Yolk steroids decline during sexual differentiation in the alligator

The leading explanation of temperature-dependent sex determination (TSD) in reptiles postulates that (1) ovarian differentiation is directed by estrogen and that (2) estrogen is synthesized in the developing gonad following induction of aromatase expression. However, the source of steroid substrate for aromatization has not yet been identified. In addition, sex ratios vary as a function of clutch, but such biases are as yet unexplained. To address these issues, we measured estradiol, testosterone, and androstenedione in yolks of the American alligator (Alligator mississippiensis) before, during, and after the period of gonadal differentiation in this TSD species. Eggs were collected from a wild population in Louisiana and were incubated at male- and female-determining constant temperatures in the lab, as well as at intermediate temperatures that produced both sexes. Steroids were assayed in yolk extracts after celite column chromatography. All three steroids were found to be in the range of nanograms/gram of yolk at stage 16. Androstenedione was the predominant steroid, 2- to 3-fold higher in concentration than estradiol and 15- to 20-fold higher than testosterone. The levels of these steroids declined (5- to 30-fold) between stages 16 and 25, most markedly between stages 21 and 23, regardless of incubation temperature. The chronology of this sharp decline in steroid levels in our study coincides with the timing of gonadal differentiation in this species, between stages 21 to 23 based on previous reports. Estradiol levels in yolks differed by 3-fold in some clutches relative to others, whereas, no clutch differences were apparent for either androstenedione or testosterone. These data demonstrate that alligator yolk contains high concentrations of two steroid substrates utilized for estrogen synthesis, as well as significant quantities of estradiol itself. We hypothesize that estradiol levels in yolk provide a steroid background, variable among and within clutches, on which gonadal development is initiated and proceeds. As a consequence, we suggest that yolk provides an epigenetic maternal contribution that modulates the effect of incubation temperature on hatchling sex.