Aromatase inhibitors administered before sexual differentiation of the gonads can induce sex reversal in female chickens. To analyze the process of sex reversal, we have followed for several months the changes induced by Fadrozole, a nonsteroidal aromatase inhibitor, in gonadal aromatase activity and in morphology and structure of the female genital system. Fadrozole was injected into eggs on day four of incubation, and its effects were examined during the embryonic development and for eight months after hatching. In control females, aromatase activity in the right and the left gonad was high in the middle third of embryonic development, and then decreased up to hatching. After hatching, aromatase activity increased in the left ovary, in particular during folliculogenesis, whereas in the right regressing gonad, it continued to decrease to reach testicular levels at one month. In treated females, masculinization of the genital system was characterized by the maintenance of the right gonad and its differentiation into a testis, and by the differentiation of the left gonad into an ovotestis or a testis; however, in all individuals, the left Müllerian duct and the posterior part of the right Müllerian duct were maintained. In testes and ovotestes, aromatase activity was lower than in gonads of control females (except in the right gonad as of one month after hatching) but remained higher than in testes of control and treated males. Moreover, in ovotestes, aromatase activity was higher in parts displaying follicles than in parts devoid of follicles. The main structural changes in the gonads during sex reversal were partial (in ovotestes) or complete (in testes) degeneration of the cortex in the left gonad, and formation of an albuginea and differentiation of testicular cords/tubes in the two gonads. Testicular cords/tubes transdifferentiated from ovarian medullary cords and lacunae whose epithelium thickened and became Sertolian. Transdifferentiation occurred all along embryonic and postnatal development; thus, new testicular cords/tubes were continuously formed while others degenerated. The sex reversed gonads were also characterized by an abundant fibrous interstitial tissue and abnormal medullary condensations of lymphoid-like cells; in the persisting testicular cords/tubes, spermatogenesis was delayed and impaired. Related to aromatase activity, persistence of too high levels of estrogens can explain the presence of oviducts, gonadal abnormalities and infertility in sex reversed females.

Aromatase inhibitors administered prior to histological signs of gonadal sex differentiation can induce sex reversal of genetic female chickens. Under the effects of Fadrozole (CGS 16949A), a nonsteroidal aromatase inhibitor, the right gonad generally becomes a testis, and the left gonad a testis or an ovotestis. We have compared the expression pattern of the genes encoding AMH (the anti-Mü llerian hormone), SF1 (steroidogenic factor 1), and SOX9 (a transcription factor related to SRY) in these sexreversed gonads with that in control testes and ovaries, using in situ hybridization with riboprobes on gonadal sections. In control males, the three genes are expressed in Sertoli cells of testicular cords; however, only SOX9 is male specific, since as observed previously AMH and SF1 but not SOX9 are expressed in the control female gonads. In addition to testicular-like cords, sexreversed gonads present many lacunae with a composite, thick and flat epithelium. We show that during embryonic and postnatal development, AMH, SF1 and SOX9 are expressed in the epithelium of testicular-like cords and in the thickened part but not in the flattened part of the epithelium of composite lacunae. AMH and SF1 but not SOX9 are expressed in follicular cells of ovotestes. Coexpression of the three genes, of which SOX9 is a specific Sertoli-cell marker, provides strong evidence for the transdifferentiation of ovarian into testicular epithelium in gonads of female chickens treated with Fadrozole.