Gonadotrophins such as luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) exert their actions on progesterone (P4) synthesis, at least in part, by regulating luteal cell phosphorylation via ser/thr kinases particularly cAMP-dependent protein kinase (PKA). Hence the aim of this study was to identify changes in P4 secretion and PKA expression during luteinization of human granulosa cells (GCs) in vitro. GCs, obtained from follicular aspirates of patients undergoing oocyte retrieval for assisted conception, were isolated by percoll gradient centrifugation and cultured in serum-supplemented medium for up to 4 days. Cells were treated with modulators of cAMP (prostaglandin E2 [PGE2], 0.01-10µM; hCG, 10-1000ng/ml; dibutyryl cyclic AMP [dbcAMP], 50-5000µM) or with an inhibitor of PKA (H89, 1-100µM) with medium being replaced every 24 hours and P4 in spent medium being measured by RIA. Cell lysates were also collected for western blotting (WB) analysis of PKA. Results show that modulators of cAMP induced concentration-dependent increases in P4 production by luteinizing human granulosa cells in vitro [10µM PGE2, 199.2%±18.2 vs basal; 1000ng/ml hCG, 237.8%±27.3; 5000µM dbcAMP, 458.2±72.7 (n=3, p<0.05)], while the PKA inhibitor H89 caused a concentration-dependent decrease in basal P4 production [100µM H89, 49.7%±22.3]. Although PGE2 hCG and dbcAMP caused concentration-dependent increases in P4 production on day 3 of culture, WB analysis of the same cells showed concomitant decreases in PKA protein expression. These results indicate that changes in PKA expression/activity (?) may play a role in the process of luteinization. This work is supported by the BBSRC.
03 - 04 Dec 2001
Society for Endocrinology