Anti-Aging Effect of Urolithin A on Bovine Oocytes In Vitro

Abstract

Oxidative stress and mitochondrial dysfunction have been associated with the age-related decline of oocyte quality and strategies for their prevention are currently quested. Urolithin A (UA) is a natural metabolite with pro-apoptotic and antioxidant effects, capable of preventing the accumulation of dysfunctional mitochondria in different aged cells. UA has never been tested in bovine oocytes. Our aim was to study the effect of UA on the developmental potential of cumulus-oocyte-complexes (COCs) and granulosa cells' (GCs) expression of important genes related to reproductive competence. Nuclear maturation progression, mitochondrial membrane potential (MMP) and developmental competence of physiologically mature (22 h) and in vitro aged oocytes (30 h of IVM) obtained from prepubertal and adult females, either supplemented with UA or not were assessed. Additionally, the amount of mRNA of several genes (NFE2L2, NQO1, and mt-DN5) and the number of mt-ND5 DNA copies were quantified in cultured GCs from prepubertal and adult females, either supplemented with UA or not. Our study confirmed the harmful effect of oocyte aging on the nuclear maturation progression, MMP, developmental competence and gene expression levels. UA treatment during in vitro maturation enhanced (p < 0.05) the maturation rate and subsequent developmental capacity of aged oocytes. A positive effect (p < 0.05) of UA on physiological maturation, MMP and embryonic development was also identified. UA also interfered on the expression profile of NFE2L2 and NQO1 genes in GCs cultures. Our findings demonstrate that UA supplementation is an effective way to prevent oocyte aging and improves the subsequent bovine embryonic development.

Keywords: Urolithin A; aging; assisted reproductive technologies; oocyte.

Figure 1

Effect of Urolithin A (UA) doses (control, 1 UA, 10 UA, 25 UA, and 50 UA, correspond to 0, 1, 10, 25 and 50 μM of UA, respectively) supplemented to the maturation medium on: (A) oocyte chromosomal configuration. Different letters indicate significant differences between groups for the same stage (CCII, condensing chromosomes II; AI/TI, Anaphase-I/Telophase-I; MII, Metaphase-II, p ≤ 0.05); (B) D7 embryo rate. Different letters indicate significant differences between groups (p ≤ 0.05). D7, day 7.

Figure 2

Effect of the Urolithin A (UA) supplementation to the maturation medium, cumulus-oocyte-complexes (COCs) aging and female age on the oocyte chromosomal configuration (prepubertal: control 22 h, UA 22 h, control aged 30 h, UA aged 30 h; adult: control 22 h, UA 22 h, control aged 30 h, UA aged 30 h groups). Different letters indicate significant differences between groups for the same stage (AI/TI, Anaphase-I/Telophase-I; MII, Metaphase-II) (p ≤ 0.05).

Figure 3

Effect of the Urolithin A (UA) supplementation to the maturation medium, cumulus-oocyte-complexes (COCs) aging and female age on mitochondrial membrane potential (MMP). The assessment of mitochondrial activity was performed using the average of the ratios (aggregate/monomers) for each oocyte analyzed in each group (prepubertal: control 22 h, UA 22 h, control aged 30 h, UA aged 30 h; adult: control 22 h, UA 22 h, control aged 30 h, UA aged 30 h groups). Different letters indicate significant differences between groups (p < 0.05). Data are expressed as the mean ratios ± standard error mean (SEM).

Figure 4

Gene expression levels of NFE2L2, NQO1 and mt-ND5 in granulosa cells (GCs) from prepubertal and adult cows (prepubertal: control, Urolithin A (UA); adult: control, UA, n = 5 for each group) supplemented with UA in the medium culture. Results are normalized to the β-actin gene. mRNA levels in control GCs for NFE2L2, NQO1 and mt-ND5 genes were set to 1. Data are expressed as the CT value mean ± standard error mean (SEM).

Figure 5

mt-ND5 copy number in granulosa cells (GCs) from prepubertal and adult cows (prepubertal: control, Urolithin A (UA); adult: control, UA, n = 5 for each group) supplemented with UA in the medium culture, assessed by qPCR using DNA. Results are normalized to their respective GCs control samples. mt-DNA copy numbers in control GCs for ND5 gene were set to 1. Data are expressed as the ratio of CT values mean ± standard error mean (SEM).