The role of ghrelin and some intracellular mechanisms in controlling the secretory activity of chicken ovarian cells

The general aim of these in-vitro experiments was to determine whether ghrelin controls the secretory activity of chicken ovarian cells and whether its action is mediated by TK-, MAPK-, CDK- or PKA-dependent intracellular mechanisms. We postulated that particular protein kinases could be considered as mediators of ghrelin action (a) if they are controlled by ghrelin, and (b) if blockers of these kinases modify the action of ghrelin. In our in-vitro experiments we investigated whether ghrelin altered the accumulation of TK, MAPK, CDK and PKA in chicken ovarian cells and whether ghrelin, with or without blockers of MAPK, CDK and PKA, affected the secretion of progesterone (P4), testosterone (T), estradiol (E2) or arginine-vasotocin (AVT). In the first series of experiments, the influence of a ghrelin 1-18 analogue (1, 10 or 100 ng/mL) was studied on the expression of TK, MAPK and PKA in cultured chicken ovarian granulosa cells. The percentage of cells containing TK/phosphotyrosine MAPK/ERK1, 2 and PKA was determined using immunocytochemistry. Ghrelin increased the expression of both TK and MAPK. The low concentration of ghrelin (1 ng/mL) increased the accumulation of PKA in ovarian cells whilst the high concentration (100 ng/mL) decreased it. The 10 ng/mL concentration had no effect. In the second series of experiments, the effects of the ghrelin analogue combined with an MAPK blocker (PD98059; 100 ng/mL), a CDK blocker (olomoucine; 1 microg/mL), or a PKA blocker (KT5720; 100 ng/mL), were tested for their effects on the secretion of hormones by cultured fragments of chicken ovarian follicular wall. P4, T, E2 and AVT secretions were measured using RIA and EIA. Ghrelin increased T and decreased E2, but did not affect P4 or AVT secretion. The PKA blocker promoted P4 secretion and suppressed E2 and AVT, but did not affect T secretion. It prevented or even reversed the effect of ghrelin on T and E2, but did not modify its effect on AVT secretion. The MAPK blocker enhanced P4 and T and reduced AVT, but did not affect E2 secretion. It was able to prevent or reverse the effect of ghrelin on T and E, and it induced a stimulatory effect of ghrelin on AVT secretion. The CDK blocker reduced the secretion of AVT, but had no effect on steroid hormone secretion. It induced the stimulatory influence of ghrelin on the secretion of P4 and AVT, but did not modify the effect of ghrelin on other hormones. These observations clearly demonstrate that ghrelin is a potent regulator of the secretory activity of ovarian cells and of TK, MAPK and PKA. Furthermore, they suggest that MAPK-, CDK- and PKA-dependent intracellular mechanisms are involved in the control of ovarian secretion and that they mediate the effects of ghrelin on these processes.