Yingfang Niu1, Jianjun Dai2, Yaning Chen1, Caifeng Wu1, Shushan Zhang1, Defu Zhang3. 1. Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences; Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai, China. 2. Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences; Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai, China. blackman0520@126.com. 3. Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences; Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai, China. zhangdefuzdf@163.com.
Abstract
BACKGROUND: The developmental potential of vitrified porcine oocytes is very lower, and apoptosis is considered as one of the key factors involved. OBJECTIVE: To investigate the effects of apoptotic inhibitor Z-VAD-FMK addition into the incubation medium after warming on apoptosis and developmental ability of vitrified porcine MII-stage oocytes. MATERIALS AND METHODS: The activities of several caspases, mitochondrial membrane potential (ΔΨm) and early apoptotic levels were measured. Parthenogenetic developmental ability and relative expression levels of apoptosis related genes were also detected. RESULTS: Caspase activity and early apoptotic level of the Z-VAD-FMK group were significantly lower than those of the group without Z-VAD-FMK addition, but were much higher than those of fresh group (P < 0.05). The ΔΨm of Z-VAD-FMK group was 1.19, higher than the vitrified group (0.91) and lower than the fresh group (1.33). The cleavage rate and blastocyst rate after parthenogenetic activation in the Z-VAD-FMK group were much higher than those in the vitrified group, and much lower than those in the fresh group (P < 0.05). Vitrified porcine oocytes exhibited increased expression of pro-apoptotic genes (caspase 3, 8, 9, TNF-α) and decreased genes expression levels of anti-apoptotic genes (Bcl-2, CuZnSOD), and the 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 CuZnSOD genes expression. CONCLUSION: The addition of apoptotic inhibitor Z-VAD-FMK into the incubation medium after warming improved the in vitro developmental ability of vitrified porcine oocytes by increasing mitochondrial function, reducing apoptotic level and changing apoptosis-elated gene expression.
BACKGROUND: The developmental potential of vitrified porcine oocytes is very lower, and apoptosis is considered as one of the key factors involved. OBJECTIVE: To investigate the effects of apoptotic inhibitor Z-VAD-FMK addition into the incubation medium after warming on apoptosis and developmental ability of vitrified porcine MII-stage oocytes. MATERIALS AND METHODS: The activities of several caspases, mitochondrial membrane potential (ΔΨm) and early apoptotic levels were measured. Parthenogenetic developmental ability and relative expression levels of apoptosis related genes were also detected. RESULTS:Caspase activity and early apoptotic level of the Z-VAD-FMK group were significantly lower than those of the group without Z-VAD-FMK addition, but were much higher than those of fresh group (P < 0.05). The ΔΨm of Z-VAD-FMK group was 1.19, higher than the vitrified group (0.91) and lower than the fresh group (1.33). The cleavage rate and blastocyst rate after parthenogenetic activation in the Z-VAD-FMK group were much higher than those in the vitrified group, and much lower than those in the fresh group (P < 0.05). Vitrified porcine oocytes exhibited increased expression of pro-apoptotic genes (caspase 3, 8, 9, TNF-α) and decreased genes expression levels of anti-apoptotic genes (Bcl-2, CuZnSOD), and the 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 CuZnSOD genes expression. CONCLUSION: The addition of apoptotic inhibitor Z-VAD-FMK into the incubation medium after warming improved the in vitro developmental ability of vitrified porcine oocytes by increasing mitochondrial function, reducing apoptotic level and changing apoptosis-elated gene expression.