Liu H, Zhang J, Krey L C, Grifo J A
Department of Obstetrics and Gynecology, New York University School of Medicine, New York 10016, USA.
Hum Reprod. 2000 Sep;15(9):1997-2002. doi: 10.1093/humrep/15.9.1997.
We evaluated whether mouse oocytes reconstructed by germinal vesicle (GV) transfer can develop to blastocyst stage. The oocytes were artificially activated with sequential treatment of A23187 and anisomycin; fertilization was then established by transfer or exchange of pronuclei with those of zygotes fertilized in vivo. Type 1 zygotes were constructed by placing the male haploid pronucleus from a zygote into the cytoplasm of an oocyte that underwent GV transfer, in-vitro maturation and activation; for type 2 zygotes, the female pronucleus was removed from a zygote and replaced with the female pronucleus of an oocyte subjected to GV transfer, in-vitro maturation and activation. Karyotypes of activated oocytes and type 2 zygotes were also subjected to analysis. When cultured in human tubal fluid (HTF) medium, reconstructed oocytes matured and, following artificial activation, consistently developed a pronucleus with a haploid karyotype; the activation rate for this medium was two- to three-fold higher than that of oocytes cultured in M199 (87% versus 30% respectively). Following transfer of a male pronucleus, only 47% of the type 1 zygotes developed to morula or blastocyst stage and embryo morphology was poor. In contrast, 73% of the type 2 zygotes developed to morula or blastocyst stage, many even hatching, with few morphological anomalies. Normal karyotypes were observed in 88% of the type 2 zygotes analysed. These observations demonstrate that the nucleus of a mouse oocyte subjected to sequential nuclear transfer at GV and pronucleus stages is, nonetheless, capable of maturing meiotically, activating normally and supporting embryonic development to hatching blastocyst stage. In contrast, the developmental potential of the cytoplasm of such oocytes appears to be compromised by these procedures.
我们评估了通过生发泡（GV）转移重建的小鼠卵母细胞是否能发育到囊胚阶段。卵母细胞通过A23187和茴香霉素的顺序处理进行人工激活；然后通过将原核与体内受精的合子的原核进行转移或交换来实现受精。1型合子是通过将来自合子的雄性单倍体原核放入经过GV转移、体外成熟和激活的卵母细胞的细胞质中构建而成；对于2型合子，从合子中去除雌性原核，并用经过GV转移、体外成熟和激活的卵母细胞的雌性原核进行替换。还对激活的卵母细胞和2型合子的核型进行了分析。当在人输卵管液（HTF）培养基中培养时，重建的卵母细胞成熟，并且在人工激活后，始终发育出具有单倍体核型的原核；该培养基的激活率比在M199中培养的卵母细胞高两到三倍（分别为87%和30%）。在转移雄性原核后，只有47%的1型合子发育到桑葚胚或囊胚阶段，胚胎形态较差。相比之下，73%的2型合子发育到桑葚胚或囊胚阶段，许多甚至孵化，形态异常很少。在分析的2型合子中，88%观察到正常核型。这些观察结果表明，在GV和原核阶段经过顺序核转移的小鼠卵母细胞的细胞核仍然能够进行减数分裂成熟、正常激活并支持胚胎发育到孵化囊胚阶段。相比之下，这些操作似乎损害了此类卵母细胞细胞质的发育潜力。
