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| CyclinA2 Gene (CycA2) Over-expression Blocks Extrusion of Porcine (Sus scrofa) Oocytes PB1 In vitro |
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Abstract During the process of oocyte maturation, CyclinA2(CycA2) controls the progression of cell cycle by activating different cyclin-dependent kinase (Cdk), which regulates the cell cycle through its expression and degradation. Eukaryotes contain 2 distinct types of CycA2 of A1 and A2. The transcription of CycA2 initiates in late G1, peaks and plateaus in mid-S, declines in G2 and totally disappears in metaphase. CycA2 is also involved in the G1/S and G2/M transitions. CycA2 is degradated by ubiquitin-proteasome system in mitosis. In recent study, it is reported that CycA2 also plays an important role in the meiotic division of female germ cells. It can promote entry into the first meiosis in mouse (Mus musculus) oocyte, furthermore, there is an unexpected role for its requirement for the sister chromatid segregation in the second meiosis. These results all suggest that CycA2 has an important role in oocyte maturation and embryo development. In order to investigate the effect of CycA2 on porcine (Sus scrofa) oocytes during in vitro maturation (IVM) in the present study, firstly, CycA2 gene was cloned from pig ovary by RT-PCR and constructed 2 eukaryotic vectors, the wild type pVenus-CycA2 and non-degradated pVenus-DN157CycA2, which were transfected into hela cells to confirm their expression and location by fluorescence microscopy observation and qRT-PCR. The cRNA of pVenus-CycA2 and pVenus-DN157CycA2 transcripted in vitro were microinjeted into porcine oocytes. Finally, the oocytes microinjected pVenus-CycA2 and pVenus-DN157CycA2 cRNA were collected for mature cultivation and the rate of the first polar body (PB1) extrusion were calculated. Besides, the CDK1 inhibitor roscovitine were used to treat the oocytes which were microinjected the DN157roscovitine cRNA and the rate of the PB1 extrusion was also examined. The results showed that the recombinant vector pVenus-CycA2 and pVenus-DN157CycA2 were successfully constructed. After transfection or microinjection, the fusion protein could express efficiently and localize accurately both in hela cells and porcine oocytes. This greatly facilitated the further study of CycA2, especially its role on oocytes maturation regulation. The rate of PB1 extrusion were decreased significantly compared with the control group(P＜0.01, P＜0.05). After treated with roscovitine, the oocytes microinjected pVenus-DN157CycA2 could extrude the PB1 and there was no significant difference in the rate of PB1 extrusion compared with the control group. In conclusion, CycA2 gene had an effect on the PB1 extrusion in a way associated with CDK1 during the porcine oocyte maturation, it provided a new direction in the research of in vitro maturation of oocytes and layed the foundation for the molecular mechanism of the CycA2 gene participates in the chromosome segregation. The study reveals an unexpected role for CycA2 in mediating PB1 extrusion in the first meiosis and also gives a platform to further explore the role of CycA2 gene in the process of the second meiotic division.
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Received: 04 December 2015
Published: 22 July 2016
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