Nuclear Replacement of In Vitro-Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method
Nuclear Replacement of In Vitro-Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method
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DOI：
10.1111/j.1439-0531.2008.01324.x
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发表时间：
2010-08-01
影响因子：
1.7
通讯作者：
Kashiwazaki, N.
中科院分区：
文献类型：
--
作者：
Maedomari, N.;Kikuchi, K.;Kashiwazaki, N.
本研究的目的是建立一种猪中期II期卵母细胞核置换的方法。用连续离心法从体外成熟的卵母细胞中分离出含有M-II染色体的核质体（K）和不含染色体的胞质体（C）。然后通过1个核质与1、2、3或4个胞质（分别为K + 1C、K + 2C、K + 3C和K + 4C）融合来重构卵母细胞。将重建的卵母细胞、没有任何胞质融合的核质体（K）和无透明带的M-II卵母细胞（对照）用于实验。孤雌激活后，各组重构卵母细胞的雌性原核形成率（58.2-77.4%）与K组和对照组（分别为58.2%和66.0%）没有差异。采用体外受精技术检测重组卵母细胞的受精能力和胚胎发育情况。胞质：核质比例不影响卵母细胞的受精状态（穿透率和雄性原核形成率）。K卵母细胞的单精受精率（p < 0.05，61.6%）明显高于其他组（18.2-32.8%）。囊胚形成率随与核质体融合的细胞质数量的增加而显著增加（p < 0.05，0.0-15.3%）。K + 4C组的囊胚率（15.3%）与对照组（17.8%）相当。K + 3C和K + 4C组的总细胞数（分别为16.0和15.3个细胞）与对照组（26.2个细胞）相当。本研究结果表明，离心-融合法是一种有效的获得具有正常受精能力和体外发育能力的M-Ⅱ染色体转移卵母细胞的方法。这表明，与核质体融合的细胞质的数量在胚胎发育中起着至关重要的作用。
The objective of the present study was to establish a method for nuclear replacement in metaphase-II (M-II) stage porcine oocytes. Karyoplasts containing M-II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro-matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona-free M-II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2-77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2-32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0-15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri-Fusion) is an effective method for producing M-II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.