126 DEVELOPMENT OF SINGLE BLASTOMERES DERIVED FROM PORCINE TWO-CELL EMBRYOS

Abstract

Monozygotic mice and calves have been produced from demi-2-cell embryos (Wang K et al. 1997 J. Reprod. Dev. 43, 91-95; Tagawa M et al. 2008 Theriogenology 69, 574-82). These studies proved that single blastomeres of 2-cell embryos could still develop properly after being separated. However, such studies are very difficult to perform in pigs due to multiple pregnancies and relatively low success of embryo transfer. The production of genetically identical animals is very useful for animal husbandry, particularly to increase the number of progeny derived from genetically valuable parents. In the present study, we compared the developmental ability to the blastocysts and their quality in terms of total cell number and gene expression of blastomere pairs derived from 2-cell embryos (pair of blastomere for short). Evenly cleaved 2-cell embryos were collected during 24 to 30 h after IVF and IVM of follicular oocytes. They were split into pairs of single blastomeres by gentle pipetting after Pronase treatment for removal of zona pellucida. Blastomeres were then cultured separately in polydimethylsiloxane (PDMS) micro-wells. Embryos were cultured in IVC-PyrLac from Days 0 to 2 (Day 0 was defined as the day of IVF) and in IVC-Glu for next 4 days (Kikuchi K et al. 2002 Biol. Reprod. 66, 1033-1041). At Day 6, single blastocysts were collected for total cell number or gene expression analysis. Eight replications were performed for each analysis. Three genes chosen for real-time PCR were high-mobility group box 1 (HMGB1); ATP synthase, H1 transporting, mitochondrial F1 (ATP5A1); and the small nuclear ribonuclear proteins (LSM2). These genes are considered as markers to identify embryos with high development competence (Withworth KM et al. 2005 Biol. Reprod. 72, 1437-1451). The expression levels were normalized to the housekeeping gene Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAG) using standard curve method. Assessment of 42 blastomere pairs at Day 6 showed that the percentages of the pairs developed to blastocysts (36.6 ± 5.3%) or degenerated (46.3 ± 10.3%) were significantly higher than those of the pairs in which one developed to blastocyst and the other degenerated (17.1 ± 7.8%, P < 0.05, 1-way ANOVA followed by Tukey test). Assessment of two blastocysts derived from one pair of blastomeres showed that one blastocyst (B#1) had better morphology than the other (B#2) in term of expansion and B#1s had significantly higher cell number (31.6 ± 2.9) than that of B#2s (19.1 ± 1.9; P < 0.05; 1-way ANOVA). Although the expression levels of ATP5A1, HMBG1, and LSM2 in B#1s (1.08 ± 0.37, 3.51 ± 1.01, and 2.63 ± 1.17, respectively) did not differ from those in B#2s (0.39 ± 0.13, 1.18 ± 0.38, and 1.68 ± 0.83, respectively), they tended to be higher in B#1s compared to B#2s (P < 0.1). In conclusion, blastomere pairs from the same origin had the same potential to develop to the blastocyst stage. However, the qualities of blastocysts in pairs were different.