Phospholipase C in mouse oocytes: characterization of beta and gamma isoforms and their possible involvement in sperm-induced Ca2+ spiking.
This study involved an investigation of the role of phospholipase C (PLC) in generating repetitive Ca2+ spikes at fertilization. Using a PCR-based strategy we have demonstrated that mouse oocytes have mRNA coding for PLC beta 1, PLC beta 3 and PLC gamma isoenzymes. Furthermore, immunodetection of PLC gamma 1 using monoclonal antibodies reveals that PLC gamma 1 protein is present in mature mouse oocytes, ruling out the possibility that mRNA was being transcribed but not expressed. We were unsuccessful at detecting the presence of PLC beta protein, but the presence of this isoform can be inferred from functional studies. The PLC inhibitor, U73122, exerted an inhibitory effect on oocytes activated by spermatozoa or acetylcholine at concentrations of 10 and 30 microM respectively, while its inactive analogue had no effect. The soluble tyrosine kinase inhibitors, genistein (100 microM), herbimycin (10 microM) and geldanamycin (0.6 microM) which could affect signalling through PLC gamma hindered but never completely inhibited Ca2+ spiking in response to fertilization. We conclude that the activation of PLC to generate InsP3 may play a critical role in fertilization.
Dupont
G
G
Babraham Institute for Molecular Signalling, Department of Zoology, University of Cambridge, U.K.
McGuinness
O M
OM
Johnson
M H
MH
Berridge
M J
MJ
Borgese
F
F
D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005306:Fertilization / Q000502:physiology; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D013094:Spermatozoa / Q000502:physiology*; D013849:Thimerosal / Q000494:pharmacology; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004963:Estrenes; D007527:Isoenzymes; D011760:Pyrrolidinones; D012333:RNA, Messenger; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D013849:Thimerosal; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D000109:Acetylcholine; D002118:Calcium