The present study assessed the effect of resveratrol on the expression of SIRT1 and mitochondrial quality and quantity in porcine oocytes. Supplementing the maturation medium with 20 µM resveratrol increased the expression of SIRT1, and enhanced mitochondrial functions, as observed from the increased ATP content and mitochondrial membrane potential. Addition of resveratrol also improved the ability of oocytes to develop into the blastocyst stage following activation. The effects of resveratrol on mitochondrial number were examined by comparing the mitochondrial DNA copy number (Mt number) between group of oocytes collected from the same donor gilt ovaries. Supplementing the maturation medium with only resveratrol did not affect the Mt number in the oocytes. However, supplementing the maturation medium with 10 µM MG132, a proteasome inhibitor, significantly increased the amount of ubiquitinated proteins and Mt number by 12 and 14%, respectively. In addition, when resveratrol was added to the medium containing MG132, the Mt number increased significantly by 39%, this effect was diminished by the addition of the SIRT1 inhibitor EX527. Furthermore, supplementing the medium with MG132 and EX527 did not affect Mt number. The mean SIRT1 expression in 20 oocytes was significantly and positively correlated with the Mt number in oocytes collected from the same donor. This study suggests that the expression of SIRT1 is associated with the Mt number in oocytes. In addition, activation of SIRT1 by resveratrol enhances the biosynthesis and degradation of mitochondria in oocytes, thereby replenishing and improving mitochondrial function and the developmental ability of oocytes.
In general, maternal age affects the quality of oocytes and embryos. The present study aimed to examine the features and age-associated gene expression profiles of bovine oocytes and embryos as well as to discover possible countermeasures against age-associated events. Comprehensive gene expression assays of germinal vesicle and metaphase II (MII)-stage oocytes and 8- to 16-cell-stage embryos were conducted using next-generation sequencing technology. The gene expression profiles of aged cows showed high expression of genes related to oxidative phosphorylation, eIF4 and p70S6K signaling, and mitochondrial dysfunction in MII-stage oocytes. Oocytes derived from aged cows, compared with those derived from their younger counterparts, exhibited high levels of abnormal fertilization and blastocysts with low total cell numbers. Levels of reactive oxygen species (ROS) and SIRT1 were higher in in vitro-matured oocytes derived from aged cows than in those derived from their younger counterparts. Supplementation of maturation medium with N-acetyl-cysteine (NAC), but not resveratrol, reduced the levels of ROS in the oocytes derived from cows of both age groups; however, resveratrol, but not NAC, improved the fertilization ratio. Conversely, EX 527, an inhibitor of SIRT1, increased the ratio of abnormal fertilization. In conclusion, gene expression profiles of oocytes and embryos derived from aged cows differ from those of oocytes and embryos derived from young cows; in particular, oocytes derived from aged cows show protein and mitochondrial dysfunction. In addition, activation of SIRT1 in oocytes may be a potential countermeasure against age-associated events in oocytes derived from aged cows.
The aim of the present study was to address the effect of resveratrol-mediated upregulation of sirtuin 1 (SIRT1) during oocyte maturation on mitochondrial function, the developmental ability of oocytes and on mechanisms responsible for blockage of polyspermic fertilization. Oocytes collected from slaughterhouse-derived ovaries were cultured in TCM-199 medium supplemented with 10% FCS and 0 or 20 µM resveratrol (Res). We examined the effect of Res on SIRT1 expression in in vitro-matured oocytes (Exp 1); fertilization and developmental ability (Exp 2); mitochondrial DNA copy number (Mt number), ATP content and mitochondrial membrane potential in matured oocytes (Exp 3); and the time required for proteinase to dissolve the zona pellucida following in vitro fertilization (as a marker of zona pellucida hardening), as well as on the distribution of cortical granules before and after fertilization (Exp 4). In Exp 1, the 20 µM Res treatment upregulated protein expression of SIRT1 in oocytes. In Exp 2, Res treatment improved the ratio of normal fertilization and the total cell number of blastocysts. In Exp 3, Res treatment significantly increased the ATP content in matured oocytes. Additionally, Res increased the overall Mt number and mitochondrial membrane potential, but the effect was donor-dependent. In Exp 4, Res-induced zona hardening improved the distribution and exocytosis of cortical granules after in vitro fertilization. In conclusion, Res improved the quality of oocytes by improving mitochondrial quantity and quality. In addition, Res added to the maturation medium enhanced SIRT1 protein expression in oocytes and improved fertilization via reinforcement of the mechanisms responsible for blockage of polyspermic fertilization.
Age-associated deterioration in both the quality and quantity of mitochondria occurs in older women. The main aim of this study was to examine the effect of age on mitochondrial DNA copy number (mtDNA number) in early developmental stage bovine embryos as well as the dynamics of mtDNA number during early embryo development. Real-time PCR was used to determine mtDNA number. In vitro-produced embryos 48 h after insemination derived from Japanese black cows, ranging in age from 25 to 209 months were categorized based on their cleavage status. There was an overall negative relationship between the age of the cow and cleavage status, to the extent that the ratio of embryos cleaved over the 4-cell stage was greater in younger cows. The mtDNA number did not differ among the cleaved status of embryos. In the next experiment, oocytes collected from each donor cow were divided into 2 groups containing 10 oocytes each, in order to compare the mtDNA number of mature oocytes and early developmental stage embryos within individuals. Upon comparing the mtDNA number between oocytes at the M2 stage and early developmental stage 48 h post insemination, mtDNA number was found to decrease in most cows, but was found to increase in some cows. In conclusion, age affects the cleaving ability of oocytes, and very old cows (> 180 months) tend to have lower mtDNA numbers in their oocytes. The change in mtDNA number during early development varied among individual cows, although overall, it showed a tendency to decrease.
Granulosa cells influence the growth and acquisition of the developmental competence of oocytes. We investigated the effects of ageing on the proliferative activity, global genomic DNA methylation, relative telomere length and telomerase activity of bovine granulosa cells. The proliferative activity of cells was examined by bromodeoxyuridine (BrdU) assay, genomic DNA methylation was examined by enzyme-linked immunosorbent assay (ELISA), and relative telomere length and telomerase activity were examined by real-time polymerase chain reaction. We first compared the proliferative activity of the granulosa cells of the medium follicles between in dominant phase ovaries and growth phase ovaries. We observed that the proliferative activity of the granulosa cells of dominant phase ovaries was significantly lower than those of growth phase ovaries. In addition, the proliferative activity of granulosa cells was inversely associated with follicular size. Based on the results, we used granulosa cells harvested from the medium follicles (3-5 mm in diameter) on the surfaces of the dominant phase ovaries collected from cows at a slaughterhouse. The proliferative activity of the granulosa cells harvested from the ovaries of old cows (N = 8; average age 165.1 months) was lower than that of the cells from young cows (N = 8; average age 30.9 months). Global loss of cytosine methylation was detected in the granulosa cells of old cows (N = 12; average age 141.0 months) compared with young cows (N = 15; average age 27.4 months). Although the relative telomere lengths of cumulus cells were similar in the two age groups, the relative telomere lengths and telomerase activity of the granulosa cells from old cows (N = 17 and 9; average age, 164.6 and 151.3 months, respectively) tended to be shorter than those of the cells from young cows (N = 17 and 10; average age 30.6 and 28.1 months, respectively); however, this difference was not significant p = 0.09 and 0.053, respectively). In conclusion, the proliferative activity and genomic global DNA methylation significantly decreased, and the relative telomere lengths and telomerase activity of granulosa cells tended to be shorter with the age of donor cows.
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