1,720,976 research outputs found
Interspecies somatic cell nuclear transfer: Advancements and problems
No full textEmbryologists working with livestock species were the pioneers in the field of reprogramming by somatic cell nuclear transfer (SCNT). Without the "Dolly experiment," the field of cellular reprogramming would have been slow and induced plutipotent cells (iPSCs) would not have been conceived. The major drive of the work in mammalian cloning was the interest of the breeding industry to propagate superior genotypes. Soon it was realized that the properties of oocytes could be used also to clone endangered mammalian species or to reprogram the genomes of unrelated species through what is known as interspecies (i) SCNT, using easily available oocytes of livestock species. iSCNT for cloning animals works only for species that can interbreed, and experiments with taxonomically distant species have not been successful in obtaining live births or deriving embryonic stem cell (ESC) lines to be used for regenerative medicine. There are controversial reports in the literature, but in most cases these experiments have underlined some of the cellular and molecular mechanisms that are incomplete during cell nucleus reprogramming, including the failure to organize nucleoli, silence somatic cell genes, activate the embryonic genome, and resume mitochondrial replication and function, thus indicating nucleus-cytoplasmic incompatibility
Dissecting the role of the germinal vesicle nuclear envelope and soluble content in the process of somatic cell remodelling and reprogramming
No full textDifferentiated nuclei can be reprogrammed/remodelled to totipotency after their transfer to enucleated metaphase II (MII) oocytes. The process of reprogramming/remodelling is, however, only partially characterized. It has been shown that the oocyte nucleus (germinal vesicle – GV) components are essential for a successful remodelling of the transferred nucleus by providing the materials for pseudo-nucleus formation. However, the nucleus is a complex structure and exactly what nuclear components are required for a successful nucleus remodelling and reprogramming is unknown. Till date, the only nuclear sub-structure experimentally demonstrated to be essential is the oocyte nucleolus (nucleolus-like body, NLB). In this study, we investigated what other GV components might be necessary for the formation of normal-sized pseudo-pronuclei (PNs). Our results showed that the removal of the GV nuclear envelope with attached chromatin and chromatin-bound factors does not substantially influence the size of the remodelled nuclei in reconstructed cells and that their nuclear envelopes seem to have normal parameters. Rather than the insoluble nuclear lamina, the GV content, which is dissolved in the cytoplasm with the onset of oocyte maturation, influences the characteristics and size of transferred nuclei
Function of atypical mammalian oocyte/zygote nucleoli and its implications for reproductive biology and medicine
No full textMammalian oocytes/zygotes contain atypical nucleoli that are composed exclusively of a dense fibrillar material. It has been commonly accepted that these nucleoli serve as a repository of components that are used later on, as the embryo develops, for the construction of typical tripartite nucleoli. Indeed, when nucleoli were removed from immature oocytes (enucleolation) and these oocytes were then matured, fertilized or parthenogenetically activated, development of the produced embryos ceased after one or two cleavages with no detectable nucleoli in nuclei. This indicated that zygotic nucleoli originate exclusively from oocytes, i.e. are maternally inherited. Recently published results, however, do not support this developmental biology dogma and demonstrate that maternal nucleoli in one-cell stage embryos are necessary only during a very short time period after fertilization when they serve as a major heterochromatin organizing structures. Nevertheless, it still remains to be determined, which other functions/roles the atypical oocyte/ zygote nucleoli eventually have
Limited reprogramming of somatic cell nuclei transferred into mouse immature oocytes
No full textSomatic cell nuclear transfer, an important approach for the analysis of certain functional changes in the genome during differentiation and for many practical applications, is in general a low-efficiency procedure, mainly due to a low effectivity in the re-establishment of the developmental program in the reconstructed embryo. The process of reprogramming is, however, poorly understood and some additional studies are clearly necessary. The aim of this study was the ultrastructural and immunofluorescent (B23-nucleophosmin) evaluation of somatic (cumulus) cell nuclei reprogramming after their transfer into intact immature mouse oocytes, kept at the germinal vesicle (GV) stage (dbcAMP) during the whole culture. Control somatic cells and nuclear transfer-reconstructed embryos (1 and 24 h after fusion induced by polyethyleneglycol) were fixed for transmission electron mcroscopy (TEM) in 2.5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated through an ethanol series, and embedded in epoxy resin. Finally, ultrathin sections were stained with uranyl acetate followed by lead citrate. The above reagents were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). In parallel, we have evaluated immunocytochemically the pattern of B23 labelling in intact and reconstructed cells. The samples were fixed in 4% paraformaldehyde, incubated with an antibody against B23 (Santa Cruz, CA, USA) and then with a biotinylated secondary antibody, and detected by fluorescein isothiocyanate (FITC)-coupled Streptavidin (Jackson ImmunoResearch, Cambridgeshire, UK). Mouse cumulus cells (12/12) contain a reticulated fibrillogranular nucleolus. The cell are also positively labeled with the anti-B23 antibody. One hour after fusion, the introduced nuclei displayed shape modifications and nuclear envelope irregularities, whereas the nucleolus still showed the typical fibrillar pattern (19/19). The volume of transferred nuclei remains unchanged. Interestingly, while the oocyte nucleolus remains negative for B23, the nucleoli in transferred somatic cells were always positively labeled (45 cells). After 24 h, the transferred nuclei increased their volume up to two-three times and displayed an irregular shape with nucleoli still possessing the unchanged reticulated pattern (17/17). As in the previous experimental interval, only the somatic cell nucleus remained labeled with the anti-B23 antibody (52 cells). The GV oocyte nucleoli remained unchanged during the whole culture period, exhibiting the typical dense-fibrillar pattern. Our results showed that the immature oocyte cytoplasm possesses a limited remodelling activity. Interestingly, the evident increase in volume of transferred somatic cells indicated some changes but TEM morphology and B23 labeling pattern remained basically unchanged. We cannot, however, exclude the beneficial effect upon reprogramming if these nuclei were subsequently used for the transfer into definitive cytoplasts.[...
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine and rabbit oocytes and nuclear donor cells of various species
No full textThe most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages
25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Scientific and technological approaches to improve SCNT efficiency in farm animals and pets
Full text linkThe birth of Dolly through somatic cell nuclear transfer (SCNT) was a major scientific breakthrough of the last century. Yet, while significant progress has been achieved across the technics required to reconstruct and in vitro culture nuclear transfer embryos, SCNT outcomes in terms of offspring production rates are still limited. Here, we provide a snapshot of the practical application of SCNT in farm animals and pets. Moreover, we suggest a path to improve SCNT through alternative strategies inspired by the physiological reprogramming in male and female gametes in preparation for the totipotency required after fertilization. Almost all papers on SCNT focused on nuclear reprogramming in the somatic cells after nuclear transfer. We believe that this is misleading, and even if it works sometimes, it does so in an uncontrolled way. Physiologically, the oocyte cytoplasm deploys nuclear reprogramming machinery specifically designed to address the male chromosome, the maternal alleles are prepared for totipotency earlier, during oocyte nuclear maturation. Significant advances have been made in remodeling somatic nuclei in vitro through the expression of protamines, thanks to a plethora of data available on spermatozoa epigenetic modifications. Missing are the data on large-scale nuclear reprogramming of the oocyte chromosomes. The main message our article conveys is that the next generation nuclear reprogramming strategies should be guided by insights from in-depth studies on epigenetic modifications in the gametes in preparation for fertilization
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