The Function Studies Of TDRG1 Protein In Human Testis

Spermatogenesis is a highly coordinated physiological process that involves thousands of properly functional genes. The regulation of this process is not well-defined since majority of the related genes has not been identified or fully characterized. So it is of great significance to identify and characterize these unknown genes to further reveal the underlying mechanism of spermatogenesis.In our department we employed bioinformatics analysis coupled with reverse transcription polymerase chain reaction (RT-PCR) to clone a novel human testis specific gene named Human Testis Development Related Gene 1, TDRG1 (GenBank Accession Number:DQ 168992). The expression of TDRG1 was exclusively detected in human testis but not in any other non-reproductive tissues. Intriguingly, TDRG1 exhibited the highest expression level at human puberty testicular tissue, but no expression in human testis in the embryonic stage and decreasing levels of expression with aging, indicating TDRG1 may have a role in human spermatogenesis and fertilization.In order to clarify the functions of TDRG1 in human testis, we have done this research project which was funded by National Natural Science Foundation of China (ID:30672090). Chapter One:Cloning and Analysis of the Homologous Sequences of Human Testis Development Related Gene 1 from different speciesObjective:To clone the homologous sequences of Human Testis Development Related Gene 1 (TDRG1) from different species and to explore the potential animal models for further researching the function ofTDRG1;Methods:The nucleotide sequence of TDRG1 was used as a query sequence to search for the mouse, rat and chimpanzee genome databases by BLASTN program to obtain the homologous sequences from these species. RT-PCR and Immunohistochemistry were used to research the homologous gene expression in the testes of these species;Results:Bioinformatics analysis indicated that no homologous sequences ofTDRG1 were found in the mouse and rat genome databases. However, there were homologous sequences in chimpanzee genome database. The percent similarity was 98%. However, RT-PCR coupled with Immunohistochemistry demonstrated that neither homologous nucleotide sequences nor homologous protein were detected in mouse and rat testes;Conclusion:No gene sequence shows to be homologous with TDRG1 in mouse and rat testes. But in chimpanzee testes, there are homologous genes of TDRG1. This study provide theoretical basis for developing TDRG1 transgenic or knockout animal models. Chapter Two:Preparation and Identification of Monoclonal Antibody against Human Testis Development Related Gene 1Objective:To construct a prokaryotic plasmid to express the TDRGl recombinant protein and obtain anti-TDRG1 mAb by immunizing mice, meanwhile identify the biological properties of the mAb;Methods:The coding sequence of TDRG1 was amplified by RT-PCR from normal human testis tissue and cloned into the vector pET21, then expressed in the E. coli BL 21(DE3) to get TDRG1 recombinant protein. The purified TDRG1 recombinant protein was injected to immunize the BALB/C mice to develop anti-TDRG1 mAb, Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted anti-TDRGl mAb were subcloned with limited dilution. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate titers and subtype of mAb. Western Blot and Immunohistochemistry were used to detect specificity of mAb;Results:The prokaryotic plasmid expressing the recombinant protein was constructed. The TDRG1 recombinant protein was expressed and purified. Two hybridoma cell lines that secreted Anti-TDRG1 mAb were obtained. The titers of the mAb in ascites were 1:1.6 X 106, and the subtype of the mAb was IgG1. Westem Blot and Immunohistochemistry analysis indicated the mAb showed specific combination with TDRG1 protein in human testes;Conclusion:The TDRG1 recombinant protein was highly purified and had strong antigenicity. The anti-TDRG1 mAb was produced successfully and can be used to further research the function of TDRG1 gene.Chapter Three:The Expression of TDRG1 Protein in Multiple Tissues in Human and Its FunctionsObjective:To further confirm the expression of TDRG1 protein in human testis and to clarify the expression profiles of TDRG1 protein in multiple human tissues and its functions;Methods:Immunohistochemistry and Western Blot were used to determine the protein expression of TDRG1 in human testis. Immunohistochemistry was employed to clarify the expression profiles of TDRG1 protein in multiple tissues of human. Real-time PCR and Immunohistochemistry were used to detect the expression level of TDRG1 in human testicle tissues with different developmental stages;Results:The TDRG1 protein exclusively expressed in human testis, not in heart, liver, spleen, gaster, large intestine and oesophagus, indicating it is a testis-specific protein. The localization of its expression is in spermatogenic cells in seminiferous tubules of human testis. Furthermore, Realtime PCR coupled with Immunohistochemistry showed TDRG1 does not express in human testicular tissues in the embryonic stage, with high expression level at human puberty and adult testicular tissues and significantly reduced levels of expression with aging;Conculsion:This study further confirmed the protein expression of TDRG1 in human testis and demonstrated that TDRGl is a testis-specific protein which is related to spermatogenesis.Chapter Four:The Expression of TDRG1 Protein in Human OrchioncusObjective:To determine the expression levels of TDRGl protein in human orchioncus and determine the pathological significance;Methods:Tissue microarray of orchioncus was stained by Immunohistochemistry with anti-TDRG1 mAb to determine the expression levels of TDRG1 in human orchioncus tissues;Results:The expression levels of TDRG1 in seminoma and teratoma tissues are significantly lower than normal testicle tissues (P<0.05). However, the expression levels of TDRG1 protein do not show significantly different from embryonal carcinoma, testicle tuberculosis and testicle atrophy tissue;Conclusion:The reduction of TDRG1 protein expression level is associated with human orchioncus, indicating that TDRG1 may function as anti-oncogene.Chapter Five:Construction of TDRG1 Eukaryotic Expression Vector and Its Expression in 293 Cell LineObjective:To construct an eukaryotic expression vector of human TDRG1 gene and identify its expression in 293 cell line;Methods:Total mRNA was extracted from normal human testis tissue, and the coding sequence of TDRG1 was obtained by RT-PCR. The TDRG1 gene was cloned into pYD5 vector and the eukaryotic expression vector of pYD5-TDRG1 was constructed and transfected into 293 cell lines. Fluorescence microscope and Western blot analysis were used to detect the expression of this vector;Results:A fragment of 5000bp and inserted fragment of 303bp were got by cutting positive recombinant plasmid of pYD5-TDRGl with BamHI/ HindIII, Automatic DNA sequence analysis demonstrated that sequence of the recombinant plasmid pYD5-TDRG1 was totally the same as the sequence of TDRG1. The expression of green fluorescence protein (GFP) was observed under the fluorescence microscope. Western Blot analysis showed a 39.8 kD protein;Conclusion:The eukaryotic expression vector of pYD5-TDRG1 was constructed and expressed successfully in 293 cell lines.