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ABSTRACT: Mammalian primary oocytes are arrested in the post-replicative G2 phase of the cell cycle. In contrast to other G2 nuclei, the nucleus of the growing mouse oocyte can reinitiate DNA synthesis after transfer by cell fusion under favorable cytoplasmic conditions, created by the parthenogenetic one-cell embryo. In the present study, we used the cell hybrid system to analyze the distribution of proteins involved in DNA re-replication in the oocyte nucleus. We show that this process is preceded by an extensive rearrangement of the insoluble fractions of minichromosome maintenance (MCM) proteins (Mcm2, -6 and 7). We also demonstrate that Cdc6 protein is present in primary growing mouse oocytes freshly isolated from the ovary, in a soluble and insoluble form. In contrast to MCM proteins, the insoluble fraction of Cdc6 was not rearranged in oocyte nuclei reinitiating DNA replication in hybrid cells. The rearrangement of MCM proteins and reinitiation of DNA synthesis occurred in the nuclei, in which the nuclear envelope remained intact. Reinitiation of DNA replication in the oocyte nucleus was sensitive to the inhibition of both CDK activity and polyadenylation of maternal mRNAs, indicating a role of proteins synthesized de novo by the embryo. These results allow us to understand better the mechanisms involved in the reinitiation of DNA replication in growing oocytes.
The International journal of developmental biology 01/2010; 54(1):21-31. · 2.16 Impact Factor
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ABSTRACT: We describe the localization of three proteins of the minichromosome maintenance (MCM) family, Mcm2, -6 and -7 in mouse ovarian oocytes. We showed that Mcm proteins are stored in two forms: soluble and insoluble. Soluble Mcm2, -6 and -7 were uniformly distributed in the nuclei of ovarian oocytes. Insoluble Mcm2 and Mcm7 (but not Mcm6) were detected in the nuclei of resting, growing and fully-grown transcribing oocytes. In transcriptionally inactive fully-grown oocytes, Mcm2 underwent redistribution and Mcm7 disappeared. A similar effect was observed when transcription in growing oocytes was inhibited with alpha-amanitin. We postulate that in mouse oogenesis, the insoluble Mcm proteins are engaged in processes related to regulation of transcription and/or chromatin organization. In oocytes preparing for meiotic maturation, aggregates of the insoluble form of Mcm2 fragmented, dispersed and ultimately disappeared from the nuclei. Numerous Mcm2-positive deposits were observed in the cytoplasm of maturing oocytes. In the one-cell embryo, insoluble Mcm2 appeared in the G1 nucleus, persisted in the S phase and was undetectable in the G2 nucleus. Such behavior of Mcm2 supports its involvement in chromatin licensing in the first embryonic cell cycle.
The International Journal of Developmental Biology 02/2007; 51(4):283-95. · 2.82 Impact Factor
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ABSTRACT: Blastocysts obtained from mice differing in pigmentation (albino versus pigmented) and the isoforms of glucose phosphate isomerase (GPI 1A versus 1B) were electrofused and those containing a single chimaeric inner cell mass (ICM) were transferred to the uterus of pseudopregnant recipients. The pups were recovered on the 20(th) day by Caesarian section and fostered by females that had littered on the previous night or 24 h earlier. Altogether nine adult animals and two pups, which died soon after delivery, were available for GPI analysis. Between 9 and 13 organs/tissues were examined and the relative contribution of the GPI 1A and 1B isoforms was estimated using an electrophoretic GPI assay. Eight adult animals were overtly chimaeric and one was chimaeric in some internal tissues only. Eight mice were males: seven were fertile, one was infertile. The ninth adult mouse was a hermaphrodite. The fertile animals produced sperm of one genotype only, i.e. derived either from the albino or from the pigmented component. This is the first report showing that adult chimaeras can be produced from two combined blastocysts, provided that fusion of the adhering trophectoderm cells is first induced and the orientation of blastocysts enables the two ICMs to integrate into a single ICM. Our results suggest that in the preimplantation blastocyst, the organisation of the ICM remains labile thus making it possible for the fused blastocysts to establish new embryonic organisation and to develop into a single organism.
The International Journal of Developmental Biology 02/2005; 49(8):909-14. · 2.82 Impact Factor
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ABSTRACT: We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.
The International Journal of Developmental Biology 02/2005; 49(7):825-32. · 2.82 Impact Factor
