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Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes.

ABSTRACT: In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Conventional in vitro maturation (IVM) impairs oocyte competence by inducing spontaneous meiotic resumption. A series of experiments were performed to determine if maintaining meiotic arrest during a pre-maturation culture phase (pre-IVM) prior to conventional IVM improves oocyte competence of juvenile-goat (2 months old) cumulus-oocyte complexes (COCs). In experiment 1, COCs were cultured with C-type natriuretic peptide (CNP; 0, 50, 100, 200 nM) for 6 and 8 h. Nuclear stage was assessed, revealing no differences in the incidence of germinal vesicle (GV) breakdown. In experiment 2, the same CNP concentrations were assessed plus 10 nM estradiol, the known upstream agonist activating expression of NPR2, the exclusive receptor of CNP. CNP (200 nM) plus estradiol increased the rate of oocytes at GV stage at 6 h compared to control group (74.7% vs 28.3%; P<0.05) with predominantly condensed chromatin configuration. In experiment 3, relative mRNA quantification revealed NPR2 expression was down-regulated after pre-IVM (6 h). In experiment 4, analysis of transzonal projections indicated that pre-IVM maintained cumulus-oocyte communication after oocyte recovery. For experiments 5 and 6, biphasic IVM (6 h pre-IVM with CNP and estradiol, plus 24 h IVM) and control IVM (24 h) were compared. Biphasic IVM increased intra-oocyte glutathione and decreased ROS, up-regulated DNA-methyltransferase 1 and pentraxin 3 expression and led to an increase in rate of blastocyst development compared to control group (30.2% vs 17.2%; P<0.05). In conclusion, a biphasic IVM, including a pre-IVM with CNP, maintains oocyte meiotic arrest for 6 h and enhances the embryo developmental competence of oocytes from juvenile goats.

SUBMITTER: Soto-Heras S

PROVIDER: S-EPMC6707569 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes.

Soto-Heras Sandra S Menéndez-Blanco Irene I Catalá Maria-Gracia MG Izquierdo Dolors D Thompson Jeremy G JG Paramio Maria-Teresa MT

PloS one 20190823 8

In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Conventional in vitro maturation (IVM) impairs oocyte competence by inducing spontaneous meiotic resumption. A series of experiments were performed to determine if maintaining meiotic arrest during a pre-maturation culture phase (pre-IVM) prior to conventional IVM improves oocyte competence of juvenile-goat (2 months old) cumulus-oocyte complexes (COCs). In experiment 1, COCs were c ...[more]

PMID: 31442286

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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:PurposeThe 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.MethodsFive 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'-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.ResultCompared to control, no significant improvement was observed in nuclear maturation in resveratrol-treated groups and at 5.0 μM concentration maturation rate decreased significantly (P < 0.05). But resveratrol treatment at the concentrations of 0.25, 0.5 μM significantly reduced intracellular ROS, and increased GSH concentrations. Oocytes treated with 0.25, 0.5 μM 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.ConclusionOur results show that resveratrol treatment at optimum concentrations (0.25 and 0.5 μM) 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.

Dataset Information

Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes.

Publications

Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes.

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