Analyses of the involvement of PKA regulation mechanism in meiotic incompetence of porcine growing oocytes.
Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. In the present study, we cloned cDNAs of cAMP-dependent protein-kinase (PKA) subunits from porcine oocytes and analyzed the involvement of the PKA regulation mechanism in the meiotic incompetence of GOs at the molecular level. We found a cAMP-independent high PKA activity in GOs throughout the in vitro culture using a porcine PKA assay system we established, and inhibition of the activity by injection of the antisense RNA of the PKA catalytic subunit (PKA-C) induced meiotic resumption in GOs. Then we examined the possibility that the amount of the PKA regulatory subunit (PKA-R), which can bind and inhibit PKA-C, was insufficient to suppress PKA activity in GOs because of the overexpression of two PKA-Rs, PRKAR1A and PRKAR2A. We found that neither of them affected PKA activity and induced meiotic resumption in GO although PRKAR2A could inhibit PKA activity and induce meiosis in cAMP-treated full-grown oocytes (FGOs). Finally, we analyzed the subcellular localization of PKA subunits and found that all the subunits were localized in the cytoplasm during meiotic arrest and that PKA-C and PRKAR2A, but not PRKAR1A, entered into the nucleus just before meiotic resumption in FGOs, whereas all of them remained in the cytoplasm in GOs throughout the culture period. Our findings suggest that the continuous high PKA activity is a primary cause of the meiotic incompetence of porcine GOs and that this PKA activity is not simply caused by an insufficient expression level of PKA-R, but can be attributed to more complex spatial-temporal regulation mechanisms.
Laboratory of Applied Genetics, Graduate School of Agriculture and Life Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Takanori
T
Nishimura
Wataru
W
Fujii
Kiyoshi
K
Kano
Koji
K
Sugiura
Kunihiko
K
Naito
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000032:analysis / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation / Q000235:genetics; D057075:Enzyme Assays / Q000379:methods; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D059471:In Vitro Oocyte Maturation Techniques; D008540:Meiosis / Q000235:genetics*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D009866:Oogenesis / Q000235:genetics* / Q000502:physiology; D021122:Protein Subunits / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D013552:Swine / Q000235:genetics / Q000378:metabolism / Q000502:physiology
D021122:Protein Subunits; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases