Role of the cyclic adenosine monophosphate-dependent protein kinase in the control of meiotic resumption in bovine oocytes cultured with thecal cell monolayers.

It has long been known that intracellular levels of cAMP are implicated in the process of meiotic resumption in bovine oocytes. The present study was undertaken to evaluate the effects of various modulators of cAMP in a coculture system with thecal cell monolayers that have been shown to promote meiotic arrest (germinal vesicle [GV] stage) in bovine oocytes. Ovaries were obtained from a slaughterhouse, and oocytes were collected by puncture of 1- to 5-mm follicles. Cumulus-oocyte complexes (COC) were cultured for 18 h in tissue culture medium 199 supplemented with 10% fetal calf serum and various modulators. When H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinole-sulfonamide) , a protein kinase A inhibitor, was used alone, a significant increase (p < 0.05) in the percentage of COC maintained in GV stage was observed. In the coculture system with thecal cell monolayers that maintained oocytes at the GV stage, H-89 significantly (p < 0.05) decreased this percentage. When oocytes were denuded of their cumulus cells, H-89 (50 microM) alone did not significantly increase the percentage of oocytes maintained at the GV stage. In addition, denuded oocytes were not significantly maintained at the GV stage when cocultured with thecal cell monolayers. In the absence of thecal cell monolayers, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (Bx), and an activator of adenylate cyclase, forskolin (Fk), significantly increased (p < 0.05) the percentage of denuded oocytes maintained at the GV stage. In contrast, Bx-Fk were not effective in maintaining COC at the GV stage. The inhibitory effect of H-89 on COC was significantly increased (p < 0.05) by Bx-Fk. Using thecal cell monolayers with Bx-Fk, the percentage of COC maintained at the GV stage was not significantly decreased. These results suggest that oocytes were maintained in meiotic arrest by inhibition of cAMP-dependent protein kinase by H-89 in cumulus cells. It seems that inhibitory factors originating from thecal cells are regulated by a cAMP-dependent protein kinase. In turn, inhibitory factors released from thecal cells may regulate a cAMP-dependent protein kinase in cumulus cells.