In Vitro Development of Reconstructed Porcine Oocytes after Somatic Cell Nuclear Transfer1

Abstract

This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 +/- 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 +/- 2.3%) (P: < 0.05), but the mean cell numbers of blastocysts were similar between the two groups. Rates of blastocyst development from NT embryos electrically pulsed at different times (2, 4, and 6 h) after electrofusion were 11.6 +/- 2.9, 6.6 +/- 2.3, and 8.1 +/- 3.3%, respectively. The mean cell numbers of blastocysts developed from NT embryos were gradually decreased (30.4 +/- 10.4 > 24.6 +/- 10.1 > 16.5 +/- 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P: < 0. 05). Nuclear-transferred embryos (9.4 +/- 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 +/- 1.9%) or parthenogenetic embryos (22.4 +/- 7.2%) (P: < 0.01). The mean cell number (28.9 +/- 11.4) of NT-derived blastocysts was smaller than that (38.6 +/- 10.4) of IVF-derived blastocysts (P: < 0. 05) and was similar to that (29.9 +/- 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo

“…To date, the developmental competence of NT embryos from nuclei of somatic cells is still lowFmany factors involved in the NT procedure could result in the low developmental competence of NT embryos (Koo et al, 2000). First, the lower developmental potential of NT embryos may come from the inappropriate cell cycle of donor nuclei, as they could have received cells which were not in the G 0 or G 1 phase of the cell cycle (Sun et al,'92;Cibelli et al,'98;Kato et al,'98;Wells et al,'99) and, as a consequence, had inadequate nuclear reprogramming from nonquiescent cells.…”

Gradual development of a genome‐wide H3‐K9 trimethylation pattern in paternally derived pig pronucleus

“…The in vitro fertilization and cultivation of matured pig oocyte was performed as reported previously (Koo et al, 2000). Especially, polyspermy was induced by using spermatozoa up to a concentration of 1 ϫ 10 6 sperm/ml.…”

DNA methylation state is preserved in the sperm-derived pronucleus of the pig zygote

“…The in vitro fertilization of matured pig oocyte was performed as reported previously (Koo et al, 2000, Koo et al, 2001. Especially, polyspermy was induced by using spermatozoa up to a concentration of 1 x 10 6 sperm/ml.…”

“…For treatment of fertilized pig oocytes with 5-aza-2'-deoxycytidine (5-azadC; Sigma), pig oocytes-spermatozoa complexes were transferred to IVF medium (Koo et al, 2000) containing either five-, 50-or 500-µM 5-azadC three hours after the commencement of in vitro fertilization, and cultured for 3 hours. The complexes were then transferred to 5-azadCcontaining IVC medium (Koo et al, 2000) and further cultured for 18 -20 hours.…”

“…The complexes were then transferred to 5-azadCcontaining IVC medium (Koo et al, 2000) and further cultured for 18 -20 hours. The fertilized embryos were fixed for immunostaining in 4% formaldehyde solution (Sigma Aldrich) for 30 minutes at room temperature.…”