In dairy cattle, oocytes isolated from ovaries with a reduced antral follicle count (AFC) have a low embryonic developmental competence. This may be related to oxidative stress, as indicated by our recent finding that ovaries with reduced AFC show a defective endothelial nitric oxide synthase/nitric oxide system. To further test this hypothesis, we evaluated whether the poor developmental competence of these oocytes was possibly due 1) to an imbalance of the reduced glutathione (GSH) system, because GSH is the major antioxidant compound stored within the oocyte and protects the zygote and early embryos from oxidative damage, and 2) to reduced mitochondrial activity. Ovaries were obtained from the abattoir, and oocytes were collected from ovaries with reduced AFC, with fewer than 10 follicles of 2 to 6 mm in diameter, and aged-matched controls, with more than 10 follicles of 2 to 6 mm in diameter. Oocyte GSH content was evaluated using the 5,5′-dithio-bis(2-nitrobenzoic acid)-GSH reductase recycling micro-GSH assay before and after in vitro maturation (IVM) in the presence or absence of 100 µM cysteamine, a GSH precursor. At the same time the developmental competence after IVF was assessed. Moreover, the mitochondrial activity during IVM was evaluated in additional oocytes from the two ovarian categories by specific MitoTracker dyes (MitoTracker FM Green and MitoTracker Orange CMTMRos, Invitrogen, Carlsbad, CA, USA) and subsequent image analysis (ImageJ software). All data were analysed by ANOVA followed by Fisher’s least significant differences test, and P-values <0.05 were considered significant. Experiments were repeated at least three times. Oocytes isolated from ovaries with a low AFC had a similar GSH content compared with oocytes isolated from control ovaries (n = 65 and 85, respectively; 4.31 ± 0.41 v. 4.51 ± 0.42 pmol oocyte–1). After IVM, oocytes from ovaries with reduced AFC showed a significantly lower GSH content compared with control oocytes (n = 55 and 65, respectively; 4.36 ± 0.31 v. 6.59 ± 0.39 pmol oocyte–1); however, cysteamine supplementation during IVM induced GSH accumulation similar to the control (n = 80 and 85, respectively; 9.88 ± 0.77 v. 10.45 ± 0.88 pmol oocyte–1). It is interesting that the increase in intracellular GSH content significantly improved the developmental competence of oocytes from ovaries with a reduced AFC (n = 196 and 201, respectively; 20.1 ± 2.9% v. 6.2 ± 1.6%), although the blastocyst rate remained lower than the control either with or without cysteamine (n = 218 and 212, respectively; 33.3 ± 3.8% and 34.2 ± 2.4%). Further, immature oocytes from ovaries with a low AFC showed a reduced mitochondrial membrane potential compared with control oocytes (n = 13 and 18, respectively; 1.74 ± 1.19 v. 2.22 ± 1.72, calculated as the ratio between the fluorescence of active and total mitochondria), whereas at the end of IVM, it declined in both categories at a comparable level (n = 17 and 24, respectively; 1.19 ± 0.10 and 1.30 ± 0.06). Our data confirmed the hypothesis that both the GSH imbalance and defective mitochondrial activity contribute to the limited developmental competence of oocytes from ovaries with a reduced AFC.
This work was supported by Dote ricerca applicata-FSE, Regione Lombardia, Italy (VL, IT).
Beneficial effects of silymarin against nitric oxide-induced oxidative stress on cell characteristics of bovine oviduct epithelial cell and developmental ability of bovine IVF embryos
