Differences in DNA methylation and mRNA levels between the breeding and non-breeding seasons of in vitro produced IVF and SCNT sheep embryos

Abstract

In this study, Small Tail Han sheep embryos were produced in the breeding and non-breeding seasons by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). Oocyte recovery and maturation rate, embryo development and blastocyst cell number were evaluated in a series of four experiments using either a 2 treatment (breeding vs. non-breeding season)×5 replicates or a 2 seasons×2 embryo types (IVF vs. SCNT)×5 replicates design. In addition, methylation levels of pluripotent genes (OCT4, SOX2 and LIN28) and imprinted genes (IGF2, IGF2R and GNAS) in matured oocytes and blastocysts, and the expression of these genes in blastocysts were assessed. The number (mean±SEM) of oocytes recovered per ovary was not significantly different between breeding seasons (3.61±0.22) and non-breeding season (3.33±0.43, P>0.05); but the percentage of acceptable oocytes and oocyte maturation rate in breeding season (56.4±5.4%, 66.4±8.7%) were significantly higher than those in non-breeding season (44.2±4.3%, 41.8±10.5%, P<0.05), and the blastocyst formation rates and the cell numbers of blastocyst were significantly higher in breeding season than those in non-breeding season (P<0.05). Methylation levels of the genes studied were not significantly different between seasons in matured oocytes. SCNT produced blastocysts had significantly higher (P<0.05 to P<0.01) methylation levels for OCT4, SOX2 and IGF2, and significantly lower (P<0.01) methylation levels for GNAS and IGF2R than IVF embryos. Methylation levels for LIN28 did not differ between embryo types. Blastocysts produced in the breeding season had significantly lower methylation levels for OCT4 (P<0.05) and IGF2 (P<0.01), and significantly higher levels for IGF2R (P<0.01). Differences in methylation levels between embryo types were not significant for SOX2, LIN28 and GNAS. IVF blastocysts had a significantly higher (P<0.05 to P<0.01) expression levels for OCT4, SOX2 and LIN28 genes, and a significantly lower expression level for the IGF2R gene (P<0.01) than SCNT blastocysts. Blastocysts produced in the breeding season had significantly higher (P<0.01) expression levels for OCT4 and SOX2 genes and a significantly lower (P<0.05) transcript level for the IGF2R gene than in the non-breeding season. No expression of IGF2R or GNAS genes was detected in IVF produced blastocysts and an extremely weak signal was observed in SCNT blastocysts. It is concluded that the season in which oocytes are collected from Small Tail Han sheep significantly affects the number recovered, their quality, maturation rate and development to blastocysts. Season also significantly affects the methylation and transcript levels of some important genes in embryos produced from these oocytes. These effects may be associated with the subsequent developmental potential of the embryos.