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 μM) were used in in vitro maturation (IVM) medium. Cell tracker blue and 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA) 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 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. Conclusion Our 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.

No abstract

Nitric oxide (NO), a free radical molecule, is produced from L-arginine by the action of nitric oxide synthase (NOS), which exists in three isoforms: inducible (iNOS), endothelial (eNOS) and neuronal (nNOS, Clementi et al. 1999). There are several reports on the putative role NO pathway plays in oocyte maturation in mice (Sengoku et al. 2001) and cattle (Viana et al. 2007). However, the role of NO and iNOS in embryo development is not clear. This study was taken up to investigate the effects of dose-dependent application of sodium nitroprusside (SNP), a NO donor, or aminoguanidine (AG), an inhibitor of iNOS, to in vitro culture (IVC) medium on the development of buffalo (Bubalus bubalis) embryos.Oocytes were aspirated from slaughterhouse buffalo ovaries, and those with an unexpanded multilayered cumulus mass and homogeneous cytoplasmic granulation were subjected to in vitro maturation (IVM) by culture in 100 ll droplets (10-15 oocytes⁄droplet) of IVM medium (TCM-199 + 10% FBS + 5 lg⁄ml pFSH + 1 lg⁄ml estradiol)17b + 0.81 mM sodium pyruvate + 50 lg⁄ml gentamycin sulphate) for 24 h in a CO 2 incubator (5% CO 2 in air) at 38.5°C. In vitro fertilization was carried out (Chauhan et al. 1998) by incubating 2-4 million spermatozoa⁄ml for 18 h in a CO 2 incubator at 38.5°C. The presumed zygotes were then divided into different groups and were cultured in IVC medium consisting of modified Charles Rosenkrans2 medium with amino acids (mCR2aa, Kumar et al. 2007,) +0.8% BSA + 10% FBS containing SNP (0, 10 )8 , 10 )6 or 10 )4 M) or AG (0, 1, 10 or 50 mM) for up to 9 days postinsemination. The IVC medium was replaced with fresh medium containing respective concentrations of SNP or AG at 48 and 96 h post-insemination. The cleavage rate was recorded at 48 h post-insemination, whereas the embryonic development was examined at days 5, 7, 8 and 9 post-insemination. The data were analysed by one way analysis of variance followed by Fisher's LSD test using SYSTAT 7.0 (SPSS Inc., Chicago, IL, USA) after arcsin transformation of percentage values.At a concentration of 10 )4 M, SNP reduced (p < 0.01) the cleavage rate to less than half that of the controls and none of the cleaved embryos developed beyond the four-to eight-cell stage, whereas at the intermediate concentration of 10 )6 M, SNP decreased (p < 0.01) day 9 hatched blastocyst production rate (Table 1). However, at the lowest concentration of 10 )8 M, SNP increased (p < 0.05) the yield of day 7 blastocysts and day 8 hatched blastocysts.Aminoguanidine (50 mM) reduced (p < 0.01) the cleavage rate and none of the cleaved embryos developed beyond the four-to eight-cell stage (Table 2). In the presence of 10 mM AG, day 5 morulae production rates were reduced (p < 0.01) and none of the 42 cleaved embryos taken developed beyond the morula stage. Similarly, the percentage of cleaved embryos that developed to morulae on day 5, to blastocysts on day 8 or to blastocysts or hatched blastocysts on day 9 postinsemination was significantly lower (p < 0.01) with 1 mM AG compared to that of con...

No abstract

No abstract