SECOND MESSENGER CONTROL OF LUTEAL STEROIDOGENESIS

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.