| As an important method for conservation of porcine genetic resources,oocyte cryopreservation gained more and more attention.Compared with human,mouse,cattle,goat and other mammals,the efficiency of cryopreservation of porcine oocytes is very low,and the studies about freezing damage mechanism are still very scarce.More deep researches need to be carried out.The apoptotic and mitochondrial damage of embryos and oocytes after cryopreservation were regarded as the main reasons for their poor survival ability after warming.However,there is no systematic studies on the apoptosis,mitochondrial damage and gene expression of porcine vitrifed oocytes.In order to improve the survival rate,it is important to clarify the main apoptotic pathways of oocytes after vitrification.In order to understand the damage degree of mitochondria after vitrification,the paper observed the mitochondrial distribution,ultrastructure,mitochondrial membrane potential(??m),ROS(reactive oxygen species)level,ATP(adenosine-5'-triphosphate)concentration and mitochondrial function related gene expression of pocine vitrified oocytes.For enucleating the apoptotic pathways of porcine vitrified-warmed oocytes,this study tested the early stage apoptotic rate by Annexin V-FITC staining and detected the key caspase3,caspase8,caspase9 and pan caspase activity by in situ fluorescence staining.Some important apoptotic related genes from death receptor mediated apoptosis pathway and mitochondrial mediated apoptosis pathway in porcine vitrified oocytes were also detected in this study.Because of the benefit of incubation after warming in the vitrified oocytes or embryos of other animals,this study tried to find out the suitable incubation time of porcine M?stage oocytes after warming by the series detections of mitochondrial function,apoptotic state and in vitro development.For the important roles of mitochondria in oocyte development and its oxidative damage in cryopreservation,this study added different concentration RES(Resveratrol,RES)in IVM medium,and tried to find out the best action concentration.The gene expression modes of matured oocytes from suitable or high concentration of RES were also dectected for understanding its action theory.On the other hand,in order to understand the effect and its action pathway of RES addition in the incubaion medium,this article also investigated the mitochondrial function,apoptotic state and apoptotic gene releated expression levels of oocytes before and after RES treatments.In order to look for the key molecules and regulatory pathways caused by cryopreservation and provide practical and theoretical guidance for the further improvement of the freezing efficiency of porcine oocytes,this study tested the global gene expression profile of the oocytes befor and after vitrification by pig genome expression profiling chips.The differentially expressed genes were selected,GO analysis and pathway analysis were carried out.This research was divided into 6 parts,the main results were as follows:Experiment 1.Changes of mitochondrial function in porcine vitrified M?-stage oocytes and their impacts on apoptosis and developmental abilityThe purpose of this study was to investigate the changes of mitochondria in porcine M?-stage oocytes after OPS(open pulled straw)vitrification and to determine their roles in apoptosis and in vitro developmental ability.The mitochondrial membrane potential(??m),ROS level,ATP concentration,mitochondrial distribution,mitochondrial ultrastructure,early-stage apoptosis with Annexin V-FITC staining,survival rate,parthenogenetic developmental ability and related gene expression were measured in the present experiments.The results showed that:(1)the mitochondrial??m of vitrified-thawed oocytes(1.05)was lower than that of fresh oocytes 1.24(P<0.05).(2)ROS level in the OPS vitrification group was much higher than that of the fresh group,while the ATP concentration was much lower than that of fresh group(P<0.05).(3)Early-stage apoptosis rate from the OPS vitrification group(57.6%)was much higher than that of fresh group(8.53%)(P<0.05),and the survival rate and parthenogenetic cleavage rate of OPS vitrified oocytes were much lower than those from fresh ones(P<0.05).(4)Vitrification not only destroyed the mitochondrial distribution of porcine M?-stage oocytes,but also damaged the mitochondrial ultrastructure.(5)After vitrification,the gene expression level of DNM1(Dynamin1)was up-regulated,and other four genes(SOD1(Cu/Zn superoxide dismutase),Mfn2(Mitofusin),BAX and Bcl2)were down-regulated.The present study suggested that not only the morphology and function of mitochondria were damaged greatly during the vitrification process,but also early-stage apoptosis was improved after vitrification.Intrinsic mitochondrial pathway could be in involved in the occurrence of apoptosis in vitrified-thawed porcine oocytes.Experiment 2.Studies on apoptotic pathways of porcine vitrified M?stage oocytesFor illuminating the appopotic pathways of porcine M?stage oocytes after vitrification.The present experiment used in situ fluorescence staining method to test the key caspase3,caspase8,caspase9 and pan caspase activities from death receptor mediated apoptosis pathway and mitochondrial mediated apoptosis pathway in vitrified porcine oocytes.Some important related genes from different pathways were also detected by RT-PCR.The results were showed as below.After vitrification,not only the caspase 8 fluorescence intensity value(32.03)from death receptor mediated apoptotic pathway,but also the caspase 9 fluorescence intensity value(16.56)from mitochondrial mediated apoptotic pathway were much higher than those of fresh oocytes(4.02 and 4.83)(P<0.05).The caspase 3 and pan caspase fluorescence intensity value(16.70 and 8.43)from both apoptotic pathways were also much higher than fresh oocytes(4.23 and 3.08)(P<0.05).The relative gene expressions of TNFa(tumor necrosis factor a),FasL(Fas ligand),CASP8 and CASP3 from death receptor mediated apoptosis pathway were increased greatly after vitrification.In mitochondrial mediated apoptosis pathway,relative gene expressions of CASP9,CASP3 and P53 were increased greatly,and the relative gene expressions of of Bcl2,BAX,SOD1 and survivin were decreased greatly(P<0.05).In conclusion,both death receptor mediated apoptosis pathway and mitochondrial mediated apoptosis pathway were involved in the apoptosis of porcine M?stage oocytes after freezing.Experiment 3.Effect of different culture time after warming on the mitochondrial function,apoptotic level and developmental ability of porcine vitrified M?stage oocytesThe purpose of this experiment was to make sure the optimal incubation culture time of porcine vitrified M?stage oocytes after warming.The oocytes were cultured in vitro for 0 h,1 h,2 h or 4 h,and mitochondrial membrane potential(??m),ROS level,ATP concentration,early-stage apoptotic rate,pan caspase activity,FDA staining survival rate and parthenogenetics developmental ability were detected.The results were showed as below.(1)The??m of 2 h and 4 h culture groups after warming were 1.19 and 1.26,which were significantly lower than that of fresh oocytes(1.34),but were much higher than that of 1 h culture group(P<0.05).(2)The ROS level of 1 h culture group after warming was the highest(69.54).With the increasing of culture time,the ROS level were decreased,and 2 h culture group was the lowest(52.81).The ATP content in 1 h culture group was the lowest,with increasing the culture time to 2 h and 4 h,the ATP content increased gradually.(3)In different culture time groups,the apoptotic rate with Annexin V-FITC staining of 2 h group was the lowest(43.18%).The pan caspase activity in 1 h culture group was also the highest(17.28).Incresing the culture time to 2 h,the lowest pan caspase activity was got(4.38).(4)FDA staining survival rate of porcine oocytes were decreased with the increasing of culture time after warming.The parthenogenetics cleavage rate were the highest(19.84).Further increasing the culture time to 4h,the parthenogenetic developmental ability began to fall.Experiment 4.Effect of RES addition in IVM medium on in vitro development,anti-freezing and gene expression of porcine M?stage oocytesIn order to investigate the effect of RES addition in IVM medium on the developmental ability of porcine oocytes,and elucidate the possible mechanism of its action on oocyte development and anti-freezing ability.This experiment compared different RES concentration in the IVM medium for studying their effects on IVM rate and developmental ability.RT-PCR was used to detect the changes of apoptotic and developmental related gene expression level of porcine matured oocytes from fresh,2?M and 10?M groups.Matured oocytes from 2?M RES addition group were vitrified with OPS method,and the development ability,mitochondrial funtion and early stage apoptotic status were used to clarify the mechanism of high anti-freezing ability.The results showed that:(1)2-5?M RES addition in IVM medium could improve porcine oocyte IVM rate and parthenogenetic developmental ability.A high RES concentration(10?M)showed a cytotoxic effect on porcine oocyte.(2)2?M RES addition in IVM medium could improve the MATER(maternal antigen that embryos require)gene expression and decline the c-mos gene expression.2?M RES group could increase the Sirtl(silent mating type information regulation 2 homolog 1)and anti-apoptotic gene expression,and decrease pro-apoptotic gene expression.When the concentration of RES was to 10?M,some of apoptotic related genes were on pro-apoptotic status.(3)After RES addition in IVM medium,the FDA survival rate and parthenogenetic cleavage rate of these oocytes after vitrification were higher than those of control group,and the early stage apoptotic rate of RES addition group was lower than that of control group.(4)After RES addition,ROS levels in those oocytes before or after vitrification were lower than those of groups without RES addition,the ATP contents and??m before or after vitrification from were higher than those of groups without RES addition.In conclusion,2-5 pM RES addition in porcine IVM medium could improve mitochondrial function,decrease apoptotic level,and improve the developmental and anti-freezing ability.High RES concentration showed a serious cytotoxic effect on porcine oocytes.Experiment 5.Effect of RES addition in incubation medium on apoptotic state and gene expression of porcine vitrifed M?stage oocytesThe purpose of this experiment was to study the effect of RES addition in incubation medium after warming on its apoptotic state and developmental ability.2?M RES was added in the incubation medium of porcine warmed oocytes,and the mitochondrial funtion(??m),ATP content,ROS level,apoptotic state,development ability and related gene expression from different apoptotic pathways were detected.The results showed that:(1)RES addition could improve the mitochondrial??m and ATP contents,decrease the ROS level of warmed oocyte.(2)The early apoptotic rate of RES addition group(39.0)decreased greatly than that of control group(46.8%)(P<0.05).The pan caspase level also decreased from 4.38 to 3.01 in RES and no RES groups.(3)RES addition significantly improved the FDA survival and parthenogentic cleavage rates of porcine warmed oocytes.(4)The results of apoptotic related gene expression showed that RES addition increased gene expression of mitochondrial function(SOD1 and DNM1).The apoptotic state was decreased and developmental ability was increased after RES treatment by decreasing the apoptotic level by both the death receptor mediated apoptosis pathway and mitochondrial mediated apoptosis pathway.Experiment 6.Analysis of global gene expression of porcine M?stage vitrified oocytesThe aim of this study was to examine the effect of the cryopreservation of porcine oocyte on their gene expression profiles and select the key pathways of these different expression genes by pig whole-genome microarrays.The results showed that:1)Compared with non-cryopreservation oocytes,171 genes for more than 2 times expression level were got,and 103 genes exhibited up-regulated and 68 genes were down-regulated.2)Response to wounding,regulation of apoptosis,regulation of programmed cell death,regulation of cell death,regulation of protein kinase,RNA splicing,nuclear mRNA splicing and mRNA metabolic were involved in the biological process GO analysis.3)Extracellular region part,basolateral plasma membrane,extracellular space,plasma membrane part,actin cytoskeleton,mitochondrial part,mitochondrion,organelle membrane and mitochondrial envelope were involved in the cellular component GO analysis.4)Toll-like receptor signaling pathway,NOD-like receptor signaling and MAPK signaling were also involved in the gene expression regulation with pathway analysis. |
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