Incubation history prior to the canonical thermosensitive period determines sex in the American alligator

Abstract

Despite the widespread occurrence of environmental sex determination (ESD) among vertebrates, our knowledge of the temporal dynamics by which environmental factors act on this process remains limited. In many reptiles, incubation temperature determines sex during a discrete developmental window just prior to and coincident with the differentiation of the gonads. Yet, there is substantial variation in sex ratios among different clutches of eggs incubated at identical temperatures during this period. Here, we test the hypothesis that temperatures experienced prior to the reported thermosensitive period for alligators (Alligator mississippiensis) can impact how the sex determination system responds to thermal cues later in development. Temperature shift experiments on eggs collected from the field within 24 h of oviposition were employed to decouple various maternal influences from thermal effects, and results demonstrate a previously undefined window of thermosensitivity occurring by stage 15 of embryonic development, six stages earlier than previously reported. We also examine the intrasexual expression of several male-and female-biased genes and show that while male-biased genes display no intrasexual differences, ovarian CYP19A1 (aromatase) transcript abundance differs by approximately twofold depending on thermal exposures experienced at early stages of embryonic development. These findings expand our understanding of the ESD in the alligator and provide the rationale for reevaluation of the temporal dynamics of sex determination in other crocodilians. Reproduction (2015) 150 279-287

Improved genome assembly of American alligator genome reveals conserved architecture of estrogen signaling

“…We could not confirm sex histologically as both gonads of each embryo were used for RNA sequencing, so we confirmed the sex of each embryo by comparing gonadal expression of CYP19A1 to AMH as in previous studies McCoy et al 2015). One embryo from clutch 13 was female despite incubation at MPT (Fig.…”

“…Due to conditions prior to egg collection, embryos can sometimes develop as a different sex than expected based on incubation temperature after collection (McCoy et al 2015). We could not confirm sex histologically as both gonads of each embryo were used for RNA sequencing, so we confirmed the sex of each embryo by comparing gonadal expression of CYP19A1 to AMH as in previous studies McCoy et al 2015).…”

“…Expression values in FPKM are useful for generating heatmaps and reporting average expression values for a gene, but Cuffdiff 2 uses raw counts rather than FPKM for differential expression analysis. For each gonad sample at the 30 d, we compared FPKMs of two genes, CYP19A1 and AMH, to verify the sex of the embryo as in previous studies McCoy et al 2015), resulting in one sample, the embryo from clutch 13 incubated at MPT, being removed from further analysis. We used an FDR-adjusted P-value reported by Cuffdiff 2 for each gene for the null hypothesis that expression levels of that gene in tissues incubated at MPT and FPT are drawn from the same distribution.…”

Oil dispersant Corexit 9500 is weakly estrogenic, but does not skew the sex ratio in Alligator mississippiensis

“…In alligators specifically, egg incubation at 30°C (female-producing temperature) during the TSP produces 100% females, whereas egg incubation at 33.0-33.5°C (maleproducing temperature, MPT) during the TSP produces 100% males (Ferguson & Joanen, 1983;Lang & Andrews, 1994;McCoy, Parrott, Rainwater, Wilkinson, & Guillette, 2015). In alligators specifically, egg incubation at 30°C (female-producing temperature) during the TSP produces 100% females, whereas egg incubation at 33.0-33.5°C (maleproducing temperature, MPT) during the TSP produces 100% males (Ferguson & Joanen, 1983;Lang & Andrews, 1994;McCoy, Parrott, Rainwater, Wilkinson, & Guillette, 2015).…”

Integrative and comparative reproductive biology: From alligators to xenobiotics

“…However, Dr. Guillette’s laboratory showed that the process of TSD at the PVT can be heavily influenced by incubation temperatures earlier than the known temperature sensitive period (TSP) in A. mississippiensis (McCoy et al, 2015). Since much of the work on TSD has been conducted in the laboratory where egg-incubation temperatures do not fluctuate as they naturally do in the field (Georges et al, 2004), further investigations into this earlier TSP, temperature fluctuation, and the molecular mechanisms driving TSD are required in A. mississippiensis , as well as other TSD species.…”