Bhak Jong-Sik, Lee Sung-Lim, Ock Sun-A, Mohana Kumar B, Choe Sang-Yong, Rho Gyu-Jin
Department of Obstetrics and Theriogenology, College of Veterinary Medicine, Gyeongsang National University, 900 Gazwa, Chinju 660-701, South Korea.
Anim Reprod Sci. 2006 Mar;92(1-2):37-49. doi: 10.1016/j.anireprosci.2005.04.016. Epub 2005 Jun 24.
Bovine oocyte activation is one of the essential elements that determine the success of nuclear transfer and the subsequent development of cloned embryos. Three methods for oocyte activation, including 5 microM ionomycin (5 min, Group 1) alone, ionomycin+1.9 mM 6-dimethylaminopurine (DMAP, 3h, Group 2), and ionomycin+10 microg/ml cycloheximide (CHX, 3h, Group 3) were compared for the development of embryos produced by somatic nuclear transfer (SCNT) to parthenotes and IVF counterparts. At 19-h post-activation/insemination (hpa/hpi), 27.5% of oocytes in Group 2 cleaved and this rate was greater (P<0.05) than other groups (Group 1, 2.1%; Group 3, 3.0%). None of the oocytes in the IVF control group cleaved at 19-22 hpi. At 24 hpa, the rates of cleavage of oocytes in Group 2 (52.1%) were greater (P<0.05) than those in Groups 1 and 3 (7 and 38.3%, respectively). Only six oocytes (3.3%) in the IVF control group cleaved at 24 hpi. The overall cleavage rates of oocytes in Group 2 (85.5%) at 48 hpa were greater (P<0.05) than other treatments, but it did not show any difference when compared with the IVF control group (75.0%). The development rate to two-cell stage embryos of Group 2 was consistently greater at all observation points followed by Groups 3 and 1. Similar results were obtained in SCNT embryos, but the rates of cleavage at 48 hpi and blastocyst development in Group 2 (68.4 and 16.3%, respectively) did not differ from Group 3 (63.0 and 13.1%, respectively). The chromosomal composition in the parthenotes and SCNT embryos differed (P<0.05) among treatments. In Groups 1 and 3, greater percentages of haploid parthenotes (86 and 71%, respectively) were observed. In contrast, 84% of parthenotes in Group 2 had abnormal ploidy (44% polyploid and 40% mixoploid). In the case of SCNT embryos, Groups 1 and 3 had greater percentages of diploid chromosomal sets (77 and 70%, respectively), whereas 54% in Group 2 were polyploid or mixoploid. These results indicate that DMAP treatment after ionomycin greatly increases the developmental rates of parthenotes, but did not differ in blastocyst development compare with CHX treatment. However, DMAP treatment increased the time-dependent cleavage rate to two-cell stage embryos. Further, it greatly enhanced the incidence of chromosomal abnormalities in parthenotes and SCNT embryos. Hence, it is concluded that CHX combined with ionomycin is more desirable than DMAP for oocyte activation during nuclear transfer in cattle.
牛卵母细胞激活是决定核移植成功及后续克隆胚胎发育的关键因素之一。比较了三种卵母细胞激活方法，包括单独使用5微摩尔离子霉素（5分钟，第1组）、离子霉素+1.9毫摩尔6-二甲基氨基嘌呤（DMAP，3小时，第2组）和离子霉素+10微克/毫升环己酰亚胺（CHX，3小时，第3组），用于比较体细胞核移植（SCNT）产生的胚胎与孤雌生殖胚胎及体外受精（IVF）胚胎的发育情况。激活/授精后19小时（hpa/hpi），第2组27.5%的卵母细胞发生分裂，该比率高于其他组（第1组为2.1%；第3组为3.0%，P<0.05）。IVF对照组的卵母细胞在19 - 22 hpi均未发生分裂。激活后24小时，第2组卵母细胞的分裂率（52.1%）高于第1组和第3组（分别为7%和38.3%，P<0.05）。IVF对照组仅6个卵母细胞（3.3%）在24 hpi发生分裂。激活后48小时，第2组卵母细胞的总体分裂率（85.5%）高于其他处理组（P<0.05），但与IVF对照组（75.0%）相比无差异。第2组双细胞期胚胎的发育率在所有观察点均始终高于第3组和第1组。SCNT胚胎也得到了类似结果，但第2组在48 hpi的分裂率和囊胚发育率（分别为68.4%和16.3%）与第3组（分别为63.0%和13.1%）无差异。孤雌生殖胚胎和SCNT胚胎的染色体组成在不同处理组间存在差异（P<0.05）。在第1组和第3组中，观察到更高比例的单倍体孤雌生殖胚胎（分别为86%和71%）。相比之下，第2组84%的孤雌生殖胚胎具有异常倍性（44%为多倍体，40%为混合倍体）。对于SCNT胚胎，第1组和第3组具有更高比例的二倍体染色体组（分别为77%和70%），而第2组54%为多倍体或混合倍体。这些结果表明，离子霉素后用DMAP处理可大幅提高孤雌生殖胚胎的发育率，但与CHX处理相比，囊胚发育无差异。然而，DMAP处理增加了双细胞期胚胎的时间依赖性分裂率。此外，它极大地提高了孤雌生殖胚胎和SCNT胚胎中染色体异常的发生率。因此，得出结论，在牛的核移植过程中，CHX联合离子霉素比DMAP更适合用于卵母细胞激活。
