猪卵母细胞体外成熟的发育潜力及核移植重构胚构建方法的研究

Studied on Porcine Oocyte’s Developmental Potential in Vitro Maturation and Reconstructed Methods in Somatic Cells Nuclear Transfer

为了提高猪体细胞核移植重构胚发育潜力,本研究对体外成熟28h、32h、36h、40h、44h、48h、52h和56h的猪卵母细胞分别进行去核构建重构胚。研究结果表明,成熟44h的卵母细胞核移植后有较高的融合率(58.99%)、卵裂率(67.52%)和囊胚率(22.78%),而成熟48h的卵母细胞则分别为56.51%、65.73%和15.96%;且卵龄为44h的卵母细胞核移植后分裂率与囊胚率显著高于卵龄为40h、36h、32h、28h的卵母细胞的分裂率与囊胚率(P<0.05)。卵龄为48h的卵母细胞融合率高于卵龄为52h卵母细胞的融合率(P<0.05)。同时我们还探讨了不同去核方法(盲吸法、Hochest33342染色法和Spindle-view system)对猪体细胞核移植重构胚发育能力的影响。研究结果发现,盲吸法、Hoechest33342染色法和Spindle-view system法的去核率分别达到76.33%,100.00%和98.40%。Hoechest染色法去核率显著高于盲吸法的去核率(P>0.05),而与Spindle-view法去核率没有差异(P>0.05)。三种方法在融合率和囊胚率方面差异不显著(P>0.05),但Hoechest染色法的分裂率较低,差异显著(P<0.05)。进一步的研究表明,细胞质内注射进行核移植构建重构胚的分裂率和囊胚率分别为68.13%和6.44%;透明带下注射法则为60.37%和8.08%,两者差异不显著(P<0.05);两者均可运用于猪体细胞的核移植,这为建立有效的猪体细胞核移植体系提供了参考。

In order to improve the developmental potential of reconstructed embryos in somatic cells nuclear transfer in porcine, we explored the reconstructed embryos about the porcine oocyte＇s in vitro maturation of different times for 28 h, 32 h, 36 h, 40 h, 44 h,48 h,52 h and 56 h by enucleated nucleus in this research, respectively. The result showed that the oocyte matured in vitro for 44 h which was transferred into the nuclear had higher the fusion rate of 58.99%, cleavage rate of 67.52% and blastocyst rate of 22.78%, while that of the 48 h respectively was 56.51%, 65.73% and 15.96%. The result also elucidated that the cleavage rate and blastocyst rate of oocytes which matured in vitro for 44 h were significantly higher than that of maturation in vitro for 40 h,36 h,32 h and 28 h. On the other hand, the fusion rate of 48 h oocytes in vitro maturation was higher than that of 52 h （P〈0.05）. Furthermore, we still have investigated the effect of different enucleated methods, including the Blindness sucking method, Hochest33342 staining method and Spindle-view system method, on the developmental capacity of reconstructed embryos in somatic cells nuclear transfer in porcine. The result also found that the enucleated rate by using Blindness sucking method, Hochest33342 staining method and Spindle-view method were 76.33%, 100.00% and 98.40%,respectively. And the enucleated rate of Hoechest33342 staining method was sharply higher than the Blindness sucking method （P〈0.05）, however, there was no significant difference compared to the enucleating using the Spin- die-view system （P〉0.05）. These three methods had little difference in the fusion rate and blastocyst rate（P〉0.05）, whenas the cleavage rate of Hoechest 33342 staining method was very lower and significantly different from others （P〈0.05）. Further result was presented that cleavage rate and the blastocyst rate of reconstructing embryos by using the method of inntracytoplasmin injection were 68.13% and 6.44%, respectively, and that of the method of injection under zona pellucid were 60.37% and 8.08%, so there were no obviously different between them （P〈0.05）. Therefore, these two methods could be applied in somatic cells nuclear transfer in porcine, which would provide the reference to establish a beneficial system of somatic cell nuclear transfer in porcine.