Study of nuclear remodeling in reconstructed mouse embryos by optical and electron microscopy

Abstract

To date, there is a large body of information on the structural composition of oocytes and embryonic cells of mammals, embryonal nucleogenesis, processes of gene expression, and specific protein synthesis during the preimplantation period. Different factors playing the key role in the transformation of the oocyte to the embryo after in the fertilization and activation of the embryonic genome [1–3] have been revealed. The number of such factors, revealed, as a rule, in the oocyte cytoplasm, constantly increases. In numerous studies on nucleus transplantation it was convincingly shown that oocytes contain specific factors causing genes highly specialized somatic cells to behave as the embryonic genome. It was established that these changes in gene expression are accompanied by considerable structural rearrangement of nuclei of these cells and their chromosome material [4–6]. As a result, the nuclei acquire the potential to direct and support embryonic development to term, though with an extremely low probability. The ability of mature oocytes to induce epigenetic reprogramming of nuclei and transform highly specialized cells ito stem cells is unique. Paradoxically, oocytes become unable to support these processes immediately after fertilization [6, 7]. Currently, the topical studies on derivation of embryonic stem cells from reconstructed embryos for therapeutic use challenge the problem of determination of the main strategy of cell reprogramming It is necessary to know in detail the spatiotemporal pattern of structural and functional rearrangements in donor nuclei and in reconstructed embryos to elucidate the mechanisms underlying these processes, and their influence on further embryo development. Among different methods of cell studies, the optical and electron microscopy are most adequate to provide a clear picture of localization of nuclei and chromatin structure and detect even very small changes in cells. Preparation of serial sections with subsequent three-dimensional reconstruction of objects on the basis of electron microscopy is a very useful approach to studying the rearrangements of nuclei in somatic cells being transferred into oocytes. No studies of this kind have been performed thus far. In this study, we obtained mouse embryos with the genome of cumulus cells and examined rearrangements in cumulus cell nuclei at early stages of their remodelling in the oocyte cytoplasm using serial ultrathin sections and three-dimensional reconstruction.