A Study Of Spermatogenesis-related Protein LM23

Spermatogenesis is a complicated and specific process of cell proliferation and differentiation including mitotic division of spermatogonia, meiotic division of spermatocytes and morphological transformation of spermatids. It is a complex process involving cell division, diffentiation and interactions between cells in the microenviroment of the seminiferous tubule. It is regulated by lots of proteins; generally, these kinds of regulation protein are expressed under precise temporal and spatial regulation in spermatogenesis specifically. Separating and identifying of proteins related to spermatogenesis and exploring of their molecular regulating mechanism at proteome level are very important for clinical diganosis and treatment of male infertility.IM23(AF492385) was a testis-specific expression gene in rat reported by our laboratory previously. LM23 mRNA was specific expression in testis, while its expression was not detected in other tissues including heart, liver, spleen, lung, kidney, brain, muscle and ovary. Real-time PCR analysis showed that the expression level of LM23 was highest in spermatocytes and very low in spermatogonia and spermatid. In situ hybridization revealed strong cytoplasmic positive signal in spermatocytes and weak signal in spermatids and spermatogonia. These results indicated LM23 possessed the testis-specific and stage-specific expression characteristics and possibly involved in rat spermatogenesis. We desired to study the feature and function of LM23 by bioinformatical methods, generating polyclonal antibodies against LM23, and utilizing animal model of LM23 knock down.??Predict the structure and function of LM23 protein by bioinformatical methodsObjective:To analyze the physical and chemical characteristics of LM23 and predict its structure and function by bioinformatical methods.Methods:Integrated bioinformatics database PredictProtein, DNAstar soft, Protfun server, expasy. HMM, CPHmodles and BLAST tools were used to analyze and predict the structure and function of LM23.Results:The results showed that LM23 was contained 312 amino acids, p1=8.76, Mr=36223.29. LM23 contained 2 N-glycosylation site,7 Protein kinase C phosphorylation site,5 Casein kinase II phosphorylation site,3 N-myristoylation site and 2 Amidation site. The LM23 protein contains 25.64%?-helix,7.05%?-pleated sheets and 67.31% loop analyzed by DNAstar sofeware. The LM23 protein contains no disulfide bonds predicted by DISULFIND. LM23 protein may be globular, but it is not as compact as a domain analyzed by PredictProtei. LM23 protein may located in the nucleus predicted by PSORT which also confirmed by immunohistochemistry analysis.LM23 may be Growth factor and may take part in translation which predicted by Protfun server tool. LM23 gene is homologous to the Mus musculus and Homo sapiens speedy homolog (Spdya,Speedy) gene analyzed by BLAST. We have succeeded to predict the 3D protein structure of LM23 protein by CPHmodles.Conclusions:Predict the structure and function of LM23 protein by bioinformatical methods successfully.??To generate and identify the rabbit polyclonal antibodies against LM23Objective:To generate and identify the rabbit polyclonal antibodies against LM23.Methods:According to the bioinformatics analysis and prediction of the possible high structure, hydrophilicity and antigenicity of LM23. The synthesized peptides were purified by reversed phase high-performance liquid chromatography (RP-HPLC), and cross-linked with keyhole limpet hemocyanin (KLH) by EDC. Rabbits were immunized with conjugated peptides for 3 times (500?g/rabbit). The polyclonal antibodies was identified by ELISA, Western blot and immunohistochemistry.Results:Two peptides (aa 1 to 20, aa 274 to291)of human LM23 were synthesized by using standard Fmoc. These two synthesized peptides with the purity of 90% were prepared respectively. The titers of purified polyclonal antibodies were 1:64,000, 1:128,000, respectively. The positive immunore activity was mainly located in spermatcoytes, and the subcellular localization of LM23 was mainly in nucleus indicated by immunohistochemistry analysis. Western blot results showed that the antibodies could recognize the protein with molecular weight of 36KD in the total nucleoprotein of rat testis.Conclusions:The polyclonal antibodies against LM23 generated and identified in the present work are specific to LM23 protein and therefore can be used in ELISA, Western blot and immunohistochemistry analysis which would be useful tools for the functional study of LM23.??To clone the LM23 gene from rat testis tissue and express LM23 protein in E. coliObjective:To clone the LM23 gene from rat testis tissue and express LM23 in E. coli for further research. Methods:The RNA of LM23 was extracted from rat testis tissue and amplified by reverse transcription PCR; the PCR products of LM23 were cloned into TA vector followed by DNA sequencing. LM23 cDNA fragments in TA vector were subcloned into pET28a (+). Analysis of the E.coli expressing LM23 fusion protein by SDS-PAGE and Western blot.Results:A DNA fragment about 900 bp was amplified and the sequencing result showed that the fragment was just rat LM23 cDNA(939bp,ORF). The recombinant pET28a (+) subcloned with LM23 cDNA was induced by IPTG for 36KD LM23 protein.Conclusions:The LM23 cDNA from rat testis tissue was successfully cloned and expressed LM23 protein in E.coli.??To explore the relationship of LM23 with cell cycle proteins, PIWIL2 and spermatogenic cell apoptosisObjective:To explore the relationship of LM23 with cell cycle proteins, PIWIL2 and spermatogenic cell apoptosis by animal model of LM2'3 knock down.Methods:Further confirm LM23 expression in the testis of LM23 gene knock down with LM23 polyclonal antibodies by Western blot and immunohistochemistry. Study the apoptosis status of the spermatogenic cell in the testis of LM23 gene knock down rats by TUNEL. Reveal the pathway of apoptosis in spermatogenic cell analyzed with Caspase 3 antibody. Exploring the relationship of LM23 with the CDK1?CDK2?CyclinAl?PIWIL2 genes down-regulated in the testis of LM23 gene knock down rats with antibodies of CDK1, CDK2, CyclinAl and PIWIL2 by Western blot and immunohistochemistry.Results:The expression of LM23 in the treated testes was significantly decreased compared with controls. These LM23 gene knock down testes contained germ cells arrested at the spermatocyte stage. The LM23 gene knock down testis showed significantly increased TUNEL positive cells compared with that of the control groups. The expression of activated caspase 3 was detected at significant levels in the spermatocytes detected by immunohistochemistry. The LM23 gene knock down testis showed significantly decreased of the CyclinAl, CDK1, CDK2, PIWIL2 compared with that of the control by Western blot and Immunohistochemical analysis.Conclusions:Collectively, these studies demonstrate that LM23 is required for meiosis in spermatogenesis. LM23 as a protein may take part in the G2/M transition during spermatogenesis through activation of CyclinA1?CDK1 and CDK2. Without LM23, germ cells would arrest at the spermatocyte stage. LM23 may also be a confactor of PIWIL2.