This study was carried out to evaluate the effect of Vero cell co-culture on developmental competence of immature oocytes. Bovine cumulus-oocyte complexes (COC) were matured in presence or absence of Vero cells. Matured oocytes were inseminated and cultured for up to 9 days. Cleavage percentages were recorded on day 2 after insemination and embryos were evaluated on a daily basis. Expanding/expanded and hatching/hatched blastocysts were used for cell number assay. Results indicated a significantly greater cleavage percentage in oocytes matured in presence of Vero cells than control (86% versus 76%, P < or = 0.05). The percentages of advanced embryos appear to be greater on a daily basis in COC matured in presence of Vero cells compared with control. However, these differences were not significant. Blastocysts derived from COC matured in the presence of Vero cells had a significantly higher (P < or = 0.05) number of inner cell mass, trophectoderm and total cell number in expanding/expanded (65.25, 224.5 and 289.7 respectively) and hatching/hatched (67.75, 289.75 and 357.5) embryos in comparison to the control (42, 203.5, 245.5 and 51.3, 265, 316.3 respectively). Results confirm that co-culture of bovine COC during in-vitro maturation, enhances their ability for cleavage and for producing blastocysts with higher quality.
"Reconstituted oocytes were rested for up to 4 h and then activated as described previously (Hosseini et al., 2006). Activated oocytes were cultured in Menezo-B2 medium (INRA, France) over verocells monolayer (Moulavi et al., 2006) for 9 days at 38.5°C, 5% O 2 , 5% CO 2 , and maximum humidity. Cryopreserved ovine fibroblast and cumulus cells prepared during a recent study (Hosseini et al., 2008) were used for intraspecies SCNT. "
[Show abstract][Hide abstract] ABSTRACT: Increased possibility of universality of ooplasmic reprogramming factors resulted in a parallel increased interest to use interspecies somatic cell nuclear transfer (iSCNT) to address basic questions of developmental biology and to improve the feasibility of cell therapy. In this study, the interactions between human somatic cells and ovine oocytes were investigated. Nuclear remodeling events were first observed 3 h post-iSCNT as nuclear swelling, chromosome condensation, and spindle formation. A time-dependent decrease in maturation promoting activity of inactivated reconstructs coincided with increased aberrations in chromosome and spindle organization of the newly developed embryos. The sequence and duration of nuclear remodeling events were irrespective of donor cell type used. Although the majority of the reconstituted embryos arrested before embryonic genome activation (8-16-cell) stage, less than 5% of them could progress beyond transcription-requiring developmental stage and formed blastocyst-like structures with distinct inner cell mass and trophectoderm at days 7 and 8 post-SCNT. Importantly, real-time assessment of three developmentally important genes (Oct4, Sox2, and Nanog) indicated their upregulation in iSCNT blastocysts. Blastocyst-derived outgrowths had alkaline phosphatase activity that was lost upon passage. Collectively, this study introduced ovine oocyte as a credible cytoplast for remodeling and reprogramming of human somatic cells back to the embryonic stage and provided a platform for further studies to unravel possible differences exist between reprogramming ability of oocytes of different mammalian species.
"Abattoirderived ovaries were used as the source of oocyte. The process of oocyte in vitro maturation was as described previously (Moulavi et al., 2006). In brief, cumulus–oocyte complexes were aspirated of antral follicles (2–8 mm) and cultured in tissue culture medium 199 (TCM199) containing 10% FCS, FSH (10 lg/mL), LH (10 lg/mL), estradiol-17b (1 lg/mL), cysteamine (0.1 mM) at 39°C, 5% CO 2 and humidified air for 18–20 h. "
[Show abstract][Hide abstract] ABSTRACT: 5-Aza-2'-deoxycytidine (AzC), trichostatin A (TSA), and its natural mimetic, sodium butyrate (NaB), are antineoplastic drugs that can modify the epigenetic status of donor cells prior to somatic cell nuclear transfer (SCNT). In this study, we used fibroblast cells treated with these drugs to investigate the direct and indirect effects of induced changes in DNA methylation and acetylation of the lysine 9 residue of histone H3 (H3K9). Additionally, we assayed cellular characteristics (cell growth, cell proliferation, cell cycle progression, and apoptosis) and SCNT efficiency in response to these drugs as well as monitoring these effects 24 h after removing the drugs. We observed the following: (1) AzC, TSA, and NaB all showed dose-dependent effects on different cellular characteristics; (2) TSA and NaB induced H3K9 hyperacetylation accompanied by DNA hypermethylation, whereas AzC induced DNA hypomethylation with no effect on H3K9 hyperacetylation; (3) TSA and NaB improved cloning efficiency, whereas AzC reduced it; and (4) unlike AzC, the effects of TSA and NaB on cellular characteristics and SCNT efficiency were reversed following drug removal. Our results indicate that somatic cells treated with TSA and NaB show better survival and recovery rates following the removal of these drugs. Moreover, H3K9 hyperacetylation (induced with TSA and NaB), but not DNA hypomethylation (induced with AzC), favors cloning efficiency.
"During this study, IVF was carried out as a normal control. The IVF procedure used was as described previously (Moulavi et al., 2006), and the presumptive zygotes were cultured in mSOFaa medium as described for cloned embryos, except that the IVF embryos were cultured in dishes without the wells. The IVF procedure used in this study has resulted in successful production of live offspring in cattle by transferring fresh and frozen blastocysts (unpublished data, www.royaninstitute.org). "
[Show abstract][Hide abstract] ABSTRACT: In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.
Molecular Reproduction and Development 08/2011; 78(8):576-84. DOI:10.1002/mrd.21344 · 2.53 Impact Factor
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