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Parthenogenetic activation of mouse eggs by microinjection of a truncated c-kit tyrosine kinase present in
C Sette1, A Bevilacqua, A Bianchini
1Dipartimento di Sanitá Pubblica e Biologia Cellulare, Sezione di Anatomia, Università di Roma Tor Vergata, Rome, Italy.
Abstract:
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.
Insights
A truncated form of the c-kit tyrosine kinase receptor (tr-kit) found in sperm can activate mouse eggs. This sperm factor triggers key events necessary for fertilization and early development.
Area of Science:
- Reproductive Biology
- Cell Signaling
- Molecular Biology
Background:
- The c-kit tyrosine kinase receptor plays a role in cell development.
- A specific truncated form (tr-kit) accumulates in late-stage sperm.
- The function of tr-kit in fertilization is not well understood.
Purpose of the Study:
- To investigate the role of tr-kit in mouse oocyte activation.
- To determine if tr-kit can trigger the events of fertilization.
Main Methods:
- Microinjection of recombinant tr-kit protein and mRNA into metaphase II-arrested oocytes.
- Assessing oocyte activation markers like cortical granule exocytosis and meiotic progression.
- Using inhibitors of calcium and phospholipase C to block activation.
Main Results:
- Microinjected tr-kit induced complete oocyte activation, including pronuclear formation and cleavage.
- Activation was dependent on calcium and phospholipase C signaling.
- tr-kit also reduced mitogen-activated protein kinase activity.
Conclusions:
- tr-kit is a potential sperm factor that triggers mouse egg activation.
- It initiates key signaling pathways necessary for fertilization.
- This finding sheds light on the molecular mechanisms of fertilization.