Parthenogenetic activation of mouse eggs by microinjection of a truncated c-kit tyrosine kinase present in spermatozoa.
A truncated form of the c-kit tyrosine kinase receptor, corresponding to the phosphotransferase portion of the cytoplasmic catalytic domain and the carboxyterminus (tr-kit), is accumulated during late mouse spermiogenesis. Here we report that tr-kit is specifically localized in the residual sperm cytoplasm, with maximal accumulation in the midpiece of the flagellum, suggesting that it can enter the egg during fertilization. Microinjection of extracts from COS cells expressing a recombinant tr-kit protein into metaphase II-arrested mouse oocytes caused complete oocyte activation, including cortical granule exocytosis, completion of the 2nd meiotic division, formation of a parthenogenetic pronucleus and progression through cleavage stages. No activation above background levels was obtained with extracts from mock-transfected COS cells. Similar results were obtained by microinjection of in vitro synthesized tr-kit mRNA into metaphase II-arrested oocytes. Tr-kit-induced parthenogenetic egg activation was completely inhibited by oocyte preincubation with the Ca2(+)-chelating agent BAPTA-AM or with a specific inhibitor of phospholipase C activity. Tr-kit-induced egg activation was associated with a decrease in activity of mitogen-activated protein kinase, an essential component of the cytostatic factor. These results candidate tr-kit as a putative sperm factor required for triggering activation of mouse eggs at fertilization.
Sette
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C
Dipartimento di Sanitá Pubblica e Biologia Cellulare, Sezione di Anatomia, Università di Roma Tor Vergata, Rome, Italy.
Bevilacqua
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A
Bianchini
A
A
Mangia
F
F
Geremia
R
R
Rossi
P
P
D000818:Animals; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis; D005260:Female; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008297:Male; D008540:Meiosis / Q000502:physiology; D051379:Mice; D008845:Microinjections; D009865:Oocytes; D010063:Ovum / Q000201:enzymology / Q000502:physiology*; D010312:Parthenogenesis / Q000502:physiology*; D051966:Phospholipase C gamma; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000737:chemistry*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D019009:Proto-Oncogene Proteins c-kit; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma