Changes in gonadal gene network by exogenous ligands in temperature-dependent sex determination

Abstract

We examined the expression of candidate sex-determining genes in the red-eared slider turtle (Trachemys scripta) during the temperature-sensitive period (TSP). Aromatase and Rspo1 were used as markers of ovarian differentiation and Sox9 was used as a marker of testicular differentiation. Eggs were incubated at a male-producing temperature (26 8C or MPT) and a female-producing temperature (31 8C or FPT). First, eggs at the beginning of the TSP (stage 16) were topically treated with the steroid hormones 17b-estradiol (E 2 ), testosterone in combination with aromatase inhibitor (AICT), the E 2 antagonist (ICI 182 780), and the androgen antagonist (flutamide). Secondly, gonads were removed at stage 16 and treated in vitro with E 2 , AICT, or hormone antagonists. At the FPT, AICT in ovo suppressed aromatase and Rspo1, while activating Sox9. At the MPT, E 2 treatment rapidly increased aromatase and Rspo1, while suppressing Sox9. Treatment with the E 2 antagonist in ovo decreased aromatase at the FPT. Treatment with the androgen antagonist in ovo increased aromatase and Rspo1 at early time points at MPT and decreased Sox9 at MPT at later developmental stages. Treatment of isolated gonads cultured in vitro with AICT at FPT decreased aromatase and Rspo1 and E 2 increased the expression of these genes at MPT. In vitro treatment with E 2 antagonist suppressed aromatase and Rspo1 expression at FPT. Overall, our results suggest that exogenous ligands dictate gonadal development by redirecting the expression of candidate sex-determining genes within the genetic cascades induced by temperature.

Dmrt1 induces the male pathway in a turtle with temperature-dependent sex determination

“…This response of Dmrt1 expression occurred at early stages of TSP, clearly preceding the first signs of morphological differentiation of testes or ovaries in T. scripta. The change of Dmrt1 transcript in response to AI treatment was earlier than that of Sox9, which occurred at stage 19 (Matsumoto et al, 2013b). By contrast, treatment of MPT eggs with estrogen caused a rapid downregulation of Dmrt1 transcript expression in T. scripta (Murdock and Wibbels, 2006), which preceded downregulation of Sox9 (Barske and Capel, 2010).…”

Transcriptomic responses to environmental temperature by turtles with temperature-dependent and genotypic sex determination assessed by RNAseq inform the genetic architecture of embryonic gonadal development

“…The first reptilian gonadal transcriptomes were recently characterized in alligator (TSD) [24], and slider turtles (TSD)[25], whereas previous studies on GSD and TSD turtles had used exclusively quantitative PCR and in situ hybridization targeting a number of genes underlying sexual development (full gene names are found in Table 1), including Wt1 [26, 27], Sf1 [28, 29], Dax1 [30, 31], Sox9 [30, 32–34], Aromatase [35, 36], Dmrt1 [30, 34, 37], Estrogen receptor [38, 39], Rspo1 [33, 40], among others (S1 Table). While these earlier turtle studies provided only fragmentary information [30, 33, 41, 42], their use of sensitive qPCR permitted the detection of differential gene expression that can pass undetected in transcriptomic analyses [43, 44], and these efforts uncovered substantial evolution of transcriptional patterns for some elements across vertebrates.…”

“…While these earlier turtle studies provided only fragmentary information [30, 33, 41, 42], their use of sensitive qPCR permitted the detection of differential gene expression that can pass undetected in transcriptomic analyses [43, 44], and these efforts uncovered substantial evolution of transcriptional patterns for some elements across vertebrates. However, the extent of evolution of the composition and expression of this gene regulatory network remains unclear.…”

Temperature Shift Alters DNA Methylation and Histone Modification Patterns in Gonadal Aromatase (cyp19a1) Gene in Species with Temperature-Dependent Sex Determination

“…In species with TSD, the expression of aromatase in embryonic gonads is female-producing temperature (FPT) specific and the expression is almost undetectable at a male-producing temperature (MPT) [12,13,15]. In red-eared slider turtles ( Trachemys scripta ), for example, an dramatic increase of aromatase mRNA expression at FPT starts close to the middle of the temperature sensitive period (TSP), at stage 19, and reaches its peak at more than 10,000-fold increase relative to baseline (stage 16) right after TSP (stage 23) [16,17]. Upon hatching, the mRNA expression drops back down to levels similar to that observed around stage 19–21.…”