Activation of porcine oocytes via an exogenously introduced rat muscarinic M1 receptor.
Thirty hours after the beginning of in vitro maturation, porcine oocytes were microinjected with mRNA coding for the rat muscarinic M1 receptor. They were then incubated for 15 h to allow sufficient time for completing maturation, translation of the mRNA, and insertion of the receptor into the plasma membrane. They were then treated with acetylcholine, the receptor's agonist, and its effect on inducing various activation-related changes was examined. Acetylcholine treatment triggered the release of Ca2+ from internal stores that could be blocked by atropine, the receptor's antagonist. The Ca2+ release was probably mediated via a G protein, since prior injection of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) totally inhibited the effect of the agonist. Pertussis toxin (PT) had no effect on the Ca2+ transients induced by acetylcholine, suggesting that the signal transduction pathway involved a PT-insensitive G protein. Electron microscopy revealed that in the injected oocytes, acetylcholine induced cortical granule exocytosis. The oocytes were released from meiotic arrest as evidenced by the decrease in H1 kinase activity measured in the oocytes during the histone H1 kinase assay. After resuming meiosis they entered interphase: 58.8% of the injected oocytes formed pronuclei after incubation with the agonist. Injection without subsequent acetylcholine treatment, or acetylcholine incubation without prior injection with the receptor mRNA, did not cause these changes. The results provide further evidence that the components of a G protein-mediated signal transduction pathway exist in porcine oocytes and that the activation of this pathway via an exogenously supplied G protein-coupled receptor results in a full complement of oocyte activation events. Whether this pathway transduces the activating signal at sperm-induced oocyte activation requires further examination.
Macháty
Z
Z
Department of Animal Sciences, University of Missouri-Columbia, 65211, USA.
Mayes
M A
MA
Kovács
L G
LG
Balatti
P A
PA
Kim
J H
JH
Prather
R S
RS
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008540:Meiosis / Q000187:drug effects / Q000502:physiology; D008845:Microinjections; D018721:Muscarinic Agonists / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism*; D037342:Pertussis Toxin; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics; D051381:Rats; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013084:Sperm-Ovum Interactions / Q000502:physiology; D013552:Swine; D013873:Thionucleotides / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D018721:Muscarinic Agonists; D012333:RNA, Messenger; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; C023427:guanosine 5'-O-(2-thiodiphosphate); D037342:Pertussis Toxin; D011494:Protein Kinases; C102333:histone H1 kinase; D019204:GTP-Binding Proteins; D000109:Acetylcholine; D002118:Calcium