281 MITOGEN-ACTIVATED PROTEIN KINASE IN PORCINE CUMULUS CELLS

Abstract

Mitogen-activated protein kinase (MAPK) is involved in many signal processes within eukaryotic organisms. Its active form is phosphorylated. For meiotic resumption in oocytes the MAPK cascade plays a central role, because it participates in the transfer of the extracellular gonadotropin signal into the nucleus. In pigs it could be shown that for a gonadotropin-induced germinal vesicle breakdown (GVBD), an activation of MAPK in oocytes is not essential, but in the surrounding cumulus cells the MAPK has to be phosphorylated (Ohashi et al. 2003 Biol. Reprod. 68, 604–609). Because cumulus cells are very important for signal transfer, the present investigation dealt with the relevance of porcine cumulus cells and the phosphorylation of MAPK for the resumption of meiosis. Oocytes of slaughtered pigs were collected and cultured (medium: TCM 199, insulin, l-glutamine, gentamycin, 20% (v/v) FCS, and with or without 2.5 μg/mL FSH and 5.0 μg/mL LH). The proteins of isolated cumulus cells and oocytes were separated by gel electrophoresis (cumulus cells of 10 cumulus-oocyte complexes and 40 oocytes per lane, respectively) followed by an immunoblot with antibodies against MAPK and p90rsk (ERK 1 (sc-94) and Rsk-1 (sc-231), respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Alternatively the nuclear maturation was determined by orceine staining. The following results were achieved: The phosphorylation of MAPK in cumulus cells began very early during the in vitro maturation period. This was demonstrated already after 0.5 h unlike in oocytes where phosphorylation of MAPK does not occur until 18 h. The phosphorylation in cumulus cells occurs both in the presence and in the absence of FSH/LH, but without FSH/LH almost no GVBD occurs (after 26 h IVM: 86.9% GV oocytes, n = 59). The phosphorylation in the absence of gonadotropins could be caused by components of FCS, but with an exchange against polyvinylpyrrolidone (0.3%), the phosphorylation without FSH/LH still existed. The specificity was examined with the MAPK kinase inhibitor U0126. A concentration of 10 μM U0126 prevented GVBD and phosphorylation of MAPK in oocytes. However, in cumulus cells the phosphorylation of MAPK was reduced only minimally. In the presence of 50 μM U0126, a distinct decrease was observed during the first hours of maturation. But after 26 h phosphorylated MAPK appeared in cumulus cells despite the high concentration of U0126. The p90rsk is an important substrate of MAPK, which is phosphorylated by activated MAPK in oocytes. In our investigations we could detect only unphosphorylated forms of p90rsk in the cumulus cells. It seems that there are different ways for phosphorylation of MAPK to occur in cumulus cells, but they do not have the same consequences. The phosphorylation of MAPK in cumulus cells is necessary for a gonadotropin induced meiotic resumption, but phosphorylation does not always lead to GVBD. Furthermore, the p90rsk appears not to have the same importance as a substrate of MAPK in cumulus cells as in oocytes.