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Although it has been suggested that in mammals the loss of female germ cells may induce the masculinization of the ovarian compartment, there has been as yet no conclusive demonstration. To directly address that question, the present study has been designed to determine the fate of follicular cells after oocyte loss. Using gamma-irradiation to selectively deplete oocytes in nongrowing follicles in female rats, we show that follicular cells in oocyte-depleted follicles survive, proliferate, and subsequently acquire morphological characteristics of Sertoli cells: elongated cytoplasm, basal location of the nucleus, and specific Sertoli cell junctions, the ectoplasmic specializations. These Sertoli-like cells express, however, the female-specific marker FOXL2 (Forkhead L2) but not the male sex-specific marker SOX-9 (Sry-type high-mobility-group box transcription factor-9) underlying the maintenance of molecular characteristics of granulosa cells. Before transdifferentiating into Sertoli-like cells, follicular cells of oocyte-depleted follicles initiate the expression of anti-Mullerian hormone and inhibin alpha-subunit that are typically synthesized by granulosa cells from the onset of follicular growth. Experimental modifications of the endocrine balance of the irradiated females show that there is a close relationship between plasma FSH levels and the occurrence of Sertoli-like cells. In addition to providing experimental evidence for the crucial role of the oocyte in granulosa cell phenotype maintenance, these results emphasize that the transdifferentiation of granulosa cells into Sertoli cells occurs in a multistep fashion, requiring the maturation of granulosa cells and depending on the endocrine milieu.

The fetal testis is not merely a precursor of the adult organ: it is indeed an endocrine gland whose function is the masculinization of the fetus. It differs physiologically and morphologically from the adult testis. In this paper, the first stages of testicular differentiation in the rat are described, with special emphasis on the ultrastructural aspects. At the stage of 13.5 days after fertilization, the first Sertoli cells differentiate; they are characterized by a voluminous and little electron dense cytoplasm, a well-developed RER formed by vesicles and short cisternae filled with a flocculent material. Progressively, they polarize and adhere to one another by adherens-like junctions and cytoplasmic interdigitations to form the differentiating seminiferous cords. In the basal part of the Sertoli cells, a mat of microfilaments differentiates under the plasmalemma, while cytoplasmic blebs protruding in the extracellular space tend to disappear. A continuous basal lamina delineating the seminiferous cords begins to appear on day 14.5 and becomes widespread on day 15.5. These observations, when compared with other data from the literature, emphasize the fact that the differentiation of the Sertoli cells is the first morphological event during testicular differentiation. A possible role of the Sertoli cells in the subsequent organogenesis of the testis is suggested.

Differentiation of the rat testis from the undifferentiated primordium begins with the appearance of a new cell type characterized by a large and clear cytoplasm. These cells aggregate, enclose germ cells, and progressively form seminiferous cords. Therefore, they were considered primordial Sertoli cells. A similar process was obtained in vitro in explants cultured in a synthetic medium. On the contrary, when fetal calf serum was added to the medium, the organization of seminiferous cords was impaired; large clear cells appeared, but they did not aggregate. Instead, they remained scattered throughout the abnormal gonad. The present experiments were undertaken to verify whether these cells are in fact Sertoli cells. The production of Müllerian inhibitor is a marker of fetal Sertoli cells. Therefore, undifferentiated gonadal primordia from 12-day 16-hr old male rat fetuses were cultured for 2 days in vitro with serum and then associated for 3 days with 14.5-day-old sex ducts from female fetuses. Müllerian ducts were inhibited as well by the abnormal cordless gonads as by those with differentiated sex cords. These experiments confirm previous views on testicular development and demonstrate that differentiation of Sertoli cells may take place quite independently of the testicular cord formation.

In previous papers (1, 2), it was shown that in the rat fetus, testicular differentiation begins 13 days after fertilization, in the depth of the primordium near to mesonephric tubules. A few cells swell and differentiate into Sertoli cells which encompass the germ cells. In the meantime they delineate locally one part of the contour of the developing seminiferous cord, which is completed by the recruitment of new cells during the next hours. Under the electron microscope the differentiation of the Sertoli cells involves cytoplasmic and organelles changes, typical junctions and microfilaments toward the external surface of the seminiferous cord. Testicular differentiation can be obtained in vitro in cultures made in a synthetic medium without or with addition of chick embryo extract. Fetal calf serum prevents the organogenesis of the sex cords or produces the disintegration of those already differentiation (on day 14). These data clearly underline some of the cellular processes involved in the differentiation of the seminiferous cords.

Lhx9 (LIM/Homeobox gene 9) encodes a transcription factor implicated in various developmental processes, including gonadogenesis. Our observations in the rat show that Lhx9 expression present in undifferentiated gonads disappears as epithelial cells differentiate into Sertoli cells and begin to express AMH. In rat and in chick testes, Lhx9 expression present in interstitial cells decreases progressively to become undetectable after birth. In the female rat, Lhx9 is highly expressed in epithelial ovigerous cords of the fetal ovary. Its expression is down-regulated as epithelial cells differentiate into granulosa cells during the process of folliculogenesis occurring at birth. If this process is impaired by the lack of oocytes, ovigerous cord organization is maintained together with Lhx9 expression. In conclusion, Lhx9 expression can be inversely correlated with the commitment into a differentiation pathway of the different categories of mesothelium-derived cells of the gonad. [Copyright &y& Elsevier]