Molecular and morphological differentiation of testes and ovaries in relation to the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina

•We define a brief thermosensitive period for sex determination in snapping turtles.•Exposure to 31 °C for 5 days of a 65-day incubation period induces ovarian development.•This temperature shift induces rapid changes in expression and splicing of WT1 mRNA.•First sex difference in WT1 +KTS:–KTS splice ratio to be reported in any vertebrate.•Model facilitates studies of specification/determination of gonad fate by temperature.

Ambient temperatures during embryonic development determine gonadal sex in many reptiles. The temperature sensitive period for sex determination has been defined by shifting eggs between female- and male-producing temperatures in a few species. This phase spans 20–35% of embryogenesis in most species, which makes it difficult to define the mechanisms that transduce temperature into a signal for ovarian versus testicular development. We present an extensive set of studies that define a brief period when high temperature specifies, and then determines, ovarian fate in a northern population of snapping turtles, Chelydra serpentina. We shifted embryos from male to female temperatures, or vice versa, at various stages of development. Gonads in embryos incubated at female temperatures commit to ovarian fate earlier (by stage 18) than gonads in embryos incubated at male temperatures commit to testicular fate (by stages 19–21). In double shift studies, embryos were incubated at a female temperature, exposed to a male temperature for set times, and shifted back to the original temperature, or vice versa. The time required to induce ovarian development (≤6 days at female temperatures) was much shorter than the time required to induce testicular formation (>20 days at male temperatures). Differentiation of the gonads at the histological level occurred after the sex-determining period. Nevertheless, we found that a change in temperature rapidly (within 24 h) influenced expression and splicing of WT1 mRNA: the absolute abundance of WT1 mRNA, the relative abundance of +KTS versus –KTS isoforms, as well as the ratio of +KTS:–KTS isoforms was higher in gonads at a male versus a female temperature. In conclusion, ovarian fate is more readily determined than testicular fate in snapping turtle embryos. The short sex-determining period in this species (6–8% of embryogenesis) will facilitate studies of molecular mechanisms for specification and determination of gonad fate by temperature.