Wenting Wang

Systematic mapping and functional analysis of a family of human epididymal secretory sperm-located proteins

The mammalian spermatozoon has many cellular compartments, such as head and tail, permitting it to interact with the female reproductive tract and fertilize the egg. It acquires this fertilizing potential during transit through the epididymis, which secretes proteins that coat different sperm domains. Optimal levels of these proteins provide the spermatozoon with its ability to move to, bind to, fuse with, and penetrate the egg; otherwise male infertility results. As few human epididymal proteins have been characterized, this work was performed to generate a database of human epididymal sperm-located proteins involved in maturation. Two-dimensional gel electrophoresis of epididymal tissue and luminal fluid proteins, followed by identification using MALDI-TOF/MS or MALDI-TOF/TOF, revealed over a thousand spots in gels comprising 745 abundant nonstructural proteins, 408 in luminal fluids, of which 207 were present on spermatozoa. Antibodies raised to 619 recombinant or synthetic peptides, used in Western blots, histological sections, and washed sperm preparations to confirm antibody quality and protein expression, indicated their regional location in the epididymal epithelium and highly specific locations on washed functional spermatozoa. Sperm function tests suggested the role of some proteins in motility and protection against oxidative attack. A large database of these proteins, characterized by size, pI, chromosomal location, and function, was given a unified terminology reflecting their sperm domain location. These novel, secreted human epididymal proteins are potential targets for a posttesticular contraceptive acting to provide rapid, reversible, functional sterility in men and they are also biomarkers that could be used in noninvasive assessments of male fertility.

Mapping of the human testicular proteome and its relationship with that of the epididymis and spermatozoa

The testis produces male gametes in the germinal epithelium through the development of spermatogonia and spermatocytes into spermatids and immature spermatozoa with the support of Sertoli cells. The flow of spermatozoa into the epididymis is aided by testicular secretions. In the epididymal lumen, spermatozoa and testicular secretions combine with epididymal secretions that promote sperm maturation and storage. We refer to the combined secretions in the epididymis as the sperm-milieu. With two-dimensional-PAGE matrix-assisted laser desorption ionization time-of-flight MS analysis of healthy testes from fertile accident victims, 725 unique proteins were identified from 1920 two-dimensional-gel spots, and a corresponding antibody library was established. This revealed the presence of 240 proteins in the sperm-milieu by Western blotting and the localization of 167 proteins in mature spermatozoa by ICC. These proteins, and those from the epididymal proteome (Li et al. 2010), form the proteomes of the sperm-milieu and the spermatozoa, comprising 525 and 319 proteins, respectively. Individual mapping of the 319 sperm-located proteins to various testicular cell types by immunohistochemistry suggested that 47% were intrinsic sperm proteins (from their presence in spermatids) and 23% were extrinsic sperm proteins, originating from the epididymis and acquired during maturation (from their absence from the germinal epithelium and presence in the epididymal tissue and sperm-milieu). Whereas 408 of 525 proteins in the sperm-milieu proteome were previously identified as abundant epididymal proteins, the remaining 22%, detected by the use of new testicular antibodies, were more likely to be minor proteins common to the testicular proteome, rather than proteins of testicular origin added to spermatozoa during maturation in the epididymis. The characterization of the sperm-milieu proteome and testicular mapping of the sperm-located proteins presented here provide the molecular basis for further studies on the production and maturation of spermatozoa. This could be the basis of development of diagnostic markers and therapeutic targets for infertility or targets for male contraception.

Cloning and identification of a novel sperm binding protein, HEL-75, with antibacterial activity and expressed in the human epididymis

BACKGROUND The HEL-75 protein is a beta-defensin that was identified by analyzing a human epididymis cDNA library. Studying its function may not only elucidate the mechanisms of host defense, but may also provide new alternatives for novel therapeutic drugs for reproductive tract infections. METHODS The HEL-75 gene was amplified by PCR, and its structure and function were predicted and analyzed with bioinformatics tools. Polyclonal serum was raised against recombinant HEL (rHEL)-75 protein. The gene expression pattern was analyzed with RT-PCR and immunofluorescent staining. Finally, the antimicrobial activity and function during fertilization of HEL-75 were analyzed using a colony-forming unit assay and IVF, respectively. RESULTS The human HEL-75 gene is located on chromosome 20p13 and encodes a 95 amino acid protein with a predicted N-terminal signal peptide of 22 amino acids. The protein has six conserved cysteine residues, characteristic of members of the beta-defensin superfamily, as well as several potential post-translational modification sites. At the transcriptional level, HEL-75 was expressed in the epididymis and lung, but only in the epididymis at the translational level. Immunofluorescent staining showed that HEL-75 protein bound spermatozoa in the epididymis. RHEL-75 protein could kill Escherichia coli in vitro in a dose- and time-dependent fashion. However, no effect was observed on sperm motility nor fertilization when spermatozoa were blocked with anti-rHEL-75 polyclonal serum. CONCLUSION HEL-75 is a new beta-defensin expressed in the epididymis and on sperm; it may play an important role in host defense.

Aged men share the sperm protein PATE1 defect with young asthenozoospermia patients

STUDY QUESTION Does a defect in the human sperm-located protein prostate and testis expressed 1 (PATE1) exist in both aged men and young asthenozoospermia patients? SUMMARY ANSWER A defect in sperm PATE1 exists in both aged men and young asthenozoospermia patients, and an antibody against PATE1 can decrease human sperm motility and zona-free hamster oocyte penetration. WHAT IS KNOWN ALREADY Both aged men and young asthenozoospermia patients have poor sperm quality. The PATE1 protein seems to mediate sperm-egg interactions; however, the mechanisms are still unknown. STUDY DESIGN, SIZE, DURATION This was a case-control study including 60 young fathers (aged 28-32 years) and 60 aged fathers (68-72 years old) who donated semen by masturbation after 7 days of sexual abstinence. Comparative sperm proteome analysis from the young fathers and aged fathers was performed to discover key proteins. The target protein PATE1 was chosen and validated by western blotting and immunohistochemistry. Quantitative assessment of sperm PATE1 protein was performed on sperm from 60 young fathers, 60 aged fathers and 110 young asthenozoospermia patients. Furthermore, an antibody against PATE1 assay was used to test whether PATE1 participated in sperm motility and penetration of zona-free hamster egg. PARTICIPANTS/MATERIALS, SETTING, METHODS Samples were pooled and separated by two-dimensional gel electrophoresis followed by identification by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Western blotting and immunohistochemistry were used to validate the confidence of proteomic data. Sperm immunofluorescence quantification experiments disclosed whether the aged men indeed shared the same PATE1 defect with 110 young asthenozoospermia patients. The sperm motility test and penetration of zona-free hamster egg assay were performed for PATE1. MAIN RESULTS AND THE ROLE OF CHANCE Twenty-two sperm proteins with significant differential expression between young adults and aged men were identified (P < 0.05, mean ratio >1.5), including 13 proteins with decreased expressions with aging. Based on bioinformatics, PATE1 was chosen for further study, and exhibited similar changes in expression level and localization on sperm from aged men and young asthenozoospermia patients. Antibody blocking revealed that PATE1 was involved in sperm-egg penetration and sperm motility. LIMITATIONS, REASONS FOR CAUTION Before any clinical application of PATE1 as a biomarker for the diagnosis of male infertility, more cases should be used to evaluate confidence in this approach. WIDER IMPLICATIONS OF THE FINDINGS This study revealed a common molecular basis underlying the decline in sperm quality in the natural aging process and in young men with asthenozoospermia. The data should greatly contribute to the development of molecular evaluation of sperm quality, and the diagnosis and treatment of asthenozoospermia. STUDY FUNDING/COMPETING INTERESTS This work was supported by grants from the National Natural Science Foundation of China (NO. 81300533, 81370013 and 81000277) and Shandong Provincial Natural Science Foundation, China (ZR2013HQ002, ZR2014HQ068). The authors declare no competing financial interests.

Comparative proteome analysis of human testis from newborn, young adult, and aged men identified spermatogenesis-associated proteins

Human testis begins to produce spermatozoa in the young adult. However, with ageing, the capacity of spermatogenesis gradually decreases including reduction in semen volume and sperm motility and changes in sperm morphology. Spermatogenesis is strictly controlled by complex protein pathways, and changes in protein expression might cause abnormal sperm functions. Here, human testicular proteomes from newborn, young adult, and aged men were compared by 2D fluorescence difference gel electrophoresis analysis to discover spermatogenesis-associated proteins. Forty-seven proteins were found increased, and 18 decreased, from newborn to young adult. Sixteen proteins were dramatically increased, and 21 decreased from young to aged organ. Proteins involved in general metabolism and structure1 functions were significantly increased from newborn to young adult testes, whereas proteins related to anti-oxidative function were dramatically downexpressed in the aged organs. All the differential proteins were confirmed by immunoblot and characterized by immunohistochemical analysis. Among these, vimentin and CCT5 located in the Sertoli cell may be related to germ cell maturation. Of the 24 sperm-associated proteins, more than 70% proteins are located in acrosome, neck, and postequatorial region, and thus might help the sperm to bind to, and fuse with, the egg. These spermatogenesis-associated proteins might serve as markers to evaluate sperm quality and deepen the knowledge of the physiological function of the human testis, which is to play an important role in male reproduction regulation.

Comparison of gene expression of the oncogenic Wnt/β-catenin signaling pathway components in the mouse and human epididymis

β-catenin is an integral part of the Wnt signaling pathway and has been linked to tumorigenesis and multiple developmental processes. The high β-catenin expression with low tumor incidence in the human epididymis is thus intriguing. In the present study, the β-catenin gene and protein was found to be highly expressed in the murine caput epididymidis, and the protein mainly localized along the lateral plasma membranes of adjacent epithelial cells throughout both human and mouse epididymides. Furthermore, the adult mouse epididymis was found to express almost all the Wnt/β-catenin signaling pathway genes that were determined previously by our group in the human organ. Despite the differences in epididymal structure, the similar location of β-catenin and the high concordance of this pathway′s components′ gene expression in both the adult human and mouse epididymides make the mouse a suitable animal model for studying the anti-tumor mechanism of the epididymis. In addition, both the mRNA and protein expression of β-catenin shared a similar spatial expression as the mRNA of Ros1, a proto-oncogene and a key developmental regulator of the initial segment of the mouse epididymis. The observations on the parallel temporal expression of β-catenin and Ros1 during postnatal development raise the possibility that the canonical Wnt signaling pathway has an additional role in the postnatal development of mouse epididymis.

In-depth quantitative proteome analysis of seminal plasma from men with oligoasthenozoospermia and normozoospermia

RESEARCH QUESTION Can seminal plasma markers for oligoasthenozoospermia be identified by comparison of the human seminal plasma proteome in men with oligoasthenozoospermia and normozoospermia? DESIGN An in-depth quantitative proteome analysis was conducted using a high-throughput method named isobaric tag for relative and absolute quantification. A total of 734 seminal plasma proteins were quantified by mass spectrometry. RESULTS Compared with the seminal plasma from men with normozoospermia, 22 upregulated proteins and 20 downregulated proteins were identified in the oligoasthenozoospermic seminal plasma. These differential seminal plasma proteins were involved in various physiological processes, including metabolism, transport, antioxidation and immune response. The confidence of some proteome data was further verified by western blot of (prostate-specific antigen [KLK3], lactotransferrin [LTF], alpha-1-antitrypsin [SERPINA1] and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). Additionally, 38% of the seminal plasma proteins identified in this study have not been reported in previously published studies on seminal plasma proteome, and 53% of our seminal plasma proteins were shared with published studies on human plasma proteome. CONCLUSIONS Our seminal plasma proteome research provides new complementary high-confidence data, and also enhances understanding of the pathogenic mechanisms in oligoasthenozoospermia.

iTRAQ-based analysis of sperm proteome from normozoospermic men achieving the rescue-ICSI pregnancy after the IVF failure

AbstractBackgroundIn the assisted reproduction, the infertile molecules of spermatozoa from normozoospermic men who underwent the unexplained failure of in vitro fertilization (IVF) due to the lack of sperm binding to the normal zona pellucida, and then achieved pregnancy with the rescue intracytoplasmic sperm injection (R-ICSI) remain unclear. More works are still necessary to explore this male infertile mechanism. MethodsNormozoospermicmen with the IVF pregnancy and normozoospermic men with the R-ICSI pregnancy after the conventional IVF failure were collected. iTRAQ-based proteomic approach were performed to reveal the new infertile causes between the IVF pregnancy men and the R-ICSI pregnancy men. To validate the confidence of proteome data, the individual samples were analyzed by western blot and immunofluorescence. Further, the spontaneous acrosome reactions were measured to evaluate the sperm quality.ResultsCompared with IVF pregnancy group, 56 sperm proteins were differentially expressed in the R-ICSI pregnancy group. Bioinformatic analyses (PANTHER, DAVID, PubMed and STRING) indicated these altered sperm proteins were involved in various molecular functions: reproduction, chromosome organization, and sperm-oocyte interaction. Moreover, the confidence of proteome data was confirmed by western blot and immunofluorescence using the individual samples, which were consistent with our proteomic data. Additionally, an increased rate of the spontaneous acrosome reaction rate was found in the R-ICSI pregnancy group.ConclusionsThe sealtered sperm proteins and the increased spontaneous acrosome reaction rate might account for this unexplained male infertility in the R-ICSI pregnancy patients. The present proteomic results will throw light on the better understanding of the unexplained infertile mechanisms underlying these normozoospermic man who achieved R-ICSI pregnancy after IVF failure.