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.

Global human tissue profiling and protein network analysis reveals distinct levels of transcriptional germline-specificity and identifies target genes for male infertility.

BACKGROUND: Mammalian spermatogenesis is a process that involves a complex expression program in both somatic and germ cells present in the male gonad. A number of studies have attempted to define the transcriptome of male meiosis and gametogenesis in rodents and primates. Few human transcripts, however, have been associated with testicular somatic cells and germ cells at different post-natal developmental stages and little is known about their level of germline-specificity compared with non-testicular tissues. METHODS: We quantified human transcripts using GeneChips and a total of 47 biopsies from prepubertal children diagnosed with undescended testis, infertile adult patients whose spermatogenesis is arrested at consecutive stages and fertile control individuals. These results were integrated with data from enriched normal germ cells, non-testicular expression data, phenotype information, predicted regulatory DNA-binding motifs and interactome data. RESULTS: Among 3580 genes for which we found differential transcript concentrations in somatic and germ cells present in human testis, 933 were undetectable in 45 embryonic and adult non-testicular tissues, including many that were corroborated at protein level by published gene annotation data and histological high-throughput protein immunodetection assays. Using motif enrichment analyses, we identified regulatory promoter elements likely involved in germline development. Finally, we constructed a regulatory disease network for human fertility by integrating expression signals, interactome information, phenotypes and functional annotation data. CONCLUSIONS: Our results provide broad insight into the post-natal human testicular transcriptome at the level of cell populations and in a global somatic tissular context. Furthermore, they yield clues for genetic causes of male infertility and will facilitate the identification of novel cancer/testis genes as targets for cancer immunotherapies.

Screening for biomarkers of spermatogonia within the human testis: a whole genome approach.

BACKGROUND: A key step in studying the biology of spermatogonia is to determine their global gene expression profile. However, disassociation of these cells from the testis may alter their profile to a considerable degree. To characterize the molecular phenotype of human spermatogonia, including spermatogonial stem cells (SSCs), within their cognate microenvironment, a rare subtype of human defective spermatogenesis was exploited in which spermatogonia were the only germ cell type. METHODS: The global expression profile of these samples was assessed on the Affymetrix microarray platform and compared with tissues showing homogeneous Sertoli-cell-only appearance; selected genes were validated by quantitative real-time PCR and immunohistochemistry on disparate sample sets. RESULTS: Highly significant differences in gene expression levels correlated with the appearance of spermatogonia, including 239 best candidates of human spermatogonially expressed genes. Specifically, fibroblast growth factor receptor 3 (FGFR3), desmoglein 2 (DSG2), E3 ubiquitin ligase c-CBL (casitas B-cell lymphoma), cancer/testis antigen NY-ESO-1 (CTAG1A/B), undifferentiated embryonic cell transcription factor 1 (UTF1) and synaptosomal-associated protein, 91 kDa homolog (SNAP91) were shown to represent specific biomarkers of human spermatogonia. CONCLUSIONS: These biomarkers, specifically the surface markers FGFR3 and DSG2, may facilitate the isolation and enrichment of human stem and/or progenitor spermatogonia and thus lay a foundation for studies of long-term maintenance of human SSCs/progenitor cells, spermatogonial self-renewal, clonal expansion and differentiation.

Highly accurate sigmoidal fitting of real-time PCR data by introducing a parameter for asymmetry.

BACKGROUND: Fitting four-parameter sigmoidal models is one of the methods established in the analysis of quantitative real-time PCR (qPCR) data. We had observed that these models are not optimal in the fitting outcome due to the inherent constraint of symmetry around the point of inflection. Thus, we found it necessary to employ a mathematical algorithm that circumvents this problem and which utilizes an additional parameter for accommodating asymmetrical structures in sigmoidal qPCR data. RESULTS: The four-parameter models were compared to their five-parameter counterparts by means of nested F-tests based on the residual variance, thus acquiring a statistical measure for higher performance. For nearly all qPCR data we examined, five-parameter models resulted in a significantly better fit. Furthermore, accuracy and precision for the estimation of efficiencies and calculation of quantitative ratios were assessed with four independent dilution datasets and compared to the most commonly used quantification methods. It could be shown that the five-parameter model exhibits an accuracy and precision more similar to the non-sigmoidal quantification methods. CONCLUSION: The five-parameter sigmoidal models outperform the established four-parameter model with high statistical significance. The estimation of essential PCR parameters such as PCR efficiency, threshold cycles and initial template fluorescence is more robust and has smaller variance. The model is implemented in the qpcR package for the freely available statistical R environment. The package can be downloaded from the author's homepage.

Cross-platform gene expression signature of human spermatogenic failure reveals inflammatory-like response.

BACKGROUND: The molecular basis of human testicular dysfunction is largely unknown. Global gene expression profiling of testicular biopsies might reveal an expression signature of spermatogenic failure in azoospermic men. METHODS: Sixty-nine individual testicular biopsy samples were analysed on two microarray platforms; selected genes were validated by quantitative real-time PCR and immunohistochemistry. RESULTS: A minimum of 188 transcripts were significantly increased on both platforms. Their levels increased with the severity of spermatogenic damage and reached maximum levels in samples with Sertoli-cell-only appearance, pointing to genes expressed in somatic testicular cells. Over-represented functional annotation terms were steroid metabolism, innate defence and immune response, focal adhesion, antigen processing and presentation and mitogen-activated protein kinase K signalling pathway. For a considerable proportion of genes included in the expression signature, individual transcript levels were in keeping with the individual mast cell numbers of the biopsies. When tested on three disparate microarray data sets, the gene expression signature was able to clearly distinguish normal from defective spermatogenesis. More than 90% of biopsy samples clustered correctly into the corresponding category, emphasizing the robustness of our data. CONCLUSIONS: A gene expression signature of human spermatogenic failure was revealed which comprised well-studied examples of inflammation-related genes also increased in other pathologies, including autoimmune diseases.

Immortalized Sertoli cell lines sk11 and sk9 and binding of spermatids in vitro.

AIM: To determine the effectiveness of the sk11, sk9 and sk11 TNUA5 Sertoli cell lines in binding germ cells in vitro. METHODS: The immortalized Sertoli cell lines sk9, sk11 and sk11 TNUA5 were used in co-culture experiments with germ cells in media with or without reproductive hormones and incubated for 44 h at 32 degrees . The number of germ cells bound to Sertoli cells was then determined and statistically analyzed. Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) studies were employed to investigate the presence of cell adhesion proteins and follicle stimulating hormone (FSH) receptor, respectively. RESULTS: No statistical difference between the number of bound step-8 spermatids and bound pre-step 8 spermatids on Sertoli cells from any of the cell lines existed. After the addition of germ cells, Sertoli cells showed more lipid accumulation in their cytoplasm, indicating active phagocytosis. Western blot analysis in the sk11 TNUA5 line indicated the expression of N-cadherin. FSH-only and testosterone-only treatments increased N-cadherin expression, regardless of germ cell addition. The addition of germ cells to the sk11 TNUA5 Sertoli cells increased the expression of espin, as did the addition of FSH with germ cells. RT-PCR studies of the sk11 TNUA5 cells indicated that the mRNA for FSH receptor decreased with successive passages. CONCLUSION: In vitro binding between isolated germ cells and sk9, sk11 or sk11 TNUA5 Sertoli cells is not feasible, and therefore these cell lines are not useful for the in vitro investigation of Sertoli-germ cell interactions and primary Sertoli cell isolates must still be used.