Liu Ji-Long, Sung Li-Ying, Barber Michele, Yang Xiangzhong
Department of Animal Science, University of Connecticut, 1390 Storrs Road, Storrs, Connecticut 06269, USA.
Biol Reprod. 2002 May;66(5):1342-9. doi: 10.1095/biolreprod66.5.1342.
Oocytes enucleated at the second metaphase stage (MII) are often used as recipient cytoplasts for nuclear transfer. The oocyte's nuclear material has been traditionally removed blindly by aspirating the first polar body (Pb1) along with a portion of the cytoplasm. However, the Pb1-guided enucleation method is unreliable because the position of the Pb1 is variable. A previous study showed that pretreatment of mouse oocytes with 3% (0.09 M) sucrose allowed visualization of the metaphase spindle and chromosomes under standard light microscopy and led to a 100% enucleation rate. The same sucrose treatment, however, did not produce the same effect in bovine oocytes. In this study, we increased the concentration of sucrose to 0.3-0.9 M in PBS containing 20% fetal bovine serum (SPF) and found that the majority of the treated bovine oocytes (75%-86%) formed a small transparent bud into the perivitelline space, as compared with the 0.1 M sucrose (6%) or the no sucrose (0%) control groups. Staining of DNA with Hoechst 33342 revealed that these projections coincided with the position of the metaphase chromosomes in 100% of sucrose-treated oocytes, whereas only 31% of oocytes showed alignment of the position of Pb1 with their nuclear materials. Furthermore, 95% of oocytes treated in 0.3 M SPF were successfully enucleated by removing a small amount of cytoplasm adjacent to the projection. This is a significantly higher enucleation rate than that obtained by conventional Pb1-guided enucleation, even when a larger amount of cytoplasm was removed. For nuclear transfer, the enucleated oocytes treated with sucrose did not differ from the control oocytes in rates of fusion, cleavage, or development to blastocysts, or in the average cell numbers in blastocysts. This study demonstrated that 0.3 M sucrose treatment of bovine oocytes facilitates the localization of metaphase chromosomes under normal light microscopy and hence increases enucleation efficiency without compromising the in vitro development potential of cloned embryos by nuclear transfer.
处于第二次减数分裂中期（MII）去核的卵母细胞常被用作核移植的受体细胞质。传统上，卵母细胞的核物质是通过吸出第一极体（Pb1）及一部分细胞质盲目去除的。然而，Pb1引导的去核方法并不可靠，因为Pb1的位置是可变的。先前的一项研究表明，用3%（0.09 M）蔗糖预处理小鼠卵母细胞可使中期纺锤体和染色体在标准光学显微镜下可见，并使去核率达到100%。然而，相同的蔗糖处理在牛卵母细胞中并未产生相同的效果。在本研究中，我们将蔗糖浓度提高至含20%胎牛血清（FBS）的PBS中0.3 - 0.9 M，发现与0.1 M蔗糖（6%）或无蔗糖（0%）对照组相比，大多数经处理的牛卵母细胞（75% - 86%）在卵周隙形成一个小的透明芽。用Hoechst 33342对DNA进行染色显示，在100%经蔗糖处理的卵母细胞中，这些突起与中期染色体的位置一致，而只有31%的卵母细胞显示Pb1位置与其核物质对齐。此外，通过去除与突起相邻的少量细胞质，95%用0.3 M FBS处理的卵母细胞成功去核。这一比传统Pb1引导去核获得的去核率显著更高，即使去除了更多的细胞质。对于核移植，经蔗糖处理的去核卵母细胞在融合率、分裂率或发育至囊胚的比率，或囊胚中的平均细胞数方面与对照卵母细胞没有差异。本研究表明，用0.3 M蔗糖处理牛卵母细胞有助于在普通光学显微镜下定位中期染色体，从而提高去核效率，而不会损害通过核移植克隆胚胎的体外发育潜力。
