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

Byungkuk Min,Kyuheum Jeon,Jung Sun Park,Yong-Kook Kang

doi:10.3389/fgene.2017.00042

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.

Highlights

X-chromosome inactivation (XCI) has evolved in female mammals to compensate for sexchromosome dosage differences by suppressing gene expression from one X chromosome, and to render all cells as functionally monosomic for the X chromosome, a process considered to be important for normal embryonic development (Monk and Harper, 1979; Penny et al, 1996).X-Gene Upregulation in Bovine BlastocystsThe current understanding of XCI during early embryogenesis largely originates from studies in mice, partially because XCI occurs during a very early developmental window when the embryo is accessible
Complementary DNA amplicons from six male and six female in-vitro fertilization (IVF)-BLs were used for RNA-seq library construction
We found that the expression of X-linked genes in female IVFBLs were almost twice that found in male IVF blastocysts (IVF-BLs) (Figure 2C)

Summary

Introduction

X-chromosome inactivation (XCI) has evolved in female mammals to compensate for sexchromosome dosage differences by suppressing gene expression from one X chromosome, and to render all cells as functionally monosomic for the X chromosome, a process considered to be important for normal embryonic development (Monk and Harper, 1979; Penny et al, 1996).X-Gene Upregulation in Bovine BlastocystsThe current understanding of XCI during early embryogenesis largely originates from studies in mice, partially because XCI occurs during a very early developmental window when the embryo is accessible. Inactivation of the paternal X chromosome occurs progressively during the first days of post-fertilization cleavage until the morula stage (Okamoto and Heard, 2006; Kalantry et al, 2009; Namekawa et al, 2010). At the late blastocyst stage, XCR is observed within cells from the inner cell mass (ICM) that will form the embryo proper (i.e., two active X chromosomes are present in these cells), whereas cells of the trophectoderm (TE), which will form the placenta, maintain imprinted inactivation of the paternal X chromosome (Mak et al, 2004; Okamoto et al, 2004; Patrat et al, 2009). At the onset of gastrulation, embryonic lineage cells randomly undergo XCI again without a parent-of-origin bias, and once random X-inactivation is initiated, all the progeny cells maintain the same X-inactivation status (Lyon, 1961). It was shown that during the early period of reprogramming in hybrid cells between human fibroblasts and mouse embryonic stem cells, human nuclei undergo a loss of XIST and XCI-associated histone marks from the inactive X chromosome to accomplish XCR, some regions on the X chromosome are refractory to reprogramming (Cantone et al, 2016)

Methods

Results

Conclusion