TEX101 is shed from the surface of sperm located in the caput epididymidis of the mouse

It is generally believed that cell-to-cell cross-talk and signal transduction are mediated by cell surface molecules that play diverse and important regulatory roles in spermatogenesis and fertilization. Recently, we identified a novel plasma membrane-associated protein, TES101-reactive protein (TES101RP, or TEX101), on mouse testicular germ cells. In this study, we investigate Tex101 mRNA expression in the adult mouse testis using in situ hybridization, and we examine the fate of TEX101 during sperm transport by immunohistochemical and Western blot analyses. Tex101 mRNA was expressed in a stage-specific manner in spermatocytes and in step 1-9 spermatids of the testis, but not in spermatogonia. Although the TEX101 protein remained on the cell surfaces of step 10-16 spermatids and testicular sperm, it was shed from epididymal sperm located in the caput epididymidis. The results of this study provide additional information on germ cell-specific TEX101 expression during spermatogenesis and post-testicular sperm maturation.     

TEX101, a germ cell-marker glycoprotein, is associated with lymphocyte antigen 6 complex locus k within the mouse testis

In adult male mice, the glycosylphosphatidyl inositol-anchored glycoprotein TEX101 is expressed only in germ cells and is thought to be involved in spermatogenesis. However, the details regarding the function of TEX101 remain to be clarified. We previously identified Ly6k as a candidate TEX101-associated protein, but as molecular probes are not currently available to detect Ly6k, we do not have conclusive evidence of the association between TEX101 and Ly6k. In this study, we confirmed the biological interaction between TEX101 and Ly6k using an established anti-mouse Ly6k polyclonal antibody (pAb). A combination of immunoprecipitation, Western blot, and immunohistochemical analyses using the pAb revealed that TEX101 is physically associated with Ly6k within the testis. In addition, these proteins simultaneously co-migrate into the detergent-resistant membrane fractions, suggesting that TEX101 collaborates with Ly6k on the cell membrane and may play a role in spermatogenesis.     

Identification, cloning, and initial characterization of a novel mouse testicular germ cell-specific antigen

A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.     

Human testis-expressed sequence 101 is limitedly distributed in germinal epithelium of testis and disappears in seminoma

Background

Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed.

Results

Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101.

Conclusions

Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.     

Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids

Monoclonal antibodies that bind the native conformation of proteins are indispensable reagents for the development of immunoassays, production of therapeutic antibodies and delineating protein interaction networks by affinity purification-mass spectrometry. Antibodies generated against short peptides, protein fragments, or even full length recombinant proteins may not bind the native protein form in biological fluids, thus limiting their utility. Here, we report the application of immunocapture coupled with selected reaction monitoring measurements (immunocapture-SRM), in the rapid screening of hybridoma culture supernatants for monoclonal antibodies that bind the native protein conformation. We produced mouse monoclonal antibodies, which detect in human serum or seminal plasma the native form of the human testis-expressed sequence 101 (TEX101) protein—a recently proposed biomarker of male infertility. Pairing of two monoclonal antibodies against unique TEX101 epitopes led to the development of an ELISA for the measurement of TEX101 in seminal plasma (limit of detection: 20 pg/ml) and serum (limit of detection: 40 pg/ml). Measurements of matched seminal plasma samples, obtained from men pre- and post-vasectomy, confirmed the absolute diagnostic specificity and sensitivity of TEX101 for noninvasive identification of physical obstructions in the male reproductive tract. Measurement of male and female serum samples revealed undetectable levels of TEX101 in the systemic circulation of healthy individuals. Immunocapture-SRM screening may facilitate development of monoclonal antibodies and immunoassays against native forms of challenging protein targets.Monoclonal antibodies that bind the native form of a protein are indispensable for the development of sensitive immunoassays, production of therapeutic antibodies and for studying protein interaction networks by affinity purification-mass spectrometry (1, 2). Large-scale purification of native proteins from biological samples may be challenging, so recombinant proteins or protein fragments are often used for antibody production. Antibodies produced against short peptides, protein fragments, or even full length recombinant proteins, however, may not bind the native protein conformation present in biological fluids, thus limiting the utility of antibodies. Rapid screening of antibody-producing hybridoma clones for native protein binders requires highly specific and sensitive assays, performed under nondenaturing conditions. Here, we report the capability of an immunocapture-SRM assay to facilitate fast screening of hybridoma cultures for monoclonal antibodies that recognize the native conformation of testis-expressed sequence 101 (TEX101)¹ protein in biological fluids.Recently, we discovered, verified, and validated two proteins, testis-specific protein TEX101 and epididymis-specific protein ECM1, as biomarkers for the differential diagnosis of azoospermia (3, 4). Combination of TEX101 and ECM1 proteins measured in seminal plasma could differentiate between normal spermatogenesis, obstructive azoospermia (OA), and nonobstructive azoospermia (NOA) with very high diagnostic sensitivity and specificity. TEX101 levels in seminal plasma also facilitated classification of NOA subtypes of hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome (5). A clinical laboratory test for TEX101 in seminal plasma may confirm the success of vasectomy or vasovasostomy, eliminate diagnostic testicular biopsies, and predict the success of sperm cell retrieval for assisted reproduction.Human TEX101 is a membrane GPI-anchored protein encoded by the TEX101 gene, located in the 19q13.31 region of chromosome 19. According to the Human Protein Atlas, TEX101 expression is restricted to testicular tissue and male germ cells, with no evidence of expression in any other human tissue or cell type (6). Investigation of the function of mouse TEX101 demonstrated its direct role in fertilization (7–9).We initially measured TEX101 levels in seminal plasma by mass spectrometry-based selected reaction monitoring (SRM) and immuno-SRM assays, with limits of detection of 120 and 5 ng/ml, respectively (4, 5). However, because of the ultra-wide range of TEX101 concentrations in seminal plasma of infertile and healthy men (0.5 ng/ml to 50,000 ng/ml) and theoretically zero levels for some azoospermic patients, a sensitive TEX101 immunoassay is required to develop a clinical laboratory test. In addition to immunoassay, monoclonal antibodies against native TEX101 would allow investigating its interactome and revealing its functional role in spermatogenesis and male fertility. Because TEX101 may emerge as a novel biomarker of male infertility, in this work we focused on the development of an ELISA for sensitive measurement of TEX101 in seminal plasma and serum.Our initial efforts to develop a TEX101 immunoassay using commercially available polyclonal antibodies were not successful. We found that commercial antibodies recognized only the denatured form of TEX101 and were useful for immunohistochemistry and Western blots, but not for the analysis of native TEX101 in seminal plasma. Here, we describe the production of mouse monoclonal antibodies against native TEX101, screening of antibody-producing clones by the two-step immunocapture and SRM assay, development of a sensitive ELISA and measurement of TEX101 in seminal plasma and serum (Fig. 1).Open in a separate windowFig. 1.Pipeline for the production of mouse monoclonal anti-TEX101 antibodies and screening of colonies using two-step immunocapture-SRM assay. Screening included the coating of microtiter plates with sheep anti-mouse IgG antibodies, the addition of hybridoma cell supernatants, incubation with seminal plasma containing the native form of TEX101 followed by trypsin digestion and SRM analysis. Two-step immunocapture followed by SRM detection facilitated rapid screening of antibody-producing colonies and provided the following advantages: no requirement for previously developed TEX101 antibodies, small scale antibody production on 96-well plates, screening of low amounts of the newly-produced antibodies and direct selection of antibodies against the native form of TEX101. Eventually, all positive clones were expanded and a sensitive immunofluorescent assay for TEX101 was developed in seminal plasma and serum.     

Conversion of midbodies into germ cell intercellular bridges

Whereas somatic cell cytokinesis resolves with abscission of the midbody, resulting in independent daughter cells, germ cell cytokinesis concludes with the formation of a stable intercellular bridge interconnecting daughter cells in a syncytium. While many proteins essential for abscission have been discovered, until recently, no proteins essential for mammalian germ cell intercellular bridge formation have been identified. Using TEX14 as a marker for the germ cell intercellular bridge, we show that TEX14 co-localizes with the centralspindlin complex, mitotic kinesin-like protein 1 (MKLP1) and male germ cell Rac GTPase-activating protein (MgcRacGAP) and converts these midbody matrix proteins into stable intercellular bridge components. In contrast, septins (SEPT) 2, 7 and 9 are transitional proteins in the newly forming bridge. In cultured somatic cells, TEX14 can localize to the midbody in the absence of other germ cell-specific factors, suggesting that TEX14 serves to bridge the somatic cytokinesis machinery to other germ cell proteins to form a stable intercellular bridge essential for male reproduction.     

Germ cell intercellular bridges

Stable intercellular bridges are a conserved feature of gametogenesis in multicellular animals observed more than 100 years ago, but their function was unknown. Many of the components necessary for this structure have been identified through the study of cytokinesis in Drosophila; however, mammalian intercellular bridges have distinct properties from those of insects. Mammalian germ cell intercellular bridges are composed of general cytokinesis components with additional germ cell-specific factors including TEX14. TEX14 is an inactive kinase essential for the maintenance of stable intercellular bridges in gametes of both sexes but whose loss specifically impairs male meiosis. TEX14 acts to impede the terminal steps of abscission by competing for essential component CEP55, blocking its interaction in nongerm cells with ALIX and TSG101. Additionally, TEX14-interacting protein RBM44, whose localization in stabile intercellular bridges is limited to pachytene and secondary spermatocytes, may participate in processes such as RNA transport but is nonessential to the maintenance of intercellular bridge stability.     

Identification and characterization of RBM44 as a novel intercellular bridge protein

Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44) as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 null male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14.     

Identification of a molecular marker for type A spermatogonia by microarray analysis using gonadal cells from pvasa-GFP transgenic rainbow trout (Oncorhynchus mykiss)

The spermatogonia of fish can be classified as being either undifferentiated type A spermatogonia or differentiated type B spermatogonia. Although type A spermatogonia, which contain spermatogonial stem cells, have been demonstrated to be a suitable material for germ cell transplantation, no molecular markers for distinguishing between type A and type B spermatogonia in fish have been developed to date. We therefore sought to develop a molecular marker for type A spermatogonia in rainbow trout. Using GFP-dependent flow cytometry (FCM), enriched fractions of type A and type B spermatogonia, testicular somatic cells, and primordial germ cells were prepared from rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa regulatory regions (pvasa-GFP rainbow trout). The gene-expression profiles of each cell fraction were then compared with a microarray containing cDNAs representing 16,006 genes from several salmonid species. Genes exhibiting high expression for type A spermatogonia relative to above-mentioned other types of gonadal cells were identified and subjected to RT-PCR and quatitative PCR analysis. Since only the rainbow trout notch1 homologue showed significantly high expression in the type A spermatogonia-enriched fraction, we propose that notch1 may be a useful molecular marker for type A spermatogonia. The combination of GFP-dependent FCM and microarray analysis of pvasa-GFP rainbow trout can therefore be applied to the identification of potentially useful molecular markers of germ cells in fish.     

A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.     

Qualitative characterization of oligosaccharide chains present on the rat zona pellucida glycoconjugates.

Zona pellucida (ZP), the extracellular glycocalyx surrounding the mammalian oocyte, is believed to mediate species-specific sperm-egg interaction. Despite numerous studies on characterization of ZP glycoconjugates in several species, little or no information is available on the number and chemical nature of the various components of the rat ZP. In this study we have attempted the biochemical characterization of the rat ZP using endo- and/or exo-glycohydrolases. Intact eggs from superovulated rats were radioiodinated by the chloramine-T method, and the labeled ZP components were resolved on SDS-PAGE under nonreducing conditions. These studies show that the rat ZP consists of three components with apparent molecular masses of 205 kDa (ZP1), 119 kDa (ZP2), and 115 kDa (ZP3). Unlike mouse ZP2 and ZP3, which resolve as distinct components on SDS-PAGE, rat ZP2 and ZP3 show substantial overlap in their molecular sizes and isoelectric points. Treatment of the rat ZP components with exo- (neuraminidase and alpha-L-fucosidase) and/or endo- (endoglycosidase H, endoglycosidase F, N-glycanase, and O-glycanase) glycohydrolases indicated the following: 1) Both rat ZP2 and ZP3 contain N-linked oligosaccharide (OS) units as indicated by their sensitivity to endoglycosidase F and N-glycanase. 2) Treatment with N-glycanase caused a reduction in size of the rat ZP2 and ZP3 components by nearly 50% and 60%, respectively, indicating that the two ZP components are highly glycosylated. 3) Rat ZP3 was sensitive to O-glycanase, suggesting that this ZP component contains O-linked OS unit(s). 4) No evidence was obtained for the presence of fucosyl or sialyl residue(s) on the O-linked OS unit(s) of rat ZP3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative Proteomic Analysis of Seminal Plasma,Sperm Membrane Proteins,and Seminal Extracellular Vesicles Suggests Vesicular Mechanisms Aid in the Removal and Addition of Proteins to the Ram Sperm Membrane

Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction‐specific proteins that show demonstrable links to sperm fertility. Many of these membrane‐bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane.     

Overexpression of Bcl-W in the testis disrupts spermatogenesis: revelation of a role of BCL-W in male germ cell cycle control

To explore physiological roles of BCL-W, a prosurvival member of the BCL-2 protein family, we generated transgenic (TG) mice overexpressing Bcl-w driven by a chicken beta-actin promoter. Male Bcl-w TG mice developed normally but were infertile. The adult TG testes displayed disrupted spermatogenesis with various severities ranging from thin seminiferous epithelium containing less germ cells to Sertoli cell-only appearance. No overpopulation of any type of germ cells was observed during testicular development. In contrast, the developing TG testes displayed decreased number of spermatogonia, degeneration, and detachment of spermatocytes and Sertoli cell vacuolization. The proliferative activity of germ cells was significantly reduced during testicular development and spermatogenesis, as determined by in vivo and in vitro 5'-bromo-2'deoxyuridine incorporation assays. Sertoli cells were structurally and functionally normal. The degenerating germ cells were TUNEL-negative and no typical apoptotic DNA ladder was detected. Our data suggest that regulated spatial and temporal expression of BCL-W is required for normal testicular development and spermatogenesis, and overexpression of BCL-W inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis.     

Verification of male infertility biomarkers in seminal plasma by multiplex selected reaction monitoring assay

Seminal plasma is a promising biological fluid to use for noninvasive clinical diagnostics of male reproductive system disorders. To verify a list of prospective male infertility biomarkers, we developed a multiplex selected reaction monitoring assay and measured the relative abundance of 31 proteins in 30 seminal plasma samples from normal, nonobstructive azoospermia and post-vasectomy individuals. Median levels of some proteins were decreased by more than 100-fold in nonobstructive azoospermia or post-vasectomy samples, in comparison with normal samples. To follow up the most promising candidates and measure their concentrations in seminal plasma, heavy isotope-labeled internal standards were synthesized and used to reanalyze 20 proteins in the same set of samples. Concentrations of candidate proteins in normal seminal plasma were found in the range 0.1-1000 μg/ml but were significantly decreased in nonobstructive azoospermia and post-vasectomy. These data allowed us to select, for the first time, biomarkers to discriminate between normal, nonobstructive azoospermia, and post-vasectomy (simulated obstructive azoospermia) seminal plasma samples. Some testis-specific proteins (LDHC, TEX101, and SPAG11B) performed with absolute or nearly absolute specificities and sensitivities. Cell-specific classification of protein expression indicated that Sertoli or germ cell dysfunction, but not Leydig cell dysfunction, was observed in nonobstructive azoospermia seminal plasma. The proposed panel of biomarkers, pending further validation, could lead to a clinical assay that can eliminate the need for testicular biopsy to diagnose the category of male infertility, thus providing significant benefits to patients as well as decreased costs associated with the differential diagnosis of azoospermia.     

Mouse germ cell clusters form by aggregation as well as clonal divisions

After their arrival in the fetal gonad, mammalian germ cells express E-cadherin and are found in large clusters, similar to germ cell cysts in Drosophila. In Drosophila, germ cells in cysts are connected by ring canals. Several molecular components of intercellular bridges in mammalian cells have been identified, including TEX14, a protein required for the stabilization of intercellular bridges, and several associated proteins that are components of the cytokinesis complex. This has led to the hypothesis that germ cell clusters in the mammalian gonad arise through incomplete cell divisions. We tested this hypothesis by generating chimeras between GFP-positive and GFP-negative mice. We show that germ cell clusters in the fetal gonad arise through aggregation as well as cell division. Intercellular bridges, however, are likely restricted to cells of the same genotype.