Progesterone produced by the mammalian corpus luteum is required for maintenance of pregnancy. Therefore, proper luteal function is essential to reproduction. A powerful approach to understanding luteal function is to observe how different steroidogenic luteal cell types function. The ovine corpus luteum contains two functionally distinct types of steroidogenic cells, small and large. Secretion of progesterone in small cells appears to be regulated by LH via a cAMP mechanism while that in large cells is not stimulated by LH or increased intracellular cAMP levels. Large cells, however, respond to prostaglandin E2 with enhanced secretion of progesterone. This effect does not seem to be mediated by cAMP. Independent lines of evidence suggest that two second messenger mechanisms involving protein phosphorylation may mediate hormonal regulation of steroidogenesis in the corpus luteum. One mechanism is initiated by cAMP activation of the cAMP-dependent protein kinase (A kinase). The other is initiated by diacylglycerol activation of the calcium, phospholipid-dependent protein kinase (C kinase). This proposal outlines experiments to determine the role of such protein phosphorylations in regulating secretion of progesterone in suspended ovine small and large luteal cells (as separated by elutriation). 1) A comparison of A and C kinase activities within each cell type and between cell types will be made. 2) Stimulation by LH of phosphorylation of specific proteins in small cells incubated with (P32)PO4 (as monitored by SDS-PAGE) will be correlated with secretion of progesterone. 3) Small and large cells will be incubated with (P32)PO4 and activators of A (dbcAMP, cholera toxin and forskolin) and C (phorbol esters) kinase. An ability of these agents to mimic LH enhancement of protein phosphorylation and progesterone secretion in small cells will be determined. 4) Proteins determined to be involved in the regulation of steroidogenesis in both cell types will be assessed in their ability to serve as specific phosphoprotein substrates for A and/or C kinase in cellular fractions. This approach will utilize Mg(gammaP32)ATP and purified catalytic subunit (A kinase) or Ca+2, phosphatidyl inositol and phorbol esters (C kinase activators). From the above observations, the role of protein phosphorylation in LH-stimulated steroidogenesis in small cells will be described. Further, a mechanism to explain the lack of regulation of steroidogenesis by cAMP in large cells will be proposed.
