Characterization of tumour necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in the adult human testis

Tumour necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of the tumour necrosis factor-alpha (TNF-alpha) family of cytokines which is known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, the presence of TRAIL and its receptors was investigated by immunohistochemistry in adult human testes. In addition, TRAIL and its receptors were studied in terms of protein and mRNA using western blot analysis and RT-PCR respectively. TRAIL and its receptors were immunodetected according to the different testicular cell types: TRAIL, DR5/TRAIL-R2 and DcR2/TRAIL-R4 were localized in Leydig cells, DR4/TRAIL-R1 was seen in peritubular and Sertoli cells whereas ligand and all receptors were detected in germ cells. Proteins and mRNA corresponding to TRAIL and its receptors were also identified in adult human testes. In conclusion, TRAIL and its receptors DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1/TRAIL-R3 and DcR2/TRAIL-R4 are expressed in the human testis, and are predominantly localized in different germ cell types.     

Human fetal testis: source of estrogen and target of estrogen action

BACKGROUND: Estrogens are involved in masculine fertility and spermatogenesis. However, little is known about estrogen involvement in human testicular organogenesis. Therefore the aim of this study was to investigate the cellular sources and targets of estrogens and their variations in the human testis during fetal development. Expression profiles of aromatase (CYP19) and estrogen receptors (ER) alpha and beta were analysed in human fetal testes at various gestational stages by immunohistochemistry and quantitative RT-PCR. METHODS: Fifty-four archival paraffin-embedded and four frozen fetal testes were studied by immunohistochemistry and real-time PCR. Tissue quality was confirmed by histology and expression of specific functional markers: androgenic enzymes for Leydig cells, anti-Müllerian hormone for Sertoli cells and Steel factor receptor for germ cells. RESULTS: We demonstrate that the human fetal testes express aromatase and ERbeta simultaneously in Sertoli, Leydig and germ cells but are devoid of ERalpha. Quantification of positive cells indicates a window of protein expression, especially between 13 and 22-24 weeks. Quantitative RT-PCR confirmed that the human fetal testis expresses CYP19 and ERbeta but not ERalpha mRNA. CONCLUSIONS: Our findings suggest that locally produced estrogens influence human testicular development through autocrine and paracrine mechanisms, most notably during the period of maximal testicular susceptibility to endocrine disruptors.     

CD147 is required for matrix metalloproteinases-2 production and germ cell migration during spermatogenesis

Spermatogenesis is a highly programmed process that requires the degradation of the extracellular matrix and the remodeling of tight junctions (TJ) to facilitate differentiating germ cell migration. Matrix metalloproteinases (MMPs) are essential in regulating Sertoli cell TJ in the testis. CD147 is known to stimulate the production of MMPs in tumor metastasis and its knockout mice are infertile. However, the functional relationship between CD147 and MMPs in spermatogenesis has not been investigated. In the present study, we examined the expression profile of CD147 and MMPs during mouse testicular development by RT-PCR, western blot and immunofluorescence staining. We also examined CD147 involvement in the production of MMP-2 and the migration of germ cells (GC-1 and GC-2 cells) using CD147 antibody or synthetic microRNA mimics-mediated knockdown. The results showed that CD147 was present at all stages of testicular development from 7 to 56 days post-partum (dpp). CD147 expression was found to increase after 21 days from moderate levels in 7 and 14 days. Of the eight MMPs studied, MMP-2, MMP-7, MMP-9 and MMP-23 were detected to have changes in expression during testicular development, with MMP-2 showing the largest change. CD147 and MMP-2 were co-localized in spermatogonia, spermatocytes and round spermatids in mouse testis, while in human testis, they were co-localized in spermatocytes and round spermatids. MMP-2 expression and migration of GC-1 and GC-2 cells were reduced by interfering with CD147 expression and function in vitro. These data suggest that CD147 regulates migration of spermatogonia and spermatocytes via induction of MMP-2 production during spermatogenesis.     

Misregulation of histone acetylation in Sertoli cell-only syndrome and testicular cancer

In many species, including humans, chromatin remodelling during spermiogenesis is initiated with a marked increase in histone acetylation in elongating spermatids. We have investigated whether this process is disturbed when spermatogenesis is defective or in human testicular tumours. For this purpose, the presence of highly acetylated histone H4 was detected on testicular sections from men with a severe impairment of spermatogenesis of several origins, as well as in different types of testicular tumours. In most tubules devoid of germinal cells (including SCO, Sertoli cell only syndromes) or lacking spermatocytes and spermatids, the Sertoli cells' nuclei showed a global increase in histone H4 acetylation. A similar observation was made in the peritumoral seminiferous tubules of testicular tumour tissues, whenever they were lacking germinal cells, with carcinoma in situ (CIS) cells being hypoacetylated. The global hyperacetylation of elongating spermatids during spermatogenesis could be part of an intercellular signalling pathway involving Sertoli cells and germinal cells, which could be disturbed in cases of severe spermatogenesis impairment, as well as in tubes surrounding germ cells in testicular tumours.     

OCT2, SSX and SAGE1 reveal the phenotypic heterogeneity of spermatocytic seminoma reflecting distinct subpopulations of spermatogonia

Spermatocytic seminoma (SS) is a rare testicular neoplasm that occurs predominantly in older men. In this study, we aimed to shed light on the histogenesis of SS by investigating the developmental expression of protein markers that identify distinct subpopulations of human spermatogonia in the normal adult testis. We analysed the expression pattern of OCT2, SSX2-4, and SAGE1 in 36 SS cases and four intratubular SS (ISS) as well as a series of normal testis samples throughout development. We describe for the first time two different types of SS characterized by OCT2 or SSX2-4 immunoexpression. These findings are consistent with the mutually exclusive antigenic profile of these markers during different stages of testicular development and in the normal adult testis. OCT2 was expressed predominantly in A(dark) spermatogonia, SSX2-4 was present in A(pale) and B spermatogonia and leptotene spermatocytes, whilst SAGE1 was exclusively present in a subset of post-pubertal germ cells, most likely B spermatogonia. The presence of OCT2 and SSX2-4 in distinct subsets of germ cells implies that these markers represent germ cells at different maturation stages. Analysis of SAGE1 and SSX2-4 in ISS showed spatial differences suggesting ongoing maturation of germ cells during progression of SS tumourigenesis. We conclude that the expression pattern of OCT2, SSX2-4, and SAGE1 supports the origin of SS from spermatogonia and provides new evidence for heterogeneity of this tumour, potentially linked either to the cellular origin of SS or to partial differentiation during tumour progression, including a hitherto unknown OCT2-positive variant of the tumour likely derived from A(dark) spermatogonia.     

Stem cell pluripotency factor NANOG is expressed in human fetal gonocytes, testicular carcinoma in situ and germ cell tumours

AIMS: NANOG is a key regulator of embryonic stem cell (ESC) self-renewal and pluripotency. Our recent genome-wide gene expression profiling study of the precursor of testicular germ cell tumours, carcinoma in situ testis (CIS), showed close similarity between ESC and CIS, including high NANOG expression. In the present study we analysed the protein expression of NANOG during normal development of human testis and in a large series of neoplastic/dysgenetic specimens. METHODS AND RESULTS: We detected abundant expression of NANOG in CIS and in CIS-derived testicular tumours with marked differences; seminoma and embryonal carcinoma were strongly positive, differentiated somatic elements of teratoma were negative. We provide evidence for the fetal origin of testicular cancer as we detected strong expression of NANOG in fetal gonocytes up to gestational week 20, with subsequent down-regulation occurring earlier than for OCT-4. We detected no expression at the protein level in normal testis. CONCLUSIONS: NANOG is a new marker for testicular CIS and germ cell tumours and the high level of NANOG along with OCT-4 are determinants of the stem cell-like pluripotency of the preinvasive CIS cell. Timing of NANOG down-regulation in fetal gonocytes suggests that NANOG may act as a regulatory factor up-stream to OCT-4.     

Environmental toxicant-induced germ cell apoptosis in the human fetal testis

BACKGROUND: Disorders of the male reproductive system are increasing in prevalence. The term testicular dysgenesis syndrome emphasizes the importance of developmental influences on the aetiology of conditions including cryptorchidism, testicular germ cell cancer and reduced spermatogenesis. Men whose mothers smoked during pregnancy have lower sperm production. Cigarette smoke contains agents acting on the aryl hydrocarbon receptor (AHR). We have investigated the presence of AHR in the developing human testis and the effects of functional activation. METHODS AND RESULTS: Immunohistochemistry determined AHR to be expressed by germ cells in the human testis between 7 and 19 week gestation, but not by other cells. Treatment of cultured fetal testis with an AHR ligand present in tobacco smoke increased markers of cell apoptosis, and this was prevented by an AHR receptor antagonist. Immunohistochemistry indicated that apoptosis was restricted to germ cells. CONCLUSIONS: Germ cells in the developing human testis are a target for regulation by AHR ligands. Activation of AHR by environmental toxicants and AHR-induced apoptotic pathways may be the mechanism of action underlying the epidemiological findings of reduced spermatogenesis in men exposed to cigarette smoke before birth, and may also be of importance in other conditions comprising the testicular dysgenesis syndrome.     

Identification of genes differentially expressed in testes containing carcinoma in situ

Virtually all testicular germ cell tumours originate from a common precursor, the carcinoma in situ (CIS) cell. The precise nature of the molecular mechanisms leading to CIS remains largely unknown. We performed the first systematic analysis of gene expression in testis with CIS compared to normal testis by the differential display (DDRT-PCR) method, with subsequent analysis by RT-PCR and in situ hybridization (ISH). In tissue containing CIS we identified overexpression of 28 mRNA, some previously reported in CIS and a number of genes not previously described in germ cell neoplasia, including the novel expressed sequence tag (EST) OIC1 (Overexpressed In CIS). The genes could be grouped functionally into genes involved in cell growth, proliferation, differentiation, immunological response, and genes with unknown biological function. Examples of overexpressed genes are SFRP1 that is involved in Wnt signalling and IGFBP6, which is of importance for fetal growth and inhibits cell growth through insulin-like growth factor-II. ISH analysis showed that both mRNA were localized to CIS cells. The results of our search for differentially expressed genes in CIS demonstrated a number of genes linked to testicular development (e.g. DCN, IGFBP6, SFRP1, SALL1), supporting our hypothesis that the origin of CIS is probably associated with disturbances of the fetal development of the testis.