Integrative and Comparative Biology Advance Access originally published online on May 9, 2008
Integrative and Comparative Biology 2008 48(3):419-427; doi:10.1093/icb/icn034
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A proposed role of the sulfotransferase/sulfatase pathway in modulating yolk steroid effects
Department of Biological Sciences, Campus Box 4120, Illinois State University, Normal, IL 61790-4120, USA
Correspondence: 1E-mail: rpaitz{at}ilstu.edu
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Steroid hormones have long been studied by behavioral ecologists as a nongenetic means whereby females can influence the development of their offspring. In oviparous vertebrates, steroids are present in the yolk at the time of oviposition and have been shown to affect numerous traits of the offspring. To date, most studies have focused on the functional relationship between yolk steroids and offspring development. In this article we used a mechanistic approach to investigate the effects of yolk steroids in an attempt to decipher how lipophilic steroids may make it from the lipid-rich yolk to the developing embryo. First, we examined the distribution of radioactive and nonradioactive estradiol following the exogenous application of each to developing eggs of the red-eared slider. Second, we quantified sulfotransferase activity in various components of the egg as a potential mechanism for the metabolism of steroids. Results indicate that exogenous estradiol is converted to a water-soluble form during the first 15 days of development, concurrent with an increase of sulfotransferase activity in the yolk and extra-embryonic membranes. Based on these data, we propose a mechanistic model based upon the sulfotransferase/sulfatase pathway as a means through which developing eggs can convert steroids to a water-soluble form that can be transported to the embryo. These sulfonated steroids may then serve as precursors for subsequent steroid production via sulfatase activity. This model utilizes a mechanism known to be important for the modulation of maternal steroid signals in placental mammals, at the same time addressing several previously unanswered questions regarding the mechanisms underlying the effects of yolk steroids.
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Steroid signals are critical for differentiation during embryonic development in vertebrates (mammals: reviewed by Gerall et al. 1992
; birds: reviewed by Balthazart and Adkins-Regan 2002; reptiles: reviewed by Lance 1997; amphibians: reviewed by Hayes 1998; fish: reviewed by Devlin and Nagahama 2002). Because of their potential to produce long-term effects on the phenotype of offspring, maternal steroids have been proposed as a nongenetic mechanism through which females can manipulate their progeny by influencing the embryonic endocrine environment (reviewed by Groothuis et al. 2005).
Variation in the embryonic endocrine environment can affect anatomical, physiological, and behavioral traits of developing offspring. Studies in litter-bearing mammals have repeatedly demonstrated that the sex of neighboring litter mates can influence a developing embryo's endocrine environment, and this can directly affect numerous traits in the offspring such as the timing of sexual maturity and mating behavior (reviewed by Ryan and Vandenbergh 2002). Similar effects can also be produced by manipulating the maternal endocrine environment which, in turn, influences the embryonic environment (reviewed by Gerall et al. 1992). Interestingly, studies that manipulate the maternal environment often report that changes in the embryonic environment are of a much smaller magnitude and in some cases the embryonic environment does not change at all (reviewed by Nathanielsz 1976). One classic example in which natural variation in maternal endocrine environment was thought to influence offsprings’ phenotype is the spotted hyena (Crocuta crocuta). Female spotted hyenas exhibit highly masculinized external genitalia and it was originally proposed that this was caused by exposure to maternal androgens in utero, based on data showing that adult female hyenas have high levels of circulating androgens, and that the hyena placenta possesses unusually low levels of aromatase which is responsible for converting androgens to estrogens (Yalcinkaya et al. 1993). Recent studies using more advanced molecular techniques now show that placental aromatase activity does not decline until after sexual differentiation and that the observed masculinization is mostly androgen independent (reviewed by Glickman et al. 2006). Data from the spotted hyena, in addition to those manipulating maternal endocrine environment, suggest that maternal effects mediated through steroids are much more complex than just a simple elevation of maternal steroid levels that are then passed on to the offspring.
Most studies investigating the effects of maternal endocrine environment on offsprings’ phenotypes have focused on placental organisms, in which the placenta serves as a buffer permitting separation of maternal and embryonic endocrine signals through the production of enzymes that modulate steroid signals (Levitz 1966; Painter and Moore 2005). In oviparous vertebrates, steroids are transferred to the yolk during folliculogenesis (Schwabl 1993) and provide a means by which females may be able to influence the embryonic endocrine environment and, thus, the phenotype of their offspring. These so-called "yolk steroids" have been found in all taxa studied to date, and have been shown to influence a suite of phenotypes (reviewed by Groothuis et al. 2005). By far the greatest amount of research in this area has been conducted on birds, where traits such as duration of incubation, muscle morphology, immune function, begging behavior, and more, have been shown to be affected by yolk steroids (reviewed by Groothuis et al. 2005). The ability of yolk steroids to influence the phenotypes of offspring has also been demonstrated in other oviparous taxa such as lizards (Lovern and Wade 2003; Uller and Olson 2003) and turtles (Bowden et al. 2000). Collectively, these data suggest that yolk steroids can have both short- and long-term effects on offspring, and that yolk steroids can affect a wide variety of phenotypic characters. Because of their apparent ability to modulate phenotypes, yolk steroids have been studied as a means whereby females can adaptively allocate resources to offspring. Behavioral ecologists have focused primarily on the ultimate causes of variation in levels of yolk steroids, and this has lead to the discovery that the transfer of yolk steroids is highly variable both within and among taxonomic groups. This approach has left researchers in this field with a large number of patterns attributable to the effects of yolk steroids, but a limited understanding of the proximate mechanisms by which they can influence offspring phenotype (reviewed by Carere and Balthazart 2007).
To date, relatively few studies have attempted to investigate the physiological mechanisms that underlie yolk steroid effects, but several studies have quantified yolk steroid levels at different points of embryonic development. These studies show that yolk steroids decline early in development and that the rate of decline far exceeds the rate at which yolk is utilized, providing evidence that steroids are not just simply absorbed along with the yolk. In a variety of birds, androgen levels in the yolk have been shown to decline significantly by day five of development (Elf and Fivizzani 2002; Eising et al. 2003; Gilbert et al. 2007), and in reptiles concentrations of yolk steroids decline during the first third of development in both the red-eared slider (Trachemys scripta) (Bowden et al. 2002) and the common snapping turtle (Chelydra serpentina), (Elf et al. 2002). Although these data clearly demonstrate that concentrations of yolk steroids decline early in embryonic development, they do not provide a mechanism that can explain this observation. Some potential explanations for this decline include metabolism of the steroids either by enzymes present in the yolk or by enzymes produced by the developing embryo and/or accessory tissues, dilution of yolk by albumen as the interface between the two regions breaks down (Gilbert et al. 2007), and active sequestration by the embryo (Bowden et al. 2002). In this study, we investigate the role that steroid metabolism may play in the decline of yolk steroid levels during embryonic development in the red-eared slider turtle.
One of the most common pathways for steroid metabolism in vertebrates is through inactivation by sulfotransferases (reviewed by Gamage et al. 2006). Steroid inactivation involves the transfer of a sulfonyl (SO3–) group to a hydroxyl group on an acceptor molecule, and this reaction is catalyzed by sulfotransferases. In the case of steroids, sulfonation results in both biological inactivation and an increase in water solubility, which is why this process serves as a primary pathway for the clearance of steroids (Gamage et al. 2006). In addition to clearance, sulfotransferase activity is also known to be very high in the placenta (Miki et al. 2002) and to play a role in buffering the fetus from maternal steroid signals (Levitz 1966).
Based upon the idea that steroids are unlikely to passively diffuse out of the yolk because they are lipophilic, and that previous experimental results demonstrate that only a very small percentage (< 1%) of exogenously applied steroid can be detected in the embryo nine days after application (Crews et al. 1991), we hypothesize that steroid metabolism is the mechanism underlying the observed decline in yolk steroids. In the first experiment, we examined the distribution of radioactive and nonradioactive estradiol throughout the egg following the exogenous administration of each as proxies for how endogenous steroids may move within the egg. In the second experiment, we quantified sulfotransferase activity in four components of the egg (yolk, albumen, extra-embryonic membranes, and embryo) as a potential mechanism to explain the distribution of radioactive and nonradioactive estradiol from the first experiment. Finally, we propose a model to explain how sulfotransferase activity may modulate signals of yolk steroids within the egg.
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Tracing steroids
Eggs were obtained from five gravid red-eared slider turtles (T. scripta) that were collected at Banner Marsh State Fish and Wildlife Area in Central Illinois. Oviposition was induced via oxytocin injection (Ewert and Legler 1978
) and experimental treatments of eggs occurred within 24 hours of oviposition. To do this, nine eggs from each clutch were divided equally into one of three treatment groups with the 10th egg frozen for measurement of initial steroid levels. The first group served as the vehicle control and these eggs received a 10 µl bolus of 95% ethanol applied to the surface of the eggshell. A second group received a 10 µl bolus of ethanol containing 10 µg of estradiol (E2). The final group received a 10 µl bolus of ethanol containing 10 000 c.p.m. of tritiated E2 ([3H]E2). Administering both radioactive and nonradioactive steroids to eggs allowed for a comparison of whether radiolabeled estradiol levels correspond with levels of exogenously applied nonlabeled estradiol in the various components of eggs, or whether radiolabeled steroids were present in a form that could not be detected by radioimmunoassay (RIA). Eggs were then incubated at 31°C and one egg from each experimental group sampled every five days of development until the experiment terminated on the 15th day of development. The first 15 days of development were the focus of this study because this is the period over which the vast majority of the decline in steroid levels of yolk takes place in T. scripta (Paitz unpublished data). Eggs were collected under IDNR permit NH07.2084 and research was conducted following the Illinois State University IACUC guidelines.
At the time of sampling, eggs were separated into shell, albumen, yolk, and embryo and each component weighed to the nearest 0.01 g before freezing at –20°C for storage until steroid quantification. For developmental day five it was not feasible to collect embryos because of their small size. Estradiol levels in the control group and the nonradiolabeled E2 group were measured using RIA. E2 levels in the yolk, albumen, and embryo were quantified by RIA using a modified version of Wingfield's and Farner's method (1975). Steroids were extracted from 50 mg of tissue using a 70 : 30 combination of diethyl ether: petroleum ether and fractionated using column chromatography. Recoveries were calculated to determine the amount of sample lost during extraction and fractionation, by the addition of a 2000 c.p.m. tracer of E2 that was initially added to each sample. The E2 fraction was then run through the competitive binding portion of the RIA in duplicate and compared to a standard curve that ranged from 5 to 750 pg/g.
To quantify radioactivity in the yolk, albumen, and embryo, steroids were extracted from 100 mg of each component diluted in 1 ml of water with a 6 ml combination of diethyl ether : petroleum ether. This is the same method of extraction used in the RIA and results in an extraction efficiency of over 80% for estradiol. The ether fraction was then dried under nitrogen gas and resuspended in 1 ml of ethanol. Radioactivity in both the water and organic fraction was counted on a Beckman© LS 6500 scintillation counter. Total radioactivity for each component of the egg was calculated by multiplying the counts detected in 100 mg by the total mass of that component. These methods resulted in the recovery of 
70% of the initial 10 000 c.p.m. that was applied.
Quantification of sulfotransferase
For this study, three clutches of T. scripta eggs were collected using the aforementioned techniques. Two eggs were immediately frozen to measure initial sulfotransferase levels and the remaining eggs were incubated at 31°C. Two eggs from each clutch were then sampled at days 2, 5, 10, and 17 of development. At the time of sampling, eggs were divided into yolk, albumen, extra-embryonic membrane, and embryo and frozen at –80°C until assay. For developmental day 2 and 5 it was not feasible to collect embryos due to their small size.
The ability of each egg component to sulfonate estradiol was quantified by measuring the conversion of tritiated E2 to a water-soluble form after the addition of the sulfonate donor, phosphoadenosine-5'-phosphosulfate (PAPS), following the method of Miki et al. (2002). At the time of assay, 100 µg of each component of the egg was homogenized in 200 µl of reaction buffer and centrifuged for 10 min at 1000 g. Sulfotransferase activity was quantified by adding the 50 µl of the supernatant to a reaction containing 40 nM [3H]E2 and 40 µM PAPS in 50 µl reaction buffer. Reactions were carried out for 30 min at 31°C and terminated with the addition of 2 ml of dichloromethane kept on ice followed by the addition of 0.4 ml 0.25 M Tris-HCl (pH 8.4), also kept on ice, to alkalinize the solution and then vortexed. Synthesis of sulfonated [3H]E2 was quantified by counting the aqueous phase on a scintillation counter. Correction for background radioactivity was made by subtracting background levels obtained from the blanks. Reaction rates were then calculated as fmole of E2 converted/milligram of egg component/minute.
Statistical analysis
To test for differences in steroid levels, we performed an ANOVA using treatment, day, and egg component as fixed factors along with their respective interactions. Any nonsignificant interactions were subsequently removed from the model. A similar ANOVA was used to test for differences in level of radioactivity, but in this case day, egg component, and extraction phase were entered as the fixed factors. Finally, differences in sulfotransferase activity within each component of the egg were examined using a mixed model ANOVA with day as a fixed factor and clutch as a random factor. For all analyses, data were appropriately transformed prior to analysis and post hoc comparisons (Tukey's HSD) were conducted to test for differences between groups. All statistical tests were performed in SAS v. 9.1 (SAS Institute, Cary, NC, USA).
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Estradiol and radioactivity distribution
Our analysis of exogenous E2 found a significant effect of treatment (F1,39 = 81.96, P < 0.0001), egg component (F1,39 = 7.48, P = 0.0093), and day of development (F2,39 = 8.87, P = 0.0007) on E2 concentrations as measured by RIA. There were no significant interactions (all P < 0.2) in the model. Eggs treated with E2 had higher concentrations in the yolk compared to control eggs while the yolk contained higher concentrations of E2 than did albumen for the treated group (Fig. 1). In the control group, E2 was undetectable in the albumen of 14/15 eggs, so albumen values from the control group were not included in the analysis. Concentrations of E2 also declined significantly from day 5 to day 15 of development in both groups (Fig. 1). Embryos did have detectable estradiol levels at days 10 and 15, but were not included in this analysis because we could not collect embryos during the first sampling period. In a separate ANOVA, neither treatment (F1,16 = 3.19, P = 0.093) nor day of development (F1,16 = 2.35, P = 0.15) had an effect on levels of E2 in the embryo.
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Analysis of the distribution of radioactivity also found significant effects of egg component (F1,48 = 8.14, P = 0.006) and extraction phase (F1,48 = 313.51, P < 0.0001). The distribution of radioactivity did not differ with day of development (F2,48 = 2.76, P < 0.073) but the interaction of the three fixed effects was significant (F7,48 = 9.07, P < 0.0001). Post hoc comparisons indicate that the aqueous phase contained significantly higher levels of radioactivity than did the organic phase for both components (yolk and albumen) at all three sampling periods (all P < 0.05) (Fig. 2). Interestingly, radioactivity could not be detected in the embryos at any point, so these samples were not included in the analysis.
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Sulfotransferase activity
Day of development had a significant effect on sulfotransferase activity in both the extra-embryonic membranes (F4,16 = 4.27, P = 0.015) and yolk (F4,17 = 5.62, P = 0.0045). For both tissues, sulfotransferase was significantly higher at day 10 compared to day 0 (Fig. 3). At day 10 of development, sulfotransferase activity was three times higher in the embryo compared to the yolk and extra-embryonic membranes but dropped significantly by day 17 of development (F1,5 = 120.11, P = 0.0001) to levels below those of the other two tissues (Fig. 3). Clutch of origin did not have a significant effect on sulfotransferase activity for any of the components (all P > 0.1). Sulfotransferase activity in the albumen was very low to undetectable throughout development, so albumen levels were not included in the analysis.
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Movement of steroids during early embryonic development
Results from the study of steroid tracing provide important insight into the movement of both exogenous and endogenous steroids during the first 15 days of development. The application of exogenous E2 did elevate levels in the yolk and albumen with the vast majority of the steroid ending up in the yolk. This finding confirms that exogenous manipulations of steroids do, indeed, elevate steroid levels in the yolk. Our results also indicate that both endogenous and exogenous E2 is converted to a water-soluble form during the first 15 days of development. All components of the egg that had detectable E2 at day five of development exhibited a decline in concentrations by day 15 (Fig. 1), demonstrating that during this early part of development, E2 levels decline throughout the egg. Additionally, only 1 of 15 control eggs had detectable E2 in the albumen, indicating that the decline of E2 from the yolk is not due to a direct relocation of steroids from the yolk.
The distribution of radioactivity after the application of tritiated E2 differed from the distribution found after the application of nontritiated E2. We detected radioactivity in both the yolk and albumen throughout the first 15 days of development (Fig. 2), with significantly higher radioactivity levels in the aqueous phase compared to the organic phase after extraction. Because our extraction technique normally pulls over 80% of E2 from the yolk and albumen into the organic phase, we would have expected to find the majority of the radioactivity in the organic phase, but our data suggest that the tritiated E2 was converted into a water-soluble form. We also saw a decrease in the amount of radioactivity in the organic phase of the yolk from day 5 to 15 which is consistent with the pattern of E2 decline we found (Fig. 1). Coincident with this decline in the organic phase of the yolk, there was an increase in the radioactivity detected in the aqueous phase of the albumen relative to the aqueous phase of the yolk at day 15. The distribution of radioactivity suggests that E2 is converted to a water-soluble form that is capable of moving out of the yolk. In turtle eggs, the water content of the yolk decreases by day 15 of development (Paitz unpublished data) and the movement of water from the yolk may account for this shift in total radioactivity detected in the aqueous phase.
Radioactivity was not detected in either the organic or aqueous extracts from whole embryos during the first 15 days of development. By day 15, the embryo only comprises about 2% of the total egg mass, and this small size may account for our inability to detect any radioactivity. Alternatively, the embryo may be buffered from steroids, and this could account for the lack of radioactivity present on day 15. Whether or not these aqueous metabolites make it to the embryo later in development remains to be determined.
Mechanism underlying movement of steroids
Sulfotransferase activity was examined as a potential mechanism to account for the conversion of E2 to a water-soluble form. Sulfotransferase was found to increase in both the yolk and extra-embryonic membranes from day 0 to 10 of development (Fig. 3). Also at day 10, we found high levels of activity in the developing embryo, indicating that numerous tissues throughout the egg are capable of converting E2 into a water-soluble form. The specific source of these sulfotransferase enzymes is not yet known; the extra-embryonic membranes and embryo are the most likely sources, but it seems unlikely that the yolk is capable of directly synthesizing these enzymes. This increase in sulfotransferase activity occurs during the same period of development in which we report a decrease in E2 concentrations. Together with the distribution of radioactivity in the aqueous phase of the yolk and albumen, these data suggest that sulfotransferase activity is responsible for the conversion of yolk E2 into an inactive water-soluble form during the first 15 days of development in the red-eared slider.
Sulfotransferase/sulfatase (SULT/STS) pathway and effects of yolk steroids
Despite the intensive investigation into yolk steroids over the past 15 years, several fundamental questions remain to be answered with regard to the physiological mechanisms that underlie the effects of yolk steroids, including (1) how do steroids get from the yolk to the embryo and (2) how do steroid signals present at oviposition influence only certain traits in the offspring without affecting other traits? Based upon the results of our experiments, we propose a model that uses the SULT/STS pathway to explain the modulation of these effects in oviparous vertebrates. According to the model, yolk E2 is converted to an inactive water-soluble form by sulfotransferases early in development. These steroids, now inactive, can then be returned to an active form by sulfatases later in development (Fig. 4). Sulfatase enzymes hydrolyze steroid sulfates into biologically active steroids and have been characterized in numerous vertebrate taxa (reveiwed by Reed et al. 2005). In humans, sulfatase activity has been detected in the testis, ovary, adrenal glands, prostate, lymphocytes, skin, brain, and bone with the highest activity found in the placenta (reviewed by Reed et al. 2005). Sulfatase activity has also been detected in numerous invertebrate taxa such as the keyhole limpet (Patella vulgata) and the Roman snail (Helix pomatia) (Ferchaud et al. 2000). The wide distribution of sulfatase activity across taxa, along with our data demonstrating the presence of sulfotransferase activity, provides support for the idea that the SULT/STS pathway is present during embryonic development in T. scripta. The model based on this pathway addresses both previously unanswered mechanistic questions by invoking a mechanism that provides a water-soluble steroid precursor that subsequently may be reactivated on a localized basis.
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The SULT/STS pathway is already known to be critical to placental mammals in dealing with maternal steroid signals (reviewed by Levitz 1966
). Our data suggest that this pathway is present in oviparous vertebrates and that it may play a critical role similar to that found in placental species, as we have demonstrated that sulfotransferase activity can be detected in various tissues in the developing egg. Several additional lines of research support the idea that oviparous vertebrates possess the enzymes necessary to modulate maternal steroid signals. First, in placental vertebrates, the fetal portion of the placenta is comprised of modified extra-embryonic membranes with the specific membrane destined to become part of the placenta dependent upon the type of placenta (e.g., chorioallantoic placenta or yolk-sac placenta) (reviewed by Blackburn 2000). These same extra-embryonic membranes that form the fetal contribution to the placenta are present in oviparous vertebrates (Carlson 1981). Second, the importance of the contribution of enzymes by the embryonic membrane to the placenta is highlighted in situations in which mutations in the fetal genome lead to the breakdown of the placental buffering system. In these studies, deficiencies in placental enzymes lead to abnormal fetal development (Epstein and Bonifas 1985) and in the case of deficiency in placental aromatase, virilization of the mother occurs due to an increased exposure to fetal androgens (Harada et al. 1992). The production of buffering enzymes by extra-embryonic membranes in oviparous vertebrates could also help explain how viviparity has evolved over 100 separate times in vertebrates, as one major barrier to the evolution of viviparity would be the separation of maternal and embryonic endocrine signals (reviewed by Blackburn 2000). In total, this model is supported by both historical data and new empirical data that suggest the SULT/STS pathway is present in oviparous vertebrates and that it may play a critical role in buffering the developing embryo from maternal steroids by allowing the embryo to modulate yolk steroid signals.
Implications of the SULT/STS model
The proposed model represents a departure from the prevailing dogma regarding the effects of yolk steroids by providing a potential mechanism by which the developing embryo may be able to modulate yolk steroid signals, which until now, has received very little attention (Muller et al. 2007). This model, while primarily focused on embryonic regulation, still allows for maternal influence on development of the offspring through the transfer of steroids to the yolk that ultimately end up as sulfonated precursors for steroid production via sulfatase activity. This scenario is consistent with the numerous studies in which differential amounts of steroids in the yolk at the time of oviposition correlate with differences in traits among the offspring (reviewed by Groothuis et al. 2005). The model also allows for differential expression of sulfatase over time and space, which may explain why similar patterns of concentrations of yolk steroids can lead to different phenotypic effects both within and between species. Additionally, differential sulfatase expression may also explain how yolk steroids influence some traits but not others within a developing embryo. Such a relationship between concentrations of yolk steroids and embryonic steroidogenic enzymes creates a situation similar to that already known for placental organisms in which embryos appear to tightly regulate maternal steroid signals.
The proposed model also alters how we interpret changes in levels of yolk steroids during development. In reptiles with temperature-dependent sex determination, it has been proposed that temperature regulates E2 levels in the yolk such that eggs incubated at female-producing temperatures maintain elevated E2 concentrations in the yolk and this E2 then triggers the production of ovaries (Elf 2003). If our model is correct, it would suggest that it is not the steroids remaining in the yolk, but those that leave the yolk, that are most important in influencing development of the offspring. Steroids that remain in the yolk would be unavailable to the developing embryo, whereas sulfonated steroids may make it to the embryo and act there as precursors for reactivation by sulfatase activity. Upon reactivation, these steroids would then be capable of binding their respective receptors to influence processes such as gene transcription in the developing embryo.
Finally, the SULT/STS pathway may be a target for endocrine disruption by chemicals persistent in the environment. Both phytoestrogens (Harris et al. 2004) and polyhalogenated aromatic hydrocarbons (Kester et al. 2002) have been shown to be potent inhibitors of sulfotransferase activity. Exposure to these chemicals during development could lead to the breakdown of the enzymatic buffer provided by sulfotransferase activity. Several studies have investigated the effect of exposing developing T. scripta eggs to endocrine-disrupting chemicals and have demonstrated that numerous chemicals are capable of feminizing the developing embryo (Willingham and Crews 1999; Gale et al. 2002). The mechanism underlying this feminizing effect remains to be elucidated. Currently, it is thought that these chemicals directly activate estrogen receptors, but it is possible that these chemicals functionally block sulfotransferase activity, thereby reducing or eliminating the steroid buffering system and consequently exposing the developing embryo to the E2 present in the egg.
Overall, this model provides a detailed explanation of how lipophilic steroids may move from the lipid-rich yolk and influence traits on a tissue-specific level. Applying the SULT/STS pathway to explain the modulation of yolk steroid effects in oviparous vertebrates addresses some previously unanswered mechanistic questions while setting up some interesting future directions of research. This model could also be modified to include other pathways that involve the conjugation/deconjugation of steroids (e.g., glucuronidation) which are similar to the SULT/STS pathway in that they involve the conversion of steroids to a water-soluble form that can serve as a precursor for steroid production. It remains to be determined whether these other pathways play a role in the modulation of yolk steroid signals. Mechanistic studies such as this are critical for furthering an understanding of how yolk steroids may be used as a means whereby females manipulate the phenotypes of their offspring.
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We would like to thank Mikael Holgersson, Heather Les, and Laura Zimmerman for assistance with the collection of eggs, Dr Rich King for the gift of radiolabeled estradiol, and the Illinois Department of Natural resources for granting access to Banner Marsh. This work was funded with support from the Beta Lambda Chapter of Phi Sigma to R.T.P. and an Illinois State University Research Grant from the College of Arts & Sciences to R.M.B.
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From the symposium "Consequences of Maternally-Derived Yolk Hormones for Offspring: Current Status, Challenges and Opportunities" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 2–6, 2008, at San Antonio, Texas.
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