摘要
颗粒细胞(CCs)对卵母细胞减数分裂成熟、受精及胚胎发育能力方面的作用在牛、羊、猪、小鼠等动物上已有报道,但是在兔上研究较少。目前仅有关于对兔卵丘细胞卵母细胞复合体(COCs)成熟培养的报道,尚未见颗粒细胞与卵母细胞相互作用的报道。兔是重要的实验动物,其卵母细胞是胚胎工程技术操作的重要材料。本论文以新西兰兔为实验动物研究了颗粒细胞对卵母细胞核成熟、胞质成熟以及卵母细胞存活和退化等方面的影响作用。采集兔 COCs 和裸卵(NOs)。将 COCs 脱去部分或全部卵丘细胞,形成放射冠包裹的卵母细胞(COs)或机械裸卵(DOs)后,分五种方式培养,即卵丘细胞包裹的卵母细胞(CEOs)的单独培养、COs 单独培养、DOs 与 COCs 共培养(DOs(COCs))、DOs与卵丘颗粒细胞共培养的(DOs (CCs)),以及 DOs 单独培养,培养 24 h 或 30 h 后,统计核成熟率。对核成熟的卵母细胞孤雌激活后用卵裂率判断卵母细胞成熟效果。得到如下结果:(1) 培养 24 h,上述五种培养方式的卵母细胞核成熟率分别为 76%、46%、13%、7%和 6%,前四种都有显著差异(P<0.05)。孤雌激活后卵裂率分别为 68%、63%、35%、21%和 17%,前两种培养方式均高于后三种培养方式。 (2) 培养 30 h, 上述五种培养方式核成熟率分别为 82%、62%、24%、16%和 7%,都有显著差异(P<0.05)。孤雌激活后的卵裂率分别为 79%、76%、61%、45%和 28%,前两种培养方式显著高于后三种培养方式(P<0.05)。(3) 培养时间从 24 h 延长至 30 h,DOs(COCs)的核成熟率显著提高(13% vs 24%,P<0.05),孤雌激活后的卵裂率也显著提高(35% vs 61%,P<0.05)。DOs(CCs)的核成熟率显著提高(7% vs 16%,P<0.05),但胞质成熟率无显著差异(P>0.05)。(4) NOs 和 NOs (CCs)成熟 24 h,核成熟率都为 2%,培养至 30 h,NOs 和 NOs (CCs)核成熟率分别为 2%和 3%,无显著差异(P>0.05)。(5) 培养 24 h,CEOs 、COs、DOs(COCs)、DOs (CCs)和 DOs 退化率分别为 7%、23%、44%、64%和 73%,相互间差异均显著(P<0.05);培养至 30 h,退化率分别为 12%、20%、41%、60%和 77%,相互间均差异显著(P<0.05)。但培养 24 h 与培养 30 h,相同培养方式下卵母细胞退化率均无显著差异(P>0.05)。以上结果表明,兔卵丘颗粒细胞直接影响卵母细胞核和细胞质的成熟,以及卵母细胞的存活;兔卵母细胞体外成熟时间从 24 h 延长到 30 h,有利于胞质成熟;裸卵不具有恢复减数分裂成熟的能力,也不能通过与颗粒细胞悬液共培养得到改善。
It has been reported that cumulus cells (CCs) play an important role in oocyte meioticmaturation, fertilization and embryo developmental potential in cattle, goat, pig and mouse,but few studies were conducted in rabbit. Previously, the reports in rabbit mainly focused onthe in vitro maturation of the cumulus-enclosed oocytes (CEOs), but the interactions betweenthe cumulus cells and oocyte haven’t been investigated. As an important experimental animal,rabbit are often used in embryo engineering. The aim of the present study was to analyze theeffects of rabbit cumulus cells on the oocyte nuclear and ooplasmic maturation, oocytesurvival and degeneration. Cumulus oocyte complexes (COCs) and naked oocytes (NOs)were recovered directly from rabbit ovaries. Corona-enclosed oocytes (COs) and DOs(denuded oocytes) were obtained from COCs freed of part or whole CCs, and then the oocyteswere cultured in the following five ways. (1) CEOs were cultured alone; (2) COs werecultured alone; (3) DOs were co-cultured with COCs (DOs(COCs)); (4) DOs wereco-cultured with CCs (DOs(CCs)); (5) DOs were cultured alone. After the oocytes were invitro cultured 24 h or 30 h, the oocyte nuclear maturation was analyzed and compared. Thecleavage was used to evaluate the oocyte maturation quality after the nuclear matured oocyteswere parthenogenetically activated. The results were as the following. (1) The percentage ofnuclear maturation of CEOs, COs, DOs(COCs), DOs(CCs) and DOs was 76%, 46%, 13%,7% and 6% respectively after 24 h incubation, and the percentage of oocyte nuclearmaturation in the former four culture ways were all significantly different with each other(P<0.05). The Cleavage rate was 68%, 63%, 35%, 21% and 17% respectively afterpathenogentic activation, and the cleavage rate in the first two culture ways were significantlyhigher than that of the others (P<0.05). (2) The percentage of oocyte nuclear maturation were82%, 62%, 24%, 16% and 7% respectively after incubation 30 h and all of them hadsignificant difference with each other (P<0.05). The cleavage rate was 79%, 76%, 61%, 45%,and 28% respectively and the first two culture ways had significantly higher ooplasmicmaturaion rate than the others (P<0.05). (3) The nuclear maturation was significantlyimproved when the culture time of DOs(COCs) was prolonged from 24 h to 30 h (13% vs24%, P<0.05) and the cleavage was also improved significantly (35% vs 61%, P<0.05).DOs(CCs) nuclear maturation was significantly improved when the culture time wasprolonged from 24 h to 30 h (7% vs 16%, P<0.05), but the ooplasmic maturation was notimproved (P>0.05). (4) The percentage of nuclear maturation of NOs incubated with orwithout cumulus cells was both 2% after 24 h incubation (P>0.05). When the culture time was
prolonged from 24 h to 30 h, the nuclear maturation was not improved (P>0.05). (5) Theoocyte degeneration rate of COCs, COs, DOs(COCs), DOs (CCs) ) and DOs wassignificantly different with each others after 24 h incubation (7%, 23%, 44%, 64%, 73%,P<0.05) and after 30 h incubation (12%, 20%, 41%, 60%, 77%, P<0.05). There was nosignificant difference in oocyte degeneration in the same groups between 24 h and 30 hincubation (P>0.05). The results suggest that rabbit cumulus cells affect the oocyte nuclearand ooplasmic maturation, survival, and the prolongation of the culture time of rabbit oocytefrom 24 h to 30 h improves the ooplasmic maturation. Rabbit NOs have no meiotic potential,which cannot be improved by co-culture with dispersed cumulus cells.
引文
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