Impact of in vitro production techniques on the expression of X‐linked genes in bovine (bos taurus) oocytes and pre‐attachment embryos

Abstract

Our previous studies showed that expression patterns of X-linked genes in cultured cells are different from those of their tissues of origin. This investigation analyses the transcription pattern of the X-linked genes BIRC4, GAB3, MECP2, RPS4X, SLC25A6, and XIST in bovine in vitro matured oocytes and in vitro fertilized embryos, and their in vivo counterparts. In vitro-derived pools of mature oocytes and pre-attachment embryos were obtained by: (a) TCM-199/serum with bovine oviductal epithelial cells as co-culture, and (b) synthetic oviductal fluid/BSA. Pools of in vivo-derived morulae and blastocysts were provided by a commercial embryo transfer operation. Total RNA was extracted for quantification of gene-specific transcript levels using real-time quantitative PCR. Statistical analysis was performed using a mixed model factorial ANOVA with alpha = 0.05. The effect of the in vitro environmental conditions on X-linked gene transcription was most evident during the fourth cell cycle, at the period of activation of the embryonic genome, and seemed to be less pronounced at later developmental stages, with the exception of BIRC4. The levels of X-linked genes transcripts in in vivo-derived embryos were lower relative to their in vitro counterparts for all genes tested. Finally, the pattern of expression of XIST in bovine oocytes and embryos was similar to that reported in humans. These results highlight the possibility that X-linked gene expression analysis is a useful tool to monitor the impact of reproductive biotechnologies on the developmental potential of embryos and aid in their improvement.

Nuclear Profiles of H3 Histones Trimethylated on Lys27 in Bovine (Bos taurus) Embryos Obtained after In Vitro Fertilization or Somatic Cell Nuclear Transfer

“…This latter observation confirms that in cultured bovine embryos, XCI-related events are detected early [60]. Notably, XIST gene expression dramatically increases from at least the 8-16-cell stage onward [60].Following SCNT, epigenetic regulation in the early bovine embryo appears to be flexible. In contrast to mouse SCNT embryos [28], the H3K27me3 epigenetic profile of the somatic donor nucleus is in bovine SCNT embryos largely remodeled.…”

Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning

“…When comparisons were made between male and female IVP embryos, the levels of expression of these X-linked genes were generally similar between the two sexes. However, no consistent trend was observed, rather the levels of gene expression varied among culture conditions and individual developmental stages (Nino-Soto et al 2007). Thus, the cause of the sexual dimorphism in early embryonic development remains unexplained.…”

“…Overall, about 85% of these differentially expressed genes were downregulated in IVP embryos. Studies conducted on a selected subset of X-linked genes in bovine in vivo and IVP embryos revealed differences in the level of expression , Nino-Soto et al 2007). However, it appears that with few exceptions, and in contrast to genes located on autosomes, the mRNA of X-linked genes was upregulated in IVP embryos compared with in vivo controls.…”

HDAC inhibition decreases XIST expression on female IVP bovine blastocysts

“…However, XCI-related mechanisms can be initiated in vitro as early as at the 2-cell stage, when low XIST expression can be detected, even though late replication can only be detected at early blastocyst stage (De La Fuente et al 1999). In addition, when the expression pattern of X-linked genes displayed by in vitro-produced (IVP) embryos is compared with their in vivo counterparts, higher levels of XIST expression are detected on IVP embryos (Wrenzycki et al 2002, Nino-Soto et al 2007. Those results suggest that mechanisms regulating XCI are affected by IVC.…”