259 DOES THE SENSITIVITY TO OXIDATIVE STRESS DEPEND ON THE SEX OF THE EMBRYO?

Male and female bovine embryos show several differences as early as the blastocyst stage. For example, differences are observed in metabolism, developmental kinetics, or gene expression that can lead to a shift in the sex ratio. Interestingly, the culture medium differentially affects male and female embryos. We previously showed that male Day 7 blastocysts present lower apoptotic rates than females (Ghys et al. 2013 Reprod. Fertil. Dev. 25, 194). The objective of the present study was to determine if such difference might be related to a differential sensitivity to oxidative stress, known to increase apoptosis in bovine blastocysts. In vitro-produced embryos were cultured in a SOF-based medium containing 0.4% BSA. At Day 5 post-insemination (pi) all the embryos were transferred in drops containing the same culture medium supplemented or not with 1 mM 2,2′-azobis(2-amidinopropane) dihydrochloride, an inducer of reactive oxygen species. Blastocysts were collected at Day 7, and apoptosis was evaluated by an immunofluorescent staining of cleaved caspase-3 (8 replicates, n = 175). Total and apoptotic cells were counted using an epifluorescence microscope. As expected, embryos cultured under stress conditions from Day 5 pi presented a lower blastocyst rate at Day 7 (10.9 ± 1.0% v. 23.1 ± 1.9% for the control group; standard least squares, P < 0.0001). The stressed blastocysts also showed fewer cells (113 ± 3 v. 139 ± 4; P < 0.0001) and higher apoptotic rates (15.3 ± 0.9% v. 9.4 ± 0.6%; P < 0.0001). As previously observed, the mean total cell number of the blastocysts was higher for males than females, whatever the culture condition (stress: males: 119 ± 4, females: 108 ± 4; control: males: 144 ± 5, females: 131 ± 6; sex effect: P = 0.005; interaction of sex × condition: P = 0.9). Interestingly, the sex ratio of the blastocysts was significantly different between control and stress conditions (χ2, P = 0.02); whereas a deviation in favour of the male embryos was observed in the control group (males: n = 57, 61%, females: n = 37, 39%; P = 0.04), it disappeared when embryos were submitted to oxidative stress (males: n = 35, 43%, females: n = 46, 57%; P = 0.22). However, oxidative stress had a similar impact on male and female blastocysts regarding the apoptotic rates (stress: males: 15.3 ± 1.3%, females: 15.4 ± 1.3%; control: males: 8.9 ± 0.7%, females: 10.2 ± 1.1%; standard least squares, sex effect: P = 0.99; interaction of sex × condition: P = 0.3). In conclusion, female embryos seem more resistant to oxidative stress than male ones when the stress is induced from Day 5 pi. Oxidative stress has a similar impact on the apoptotic rates in male and female blastocysts. The higher rate of apoptosis previously observed in female blastocysts can thus not be explained by a higher sensitivity of female embryos to oxidative stress. This is in accordance with the higher level of expression of several X-linked genes related to antioxidant pathways in female blastocysts.