Submission Number: STE-4-19-1

Abstract Number: 423

EVIDENCE THAT THE GAP JUNCTION COUPLING PATHWAY, AND NOT A PARACRINE FACTOR, MEDIATES THE MEIOSIS-INDUCING ACTION OF GONADOTROPIN IN ISOLATED MOUSE OOCYTES.

Stephen M Downs

Biology Dept, Marquette Univ, Milwaukee, WI ¹

Abstract:

The role of a secreted paracrine factor versus that of a gap junction-transmitted signal in meiotic induction has been studied in oocytes from eCG-primed mice. Oocyte-cumulus cell complexes (OCC) were pretreated for 3 h with 0.3 mM dbcAMP, 2 mM dbcAMP or 0.3 mM dbcAMP+FSH, and the oocytes then cultured for 17-18 h in 40-microliter drops containing either 0.3 mM dbcAMP or 4 mM hypoxanthine (HX) as: (1) intact cumulus cell-enclosed oocytes (CEO); (2) denuded oocytes (DO), cultured after removal of cumulus cells; or (3) cocultured cumulus cells and oocytes (CC/DO), where cumulus cells were removed in the same drop and cultured with the oocytes. When pretreated with high dbcAMP or FSH, germinal vesicle breakdown (GVB) was stimulated in CEO by 41.4-53.7%. Pretreatment failed to affect maturation in DO. DO maturation was not appreciably altered by cocultured cumulus cells when arrest was maintained with dbcAMP. However, an increase in maturation of 21-23% was observed in CC/DO in the HX-containing cultures that was not dependent on prior treatment with a meiosis-inducing stimulus. When DO were cocultured with intact, FSH-treated OCC, there was no evidence of a positive factor secreted by the complexes, even though oocytes within the OCC were induced to resume maturation. In a second series of experiments the gap junction inhibitor, 18-glycerrhetinic acid (GA), was utilized. When HX-arrested CEO and DO were cultured 17-18 h in medium containing GA, meiotic maturation was induced in CEO but not DO, suggesting that the cumulus cells provided a positive stimulus in the absence of gap junctional communication. No effect of GA was seen in dbcAMP-arrested oocytes. A kinetics experiment showed that when CEO were cultured in dbcAMPFSH, meiotic induction was initiated after 3 h and GVB reached 60% by 6 h. When GA was added to the cultures at different times after the initiation of culture (0, 2, 3, 4, and 5 h), meiotic induction was immediately blocked. In addition, no reduction in coupling occurred during this induction period. Thus, although cumulus cells secrete a positive factor, it is normally overridden by inhibitory influences transmitted through the gap junction pathway. Furthermore, upon FSH stimulation, a positive gap junction-mediated signal predominates to trigger GVB and is utilized throughout the induction period. .

Keywords: Coculture, meiotic induction, hypoxanthine, dbcAMP

This abstract is being presented at: 2:30 PM in session:
SESSION 15: OOCYTE DEVELOPMENT