The Role Of TDRG1 Protein In Asthenospermia And Its Mechanism

Asthenospermia,also known as inadequate sperm motility,is mainly characterized by low motility of sperm,which may be caused by abnormal structure of axons and fibrous sheaths in the tail of sperm.Epidemiological survey found that asthenospermia accounted for 15%-25% of male infertility,and showed an upward trend year by year.Therefore,the study of the occurrence and development of asthenospermia is of great significance to the clinical diagnosis and treatment of male infertility.There are many pathogenic factors of asthenospermia,including genital tract infection,varicocele,endocrine disorders,abnormal gene expression and so on.Current studies on genes related to asthenospermia have shown that abnormal expressions of genes encoding spermatogenic flagellin and energy metabolism related proteins decrease sperm motility,and some of these genes are expressed specifically in testis.This suggests that testicle-specific genes may be involved in regulating sperm motility.I have been committed to the study of Testis Development Related Gene 1(TDRG1)which is a testis-specific expressed Gene(Gen Bank accession No.DQ168992).Our previous results have confirmed that TDRG1 is a primate specific gene,which has no homologous gene expression in the testicles of mice and rats,but has homologous gene expression in the testicles of rhesus monkeys and chimpanzees.It was also confirmed that TDRG1 was expressed in the cytoplasm of human testicular spermatogenic cells,and the content of TDRG1 increased with the maturation of spermatogenic cells.Therefore,we preliminarily hypothesized that TDRG1 have an effect in the regulation of the function of human mature spermatozoa.Thorough research was carried on in order to prove this hypothesis,and supported by the National Natural Science Fundation of China(No.81501317 and No.81871207).The project is made up of 4 parts as following.Chapter One: The expression and localization of primate testicular specific protein TDRG1 in mature human spermatozoaObjective: To investigate the expression and localization of primate testicular specific protein TDRG1 in mature human spermatozoa.Methods: Immunohistochemical staining,RT-PCR,Western Blot and Immunofluorescence were used to determine the the expression and localization of primate testicular specific protein TDRG1 in mature human sperm.Results: The result of Immunohistochemical staining showed that the content of TDRG1 increased with the maturation of spermatogenic cells.The results of RT-PCR and Western Blot showed that TDRG1 was expressed in mature human sperm.The results of Immunofluorescence confirmed that TDRG1 was mainly located in the tail of spermatozoa(including midpiece,principal piece and flagellum).Conclusion: The expression and localization of TDRG1 in the tail of mature human spermatozoa provides a theoretical basis for the future study of the function of TDRG1 in human mature spermatozoa.Chapter Two: Recombinant protein TDRG1 was successfully introduced into human mature spermatozoa by cell-penetrating peptide.Section One: cell-penetrating peptide TAT can carry EGFP into human mature spermatozoa.Objective: To verify that cell-penetrating peptide TAT can carry the recombinant protein EGFP into human mature spermatozoa without side-effects.Methods: Firstly,the prokaryotic expression vectors of recombinant plasmids p ET28a-TAT-EGFP and p ET28a-EGFP-TAT were constructed.Secondly,the fusion proteins TAT-EGFP and EGFP-TAT were expressed and purified.Finally,the fusion proteins were incubated with normal human sperm.The changes of sperm motility parameters were observed under fluorescence microscope.Results: The expression vectors p ET28a-TAT-EGFP and p ET28a-EGFP-TAT have been successfully constructed and the fusion proteins p ET28a-TAT-EGFP and EGFP-TAT have been expressed and purified.After the sperm was incubated with the fusion protein,green fluorescence was observed in the sperm under the fluorescence microscope.There was no significant change in sperm viability and motility parameters.Conclusion: The fusion protein EGFP carried by cell-penetrating peptide TAT can be introduced successfully into spermatozoa,and has no obvious toxic effects on spermatozoa,providing an experimental basis for the study of sperm cytoplasmic protein;Section Two:cell-penetrating peptide TAT can carry TDRG1 into human mature spermatozoa.Objective: To verify that cell-penetrating peptide TAT can carry the recombinant protein TDRG1 into human mature spermatozoa and to observe its transmembrane effect.Methods: Firstly,the prokaryotic expression vector of recombinant plasmid p ET28a-TAT-TDRG1 was constructed.Secondly,the fusion protein TAT-TDRG1 was expressed and purified.Finally,the fusion protein was incubated with normal human sperm.The effect of transmembrane and optimal concentration of TAT-TDRG1 were detected by flag tag antibody and TDGR1 antibody combined with Western Blot.Results: The expression vectors p ET28a-TAT-TDRG1 has been successfully constructed and the fusion proteins TAT-TDRG1 has been expressed and purified.After the sperm was incubated with the fusion protein,Western Blot test results confirmed that exogenous TDRG1 had been successfully introduced into human spermatozoa,and the optimum penetration concentration was 80?g/ml.Conclusion: The fusion protein TDRG1 carried by cell-penetrating peptide TAT can be successfully introduced into spermatozoa,which provides an experimental basis for the future study of the function of TDRG1 in human mature spermatozoa.Chapter Three: The correlation between TDRG1 protein and asthenospermiaObjective: To elucidate the relationship between TDRG1 protein and asthenospermia.Methods: RT-PCR and Western Blot were used to elucidate the correlation between TDRG1 and asthenospermia.At the same time,asthenospermic samples was incubated with the fusion protein TAT-TDRG1 involved in chapter 2,the motility of sperm incubated with fusion protein was compared with the control group,as a positively evaluation that reflected the function of TDRG1 protein in asthenospermia sperm and further clarified the correlation between TDRG1 and asthenospermia.Results: The amount of TDRG1 in both nucleic acid and protein levels were on the low side in asthenospermic samples.The result of Immunofluorescence showed that the distribution of TDRG1 in asthenospermia sperm decreased or even disappeared.There was a positive linear correlation between TDRG1 content and progressive motility of human sperm.The fusion protein TAT-TDRG1 could not only increase the PR of normal sperm(about 10%),but also significantly increase the PR of asthenospermic sperm(40%).Conculsion: There was a correlation between TDRG1 protein and asthenospermia,that is,the content of TDRG1 was significantly decreased in asthenospermic sperm,and it had a positive correlation with the progressive motility.Fusion protein TAT-TDRG1 significantly increased the progressive motility of asthenospermic sperm.Chapter Four: The mechanism of TDRG1 protein in asthenospermiaObjective: To explore the possible mechanism of TDRG1 protein in asthenospermiaMethods: The possible interaction proteins of TDRG1 were searched by non-standard quantitative and quantitative proteomics research strategy of high resolution liquid chromatography-mass spectrometry,and the possible interaction proteins of TDRG1 were verified by Co-immunoprecipitation.Results: 5298 proteins were successfully identified by non-standard quantitative analysis and quantitative proteomics research strategy of high resolution liquid chromatography-mass spectrometry,632 differentially expressed proteins were identified,including 370 upregulated(including 41 reproductive-related proteins)and 262 down-regulated(including 31 reproductive-related proteins).In the down-regulated differentially expressed proteins,we analyzed and predicted the interaction between CATSPER1 and TDRG1.Finally,the results of Co-immunoprecipitation confirmed the interaction between TDRG1 and CATSPER1.Conclusion: TDRG1 may regulate human sperm motility through the CATSPER-Ca²⁺ pathway.