A Role of the TEX101 Interactome in the Common Aetiology Behind Male Subfertility and Testicular Germ Cell Tumor.

Patients who develop testicular germ cell tumours (TGCT) are at higher risk to be subfertile than the general population. The conditions are believed to originate during foetal life, however, the mechanisms behind a common aetiology of TGCT and male subfertility remains unknown. Testis-expressed 101 (TEX101) is a glycoprotein that is related to male fertility, and downregulation of the TEX101 gene was shown in pre-diagnostic TGCT patients. In this review, we summarize the current knowledge of TEX101 and its interactome related to fertility and TGCT development. We searched literature and compilation of data from curated databases. There are studies from both human and animals showing that disruption of TEX101 result in abnormal semen parameters and sperm function. Members of the TEX101 interactome, like SPATA19, Ly6k, PICK1, and ODF genes are important for normal sperm function. We found only two studies of TEX101 related to TGCT, however, several genes in its interactome may be associated with TGCT development, such as PLAUR, PRSS21, CD109, and ALP1. Some of the interactome members are related to both fertility and cancer. Of special interest is the presence of the glycosylphosphatidylinositol anchored proteins TEX101 and PRSS21 in basophils that may be coupled to the immune response preventing further development of TGCT precursor cells. The findings of this review indicate that members of the TEX101 interactome could be a part of the link between TGCT and male subfertility.

TMT-based quantitative N-glycoproteomic analysis reveals glycoprotein protection can improve the quality of frozen bovine sperm.

Cryopreservation of bovine semen plays a vital role in accelerating genetic improvement and elite breeding, but it has a detrimental effect on sperm quality, resulting in the decline of the reproductive efficiency. The glycosylation modification of protein has irreplaceable roles in spermatozoa. Herein, the effect of cryopreservation on glycoproteins of bovine spermatozoa has been studied for the first time using a tandem mass tag (TMT)-labeled quantitative glycoproteome. A total of 2598 proteins and 492 glycoproteins were identified, including 83 different expression proteins (DEPs) and 44 different expression glycosylated proteins (DEGPs) between fresh and frozen spermatozoa. Thirty-three DEPs are glycoproteins, which demonstrates that glycoproteins of bovine sperm were seriously affected by cryopreservation. Moreover, the effects include glycoprotein expression, glycosylation modification, and substructure localization for proteins such as glycoproteins TEX101, ACRBP, and IZOMU4. The biologic functions of the 115 changed proteins are mainly involved in sperm capacitation, migration in female genitalia, and sperm-egg interaction. Mostly key regulators were identified to be glycoproteins, which confirms that glycosylated proteins played important roles in bovine sperm. This comprehensive study of sperm glycoproteins helps to unravel the cryoinjury mechanisms, thus implying that glycoprotein protection should be an effective way to improve the quality of frozen sperm.

Biochemical Assessments of Seminal Plasma Zinc, Testis-Expressed Sequence 101 and Free Amino Acids and Their Correlations with Reproductive Hormones in Male Infertility.

The role of the male factors in the couple's infertility has been significantly increased in recent years due to a sententious assessment of male reproductive functions and enhanced diagnostic tools. We investigated the correlations among the seminal plasma (SP) levels of each of zinc, testis-expressed sequence 101 (TEX101), and free amino acids levels with reproductive hormones in adult fertile and infertile men. The study included 100 infertile men categorized into 50 non-obstructive azoospermic patients and 50 patients with idiopathic oligoasthenoteratozoospermia (iOAT), in addition to 50 fertile controls. Semen analyses, serum ELISA assays for male reproductive hormones (follicle-stimulating hormone (FSH), luteinizing hormone (LH), total testosterone, and prolactin), colorimetric assays of SP zinc and total proteins, SP free amino acids using high-performance liquid chromatography (HPLC), and ELISA assays of SP TEX101 were performed for all subjects. Infertile men with azoospermia had significantly lower SP median levels of zinc, TEX101, and many SP free amino acids compared to both men with iOAT and fertile controls (P Ë‚ 0.05 for all). There were lower SP levels of zinc and some free amino acids among men with iOAT compared to the fertile controls (P Ë‚ 0.05 for all) with non-significant difference regarding to SP TEX101 (P Ëƒ 0.05). Azoospermic men exhibited negative correlations between FSH, LH, and prolactin with some SP free amino acids (P Ë‚ 0.05 for all), and a positive correlation between glycine with total testosterone (P Ë‚ 0.05). Among iOAT patients, LH and FSH were positively correlated with SP zinc, TEX101, and some measured free amino acids (P Ë‚ 0.05 for all). Total testosterone was positively correlated with some amino acids, while prolactin was negatively correlated with glycine (P Ë‚ 0.05 for all). iOAT and azoospermic men exhibited low SP zinc and some free amino acids levels that were more pronounced in azoospermic men and were significantly associated with the reproductive hormones. TEX101 could be a helpful confirmatory test for azoospermia.

Role of the Glycosylphosphatidylinositol-Anchored Protein TEX101 and Its Related Molecules in Spermatogenesis.

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) on the plasma membrane are involved in several cellular processes, including sperm functions. Thus far, several GPI-APs have been identified in the testicular germ cells, and there is increasing evidence of their biological significance during fertilization. Among GPI-APs identified in the testis, this review focuses on TEX101, a germ cell-specific GPI-AP that belongs to the lymphocyte antigen 6/urokinase-type plasminogen activator receptor superfamily. This molecule was originally identified as a glycoprotein that contained the antigen epitope for a specific monoclonal antibody; it was produced by immunizing female mice with an allogenic testicular homogenate. This review mainly describes the current understanding of the biochemical, morphological, and physiological characteristics of TEX101. Furthermore, future avenues for the investigation of testicular GPI-Aps, including their potential role as regulators of ion channels, are discussed.

Discovery of a Human Testis-specific Protein Complex TEX101-DPEP3 and Selection of Its Disrupting Antibodies.

TEX101 is a testis-specific protein expressed exclusively in male germ cells and is a validated biomarker of male infertility. Studies in mice suggest that TEX101 is a cell-surface chaperone which regulates, through protein-protein interactions, the maturation of proteins involved in spermatozoa transit and oocyte binding. Male TEX101-null mice are sterile. Here, we identified by co-immunoprecipitation-mass spectrometry the interactome of human TEX101 in testicular tissues and spermatozoa. The testis-specific cell-surface dipeptidase 3 (DPEP3) emerged as the top hit. We further validated the TEX101-DPEP3 complex by using hybrid immunoassays. Combinations of antibodies recognizing different epitopes of TEX101 and DPEP3 facilitated development of a simple immunoassay to screen for disruptors of TEX101-DPEP3 complex. As a proof-of-a-concept, we demonstrated that anti-TEX101 antibody T4 disrupted the native TEX101-DPEP3 complex. Disrupting antibodies may be used to study the human TEX101-DPEP3 complex, and to develop modulators for male fertility.

Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility.

BACKGROUND: TEX101 is a cell membrane protein exclusively expressed by testicular germ cells and shed into seminal plasma. We previously verified human TEX101 as a biomarker for the differential diagnosis of azoospermia, and developed a first-of-its-kind TEX101 ELISA. To demonstrate the clinical utility of TEX101, in this work we aimed at evaluating ELISA performance in a large population of fertile, subfertile, and infertile men. METHODS: Mass spectrometry, size-exclusion chromatography, ultracentrifugation, and immunohistochemistry were used to characterize TEX101 protein as an analyte in seminal plasma. Using the optimized protocol for seminal plasma pretreatment, TEX101 was measured by ELISA in 805 seminal plasma samples. RESULTS: We demonstrated that TEX101 was present in seminal plasma mostly in a free soluble form and that its small fraction was associated with seminal microvesicles. TEX101 median values were estimated in healthy, fertile pre-vasectomy men (5436 ng/mL, N = 64) and in patients with unexplained infertility (4967 ng/mL, N = 277), oligospermia (450 ng/mL, N = 270), and azoospermia (0.5 ng/mL, N = 137). Fertile post-vasectomy men (N = 57) and patients with Sertoli cell-only syndrome (N = 13) and obstructive azoospermia (N = 36) had undetectable levels of TEX101 (≤0.5 ng/mL). A cut-off value of 0.9 ng/mL provided 100% sensitivity at 100% specificity for distinguishing pre- and post-vasectomy men. The combination of a concentration of TEX101 > 0.9 ng/mL and epididymis-specific protein ECM1 > 2.3 µg/mL provided 81% sensitivity at 100% specificity for differentiating between non-obstructive and obstructive azoospermia, thus eliminating the majority of diagnostic testicular biopsies. In addition, a cut-off value of ≥0.6 ng/mL provided 73% sensitivity at 64% specificity for predicting sperm or spermatid retrieval in patients with non-obstructive azoospermia. CONCLUSIONS: We demonstrated the clinical utility of TEX101 ELISA as a test to evaluate vasectomy success, to stratify azoospermia forms, and to better select patients for sperm retrieval.

Quantitative Glycopeptide Changes in Rat Sperm During Epididymal Transit.

Mammalian spermatozoa acquire fertilizing potential as they undergo a series of changes during epididymal transit. One major facet of such is the alterations in the sperm glycome. Modifications of the sialic acid content within glycan moieties are known to regulate epitope presentation and cellular adhesion and signaling, all of which may be critical for sperm to successfully reach and fertilize the egg. To date, there is paucity of information regarding the sialic acid changes that occur on spermatozoa during epididymal transit. Therefore, the aim of this study was to identify N-linked sialylated glycoproteins in rat epididymal sperm and investigate whether they are regulated during epididymal transit. Sialylated glycopeptides from caput, corpus, and cauda spermatozoa were enriched using titanium dioxide beads. Bound N-linked glycopeptides were released by enzymatic deglycosylation using PNGase F and then analyzed by liquid chromatography tandem-mass spectrometry. A total of 92 unique N-linked sialylated glycopeptides were identified from 65 different proteins. These included members of the disintegrin and metalloproteinase domain-containing protein family (ADAM), Basigin, and Testis-expressed protein 101 (TEX101). Remarkably, label-free quantification showed that more than half of these peptides (48/92) were regulated during epididymal transit. Of interest, the protein TEX101 exhibited PNGase F-resistant deglycosylation under the conditions used in this study. The results from this study showed that changes in the N-linked sialoglycoprotein profile is a major hallmark of sperm maturation in rats.

Transcriptome analysis of the cancer/testis genes, DAZ1, AURKC, and TEX101, in breast tumors and six breast cancer cell lines.

Breast cancer is the most frequent cancer with second mortality rate in women worldwide. Lack of validated biomarkers for early detection of breast cancer to warranty the diagnosis and effective treatments in early stages has directed to the new therapeutic approach. Cancer/testis antigens which have restricted normal expression in testis and aberrant expression in different cancers are promising targets for generating cancer vaccines, monoclonal antibodies, or dendritic cell-based immunotherapy. In this context, we investigated the expression of two known cancer testis genes, Aurora kinase C (AURKC) and testis expressed 101 (TEX101), and one new candidate, deleted in azoospermia 1 (DAZ1), in six breast cancer cell lines including two ductal carcinomas, T47D and BT-474, and four adenocarcinomas, MDA-MB-231, MDA-MB-468, MCF7, and SKBR3 as well as 50 breast cancer tumors in comparison to normal mammary epithelial cells using quantitative real-time reverse transcription PCR (RT-PCR). Results showed significant overexpression (p = 0.000) of all three genes in BT474, DAZ1 in MDA-MB-231, and AURKC and DAZ1 in SKBR3 and significant downregulation (p = 0.000) of AURKC in MCF7 cell line relative to normal breast epithelial cells. Breast tumors showed significant overexpression of AURKC in comparison to normal breast tissues (p = 0.016). The results are noticeable especially in the case of AURKC; however, there is a little knowledge about the nature, causes, consequences, and effects of cancer/testis antigens activation in different cancers. It is suggested that AURKC has effects on cell division via its serin/threonin kinases activity and organizing microtubules in relation to centrosome/spindle function during mitosis.

An Emerging Role of TEX101 Protein as a Male Infertility Biomarker.

Infertility is an important aspect of human reproduction. It affects up to 15% of couples, with the male factor contributing to approximately 50% of all cases. Azoospermia is one of the most severe forms of male infertility, which is characterized by the absence of sperm in semen. The mechanisms underlying male infertility remain unknown. Currently, clinicians rely on semen analysis to predict the reproductive potential of a male, and testicular biopsy is the only reliable method to diagnose different subtypes of azoospermia. Recently, advances in proteomics encouraged the search for novel male infertility biomarkers in seminal plasma. In this review, we focus on TEX101, a testicular germ cell-specific protein, one of the most promising male infertility biomarkers. We discuss its role in spermatogenesis and fertilization and summarize our current knowledge about this new potential biomarker.

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.

Gene suppression of mouse testis in vivo using small interfering RNA derived from plasmid vectors.

We evaluated whether inhibiting gene expression by small interfering RNA (siRNA) can be used for an in vivo model using a germ cell-specific gene (Tex101) as a model target in mouse testis. We generated plasmid-based expression vectors of siRNA targeting the Tex101 gene and transfected them into postnatal day 10 mouse testes by in vivo electroporation. After optimizing the electroporation conditions using a vector transfected into the mouse testis, a combination of high- and low-voltage pulses showed excellent transfection efficiency for the vectors with minimal tissue damage, but gene suppression was transient. Gene suppression by in vivo electroporation may be helpful as an alternative approach when designing experiments to unravel the basic role of testicular molecules.

Expression of Testis Specific Genes TSGA10, TEX101 and ODF3 in Breast Cancer.

BACKGROUND: Breast cancer is the most common malignancy in women around the world so finding new biomarkers for early detection and also study on molecular aspects of breast cancer is valuable. Cancer testis genes are a group of genes expressed solely in testis and in a range of human malignancies. OBJECTIVES: In this study we determined the expression of cancer testis genes Tsga10, TEX101 and ODF3 in patients with breast cancer. MATERIALS AND METHODS: Fifty patients with breast cancer were enrolled in this study. Breast cancer cell lines MCF-7 and MDA-MB-231 were also used to determine the expression of testis cancer genes. For both patients and cell lines, cancer testis genes of TSGA10, TEX101 and ODF3 were determined by RT-PCR. The presence of auto antibody against these genes in patients' serums was carried on by ELISA method. RESULTS: Seventy percent of patients showed TSGA10 expression but none of them showed expression of TEX101 and ODF3. Fourteen percent of patients were positive for anti TSGA10 but all patients were negative for anti TEX101 and anti ODF3. Both of breast cancer cell lines exhibited very strong expression of TSGA10. CONCLUSIONS: Because of the important roles of Tsga10 in cell proliferation, we concluded that this gene may have a role in proliferation and survival of breast cancer cells and could be used for diagnosis and immunotherapy of breast cancer.