The Mechanism Of CCP1 And Its Interacting Protein MYH11 In Mouse Testis Development

Background Cytosolic Carboxypeptidase 1(CCP1)belongs to the M14 subfamily of the cytosolic carboxypeptidase family.The autosomal recessive mutation of CCP1 causes the Purkinje cell decline,male sterility and female fertility decline in pcd mouse.The study found that in normal mouse brain tissue,CCP1 protein can remove the polyglutamic acid of α and β-tublin protein,but the function of CCP1 protein in mouse testis remains unclear.Recent studies have reported that CCP1 can cause Purkinje cell degeneration by activating NF-κB signaling pathway,but the role of this signaling pathway in the development of male reproductive system remains unclear.studies have shown that MAPK signaling pathway is involved in the differentiation and regulation of testicular spermatogenic cells Death,but whether the loss of function of CCP1 has led to the detection of this signaling pathway leading to male sterility in pcd mouse has not been reported;Myosin heavy polypeptide 11(MYH11)is a member of myosin Myosin family.Studies have shown that Myosin is involved in acrosome formation,vesicle trafficking,gene transcription and nuclear formation,and actin Actin and myosin Myosin cooperate to promote spermatogenesis during spermatogenesis;our previous study used LC-MS/MS to find that MYH11 is an important protein of CCP1 in mouse testis,so we understand that CCP1 and its interacting protein MYH11 are The mechanism of action in mouse testis is important for revealing its mechanism of action in spermatogenesis and male sterility.Objective By detecting key proteins ERK1/2,phospho-ERK1/2(p-ERk1/2),p38 MAP Kinase(p38),NF-κB p65(p65)and phospho-NF-κB p65 in MAPK and NF-κB signaling pathways(Ser536)(p-p65)expression in testicular tissues of pcd mouse at different developmental stages,exploring the role and molecular mechanism of CCP1 gene in mouse spermatogenesis;further study of CCP1 and its interacting proteins by bioinformatics methods The role and relationship of MYH11 in mouse testis,explore the molecular mechanism of male sterility in pcd mouse.Method Part1: 1.Western blot analysis of MAPK and NF-κB signaling pathway-related proteins(ERK1/2,p-ERK1/2,p38,p65 and p-p65)in testis and CCP1 knockdown of pcd mouse at different developmental stages Expression in GC-1 spermatogonia.2.Western blot was used to detect the expression of tubulin polyglutamine in pcd mouse testis tissue and CCP1 knockdown of GC-1 type B spermatogonia.Part 2: 1.Immunohistochemistry,RT-PCR and Western blot were used to detect the expression of MYH11 in wild type mouse testis and three testicular cell lines cultured in vitro.2.q PCR,Western blot and immunohistochemistry were used to detect the expression of MYH11 in pcd spermatogonial cells in pcd mouse testis and CCP1 knockdown.3.Using bioinformatics methods to analyze the molecular functions,biochemical processes,cellular components,signaling pathways and interaction networks of MYH11 interacting proteins,and screen proteins that may interact with MYH11 in mouse testis.4.q PCR,Western blot and immunohistochemistry were used to detect the expression of ACTG2 in pcd mouse testis and CCP1 knockdown of GC-1 type B spermatogonia.Results Part 1: 1.The results of Western blot showed that the expression of ERK1/2,p65 and phosphorylated p65 protein was up-regulated in testicular tissues of pcd mouse 15 days after birth,and pcd mouse were 18 days after birth.The expression of ERK1/2,p65 and phosphorylated p65 protein in the testis was down-regulated,while the expression of phosphorylated ERK1/2,p65 and phosphorylated p65 protein was up-regulated in the testis of adult pcd mouse.In the CCP1 knockdown of GC-1spermatogonia,the results showed that ERK1/2 protein was up-regulated in CCP1 knockdown of GC-1 type B spermatogonia compared to the negative control group(CCP1 normal expression),phosphorylation of ERK1 /2 and phosphorylated p65 protein expression was down-regulated.2.Compared with wild-type mice,the expression level of tubulin polyglutamine was increased in the testis tissue of pcd mouse;compared with the negative control group(CCP1 normal expression),CCP1 knockdown Increased expression of tubulin polyglutamine in GC-1 type B spermatogonia.Part 2: 1.Immunohistochemistry results showed that MYH11 was expressed in the supporting cells and spermatogenic cells of wild-type mouse testis.MYH11 was cultured in three mouse cultured Leydig cells(TM3 cells)and mouse testicular support cells(15P-1 cells)and mouse type B spermatogonia(GC-1 cells)were expressed in agreement with the expression of MYH11 in mouse testis tissue.2.MYH11 is down-regulated in pcd mouse testis tissue and CCP1 knockdown of GC-1 type B spermatogonia.3.The first 100 proteins interacting with MYH11 with a confidence greater than 0.400 were analyzed by String database.The results showed that these proteins are mainly related to protein binding activity and structural molecular activity,mainly involved in multicellular biological processes,cell processes and development.In the process,these proteins are mainly expressed in the cytoplasm and cytoskeleton,and partly in the organelles such as the nucleus and mitochondria.Signal pathway analysis showed that proteins interacting with MYH11 are mainly involved in three signaling pathways,of which 23 proteins are involved in chemokines and cytokine-mediated inflammatory signaling pathways,and 22 proteins are involved in Rho GTPase-regulated cytoskeleton.The signaling pathway,17 proteins involved in the nicotinic acetylcholine receptor signaling pathway,is notable,including actin family members such as ACTG1,ACTG2,ACTA1,ACTA2,ACTC1,and ACTB in all three major signaling pathways.The actin ACTG2,which was expressed in mouse testis and had the highest correlation coefficient with MYH11(r=0.636),was analyzed and combined with String and COXPRESdb database.4.ACTG2 was down-regulated in pcd mouse testis tissue and CCP1 knockdown of GC-1 type B spermatogonia.Immunohistochemistry showed that ACTG2 was expressed in the spermatogenic cells,supporting cells and Leydig cells of testis of wild-type and pcd mouse,which was consistent with the expression of MYH11 in the testis of pcd mouse.Conclusions CCP1 may cause abnormal spermatogenesis in pcd mouse by regulating NF-κB and MAPK signaling pathways.Polyglutamination of tubulin in the testis tissue of pcd mouse.ACTG2 may be an interacting protein of MYH11 in mouse testis.Loss of function of CCP1 results in down-regulation of MYH11 and ACTG2 expression.