Treatment of buffalo (Bubalus bubalis) SCNT embryos with microRNA-21 mimic improves their quality and alters gene expression but does not affect their developmental competence

Abstract

Application of cloning technology on a wide scale is severely limited by the very low live birth rate obtained with cloned embryos. Embryo quality is an important factor which affects the conception and live birth rate of cloned embryos. microRNA-21 (miR-21) has been implicated in the regulation of apoptosis and the expression level of several important genes which control apoptosis. We examined the effects of treatment of reconstructed buffalo embryos, produced by Hand-made cloning, with miR-21 mimic on developmental competence, quality and gene expression of cloned embryos. Expression level of miR-21, which increased from 2-cell to 8-cell stage and then decreased at the blastocyst stage, showed a similar pattern in cloned and IVF embryos. It was lower in cloned than in IVF embryos at 2-, 4- and 8-cell (P < 0.001) and blastocyst (P < 0.05) stages but not at morula stage. Treatment of reconstructed embryos with miR-21 mimic for 1 h after 1 h of electrofusion, increased (P < 0.05) the total cell number (251.3 ± 10.7 vs 181.5 ± 2.13). Blastocysts produced from miR-21-treated reconstructed embryos had lower (P < 0.05) apoptotic index than controls and IVF blastocysts (2.01 ± 0.17, 5.46 ± 0.26 and 4.19 ± 0.15, respectively). The treatment also improved the inner cell mass:trophectoderm cell number ratio of blastocysts than in controls (0.21 ± 0.01 vs 0.11 ± 0.003) to values observed in IVF blastocysts (0.20 ± 0.008). However, miR-21 mimic treatment did not affect the blastocyst rate, which was similar for treatment, control and negative control groups (36.58 ± 3.64, 36.58 ± 3.64 and 32.2 ± 2.86%, respectively). miR-21 mimic treatment increased (P < 0.01) the expression level of apoptosis- (BCL2 and PTEN), pluripotency- (OCT4 and SOX2) and development-related genes (GLUT1, FGF4 and P53), but not that of CASPASE3 than in untreated controls in blastocysts. These results suggest that treatment of reconstructed embryos with miR-21 mimic improves blastocyst quality, reduces apoptosis and alters gene expression without improving the blastocyst rate.