3 SIRT1—A POSSIBLE MARKER FOR REPRODUCTIVE AGING OF IN VIVO-DERIVED BOVINE OOCYTES?

Abstract

Maternal aging-associated reduction of oocyte viability is a common feature in mammals. Effective measures to counteract this process have not yet been developed. Cows are commonly used as a model of early human development, including maternal aging, because both species share a very high degree of similarity, including follicle selection, cleavage and blastocyst formation and a long reproductive lifespan. SIRT1, a member of the Sirtuin family, deacetylates transcriptional regulators localised in the nucleus and cytoplasm by a NAD+-dependent mechanism. Resveratrol (3,4′,5-trihydroxystilbene) is an antioxidant identified in various plant species and red wine which enhances SIRT1 activity. Based on these observations, the goal of the present study was to examine, if SIRT1 gene and protein expression is either affected by maternal age and/or can be modulated by resveratrol. Cumulus-oocyte-complexes of prepubertal (5–6 months old) and adult/aged (2 to 8 lactation) cows were collected by ovum pick-up twice a week. Medium for in vitro maturation (TCM 199) and in vitro fertilization (FertTalp) was supplemented with 20 µL of Resveratrol^® (Sigma-Aldrich, Buchs, Switzerland) to get a final concentration of 2 µM Resveratrol respectively. Standard (TCM 199 and FertTalp) media without Resveratol were used as control. Cleavage rates and blastocyst formation were evaluated. Comprehensive gene expression assays of germinal vesicle and metaphase II (MII)-stage oocytes and blastocyst were conducted using next-generation sequencing technology. Finally, SIRT1 protein expression in oocytes and blastocysts were analysed by fluorescence immunostaining under a confocal microscope (LSM510, Zeiss, Germany) and relative fluorescent intensity was calculated. The cleavage rates of adult and prepubertal donors did not differ significantly among the treatments (standard protocol: 56.5 ± 5.4% for adult and 53.0 ± 4.7% for prepubertal donors, Resveratrol supplemented protocol: 62.1 ± 4.3% for cows and 63.6 ± 3.9% for calves). The blastocyst rates were slightly enhanced in the Resveratrol supplemented groups (cows: 34.2 ± 3.8% and calves: 33.1 ± 4.2%) compared to those of standard protocol (cows: 27.5 ± 4.8% and calves: 26.4 ± 3.3%). Relative mRNA abundance levels of SIRT1 were lower in oocytes and blastocysts derived from cows than in those derived from their younger counterparts (2.8-fold change; P = 0.05), but did not differ significantly among treatment groups. Protein expression profiles revealed that bovine SIRT1 was localised in the nucleus. The relative fluorescence levels of SIRT1 were significantly lower (221 ± 34 FIU) in control groups compared to the resveratrol treated groups (865 ± 45 FIU, respectively; P = 0.05). Additionally, SIRT1 protein levels were significantly higher in MII-oocytes (1255 ± 56 FIU) and blastocysts (984 ± 26 FIU) derived from calves compared with their older counterparts (442 ± 37 FIU and 310 ± 23 FIU, respectively, P = 0.05). In conclusion, these results indicate that resveratrol affects SIRT1 protein expression in oocytes and blastocysts of donors in different age. Thus, we hypothesise that SIRT1 is a reliable marker for reproductive aging, which could also be useful for better understanding of human infertility caused by aging.