GH regulates secretory activity and apoptosis in cultured bovine granulosacells through the activation of the cAMP/protein kinase A system

We have studied the action of GH on the production of hormones, growth fact ors, growth factor-binding protein and the occurrence of apoptosis in bovin e ovarian granulosa cells, as well as the role of cAMP-stimulated protein k inase A (PKA) in the mediation of these effects. For this purpose we invest igated the effects of exogenous bovine GH (0.001-10 mu g/ml), PKA blockers KT5720 (100 ng/ml) and adenosine-3',5'-monophosphothiodate (Rp-cAMPS) (1 mu mu mol), alone and in combination, on IGF-I, IGF-binding protein (IGFBP)-3 , oxytocin, progesterone and estradiol secretion, cAMP and PKA content and the occurrence of apoptosis. The secretion of hormones, IGF-I and IGFBP-3 into the culture medium was me asured using RIA/IRMA. The presence of PKA was detected using immunocytoche mistry and Western immunoblotting. The presence of cAMP in cells was demons trated using immunocytochemistry, whilst the proportion of apoptotic cells was determined by the TUNEL method. It was found that the addition of GH to the culture medium strongly (P < 0. 05) stimulated IGF-I (at a concentration of 0.001-10 mu g GH/ml medium), IG FBP-3 (0.001-1 mu g GH/ml) and oxytocin (0.01-10 mu g GH/ml) secretion. Low concentrations (1-100 ng/ml) of GH stimulated, whilst a higher concentrati on (10 mu g/ml) inhibited estradiol output. GPI slightly (P < 0.05) inhibit ed progesterone (1-100 ng GH/ml) secretion and significantly (P < 0.05) dec reased the incidence of apoptosis (0.01-1 mu g GH/ml) in cultured cells. Th e addition of GH (100 ng/ml) caused a dramatic (P < 0.05) increase in the p roportion of cells possessing the immunoreactive catalytic subunit of PKA a nd a slight decrease in the proportion of cells containing the regulatory P KA subunit. PKA blockers KT5720 and Rp-cAMPS significantly (P < 0.05) reduced the propo rtion of granulosa cells containing cAMP, and the catalytic and (in the cas e of KT5720) regulatory subunits of PKA. KT5720 given alone significantly ( P < 0.05) inhibited the secretion of IGFBP-3, but not that of IGF-I or prog esterone. Rp-cAMPS decreased (P < 0.05) the secretion of oxytocin but not t hat of estradiol output or the occurrence of apoptosis. KT5720 and Rp-cAMPS fully or partially prevented the GH effect on IGF-I, IGFBP-3, oxytocin, pr ogesterone, estradiol and apoptosis. These observations suggest the involvement of GH and a cAMP/PKA-dependent i ntracellular cascade in the control of IGF-I, IGFBP-3, oxytocin, progestero ne, estradiol, cAMP and apoptosis in bovine ovarian granulosa cells. The st imulation of PKA by GH and the prevention of GH-induced effects by PKA bloc kers suggest that the observed GH effects on bovine ovarian cells are proba bly mediated by the cAMP/PKA system.