Sex reversal and aromatase in the European pond turtle: Treatment with letrozole after the thermosensitive period for sex determination

In the European pond turtle (Emys orbicularis), gonadal sex differentiation is temperature-dependent. The temperature sensitive period (TSP) of gonado genesis lies between stages 16 and 22 of embryonic development. Previous st udies have shown that embryos incubated at 30 degreesC, a temperature yield ing 100% phenotypic females, can be sex reversed by treatments with an arom atase inhibitor administered during TSP or even somewhat after TSP (as of s tage 22+). The goal of the present study was to determine whether the ovary still retains male potential at later stages of embryonic development and whether the induced male characters persist after hatching. For this purpos e, eggs of E. orbicularis were treated with letrozole, a nonsteroidal aroma tase inhibitor, at or as of stages 23, 24 or 25, then gonadal aromatase act ivity in each individual and the related gonadal structure were studied at hatching (stage 26) and for one year after hatching. Two kinds of treatment s were carried out: 1) repeated applications of 10 mug of letrozole in etha nolic solution onto the eggshell; and 2) a single injection of 10 mug of le trozole in olive oil. Similar results were obtained with either application or injection of the aromatase inhibitor. In treatments as of or at stage 2 3, individuals with gonadal aromatase activity lower than 20 fmoles/hour/go nad had ovotestes, i.e., 22% of the treated individuals. At hatching, the i nner part of these ovotestes contained testicular cords and also mixed lacu nae presenting various degrees of transdifferentiation of the epithelium in to a Sertolian epithelium. The cortex was maintained, although some germ ce lls degenerated within it. These processes continued after hatching. Howeve r, at 12 months, gonads were still ovotestes displaying some follicles with a growing oocyte in the remaining parts of the cortex. In treatments as of or at stages 24 or 25, only a few individuals were masculinized. One had o votestes; in others, the cortex was absent in some parts and when it was pr esent oocytes were degenerating. These results show that in the European po nd turtle, differentiation of ovotestes from ovaries can be induced by trea tment with an aromatase inhibitor starting at late stages of embryonic deve lopment (between the end of TSP and hatching), although such differentiatio n is less frequent as embryonic development proceeds. Sex reversal persists for at least one year after hatching. J. Exp. Zool. 290:490-497, 2001. (C) 2001 Wiley-Liss, Inc.