Involvement of Rabphilin-3A in cortical granule exocytosis in mouse eggs.
Rabphilin-3A is a putative target protein for Rab3A, a member of the small GTP-binding protein superfamily that has been suggested to play a role in regulated exocytosis in presynapses. In this study we determined the expression and the function of Rabphilin-3A in mouse eggs at fertilization. Rabphilin-3A mRNA and protein were detected by reverse transcriptase-PCR and immunoblot analysis, respectively, in metaphase II mouse eggs. Immunofluorescence analysis showed that Rabphilin-3A protein was distributed in the cortical region in eggs. Sperm induces cortical granule (CG) exocytosis via an increase in cytosolic Ca2+ at fertilization. We microinjected the NH2- or COOH-terminal fragment of recombinant Rabphilin-3A into metaphase II eggs. Neither treatments altered the sperm-induced cytosolic Ca2+ increase, but both inhibited CG exocytosis in a dose-dependent manner. The NH2-terminal fragment was more effective than the COOH-terminal fragment. Full-length Rabphilin-3A did not affect CG exocytosis, but it attenuated the inhibition of CG exocytosis by the NH2-terminal fragment. These results show that Rabphilin-3A is involved in Ca(2+)-dependent CG exocytosis at fertilization in mouse eggs.
Masumoto
N
N
Department of Obstetrics and Gynecology, Osaka University Medical School, Japan. masumoto@gyne.med.osaka-u.ac.jp
Sasaki
T
T
Tahara
M
M
Mammoto
A
A
Ikebuchi
Y
Y
Tasaka
K
K
Tokunaga
M
M
Takai
Y
Y
Miyake
A
A
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000502:physiology*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005260:Female; D005306:Fertilization / Q000502:physiology; D019204:GTP-Binding Proteins / Q000032:analysis / Q000235:genetics*; D015870:Gene Expression / Q000502:physiology; D005982:Glutathione Transferase / Q000494:pharmacology; D006388:Hemagglutinins / Q000494:pharmacology; D008297:Male; D008677:Metaphase / Q000502:physiology; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics*; D010063:Ovum / Q000737:chemistry* / Q000166:cytology / Q000502:physiology; D010446:Peptide Fragments / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium; D019204:GTP-Binding Proteins; D005982:Glutathione Transferase; D006388:Hemagglutinins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000097624:Rabphilin-3A