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.

In many reptile species, sexual differentiation of gonads is sensitive to temperature during a critical period of embryonic development (thermosensitive period, TSP). Experiments carried out with different models among which turtles, crocodilians and lizards have demonstrated the implication of estrogens and the key role played by aromatase (the enzyme complex that converts androgens to estrogens) in ovary differentiation during TSP and in maintenance of the ovarian structure after TSP. In some of these experiments, the occurrence of various degrees of gonadal intersexuality is related to weak differences in aromatase activity, suggesting subtle regulations of the aromatase gene at the transcription level. Temperature could intervene in these regulations. Present studies deal with cloning (complementary DNAs) and expression (messenger RNAs) of genes that have been shown, or are expected, to be involved in gonadal formation and/or differentiation in mammals. Preliminary results indicate that homologues of AMH, DAX1, SF1, SOX9 and WT1 genes with the same function(s) as in mammals exist in reptiles. How these genes could interact with aromatase is being examined.

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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.

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Emys orbicularis is a freshwater turtle with temperature-dependent sex determination. Estrogens play a major role in gonadal differentiation; when they are produced at high levels during the thermosensitive period (TSP), ovaries differentiate; when their synthesis is very low, testes differentiate. Estrogens are synthesized from androgens through the activity of aromatase. We examine here two aspects of gonadal differentiation, intersexuality and growth, in E. orbicularis. For gonadal intersexuality, we studied the relationship between gonadal aromatase activity and gonadal structure at 28.5°C (pivotal temperature), from the beginning of TSP to hatching, and compared results to those obtained at 30°C (producing 100% females) and 25°C (producing 100% males). At 28.5°C, both males and females are obtained. However, histological differentiation of gonads is delayed compared to that at 25°C and 30°C, and an ovarian-like cortex of various thicknesses often develops at the surface of the male gonads; thus, several individuals display ovotestes at hatching. Despite important individual variations, the aromatase activity in ovaries differentiating at 28.5°C increases during development as in ovaries differentiating at 30°C. In most cases, however, activity is slightly lower than at 30°C, and at the end of embryonic life, it becomes similar to that at 30°C. In testes or ovotestes differentiating at 28.5°C, aromatase activity remains low but is generally slightly higher than in testes at 25°C; however, at the end of embryonic development, it becomes similar to that at 25°C. Oocytes in the cortex of ovotestes begin to degenerate around hatching and continue to degenerate after hatching. Therefore, ovotestes evolve as testes. However, some oocytes may persist at the surface of testes up to the adult age.To estimate gonadal growth, the protein content was measured at different embryonic stages at 25°C and at 30°C. Testis growth is fast during TSP, somewhat slower after TSP, and decreases around hatching. Ovary growth is much slower than testis growth during TSP and then accelerates up to the end of embryonic development. This differential growth is well correlated with gonadal aromatase activity-much higher at 30°C than at 25°C-and can be explained by the fact that during TSP, testicular cords develop at 25°C whereas they are inhibited at 30°C; the ovarian cortex begins to form during this period but grows chiefly after TSP.Both inhibition of testicular cord development and stimulation of cortex development are under the control of endogenous estrogens. In the case of ovotestes, slight increases in estrogen synthesis, compared to that in typical testes, are sufficient to induce the transient formation of an ovarian-like cortex although they do not inhibit the development of testicular cords.

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In the European pond turtle (Emys orbicularis), gonadal sex differentiation is temperature-dependent. The temperature sensitive period (TSP) of gonadogenesis lies between stages 16 and 22 of embryonic development. Previous studies have shown that embryos incubated at 30 degrees C, a temperature yielding 100% phenotypic females, can be sex reversed by treatments with an aromatase inhibitor administered during TSP or even somewhat after TSP (as of stage 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 purpose, eggs of E. orbicularis were treated with letrozole, a nonsteroidal aromatase inhibitor, at or as of stages 23, 24 or 25, then gonadal aromatase activity in each individual and the related gonadal structure were studied at hatching (stage 26) and for one year after hatching. Two kinds of treatments were carried out: 1) repeated applications of 10 microg of letrozole in ethanolic solution onto the eggshell; and 2) a single injection of 10 microg of letrozole in olive oil. Similar results were obtained with either application or injection of the aromatase inhibitor. In treatments as of or at stage 23, individuals with gonadal aromatase activity lower than 20 fmoles/hour/gonad had ovotestes, i.e., 22% of the treated individuals. At hatching, the inner part of these ovotestes contained testicular cords and also mixed lacunae presenting various degrees of transdifferentiation of the epithelium into a Sertolian epithelium. The cortex was maintained, although some germ cells degenerated within it. These processes continued after hatching. However, 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 ovotestes; in others, the cortex was absent in some parts and when it was present oocytes were degenerating. These results show that in the European pond turtle, differentiation of ovotestes from ovaries can be induced by treatment with an aromatase inhibitor starting at late stages of embryonic development (between the end of TSP and hatching), although such differentiation is less frequent as embryonic development proceeds. Sex reversal persists for at least one year after hatching. J. Exp. Zool. 290:490-497, 2001.