Predetermination of sexual fate in a turtle with temperature-dependent sex determination

Egg incubation temperature determines offspring sex in many reptilian species, including red-eared slider turtles, where embryos incubated at low temperatures during the initial stages of gonad formation develop as males, while those kept at higher temperatures develop as females. Incubation at the threshold, or pivotal, temperature (PvT) yields an even ratio of males and females. This strong susceptibility to temperature indicates that each embryo of this species is competent to develop as a male or a female. However, the mechanism that determines sexual fate at the PvT has not been identified. One possibility is that sexual fate is stochastic at the PvT, but coordinated by systemic signals within a single embryo. If this is the case, gonads explanted separately to culture should not coordinate their fate. Here we show that gonad pairs from embryos incubated at the PvT share a strong predisposition for one sex or the other when cultured in isolation, indicating that they were affected by shared genetic signals, maternally-deposited yolk hormones or other transient influences received prior to the stage of dissection. In ovo studies involving shifts from the male- or female-producing temperature to the PvT further indicate that embryos adopt a sexual differentiation trajectory many days prior to the onset of morphological differentiation into testes or ovaries and usually maintain this fate in the absence of an extreme temperature signal favoring the development of the other sex. Our findings therefore suggest that the outcome of sex determination in these reptiles is heavily influenced (i) by an inherent predisposition at the PvT and (ii) by the sexual differentiation trajectory established early in gonad development under male- or female-producing temperatures.