Can Vero cell co-culture improve in-vitro maturation of bovine oocytes?

Royan Institute, Tehran, Iran.
Reproductive biomedicine online (Impact Factor: 3.02). 10/2006; 13(3):404-11. DOI: 10.1016/S1472-6483(10)61446-0
Source: PubMed


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.

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    • "Presumptive zygotes in groups of ten were then cultured in 20 ml droplets of mSOF. Developed blastocysts in different groups were used for differential staining as described byMoulavi et al. (2006). "
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    ABSTRACT: The effect of technical steps of somatic cell nuclear transfer (SCNT) on different aspects of cloned embryo development was investigated in sheep. In vitro matured oocytes were enucleated in presence or absence of zona and reconstituted by three different SCNT techniques: conventional zona-intact (ZI-NT), standard zona-free (ZF-NT), and intracytoplasmic nuclear injection (ICI-NT). Step-wise alterations in nuclear remodeling events and in mRNA abundances, throughput and efficiency of cloned embryo development and cell allocation of the resulted blastocysts were assessed. Early signs of nuclear remodeling were observed as soon as 2 h post reconstitution (hpr) in fusion based methods of nuclear remodeling (ZI-NT and ZF), but were not observable until 4 hpr in ICI-NT method. The relative mRNA abundances of HSP90, NPM2, and ATPase genes were not affected by i) presence or absence of zona, ii) oocyte enucleation method, and iii) nuclear transfer method. After reconstitution, the relative mRNA contents of OCT4 in ZI-NT and ZF-NT methods and of PAP in ZF-NT were significantly lower than ICI-NT method. Zona removal doubled the throughput of cloned blastocyst development in ZF-NT technique over ZI-NT and ICI-NT. Cleavage rates were not affected by the SCNT protocol, whereas blastocyst yield rate in ICI-NT technique (17.0±1.0%) was significantly higher than ZF-NT (7.1±1.5%). Despite similarities in total cell number, SCNT protocol changed the distribution of cells in the blastocysts, as ZF-NT cloned blastocysts had significantly smaller inner cell mass than ZI-NT. These results implicate that technical aspects of cloning may result in the variety of cloning phenotypes.
    Full-text · Article · Feb 2013 · Reproduction
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    • "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. "
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    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.
    Full-text · Article · Mar 2012
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    • "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. "
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    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.
    Full-text · Article · Sep 2011
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