Characterization of X-Chromosome Gene Expression in Bovine Blastocysts Derived by In vitro Fertilization and Somatic Cell Nuclear Transfer

Abstract

To better understand X-chromosome reactivation (XCR) during early development, we analyzed transcriptomic data obtained from bovine male and female blastocysts derived by in-vitro fertilization (IVF) or somatic-cell nuclear transfer (SCNT). We found that X-linked genes were upregulated by almost two-fold in female compared with male IVF blastocysts. The upregulation of X-linked genes in female IVFs indicated a transcriptional dimorphism between the sexes, because the mean autosomal gene expression levels were relatively constant, regardless of sex. X-linked genes were expressed equivalently in the inner-cell mass and the trophectoderm parts of female blastocysts, indicating no imprinted inactivation of paternal X in the trophectoderm. All these features of X-linked gene expression observed in IVFs were also detected in SCNT blastocysts, although to a lesser extent. A heatmap of X-linked gene expression revealed that the initial resemblance of X-linked gene expression patterns between male and female donor cells turned sexually divergent in host SCNTs, ultimately resembling the patterns of male and female IVFs. Additionally, we found that sham SCNT blastocysts, which underwent the same nuclear-transfer procedures, but retained their embryonic genome, closely mimicked IVFs for X-linked gene expression, which indicated that the embryo manipulation procedure itself does not interfere with XCR in SCNT blastocysts. Our findings indicated that female SCNTs have less efficient XCR, suggesting that clonal reprogramming of X chromosomes is incomplete and occurs variably among blastocysts, and even among cells in a single blastocyst.

Initiation of X Chromosome Inactivation during Bovine Embryo Development

“…However, in bovine embryos, we found XIST accumulation did not lead to globally reduced expression of X-linked genes, similarly to what has been reported for human embryos [9]. In agreement with our findings, microarray and RNA-seq analyses in bovine blastocysts demonstrated enhanced expression of X-chromosome-linked genes in intact female compared with male blastocysts, which indicates that dosage compensation had not initiated at this stage [21,56]. Interestingly, we did observe a significant increase of intense H3K27me3 spots within nuclei from the morula to the blastocyst stage, indicating that this histone modification also did not lead to global downregulation of X-linked genes.…”

“…Despite the XIST coating of the inactivated X chromosome, several X-linked genes escape silencing and are still expressed, as has been demonstrated in human and rabbit embryos [9]. In bovine blastocysts, X-linked genes were expressed at higher levels in females compared with males, suggesting that X-chromosome inactivation was not yet operational [21]. These data suggest that downregulation of X-linked genes after XCI initiation is not uniformly conserved among species.Here, we examined the timing of XCI, its initiation in different lineage segregation, and regulation of X-linked gene expression during bovine embryo development.…”

Determination of Oocyte-Manipulation, Zygote-Manipulation, and Genome-Reprogramming Effects on the Transcriptomes of Bovine Blastocysts

“…The comparisons enabled us to separately examine the effects of various NT-involved factors such as the manipulation, partial removal of oocyte/zygote cytoplasm, and donor genome reprogramming (Figure 1A ). We analyzed previous Pip-seq data (Min et al, 2017 ) obtained from individual blastocysts after PCR-mediated amplification of embryonic transcripts by pico-profiling (Min et al, 2015 , 2016 ). Each blastocyst group comprised 12 blastocysts, with six males and six females (with the exception of five female shNTs).…”

“…A total of 35 RNA-seq libraries (12 IVF, 12 scNT, and 11 shNT blastocysts) were constructed using TruSeq DNA Sample Preparation kit (Illumina) (Min et al, 2015 , 2017 ). Two hundred nano grams of pico-profiled amplicons were applied to the End-repair reactions by incubating with End Repair Mix (Illumina) at 30°C for 2 h. The reactions were purified with AMPure XP beads (Beckman).…”

Dysregulated gene expression of imprinted and X-linked genes: a link to poor development of bovine haploid androgenetic embryos

“…In cattle, the presence of XIST transcripts has been reported as early as the 2-cell stage (Mendonca et al, 2019). Microarray and RNA-seq analyses of bovine blastocysts demonstrated higher expression of X-linked genes in female compared with male embryos, indicating that dosage compensation initiates later (Bermejo-Alvarez et al, 2010; Min et al, 2017). A recent report has indicated that XIST accumulation and XCI in bovine embryos starts at the morula stage.…”