影响猪体细胞核移植重构胚体外发育的若干因素

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56.7%,发育至桑椹胚达11.7%、孵化囊胚率为6.7%,显著高于成纤维细胞组成的重构胚(p<0.05)。我们研究了卵母细胞的采集方法,激活方法和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至GO或G1期,抽吸法/解剖法采集卵母细胞,体外培养33或44 h,将卵丘细胞置于去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电脉冲结合6-DMAP激活处理,体外培养6天,结果表明,卵母细胞采集方法、激活液中细胞松弛素(CB)并不影响重构胚的发育(以卵龄44h的卵母细胞为受体);而以电脉冲结合6-DMAP激活处理能提高重构胚发育能力(以卵龄33 h的卵母细胞为受体)(p<0.05)。本研究显示,以电脉冲结合6-DMAP激活卵丘细胞重构胚,能在体外发育至囊胚

SOME FACTORS AFFECTING IN VITRO DEVELOPMENT OF PORCINE EMBRYO RECONSTITUTION FROM SOMATIC CELLS NUCLEAR TRANSFER

In this paper, a study on reconslitution of porcine oocytes by using nuclear transfer with cumulus cells(CC) and fibroblast cells(FC) was carried out. Reconstituted oocytes which were the fusion with CC and showed a cleavage rate of 56.7% , developed to morula( 11.7%) and blastocysls(6.7% ) phases which were higher than those derived from the fusion with FC( p<0. 05) . The results of this study also involved the effects of oocyte collection method and maturational age of recipient oocytcs during the in vitro develpoment of nuclear-transfer embryos which were reconstructed with cuhured cumulus cells. The cumulus cells sync hronized in G0/G1 phases through serum-starvation culture, were transferred into enucleated oocytes which were collected by aspiration or dissection method and culinred for 33 or 44 h. Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulsation and 6-DMAP, and cultured for 6 days. As for the oocytes collection methods, activation treatment in the presence of cytochalasm H did not affect the developmental rate of embryos reconstituted with 44-h-mature recipients. However, the development rate of reconstituted embryos with 33-h-mature recipients was significantly higher( p<0. 05) by activation with the combination of electric pulsation and 6-DMAP. These results suggest that reconstiluted porcine embryos derived from cultured cumulus cells can develop to the blastocyst stage and that the development of the former could be improved by reconstruction with young oocyte cytoplasl after the activation with the combination of electric pulsation and 6-DMAP.