a variety of water-soluble estrogen sulfates that reside in the yolk and extraembryonic fluids until late in development. Within the final stages of development, we observe a significant decline in the total amount of metabolites present in the yolk and extraembryonic fluids and a significant increase in the amount of metabolites present in the embryo. While estradiol metabolism occurs during the early stages of development, the later stages appear to be the most dynamic with regards to the movement of estradiol metabolites. Our findings have important implications for studies investigating the effect of maternally derived steroids on offspring development.

Biological activity of oestradiol sulphate in an oviparous amniote: implications for maternal steroid effects.

Understanding the many factors that underlie phenotypic variation is of profound importance to evolutionary biologists. The embryonic endocrine environment is one such factor that has received much attention. In placental amniotes, the dynamic interaction of maternal and embryonic steroid production and metabolism is critical to regulating the endocrine environment. Less is known about how embryos of oviparous amniotes regulate their endocrine environment because most studies have focused on relating initial steroid levels in the yolk at oviposition to offspring phenotype. We tested the hypothesis that embryos of oviparous amniotes regulate their endocrine environment by conjugating maternal steroids and subsequently using the metabolites as precursors for steroid production later in development. Using the red-eared slider turtle (Trachemys scripta), we first characterized the conjugation of exogenous oestradiol to either oestradiol glucuronide or oestradiol sulphate (E(2)-S) in ovo during the first 15 days of development. Results show that oestradiol is primarily conjugated to E(2)-S. We then examined whether E(2)-S influenced sex determination and report that E(2)-S increases the production of female offspring. These data demonstrate that oviparous amniotes can both sulphonate steroids and respond to sulphonated steroids during embryonic development in a manner similar to placental amniotes.

Embryonic modulation of maternal steroids in European starlings (Sturnus vulgaris).

In birds, maternally derived yolk steroids are a proposed mechanism by which females can adjust individual offspring phenotype to prevailing conditions. However, when interests of mother and offspring differ, parent-offspring conflict will arise and embryonic interests, not those of the mother, should drive offspring response to maternal steroids in eggs. Because of this potential conflict, we investigated the ability of developing bird embryos to process maternally derived yolk steroids. We examined how progesterone, testosterone and oestradiol levels changed in both the yolk/albumen (YA) and the embryo of European starling eggs during the first 10 days of development. Next, we injected tritiated testosterone into eggs at oviposition to characterize potential metabolic pathways during development. Ether extractions separated organic and aqueous metabolites in both the embryo and YA homogenate, after which major steroid metabolites were identified. Results indicate that the concentrations of all three steroids declined during development in the YA homogenate. Exogenous testosterone was primarily metabolized to an aqueous form of etiocholanolone that remained in the YA. These results clearly demonstrate that embryos can modulate their local steroid environment, setting up the potential for parent-offspring conflict. Embryonic regulation must be considered when addressing the evolutionary consequences of maternal steroids in eggs.

Progesterone metabolites, "xenobiotic-sensing" nuclear receptors, and the metabolism of maternal steroids.

During development, embryos utilize steroid signals to direct sexual differentiation of tissues necessary for reproduction. Disruption of these signals by exogenous substances (both natural and synthetic) frequently produce phenotypic effects that can persist into adulthood and influence reproduction. This paper reviews the evidence that during embryonic development, progesterone metabolites and xenobiotic-sensing nuclear receptors may interact to increase the expression of numerous enzymes responsible for steroid metabolism in oviparous and placental amniotes. In these groups, embryonic development is characterized by (1) elevated progesterone concentrations, (2) 5 beta reduction being the primary metabolic pathway of progesterone, (3) the presence of xenobiotic-sensing nuclear receptors that can bind 5 beta metabolites of progesterone, and (4) increased expression of a suite of enzymes responsible for the metabolism of multiple steroids. We propose that xenobiotic-sensing nuclear receptors initially evolved to buffer the developing embryo from the potentially adverse effects of various maternal steroids on sexual differentiation.

Rapid decline in the concentrations of three yolk steroids during development: is it embryonic regulation?

Maternally derived yolk steroids have been found to elicit both short-term and long-term effects on offspring phenotype. Paradoxically, their effects can be strikingly specific given the often substantial concentrations present at oviposition, and they do not appear to uniformly affect all steroid-sensitive processes. To better understand the dynamics of yolk steroids across embryonic development, we quantified levels of progesterone, testosterone, and estradiol at 5-day intervals throughout development in eggs of the red-eared slider turtle (Trachemys scripta) incubated at both male- and female-producing temperatures. We also assessed the effect of season on yolk steroid levels. For all steroids assayed, the concentrations in yolk declined significantly by day 15 of embryonic development despite large differences in initial concentrations among steroids. We found that estradiol was the only steroid whose initial concentration varied significantly with season, while only the decline in testosterone was affected by incubation temperature. These findings illustrate the complex nature of yolk steroid dynamics and suggest that maternal steroids may be rapidly degraded or subject to embryonic processing, emphasizing the need for studies aimed at understanding the mechanisms through which yolk steroids may elicit their effects.

A modified yolk biopsy technique improves survivorship of turtle eggs.

Nongenetic maternal contributions, such as steroid hormones, have received much attention in recent years because they have the potential to influence offspring phenotype. Research in oviparous taxa has demonstrated that there is among-species variability in their response to these maternal contributions. However, studies in chelonians and crocodilians have been hampered by the fact that techniques involving egg manipulations that breach the eggshell routinely result in massive egg mortality. In this study, we present an improved yolk manipulation technique that resulted in increased egg survival (in excess of 70% survival) in the red-eared slider turtle (Trachemys scripta) and that may be broadly applicable to other species. By elevating survival to a level on par with other oviparous taxa, this method permits a more thorough exploration of reptilian egg physiology and allows for studies that examine traits in both the egg and the resulting hatchling.

Long-term sex reversal by oestradiol in amniotes with heteromorphic sex chromosomes.

Oestradiol application during embryonic development reverses the sex of male embryos and results in normal female differentiation in reptiles lacking heteromorphic sex chromosomes, but fails to do so in birds and mammals with heteromorphic sex chromosomes. It is not clear whether the evolution of heteromorphic sex chromosomes in amniotes is accompanied by insensitivity to oestradiol, or if the association between oestradiol insensitivity and heteromorphic sex chromosomes can be attributable to phylogenetic constraints in these taxa. Turtles provide an ideal system to examine the potential relationship between oestradiol insensitivity and sex chromosome heteromorphy, since there are species with heteromorphic sex chromosomes that are closely related to species lacking heteromorphic sex chromosomes. We investigated this relationship by examining the long-term effects of oestradiol-17beta application on sex determination in Staurotypus triporcatus and Staurotypus salvinii, two turtle species with male heterogamety. After raising the turtles in the lab for 3 years, we found follicular and Müllerian duct morphology in oestradiol-treated turtles that was identical to that of untreated females. The lasting sex reversal suggests that the evolutionary transition between systems lacking heteromorphic sex chromosomes and those with heteromorphic sex chromosomes is not constrained by a fundamental mechanistic difference.

Hormone levels in yolk decline throughout development in the red-eared slider turtle (Trachemys scripta elegans).

This study investigates the potential effects of maternally derived hormones present in the yolk of reptile eggs. Specifically, we ask when are these hormones utilized by developing red-eared slider turtles (Trachemys scripta elegans), a species with temperature-dependent sex determination (TSD). Eggs were incubated at 27 degrees C, a male-producing temperature, and at 31 degrees C, a female-producing temperature. Concentrations of progesterone, testosterone, and 17beta-estradiol were measured at four points during development: at oviposition, at the start of the temperature sensitive period (TSP), at the end of the TSP, and at hatching. No effects of incubation temperature on yolk hormone concentrations were detected. The highest concentrations of all three hormones were measured at oviposition. Hormone-specific patterns of decline occurred throughout development. Each hormone declined between oviposition and the early TSP. Although estradiol was present in detectable quantities at oviposition, it was virtually undetectable by the early TSP. Testosterone showed no further decline after the early TSP. Progesterone continued to decline between the early and post-TSP. These results demonstrate that maternally derived yolk hormones decline at different rates. Alternative explanations for the disappearance of these yolk hormones are presented.

Maternally derived yolk hormones vary in follicles of the painted turtle, Chrysemys picta.

The transfer of hormones from a female to her offspring is known to occur in egg laying vertebrates, and the potential for these early, maternally derived hormones to influence sex determination in reptiles with temperature-dependent sex determination is intriguing. In the present study, we examine variation in the concentrations of progesterone, testosterone, and estradiol among three follicle size classes within a female painted turtle (Chrysemys picta) and among females across four periods that span the pre- to post-nesting season. Females were collected, and both follicles and shelled eggs (when present) were harvested for hormone analysis. Progesterone levels did not vary seasonally. However, the concentration of progesterone did vary among and within follicle classes, and was primarily dependent upon ovulatory state: Recently ovulated follicles (as yolks within shelled eggs) contained significantly more progesterone than unovulated follicles. Concentrations of testosterone were low and did not vary either among size classes or across the season. Estradiol levels decreased with increasing follicle size and were higher later in the nesting season. Thus, hormone concentrations varied among follicle sizes and states but in patterns that differed among hormones. This variation has the potential to influence sex determination.