Effects of Donor Fibroblasts Expressing OCT4 on Bovine Embryos Generated by Somatic Cell Nuclear Transfer

Abstract

The production of healthy, live, cloned animals by somatic cell nuclear transfer (SCNT) has been hampered by low efficiencies. Significant epigenetic changes must take place to ensure proper chromatin remodeling in SCNT. We hypothesized that exogenous expression of OCT4 in donor fibroblasts prior to its fusion with enucleated oocytes would facilitate SCNT reprogramming. We infected bovine adult fibroblasts with retroviral vectors containing yellow fluorescent protein (YFP) only, or the OCT4 gene fused to YFP (YO). We found that development to the blastocyst stage was not different between NT-YFP and NT-YO groups. NT-YFP embryos had the fewest trophoblast cells, measured by numbers of CDX2-positive cells. Fibroblasts expressing OCT4 had reduced levels of histone 3 lysine 9 or 27 trimethylation (H3K9me3 and H3K27me3, respectively). NT-YO blastocysts displayed higher H3K9me3 levels than IVF and NT-YFP embryos; however, they did not have different H3K27me3 levels. Levels of XIST mRNA expression in NT-YO and NT-YF were higher when compared to in vitro-fertilized blastocysts. We observed no differences in the expression of SOX2, NANOG, and CDX2. Although overexpression of OCT4 in donor cells increased H3K9me3 and did not reduce XIST gene expression, we show that a single transcription factor can affect the number of trophectoderm cells in bovine SCNT embryos.

Exogenous expression of OCT4 facilitates oocyte‐mediated reprogramming in cloned porcine embryos

“…For example, the implantation of blastocysts and maintenance of pregnancy require the silencing of OCT4 in the trophectoderm (Liu et al, ), which is prerequisite for the expression of CDX2 . Yet, an increase in CDX2 expression in SCNT embryos reconstructed with OCT4 ‐overexpressing donor fibroblasts in bovine was reported (Goissis et al, ), and we observed a significant elevation in CDX2 expression in OCT4 ‐SCNT blastocysts (Fig. F).…”

“…Notably, the overexpression of exogenous OCT4 was transient in both studies, meaning that exogenous OCT4 is only necessary at initial stages of development. On the other hand, Goissis et al () reported no difference in the development rate when fibroblasts expressing OCT4 were used as donor cells in bovine SCNT. Further, ectopic expression of OCT4 can cause dysplasia in epithelial tissues in adult mice (Hochedlinger et al, ), suggesting that constant overexpression of OCT4 could interfere with normal development.…”

“…Cloned embryos are also frequently plagued by high XIST expression levels, which are associated with incomplete reprogramming of the somatic nuclei. For example, increased XIST levels were observed in SCNT embryos in bovine (Goissis et al, ) and mouse (Nolen et al, ; Inoue et al, ), while knocking out XIST significantly improves term development of cloned mouse embryos (Inoue et al, ). We found the XIST levels in OCT4 ‐SCNT porcine blastocysts were below the detection limit (Fig.…”

DNA methylation regulated by ascorbic acids in yak preimplantation embryo helps to improve blastocyst quality

“…The interaction between the regulation of pluripotency genes and DNA methylation is critical for early embryonic development (Goissis, Suhr, & Cibelli, ; Hattori et al, ). Previous studies have found that the pluripotency genes SOX2 and CDX2 are at a low methylation level at all stages of porcine IVF embryo development (Zhao et al, ).…”

“…Immunofluorescence staining analysis of 5mC to 5hmC is crucial for the study of DNA demethylation dynamics during early embryonic development. Previous studies have found that during The interaction between the regulation of pluripotency genes and DNA methylation is critical for early embryonic development (Goissis, Suhr, & Cibelli, 2013;Hattori et al, 2007). Previous studies have found that the pluripotency genes SOX2 and CDX2 are at a low methylation level at all stages of porcine IVF embryo development .…”