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. 2020 Apr 19;9(4):1016.
doi: 10.3390/cells9041016.

Initiation of X Chromosome Inactivation during Bovine Embryo Development

Bo Yu  1 , Helena T A van Tol  1 , Tom A E Stout  2 , Bernard A J Roelen  3
Affiliations

Initiation of X Chromosome Inactivation during Bovine Embryo Development

Bo Yu et al. Cells. .

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.

Keywords: H3K27me3; XCI; XIST; bovine; embryo; inner cell mass.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The relative expression of XCI related genes during in vitro bovine embryo development, as determined by quantitative RT-PCR. (A) XIST, (B) HPRT1, (C) EED, (D) EZH2, (E) HNRNPK, (F) HNRNPU, (G) RING1, (H) JPX. GV, MII, PN, 2C, 4C, 8C, MO, and BL refer to germinal vesicle, metaphase II, pronuclear, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages, respectively. Embryos were derived by fertilization with non-sexed sperm. Relative expression in GV oocytes set at 1. Significant differences between bars are indicated by different letters above bars (p < 0.05). Error bars indicate standard deviations of three independent biological replicates.
Figure 2
Figure 2
The relative expressions of XCI related genes in female (red bars) and male (blue bars) bovine embryos from 8-cell stage to day 8 blastocysts, as determined by quantitative RT-PCR. (A) XIST, (B) HPRT1, (C) EED, (D) EZH2, (E) HNRNPK, (F) HNRNPU, (G) RING1, (H) JPX. Relative expression from female morulae set at 1; * (p < 0.05) and ** (p < 0.005) indicate significant differences between females and males. Significant differences among embryos with same gender are indicated by different letters with the same color (p < 0.05). Error bars indicate standard deviations of three independent biological replicates.
Figure 3
Figure 3
Combined XIST RNA FISH and H3K27me3 immunofluorescence in bovine oviduct epithelial cells and female embryos. XIST RNA FISH combined with H3K27me3 immunofluorescence in oviduct cells and the 8-cell embryos up to day (D) 9 blastocysts (A). XIST and H3K27me3 colocalization are indicated (arrow), scale bar = 50 µm. White boxes indicate areas presented in the right column at higher magnification. Percentages of cells lacking a XIST spot (B), 1 XIST spot (C), 2 XIST spots (D) from 8-cell embryos up to D9 blastocysts. Percentages of XIST and H3K27me3 colocalization from morula to day 9 blastocyst (E). Significant differences between boxes are indicated by different letters (p < 0.05). BL = blastocyst.
Figure 4
Figure 4
The relative expression of XCI related genes in inner cell mass (ICM) cells (black bars) and trophectoderm (TE) cells (white bars), and XIST RNA FISH combined with immunofluorescence in female embryos. The relative expression of XCI related genes (A). Relative expression in ICM set at 1; * (p < 0.05) and ** (p < 0.05) indicate significant differences. Error bars indicate standard deviations of three biological replicates. XIST RNA FISH combined with double staining of H3K27me3 and CDX2 (B) or NANOG (C) from day 7 (D7) to day 9 (D9) blastocysts, scale bar = 50 µm. BL = blastocyst.
Figure 5
Figure 5
Percentages of nuclei with different numbers of XIST spots in CDX2 and NANOG positive or negative cells, and percentages of colocalization of XIST and H3K27me3 from day 7 to day 9 female blastocysts. Absence of an XIST spot (A), one XIST spot (B), two XIST spots (C), and colocalization of XIST and H3K27me3 (D) in day 7 (D7) blastocysts; absence of an XIST spot (E), one XIST spot (F), two XIST spots (G), and colocalization of XIST and H3K27me3 (H) in day 8 (D8) blastocysts; absence of an XIST spot (I), one XIST spot (J), two XIST spots (K), and colocalization of XIST and H3K27me3 (L) in day 9 (D9) blastocysts. Presence (purple box) and absence (red box) of CDX2 are indicated by + and -, respectively. Presence (orange box) and absence (blue box) of NANOG are indicated by + and -, respectively. Significant differences between boxes are indicated by different letters (p < 0.05). BL = blastocyst.
Figure 6
Figure 6
Proportional distribution of X-linked gene expression in blastocysts versus morulae and inner cell mass cells versus trophectoderm cells. Proportional distribution of upregulated (red), downregulated (blue), and equally expressed (green) X-linked genes in blastocysts versus morulae (A) and inner cell mass cells versus trophectoderm cells (C). The relative expression (Log2) of X-linked genes in blastocysts versus morulae (B) and inner cell mass cells versus trophectoderm cells (D) are plotted according to their locations on the X chromosome.
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References

    1. Heard E., Clerc P., Avner P. X-chromosome inactivation in mammals. Annu. Rev. Genet. 1997;31:571–610. doi: 10.1146/annurev.genet.31.1.571. - DOI - PubMed
    1. Lyon M.F. Gene action in the X-chromosome of the mouse (Mus musculus L.) Nature. 1961;190:372–373. doi: 10.1038/190372a0. - DOI - PubMed
    1. Sado T., Brockdorff N. Advances in understanding chromosome silencing by the long non-coding RNA Xist. Philos. Trans. R. Soc. B. 2013;368 doi: 10.1098/rstb.2011.0325. - DOI - PMC - PubMed
    1. Yue M.H., Richard J.L.C., Yamada N., Ogawa A., Ogawa Y. Quick Fluorescent in Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation. JoVE-J. Vis. Exp. 2014:e52053. doi: 10.3791/52053. - DOI - PMC - PubMed
    1. Deng X., Berletch J.B., Nguyen D.K., Disteche C.M. X chromosome regulation: Diverse patterns in development, tissues and disease. Nat. Rev. Genet. 2014;15:367–378. doi: 10.1038/nrg3687. - DOI - PMC - PubMed

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