Open AccessEffect of H89 on the meiotic resumption of pig oocytes
Author(s) -
CayoColca Ilse Silvia,
Harayama Hiroshi,
Miyano Takashi
Publication year - 2011
Publication title -
reproductive medicine and biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.005
H-Index - 22
eISSN - 1447-0578
pISSN - 1445-5781
DOI - 10.1007/s12522-010-0073-2
Subject(s) - germinal vesicle , oocyte , immunocytochemistry , protein kinase a , phosphorylation , kinase , chemistry , meiosis , blot , protein kinase c , microbiology and biotechnology , biology , biochemistry , endocrinology , embryo , gene
Purpose We studied the effect of H89, an inhibitor of protein kinase A (PKA), on the meiotic resumption of pig oocytes. Methods Pig cumulus–oocyte complexes (COCs) and denuded oocytes (DOs) were cultured for 27 h to induce meiotic resumption. COCs and DOs were exposed to H89 for different periods. Oocyte PKA activity was assessed by in vitro kinase assay and immunocytochemistry using an antibody against fully active PKA catalytic subunits. Oocyte serine/threonine (Ser/Thr)‐phosphorylated proteins were detected by Western blotting and immunocytochemistry using an anti‐pSer/pThr PKA substrate antibody. Results H89 suppressed germinal vesicle break down (GVBD) in COCs and DOs. To determine whether the suppression was due to inhibition of oocyte PKA, we analyzed oocyte PKA. Kinase assay showed that both types of oocytes possessed PKA activity throughout the culture period. Immunocytochemistry showed that fully active PKA catalytic subunits and Ser/Thr phosphorylated proteins were present in the oocytes at the GV stage and after GVBD. Western blotting indicated that both types of oocytes contained Ser/Thr phosphorylated proteins at the GV stage, and that several proteins became phosphorylated after GVBD. Conclusions Pig oocytes contain active PKA during the occurrence of GVBD, and H89 suppresses the GVBD.