A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic induction in isolated mouse oocytes.
This study was carried out to compare the possible role of a secreted paracrine factor versus that of a gap-junction-transmitted signal in mediating meiotic induction in isolated mouse oocytes from PMSG-primed, immature mice. In the first set of experiments, oocyte-cumulus cell complexes (OCC) were pretreated for 3 h with 2 mM dbcAMP or FSH, washed, and the oocytes then cultured for 17-18 h in 40 microl drops containing either 300 microM dbcAMP or 4 mM hypoxanthine (HX). Each set of pretreated oocytes was cultured under three different conditions: (1) intact cumulus-cell-enclosed oocytes (CEO); (2) denuded oocytes (DO), cultured alone after removal of cumulus cells; and (3) co-cultured cumulus cells and oocytes (CC/DO), where the cumulus cells were removed in the same drop with a mouth-operated pipette and cultured alongside the oocytes. When pretreated with high dbcAMP or FSH, maturation was stimulated in CEO when cultured in either inhibitor (by 41.4-53.7%). Pretreatment failed to affect the maturation rate in DO. DO maturation was not altered appreciably by co-cultured 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 co-cultured with intact, FSH-treated OCC, there was no evidence of a positive factor secreted by the stimulated complexes, despite the fact that oocytes within the OCC were induced to resume maturation. In a second series of experiments the gap junction inhibitor, 18alpha-glycyrrhetinic acid (GA), was utilised. An initial experiment determined that GA dose-dependently blocked OCC metabolic coupling (0.2% coupling at 10 microM compared with 13.6% in controls). When HX-arrested CEO and DO were cultured for 17-18 h in medium containing increasing concentrations of GA, meiotic maturation was induced in CEO but not DO, suggesting that the cumulus cells provided a positive stimulus in the absence of functional gap junctional communication. No effect of GA was seen in dbcAMP-arrested oocytes. A kinetics experiment showed that when CEO were cultured in dbcAMP +/- FSH, meiotic induction was initiated after 3 h and germinal vesicle breakdown 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, measurement of OCC coupling revealed that no reduction in coupling occurred during this induction period in the absence of GA. It is concluded that cumulus cells can secrete a positive factor, but that this is normally overridden by inhibitory influences transmitted through the gap junction pathway in intact complexes. Furthermore, upon exposure of complexes to a meiosis-inducing stimulus, a positive gap-junction-mediated signal now predominates to trigger germinal vesicle breakdown, and this signal is utilised throughout the induction period.
Downs
S M
SM
Biology Department, Marquette University, Milwaukee, Wisconsin 53233, USA. stephen.downs@marquette.edu
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D018920:Coculture Techniques; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D006034:Glycyrrhetinic Acid / Q000494:pharmacology; D019271:Hypoxanthine / Q000494:pharmacology; D007700:Kinetics; D008540:Meiosis / Q000187:drug effects; D051379:Mice; D008815:Mice, Inbred Strains; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006080:Ovarian Follicle / Q000166:cytology; D019899:Paracrine Communication; D015398:Signal Transduction
D019271:Hypoxanthine; D003994:Bucladesine; D005640:Follicle Stimulating Hormone; D006034:Glycyrrhetinic Acid