Changes in cAMP-dependent protein kinase (PKA) and progesterone secretion in luteinizing human granulosa cells

Abstract

Luteinization of follicular granulosa cells leads to an increase in progesterone secretion that is regulated by luteinizing hormone (LH). LH acts mainly by elevating intracellular cyclic 3 ,5 -adenosine monophosphate (cAMP) and activating cAMP-dependent protein kinase (PKA). In this study, we have examined the role of PKA in relation to progesterone output by luteinizing human granulosa cells. Human granulosa cells were obtained by percoll gradient centrifugation of follicular aspirates of patients undergoing oocyte retrieval for assisted conception. Cells were cultured in serum-supplemented medium for up to 3 days in the presence and/or absence of human (h)LH and other cAMP-elevating agents. Spent medium was assayed for cAMP and progesterone content by specific RIA. Cell lysates were collected and assessed for PKA regulatory (R)II /catalytic (C) expression by Western blotting. Although basal progesterone secretion increased progressively throughout culture, cAMP levels remained unchanged. Under basal conditions, PKA RII /C expression appeared to increase throughout the 3-day culture period. In the presence of hLH and other cAMP-elevating agents, progesterone secretion increased in a dose-dependent manner coincident with an increase in cAMP. However, despite the increase in both progesterone secretion and cAMP accumulation, there was a dose-dependent decrease in both PKA RII and C expression. Thus, data presented in this study show that increases in progesterone secretion in luteinizing human granulosa cells can be dissociated from increases in PKA expression. This notion implies that progesterone secretion may be regulated by PKA-dependent as well as PKAindependent mechanisms.

Transient expression of progesterone receptor and cathepsin-l in human granulosa cells during the periovulatory period

“…This increases intracellular cAMP, thereby activating protein kinase A and inducing expression of PR (29,30). Our in vivo study also indicates that the PKA protein is activated by hCG and that in vitro H89 reduces significantly PR expression.…”

Distribution and function of 3′,5′-Cyclic-AMP phosphodiesterases in the human ovary

“…In preantral follicles PDE4D and PDE3A were expressed to a significantly higher extent than in the GCs of later follicle stages as indicated by microarray data. As shown previously (Chin et al, 2004), when GCs isolated after ovulation induction were cultured in the presence of forskolin the cells were stimulated via cAMP to produce progesterone. Inhibition of PDE4 (i.e., by roflumilast) further increased the forskolininduced progesterone production, while there was no effect on the non-forskolin stimulated GCs.…”

Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation

“…Since we have previously shown that gonadotrophins, as well as other agents that raise intracellular cAMP, were able to increase progesterone secretion during the luteinization of human granulosa cells (Chin et al 2004), we went on to investigate the effect of PKA inhibition on agonist-induced progesterone secretion. Granulosa cells were incubated on days 1 (0-24 h) and 3 (48-72 h) of culture in the presence of hLH (100 ng/ml), the adenylate cyclase activator forskolin (10 µM) or the cell-permeable cAMP analogue, dbcAMP (5000 µM), with or without increasing doses of the PKA inhibitor, H89 (0·1-10 µM).…”

“…We have previously shown that, during luteinization of human granulosa cells, gonadotrophin-induced progesterone secretion is coincident with a decrease in the expression of the active catalytic subunit of PKA (Chin et al 2004), a finding which suggests that, during luteinization, increases in progesterone secretion can be dissociated from increases in PKA expression/activity; that is, steroid synthesis becomes less dependent upon PKA as luteinization proceeds. Both cAMP-GEFs (I and II) and the effector Rap are expressed in granulosa cells at the level of mRNA (Gonzalez-Robayna et al 2000).…”