GAMETE BIOLOGY AND GAMETOGENESIS - B
Tuesday, August 3, 2004
10:30 AM–12:30 PM
(417) AMPK (5′AMP-ACTIVATED PROTEIN KINASE) IS CONTROLLING SPONTANEOUS MEIOTIC RESUMPTION VIA CUMULUS CELLS IN PORCINE OOCYTES.
Laforest, Martin 1, Pouliot, Éric 1, Cazet , Aurélie 1, Coulombe, Zoé 1, François , Richard.1, 1 Centre de Recherche en Biologie de la reproduction (C.R.B.R.), Ste-Foy, Québec, Canada
ABSTRACT- The meiosis of mammalian oocytes begins during the foetal life. It progresses through prophase-I to stop at dictyate stage. LH surge in vivo or culturing oocytes in vitro allows spontaneous meiotic resumption. In mouse oocyte, AMPK (5′AMP-activated protein kinase) is a kinase involved in meiotic resumption (Downs et al. 2002, Dev Biol 245: 200). This ser/thr kinase is implicated in several signaling pathways like glycolysis, lipolysis and sterols synthesis. This study has assessed the role of AMPK on nuclear maturation in porcine oocytes. Our first objective investigated whether activation of AMPK would reverse the inhibitory effect of phosphodiesterase 3 inhibitor, on spontaneous meiotic resumption. Cumulus-oocytes complexes (COC) and denuded oocytes (DO) were collected from gilt ovaries obtained at a local slaughterhouse. The maturation medium was NCSU23 supplemented with cystein (0,1 mg/ml), penicillin (65 g/ml), streptomycin (50 g/ml), b-mercaptoethanol (10 mM) and 10% of porcine follicular fluid, then fixed and classified according to the nuclear maturation stage. Oocytes were incubated in presence of the pharmacological compounds: PDE3 inhibitor, cilostamide (20 M) or the unspecific PDE inhibitor IBMX (500 M) and a potent activator of AMPK, AICAR (5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside; 0-1mM), for 24h or 48h. Results showed that instead of stimulating nuclear maturation, AICAR reversibly blocked spontaneous meiotic resumption and downregulated cumulus expansion in a dose-dependant manner, with an efficient concentration (EC50) of 0.4135 ± 1.17 mM in COC. The inhibitory effect was not observed in DO suggesting that AMPK is efficient in cumulus cells of porcine oocyte. Our second objective studied the efficient half-live of AICAR on COC. When cultured for 48h in presence of AICAR they resumed meiosis. However, when COC were transferred with newly prepared AICAR solution after the first 24h of culture in presence of AICAR, oocytes remained in GV stage. In presence of gonadotropin or forskolin, COC spontaneously resumed meiosis. In conclusion, these results suggest that the control of spontaneous meiotic resumption in porcine oocyte is involving AMPK pathway in cumulus cells.
KEY WORDS: Phosphodiesterases, nuclear maturation, AMPK (5′AMP-activated protein kinase) , oocyte