Multiple hormonal controls are required to modulate the primary actions of gonadotropin in the Leydig cell (i.e. estrogen, intracellularly; CRF and angiotensin II, at the membrane level). The presence of a unique CRF receptor and its mechanisms of action in the Leydig cell has been elucidated. CRF acts rapidly (minutes) in the Leydig cell to exert highly effective negative autocrine regulation on Leydig cells. The inhibitory actions of CRF on cAMP generation and steroidogenesis are mediated by interaction of the peptide with a distinct high affinity receptor subtype. Although, present in low abundance in the Leydig cells (500 to 800/cell for CRF vs 20,000 for LH/HCG and 2,000/cell for AII) CRF receptors are highly coupled to a G-unit other than Gs and Gi (possibly Gp). CRF caused marked inhibition of Mn 2+ and forskolin stimulated adenylate cyclase activity in detergent solubilized membranes indicating that the site of inhibition indicated by the peptide is the catalytic subunit of adenylate cyclase. The inhibitory action of CRF is prevented by inhibitors of PKC and the action of the peptide was not observed in cells depleted of PKC. Furthermore, CRF caused rapid translocation of PKC. Thus, these studies demonstrate that the inhibitory action of CRF is exerted through direct or indirect actions of PKC on the catalytic unit of adenylate cyclase. Regulation of CRF release from the Leydig cell is modulated directly or indirectly through the gonadotropin stimulus, which releases the peptide in a dose-dependent fashion. The action of the trophic hormone is reproduced by cyclic AMP analogs. In contrast, norepinephine, while effective in stimulating cAMP and testosterone production, did not cause CRF release and forskolin was less effective than the trophic stimulus. This indicates that gonadotropin stimulation of CRF release is highly compartmentalized. Studies using the alpha-helical CRF (9-41) antagonist revealed tonic suppression of basal and gonadotropin-stimulated testosterone production by endogenously produced CRF. The CRF antagonist, and also an antibody to CRF, increased cell sensitivity to the trophic hormone. The intracellular pool of CRF in the cells is large and the minor proportion of peptide released during stimulation acts as a potent autocrine regulator of gonadotropin action. These findings have led to the proposal of CRF as a local antireproductive hormone responsible for certain disorders of androgen production and some forms of infertility in men. In other studies, we have provided evidence of yet another effect of forskolin in Leydig cells by the demonstration of an inhibitory action of high doses of this diterpene on hCG-stimulated steroidogenesis while potentiating hCG-stimulated adenylate cyclase activity and cAMP production. Further, this inhibitory effect was cAMP-independent and located beyond pregnenolone synthesis. The site of inhibition of forskolin seems to reside at the conversion of androstenedione to testosterone, possibly with reduced affinity of 17alpha-hydroxysteroid dehydrogenase.
