Relationship between mitochondrial DNA Copy Number and SIRT1 Expression in Porcine Oocytes

<div><p>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.</p></div

Summary of the effects of resveratrol, MG132, and EX527 on mitochondrial DNA copy number.

<p>Group 1: Supplementing the maturation medium with resveratrol did not affect mitochondrial DNA copy number (Mt number), which may be due to mitochondrial turnover via biogenesis and degradation. Group 2: MG132 significantly increased relative Mt number in oocytes by inhibiting mitochondrial degradation. Group 3: Supplementing media containing MG132 with resveratrol increased Mt number, which may be due to upregulated mitochondrial biogenesis. Group 4: Adding EX527 to the media containing MG132 diminished the effect of resveratrol on Mt number, suggesting that the upregulation of SIRT1 by resveratrol increased Mt number. Group 5 and Group 6: The addition of Ex527, an inhibitor of SIRT1, suppresses both mitochondrial generation and degeneration, which results in no difference between EX527 and vehicle or between EX527+MG132 and vehicle. V; Vehicle, R; Resveratrol, M; MG132, E; EX527, Bold line; mean of relative Mt number.</p

Effect of MG132 on amount of ubiquitinated protein in oocytes.

<p>Oocytes were cultured in medium containing 0 µM or 10 µM MG132. Comparison of the mean lane intensity between the two MG132 concentrations. Average intensity data were normalized to the value of 1 for controls. *,Letters indicate a significant difference (P<0.05).</p

The relationship between SIRT1 expression and mitochondrial DNA copy number.

<p>The relationship between the mean SIRT1 expression and mitochondrial DNA copy number (Mt number) in cohort oocytes collected from the same gilt. The correlation coefficient is 0.413 (P<0.05).</p

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

<div><p>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-<i>m</i>- chlorophenylhydrazone (CCCP), or a vehicle control, after which cumulus oocyte complexes were subjected to <i>in vitro</i> 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.</p></div

Effect of MG132 and CCCP on MtDNA copy number in oocytes.

<p>(A) Twenty COCs collected from individual cows were divided into two groups, and incubated in medium containing vehicle or 10 μM MG132 for 21 h, after which mitochondrial DNA copy number in oocytes was assayed. (B) Twenty COCs collected from individual cows were divided into two groups and treated with the vehicle or 10 μM CCCP for 2 h, then cultured for 19 h in IVM medium. Two IVM media were used in this experiment; an IVM medium where both mitochondrial biogenesis and degradation occur and an IVM medium containing MG132 in which only mitochondrial biogenesis occurs.</p

Effect of CCCP treatment on ROS levels in oocytes derived from young and aged cows.

<p>Oocytes were treated with CCCP or vehicle (Control) and cultured for 19 h. ROS content was assayed immediately after CCCP treatment or following restoration culture (19 h). (A) ROS content in oocytes. Y axis: relative ROS content in oocytes. Average fluorescence intensity of vehicle-treated oocytes from young cows was set to 1.0 and values represent the fold difference in fluorescence intensity. Data is expressed as mean ± SE. a−c, P < 0.05. (B) Representative fluorescent images of cultured oocytes treated with or without CCCP treatment followed by staining with ROS detection reagents are shown. (N = 7, described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188099#pone.0188099.t001" target="_blank">Table 1</a>).</p

Summary of the effect of CCCP treatment on MtDNA copy number in oocytes.

<p>MG132, which inhibits mitochondrial degeneration, increased MtDNA copy number in the oocytes of young cows (1 vs. 2), but not in the oocytes of aged cows (5 vs. 6), indicating that there was less mitochondrial biogenesis and degeneration in the oocytes of aged cows. CCCP treatment of oocytes did not affect mitochondrial DNA copy number when oocytes were incubated in IVM medium without MG132 (1 vs. 3). However, in medium containing MG132, CCCP treatment upregulated mitochondrial biogenesis in the oocytes of young cows (2 vs. 4), but did not affect mitochondrial biogenesis in oocytes of aged cows (6 vs. 8). White arrows indicate degradation, black arrows indicate biogenesis and dotted arrows indicate inhibition of degeneration.</p

Number of donor and oocytes used in experiments.

<p>Number of donor and oocytes used in experiments.</p

Effect of CCCP treatment on ATP content of oocytes derived from young and aged cows.

<p>COCs from young and aged cows were treated with CCCP or vehicle (Control) for 2 h, and then ATP content in the oocyte was determined. Data are presented as mean ± SE. a−c indicate statisticaly significantly differences, P < 0.001. (N = 8, described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188099#pone.0188099.t001" target="_blank">Table 1</a>).</p