Based On The Mechanism Of Mice Infertility Caused By Abnormal Expression Of Catsper To Study The Therapymechanism Of Cuyushengjingfang

Aim: The research is aimed to study the transcription mechanism of Catsper1 gene through cAMP-responsive element modulator(CREM).It also aimed to investigate the effects and mechanism of Cuyushengjingfang on treating infertility of mice.Methods: 1.Study the effects of silence of CREM on the expressions of Catsper1 mRNA and protein.Method: The shRNA lentivirus vector targeting CREM was constructed and it was used to transfect the spermatocyte named GC-2spd cells.The fluorescence microscope was used to detect the transfection efficiency of different gradients lentivirus vector and then chosing the optimum transfection concentration from it.Extracting the total RNA and total protein from GC-2spd cells after 72 hours,Real-time PCR(RT-PCR)and Western blot was performed to detect the silent efficiency of CREM and the expressions of Catsper1 mRNA and protein.2.Detect the expression quantitis of CREM-related mRNA and protein and analyze the effects of CREM silencing on its related protein.Method: After silencing CREM gene,RT-PCR and Western blot were performed to detect the expressions of activator of CREM in testis(ACT)and CREB binding protein(CBP).3.Research that whether there is a gene locus on the Catsper1 promoter which can bind to CREM transcription.Method: Extracting the nucleoprotein in GC-2spd cells and projecting and compounding the gene probe that covered the CRE locus in Catsper1 promotor according the literature report,then the probe were labeled by biotin.Using Electrophoretic mobility shift assay technology to analyze that whether here is a gene locus on the Catsper1 promoter which can bind to CREM transcription.4.Study the influence of CREM-CBP dimer inhibitor on the expressions of Catsper1 mRNA and protein.Method: The CREM-CBP dimer inhibitor 666-15 was used to block the combination between CREM and CBP.Extracting the total RNA and total protein from GC-2spd cells after 24 hours,RT-PCR and Western blot was performed to detect the expretion of Catsper1 gene in GC-2spd cells.5.Investigate the effects and mechanism of Cuyushengjingfang on treating infertility of mice.Method: Fifty male mice were randomly divided into 5 groups,respectively are blank control group(CG),model group(MG),drug-intervention groups(low-dose group(SG)),middle-dose group(MDG),high-dose group(HG)),10 mice in each group.The mice were The oligoasthenospermia mice model was constructed by injecting cyclophosphamide(CP)with the concentration of 60mg/kg into abdominal cavity for 5 days,and the CG group was injected the normal saline.Then oligoasthenospermia mice were treated with Cuyushengjingfang in different concentration(0.45,0.90 and 1.80g/kg)by gastric perfusion for 35 days.In the end,the sperm density and sperm motility were observed by optical microscope.RT-PCR and Western blot was performed to detect the expression of ACT?CREM and Catsper1 in testicular tissue.Results: 1.After transfecting CREM shRNA into the spermatocyte(GC-2spd cells),the expressions of Catsper1 mRNA and protein are observably reduced(P<0.01).2..After transfecting CREM shRNA into the spermatocyte(GC-2spd cells),the mRNA and protein expressions of ACT and CBP were also detected,the result is that the expressions of ACT mRNA and protein are observably reduced(P<0.01)while the expressions of CBP mRNA and protein are observably rise(P<0.05).3.The probe which was projected that covered the CRE site can bind with CREM transcription in vitro.4.The expressions of Catsper1 mRNA and protein were observably rise(P<0.05)after adding CREM-CBP dimer inhibitor 666-15 into GC-2spd cells.5.At animal level,the sperm density and sperm motility are significantly lower in MG group than those in CG group(P<0.05)and the expressions of ACT?CREM and Catsper1 mRNA and proteins in MG group are also lower than those in CG group(P<0.05).It demonstrated that the oligoasthenospermia mice model is constructed successfully.The sperm density in SG ? MDG and HG groups were higher than that in MG group(P<0.05),what'more,the sperm in HG group has achieved the maximum density.The sperm motility in SG?MDG and HG groups were better than that in MG group(P<0.05)and the sperm motility in SG group was best.Comparing with MG group,the expressions of ACT?CREM and Catsper1 mRNA and proteins have significantly rised(P<0.05)and the MDG group has achieved the best effect.Conclusion: 1.The transcription factor CREM can regulate the expressions of Catsper1 mRNA and protein and the CREM monomer directly combines with the CRE site that from the promoter of Catsper1 gene is the way for it to work on.Furthermore,the form of CREM-CBP dimer can't up-regulate the expression of Catsper1.2.Cuyushengjingfang can improve the number of sperm and the quality of sperm in oligoasthenospermia mice,furthermore,it can up-regulate the expressions of ACT,CREM and Catsper1 mRNA and proteins,these demonstrate that maybe up-regulate the expressions of ACT,CREM and Catsper1 mRNA and proteins is one of the ways that Cuyushengjingfang improving the sperm quality.