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Resveratrol treatment during goat oocytes maturation enhances developmental competence of parthenogenetic and hand-made cloned blastocysts by modulating intracellular glutathione level and embryonic gene expression.

ABSTRACT: PURPOSE: The aim of the present study was to determine whether supplementation of resveratrol, a stilbenoid antioxidant with therapeutic significance, influences goat (Capra hircus) oocyte maturation and subsequent embryonic development and expression of apoptosis and early embryonic development-related genes. METHODS: Five different concentrations of resveratrol (0.1, 0.25, 0.5, 2.0 and 5.0 ?M) were used in in vitro maturation (IVM) medium. Cell tracker blue and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent stains were used to assay intracellular glutathione and reactive oxygen species levels in mature oocytes. Parthenogenetic activation and hand-made cloning were performed to check the developmental potential following resveratrol treatment. We used quantitative real-time PCR to analyze embryonic gene expression. RESULT: Compared to control, no significant improvement was observed in nuclear maturation in resveratrol-treated groups and at 5.0 ?M concentration maturation rate decreased significantly (P < 0.05). But resveratrol treatment at the concentrations of 0.25, 0.5 ?M significantly reduced intracellular ROS, and increased GSH concentrations. Oocytes treated with 0.25, 0.5 ?M resveratrol when subsequently used for PA and HMC, higher extent of blastocyst yields were observed. Expression analysis of proapoptotic (Bax) gene in mature oocytes, cumulus cells, and HMC-derived blastocysts revealed lesser transcript abundances in various resveratrol-treated groups., however no change in the same was observed for antiapoptotic gene (Bcl2). Differential expression of genes associated with developmental competence and nuclear reprogramming was also observed in HMC-derived blastocysts. CONCLUSION: Our results show that resveratrol treatment at optimum concentrations (0.25 and 0.5 ?M) during IVM produced beneficial microenvironment within oocytes by increasing the intracellular GSH, decreasing ROS level and this in turn, stimulated embryonic development and regulated gene expression.

SUBMITTER: Mukherjee A

PROVIDER: S-EPMC3933608 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Resveratrol treatment during goat oocytes maturation enhances developmental competence of parthenogenetic and hand-made cloned blastocysts by modulating intracellular glutathione level and embryonic gene expression.

Mukherjee Ayan A Malik Hrudananda H Saha Ambika P AP Dubey Amit A Singhal Dinesh Kumar DK Boateng Samuel S Saugandhika Shrabani S Kumar Sudarshan S De Sachinandan S Guha Shyamal Kanti SK Malakar Dhruba D

Journal of assisted reproduction and genetics 20131204 2

<h4>Purpose</h4>The aim of the present study was to determine whether supplementation of resveratrol, a stilbenoid antioxidant with therapeutic significance, influences goat (Capra hircus) oocyte maturation and subsequent embryonic development and expression of apoptosis and early embryonic development-related genes.<h4>Methods</h4>Five different concentrations of resveratrol (0.1, 0.25, 0.5, 2.0 and 5.0 μM) were used in in vitro maturation (IVM) medium. Cell tracker blue and 2',7'-dichlorodihyd ...[more]

PMID: 24305840

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Project description:BackgroundDNA methylation is one the epigenetic mechanisms, which is critically involved in gene expression. This phenomenon is mediated by DNA methyl-transferases and is affected by environmental stress, including in vitro maturation (IVM) of oocytes. Melatonin, as an antioxidant, may theoretically be involved in epigenetic regulation via reductions of reactive oxygen species. This study was performed to investigate DNA methylation and the possibility of goat oocyte development after treatment with different concentrations of melatonin.Materials and methodsThis experimental study was performed to investigate DNA methylation and the possibility of goat oocyte development after treatment with different concentrations of melatonin. For this purpose, oocytes with granulated cytoplasm were selected and co-cultured with at least two layers of cumulus cells in maturation medium with 10-6 M, 10-9 M, 10-12 M and 0-M (as control group) of melatonin. Nucleus status, glutathione content and developmental competence of the oocytes in each experimental group were assessed. Also, expression of genes associated with DNA methylation, including DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3b (DNMT3b) and DNA methyltransferase 3a (DNMT3a) was evaluated by quantitative real time-polymerase chain reaction (RT-PCR).ResultsAccording to our findings, the percentage of oocytes that reached the M-II stage significantly increased in the 10-12 M group (P<0.05). Also, a significant elevation of glutathione content was observed in melatonin-treated oocytes (P<0.05). Analysis of blastocyst formation revealed that developmental competence of the oocytes was higher than the control group (P<0.05). It was observed that melatonin treatment decreased expression levels of DNA methyltransferases (DNMTs) and global DNA methylation (P<0.05). In addition, the expression of melatonin receptor1A (MTNR1A) was detected in both cumulus and oocyte by RT-PCR.ConclusionThe results suggested that in goat model melatonin affects DNA methylation pattern, leading to an improvement in the developmental competence of the oocytes.

Project description:To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P<0.05) embryos cultured with EAA (12.8%) reached the blastocyst stage as compared with the control group (25.6%) and NEAA group (30.3%). Based on these findings, we identified genes with altered expression in parthenogenetic embryos exposed to medium with or without EAAs. The results indicated that EAA influenced gene expression patterns, particularly those of imprinted genes (e.g., H19, IGF2R, PEG1, XIST). However, NEAAs did not affect impaired imprinted gene expressions induced by EAA. The results also showed that mechanistic target of rapamycin (MTOR) mRNA expression was significantly increased by EAA alone as compared with control cultures, and that the combined treatment with NEAA and EAA did not differ significantly from those of control cultures. Our results revealed that gene transcription levels in porcine embryos changed differentially depending on the presence of EAA or NEAA. However, the changes in the H19 mRNA observed in the parthenogenetic blastocysts expression level was not related to the DNA methylation status in the IGF2/H19 domain. The addition of exogenous amino acid mixtures affected not only early embryonic development, but also gene transcription levels, particularly those of imprinted genes. However, this study did not reveal how amino acids affect expression of imprinted genes under the culture conditions used. Further studies are thus required to fully evaluate how amino acids affect transcriptional regulation in porcine embryos.

Dataset Information

Resveratrol treatment during goat oocytes maturation enhances developmental competence of parthenogenetic and hand-made cloned blastocysts by modulating intracellular glutathione level and embryonic gene expression.

Publications

Resveratrol treatment during goat oocytes maturation enhances developmental competence of parthenogenetic and hand-made cloned blastocysts by modulating intracellular glutathione level and embryonic gene expression.

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