35 RELATIONSHIP BETWEEN THE ONSET OF ENUCLEATION DURING MEIOTIC MATURATION OF RECIPIENT OOCYTES AND DEVELOPMENTAL ABILITY OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN PIGS

Abstract

In somatic cell nuclear transfer (SCNT), maturation promoting factor (MPF) is believed to be one of the factors involved with nuclear envelope breakdown and chromatin condensation of the transferred nucleus. Although MPF activity is high both in metaphase-I or -II oocytes (M-I and M-II, respectively), only M-II oocytes have been used exclusively as recipient cytoplasts in SCNT. In this study, we examined the effect of different onset of (1) enucleation of recipient oocytes at the M-I and M-II stages, and (2) fusion and activation of the couplets on their developmental ability to the blastocyst stage in pigs. The primary cultured cumulus cells were used as donor karyoplasts, and recipient cytoplasts were prepared by enucleation of in vitro-matured oocytes using gradient centrifugation in percoll solution. A karyoplast and a cytoplast were fused by 2 DC pulses of 1.5 kV cm^-1 for 20 µs, and then the couplets were activated by 2 DC pulses of 0.8 kV cm^-1 for 30 µs. The reconstructed embryos were cultured according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) except for the addition of 5% FCS to NCSU-37 during Days 2–7 (Day 0 is the day of SCNT) of embryo culture using the WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 258–264). Some of the embryos were fixed at 1, 10, and 24 h after activation and examined for morphology of nuclei. After 30 h of IVM, oocytes (mainly at the M-I stage) were enucleated. Then the couplets were fused immediately (Group A) or at 48 h after the onset of IVM (Group B); activation was conducted at 48 h of IVM (Group A) or at 1 h after fusion (Group B). As a control group, oocytes were enucleated after 48 h of IVM and then the couplets were fused and activated. None of the embryos in Group B developed to the blastocyst stage. However, a few of the embryos [2/117 (1.7%)] in Group A developed to the blastocyst stage; however, the rate was significantly lower than that of the control group [10/112 (8.9%); chi-square; P = 0.03]. The rates of embryos undergoing premature chromosome condensation (PCC) in Group B at 1 h and 10 h after activation were significantly lower than those in Group A [1 h: 51/69 (73.9%) vs. 76/76 (100%); 10 h: 24/76 (31.6%) vs. 45/91 (49.5%), respectively); some of them had pseudo-pronuclei. By 24 h after activation there were no detectable differences in the rates of cleavage [2/70 (2.9%) vs. 2/61 (3.3%)]; however, the rates were significantly lower than that of the control group [23/90 (25.6%); chi-square; P < 0.05]. These results suggest that MPF activity might be changed in oocytes without nucleus during the maturation culture. Thus, a specific nucleus-associated factor(s) that may present in the cytoplasm seems to be essential for the successful remodeling of the transferred nucleus and the development of SCNT embryos to the blastocyst stage.