CYCLIC ADENOSINE-MONOPHOSPHATE SIGNALING IN THE PRIMATE CORPUS-LUTEUM- MAINTENANCE OF PROTEIN-KINASE-A ACTIVITY THROUGHOUT THE LUTEAL-PHASE OF THE MENSTRUAL-CYCLE

Recent studies from our laboratory (Endocrinology 136:4762-4768, 1995) demonstrating that the expression of cAMP-dependent nuclear transcrip tion factor CREB (cAMP response element binding protein, is lost follo wing ovulation in macaques has revealed a novel mechanism by which the cytoplasmic and nuclear actions the cAMP-protein kinase A (PKA) intra cellular signaling system may be regulated independently. Implicit in this hypothesis is the assumption that PICA activity is maintained thr oughout the luteal phase of the menstrual cycle, yet to date there hav e been no published reports regarding PKA activity in the primate corp us luteum. PKA activity was assessed by the incorporation of P-32 from radiolabeled ATP into a PKA-specific peptide substrate (kemptide) in the presence or absence of cAMP. Luteal cytosolic fractions were obtai ned from corpora lutea collected during the spontaneous luteal phase ( days 3-5, 7-8, 10-11, 13-15, and postmenses) or obtained from animals on days 11 or 16 of the luteal phase after the animals received seven days of exogenous human CG (hCG) treatment. Examination of PKA activit y in luteal slices from various aged CL maintained in short-term organ culture in the presence or absence of recombinant cynomolgus monkey L H was also performed. There were no significant differences in basal o r cAMP-stimulated PKA activities in corpora lutea collected throughout the spontaneous luteal phase. Further, Western immunoblot analyses of the catalytic subunit of PKA (PKA C alpha) in corpora lutes collected throughout the luteal phase revealed immunoreactive protein bands wit h similar intensities. In vitro addition of recombinant cynomolgus LH and dibutyryl cAMP stimulated PKA activity in corpora lutea collected during the early, mid, and late luteal phases. In corpora lutea obtain ed from animals treated with hCG during the midluteal phase, basal PIC A activity was decreased 65% as compared with untreated day 11 control s and in late luteal phase, hCG-exposed CL basal PKA activity was decr eased 30% as compared with untreated day 16 controls. However, there w ere no measurable differences in cAMP-stimulated PKA activity in CL ex posed to prior hCG treatment in vivo and Western immunoblot analyses f or PKA C alpha in these tissues revealed immunoreactive protein bands that were comparable with corpora lutea collected from untreated anima ls. Further, immunoblot analyses for CREB in corpora lutea collected f rom hCG-treated animals revealed that CREB immunoreactivity remained u ndetectable following a treatment regimen with hCG that mimics early p regnancy. These results demonstrate that, although CREB expression cea ses following ovulation, PKA activity is maintained throughout the lut eal phase, which provides a mechanism by which the acute steroidogenic actions of LPI may be separated from longer term trophic actions that may rely the transcriptional activity of CREB.