Activation treatment is one of the important factors that affect the development of somatic cell nuclear transfer (SCNT) embryos. We examined the effect of post-activation (PA) treatment on the change in donor nucleus and SCNT embryo development in pig. Cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and in fresh hormone-free medium for 18 h. After 40 h of IVM, oocytes with a polar body were enucleated, injected with a donor cell (ear skin fibroblasts bearing the human decay accelerating factor gene), electrically fused, and activated 1 h after fusion. Then, SCNT embryos were cultured in a modified NCSU-23 medium (Park et al. 2005 Zygote 13, 269–275) containing no additives (control), 5 �g mL-1 cytochalasin B (CB), 0.4 �g mL-1 demecolcine (D), or CB+D for 4 h. CB and D were prepared from stock solutions of 5 mg mL-1 CB in DMSO and 10 �g mL-1 D in Hank's balanced salt solution (HBSS), respectively. After PA treatment, SCNT embryos were cultured in a modified NCSU-23 medium for 6 days. The embryos (n = 188, 189, 187, and 186 for control, CB, D, and CB+D, respectively) were examined for cleavage and blastocyst (BL) formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). Cell number of BL was examined by counting the number of nuclei stained with Hoechst 33342 under fluorescence. To assess the nuclear structure, some of the fused oocytes were fixed at 12 h after PA and stained with aceto-orcein (n = 42, 44, 43, and 45 for control, CB, D, and CB+D, respectively). Nuclear state was classified as 1 pseudopronucleus (PPN), multi-PPN, and others. Data were analyzed by ANOVA (GLM procedure) in SAS (SAS Institute, Inc., Cary, NC, USA). PA treatment with D and CB+D significantly (P < 0.05) increased 1 PPN formation (84 and 80%, respectively) compared to control and CB (62 and 64%, respectively). Conversely, a higher (P < 0.001) rate of multi-PPN was observed in control and CB (31 and 36%, respectively) than in D and CB+D (9 and 7%, respectively). This result was in contrast with the finding in mouse that nocodazole, another microtubule depolymerizing agent, induced multi-PPN in reconstructed zygotes. Pig meiotic spindles differ at their poles from those in mice by lacking γ-tubulin. Absence of γ-tubulin in pig oocytes would make spindle dynamics more sensitive to depolymerization, which might lead to a different result in this study. Embryo cleavage (77–85%) was not altered by PA treatments, but BL formation was significantly (P < 0.05) increased by CB, D, or CB+D (26, 28, and 28%, respectively) compared to control (16%). Total cell number of BL (36–40 cells/BL) was not different among groups. These results indicate that PA treatment with CB and/or D improved in vitro development of SCNT pig embryos and that D treatment effectively prevented the formation of multi-PPN.
This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.
The influence of donor nucleus source on the outcome of zebrafish somatic cell nuclear transfer
