Bovine blastocyst development from ocytes injected with freeze-dried spermatozoa
Bovine blastocyst development from ocytes injected with freeze-dried spermatozoa
复制标题
DOI：
10.1095/biolreprod67.2.409
复制
发表时间：
2002-08-01
影响因子：
3.6
通讯作者：
Brackett, BG
中科院分区：
文献类型：
--
作者：
Keskintepe, L;Pacholczyk, G;Brackett, BG
用冷冻干燥精子进行卵胞浆内单精子注射，对牛受精卵的原核形成、染色体组成和发育能力进行了评价。选择冻融精子，冷冻干燥，并在4 ℃下保存备用。体外成熟22-24小时后，剥除卵母细胞，并单独注射冻干精子。注射的卵母细胞通过单独用10 μ M离子霉素处理（5分钟）和与1.9mM6-二甲基氨基嘌呤（DMAP）组合处理4小时来活化。离子霉素+DMAP激活组精子注射的卵母细胞中有两个原核的比例显著高于离子霉素单独激活组（74% vs.56%; P < 0.03）。注射精子的卵母细胞经离子霉素+DMAP激活后的卵裂率、桑椹胚发育率和囊胚发育率分别为63.3%、34.2%和29.6%，高于单用离子霉素激活后的44.7%、18.7%和10.6%（P < 0.05）。用冷冻干燥的精子生产的囊胚有73%是二倍体。这些结果表明，在体外成熟的牛卵母细胞可以与冷冻干燥的精子细胞受精，并得到的受精卵可以发育成核型正常的囊胚。
Pronuclear formation, and the chromosomal constitution and developmental capacity of bovine zygotes formed by intracytoplasmic sperm injection with freeze-dried (lyophilized) spermatozoa were evaluated. Frozen-thawed spermatozoa were selected, freeze-dried, and stored at 4degreesC until use. After 22-24 h of in vitro maturation oocytes were denuded and injected singly with a lyophilized spermatozoon. Injected oocytes were activated by treatment with 10 muM ionomycin (5 min) alone and in combination with 1.9 mM 6-dimethylaminopurine (DMAP) for 4 h. lonomycin plus DMAP activation treatment resulted in a significantly higher proportion of sperm-injected oocytes with two pronuclei than was found after activation with ionomycin alone (74% vs. 56%; P < 0.03). The rates of cleavage, morula, and blastocyst development of sperm-injected oocytes treated with ionomycin plus DMAP were higher than after activation with ionomycin alone (63.3%, 34.2%, and 29.6% vs. 44.7%, 18.7%, and 10.6%, respectively; P < 0.05). Seventy-three percent of blastocysts produced with lyophilized sperm were diploid. These results demonstrate that in vitro-matured bovine oocytes can be fertilized with freeze-dried sperm cells, and that resultant zygotes can develop into karyotypically normal blastocysts.