Rho G J, Kawarsky S, Johnson W H, Kochhar K, Betteridge K J
Departments of Biomedical Sciences, Ontario Veterinary College, Iniversity of Guelph, Ontario, Canada.
Biol Reprod. 1998 Oct;59(4):918-24. doi: 10.1095/biolreprod59.4.918.
This study assessed pronuclear formation, the chromosomal constitution, and the developmental capacity of bovine zygotes formed by intracytoplasmic injection of oocytes with sperm, treated or not with dithiothreitol (DTT). Oocytes were matured in vitro for 22-24 h and then centrifuged so that sperm, prepared by swim-up in the presence or absence of 5 mM DTT, could be injected into the cleared area of the ooplasm. Injected oocytes were activated by treatment with 5 microM ionomycin (5 min) and, after a 3-h interval, with 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h. They were then cocultured with bovine oviductal epithelial cells in M199. Sperm treatment resulted in a significantly higher proportion of male pronucleus formation 16 h after injection (40% vs. 11%; p < 0.0001) and a significantly higher rate of blastocyst development (24% vs. 10%; p < 0.005). Sixty-one percent of blastocysts produced with treated sperm were diploid. Of 12 blastocysts produced with treated sperm and sexed by a polymerase chain reaction, 4 were male and 7 female, and in one a definite diagnosis could not be made. Embryo transfer (2 embryos per heifer) resulted in pregnancies in 6 of 16 recipients at Day 49, but none was carried to term. These results show that the efficiency of bovine intracytoplasmic sperm injection can be improved by sperm pretreatment with DTT and by oocyte activation with ionomycin plus DMAP, although the developmental capacity of the resulting embryos remains limited.
本研究评估了通过将精子注入经或未经二硫苏糖醇（DTT）处理的卵母细胞胞质内而形成的牛受精卵的原核形成、染色体组成及发育能力。卵母细胞在体外成熟22 - 24小时，然后离心，以便将在有或无5 mM DTT存在的情况下通过上浮法制备的精子注入卵质的清亮区域。注入后的卵母细胞先用5 microM离子霉素处理5分钟进行激活，间隔3小时后，再用1.9 mM 6 - 二甲基氨基嘌呤（DMAP）处理3小时。然后将它们与牛输卵管上皮细胞在M199中共同培养。精子处理导致注射后16小时雄性原核形成的比例显著更高（40%对11%；p < 0.0001），囊胚发育率也显著更高（24%对10%；p < 0.005）。用经处理的精子产生的囊胚中有61%是二倍体。在12个用经处理的精子产生并通过聚合酶链反应进行性别鉴定的囊胚中，4个为雄性，7个为雌性，还有1个无法做出明确诊断。胚胎移植（每头小母牛移植2个胚胎）在第49天导致16头受体中有6头怀孕，但无一足月分娩。这些结果表明，虽然所得胚胎的发育能力仍然有限，但通过用DTT对精子进行预处理以及用离子霉素加DMAP对卵母细胞进行激活，可以提高牛胞质内精子注射的效率。
