h-89 and phorbol-12-13-didecanoate

The purpose of this investigation was to determine if precocious oocyte maturation could be induced by modulating ovarian cAMP-dependent protein kinase (PKA) or protein kinase C (PKC) signal transduction pathways in the intact hamster. The following inhibitors and stimulators were injected into the ovarian bursal cavity of the anesthetized hamster: N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a relatively selective inhibitor of PKA phosphorylations; a structurally related compound, H-7, a less potent and selective inhibitor used to alter PKA and PKC pathways; phorbol 12, 13-didecanoate (PDD beta), an active stimulator of PKC and the inactive analog, 4 alpha-phorbol 12, 13-didecanoate (PDD alpha); and GF109203x, a potent and selective inhibitor of PKC phosphorylations. The experimental design was to inject the modulator into the bursal cavity of one ovary and control solution of diluent or inactive compound into the contralateral bursal cavity. After 1 hr oocytes were collected and evaluated microscopically for the presence or absence of a germinal vesicle. Only oocytes recovered from H-89 treated ovaries (> 50 microM) showed significantly greater frequency of meiotic resumption. Exposure of ovaries to H-7 (< or = 150 microM), PDD beta (< or = 100 microM), or GF109203x (< or = 100 microM) did not significantly affect oocyte maturation state. These results suggest that ovarian protein phosphorylations carried out by PKA are necessary for the maintenance of oocyte meiotic arrest in situ.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cricetinae; Cyclic AMP-Dependent Protein Kinases; Female; Indoles; Isoquinolines; Maleimides; Mesocricetus; Oocytes; Phorbol Esters; Protein Kinase C; Signal Transduction; Sulfonamides

Four hypotheses were tested using isolated bovine oocytes. (1) Cumulus oocyte complexes (COCs) or denuded oocytes (DOs) were cultured with the protein kinase A (PKA) inhibitor, H-89, to test if meiotic arrest induced by forskolin or IBMX was due to cAMP-stimulated PKA activity or nonspecific effects of these cAMP elevators. (2) COCs were cultured with a protein kinase C (PKC) stimulator (PDD beta) or inhibitor (GF109203x) to test if PKC modulation altered oocyte maturation. (3) COCs were prestimulated for 15 min with (a) PDD beta followed by cotreatment with forskolin, or (b) with H-89 or H-7 followed by cotreatment with GF109203x, to test for interaction between the PKA and PKC signal transduction pathways. (4) H-89 was added to spontaneously maturing COCs at intervals of 0-18 hr to test if H-89 interfered with the transition between meiosis I and II. The results were as follows: H-89 interfered with forskolin or IBMX arrested oocytes in dose-response manner (IBMX ED50 = 41 microM for COCs; forskolin ED50 = 9 microM for denuded oocytes). Prestimulation with PKC induced meiotic resumption in COCs in spite of the presence of forskolin [PDD beta followed by PDD beta + forskolin: 41-47% germinal vesicle (GV) oocytes; forskolin alone: 90-95% GV], while PKC inhibition induced meiotic arrest to a similar extent as forskolin (GF109203x, 85% GV; forskolin, 67-80% GV). Additionally, pretreatment of COCs with H-89 interfered with GF109203x induced arrest (41% vs. 90% GV, respectively). Finally, H-89 interfered with the timely progression of COCs from meiosis I and II. These results indicate that the PKA and PKC pathways can modulate the maturation of bovine oocytes in vitro.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Cattle; Cells, Cultured; Colforsin; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Female; Indoles; Isoquinolines; Maleimides; Meiosis; Oocytes; Phorbol Esters; Protein Kinase C; Signal Transduction; Sulfonamides; Time Factors