Exogenous application of estradiol to eggs unexpectedly induces male development in two turtle species with temperature-dependent sex determination

Abstract

Steroid hormones affect sex determination in a variety of vertebrates. The feminizing effects of exposure to estradiol and the masculinizing effects of aromatase inhibition during development are well established in a broad range of vertebrate taxa, but paradoxical findings are occasionally reported. Four independent experiments were conducted on two turtle species with temperature-dependent sex determination (Chrysemys picta and Chelydra serpentina) to quantify the effects of egg incubation temperature, estradiol, and an aromatase inhibitor on offspring sex ratios. As expected, the warmer incubation temperatures induced female development and the cooler temperatures produced primarily males. However, application of an aromatase inhibitor had no effect on offspring sex ratios, and exogenous applications of estradiol to eggs produced male offspring across all incubation temperatures. These unexpected results were remarkably consistent across all four experiments and both study species. Elevated concentrations of estradiol could interact with androgen receptors or inhibit aromatase expression, which might result in relatively high testosterone concentrations that lead to testis development. These findings add to a short list of studies that report paradoxical effects of steroid hormones, which addresses the need for a more comprehensive understanding of the role of sex steroids in sexual development. KeywordsAromatase inhibitor, Chelydra serpentina, Chrysemys picta, Environmental sex determination, Sex steroids, Sexual differentiation Disciplines Aquaculture and Fisheries | Ecology and Evolutionary Biology | Other Ecology and Evolutionary Biology | Population Biology | ZoologyComments NOTICE: this is the author's version of a work that was accepted for publication in General and Comparative Endocrinology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in General and Comparative Endocrinology, 206 (2014) ABSTRACTSteroid hormones affect sex determination in a variety of vertebrates. The feminizing effects of exposure to estradiol and the masculinizing effects of aromatase inhibition during development are well established in a broad range of vertebrate taxa, but paradoxical findings are occasionally reported. Four independent experiments were conducted on two turtle species with temperature-dependent sex determination (Chrysemys picta and Chelydra serpentina) to quantify the effects of egg incubation temperature, estradiol, and an aromatase inhibitor on offspring sex ratios. As expected, the warmer incubation temperatures induced female development and the cooler temperatures produced primarily males. However, application of an aromatase inhibitor had no effect on offspring sex ratios, and exogenous applications of estradiol to eggs produced ...

Seasonal shifts in sex ratios are mediated by maternal effects and fluctuating incubation temperatures

“…to the outside of the egg result in female sex determination, presumably due to their conversion into oestrogens, whereas nonaromatizable androgens result in the production of males (Wibbels & Crews 1992, 1995Crews et al 1994Crews et al , 1995. However, dosing studies generally lack biological realism Crews 1996;Warner et al 2014) necessitating the use of correlational studies utilizing natural variation in maternal steroids, like the present one. Designing experiments based on natural variation in maternal oestrogens ensures that the entire suite of oestrogens, their metabolites, and aromatizable androgens that may act as oestrogen sources is captured.…”

“…However, dosing studies generally lack biological realism (Crews, Bull & Wibbels ; Crews ; Warner et al . ) necessitating the use of correlational studies utilizing natural variation in maternal steroids, like the present one. Designing experiments based on natural variation in maternal oestrogens ensures that the entire suite of oestrogens, their metabolites, and aromatizable androgens that may act as oestrogen sources is captured.…”

“…However, due to the timing of dosing and the supraphysiological doses used to alter sex and other phenotypic traits, our understanding of how natural maternal steroid allocation influences offspring is lacking (Crews, Bull & Wibbels ; Crews ; Warner et al . ). Previous research has found that endogenous (yolk) steroids are also correlated with offspring sex (Bowden, Ewert & Nelson ; Lovern & Wade ) as in green anole lizards ( Anolis anolis ) where maternally derived yolk testosterone is higher in male‐producing eggs (Lovern & Wade ).…”

“…But unlike birds and mammals, where evidence for facultative primary sex ratio skew is relatively rare (Ewen, Cassey & Moller 2004), exposure to exogenous steroids can directly affect sex in reptiles, even those with genetic sex determination (Bull, Gutzke & Crews 1988;Lovern & Wade 2003;Freedberg et al 2006), thus highlighting the value of utilizing reptiles to investigate sex-specific maternal effects. However, due to the timing of dosing and the supraphysiological doses used to alter sex and other phenotypic traits, our understanding of how natural maternal steroid allocation influences offspring is lacking Crews 1996;Warner et al 2014). Previous research has found that endogenous (yolk) steroids are also correlated with offspring sex (Bowden, Ewert & Nelson 2000;Lovern & Wade 2003) as in green anole lizards (Anolis anolis) where maternally derived yolk testosterone is higher in male-producing eggs (Lovern & Wade 2003).…”

Ecotoxicological Perspectives of Sex Determination

“…In fact, exposure to an aromatase inhibitor after the thermosensitive period sex-reversed developing embryos of the European pond turtle, Emys orbicularis , incubated at a female-producing temperature, revealing that sex differentiation was still labile, albeit harder to override, since the gonadal aromatase and estrogen levels are already well established at this developmental stage [Belaid et al, 2001]. Yet, a high dose of estradiol-17β in C. picta and C. serpentina yielded, opposite to expectation, a high percentage of males at both male-and female-producing temperatures, perhaps because a high concentration of estradiol permitted the interaction of estrogen with androgen receptors or caused aromatase inhibition [Warner et al, 2014].…”