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

Abstract

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.

Mitochondrial biogenesis and degradation are induced by CCCP treatment of porcine oocytes

“…To highlight mitochondrial biogenesis during IVM, two cultures were used: standard IVM medium and IVM medium containing the proteasome inhibitor MG132. The addition of MG132 to the IVM medium inhibits protein degradation, as previously confirmed by the accumulation of ubiquitinated proteins in oocytes analysed by western blotting (Sato et al 2014) as well as by immunostaining against ubiquitinated proteins in the present study (Supplementary Figure 1, see section on supplementary data given at the end of this article). In the medium containing MG132, mitochondrial degradation is also inhibited and culturing oocytes in the medium for 44 h thus significantly increased the Mt number (Sato et al 2014).…”

“…This result indicates that mitochondrial kinetics depend on the oocyte developmental stage, which may reflect the progression of the follicle phase in individual donors. In our previous reports, mitochondrial number determined using ten oocytes was almost identical to that determined using other cohorts of ten oocytes derived from the same donor gilts, at both the GV and M2 stages (Sato et al 2014), indicating that comparison within cohort oocytes may avoid the bias by mixing oocytes from differential follicular phase. Sato et al (2014) collected 20 oocytes from individual donor gilts, divided them into two groups and cultured the oocytes in a medium containing a proteasome inhibitor (MG132) or vehicle.…”

“…Mitochondrial DNA copy numbers differ a great deal among donors, but the Mt number predicted by ten oocytes closely reflects the Mt number of other cohort oocytes collected from the same donors in cows and pigs (Iwata et al 2011, Sato et al 2014. Thus, in the present experiments, 20 oocytes were collected from a donor and divided into two groups: ten oocytes were treated with 10 mM CCCP and the other ten were treated with 0 mM CCCP (vehicle control).…”

“…The oocyte number used for Mt number assays was based on previous reports that demonstrated that the Mt number determined from ten oocytes collected from a donor closely resembles the Mt number obtained by another group of ten cohort oocytes collected from the same donor (Sato et al 2014). Oocytes were denuded from granulosa cells and the DNA extraction and PCR protocols procedures were performed according to previously described methods (Iwata et al 2011).…”

“…Despite the presence of both de novo synthesis and degradation pathways in somatic cells, mitochondrial turnover during oocyte maturation has not been investigated. In this context, Sato et al (2014) reported that when oocytes were cultured in a medium containing a proteasome inhibitor, mitochondrial DNA copy number (Mt number) increased during oocyte maturation, suggesting that proteasomes play a role in mitochondrial removal. Using the abovementioned culture conditions, the authors further showed that resveratrol upregulates mitochondrial biosynthesis.…”