Limited distribution of TEX101 in testis

To elucidate the expression profile of TEX101, 28 types of normal human organs were collected and subjected to immunohistochemistry with a high-quality anti-TEX101 antibody. Analysis of the sections showed that testis was the only organ in which TEX101 was highly expressed (Figure 1). The staining for TEX101 was not detected in the other 27 types of organs, including ovary (Figure 1). These results confirmed that TEX101 was a testis-abundant protein. This marker has been utilized to diagnose male infertility [16, 17].

Expression characteristics of TEX101 in testis

Further detailed analysis revealed the expression characteristics of TEX101 in testis. The high magnification view showed that the connective tissue beyond the seminiferous tubules presented negative staining for TEX101. Although TEX101 presented very strong staining in the seminiferous tubules, the negative staining was also observed in some types of cells (Figure 2). Analysis of the sections showed that TEX101 was highly expressed in spermatocytes and spermatids but relatively expressed at low levels in spermatogonia that were near the outer edge of the seminiferous tubules (Figure 2). It is known that the maturation of male germ cells follows the course from spermatogonia to spermatocytes and finally spermatids. Our finding confirmed TEX101 should be related to the maturation process of germ cells and be considered a biomarker for the later stage of development.

Western blotting analysis of TEX101 in testis

Western blotting was also performed to analyze the expression features of TEX101. The tissue proteins from three different individuals were subjected to Western blotting and showed accordant features. For human testis tissues, a single, specific band of 38 kDa was detected by a polyclonal antibody of anti-TEX101 (Figure 3). However, no band was detected in mouse testis examined under the same experimental conditions (Figure 3). It is interesting that no band appeared at 21 kDa, which is the predicted molecular weight of the mature TEX101 protein. These results disclosed the actual molecular weight of TEX101 in human testis tissues and confirmed the human specificity of this TEX101 antibody.

Structure prediction showed that human and mouse TEX101 both possess a UPAR/Ly6 domain. However, only 60% homology was found in the amino acid sequences of the two proteins. This relatively low homology suggested that there was a greater evolutionary variation and there could be different functions between human and mouse TEX101. The actual molecular weight of TEX101 was determined to be 38 kDa, which is much larger than its predicted molecular weight, and this difference in size could result from post-translational modification [18, 19]. Bioinformatics analysis showed that the human TEX101 protein contains one GPI anchor site (Asn-222) and two glycosylation sites (Asn-45 and Asn-159). These modifications could lead to variations in structure and electrical charge, which might also contribute to the increased mobility of TEX101 on the gel [20, 21].

Disappearance of TEX101 in testicular cancer

Because high expression of TEX101 was detected in testis, we also wanted to know if its expression changed in testicular cancer. Thirty-eight testicular cancer tissues, including 36 samples of seminoma and two samples of yolk sac tumor and embryonal carcinoma, were subjected to immunohistochemistry. The sections showed that none of the testicular cancer tissues showed staining for TEX101, resulting in 100% negative expression (Figure 4). In contrast, all 10 samples of normal testis tissues that were collected in this study were confirmed to possess strong staining of TEX101, resulting in 100% positive expression (Figure 4).

Previous studies demonstrated that TEX101 could reduce the activities of uPA, MMPs and cathepsin B, leading to the suppression of cell growth and migration [12]. We speculated that the loss of TEX101 could disrupt the metabolic balance of the above-mentioned enzymes in the germinal epithelium, which could contribute to the growth and migration of mutant cancer cells. Therefore, our research not only found a potential biomarker for the diagnosis of testicular cancer but also provided a potential molecular mechanism for the progression of testicular cancer. However, for testicular cancer in men who do not have a germ cell line (e.g. Sertoli cell only syndrome), TEX101 should not be a good marker. Other studies found the expression of TEX101 in head and neck squamous cell carcinoma (HNSCC) as well as in nasopharyngeal carcinoma and chronic myeloid leukemia [10–12], which indicated that TEX101 could be related to cancer progression.

Materials

The primary antibodies were purchased from Atlas (rabbit anti-TEX101, R38320) and Santa Cruz (mouse anti-beta-actin, sc-81178). The secondary antibodies, including goat anti-rabbit IgG (ZB-2301) and goat anti-mouse IgG (ZB-2305), were purchased from ZSGB-Bio. The Immobile Western chemiluminescent HRP substrate (WBKLS01000) was purchased from Millipore. The antibodies used for immunohistochemistry (biotinylated anti-rabbit IgG, ZB-2010) and horseradish peroxidase-labeled streptavidin (ZB-2404) were purchased from ZSGB-Bio.

Animal and human tissue samples

Three BALB/c mice were obtained from the Animal Center of Sichuan University. A total of 38 samples of testicular cancer tissue, 10 samples of normal testis tissue, and different organs samples from 3 different human individuals were from West China Hospital of Sichuan University. Collected tissues samples were numbered and fixed in formalin (4%) for 3 days, and the fixed samples were then washed with water for 2 h, and dehydrated with a graded ethanol series (75%, 85%, 95% and 100%) for 1 h each. Then, the samples were infiltrated with xylene for 1 h and embedded in paraffin blocks. The tissue was cut into 4 μm-thick sections, which were then placed on highly adhesive slides. The sections were stored at 4°C until the subsequent experiments were performed. This study was approved by the institutional ethics committee of Sichuan University. All of the patients assented to participate in the study after providing informed consent.

Immunohistochemistry

Immunohistochemistry was performed using the SP (streptavidin-peroxidase) method. Briefly, the sections were pretreated with 10 mM citrate buffer (pH 6.0), incubated with 3% H₂O₂ for 10 min, and blocked in 5% BSA for 10 min at room temperature. The sections were then probed with the primary antibody against TEX101 (1:300, diluted with PBS) overnight at 4°C. Subsequently, the sections were washed with PBS three times and incubated with biotinylated anti-rabbit IgG for 30 min at 37°C. After washing three times, the sections were incubated with horseradish peroxidase-labeled streptavidin for 30 min at 37°C and then washed four times with PBS. Finally, the sections were visualized using diaminobenzidine (DAB). Images were captured (200× and 400×) using high-resolution microscopy (Leica DM2500 microscope) and analyzed using Leica Application Suite software.

Western blotting

The collected tissue samples of human and mouse testis were ground in liquid nitrogen, lysed in RIPA Buffer for 30 min on ice, and centrifuged at 16,000 g for 30 min at 4°C. After measurement using a Protein Assay kit (Bio-Rad), the proteins were separated on 12% SDS-PAGE gels and transferred to PVDF membranes (Bio-Rad, 162–0177). The membranes were blocked in TBST (20 mM Tris (pH 7.5), 150 mM NaCl, and 0.1% Tween 20) that contained 5% skimmed milk for 3 h at 37°C and then incubated with the primary antibodies against TEX101 (1:1000, diluted by TBST) for 2 h at 37°C. Subsequently, the membranes were washed three times with TBST and probed with secondary antibodies conjugated to HRP (1:15000, diluted by TBST) for 1 h at 37°C. After washing three times, the membranes were developed using the Immobilon Western kit (chemiluminescent HRP substrate) (Millipore).