Gonads from sexually undifferentiated 16- and 17-day rabbit embryos were maintained in organ culture in a serum-free medium for 24–96 h. The interstitial cells of fetal testes developed from undifferentiated to well differentiated Leydig cells after 4 days of culture. The enzymatic reactions necessary to convert [7α-3H]dehydroepiandrosterone to androstenedione and testosterone also developed in culture at the expected time (corresponding to day 18 in vivo) in the absence of gonadotropins. Increased cAMP synthesis in response to human (h) CG first became apparent at day 17.5 and reached a maximum at day 20. The increased cAMP response was accompanied by an increase in LH/hCG receptor content and the appearance of 3β-hydroxysteroid dehydrogenase- Δ4, 5-isomerase activity. Testosterone synthesis, however, was not stimulated by hCG until day 20. Fetal ovaries were unresponsive to hCG at all times studied. The correspondence between the development of LH/hCG receptors, cAMP responsiveness, and the appearance of the enzymatic capacity to synthesize testosterone from C21 precursors suggests that the development of these functions is closely linked during Leydig cell maturation. However, the delay of 2 or more days in coupling between LH/hCG receptors and the ability to increase testosterone synthesis in response to added hCG suggests that the initiation of androgen synthesis at the time of male phenotypic differentiation in the rabbit embryo is independent of extragonadal hormone stimulation.
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