Initiation of X Chromosome Inactivation during Bovine Embryo Development

Abstract

X-chromosome inactivation (XCI) is a developmental process that aims to equalize the dosage of X-linked gene products between XY males and XX females in eutherian mammals. In female mouse embryos, paternal XCI is initiated at the 4-cell stage; however, the X chromosome is reactivated in the inner cell mass cells of blastocysts, and random XCI is subsequently initiated in epiblast cells. However, recent findings show that the patterns of XCI are not conserved among mammals. In this study, we used quantitative RT-PCR and RNA in situ hybridization combined with immunofluorescence to investigate the pattern of XCI during bovine embryo development. Expression of XIST (X-inactive specific transcript) RNA was significantly upregulated at the morula stage. For the first time, we demonstrate that XIST accumulation in bovine embryos starts in nuclei of female morulae, but its colocalization with histone H3 lysine 27 trimethylation was first detected in day 7 blastocysts. Both in the inner cell mass and in putative epiblast precursors, we observed a proportion of cells with XIST RNA and H3K27me3 colocalization. Surprisingly, the onset of XCI did not lead to a global downregulation of X-linked genes, even in day 9 blastocysts. Together, our findings confirm that diverse patterns of XCI initiation exist among developing mammalian embryos.

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

“…Finally, we performed a gene specific bisulfite sequencing in order to analyze whether DMR methylation patterns were related to the high expression of XIST and KCNQ1OT1. As for the XIST DMR, all groups were demethylated, which is in accordance with its biallelic expression at during the early stages of bovine embryogenesis (Bermejo-Alvarez et al, 2010; Yu et al, 2020). In bovine sperm cells, the XIST gene does not appear methylated (Mendonca et al, 2019), suggesting that XIST would be expressed in androgenetic cells It is likely that the XIST DMR undergoes methylation later during embryonic development, as the onset of XCI initiates at blastocyst stage (Yu et al, 2020).…”

“…A recent report has indicated that XIST accumulation and XCI in bovine embryos starts at the morula stage. However, XIST colocalization with repressive marks (H3 lysine 27 trimethylation) on histones was only detected by day 7 blastocysts, indicating that complete XCI is only partially achieved at the blastocyst stage (Yu et al, 2020). Although XIST accumulation did not lead to globally reduced expression of X-linked genes, and X-Chr inactivation is only partially achieved at the blastocyst stage (Bermejo-Alvarez et al, 2010; Yu et al, 2020), the effects of the dysregulated expression of this long-noncoding RNA in haploid androgenetic morulas and blastocyst stages needs further investigation to clarify its impacts on chromosome-wide downregulation of gene expression.…”

“…However, XIST colocalization with repressive marks (H3 lysine 27 trimethylation) on histones was only detected by day 7 blastocysts, indicating that complete XCI is only partially achieved at the blastocyst stage (Yu et al, 2020). Although XIST accumulation did not lead to globally reduced expression of X-linked genes, and X-Chr inactivation is only partially achieved at the blastocyst stage (Bermejo-Alvarez et al, 2010; Yu et al, 2020), the effects of the dysregulated expression of this long-noncoding RNA in haploid androgenetic morulas and blastocyst stages needs further investigation to clarify its impacts on chromosome-wide downregulation of gene expression. Latham et al (2002) reported elevated expression of Xist RNA in haploid mouse androgenotes, and a similar pattern for the PGK1 gene, suggesting that haploid androgenotes may undergo deficient XCI, or that the embryos that initiate the XCI process begin to die soon thereafter.…”

Cross-species examination of X-chromosome inactivation highlights domains of escape from silencing

“…At the blastocyst stage, human as well as rabbit express XIST (the RNA that initiates the silencing cascade) from both alleles, while mouse has exclusively paternal Xist expression [ 1 ]. Cow has been observed to upregulate XIST at a similar stage to human and rabbit [ 12 ]. Human and rabbit also showed later inactivation timing than mouse [ 1 ].…”

Cross-species examination of X-chromosome inactivation highlights domains of escape from silencing

“…At the blastocyst stage, human as well as rabbit express XIST (the RNA that initiates the silencing cascade) from both alleles, while mouse has exclusively paternal Xist expression [1]. Cow has been observed to upregulate XIST at a similar stage to human and rabbit [12]. Human and rabbit also showed later inactivation timing than mouse [1].…”