Effection Of ASF1A On Pig Oocyte Meiotic Maturation

To investigate the effect of histone chaperone ASF1A(Anti-Silence Function1A) onmeiotic porcine oocyte cultured in vitro, pig ovarys are used as material, the morphologicalchanges of chromosomes during various periods of porcine oocyte in vitro maturation wereobserved, and determined the porcine oocyte during the specific time point by aceto-orceinstaining technique. The ASF1A gene was cloned from fibroblasts by RT-PCR.ASF1A-Venus eukaryotic expression vector was constructed, and transcripted to RNA in vitro.The expression of ASF1A mRNA were examined by RT-PCR and the real time QPCR, then weexpressed ASF1A tagged with Venus via mRNA microinjection, to determine its localization duringpig oocyte maturation. Last, ASF1A cRNA and ARF1SiRNA were respectively microinjected intoporcine oocytes to verify the effection of superexpression and lacking ASF1A on oocyte invitro mature.The results are as follows:1. We used aceto-orcein staining technique, examined chromosome morphologicalchanges of porcine oocyte in vitro maturation, and established the oocytes chromosomedevelopmental process of the timetable. At each time point, the chromosomal status ofoocytes was evaluated, frequencies were computed, and the time spent on each stage wasdetermined. The germinal vesical(GV)was spent from0to17.56h, germinal vesicalbreakdown~pre-metaphase Ⅰ (GVBD~pre-MⅠ) at17.56to25.98h, metaphase Ⅰat25.98to31.56h, anaphase Ⅰ at31.56to33.91h, telophase Ⅰ at33.91to37.73, and metaphaseⅠat37.73to42h.2. We successfully cloned ASF1A gene and constructed the recombinant vectorpASF1A-Venus. After transfection, the fusion protein could be expressed efficiently in Helacells. We also successfully got ASF1A-Venus RNA cRNA by transcription in vitro.3.There are ASF1A during different stages of oocyte in vitro mature at mRNA level;ASF1A are expressed in the cytoplasm and nucleus at protein level during GV stage, andmainly in nucleus. ASF1A uniformly distributed in nucleus and cytoplasm during MII.Weobserved no influence of overexpression of ASF1A on emission of the first polarbody(P>0.05). Depletion of ASF1A extremely signifcant inhibited the emission of the firstpolar body(P<0.01), but GVBD were not be effected(P>0.05), and the morphology of spindle is normal.Conclusion: The present study established the pig oocytes meiotic developmentalprocess of the timetable; We examined the expression of ASF1A and subcellular localization,and deletion of it inhibited the emission of the first polar body. ASF1A is essential for meioticmaturation of pig oocyte.