| In mammals,SUMO(small ubiquitin-like modifier)was an important post-translational modification that exists in many cellular processes.It includes 4 SUMO molecules,SUMO-1,SUMO-2,and SUMO-3 and SUMO-4.Among them,SUMO-1 was involved in biological processes such as intracellular protein decomposition,cell cycle,cell proliferation,gene expression,cell apoptosis,DNA replication and repair,nucleoplasm transport,signal transduction and transcription.SUMOylation was catalyzed by E1,E2,and E3 enzymes and binds to substrate proteins.Reactive oxygen,as an indispensable signal molecule outside and inside the cell,will carry out a series of gene expression regulation or protein transcription.post-translational modification regulation in the cell.It can regulate SUMO at the level of binding or debinding.However,during the in vitro maturation of porcine oocytes and the in vitro development of embryos,the molecular mechanism of SUMOylation was still unclear.Therefore,the main purpose of this study was to explore the effects of the SUMOylated E1 activating enzymes UBA2,E2 conjugating enzyme UBC9 and hydrogen peroxide on the in vitro maturation and apoptosis of porcine oocytes,sperm-oocytes binding,parthenogenetic activation and embryo development after in vitro fertilization.The test was divided into three parts and the results are as follows:Test 1:Effects of ubiquitin-like El activating enzyme UBA2 on porcine oocyte maturation,apoptosis and parthenogenesis activation in vitro1.Determination of SUMO-1 protein content in follicular fluid proteins extracted from follicles of different diameters.The content of SUMO-1 protein in follicular fluid with diameters less than 3 mm and 3-6 mm was significantly higher than that with diameters greater than 6 mm Treatment group.2.Add UBA2 to the oocyte maturation solution in vitro to extract mature oocyte protein.The SUMO-1 protein bands appeared at molecular weights of approximately 67,96,and 123 ku,and the SUMO-1 protein content in the 10 ?g/mL UBA2 treatment group increased significantly.3.After adding UBA2 to the in vitro maturation culture medium of porcine oocytes,the maturation rate of oocytes in the 10 ?g/mL UB A2 treatment group increased significantly4.Select high-quality cumulus cells for Annexin v-FITC/PI double staining,and use flow cytometry to detect the apoptosis of cumulus cells.The apoptosis rates of cells were:3.50%,3.31%,0.66%And 0.42%,after treatment with 10 ?g/mL UBA2 treatment group,the apoptosis of cells was significantly reduced5.Pick out high-quality mature oocytes and extract RNA,and use RT-qPCR to detect apoptosis-related genes.The gene levels were divided into two trends.In the 5?g/mL UBA2 treatment group,Bcl-2 gene expression was significantly up-regulated,While the Bax gene was significantly down-regulated,the Caspase3 gene was significantly down-regulated in the 10 ?g/mL UBA2 treatment group.6.After the oocytes added with UBA2 were mature and parthenogenetic,the cleavage rate of the 5?g/mL UBA2 treatment group increased significantly,while the blastocyst rate of the 10 ?g/mL UBA2 treatment group increased significantlyTest 2:Effects of ubiquitin-like E2 conjugating enzyme UBC9 on porcine oocyte maturation,apoptosis and embryonic development in vitro1.UBC9 was added to the in vitro maturation solution of oocytes to extract mature oocyte proteins.The SUMO-1 protein bands appeared at the molecular weights of about 59 and 71ku,and the SUMO-1 protein content in the 10 ?g/mL UBC9 treatment group was significantly reduced2.After UBC9 was added to the maturation medium of oocytes for maturation,the maturation rate of oocytes in the 5 ?g/mL UBA2 treatment group decreased significantly.3.Select high-quality cumulus cells for Annexin v-FITC/PI double staining,and use flow cytometry to detect the apoptosis of cells.The apoptosis rates of cells were:0.47%,15.96%,21.63%and 25.79,respectively,5 ?g/mL UBC9 treatment group significantly promoted cell apoptosis,and 10 and 15 ?g/mL UBC9 treatment groups significantly promoted cell apoptosis4.RT-qPCR method was used to detect apoptosis-related genes.In the 10 ?g/mL UBC9 treatment group,Bcl-2 gene expression was significantly down-regulated.The Bax and Caspase3 genes were significantly up-regulated when 5?g/mL was added.5.After oocytes were matured and fertilized in vitro,the 10 ?g/mL UBC9 treatment group significantly reduced the embryo cleavage rate.Test 3:Effect of hydrogen peroxide on ubiquitin-like modification and sperm-oocyte binding of porcine oocytes1.Add different concentrations of H2O2 to the in vitro maturation medium.In the 75 ?g/mL H2O2 treatment group,the maturation rate of oocytes was significantly reduced.It can be seen that the addition of high concentration of H2O2 reduces the maturation rate of oocytes,while the low concentration of H2O2 has no significant effect on the maturation rate of oocytes2.Adding different concentrations of H2O2 to the mature culture medium of oocytes,after immunoblotting analysis,SUMO-1 markers appeared at 18 and 77 ku,and the expression of SUMO-1 protein in the 75 ?g/mL H2O2 treatment group was significantly reduced,H2O2 can inhibit the SUMO-1 protein in the maturation of porcine oocytes in vitro.3.After adding different concentrations of H2O2 for 44h,the apoptosis of cumulus cells were detected by flow cytometry.The apoptotic rates of cells were 6.26%,8.22%,22.85%,40.13%,and 43.45%.The apoptotic rates increased gradually,and after 75 ?g/mL H2O2 treatment,the cell apoptosis was significantly promoted.4.After adding different concentrations of H2O2,high-quality mature oocytes were picked and RNA was extracted,and RT-qPCR method was used to detect apoptosis-related genes.Results In the 75 ?g/mL H2O2 treatment group,Bcl-2 gene expression was significantly down-regulated and Caspase3 gene expression was significantly up-regulated.The Bax gene was significantly up-regulated when the 50?g/mL H2O2 treatment group was added.5.After adding H2O2 to the oocyte maturation medium in vitro,the 75?g/mL H2O2 treatment group significantly reduced the solution of ZP.6.After the sperm-oocyte combination,the addition of 75 ?g/mL H2O2treatment began to reduce the blue fluorescence,significantly reducing the number of sperm attached to the oocyteIn summary,UBC9 and H2O2 reduced the in vitro maturation rate of porcine oocytes,the SUMO-1 protein content,and increased apoptosis of cumulus cells and the expression of apoptotic genes.Moreover,UBC9 reduced embryo development after in vitro fertilization,while H2O2 prolonged the solubility of ZP and reduced the number of sperm attached to the oocyte.UBA2 improves the maturation rate of oocytes in vitro,the content of SUMO-1 protein and the embryonic development after parthenogenetic activation,reduces the expression of apoptotic genes and reduces apoptosis of cumulus cells.The results of this study will provide more theoretical basis for ubiquitin-like modification of oocytes and provide more theoretical basis for future reproductive production... |
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