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.

[In vitro culture of the testis tissue of BALB/c mice with spermatogenic dysfunction].

OBJECTIVE: To explore the pathways of development and maturation of the testis tissue in mice with spermatogenic dysfunction in vitro. METHODS: Sixty-eight 8-week-old BALB/c male mice were randomly divided into four groups of equal number, normal control, Sertoli cell only syndrome (SCOS), severe hypo-spermatogenesis (H-S1), and mild hypo-spermatogenesis (H-S2), and the models were established in the latter three groups by intraperitoneal injection of busulfan at 40 mg/kg for 4, 6 and 8 weeks, respectively. The testis tissues of the mice were cultured with the agarose gel method in vitro till the 4th week, followed by determination of the expressions of the marker proteins STRA8 at meiotic initiation, SCP3 during meiosis, and TNP1 after meiosis by immunohistochemistry. The development and maturation of the germ cells during the in vitro culture were evaluated, and their apoptosis detected by TUNEL. RESULTS: The more severe the testicular tissue injury, the lower the expression of the STRA8 protein in the SCOS, H-S1 and H-S2 groups as compared with the normal control before in vitro culture on agarose gel (P < 0.05), and the STRA8 expression was significantly upregulated in the former three groups after 4 weeks of culturing (P < 0.05). The expression of SCP3 was the lowest in the SCOS but the highest in the H-S2 group before culturing (P < 0.05), and was not as high as that in the control, though increased after 4 weeks of culturing. TNP1 was positively expressed in all the mice of the control, some individuals of the H-S1 and H-S2 groups (P< 0.05), but not in the SCOS group at 4 weeks. The apoptosis of germ cells was significantly increased in the SCOS but decreased in the H-S groups compared with that in the normal control after 4 weeks of culturing (P< 0.05). CONCLUSIONS: In vitro culture on agarose gel induces the meiosis of the testis tissue in BALB/c mice with spermatogenic dysfunction, and the effect is even better in those with mild spermatogenic dysfunction.

Expression patterns of HENMT1 and PIWIL1 in human testis: implications for transposon expression.

This study aimed to define the expression patterns of HENMT1 and PIWI proteins in human testis and investigate their association with transposon expression, infertility sub-type or development of testicular germ cell tumours (TGCTs). Testis biopsies showing normal spermatogenesis were used to identify normal localisation patterns of HENMT1 and PIWIL1 by immunolocalisation and RT-PCR after laser microdissection. 222 testis biopsies representing normal spermatogenesis, hypospermatogenesis, spermatogenic arrests, Sertoli cell-only (SCO) tumours and TGCTs were analysed by RT-qPCR for expression of HENMT1/PIWIL1/PIWIL2/PIWIL3/PIWIL4 and LINE-1 Additionally, HENMT1-overexpressing TCam2 seminoma cell lines were analysed for the same parameters by RT-qPCR. We found that HENMT1 and PIWIL1 are coexpressed in pachytene spermatocytes and spermatids. Expression of HENMT1, PIWIL1 and PIWIL2 was mainly dependent on germ cell content but low levels of expression were also detected in some SCO samples. Levels of HENMT1, PIWIL1 and PIWIL2 expression were low in TGCT. Samples with HENMT1, PIWIL2 and PIWIL4 expression showed significantly (P < 0.05) lower transposon expression compared to samples without expression in the same histological group. HENMT1-overexpressing TCam2 cells showed lower LINE-1 expression than empty vector-transfected control lines. Our findings support that the transposon-regulating function of the piRNA pathway found in the mouse is conserved in adult human testis. HENMT1 and PIWI proteins are expressed in a germ-cell-specific manner and required for transposon control.

Expression of katanin p80 in human spermatogenesis.

OBJECTIVE: To define the stage-by-stage expression of KATNB1 during human spermatogenesis. DESIGN: Gene expression analysis, histologic and immunohistochemical evaluation. SETTING: University research laboratories and andrological clinic. PATIENT(S): Eighty human testicular biopsy samples: 43 showing normal spermatogenesis, 9 with maturation arrest at level of spermatocytes, 8 with maturation arrest at level of spermatogonia, and 20 with a Sertoli cell only syndrome. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Evaluation of katanin p80 expression in normal as well as impaired spermatogenesis on mRNA (RT-PCR, RT-qPCR, and in situ hybridization) and protein level (immunohistochemistry/immunofluorescence). RESULT(S): KATNB1 messenger RNA is exclusively expressed in germ cells, and quantitatively reduced in maturation arrests at the level of spermatogonia. The KATNB1 protein was detected in type B spermatogonia entering meiosis and in the Golgi complex of pachytene spermatocytes. Immediately before the first meiotic division, it is colocalized with the cleaving centriole. It was also detected in early round spermatids in the dictyosome. CONCLUSION(S): The expression and localization of KATNB1 support a role in spindle formation. The localization of KATNB1 in early round spermatids suggests an involvement in the formation of microtubule-based structures during spermiogenesis (manchette and flagellum). These data are consistent with the demonstrated role of KATNB1 in mouse meiosis, nuclear shaping, and flagellum formation of sperm and suggest the strong conservation of function even between distantly related species.

Altered gene expression signature of early stages of the germ line supports the pre-meiotic origin of human spermatogenic failure.

The molecular basis of spermatogenic failure (SpF) is still largely unknown. Accumulating evidence suggests that a series of specific events such as meiosis, are determined at the early stage of spermatogenesis. This study aims to assess the expression profile of pre-meiotic genes of infertile testicular biopsies that might help to define the molecular phenotype associated with human deficiency of sperm production. An accurate quantification of testicular mRNA levels of genes expressed in spermatogonia was carried out by RT-qPCR in individuals showing SpF owing to germ cell maturation defects, Sertoli cell-only syndrome or conserved spermatogenesis. In addition, the gene expression profile of SpF was compared with that of testicular tumour, which is considered to be a severe developmental disease of germ cell differentiation. Protein expression from selected genes was evaluated by immunohistochemistry. Our results indicate that SpF is accompanied by differences in expression of certain genes associated with spermatogonia in the absence of any apparent morphological and/or numerical change in this specific cell type. In SpF testicular samples, we observed down-regulation of genes involved in cell cycle (CCNE1 and POLD1), transcription and post-transcription regulation (DAZL, RBM15 and DICER1), protein degradation (FBXO32 and TM9SF2) and homologous recombination in meiosis (MRE11A and RAD50) which suggests that the expression of these genes is critical for a proper germ cell development. Interestingly, a decrease in the CCNE1, DAZL, RBM15 and STRA8 cellular transcript levels was also observed, suggesting that the gene expression capacity of spermatogonia is altered in SpF contributing to an unsuccessful sperm production. Altogether, these data point to the spermatogenic derangement being already determined at, or arising in, the initial stages of the germ line.

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.

Spermatogonial stem cell functions in physiological and pathological conditions.

Sperm have a vital role in the continuity of a species by contributing genetic information to the next generation. Production of these specialized gametes in numbers sufficient to confer normal fertility occurs via cycling of the spermatogenic lineage, a process referred to as spermatogenesis. Continuity relies on the activities of a self-renewing reservoir of spermatogonial stem cells (SSCs) from which progenitors will arise that transiently amplify in number before committing to a pathway of terminal differentiation. A primary population of SSCs is established during neonatal development from a pool of quiescent gonocyte precursors that forms in embryogenesis. Disruption of this process has dire consequences on maintenance of a cycling spermatogenic lineage in adulthood. At present, the molecular mechanisms underlying initial formation of the SSC pool are largely undefined. However, several transcription factors and posttranscriptional regulators have been identified as important regulators of SSC self-renewal from studies with mutant mouse models and experimental manipulation within primary cultures of mouse SSCs. Importantly, loss of function of these self-renewal factors may be underlying causes of infertility. Furthermore, disruption in the establishment of the SSC state within gonocytes or misregulation of self-renewal may manifest as testicular germ cell tumors in postnatal life.

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.

Sperm retrieval and live birth rates in presumed Sertoli-cell-only syndrome in testis biopsy: a single centre experience.

We aimed to investigate sperm retrieval rates (SRR) by testicular sperm extraction (TESE), factors affecting SRR, and fertilization rate (FR), implantation rate (IR), clinical pregnancy rate (CPR) and live birth rate (LBR) in patients with presumed Sertoli-cell-only syndrome in testis biopsy (SCOS). We retrospectively evaluated files of 134 patients with SCOS who underwent TESE. Group I were patients in whom spermatozoa were retrieved and Group II were patients in whom no spermatozoa could be retrieved. SRR, Follicle stimulating hormone (FSH), Luteinizing hormone (LH), and testosterone levels, and the volume of testicles were compared between groups. In addition, FR, IR, CPR and LBR were determined. Sperm retrieval was achieved in 37 (27.6%) patients (Group I), and the remaining 97 (72.4%) patients made Group II. There were no significant differences in age, infertility time, testicular volume, serum FSH, LH and testosterone levels between Groups I and II (p > 0.05). Intracytoplasmic sperm injection (ICSI) was performed in 36 patients. FR, IR, and CPR were 60.86 ± 23.03, 36.53 ± 41.78 and 51.3% respectively. Cycle and patient based LBRs were 37.8 and 45.1% respectively. SRR in SCOS is lower than patients with non-obstructive azoospermia (NOA) in general. No parameters to predict spermatozoa retrieval were determined. In patients with SCOS, ICSI achieves similar live birth rate to other patients with NOA.

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.

Androgen decline in patients with nonobstructive azoospemia after microdissection testicular sperm extraction.

OBJECTIVES: Microdissection testicular sperm extraction (TESE) is the ideal procedure for obtaining a high sperm retrieval rate. However, few studies of the postoperative endocrinologic course have been reported. We evaluated the endocrinologic course for 1 year after microdissection TESE and compared the results with the testicular histologic findings. METHODS: A total of 69 patients with nonobstructive azoospermia who had undergone microdissection TESE were included. The overall sperm retrieval rate was 50.7%. The endocrinologic data were evaluated before and 3, 6, and 12 months after surgery. RESULTS: The mean serum total testosterone level in patients with hypospermatogenesis decreased postoperatively and had recovered by 12 months (102%). The mean serum total testosterone level in patients with Klinefelter syndrome also decreased postoperatively but had recovered to only 50% of the baseline value at 12 months after microdissection TESE. At 12 months, the mean serum total testosterone level in patients with maturation arrest was 93.1% of the preoperative level and that in patients with Sertoli cell-only syndrome was 80.6% of the preoperative level. The preoperative serum luteinizing hormone and follicle-stimulating hormone in patients with Klinefelter syndrome was high and remained high after microdissection TESE. The mean serum luteinizing hormone and follicle-stimulating hormone levels in patients with hypospermatogenesis did not change, and those in patients with maturation arrest increased continuously after microdissection TESE. Finally, those in patients with Sertoli cell-only syndrome increased up to 6 months after surgery and decreased after that. CONCLUSIONS: The results of our study indicate that long-term endocrinologic follow-up is necessary after microdissection TESE, particularly for patients with Klinefelter syndrome to detect hypogonadism.

Is there a role for the nuclear export factor 2 gene in male infertility?

OBJECTIVE: To determine the presence of mutations in the NXF2 gene of patients with nonobstructive azoospermia. DESIGN: Molecular analysis of male infertility. SETTING: University genetic laboratory and reproductive clinic. PATIENT(S): Sixty-five patients with Sertoli cell-only syndrome (SCOS) and 20 control men. INTERVENTION(S): Polymerase chain reaction, sequencing analysis, RNA extraction, and reverse transcription polymerase chain reaction. MAIN OUTCOME MEASURE(S): Expression of NXF2 messenger RNA and analysis of the NXF2 gene for the presence of mutations and polymorphisms. RESULT(S): Messenger RNA derived from the NXF2 gene could be amplified from normal human testicular tissue. Sequencing analysis showed the presence of two polymorphisms in the NXF2 gene. A first alteration, c.1779C>T, was observed in one man who had complete SCOS. Although it is located near an intron-exon boundary, this change probably does not affect splicing. The second alteration, c.1857A>G, was detected in 22 patients with complete SCOS and in 13 patients with incomplete SCOS. Also, 15 of 20 men with normal spermatogenesis had this alteration. Neither of these alterations causes a change at the amino acid level. CONCLUSION(S): No mutations were detected in the NXF2 gene, from which we concluded that there is no need to screen for mutations in the NXF2 gene in a routine IVF program.

Expression and distribution of laminin chains in the testis for patients with azoospermia.

The aim of our study was to investigate the relationships between the expression of laminin in the testis and spermatogenesis, and the basement membrane (BM) of testicular tubules in fertile and infertile men. Testicular tissue samples were collected from the testes of 9 patients with obstructive azoospermia (OA), 9 patients with maturation arrest (MA), and 15 patients with Sertoli cell-only syndrome (SCO). In testicular tissue, laminin was identified by staining with polyclonal antibodies. Serum follicle-stimulating hormone (FSH), lutenizing hormone (LH), and testosterone were determined by chemiluminescence assays. In seminal plasma, laminin was estimated using a double-antibody enzyme immunoassay. BM thickness was significantly correlated with testicular tubule diameter (r = -0.49, P = .004) and FSH (r = 0.52, P = .008). The beta2 chain of laminin was most expressed on the inner BM of testicular tubules. The laminin index for the beta2 chain in SCO was significantly higher than in OA (P < .0001) and MA (P = .03). The mean seminal laminin levels in SCO were significantly lower than in OA (P < .001). We demonstrated that overabundance of the beta2 chain of laminin is associated with increased BM thickness and is possibly related to spermatogenic dysfunction.