Analyses of the involvement of PKA regulation mechanism in meiotic incompetence of porcine growing oocytes

Abstract

Mammalian growing oocytes (GOs) lack the ability to resumemeiosis, although the molecular mechanism of this limitation isnot fully understood. In the present study, we cloned cDNAs ofcAMP-dependent protein-kinase (PKA) subunits from porcineoocytes and analyzed the involvement of the PKA regulationmechanism in the meiotic incompetence of GOs at themolecular level. We found a cAMP-independent high PKAactivity in GOs throughout the in vitro culture using a porcinePKA assay system we established, and inhibition of the activityby injection of the antisense RNA of the PKA catalytic subunit(PKA-C) induced meiotic resumption in GOs. Then we examinedthe possibility that the amount of the PKA regulatory subunit(PKA-R), which can bind and inhibit PKA-C, was insufficient tosuppress PKA activity in GOs because of the overexpression oftwo PKA-Rs, PRKAR1A and PRKAR2A. We found that neither ofthem affected PKA activity and induced meiotic resumption inGO although PRKAR2A could inhibit PKA activity and inducemeiosis in cAMP-treated full-grown oocytes (FGOs). Finally, weanalyzed the subcellular localization of PKA subunits and foundthat all the subunits were localized in the cytoplasm duringmeiotic arrest and that PKA-C and PRKAR2A, but not PRKAR1A,entered into the nucleus just before meiotic resumption inFGOs, whereas all of them remained in the cytoplasm in GOsthroughout the culture period. Our findings suggest that thecontinuous high PKA activity is a primary causeof th meioticincompetence of porcine GOs and that this PKA activity is notsimply caused by an insufficient expression level of PKA-R, butcan be attributed to more omplex spatial-temporal regulationmechanisms. © 2012 by the Society for the Study of Reproduction, Inc.