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00 X C I 1 5 3 00 X rar



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X-chromosome inactivation is established during early embryonic development and maintained thereafter [5]. In the mouse, imprinted XCI is initiated at the 4-cell stage embryo stage with exclusive inactivation of the paternal X chromosome (Xp). Indeed, the paternal X chromosome remains inactive in trophectoderm (TE) cells during and after formation of the mouse blastocyst [6,7]. In the inner cell mass (ICM) of female embryos, however, the inactive Xp is reactivated, resulting in two active X chromosomes (XaXa). At around the time of implantation when the epiblast is being established, XCI is re-established with either maternal or paternal X chromosomes inactivated randomly in different cells, a process known as random X chromosome inactivation [8]. Although the initiation of XCI has been extensively explored in mouse embryos, the pattern of XCI in other mammalian species is less clear. In rabbits and humans for example, XCI starts later at the morula and blastocyst stages, respectively, and is not subject to imprinting [9]. Furthermore, large XIST clouds have been detected around both X chromosomes in cells of rabbit and human embryos, even in the ICM [9,10]. It appears, therefore, that XCI does not follow a uniform pattern in mammals.


In female mice, one of the defining characteristics of the naïve pluripotent state is the presence of two active X chromosomes; i.e., XaXa [11]. Similarly to the ICM, the absence of XIST expression from both X chromosomes has been observed in female mouse embryonic stem (ES) cells and induced pluripotent stem cells [12]. Moreover, the pluripotency factors OCT4, SOX2, and NANOG have been implicated in suppression of XIST expression, by binding to its first intron [13]. Upon pluripotent cell differentiation, the pluripotency factors are transcriptionally downregulated and XIST expression increases, leading to random XCI [14]. In human cells, however, the correlation between pluripotency state and XCI is less clear [14]. Even though the XaXa state has been reported in female human ES cells, these cell lines were shown to be very unstable during passages [15]. Meanwhile, inactive X chromosomes (Xi) were detected in other human ES and induced pluripotent stem cells, with XIST coating and accumulation of heterochromatin markers on the Xi [16]. Interestingly, female mouse epiblast stem cells, considered to be at a primed pluripotency state, also exhibit random XCI and share several morphological and molecular similarities with human ES cells. It has been hypothesized that human ES cells are at a primed pluripotent state, and that the presence of XaXa in female cells may still be a hallmark of naïve pluripotency in human cells [16,17,18]. So far, however, the connection between pluripotency and XCI state in other mammals has not been investigated in depth. Interestingly, stable primed pluripotent embryonic stem cell lines have recently been established from bovine embryos; however, their X chromosome activation states have not yet been reported [19].


One of the major consequences of XCI is the downregulation of X-linked gene expression on the inactivated X chromosome. During mouse development, silencing of X-linked genes follows the XIST coating [8,20]. 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.


For subsequent analysis, germinal vesicle (GV) oocytes and metaphase II (MII) oocytes were collected immediately after COC recovery and after 23 h in vitro maturation culture, respectively. Zygotes, and 2, 4, and 8 cell embryos were collected at 20, 32, 38 and 56 h after the start of fertilization respectively, and morulae and blastocysts were collected after 5 and 8 days, respectively of in vitro culture.


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


We also investigated gene expression for two members of the polycomb repressive complex 2 (PRC2); namely, EED and EZH2. PRC2 has been reported to accumulate on the inactive X chromosome and to be required for establishing histone methylation [27,28]. Expression of EED was relatively constant throughout embryonic development, but was significantly elevated at the morula stage (Figure 1C). EZH2 expression was stable until the 8-cell stage, after which expression decreased gradually (Figure 1D).


HNRNPK can bind to XIST to recruit polycomb repressive complex 1 (PRC1) [29]. HNRNPU, another XIST RNA binding protein, was previously reported to be essential for XIST recruitment to the inactive X chromosome (Xi) [30]. HNRNPK (Figure 1E) exhibited a similar expression pattern to EED. The expression of HNRNPU was relatively constant throughout embryo development (Figure 1F).


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


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


We then addressed localization of XIST RNA and H3K27me3 in embryos from the 8-cell stage up to day 9 blastocysts. The sexes of individual embryos were determined by PCR, with the exception of 8-cell stage embryos, for which insufficient genomic DNA was available. In agreement to what we found by qRT-PCR (Figure 1A and Figure 2A), there was no expression of XIST in any 8-cell stage embryo we examined by RNA FISH; instead, spots of XIST were first detected in female embryos at the morula stage (Figure 3A, Figure S1B).


In fact, we observed three different patterns of XIST staining (no spot, one spot, or two spots) in embryonic nuclei. Nuclei without any XIST spots were found at all stages from the 8-cell stage embryo up to the day 9 female blastocyst. However, the percentage of nuclei without a XIST spot decreased markedly from 100% at the 8-cell stage to 60% in the morulae, and 36% in day 7 blastocysts (Figure 3B). Conversely, the percentage of nuclei with one XIST spot increased significantly from 0% at the 8-cell stage to 32% at the morula stage and 50% in day 7 blastocysts (Figure 3C). Nuclei with two XIST spots represented only small proportions of the total nuclei; namely, 9% at the morula stage and 15% in day 7 blastocysts (Figure 3D). Interestingly, the percentages of nuclei displaying the three different numbers of XIST spots were relatively stable across day 7 to day 9 blastocysts, indicating that the accumulation of XIST RNA was already established at day 7.


To confirm XIST expression in ICM cells of the ICM, we examined ICM and TE cells separated mechanically. This was performed on embryos produced with unsorted sperm, since X and Y sorted sperm yield far fewer embryos, whereas we needed large numbers of blastocysts to obtain sufficient RNA from embryo fragments. The specificity of isolated ICM and TE fragments was determined by relative expression of OCT4, SOX2, and CDX2, with similar results to those reported previously [22] (Figure 4A). Interestingly, expression of XIST in ICM cells was at a higher level in TE cells, but this difference did not reach statistical significance (p > 0.361). In addition, other XCI related genes were expressed at similar levels in the ICM and TE (Figure 4A). 041b061a72


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