Differential Incubation Temperatures Result in Dimorphic DNA Methylation Patterning of the SOX9 and Aromatase Promoters in Gonads of Alligator (Alligator mississippiensis) Embryos1

Abstract

Environmental factors are known to influence sex determination in many nonmammalian vertebrates. In all crocodilians studied thus far, temperature is the only known determinant of sex. However, the molecular mechanisms mediating the effect of temperature on sex determination are not known. Aromatase (CYP19A1) and SOX9 play critical roles in vertebrate sex determination and gonadogenesis. Here, we used a variety of techniques to investigate the potential roles of DNA methylation patterning on CYP19A1 and SOX9 expression in the American alligator, an organism that relies on temperature-dependent sex determination. Our findings reveal that developing gonads derived from embryos incubated at a male-producing temperature (MPT) show elevated CYP19A1 promoter methylation and decreased levels of gene expression relative to incubation at a female-producing temperature (FPT). The converse was observed at the SOX9 locus, with increased promoter methylation and decreased expression occurring in embryonic gonads resulting from incubation at FPT relative to that of MPT. We also examined the gonadal expression of the three primary, catalytically active DNA methyltransferase enzymes and show that they are present during critical stages of gonadal development. Together, these data strongly suggest that DNA methylation patterning is a central component in coordinating the genetic cascade responsible for sexual differentiation. In addition, these data raise the possibility that DNA methylation could act as a key mediator integrating temperature into a molecular trigger that determines sex in the alligator.

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

“…In T. scripta, the DNA methylation level of the gonadal Cyp19a1 promoter was higher at MPT than FPT by stage 19 onwards, and was reduced in response to temperature shifts from MPT to FPT (Matsumoto et al, 2013a). Similarly, differential incubation temperatures resulted in dimorphic DNA methylation patterning of the Cyp19a1 and Sox9 promoters in gonads of American alligator embryos (Parrott et al, 2014). We show that the DNA methylation status of the Dmrt1 promoter in gonads of T. scripta displayed significant temperature-dependent dimorphism from stage 15 onwards, and responded rapidly to temperature shifts both from MPT to FPT and FPT to MPT.…”

“…Epigenetic mechanisms, including DNA methylation, histone modification and non-coding RNAs, can integrate environmental information with the regulation of gene expression, and have been emerging as a promising regulatory mechanism for TSD (Matsumoto et al, 2013a;Navarro-Martín et al, 2011;Parrott et al, 2014;Piferrer, 2013;Venegas et al, 2016). The first example of an epigenetic mechanism mediating temperature effects on sexual development in a vertebrate came from a study on the European sea bass, a fish with a polygenic system of sex determination where temperature and genetics contribute equally to sexual fate.…”

Simultaneous exposure to estrogen and androgen resulted in feminization and endocrine disruption

“…Plenty of experiment results suggested that epigenetic regulation plays multiple crucial roles in the gonadal differentiation of teleosts and other vertebrates (Navarro-Martin et al 2011, Zhang et al 2013, Parrott et al 2014, Shao et al 2014. Neonatal exposure to the synthetic estrogen diethylstilbestrol caused abnormal demethylation of the CpG sites upstream of the estrogen-response element of the lactoferrin promoter (Li et al 1997).…”

“…Genes in the sex determination pathways are the major targets of substantial methylation modification (Navarro-Martin et al 2011, Zhang et al 2013, Parrott et al 2014, Shao et al 2014. The different expression patterns of DNA methyltransferases (DNMTs) in gonads are probably essential for the acquisition of a sex-specific methylation pattern (La Salle et al 2004).…”

Epigenetic regulation of sex ratios may explain natural variation in self-fertilization rates

“…PCRs for cyp19a and dmrt1 were carried out using primers designed for Japanese flounder as described by Shao et al [36]. PCRs for Sox9a were carried out using primers designed for alligator by Parrott et al [37]. .…”