The Application Of Apoptotic Inhibitors On Apoptosis Pattern In Porcine Oocyte After Vitrification

Due to the large amount of cytoplasmic lipid, cryopreservation of porcine oocytes is still very difficult, which primarily manifested in the lower potential of continued development. Therefore, studies on injury mechanism are very important. In conpreservation of animal oocyte, apoptosis is considered one of main reasons for declined developmental potential. And there are little researches on apoptosis of vitrified porcine oocyte, especially for cryopreservation induced apoptotic pathway in vitrified porcine oocyte. In this experiement, we studied apoptotic pathway of vitrified porcine oocyte by adding inhibitors of key apoptotic factors casapse8 and caspase9 in receptor mediated and mitochondrial mediated apoptotic pathway during incubation, and pan-caspase inhibitor in both pathways, in which we also clarified positive effect of apoptotic inhibitor on its development. This study deepened the awareness of apoptotic mechanism in vitrified porcine oocyte and provided theoretical and experimental basis for improving development of porcine oocytes after vitrification. The experiment includes two parts:The first part explored the possible apoptotic pathways of MΠ stage porcine oocyte after vitrification by applying of specific apoptotic inhibitors. Apoptosis is one of the main reasons for declined developmental potential in porcine oocyte after vitrification, but the apoptotic pathway is poorly understood. Z-IETD-FMK, Z-LEHD-FMK and Z-DEVD-FMK are specific inhibitors of caspase8, caspase9 and caspase3 in death-receptor, mitochondrial apoptotic pathway and their commom pathway. To further study apoptotic pathway of MΠ stage porcine vitrified oocyte, this study detected activities and expressions of key proteins and molecular using in situ fluorescence staining and Real-time PCR(RT-PCR). Survival and parthenogenetic development were also examined. Results showed: 1) compared with vitrified group, pan-caspase, caspase3, caspase8, caspase9 activities and early apoptotic rate in Z-IETD-FMK, Z-LEHD-FMK and Z-DEVD-FMK groups were significantly decreased(P < 0.05); 2) after vitrification, mitochondrial △Ψm was decreased from 1.33 to 0.90, while Z-IETD-FMK, Z-LEHD-FMK and Z-DEVD-FMK significantly increased mitochondrial △Ψm in vitrified oocyte(1.09, 1.05 and 1.11, P < 0.05); 3) Z-IETD-FMK, Z-LEHD-FMK and Z-DEVD-FMK were beneficial for relative gene expressions in death-receptor pathway(caspase 8 and TNF-α) and mitochondrial pathway(caspase 9, Bcl-2 and Cu Zn SOD); 4) suvivial, cleavage and blastocyst rate were decreased in vitrified group, and they were significantly increased in Z-IETH-FMK, Z-LEHD-FMK and Z-DEVD-FMK groups(P < 0.05).The second part was analysised the effect of pan-caspase inhibitor Z-VAD-FMK on apoptosis and devdloment ability in porcine vitrified-thawed MII stage oocytes. Developmental potential of vitrified oocytes is very lower in porcine, and apoptosis is considered as one of the key factors. Z-VAD-FMK is a pan-caspasse inbihitor. In order to investigate the effects of apoptotic inhibitor Z-VAD-FMK addition in incubation medium after warming on apoptosis and developmental ability of porcine vitrified MII-stage oocytes. This study measured several caspase activities, mitochondrial membrane potential(ΔΨm) and early aopptotic levels of oocytes with different fluorescent staining. Parthenogenetic developmental ability and relative expression levels of apoptosis related genes were also detected. The caspase activity and early apoptotic level from Z-VAD-FMK addition group were significantly lower than those from vitrified group without Z-VAD-FMK addition, and were much higher than those from fresh group(P < 0.05). The mitochondrial ΔΨm of Z-VAD-FMK group was 1.19, which was significantly higher than vitrified group(0.91), and lower than fresh group(1.33). The cleavage rate and blastocyst rate after parthenogenetic activation in Z-VAD-FMK group were much higher than those in vitrified group, and which were much lower than those in fresh group(P < 0.05). Porcine vitrified oocytes exhibited increased gene expression levels of pro-apoptotic genes(caspase 3, 8, 9, TNF-α) and decreased genes expression levels of anti-apoptotic genes(Bcl-2, Cu Zn SOD), and Z-VAD-FMK addition in incubaiton medium significantly decreased the transcripts levels of caspase 3,8,9, Bax, TNF-α and increased Bcl-2 and Cu Zn SOD genes expression.Conclusion:In summary, it is proved in another way that death-receptor and mitochondrial apoptotic pathway may both participated in apoptosis of MΠ stage porcine vitrified oocyte; The addition of apoptotic inhibitor Z-VAD-FMK in incubation medium after warming improved the in vitro developmental ability of vitrified porcine oocytes by increasing mitochondrial function, reducing apoptosis level and changing apoptosis related gene expression levels.