| The cryopreservation technique of gametes and embryos plays an important role in assisted reproductive field and the conservation of human fertility,and also plays an important role in the intercultural exchange of germplasm resources.After several decades of development,vitrification technology has been greatly improved,but organelles,cytoskeleton,DNA damage and apoptosis are still the main reasons for the decline of embryonic development after vitrification.This study detected the blastocyst development and apoptotic level of porcine vitrifiedparthenogenetic blastocysts.And the effects of resveratrol and Pan-caspase inhibitor on blastocyst development and apoptosis after cryopreservation were also evaluated during the whole process of blastocyst culture or the incubation solution after warming.The objective of the first part was to evaluate the effect of vitrification on apoptosis of porcine parthenogenetic blastocysts.The blastocoel recovery,mitochondrial membrane potential,early apoptotic level and activities of several caspases from different pathways of vitrified-thawed blastocysts were measured,and the mRNA expression levels of apoptosis related genes involved in different apoptotic pathways were also detected.The results showed that the blastocoel recovery rate and total cellsof vitrified blastocysts were significantly lower than those of fresh blastocysts(P<0.05).The mean mitochondrial ??m of vitrified blastocysts was 0.46,which was significantly lower than that of fresh blastocysts(1.02,P<0.05).The rate of apoptotic cells in vitrified blastocysts after TUNEL assay was much higher than that in fresh blastocysts(P<0.05).The pan-caspase,caspase-3,caspase-8 and caspase-9 activities from different apoptotic pathways in vitrified blastocysts were significantly higher than those in fresh blastocysts(P<0.05).qRT-PCR results showed that the expression levels of Caspase-8 and TNF-? from death receptor apoptotic pathway,and Caspase-9 from mitochondrial apoptotic pathway in vitrified group were significantly higher than those in fresh group(P<0.05),The expression levels of Bcl-2 and SOD-1 from mitochondrial pathway in vitrified group were significantly lower than those in fresh group(P<0.05).This study concluded that the in vitro developmental competence of blastocysts after vitrification was reduced by the decreasing of mitochondrial function and the increasing of apoptotic levels mediated by both mitochondria and death receptor apoptotic pathways.Resveratrol were added during culture,and the rate of blastocoel recovery of frozen thawed blastocysts were observed,mitochondrial membrane potential,early apoptotic levelsand the activities of severalcaspases were measured,and the mRNA expression levels of genes involved in different apoptotic pathways were measured by qRT-PCR method.The results showed that the cleavage rate and the blastocyst rate of RES addition group(95.18%;51.85%)were significantly higher than fresh group(85.79%;41.46%;P<0.05).The rate of re-expanded blastocysts and total cells of RES vitrified blastocysts were significantly lower than vitrification group(P<0.05).The mitochondrial ??m of RES vitrification groupwas significantly higher than vitrification group(P<0.05),but they all lower than fresh blastocyst.TUNEL assay combined with Hoechst33342 staining revealed a significantly higher apoptotic ratein the two vitrification group than that of fresh group(P<0.05),and the apoptotic level of RES vitrification group was significantly decreased than the vitrification group without RES addition.The fluorescence intensity of Pan-caspase,Caspase-3,Caspase-8 and Caspase-9 activities of RES vitrification groupwere significantly lower than vitrification group(P<0.05),but they were all higher than fresh blastocysts(P<0.05).qRT-PCR results showed that the genes Caspase-8,Caspase-9 and TNF-? in RES vitrification group were higher than fresh group(P<0.05),but were significantly lower than vitrification group(P<0.05).The genes Bcl-2 and SOD-1 in RES vitrification group were significantly lower than fresh group(P<0.05),but were higher than vitrification group(P<0.05).In conclusion,after RES addition before and after vitrification,the mitochondrial function was improved,apoptotic level was reduced,and then the developmental ability of blastocyst after warming was increased.Z-VAD-FMK was added during incubation after warming.The blastocoel recovery,mitochondrial membrane potential,early apoptotic level and activities of several caspases from different pathways of vitrified-thawed blastocysts were measured,and the mRNA expression levels of apoptosis related genes involved in different apoptotic pathways were also detected.The results showed that the rate of re-expanded blastocysts and total cells of Z-VAD-FMK group were significantly higher than vitrification group(P<0.05).The mitochondrial ??m of Z-VAD-FMK group was also higher than vitrification group(P<0.05).TUNEL assay combined with Hoechst33342 staining revealed a significantly higher rate of DNA fragmentation in Z-VAD-FMK group and vitrification group than fresh group(P<0.05),and the apoptosis level was significantly decreased by adding Z-VAD-FMK after warming.The fluorescence intensity of Pan-caspase,Caspase-3,Caspase-8 and Caspase-9 activities of Z-VADFMK group were significantly lower than vitrification group(P<0.05),but they all higher than fresh blastocyst(P<0.05).qRT-PCR results showed that the genes Caspase-8,Caspase-9 and TNF-? in Z-VAD-FMK group were higher than fresh group(P<0.05),but were significantly lower than vitrification group(P<0.05).The genes Bcl-2 and SOD-1 in Z-VAD-FMK group were significantly lower than fresh group(P<0.05),but were higher than vitrification group(P<0.05).In conclusion,after Z-VAD-FMK addition in the incubation medium,the mitochondrial function was improved,apoptotic level was reduced,and then the developmental ability of blastocyst after warming was increased. |
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