Dataset Information

Functional analysis of Rex1 during preimplantation development.

ABSTRACT: Rex1/Zfp42 is a nuclear protein that is highly conserved in mammals, and widely used as an embryonic stem (ES) cell marker. Although Rex1 expression is associated with enhanced pluripotency, loss-of-function models recently described do not exhibit major phenotypes, and both preimplantation development and ES cell derivation appear normal in the absence of Rex1. To better understand the functional role of Rex1, we examined the expression and localization of Rex1 during preimplantation development. Our studies indicated that REX1 is expressed at all stages during mouse preimplantation development, with a mixed pattern of nuclear, perinuclear, and cytoplasmic localization. Chromatin association seemed to be altered in 8-cell embryos, and in the blastocyst, we found REX1 localized almost exclusively in the nucleus. A functional role for Rex1 in vivo was assessed by gain- and loss-of-function approaches. Embryos with attenuated levels of Rex1 after injection of zygotes with siRNAs did not exhibit defects in preimplantation development in vitro. In contrast, overexpression of Rex1 interfered with cleavage divisions and with proper blastocyst development, although we failed to detect alterations in the expression of lineage and pluripotency markers. Rex1 gain- and loss-of-function did alter the expression levels of Zscan4, an important regulator of preimplantation development and pluripotency. Our results suggest that Rex1 plays a role during preimplantation development. They are compatible with a role for Rex1 during acquisition of pluripotency in the blastocyst.

SUBMITTER: Climent M

PROVIDER: S-EPMC3549625 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Functional analysis of Rex1 during preimplantation development.

Climent María M Alonso-Martin Sonia S Pérez-Palacios Raquel R Guallar Diana D Benito Alfredo A AA Larraga Ana A Fernández-Juan Marta M Sanz Marta M de Diego Alicia A Seisdedos María T MT Muniesa Pedro P Schoorlemmer Jon J

Stem cells and development 20121009 3

Rex1/Zfp42 is a nuclear protein that is highly conserved in mammals, and widely used as an embryonic stem (ES) cell marker. Although Rex1 expression is associated with enhanced pluripotency, loss-of-function models recently described do not exhibit major phenotypes, and both preimplantation development and ES cell derivation appear normal in the absence of Rex1. To better understand the functional role of Rex1, we examined the expression and localization of Rex1 during preimplantation developmen ...[more]

PMID: 22897771

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Project description:The degradation of maternal proteins is one of the most important events during early development, and it is presumed to be essential for embryonic genome activation (EGA), but the precise mechanism is still not known. It is thought that a large proportion of the degradation of maternal proteins is mediated by the ubiquitin-proteolytic system. In this study we focused on the expression of the Skp1-Cullin1-F-box (SCF) complex, a modular RING-type E3 ubiquitin-ligase, during bovine preimplantation development. The complex consists of three invariable components--Cul1, Skp1, Rbx1 and F-box protein, which determines the substrate specificity. The protein level and mRNA expression of all three invariable members were determined. Cul1 and Skp1 mRNA synthesis was activated at early embryonic stages, at the 4c and early 8c stage, respectively, which suggests that these transcripts are necessary for preparing the embryo for EGA. CUL1 protein level increased from MII to the morula stage, with a significant difference between MII and L8c, and between MII and the morula. The CUL1 protein was localized primarily to nuclei and to a lesser extent to the cytoplasm, with a lower signal in the inner cell mass (ICM) compared to the trophectoderm (TE) at the blastocyst stage. The level of SKP1 protein significantly increased from MII oocytes to 4c embryos, but then significantly decreased again. The localization of the SKP1 protein was analysed throughout the cell and similarly to CUL1 at the blastocyst stage, the staining was less intensive in the ICM. There were no statistical differences in RBX1 protein level and localization. The active SCF-complex, which is determined by the interaction of Cul1 and Skp1, was found throughout the whole embryo during preimplantation development, but there was a difference at the blastocyst stage, which exhibits a much stronger signal in the TE than in the ICM. These results suggest that all these genes could play an important role during preimplantation development. This paper reveals comprehensive expression profile, the basic but important knowledge necessary for further studying.

Project description:Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4-8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS) to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF) cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos provides insight into the cytogenetic mechanisms underlying their formation and the consequences for human fertility.

Dataset Information

Functional analysis of Rex1 during preimplantation development.

Publications

Functional analysis of Rex1 during preimplantation development.

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