Testicular Somatic Cells Research Articles

Reassembly of somatic cells and testicular organogenesis in vitro

Testicular organogenesis in vitro requires an environment allowing a reassembly of testicular cell types. Previous in vitro studies using male murine germ cells cultured in a defined three-dimensional environment demonstrated tubulogenesis and differentiation into spermatozoa. Combining scaffolds as artificial culture substrates with testicular cell culture, we analysed the colonization of collagen sponges by rat testicular cells focusing on cell survival and reassembly of tubule-like-structures in vitro. Isolated testicular cells obtained from juvenile Sprague Dawley and eGFP transgenic rats were cultured on collagen sponges (DMEM high glucose+Glutamax, 35°C, 5% CO2 with or without gonadotropins). Live cell imaging revealed the colonization of cells across the entire scaffold for up to 35 days. After two days, histology showed cell clusters attached to the collagen fibres and displaying signs of tubulogenesis. Clusters consisted mainly of Sertoli and peritubular cells which surrounded some undifferentiated spermatogonia. Flow cytometry confirmed lack of differentiation as no haploid cells were detected. Leydig cell activity was detected by a rise of testosterone after gonadotropin stimulation. Our approach provides a novel method which is in particular suitable to follow the somatic testicular cells in vitro an issue of growing importance for the analysis of germ line independent failure of spermatogenesis.

Mesenchymal origin of multipotent human testis-derived stem cells in human testicular cell cultures

In contrast to mouse germ cell-derived pluripotent stem cells, the pluripotent state of human testis-derived embryonic stem cell (ESC)-like that spontaneously arise in primary testicular cell cultures remains controversial. Recent studies have shown that these cells closely resemble multipotent mesenchymal stem cells (MSCs), raising the question of their origin and designating these cell populations as multipotent human testis-derived stem cells (mhtSCs) rather than truly ESC-like cells. Here, we evaluate the origin of mhtSCs in vitro by culturing selected testicular cell types. We demonstrate that mhtSCs can be obtained equally efficiently in cultures of pure testicular somatic cells devoid of germ cells. Conversely, cultures with a purified population of germ cells/spermatogonia do not produce any mhtSCs. Based on common molecular characteristics of the somatic starting population and mhtSCs, we conclude that mhtSCs colonies originate from somatic mesenchymal progenitors present in primary testicular cell cultures and do not arise from germ cells undergoing incomplete reprogramming in vitro.

A combined approach facilitates the reliable detection of human spermatogonia in vitro

Does a combined approach allow for the unequivocal detection of human germ cells and particularly of spermatogonia in vitro? Based on our findings, we conclude that an approach comprising: (i) the detailed characterization of patients and tissue samples prior to the selection of biopsies, (ii) the use of unambiguous markers for the characterization of cultures and (iii) the use of biopsies lacking the germ cell population as a negative control is the prerequisite for the establishment of human germ cell cultures. The use of non-specific marker genes and the failure to assess the presence of testicular somatic cell types in germ cell cultures may have led to a misinterpretation of results and the erroneous description of germ cells in previous studies. Testicular biopsies were selected from a pool of 264 consecutively obtained biopsies. Based on the histological diagnosis, biopsies with distinct histological phenotypes were selected (n = 35) to analyze the expression of germ cell and somatic cell markers. For germ cell culture experiments, gonadotrophin levels and clinical data were used as selection criteria resulting in the following two groups: (i) biopsies with qualitatively intact spermatogenesis (n = 4) and (ii) biopsies from Klinefelter syndrome Klinefelter patients lacking the germ cell population (n = 3). Quantitative real-time PCR analyses were performed to evaluate the specificity of 18 selected germ cell and 3 somatic marker genes. Cell specificity of individual markers was subsequently validated using immunohistochemistry. Finally, testicular cell cultures were established and were analyzed after 10 days for the expression of germ cell- (UTF1, FGFR3, MAGE A4, DDX4) and somatic cell-specific markers (SMA, VIM, LHCGR) at the RNA and the protein levels. Interestingly, only 9 out of 18 marker genes reflected the presence of germ cells and cell specificity could be validated using immunohistochemistry. Furthermore, VIM, SMA and LHCGR were found to reflect the presence of testicular somatic cells at the RNA and the protein levels. Using this validated marker panel and biopsies lacking the germ cell population (n = 3) as a negative control, we demonstrated that germ cell cultures containing spermatogonia can be established from biopsies with normal spermatogenesis (n = 4) and that these cultures can be maintained for the period of 10 days. However, marker profiling has to be performed at regular time points as the composition of testicular cell types may continuously change under longer term culture conditions. There are significant differences regarding the spermatogonial stem cell (SSC) system and spermatogenesis between rodents and primates. It is therefore possible that marker genes that do not reflect the presence of spermatogonia in the human are specific for spermatogonia in other animal models. While some studies have reported that human SSCs can be maintained in vitro and show characteristics of pluripotency, the germ cell origin and the differentiation potential of these cells were subsequently called into question. This study provides critical insights into possible sources for the misinterpretation of results regarding the presence of germ cells in human testicular cell cultures and our findings can therefore help to avoid conflicting reports in the future. This project was supported by the Stem Cell Network North Rhine-Westphalia and the Innovative Medical Research of the University of Münster Medical School (Grant KO111014). In addition, it was funded by the DFG-Research Unit FOR 1041 Germ Cell Potential (GR 1547/11-1 and SCHL 394/11-2), the BMBF (01GN0809/10) and the IZKF (CRA 03/09). The authors declare that there is no conflict of interest. Not applicable.

7: Cell-specific action of extracellular HMGB1 in sterile inflammation of rat testis

High mobility group box protein 1 (HMGB1), the non-histone chromosomal protein, plays an important role in onset and chronification of autoimmune diseases once released from the nuclei. In this study, we analyzed how HMGB1 can regulate inflammatory reactions in vivo in a rat model of experimental autoimmune orchitis (EAO) and in vitro in primary testicular cells. HMGB1 was translocated from the nuclei in EAO testis and in the testis of infertile men with leukocytic infiltrates. HMGB1 levels in EAO testis were elevated at late chronic phase of disease as compared to early proinflammatory cytokines such as IL-6 and TNF- α . We found that testicular somatic cells show a cell-specific expression profile of HMGB1 receptors TLR4 and receptor for advanced glycation end products (RAGE). The highly sensitive and specific proximity ligation assay was used to analyze HMGB1 receptor binding in testicular cells. HMGB1-TLR4 binding was dominant in testicular macrophages. However, Sertoli and peritubular cells showed higher levels of HMGB1-RAGE interaction. In support, HMGB1 triggered RAGE-dependent ERK1/2 MAPK and CREB activation in Sertoli and peritubular cells, whilst in testicular macrophages HMGB1 induced TLR4-signaling as evidenced by p38 MAPK and p65 NF-?B phosphorylation which stimulated an increase in mRNA levels of TNF- α and IL-6. Recent data shows that RAGE induced ERK activation leads to enhanced autophagy levels. In line with these data, extracellular HMGB1 triggered formation of autophagosomes in Sertoli cells. Increased autophagy levels in Sertoli cells might explain how these cells survive the inflammatory environment in EAO testis. Considering HMGB1’s late phase of action, inhibition of HMGB1 may be a putative target for therapeutic intervention in treatment of chronic testicular inflammation.

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1. Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses. Various strategies such as Staput, centrifugal elutriation, and flow cytometry (FC) have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates--which tend to form because of the syncytial nature of the seminiferous epithelium--and lack cell clumps . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones. On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types . The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting, and ulterior gene expression studies.

Stem Cell Origin of Testicular Seminoma

IntroductionA major question concerning cancer is its cells of origin. We hypothesized that distinct cancer subtypes arise from unique cancer-initiating cells. By performing a microarray meta-analysis of seminomas and spermatogonial stem cells, we investigated a putative cell of origin for seminoma. Materials and MethodsWe obtained published microarray data for 6 human adult germ cell lines, 16 embryonic stem cell lines, 3 normal testicular tissue samples, and 40 seminomas from the Gene Expression Omnibus database. By assessing correlations between various tissue microarrays, we determined the number of transitional events and the distance between seminomas and human spermatogonial stem cells. ResultsOur meta-analysis showed that spermatogonial stem cells correlated similarly with seminoma (95% CI of Spearman ρ, 0.33-0.44) and with normal somatic testicular tissue cells (95% CI, 0.39-0.40), which suggests parallel paths of cellular origins. ConclusionAnalysis of our results suggests that a unique cancer subtype, namely seminoma, may have originated from an undifferentiated cell with stemness features rather than from a differentiated cell that acquired stemness features.

Isolation and culture of mouse spermatogonial stem cells and determination of the related markers

To establish a simple and highly effective isolation and culture system of mouse spermatogonial stem cells(SSCs)and detect the expression of stem cell-related markers in the isolated cells. The structures of seminiferous tubules of neonatal(6-8 days of age)and adult(26-28 weeks)DBA/2 mice were compared using histochemical examination. Testes of neonatal mice were selected for preparing primary cells. The digestive efficiency of different enzymes was compared. SSCs were isolated according to the different binding abilities of testicle somatic cells and SSCs to gelatin matrix. The effects of different base culture media such as StemPro34 and α-MEM,gelatin,and serum on the SSCs binding activity and growth were studied. The cell morphology was observed during the culture process. Immunofluorescence was used to detect the expression of SSCs and cancer stem cells(CSCs)-related markers in SSCs. The content of SSCs in the testes of neonatal mice was relatively higher than that in adult mice. Trypsin showed the highest digestive efficiency. In StemPro34 supplemented with 1% fetal bovine serum and on the gelatin matrix,testicular somatic cells could bind with the plate efficiently. Spermatogonial cells grew well when using mitomycin C-treated testicular somatic cells as feeder cells and showed typical characteristic of SSCs. After 13 days of culture,spermatogonial cells formed cell clusters. Immunofluorescence assay showed that SSCs markers glial cell line-derived neurotrophic factor(GDNF)family receptor α1(GFRα1)and VASA protein were highly expressed in the cell clusters. CSCs marker CD44 was expressed in the As,Apr,Aal and the inner cells of the cell clusters,while seldom expressed in the somatic cells. An isolation and culture system of SSCs derived from DBA/2 mice was established. CD44 is highly expressed in the early stage of spermatogonial cell development.

Genomic and post-genomic leads toward regulation of spermatogenesis

Declining male fertility without sign of any recovery and limited understanding about mechanisms involved in the intra-testicular regulation of spermatogenesis, withholding clinicians from delivering appropriate line of treatment, are serious causes of concern. Several infertile men are not amenable to treatment because hormonal deficiency or physical obstruction is not the underlying cause. A hope has been generated in the post genomic era where we can have information about the testicular genes and proteins which regulate germ cell division, differentiation and maturation in an interactive manner. Expression of some of these genes and proteins may be governed by classical hormones. However, if the genes are defective (naturally or acquired later in life), mere treatment with hormone(s), as is opted presently by clinicians, would not result into production of sperm. High throughput techniques and post genomic endeavors have generated plethora of data for fundamental and clinical andrology. Appropriate analyses and interlinking of these datasets may provide access to very precise information on a myriad of somatic and germ cell specific genes and proteins. Studies of functional genomics involving cell and age specific expression of some of these testicular genes will not only pin point precise role of certain biomolecules in various steps of spermatogenesis but it will also provide strong basis for the diagnosis and treatment of male infertility. In this review, we present some transcriptomic and proteomic information from various testicular somatic and germ cell studies and discuss how a systems biology approach may be brought in to meaningfully utilize the available information.

Hidden gems in the niche: a new approach to the study of spermatogonial stem cells

Hidden gems in the niche: a new approach to the study of spermatogonial stem cells

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

What are the reference values for delineating altered somatic cell gene expression from transcript enrichment/dilution in gene expression studies of human spermatogenesis? 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. 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. 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. 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. 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. 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. 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. Financial support was from grant Sp721/1-3 of the German Research Foundation. There are no competing interests to be declared.

Profiling of Androgen Response in Rainbow Trout Pubertal Testis: Relevance to Male Gonad Development and Spermatogenesis

The capacity of testicular somatic cells to promote and sustain germ cell differentiation is largely regulated by sexual steroids and notably androgens. In fish species the importance of androgens is emphasized by their ability to induce sex reversal of the developing fries and to trigger spermatogenesis. Here we studied the influence of androgens on testicular gene expression in trout testis using microarrays. Following treatment of immature males with physiological doses of testosterone or 11-ketotestosterone, 418 genes that exhibit changes in expression were identified. Interestingly, the activity of testosterone appeared stronger than that of 11-ketotestosterone. Expression profiles of responsive genes throughout testis development and in isolated germ cells confirmed androgens to mainly affect gene expression in somatic cells. Furthermore, specific clusters of genes that exhibit regulation coincidently with changes in the natural circulating levels of androgens during the reproductive cycle were highlighted, reinforcing the physiological significance of these data. Among somatic genes, a phylogenetic footprinting study identified putative androgen response elements within the proximal promoter regions of 42 potential direct androgen target genes. Finally, androgens were also found to alter the germ line towards meiotic expression profiles, supporting the hypothesis of a role for the somatic responsive genes in driving germ cell fate. This study significantly increases our understanding of molecular pathways regulated by androgens in vertebrates. The highly cyclic testicular development in trout together with functions associated with regulated genes reveal potential mechanisms for androgen actions in tubule formation, steroid production, germ cell development and sperm secretion.

Necrosis Is the Dominant Cell Death Pathway in Uropathogenic Escherichia coli Elicited Epididymo-Orchitis and Is Responsible for Damage of Rat Testis

Male infertility is a frequent medical condition, compromising approximately one in twenty men, with infections of the reproductive tract constituting a major etiological factor. Bacterial epididymo-orchitis results in acute inflammation most often caused by ascending canalicular infections from the urethra via the continuous male excurrent ductal system. Uropathogenic Escherichia coli (UPEC) represent a relevant pathogen in urogenital tract infections. To explore how bacteria can cause damage and cell loss and thus impair fertility, an in vivo epididymo-orchitis model was employed in rats by injecting UPEC strain CFT073 into the vas deference in close proximity to the epididymis. Seven days post infection bacteria were found predominantly in the testicular interstitial space. UPEC infection resulted in severe impairment of spermatogenesis by germ cell loss, damage of testicular somatic cells, a decrease in sperm numbers and a significant increase in TUNEL (+) cells. Activation of caspase-8 (extrinsic apoptotic pathway), caspase-3/−6 (intrinsic apoptotic pathway), caspase-1 (pyroptosis pathway) and the presence of 180 bp DNA fragments, all of which serve as indicators of the classical apoptotic pathway, were not observed in infected testis. Notably, electron microscopical examination revealed degenerative features of Sertoli cells (SC) in UPEC infected testis. Furthermore, the passive release of high mobility group protein B1 (HMGB1), as an indication of necrosis, was observed in vivo in infected testis. Thus, necrosis appears to be the dominant cell death pathway in UPEC infected testis. Substantial necrotic changes seen in Sertoli cells will contribute to impaired spermatogenesis by loss of function in supporting the dependent germ cells.

74 GERM CELLS AND TESTICULAR SOMATIC CELLS HAVE DIFFERENT SENSITIVITY TO CRYOPRESERVATION

Spermatogonial stem cells (SSC) are the foundation of spermatogenesis. Undifferentiated spermatogonia, containing SSC, represent only 2 to 5% of cells recovered from immature mammalian testis. Cryopreservation in liquid nitrogen allows for long-term storage of cells. Preservation of germ cells can serve as a means of genetic preservation from immature males when sperm storage is not an option. Studies have investigated the effects of cryopreservation on the spermatogenic potential of SSC and the efficiency of various cryopreservation protocols. Preliminary observations indicated that germ cells may survive cryopreservation better than testicular somatic cells, resulting in a post-thaw cell population enriched in germ cells. However, this has not been critically evaluated. The objective of this study was to test the hypothesis that germ cells are less susceptible to cryo-damage than testicular somatic cells. Cells were harvested from the testes of 1-wk-old piglets by 2-step enzymatic digestion. The initial cell suspension was subjected to differential adhesion to enrich the cell population for germ cells. Cells were plated in DMEM + 5% fetal bovine serum and incubated at 37°C in 5% CO2 in air. After 18 h, cells in suspension and cells slightly attached were recovered by trypsinization (1 : 10 trypsin-ethylenediaminetetraacetic acid) for 30 s and replated. This was repeated 24 and 36 h after initial plating. The enriched population was placed into cryovials at a concentration of 30 × 106 cells in freezing media (70% DMEM + 20% fetal bovine serum + 10% dimethyl sulfoxide), kept for 24 h at –80°C in a cryogenic freezing container and transferred to liquid nitrogen for 1 week. Aliquots of cells before freezing and after thawing at 37°C followed by incubation at 37°C in 5% CO2 in air for 1 h were analyzed for viability by propidium iodide (PI) exclusion and immunofluorescence for the germ cell marker VASA to identify viable germ cells (VASA+/PI–), nonviable germ cells (VASA+/PI+), viable somatic cells (VASA–/PI–), and nonviable somatic cells (VASA–/PI+). The percentage of viable germ cells after freezing and thawing was compared to the percentage of viable somatic cells by ANOVA. After enrichment by differential plating, the cell population had 95.6 ± 0.9% viability and contained 27.1 ± 7.4% germ cells (n = 3 replicates). After cryopreservation, the overall cell viability was 77.5 ± 1.6%, and 25.8 ± 8.0% were germ cells. The overall viability after cryopreservation could potentially have benefited from the 1-h incubation prior to analysis. The viability of the germ cell population after freezing and thawing was higher (92.1 ± 3.1%) than somatic cell viability (72.3 ± 1.7%; P < 0.01). These results indicate that porcine germ cells survive cryopreservation better than do testicular somatic cells. Therefore, cryostorage of germ cells can be an efficient means for preservation of male genetic material. Supported by NIH ORIP/DCM RR17359.

Localization of cathepsin D in mouse reproductive tissues and its acquisition onto sperm surface during epididymal sperm maturation

Sperm maturation in the epididymis involves multiple complex events, that include the adsorption of epididymal secretory proteins, re-organization and removal of sperm surface ligands. In this study, we investigated the existence and distribution of cathepsin D (CAT-D) transcripts and proteins in mouse reproductive tissues and proposed a transfer mechanism of CAT-D to the sperm surface. CAT-D transcripts were highly expressed in cultured Sertoli cells, but not in germ cells. The transcriptional level was relatively higher in the caput epididymis (CP) than in the cauda epididymis (CD). At the translational level, CAT-D was detected in testicular somatic cells and in the principal and basal cells in the CP. The expression of CAT-D was fairly specific to the clear cells in the CD. All forms of CAT-D were detected in ultracentrifuged epididymosomes. In conjunction with the expression levels in epididymal epithelium and epididymosomes, CAT-D expression level on the sperm surface was relatively high in CP sperm, but gradually declined toward the CD. Overall, our results indicated that CAT-D was not inherent to sperm themselves, but rather of epididymal origin and was presumably transported to the sperm surface via epididymosomes.

Combining next‐generation sequencing with microarray for transcriptome analysis in rainbow trout gonads

Microarray technology is a powerful tool for studying genome-wide gene expression. As the genome of many fish has not yet been determined, however, cDNA microarrays can only be designed from limited expressed sequence tag data. In this study, we designed a microarray based on the sequencing data (337,466 reads) obtained by next-generation sequencing of RNA extracted from rainbow trout (Oncorhynchus mykiss) embryonic genital ridge, testis, and ovary. These data (307,264 reads) were assembled into 28,668 contigs; 3,298 reads could not be assembled and 26,904 reads were unique sequences that did not cluster with other reads. Based on this information, 55,928 microarray probes were designed for a microarray, which was validated by hybridization experiments with RNA extracted from type A spermatogonia (A-SG) and testicular somatic cells. Expression of known spermatogonial markers was confirmed to be higher in A-SG than in testicular somatic cells whereas supporting-cell markers were expressed at higher levels in testicular somatic cells. This microarray analysis revealed that 8,068 transcripts showed at least fourfold higher signal in A-SG than testicular somatic cells. Fourteen of 17 randomly selected transcripts were expressed at significantly higher-levels in A-SG than somatic cells, by quantitative RT-PCR. In addition, three transcripts analyzed with in situ hybridization showed A-SG-specific signals in immature trout testis, with one of them exhibiting a heterogeneous expression pattern in A-SG. The rainbow trout gonad microarray developed in this study therefore appears to be a useful tool to understand gametogenesis in rainbow trout.

Is matrix metalloproteinase required in postnatal testicular tubules for germ cell maturation?

Background/AimCryptorchidism may cause infertility by failed transformation of neonatal gonocytes into adult dark spermatogonia, the putative stem cells for spermatogenesis. Gonocytes migrate centrifugally to the tubular basement membrane to become adult dark spermatogonia. Regulation of this transformation remains unknown. We aimed to investigate neonatal rodent testis matrix metalloproteinase (MMP) production to see whether MMPs loosen extracellular matrix between Sertoli cells to facilitate gonocyte movement. MethodsSprague-Dawley rat testes (n = 4-6 per group) were collected at embryonic day 19 (E19) and postnatal (P) days P0 to 10 for immunohistochemistry. Immunofluorescent confocal images were captured for presence of membrane type 1 MMP (MT1-MMP), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 2 (TIMP2), mouse VASA homologue, anti-Müllerian hormone, and androgen receptor in tissue sections. Testicular proteins were analyzed by immunoblotting. ResultsMembrane type 1 MMP was strongly present in gonocytes at E19 then decreased, whereas it increased in testicular somatic cells from P0 to P10. Testicular protein levels of MT1-MMP, MMP2, and androgen receptor were constant from E19 to P10. Anti-Müllerian hormone protein sharply decreased after P2, whereas TIMP2 gradually increased from E19 to P10. Gonocytes migrated to basement membrane at P2 to P6. ConclusionMembrane type 1 MMP, MMP2, and TIMP2 were present in testis from E19 to P10 during gonocyte migration and transformation into spermatogenic stem cells. Increased knowledge about germ cell development may aid efforts to improve fertility in cryptorchidism.

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

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. 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. 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. 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.

P086 HMGB1 modulates immune responses in a cell-specific manner in the rat autoimmune orchitis

Introduction High mobility group box protein 1 (HMGB1) is a non-histone nucleosomal protein. Under inflammatory conditions HMGB1 is secreted from activated immune cells or released from necrotic cells and acts as an endogenous danger signal. Despite its immune privileged status, the testis is prone to inflammatory infertility. In this study we analyzed the role of HMGB1 in mediating autoimmune responses in experimental autoimmune orchitis (EAO) – a rodent model of chronic testicular inflammation. Methods EAO was induced in WKY rats by immunization with testicular homogenate in complete Freund’s adjuvant. HMGB1 localization was analyzed in EAO and adjuvant control rat testes by immunofluorescence staining. Levels of HMGB1, IL-6 and TNF- α were measured in testes and serum using ELISA. Isolated testicular macrophages (TM), Sertoli cells (SC) and peritubular cells (PTC) were stimulated with recombinant HMGB1. Cytokine mRNA levels were measured by qRT-PCR. Testosterone production was evaluated using RIA assay in HMGB1 treated Leydig cells (LC). Expression of RAGE as well as activation of NF-?B (p65), MAPK (p38, ERK1/2) and Akt pathway were investigated in TM, SC and pPTC by Western blot. Possible binding of HMGB1 to TLR4 was investigated in isolated testicular cells using Duolink PLA assay. Results In control testes, HMGB1 was expressed mainly in the nucleus of testicular somatic cells. In contrast, EAO testes showed translocation of HMGB1 from the nucleus into the cytoplasm. Concentrations of IL-6 and TNF- α were increased 50 days after first immunization in EAO testes, whilst HMGB1 levels increased 80 days post-immunization. RAGE was highly expressed in SC as well as PTC and to a lower level in TM. In contrast, TLR4 levels were higher in TM. Our preliminary results show that HMGB1 binding to TLR4 is significantly higher in TM than in PTC. In vitro stimulation of isolated TM using recombinant HMGB1 activates phosphorylation of p38 and p65. However, HMGB1 treated PTC and SC activated ERK1/2. HMGB1 induced a significant increase in mRNA expression of TNF- α and IL-6 in TM. In HMGB1 treated SC the levels of IL-6 and TNF- α mRNA did not show a statistically significant difference. HMGB1 induced a significant increase in testosterone production by LC. Conclusion In control testis, HMGB1 was expressed mainly in the nucleus of testicular somatic cells. In contrast, EAO testes showed translocation of HMGB1 from the nucleus into the cytoplasm. Concentrations of IL-6 and TNF- α were increased 50 days after first immunization in EAO testis, whilst HMGB1 levels increased 80 days post-immunization. RAGE was highly expressed in SC as well as PTC and to a lower level in TM. In contrast, TLR4 levels were higher in TM. Our preliminary results show that HMGB1 binding to TLR4 is significantly higher in TM than in PTC. In vitro stimulation of isolated TM using recombinant HMGB1 activates phosphorylation of p38 and p65. In contrast, HMGB1 treated PTC and SC activated ERK1/2. HMGB1 induced a significant increase in mRNA expression of TNF- α and IL-6 in TM. In HMGB1 treated SC the levels of IL-6 and TNF- α mRNA did not show a statistically significant difference. HMGB1 induced a significant increase in testosterone production by LC.

Altered LKB1/AMPK/TSC1/TSC2/mTOR signaling causes disruption of Sertoli cell polarity and spermatogenesis

Male patients with Peutz-Jeghers syndrome (PJS) have defective spermatogenesis and are at increased risk of developing Sertoli cell tumors. Mutations in the Liver Kinase B1 (LKB1/STK11) gene are associated with the pathogenesis of PJS and have been identified in non-PJS patients with sporadic testicular cancers. The mechanisms controlled by LKB1 signaling in Sertoli cell functions and testicular biology have not been described. We have conditionally deleted the Lkb1 gene (Lkb1(cko)) in somatic testicular cells to define the molecular mechanisms involved in the development of the testicular phenotype observed in PJS patients. Focal vacuolization in some of the seminiferous tubules was observed in 4-week-old mutant testes but germ cell development appeared to be normal. However, similar to PJS patients, we observed progressive germ cell loss and Sertoli cell only tubules in Lkb1(cko) testes from mice older than 10 weeks, accompanied by defects in Sertoli cell polarity and testicular junctional complexes and decreased activation of the MAP/microtubule affinity regulating and focal adhesion kinases. Suppression of AMP kinase and activation of mammalian target of rapamycin (mTOR) signaling were also observed in Lkb1(cko) testes. Loss of Tsc1 or Tsc2 copies the progressive Lkb1(cko) phenotype, suggesting that dysregulated activation of mTOR contributes to the pathogenesis of the Lkb1(cko) testicular phenotype. Pten(cko) mice had a normal testicular phenotype, which could be explained by the comparative lack of mTOR activation detected. These studies describe the importance of LKB1 signaling in testicular biology and the possible molecular mechanisms driving the pathogenesis of the testicular defects observed in PJS patients.