Developmentally‐regulated changes of Xist RNA levels in single preimplantation mouse embryos, as revealed by quantitative real‐time PCR

Abstract

Xist RNA localizes to the inactive X chromosome in cells of late cleavage stage female mouse embryos (Sheardown et al., 1997: Cell 91:99-107). Fluorescence in situ hybridization (FISH), however, does not quantify the number of Xist transcripts per nucleus. We have used real-time reverse transcription-polymerase chain reaction (RT-PCR) to measure Xist RNA levels in single preimplantation embryos and to establish developmental profiles in both female and male samples. The gender of each embryo was readily established based on Xist RNA levels, by counting Xist gene copies per cell, and by independent detection of the presence/absence of Sry, a Y chromosome-specific gene. Xist expression in males was found to be very low at all stages, as suggested by FISH. In contrast, female embryos contained measurable levels of Xist mRNA starting at the late 2-cell stage and rapidly accumulated Xist transcripts until morula stage. Xist RNA accumulation per embryo then reached a plateau, while cell division continued. We propose that during early cleavage high enough levels of Xist mRNA are transcribed to generate a pool of unbound molecules. This pool would serve to temporarily maintain X chromosome inactivation without additional transcription while the trophectoderm and inner cell mass (ICM) differentiate. The ICM would then loose the paternally imprinted pattern of X inactivation originally present in all embryonic cells.

Transcriptional sexual dimorphism during preimplantation embryo development and its consequences for developmental competence and adult health and disease

“…However, during the preimplantation period, X inactivation is a reversible dynamic process; both X-chromosomes are active after embryonic genome activation and XCI is not fully accomplished during early development, which leads to a higher expression of X-linked genes in female embryos (Kobayashi et al 2006, Bermejo-Alvarez et al 2010a. In this sense, a higher expression of selected X-linked genes such as XIST, glucose-6-phosphate dehydrogenase (G6PD), HPRT1, PGK, XIAP and MAOA was reported for mouse (Kay et al 1994, Hartshorn et al 2002, bovine (Gutierrez-Adan et al 2000, Peippo et al 2002, Wrenzycki et al 2002, Jimenez et al 2003, Morton et al 2007) and human (Taylor et al 2001) embryos. The developmental stage at which XCI is accomplished is not clear and may differ greatly between species (Okamoto & Heard 2009), but two global transcriptional studies reported that many X-linked genes are upregulated in female blastocysts in mice (Kobayashi et al 2006) and especially in cattle, where most of them (w90%) are highly expressed in females (Bermejo-Alvarez et al 2010a).…”

Evaluation of the Quality of Porcine Somatic Cell Nuclear Transfer Embryo by Gene Transcription Profiles

“…The FGF receptor family [14,15], IL6 and IL6r [16,17] are strongly expressed at the blastocyst stage and are known to play critical roles in postblastocyst development and implantation. Xist is required for X inactivation [18] and is also expressed strongly in the blastocyst stage [19][20][21]. The transcription level of Xist in female embryos was substantially higher than that of male embryos [19,21].…”

“…Xist is required for X inactivation [18] and is also expressed strongly in the blastocyst stage [19][20][21]. The transcription level of Xist in female embryos was substantially higher than that of male embryos [19,21]. It has been reported that in cattle these genes are useful markers for assessing the developmental potency of nuclear transfer embryos at the blastocyst stages [7][8][9].…”

“…The c-kit ligand gene is known to have an important function in early embryonic development, especially in the blastocyst stage [17,22,23]. All of these genes start to express at the 2 cell stage [7,17,19,22], that is before genome activation in porcine embryos [24,25].…”

The evolution of sex chromosomes

“…Breaching Lyon's hypothesis, marsupials (and maybe mice) preferentially inactivate the paternally derived X chromosome via an imprint (Takagi and Sasaki, 1975;Zuccotti and Monk, 1995). Mice are now thought to lose their paternal imprint after the first dozen rounds of embryonic cleavage, randomising inactivation during the differentiation of the inner cell mass (Hartshorn et al, 2002).…”