Testis cell pyroptosis mediated by CASP1 and CASP4: possible sertoli cell-only syndrome pathogenesis.

BACKGROUND: Sertoli cell-only syndrome (SCOS) is the most serious pathological type of non-obstructive azoospermia. Recently, several genes related to SCOS have been identified, including FANCM, TEX14, NR5A1, NANOS2, PLK4, WNK3, and FANCA, but they cannot fully explain the pathogenesis of SCOS. This study attempted to explain spermatogenesis dysfunction in SCOS through testicular tissue RNA sequencing and to provide new targets for SCOS diagnosis and therapy. METHODS: We analyzed differentially expressed genes (DEGs) based on RNA sequencing of nine patients with SCOS and three patients with obstructive azoospermia and normal spermatogenesis. We further explored the identified genes using ELISA and immunohistochemistry. RESULTS: In total, 9406 DEGs were expressed (Log2|FC|≥ 1; adjusted P value < 0.05) in SCOS samples, and 21 hub genes were identified. Three upregulated core genes were found, including CASP4, CASP1, and PLA2G4A. Thus, we hypothesized that testis cell pyroptosis mediated by CASP1 and CASP4 might be involved in SCOS occurrence and development. ELISA verified that CASP1 and CASP4 activities in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenesis. Immunohistochemical results showed that CASP1 and CASP4 in the normal spermatogenesis group were mainly expressed in the nuclei of spermatogenic, Sertoli, and interstitial cells. CASP1 and CASP4 in the SCOS group were mainly expressed in the nuclei of Sertoli and interstitial cells because of the loss of spermatogonia and spermatocytes. CASP1 and CASP4 expression levels in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenisis. Furthermore, the pyroptosis-related proteins GSDMD and GSDME in the testes of patients with SCOS were also significantly higher than those in control patients. ELISA also showed that inflammatory factors (IL-1 ß, IL-18, LDH, and ROS) were significantly increased in the SCOS group. CONCLUSIONS: For the first time, we found that cell pyroptosis-related genes and key markers were significantly increased in the testes of patients with SCOS. We also observed many inflammatory and oxidative stress reactions in SCOS. Thus, we propose that testis cell pyroptosis mediated by CASP1 and CASP4 could participate in SCOS occurrence and development.

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.

A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human-induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell-only syndrome.

BACKGROUND: The most serious condition of male infertility is complete Sertoli cell-only syndrome (SCOS), which refers to the lack of all spermatogenic cells in the testes. The genetic cause of SCOS remains to be explored. We aimed to investigate the genetic cause of SCOS and assess the effects of the identified causative variant on human male germ cells. METHODS: Whole-exome sequencing was performed to identify potentially pathogenic variants in a man with complete SCOS, and Sanger sequencing was performed to verify the causative variant in this man and his father and brother. The pathogenic mechanisms of the causative variant were investigated by in vitro differentiation of human-induced pluripotent stem cells (hiPSCs) into germ cell-like cells. RESULTS: The homozygous loss-of-function (LoF) variant p.His244ArgfsTer31 (c.731_732delAT) in PIWIL2 was identified as the causative variant in the man with complete SCOS, and the same variant in heterozygosis was confirmed in his father and brother. This variant resulted in a truncated PIWIL2 protein lacking all functional domains, and no PIWIL2 expression was detected in the patient's testes. The patient and PIWIL2-/- hiPSCs could be differentiated into primordial germ cell-like cells and spermatogonial stem cell-like cells (SSCLCs) in vitro, but the formation and maintenance of SSCLCs were severely impaired. RNA-seq analyses suggested the inactivation of the Wnt signaling pathway in the process of SSCLC induction in the PIWIL2-/- group, which was validated in the patient group by RT-qPCR. The Wnt signaling pathway inhibitor hindered the formation and maintenance of SSCLCs during the differentiation of normal hiPSCs. CONCLUSIONS: Our study revealed the pivotal role of PIWIL2 in the formation and maintenance of human spermatogonial stem cells. We provided clinical and functional evidence that the LoF variant in PIWIL2 is a genetic cause of SCOS, which supported the potential role of PIWIL2 in genetic diagnosis. Furthermore, our results highlighted the applicability of in vitro differentiation models to function validation experiments.

MicroRNA expression profiles in the seminal plasma of nonobstructive azoospermia patients with different histopathologic patterns.

OBJECTIVE: To investigate microRNA (miRNA) expression profiles in the seminal plasma of nonobstructive azoospermia (NOA) patients with different histopathologic patterns and evaluate potential noninvasive diagnostic biomarkers of NOA. DESIGN: Sequencing and validation using quantitative reverse transcription polymerase chain reaction (qRT-PCR). SETTING: Reproductive center and research institute. PATIENT(S): Thirteen patients with NOA (7 Sertoli cell-only syndrome [SCOS] and 6 hypospermatogenesis to spermatogenesis arrest [SA]) and 7 normal fertile controls for sequencing, six samples per group for validation; 54 patients with NOA (27 SCOS and 27 SA) and 19 normal fertile controls for large-sample qRT-PCR analysis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): MicroRNA expression profiles in the seminal plasma of patients with NOA with different histopathologic patterns were assessed using high-throughput sequencing and validated using qRT-PCR. RESULT(S): There were 78 overexpressed and 132 underexpressed miRNAs in patients with SCOS and 32 up-regulated and 90 down-regulated miRNAs in patients with SA compared with fertile men with normozoospermia. Two down-regulated and one up-regulated miRNA were validated using qRT-PCR, which indicated that the qRT-PCR and sequencing results were basically consistent. Hsa-miR-34c-5p expression was significantly lower in the seminal plasma of patients with NOA than normal fertile controls. The area under the receiver operating characteristic curve(AUC) for hsa-miR-34c-5p was 0.979 and 0.987 in the seminal plasma of patients with SA and patients with SCOS, respectively, compared with normal fertile controls. The AUC was 0.799 for hsa-miR-34c-5p in the seminal plasma between patients with SA and patients with SCOS. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differentially expressed miRNA target genes revealed that the Notch signaling pathway was one of the most abundant signaling pathways. The expression of Hes5, an effector of the Notch signaling pathway, was significantly higher in the seminal plasma of patients with NOA than normal fertile controls. CONCLUSION(S): MicroRNA expression profiles in seminal plasma were altered in patients with NOA compared with normal fertile controls. The profiles differed in patients with NOA with different pathologic patterns. We speculate that miR-34c-5p in seminal plasma could be a potential noninvasive biomarker to diagnose patients with NOA and distinguish different pathologic types of NOA. The Notch signaling pathway may be involved in the pathogenesis of NOA.

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.

Expression and localization of retinoid receptors in the testis of normal and infertile men.

Retinoic acid (RA), the active metabolite of vitamin A, is one of the most important factors regulating spermatogenesis. RA activates downstream pathways through its receptors (retinoic acid receptor alpha [RARA], retinoic acid receptor beta, and retinoic acid receptor gamma [RARG]) and retinoid X receptors (retinoid X receptor alpha [RXRA], retinoid X receptor beta [RXRB], and retinoid X receptor gamma [RXRG]). These receptors may serve as therapeutic targets for infertile men. However, the localization and expression of retinoid receptors in normal and infertile men were unknown. In this study, we found RARA and RARG were mostly localized in spermatocytes and round spermatids, RXRB was mainly expressed in Sertoli cells, and RXRG was expressed in most cell types in the fertile human testis. The localization of RARA, RARG, RXRB, and RXRG in men with hypospermatogenesis (HYPO) was similar to that of men with normal fertility. In addition, the messenger RNA expression levels of RARA, RARG, RXRA, RXRB, and RXRG were significantly decreased in men with Sertoli cell-only syndrome (SCOS) and maturational arrest (MA), but not in men with HYPO. These results suggest that reduced levels of RARA, RARG, RXRB, RXRA, and RXRG are more closely associated with SCOS and MA spermatogenetic failure. These results could contribute to the development of new molecular indicators of spermatogenic dysfunction and might provide novel therapeutic targets for treating male infertility.

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.

Induction of Sertoli-like cells from human fibroblasts by NR5A1 and GATA4.

Sertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). Here, we report the in vitro reprogramming of fibroblasts to human induced Sertoli-like cells (hiSCs). Initially, five transcriptional factors and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles and cellular properties that are similar to those of primary human Sertoli cells. Moreover, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. hiSCs suppress the proliferation of human T lymphocytes and protect xenotransplanted human cells in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli cell only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells.

Aberrant gene expression by Sertoli cells in infertile men with Sertoli cell-only syndrome.

Sertoli cell-only (SCO) syndrome is a severe form of human male infertility seemingly characterized by the lack all spermatogenic cells. However, tubules of some SCO testes contain small patches of active spermatogenesis and thus spermatogonial stem cells. We hypothesized that these stem cells cannot replicate and seed spermatogenesis in barren areas of tubule because as-of-yet unrecognized deficits in Sertoli cell gene expression disable most stem cell niches. Performing the first thorough comparison of the transcriptomes of human testes exhibiting complete spermatogenesis with the transcriptomes of testes with SCO syndrome, we defined transcripts that are both predominantly expressed by Sertoli cells and expressed at aberrant levels in SCO testes. Some of these transcripts encode proteins required for the proper assembly of adherent and gap junctions at sites of contact with other cells, including spermatogonial stem cells (SSCs). Other transcripts encode GDNF, FGF8 and BMP4, known regulators of mouse SSCs. Thus, most SCO Sertoli cells can neither organize junctions at normal sites of cell-cell contact nor stimulate SSCs with adequate levels of growth factors. We propose that the critical deficits in Sertoli cell gene expression we have identified contribute to the inability of spermatogonial stem cells within small patches of spermatogenesis in some SCO testes to seed spermatogenesis to adjacent areas of tubule that are barren of spermatogenesis. Furthermore, we predict that one or more of these deficits in gene expression are primary causes of human SCO syndrome.

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.

[Expression of CLAUDIN-11 in the testicular tissue of the patient with non-obstructive azoospermia and its clinical significance].

OBJECTIVE: To study the expression of CLAUDIN-11 in the testis tissue of non-obstructive azoospermia (NOA) patients with different severities and investigate its clinical significance. METHODS: Sixty-two NOA patients were divided into a hypospermatogenesis (HS) group (n = 30) and a Sertoli cell only syndrome (SCO) group (n =32). The expression of CLAUDIN-11 in the testicular tissue of the patients was detected by immunohistochemistry, that of CLAUDIN-11 mRNA determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and the levels of serum reproductive hormones measured by chemiluminescent immunoassay. RESULTS: Immunohistochemistry showed that the expression of CLAUDIN-11 was mainly in the cytoplasm of the Sertoli cells around the seminiferous tubule wall in the HS group, but diffusely distributed in the membrane of the Sertoli cells in the SCO group. RT-qPCR revealed a significantly lower expression of CLAUDIN-11 mRNA in the HS than in the SCO group (0.008 ± 0.001 vs 0.013 ± 0.002, t = 10.616, P<0.01). The level of serum luteotropic hormone (LH) was also markedly lower in the HS than in the SCO group (ï¼»3.62 ± 1.34ï¼½ vs ï¼»4.96 ± 3.10ï¼½ IU/L, P<0.05) and so was that of follicle-stimulating hormone (FSH) (ï¼»5.36 ± 2.80ï¼½ vs ï¼»10.65 ± 9.18ï¼½ IU/L, P<0.05). CONCLUSIONS: The up-regulated expression of CLAUDIN-11 in Sertoli cells may play an important role in the development and progression of spermatogenic dysfunction in NOA patients.

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.

Abnormal Accumulation of Collagen Type I Due to the Loss of Discoidin Domain Receptor 2 (Ddr2) Promotes Testicular Interstitial Dysfunction.

BACKGROUND: Loss of functional allele for discoidin domain receptor 2 (Ddr2) results in impaired Leydig cell response to luteinizing hormone (LH), low testosterone production and arrested spermatogenesis in older male Ddr2slie/slie mice. However, the underlying mechanism responsible for this phenotype remains unknown. Herein, we reported for the first time that the deregulated expression of Ddr2 cognate ligand, namely collagen type I (COL1), may account for the disruption of the testicular steroidogenesis in Ddr2slie/slie mutant testes. METHODOLOGY/PRINCIPAL FINDINGS: Expression of Ddr2 increased gradually along postnatal development, whereas COL1 expression became negligible from adulthood onwards. In Ddr2slie/slie mutant testis, however, in contrast to the undetectable staining of Ddr2, COL1 expression was constantly detected, with the highest values detected during adulthood. In the experimental vasectomy model, Ddr2slie/slie mutant mice exhibited an early androgen deficiency than wild-type mice, along with the accumulation of fibrotic tissue in the interstitium. Functionally, ablation of endogenous Ddr2 resulted in a significant decrease of testosterone (T) level in TM3 cells in the presence of higher concentration of COL1 treatment. Conversely, overexpression of Ddr2 could help TM3 cells to maintain a normal testicular steroidogenesis even in the presence of high concentration of COL1. Additionally, attenuated expression of Ddr2 correlates to the deregulated level of serum T levels in human pathological testes. CONCLUSIONS: Abnormal accumulation of interstitial COL1 may be responsible for the steroidogenic dysfunction in Ddr2slie/slie mutant testes.

PRPS2 Expression Correlates with Sertoli-Cell Only Syndrome and Inhibits the Apoptosis of TM4 Sertoli Cells.

PURPOSE: Sertoli-cell only syndrome is one of the reasons for male infertility but its pathogenesis remains unclear. PRPS2, a subset of PRS, is reported to be a potential protein associated with Sertoli-cell only syndrome. In this study we further investigated the correlation between PRPS2 and Sertoli-cell only syndrome, and evaluated the effect of PRPS2 expression on apoptosis of TM4 Sertoli cells. MATERIALS AND METHODS: PRPS2 expression was detected in patients with Sertoli-cell only syndrome and normal spermatogenesis, and in Sertoli-cell only syndrome mouse models by immunohistochemistry, quantitative reverse transcription-polymerase chain reaction and Western blot. PRPS2 expression in TM4 Sertoli cells was then down-regulated and up-regulated by lentivirus vectors. The effect of PRPS2 expression on cell apoptosis and cell cycle transition was evaluated by flow cytometry. RESULTS: PRPS2 expression in patients with Sertoli-cell only syndrome was significantly greater than in those with normal spermatogenesis. A significant increase in PRPS2 expression was observed in Sertoli-cell only syndrome mouse models. PRPS2 over expression significantly inhibited cell apoptosis and promoted cell cycle transition in TM4 Sertoli cells. However, PRPS2 down-regulation showed a reverse effect. Moreover, results revealed that PRPS2 over expression inhibited cell apoptosis via the p53/Bcl-2/caspase-9/caspase-3/caspase-6/caspase-7 signaling pathway. CONCLUSIONS: PRPS2 expression correlates with Sertoli-cell only syndrome and inhibits the apoptosis of TM4 Sertoli cells via the p53/Bcl-2/caspases signaling pathway.

Decreased expression of SAM68 in human testes with spermatogenic defects.

OBJECTIVE: To assess the expression patterns of SAM68 in the testes of azoospermic patients with normal and abnormal spermatogenesis. DESIGN: Retrospective study and in vitro study. SETTING: University hospital. PATIENT(S): Testicular biopsies of azoospermic men with normal spermatogenesis (OAZ; n=20), with maturation arrest at the spermatocyte stage (MA; n=20), and with Sertoli cell-only syndrome (SCOS; n=10). INTERVENTION(S): No interventions with patients. Knockdown of Sam68 was performed in the GC-2spd(ts) cell line. MAIN OUTCOME MEASURE(S): SAM68 expression was analyzed using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry analysis in tissues. Moreover, Sam68 was knocked down in GC-2spd(ts) cells. Cell viability was measured using the MTT assay, and the apoptosis rate was detected using flow cytometry with the Annexin V-FITC kit. RESULT(S): Using qRT-PCR, the expression level of testicular SAM68 mRNA in MA and SCOS patients was statistically reduced compared with in OAZ patients. In addition, using qRT-PCR, Western blot, and immunohistochemistry analyses, mRNA and protein expressions of SAM68 were absent or barely detectable in testicular tissues in 45% (9 of 20) of patients with MA and in all patients with SCOS. Furthermore, decreased expression of Sam68 suppressed germ cell proliferation and induced apoptosis in transfected GC-2spd(ts) cells. CONCLUSION(S): Deficient SAM68 expression was observed in the human testis with MA at the spermatocyte stage and SCOS. These results may offer new perspectives on the molecular basis of abnormal spermatogenesis.

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.

Up-regulation of SOX9 in sertoli cells from testiculopathic patients accounts for increasing anti-mullerian hormone expression via impaired androgen receptor signaling.

BACKGROUND: Testosterone provokes Sertoli cell maturation and represses AMH production. In adult patients with Sertoli-cells-only syndrome (SCOS) and androgen insensitivity syndrome (AIS), high level of AMH expression is detected in Sertoli cells due to defect of androgen/AR signaling. OBJECTIVE: We postulated that up-regulation of SOX9 due to impairment of androgen/AR signaling in Sertoli cells might explain why high level of anti-Mullerian hormone (AMH) expression occur in these testiculopathic patients. METHODS: Biological research of testicular specimens from men with azoospermia or mouse. The serum hormone levels were studied in 23 men with obstructive azoospermia, 33 men with SCOS azoospermia and 21 volunteers with normal seminograms during a period of 4 years. Immunohistochemical staining and reverse-transcription PCR were used to examine the relationships among AR, SOX9 and AMH expression in adult human and mouse testes. The ability of AR to repress the expression of SOX9 and AMH was evaluated in vitro in TM4 Sertoli cells and C3H10T1/2 cells. RESULTS: SCOS specimens showed up-regulation of SOX9 and AMH proteins but down-regulation of AR proteins in Sertoli cells. The mRNA levels of AR were significantly lower and the SOX9, AMH mRNA levels higher in all SCOS patients compared to controls (P< 0.05). The testosterone levels in the SCOS patients were within the normal range, but most were below the median of the controls. Furthermore, our in vitro cell line experiments demonstrated that androgen/AR signaling suppressed the gene and protein levels of AMH via repression of SOX9. CONCLUSIONS: Our data show that the functional androgen/AR signaling to repress SOX9 and AMH expression is essential for Sertoli cell maturation. Impairment of androgen/AR signaling promotes SOX9-mediated AMH production, accounts for impairments of Sertoli cells in SCOS azoospermic patients.

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.