DDX58 expression promotes inflammation and growth arrest in Sertoli cells by stabilizing p65 mRNA in patients with Sertoli cell-only syndrome.

Sertoli cell -only syndrome (SCOS) is a type of testicular pathological failure that causes male infertility and no effective treatment strategy, is available for this condition. Moreover, the molecular mechanism underlying its development remains unknown. We identified DExD/H-Box helicase 58 (DDX58) as a key gene in SCOS based on four datasets of testicular tissue samples obtained from the Gene Expression Synthesis database. DDX58 was significantly upregulated in SCOS testicular Sertoli cells. Moreover, high expression of DDX58 was positively correlated with the expression of several testicular inflammatory factors, such as IL -1ß, IL-18, and IL-6. Interestingly, DDX58 could be induced in the D-galactose (D-gal)-stimulated TM4 cell injury model. Whereas silencing of DDX58 inhibited D-gal -mediated p65 expression, inflammatory cytokine release, and growth arrest. Mechanistically, we found that DDX58 acts as an RNA-binding protein, which enhances p65 expression by promoting mRNA stability. Furthermore, p65 gene silencing decreased the expression of inflammatory cytokines and inhibition of cell growth in D-gal-induced cells. In conclusion, our findings demonstrate that DDX58 promotes inflammatory responses and growth arrest in SCOS Sertoli cells by stabilizing p65 mRNA. Accordingly, the DDX58/p65 regulatory axis might be a therapeutic target for SCOS.

Testicular steroid sulfatase overexpression is associated with Leydig cell dysfunction in primary spermatogenic failure.

BACKGROUND: Decreased testosterone (T) to LH ratio and increased 17ß-estradiol (E2) serum concentrations represent a common finding among patients with severe spermatogenic failure, suggesting a concurrent Leydig cell steroidogenic dysfunction. Aromatase overexpression has been associated with increased serum and intratesticular E2 in these patients. However, it is unknown whether the sulfatase pathway contributes to the increased availability of active estrogens in patients with primary spermatogenic failure. OBJECTIVES: To assess estrogen sulfotransferase (SULT1E1) and steroid sulfatase (STS) mRNA abundance in testicular tissue of patients with Sertoli cell-only syndrome (SCOS) and normal tissues, its association with serum and intratesticular hormone levels, and to explore the mRNA and protein testicular localization of both enzymes. MATERIALS AND METHODS: Testicular tissues of 23 subjects with SCOS (cases) and 22 patients with obstructive azoospermia and normal spermatogenesis (controls) were obtained after biopsy. SULT1E1 and STS transcripts accumulation was quantified by RT-qPCR. For mRNA and protein localization, we performed RT-qPCR in Leydig cell clusters and seminiferous tubules isolated by laser-capture microdissection and immunofluorescence in testicular tissues. Serum and intratesticular hormones were measured by immunoradiometric assays. RESULTS: SULT1E1 mRNA accumulation was similar in both groups. The amount of STS mRNA was higher in cases (p = 0.007) and inversely correlated with T/LH ratio (r = -0.402; p = 0.02). Also, a near significant correlation was observed with intratesticular E2 (r = 0.329, p = 0.057), in agreement with higher intratesticular E2 in cases (p < 0.001). Strong STS immunoreaction was localized in the wall of small blood vessels but not in Leydig cells. Both SULT1E1 and STS mRNA abundance was similar in Leydig cell clusters and the tubular compartment, except for lower SUTL1E1 mRNA in the seminiferous tubules of SCOS patients (p = 0.001). CONCLUSIONS: Our results suggest that an unbalance of the STS/SULT1E1 pathway contributes to the testicular hyperestrogenic microenvironment in patients with primary spermatogenic failure and Leydig cell dysfunction.

Characterization of the stem cell niche components within the seminiferous tubules in testicular biopsies of Klinefelter patients.

OBJECTIVE: To characterize the tubular environment in testicular biopsy tissues from patients with Klinefelter syndrome (KS). DESIGN: Observational immunohistochemical study. SETTING: Academic research unit. PATIENT(S): Males with KS and controls at different developmental time points: fetal, prepubertal, peripubertal, and adult. INTERVENTION(S): Immunohistochemical analysis of testicular biopsies samples to characterize maturation of Sertoli cells and tubular wall components-peritubular myoid cells (PTMC) and extracellular matrix (ECM) proteins. MAIN OUTCOME MEASURE(S): Intensity of antimüllerian hormone staining; proportion of Sertoli cells expressing androgen receptor (AR); and expression of tubular wall markers as characterized by identifying abnormal staining patterns. RESULT(S): Decreased expression for alpha smooth muscle actin 2 (ACTA2) was observed in peripubertal and adult KS as well as in Sertoli cell only (SCO) patients. Altered expression patterns for all ECM proteins were observed in SCO and KS biopsy tissues compared with controls. Only for collagen I and IV were altered expression patterns observed between KS and SCO patients. In peripubertal samples, no statistically significant differences were observed in the maturation markers, but altered ECM patterns were already present in some samples. CONCLUSION(S): The role of loss of ACTA2 expression in PTMC in the disintegration of tubules in KS patients should be further investigated. Future research is necessary to identify the causes of testicular fibrosis in KS patients. If the mechanism behind this fibrotic process could be identified, this process might be altered toward increasing the chances of fertility in KS patients.

Ran-binding protein 3 is associated with human spermatogenesis and male infertility.

Ran-binding protein 3 (RanBP3) is a Ran-interacting protein, which participates in the Ran GTPase system in cancer cell biology. However, the expression pattern and physiological role of RanBP3 remain largely unknown. In this study, we found that RanBP3 was expressed in human testes and localised to spermatogonium and spermatocyte of germ cells. In subcellular structure, its localisation is in the nucleus and cytoplasm. Interestingly, compared with normal groups, RanBP3 expression was lower in groups of patients with Maturation Arrest (MA) and Sertoli cell-only syndrome (SCO) when considered by the Johnson Score. RanBP3 expression in the MA group and SCO groups was dramatically lower than that in the normal control group. Studies have shown that RanBP3, which is one of the helper factors of Ran, is mainly participate in the nucleocytoplasmic transport of cells. RanBP3 helps Ran to achieve some functions such as nucleocytoplasmic transport, spindle assembly during mitosis and nuclear assembly after mitosis. Consequent changes in the expression of RanBP3 may associate with human spermatogenesis disorders and male infertility. The identification and characterisation of RanBP3 enhances our understanding of the molecular mechanisms underpinning its function in human spermatogenesis and male infertility.

Fibroblast growth factor-5 promotes spermatogonial stem cell proliferation via ERK and AKT activation.

BACKGROUND: Sertoli cells are the most important somatic cells contributing to the microenvironment (named niche) for spermatogonial stem cells (SSCs). They produce amounts of crucial growth factors and structure proteins that play essential roles in the complex processes of male SSCs survival, proliferation, and differentiation. It has been suggested that Sertoli cell abnormalities could result in spermatogenesis failure, eventually causing azoospermia in humans. However, to the end, the gene expression characteristics and protein functions of human Sertoli cells remained unknown. In this study, we aimed to evaluate the effect of fibroblast growth factor-5 (FGF5), a novel growth factor downregulated in Sertoli cells from Sertoli cell-only syndrome (SCOS) patients compared to Sertoli cells from obstructive azoospermia (OA) patients, on SSCs. METHODS: We compared the transcriptome between Sertoli cell from SCOS and OA patients. Then, we evaluated the expression of FGF5, a growth factor which is downregulated in SCOS Sertoli cells, in human primary cultured Sertoli cells and testicular tissue. Also, the proliferation effect of FGF5 in mice SSCs was detected using EDU assay and CCK-8 assay. To investigate the mechanism of FGF5, Phospho Explorer Array was performed. And the results were verified using Western blot assay. RESULTS: Using RNA-Seq, we found 308 differentially expressed genes (DEGs) between Sertoli cells from SCOS and OA patients. We noted and verified that the expression of fibroblast growth factor-5 (FGF5) was higher in Sertoli cells of OA patients than that of SCOS patients at both transcriptional and translational levels. Proliferation assays showed that rFGF5 enhanced the proliferation of mouse SSCs line C18-4 in a time- and dose-dependent manner. Moreover, we demonstrated that ERK and AKT were activated and the expression of Cyclin A2 and Cyclin E1 was enhanced by rFGF5. CONCLUSION: The distinct RNA profiles between Sertoli cells from SCOS and OA patients were identified using RNA-Seq. Also, FGF5, a growth factor that downregulated in SCOS Sertoli cells, could promote SSCs proliferation via ERK and AKT activation.

Leydig cell dysfunction is associated with post-transcriptional deregulation of CYP17A1 in men with Sertoli cell-only syndrome.

STUDY QUESTION: Is the expression of steroidogenic enzyme 17α-Hydroxylase/17,20-Lyase (CYP17A1) down-regulated in Leydig cells (LCs) of men with spermatogenic failure and compensated impairment of LC function, i.e. a low testosterone to LH (T/LH) ratio? SUMMARY ANSWER: Although the transcriptional expression of CYP17A1 is increased, its protein expression is decreased, in isolated LCs of men with spermatogenic failure and reduced serum T/LH. WHAT IS KNOWN ALREADY: Primary spermatogenic defects have been associated with functional and morphological abnormalities of LCs, characterized by decreased serum testosterone (T) levels, decreased T/LH, increased 17ß-estradiol (E2) and E2/T ratio, and larger clusters of LCs (LC hyperplasia). CYP17A1 is a key enzyme in the testosterone pathway and has been implicated in the steroidogenic lesion produced by E2 stimulation. STUDY DESIGN, SIZE, DURATION: We studied 18 azoospermic patients with Sertoli cell-only syndrome (SCOS) and signs of LC dysfunction (cases) and 10 obstructive azoospermic/oligozoospermic men with normal spermatogenesis (controls). The SCOS patients were sub-grouped into 9 cases with T/LH <2 and 9 cases with T/LH ≥2. All of the men underwent testicular biopsy for sperm retrieval at the Reproductive Unit of a University Hospital. PARTICIPANTS/MATERIALS, SETTING, METHODS: The transcriptional expression of CYP17A1 and SF-1 (steroidogenic factor 1) was quantified by SYBR®Green-based qPCR in LCs isolated by laser capture microdissection (LCM), and relative expression to the control pool was assessed. CYP17A1 protein expression was semi-quantified by indirect immunofluorescence (IFI) using Image-Pro Plus v7.0 (Media Cybernetics) in testicular tissue. FSH and LH serum concentrations, and serum and intratesticular T (ITT) and E2 (ITE2) were measured by IRMA and RIA, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Relative CYP17A1 mRNA expression was increased in cases with T/LH <2 compared to cases with T/LH ≥2, by a mean of 3.3-fold (P = 0.002). No corresponding increase in protein expression was found; in fact, CYP17A1 immunostaining intensity assessed by the Integrated Optical Density (IOD) parameter was lower in the cases with T/LH <2 compared to controls (P = 0.008). Relative SF-1 mRNA expression was similar in both case subgroups. CYP17A1 mRNA expression correlated with ITE2 and intratesticular E2/T (r = 0.536; P = 0.026 and r = 0.542; P = 0.016, respectively), while an inverse association was observed for ITE2 and protein level expression (r = -0.421; P = 0.05). LARGE SCALE DATA: Not applicable. LIMITATIONS REASONS FOR CAUTION: We should interpret the results of the semi-quantification of immunofluorescent staining by Image-Pro Plus software with caution, because it is a semi-quantitative method that may have certain difficulties regarding the disposition of protein in the cells. However, it is not influenced by variations in the number of cells that express the protein, as could be the case of western blot analysis in testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: Dysfunctional LCs of men with SCOS show post-transcriptional deregulation of CYP17A1, with increased mRNA and decreased protein expression, which may be modulated by increased ITE2 levels. In addition, transcriptional expression of CYP17A1 was not associated with changes in SF-1 mRNA expression. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Fund for Scientific and Technological Development (FONDECYT) of Chile to A.C. [grant number 1120176]. The authors declare no conflict of interest.

Overexpression of CYP19A1 aromatase in Leydig cells is associated with steroidogenic dysfunction in subjects with Sertoli cell-only syndrome.

Several observational studies have showed a combination of lower testosterone (T) to LH ratio and higher estradiol (E2 ) to T ratio in secretory infertile men compared to men with normal spermatogenesis, suggesting a steroidogenic dysfunction of Leydig cells (Lc) that may involve increased aromatase activity. Low T/LH ratio is associated with Lc hyperplasia, which together with LH hyperstimulation may represent compensation for impaired T production. Aromatase expression and oestrogen production are mainly detected in Lc of the testis, although Sertoli and germ cells also contribute to testicular aromatase activity. The aim of this study was to assess the transcriptional expression of CYP19A1 (aromatase) in isolated Lc of subjects with Sertoli cell-only syndrome (SCOS) and signs of Lc impairment. Nineteen patients with SCOS and 10 controls with normal spermatogenesis who had medical indication of testicular biopsy for sperm retrieval were studied. Leydig cells were isolated by Laser Capture Microdissection (LCM) and CYP19A1 mRNA expression was quantified by SYBR® Green-based qPCR. In addition, testicular T and E2 and serum hormonal levels were measured. Relative to control group, CYP19A1 was overexpressed more than twofold in 10/19 cases (2.3-12.2-fold increase), showing a significant increment in cases with low T/LH ratio (T/LH < 2) compared to cases with T/LH ≥ 2 (p = 0.038, REST® ). Moreover, Rq data for CYP19A1 had a direct correlation with testicular levels of E2 and the E2 /T ratio (r = 0.869; p < 0.001 and r = 0.633; p = 0.005). In summary, Lc from infertile patients with signs of Lc dysfunction overexpressed aromatase and showed an increment of testicular E2 . Our results suggest that increased expression of aromatase in Lc leads to higher E2 production and may account for the functional impairment of the Lc in patients with SCOS.

Melatonin in testes of infertile men: evidence for anti-proliferative and anti-oxidant effects on local macrophage and mast cell populations.

Melatonin acting through the hypothalamus and pituitary regulates testicular function. In addition, direct actions of melatonin at the testicular level have been recently suggested. We have described that melatonin inhibits androgen production in hamster Leydig cells via melatonin subtype 1a (mel1a) receptors and the local corticotrophin-releasing hormone (CRH) system. The initial events of the melatonin/CRH signalling pathway have also been established. Melatonin and all components of the melatonergic/CRH system were also detected in Leydig cells of infertile men. This study attempted to search for additional targets of melatonin in the human testis, and to investigate the effects of melatonin on proliferation and the oxidative state in these novel target cells. To this aim, evaluation of human testicular biopsies of patients suffering from hypospermatogenesis or Sertoli cell only syndrome and cell culture studies were performed. Melatonergic receptors were found in macrophages (MACs) and mast cells (MCs) of the human testis. In biopsies of patients suffering idiopathic infertility, melatonin testicular concentrations were negatively correlated with MAC number per mm(2) and TNFα, IL1ß and COX2 expression, but positively correlated with the expression of the anti-oxidant enzymes SOD1, peroxiredoxin 1 and catalase. Melatonin inhibited proliferation and the expression of pro-inflammatory cytokines and cyclooxygenase 2 (COX2) in both the human non-testicular THP-1 MAC cell line and primary cell cultures of hamster testicular MACs. In the human HMC-1 MC line, melatonin increased the expression of anti-oxidant enzymes and decreased reactive oxygen species (ROS) generation. The results reveal new testicular targets of melatonin and describe anti-proliferative and anti-inflammatory effects of this hormone on testicular MACs. Furthermore, melatonin might provide protective effects against oxidative stress in testicular MCs.

Fold-change correction values for testicular somatic transcripts in gene expression studies of human spermatogenesis.

STUDY QUESTION: What are the reference values for delineating altered somatic cell gene expression from transcript enrichment/dilution in gene expression studies of human spermatogenesis? SUMMARY ANSWER: We have designed a crosstable and rule-of-thumb values for different stages of spermatogenic impairment that define the reference cut-off values for altered gene expression in Sertoli and Leydig cells in the context of impaired spermatogenesis. WHAT IS KNOWN ALREADY: Morphometrical studies have shown that on the cellular level, impaired spermatogenesis results in a relative enrichment of somatic cell types. However, until now it is not known how this affects transcript levels in gene expression studies. STUDY DESIGN, SIZE DURATION: In this study, 31 testis biopsies from men with different stages of spermatogenic impairment (full spermatogenesis, hypospermatogenesis, meiotic arrest, spermatogonial presence, Sertoli cell-only syndrome, complete tubular atrophy) were used to define reference ratios of somatic transcript enrichment/dilution. The reference ratios were validated on an independent test set of 28 samples and on gene expression data from men with Y-chromosomal microdeletions. PARTICIPANTS/MATERIAL, SETTING, METHODS: High-quality microarray data were filtered with respect to Sertoli- and Leydig-cell-specific genes. General reference enrichment/dilution factors for these two cell types for all combinations of spermatogenic impairment were calculated using robust permutation statistics. To validate the specificity of the filtered transcripts, we calculated ratios for an independent test set of spermatogenic impairment and for transcriptional data from men with Y-chromosomal microdeletions, and checked the functional enrichment (gene ontology) and cellular localization of the corresponding proteins in a histological database and assessed their correlation with testicular size. MAIN RESULTS AND THE ROLE OF CHANCE: Filtering of Sertoli- and Leydig-cell-specific genes resulted in a set of 54 and 332 transcripts, respectively. These were used in defining robust reference dilution/enrichment factors of somatic transcripts for all spermatogenic levels and were compiled in a reference crosstable. Validation on an independent test set showed ratios within 0.5 units of our reference crosstable. Analysis of the resulting transcripts with respect to functional enrichment for Sertoli- and Leydig-cell-specific functions and protein expression, as obtained from an immunohistochemical database, indicated filtering of data sets highly enriched for Sertoli and Leydig cell function. The dilution/enrichment ratios differed significantly when transcripts were interrogated in samples with Y-chromosomal microdeletions, pointing to an overall decreased expression of somatic markers in a genetically altered background. LIMITATIONS, REASONS FOR CAUTION: The defined reference ratios might apply with some restrictions in samples that display very heterogeneous histology (e.g. Sertoli cell only with a significant proportion of spermatogenic foci) or when spermatogenic impairment is a consequence of an altered genetic background. WIDER IMPLICATIONS OF THE FINDINGS: The reference dilution/enrichment values for somatic testicular transcripts as defined in this study are to be seen as cut-off values for discriminating between simple transcript dilution/enrichment as a consequence of an altered germ cell composition and actual transcriptional regulation. Future studies dealing with transcriptional changes in testicular somatic cells in a background of altered germ cell quantities should consider these correction factors in order to avoid the description of transcriptional changes that are based simply on shifts in somatic cellular quantities. STUDY FUNDING/COMPETING INTEREST(S): Financial support was from grant Sp721/1-3 of the German Research Foundation. There are no competing interests to be declared.

Presence of metastasis-associated protein 1 in Sertoli cells is required for proper contact between Sertoli cells and adjacent germ cells.

OBJECTIVE: To investigate whether the normal expression of metastasis-associated protein 1 (MTA1) in Sertoli cells (SCs) is associated with adjacent germ cells (GCs) and to provide the functional relevance of MTA1 in this somatic cell. METHODS: The expression pattern of MTA1 in the SCs of impaired human spermatogenesis was determined using immunohistochemistry. The effect of the depletion of GCs on the expression of MTA1 in isolated SCs was evaluated using reverse transcriptase polymerase chain reaction in murine testes treated with busulphan. Finally, using multiple assays, the functional investigation of MTA1 by its specific knockdown was performed in SC-GC co-cultures. RESULTS: SCs were negatively immunolabeled in the tubules with impaired spermatogenesis. Depletion of murine GCs by treatment with busulphan resulted in a dramatic decrease of the MTA1 transcripts level in the isolated SCs on the 15th day of treatment and thereafter had totally abolished MTA1 expression by the 30th day of treatment, respectively. The addition of isolated round spermatids into SC culture could partially elevate MTA1 expression in the latter. Furthermore, MTA1 is crucial to maintain the GC nursery function and normal anchoring junction formation in SCs because ablation of MTA1 by siRNA induced extensive defects of genes related to SC homeostasis. CONCLUSION: We propose a novel role for SC-expressing MTA1, which is determined by the presence of surrounding GCs, in mediating the crosstalk between SCs and GCs by influencing a broad spectrum of gene changes.

HnRNPL as a key factor in spermatogenesis: Lesson from functional proteomic studies of azoospermia patients with sertoli cell only syndrome.

Sertoli cell only syndrome (SCOS) is one of the main causes leading to the abnormal spermatogenesis. However, the mechanisms for abnormal spermatogenesis in SCOS are still unclear. Here, we analyzed the clinical testis samples of SCOS patients by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to find the key factors contributing to SCOS. Thirteen differential proteins were identified in clinical testis samples between normal spermatogenesis group and SCOS group. Interestingly, in these differential proteins, Heterogeneous nuclear ribonucleoprotein L(HnRNPL) was suggested as a key regulator involved in apoptosis, death and growth of spermatogenic cells by String and Pubgene bioinformatic programs. Down-regulated HnRNPL in testis samples of SCOS patients was further confirmed by immunohistochemical staining and western blotting. Moreover, in vitro and in vivo experiments demonstrated that knockdown of HnRNPL led to inhibited proliferation, increased apoptosis of spermatogenic cell but decreased apoptosis of sertoli cells. Expression of carcinoembryonic antigen-related cell adhesion molecule 1 in GC-1 cells or expression of inducible nitric oxide synthases in TM4 sertoli cells, was found to be regulated by HnRNPL. Our study first shows HnRNPL as a key factor involved in the spermatogenesis by functional proteomic studies of azoospermia patients with sertoli cell only syndrome. This article is part of a Special Issue entitled: Proteomics: The clinical link.

A novel application of cell-free seminal mRNA: non-invasive identification of the presence of germ cells or complete obstruction in men with azoospermia.

BACKGROUND: Cell-free seminal mRNA (cfs-mRNA) exists in human ejaculate at high concentrations and with high stability, and contains many tissue-specific transcripts secreted from the male reproductive system. Owing to the sensitivity of RNA technology, cfs-mRNAs are ideal candidates for non-invasive biomarkers of physiopathological conditions. This study applied cfs-mRNA in identifying the presence of either germ cells or complete obstruction in men with azoospermia. METHODS: RT-PCR was performed to amplify the germ cell-specific (DDX4), seminal vesicle-specific (SEMG1) and prostate-specific (TGM4) mRNAs from cfs-mRNAs, which were isolated from the seminal plasma of men with non-obstructive azoospermia (NOA) or obstructive azoospermia (OA). The 39 patients with NOA, diagnosed by testicular biopsy, included 8 men with maturation arrest (MA), 3 men with incomplete sertoli cell only (iSCO) syndrome and 28 men with complete SCO (cSCO). The 29 patients with OA, confirmed by the presence of sperm in the testis or epididymis, included 8 men with congenital bilateral absence of the vas deferens (CBAVD) and 21 men with non-CBAVD. Healthy individuals and vasectomized men were enrolled as controls. RESULTS: TGM4 was detected in all participants. Consistent with their diagnosis, DDX4 was detected in all patients with MA or iSCO but was absent in most cases of cSCO (n = 21, 75.0%) or non-CBAVD (n = 18, 85.7%), and in all men with vasectomy or CBAVD. The presence of DDX4 in the other seven men with cSCO and three patients with non-CBAVD suggests the presence of germ cells in the testis, and incomplete obstruction, respectively. SEMG1 was undetectable in three patients with CBAVD with bilateral absence of the seminal vesicles, and in two non-CBAVD cases with low ejaculate volume. CONCLUSIONS: These results suggest that, with high sensitivity and representativity, cfs-mRNA could be non-invasive biomarkers for identifying the presence of germ cells or complete obstruction in azoospermia.

Up-regulation of NDRG2 through nuclear factor-kappa B is required for Leydig cell apoptosis in both human and murine infertile testes.

Many pro-apoptotic factors, such as nuclear factor-kappa B (NF-κB) and Fas, play crucial roles in the process of Leydig cell apoptosis, ultimately leading to male sterility, such as in Sertoli cell only syndrome (SCO) and hypospermatogenesis. However, the molecular mechanism of such apoptosis is unclear. Recent reports on N-myc downstream-regulated gene 2 (ndrg2) have suggested that it is involved in cellular differentiation, development, and apoptosis. The unique expression of NDRG2 in SCO and hypospermatogenic testis suggests its pivotal role in those diseases. In this study, we analyzed NDRG2 expression profiles in the testes of normal spermatogenesis patients, hypospermatogenesis patients, and SCO patients, as well as in vivo and in vitro models, which were Sprague-Dawley rats and the Leydig cell line TM3 treated with the Leydig cell-specific toxicant ethane-dimethanesulfonate (EDS). Our data confirm that NDRG2 is normally exclusively located in the cytoplasm of Leydig cells and is up-regulated and translocates into the nucleus under apoptotic stimulations in human and murine testis. Meanwhile, transcription factor NF-κB was activated by EDS administration, bound to the ndrg2 promoter, and further increased in expression, effects that were abolished by NF-κB inhibitor Pyrrolidine dithiocarbamate (PDTC). Furthermore, siRNA knock-down of ndrg2 led to increased proliferative or decreased apoptotic TM3 cells, while over-expression of ndrg2 had the reverse effect. This study reveals that ndrg2 is a novel gene that participates in Leydig cell apoptosis, with essential functions in testicular cells, and suggests its possible role in apoptotic Leydig cells and male fertility.

Apoptosis-inhibitor Aven is downregulated in defective spermatogenesis and a novel estrogen target gene in mammalian testis.

OBJECTIVE: To study the expression and localization of Aven in rat and human testis from azoospermic patients with different etiologies and its regulation by estrogens. DESIGN: Experimental study. SETTING: University research center and private IVF clinic. PATIENT(S): Six men with obstructive azoospermia, five with hypospermatogenesis, and six with Sertoli cell-only syndrome; male Wistar rats. INTERVENTION(S): Testicular biopsies and rat seminiferous tubules (SeT) cultured in the presence or absence of 17ß-estradiol (E(2)). MAIN OUTCOME MEASURE(S): Testicular cell localization of Aven protein was analyzed by immunohistochemistry. Expression levels of Aven in testicular biopsies and cultured SeT, in the presence or absence of 17ß-estradiol, were determined by quantitative reverse transcription-polymerase chain reaction and Western blot. RESULT(S): Aven is expressed in Sertoli cells, spermatocytes, and spermatogonia of both rat and human testis. Aven is underexpressed in the testis of men with nonobstructive azoospermia, and its expression levels correlate with severity of spermatogenic status. Aven expression is regulated by E(2) in rat SeT cultured ex vivo. CONCLUSION(S): The results suggest that deregulation of the expression of the apoptosis inhibitor Aven may be related to male factor infertility. Moreover, Aven is an estrogen target gene and may be involved in the mechanism of testicular apoptosis control by estrogens.

Recognising the Sertoli-cell-only (SCO) syndrome: a case study.

Total Sertoli-cell-only (SCO) syndrome is often confused with a focal SCO picture, in which testicular illness caused damage to seminiferous tubules and compromised the Sertoli cell range of maturation and functions, but from which still some spermatozoa can be retrieved for assisted reproductive techniques. Here, a possibly new SCO syndrome phenotype is reported exhibiting complete lack of germ cells despite normal architecture of the seminiferous tubules with presence of mature Sertoli cells and normal Leydig cells in the intertubular tissue. Sertoli cells are immunonegative for the prepubertal differentiation markers cytokeratin-18, anti-Muellerian hormone and M2A antigen, but reveal a positive signal for the gap junctional protein connexin 43 known to be expressed in Sertoli cells with an adult type of differentiation. The complete lack of germ cells in combination with fully differentiated adult-type Sertoli cells in this case is in contradiction with known SCO subtypes and with the current hypothesis of reciprocal regulation of Sertoli and germ cell differentiation.

14-3-3 beta in the healthy and diseased male reproductive system.

BACKGROUND: Dysfunction of cellular processes in the testes can lead to infertility, tumourigenesis or other testicular disorders. 14-3-3 proteins are known to play pivotal roles in cellular communication, signal transduction, intracellular trafficking, cell-cycle control, transcription and cytoskeletal structure and have been implicated in several diseases including tumourigenesis. Here we investigated the expression of the 14-3-3 beta isoform in healthy testicular tissues of humans, rats and mice as well as in tissues of Sertoli-cell-only (SCO) syndrome, intratubular germ cell neoplasia (IGCN) and classical seminoma. METHODS: Samples of healthy and diseased testes from humans, rats and mice were analysed by immunohistochemistry. For PCR, human testis cell lysates were used. Immunoblot analyses of rats and humans healthy testes were performed. Duolink proximity ligation assay (PLA) and co-immunoprecipitation (Co-IP) were carried out to investigate interactions between 14-3-3 beta and vimentin in human, rat and mouse testes. RESULTS: In healthy testes and SCO syndrome, strong 14-3-3 beta-positive cells could be identified as Sertoli cells. Furthermore, 14-3-3 beta proteins were detected in cells of the peritubular stroma. In samples of IGCN and classical seminoma, the malignant transformed cells stained positive for 14-3-3 beta antigen. Immunoblot analyses revealed the presence of 14-3-3 beta in healthy testicular tissues. 14-3-3 beta mRNA transcripts were detected in cell lysates of healthy human testes. Interaction of 14-3-3 beta with the intermediate filament vimentin was revealed by Duolink PLA and Co-IP. Co-IP experiments identified tubulin as another 14-3-3 beta binding partner. CONCLUSIONS: Our data suggest that 14-3-3 beta expression is essential for normal spermatogenesis by interacting with vimentin in Sertoli cells. Additionally, 14-3-3 beta expression in malignant transformed cells in IGCN and classical seminoma may lead to tumourigenesis and cell survival.

SCF and c-kit expression profiles in male individuals with normal and impaired spermatogenesis.

The transcription levels of stem cell factor (SCF) and c-kit were examined using real-time RT PCR in interstitial and intratubular cell fractions, as well as in tissue homogenates from normal, azoospermic and neoplasmic patients. Peripheral blood mononuclear cells (PBMC) were used as a systemic control. The observed level of c-kit expression in all investigated groups was generally higher than the expression of SCF. The highest (statistically significant) level of c-kit was noted in testicular tumours (the greater part of which were represented by seminomas) in contrast to SCF mRNA, which may indicate an association between c-kit overexpression and seminoma development. In Sertoli cell only syndrome, almost equal levels of SCF and c-kit transcripts were noted. These results may indicate Leydig cells as the alternative source of c-kit gene transcription. SCF transcript values were low and comparable among the analysed subgroups except that in maturation arrest at spermatocyte stage, the SCF gene expression was statistically higher than in testicular tumours. It appears from the study that c-kit has been a dynamic gene, changing its activity in a variety of testicular pathologies while being expressed in all testicular compartments but clearly overexpressed in testicular tumours of seminomatous origin.

Cyclooxygenase-2 in testes of infertile men: evidence for the induction of prostaglandin synthesis by interleukin-1ß.

As we previously reported, testes of men suffering from hypospermatogenesis and germ cell arrest or Sertoli cell-only syndrome show a major increase in the number of macrophages expressing interleukin-1ß (IL-1ß) and abundant expression of cyclooxygenase-2 (COX-2), the inducible isoform of the key enzyme in the biosynthesis of prostaglandins (PGs), in Leydig cells. In the present study we report [1] a positive correlation between IL-1ß levels and COX-2 expression in testes of infertile patients, [2] the induction of COX-2 by IL-1ß in mouse Leydig cells (TM3) and human macrophages (THP-1), and therefore [3] evidence for an IL-1ß-dependent induction of testicular inflammatory states.

Immunohistological profile of the Ras homologous B protein (RhoB) in human testes showing normal spermatogenesis, spermatogenic arrest and Sertoli cell only syndrome.

Ras homologous B protein (RhoB) belongs to the Ras homologous subfamily which consists of low molecular weight (21 kDa) GTP-binding proteins. Rho proteins are regulatory molecules associated with various kinases and as such they mediate changes in cell shape, contractility, motility and gene expression. To date, no data are available about the expression pattern of RhoB protein in the human testis showing normal and abnormal spermatogenesis. The present study addresses these issues. Human testicular biopsy specimens were obtained from patients suffering from post-testicular infertility (testis showing normal spermatogenesis, 10 cases) and testicular infertility (testis showing Sertoli cell only syndrome and spermatogenic arrest, 10 patients each). The expression of RhoB was examined using in situ immunofluorescent staining methods. In testes showing normal spermatogenesis, RhoB had a strong expression in the seminiferous epithelium (cytoplasm of Sertoli-cells, spermatogonia and spermatocytes) and in the interstitium (Leydig cells). RhoB expression was weak in the myofibroblasts and absent in the spermatids and sperms. In the testes showing abnormal spermatogenesis, RhoB expression was moderate in the seminiferous epithelium (cytoplasm of Sertoli cells, spermatogonia and spermatocytes) and was completely absent in the Leydig cells, myofibroblasts, spermatids and sperms. To the best of our knowledge, this study provides the first morphological indication that RhoB protein is expressed in human testis and its expression undergoes testicular infertility associated changes. These findings suggest the involvement of RhoB in the process of spermatogenesis in human and their possible therapeutic ramifications in testicular infertility are open for further investigations.

Comparing expression of progesterone and estrogen receptors in testicular tissue from men with obstructive and nonobstructive azoospermia.

The objective of this study was to identify and compare the expression profiles of progesterone receptor (PR) and estrogen receptor alpha (ERalpha) in the testes of men with obstructive azoospermia (OA), maturation arrest (MA), and Sertoli cell-only (SCO) histology. Testicular biopsies were obtained from 10 patients with OA, 10 patients with MA (either early or late arrest), and 8 patients with SCO who did not have hormonal abnormalities and varicoceles. Expression of PR and ERalpha was detected by immunofluorescence and Western blot. PR was expressed in the spermatogenic, Leydig, and Sertoli cells in the testes of OA patients. In the MA and SCO patients, the expression of PR was reduced in all cell types as compared with that in the OA patients. Western blot demonstrated that both the full-size (120 KDa) and the truncated (52 KDa) isoforms of the PR were expressed in the OA and MA testes. However, in the SCO testes, only the truncated isoform of PR (52 KDa) was expressed. ERalpha (66 KDa) was expressed principally in the spermatogenic and Leydig cells in the OA testes. By immunohistochemistry staining, expression of ERalpha was decreased in the spermatogenic and Leydig cells of the MA testes, whereas its expression was enhanced in the Leydig cells of the SCO testes. However, by Western blot, expression of ERalpha was significantly reduced in the SCO testes as compared with that in the OA and MA testes. We conclude that PR and ERalpha may play a role in the pathogenesis of the MA and SCO phenotype in patients with infertility.