Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes.
Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes.
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DOI：
10.1095/biolreprod59.4.918
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发表时间：
1998-10
影响因子：
3.6
通讯作者：
G. Rho;S. Kawarsky;W. Johnson;Kanwal Kochhar;K. Betteridge
中科院分区：
文献类型：
--
作者：
G. Rho;S. Kawarsky;W. Johnson;Kanwal Kochhar;K. Betteridge
本研究评估了卵母细胞胞浆内注射精子形成的牛受精卵的原核形成、染色体构成和发育能力，无论是否用二硫苏糖醇（DTT）处理。卵母细胞体外成熟22-24 h后离心，在5 mM DTT存在或不存在的情况下，通过游动制备的精子可注射到卵浆的清除区。注射的卵母细胞分别用5 μ m离子霉素（5min）和1.9 μ m 6-二甲氨基嘌呤（DMAP）活化3 h，然后在M199中与牛输卵管上皮细胞共培养。精子处理导致注射后16小时雄性原核形成比例显著提高（40%比11%，p < 0.0001），囊胚发育率显著提高（24%比10%，p < 0.005）。经处理的精子产生的囊胚有61%是二倍体。在用处理过的精子产生的12个囊胚中，通过聚合酶链反应对其进行了性别鉴定，其中4个为男性，7个为女性，其中1个不能做出明确的诊断。胚胎移植（每头小母牛2个胚胎）在第49天导致16个受者中有6个怀孕，但没有一个足月。上述结果表明，DTT预处理精子和离子霉素+ DMAP激活卵母细胞可以提高牛胞浆内单精子注射效率，但胚胎发育能力仍然有限。
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.