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 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 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 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

Effect of MG132 on ubiquitinated protein levels in oocytes.

<p>Oocytes from young and aged cows were incubated with or without MG132 for 21 h and then immunostained for ubiquitinated proteins. Average fluorescence intensity of oocytes is shown with values of vehicle-treated oocytes from young cows being set to 1.0 (Y axis). Data are expressed as mean ± SE. a-b, P < 0.05. (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

Effect of CCCP treatment on developmental competence in oocytes derived from young and aged cows.

<p>Effect of CCCP treatment on developmental competence in oocytes derived from young and aged cows.</p

Ratio of MtDNA copy number of CCCP-treated to vehicle-treated oocytes derived from young and aged cows.

<p>Oocytes were collected from individual cows and were treated with CCCP (dark bars) or vehicle (white bars) and then cultured in IVM medium for 19 h. After incubation, the MtDNA copy number was assayed. A−B) After CCCP treatment, oocytes derived from young (A; N = 10) and aged cows (B; N = 10) were cultured in IVM medium. C−D) After CCCP treatment, oocytes derived from young (C; N = 12) and aged (D; N = 13) were cultured in medium containing MG132. Mitochondrial DNA copy number of vehicle-treated oocytes from each donor was set to 1.0 and the average of relative mitochondrial DNA copy number of CCCP-treated oocytes to that of vehicle-treated oocytes is presented. Data are expressed as mean ± SE. a-b, P < 0.05.</p

Effect of CCCP treatment on SIRT1 expression in oocytes.

<p>Oocytes were treated with CCCP for 2 h and then incubated in IVM medium. Immediately following CCCP treatment (A) and 3 h after treatment (B) oocytes were immunostained for SIRT1, and expression levels of fluorescent intensity were presented. Average expression levels of SIRT1 in vehicle treated oocytes from young cows were set to 1.0. Data are expressed as mean ± SE. a-b, P < 0.05. Oocyte from 32 young and 25 aged cows were used in this experiment.</p