Maternal aging affects oocyte resilience to carbonyl cyanide-m-chlorophenylhydrazone -induced mitochondrial dysfunction in cows.

Mitochondrial quality control is important for maintaining cellular and oocyte viability. In addition, aging affects mitochondrial quality in many cell types. In the present study, we examined how aging affects oocyte mitochondrial biogenesis and degeneration in response to induced mitochondrial dysfunction. Cumulus oocyte complexes were harvested from the ovaries of young (21‒45 months) and aged (≥120 months) cows and treated for 2 hours with 10 µM carbonyl cyanide-m- chlorophenylhydrazone (CCCP), or a vehicle control, after which cumulus oocyte complexes were subjected to in vitro fertilization and culture. CCCP treatment reduced ATP content and increased reactive oxygen species (ROS) levels in the oocytes of both young and aged cows. When CCCP-treated cumulus oocyte complexes were subsequently cultured for 19 hours and/or subjected to fertilization, high ROS levels in oocytes and a low rate of blastocyst development was observed in oocytes derived from aged cows. In addition, we observed differential responses in mitochondrial biogenesis to CCCP treatment between young and aged cows. CCCP treatment enhanced mitochondrial biogenesis concomitant with upregulation of SIRT1 expression in oocytes of young, but not aged, cows. In conclusion, aging affects mitochondrial quality control and recuperation of oocytes following CCCP-induced mitochondrial dysfunction.

Age-associated deterioration in follicular fluid induces a decline in bovine oocyte quality.

Maternal age affects the quality of oocytes. The present study examined whether follicular fluid (FF) is a casual factor for age-associated decline in oocyte quality. First, we measured the concentration of advanced glycation end-products (AGE) in FF derived from young (21-45 months; Young-FF) and aged (≥120 months; Aged-FF) cows and found significantly higher concentrations of AGE in Aged-FF than Young-FF. Second, oocytes were collected from ovaries of young or aged cows and cultured in maturation medium containing 10% FF derived from young or aged cows. Regardless of oocyte origin, Aged-FF accelerated nuclear maturation progression and gap junction closure between oocytes and cumulus cells, increased reactive oxygen species (ROS) content and the rate of abnormal fertilisation of oocytes and decreased blastulation rate compared with Young-FF. Furthermore, supplementation of maturation medium with AGE induced similar age-associated events in oocytes derived from young cows, in that AGE accelerated the progression of nuclear maturation, increased ROS content in oocytes, increased the rate of abnormal fertilisation and decreased blastulation rate. In conclusion, maternal aging increased the concentration of AGE in FF, and both AGE and Aged-FF accelerated nuclear maturation and reduced the developmental competence of oocytes.

Liver condition of Holstein cows affects mitochondrial function and fertilization ability of oocytes.

The aim of the present study was to examine the fertilization ability and mitochondrial function of oocytes derived from cows with or without liver damage. Oocytes were collected from the ovaries of cows with damaged livers (DL) and those of cows with healthy livers (HL), subjected to in vitro maturation, and fertilized in vitro. A significantly high abnormal fertilization rate was observed for oocytes from DL cows compared to oocytes from HL cows. The time to dissolve the zona pellucida by protease before fertilization was similar between the two liver conditions, whereas after fertilization treatment this time was shorter for DL cows than for HL cows. The percentage of oocytes with equivalent cortical granule distributions underneath the membrane was greater for in vitro matured oocytes from HL cows, whereas an immature distribution pattern was observed for oocytes from DL cows. In addition, a greater percentage of oocytes derived from HL cows released cortical granules following fertilization compared with oocytes from DL cows. Mitochondrial function determined by ATP content and membrane potential were similar at the germinal vesicle stage, but post-in vitro maturation, the oocytes derived from HL cows showed higher values than DL cows. The mitochondrial DNA copy number in oocytes was similar between the two liver conditions for both the germinal vesicle and post-in vitro maturation oocytes. In conclusion, liver damage induces low fertilization, likely because of incomplete cortical granule distribution and release, and the maturation of oocytes from DL cows contain low-functioning mitochondria compared to their HL counterparts.

Effect of 5-aminoimidazole-4-carboxamide ribonucleoside on the mitochondrial function and developmental ability of bovine oocytes.

Oocyte nuclear maturation depends on sufficient energy supply through oxidative phosphorylation and ß-oxidation. AMP-activated protein kinase (AMPK) is an energy sensor controlling the oocyte energy metabolism. The main aim of this study was to examine the effect of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a potent activator of AMPK, on the ATP content and mitochondrial DNA copy number (Mt-number) of bovine oocytes and on their developmental ability. Oocytes were collected from slaughterhouse-derived bovine ovaries. When these oocytes were cultured in a maturation medium containing 0-, 50-, 250-, and 500-µM AICAR, higher AICAR concentrations reduced the rate of meiotic maturation and the ATP content in oocytes, whereas lower AICAR increased the ATP content in oocytes without affecting the maturation rate. Supplementation of the maturation medium with a low concentration of AICAR (50 and 250 µM) increased phospho-AMPK expression level, as determined by immunostaining. In addition, AICAR treatment increased the ATP content in oocytes, which remained elevated for as long as 2 days after fertilization. On culturing the oocytes with AICAR (250 µM), the fertilization outcome, rate of blastulation, and total cell number of the blastocysts significantly improved. When the proteosomal mitochondrial degradation was inhibited by supplementing the maturation medium with MG132, the Mt-number, as determined by real-time polymerase chain reaction, significantly increased. However, the treatment of oocytes with AICAR did not affect the Mt-number in the presence or absence of MG132. From these data, we conclude that low concentrations of AICAR improved the embryonic developmental ability, presumably via the upregulation of the ATP content in oocytes, but the increase in the ATP content was not due to the upregulation of mitochondrial biogeneration.

Relationship between mitochondrial DNA copy number and SIRT1 expression in porcine oocytes.

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.

Resveratrol improves the mitochondrial function and fertilization outcome of bovine oocytes.

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.

Effect of maternal age on the ratio of cleavage and mitochondrial DNA copy number in early developmental stage bovine embryos.

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.

Effect of bovine age on the proliferative activity, global DNA methylation, relative telomere length and telomerase activity of granulosa cells.

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.