The application of apoptotic inhibitor in apoptotic pathways of MII stage porcine oocytes after vitrification

Abstract

Apoptosis is one of the main drivers of the decline in developmental potential of porcine oocytes after vitrification. However, which apoptotic pathways are engaged after vitrification remains poorly understood. To distinguish among the possible apoptotic pathways induced by vitrification of MII stage porcine oocytes, this study detected activity and expression levels of several key proteins and genes in both the death receptor and mitochondrial pathways using in situ fluorescence staining and real-time PCR (RT-PCR) following the addition of specific inhibitors of either the death receptor or the mitochondrial apoptotic pathway (Z-IETD-FMK or Z-LEHD-FMK, respectively) into the incubation solution. Survival and parthenogenetic developmental ability were also examined. The results showed the following: (i) compared with the vitrified group, the activities of pan-caspase, caspase 3, caspase 8 and caspase 9 as well as the early apoptotic rate were significantly lower in the Z-IETD-FMK and Z-LEHD-FMK groups (p < .05); (ii) After vitrification, mitochondrial ΔΨm decreased from 1.33 for fresh oocytes to 0.90 for vitrified oocytes, while the addition of Z-IETD-FMK or Z-LEHD-FMK following vitrification increased mitochondrial ΔΨm to 1.09 and 1.05, respectively (p < .05); (iii) Relative expression levels of apoptotic-related genes from both the death receptor pathway (caspase 8 and TNF-α) and the mitochondrial pathway (caspase 9, Bcl-2 and CuZnSOD) changed substantially after the addition of Z-IETD-FMK or Z-LEHD-FMK into the incubation solution; (iv) Survival, cleavage and blastocyst rates were higher in the Z-IETH-FMK or Z-LEHD-FMK groups than in the vitrified group (p < .05). In summary, both the death receptor and the mitochondrial apoptotic pathways participate in the apoptosis of MII stage porcine oocytes after vitrification.