Adult Human Testis Research Articles

A Determination of p97/VCP (Valosin Containing Protein) and SVIP (Small VCP Interacting Protein) Expression Patterns in Human Testis.

Background and Objectives: The ubiquitin proteosome system (UPS) is a non-lysosomal pathway that functions in all eukaryotes. The transport of polyubiquitinated proteins to proteosomes takes place via the p97/Valosin-containing protein (VCP) chaperone protein. The p97/VCP binds to polyubiquitinated proteins, allowing these proteins to reach the proteasome and, thus, their destruction. In the case of p97/VCP deficiency, ubiquitinated proteins accumulate in the cell cytoplasm, and their subsequent failure to break down produces various pathological conditions. Small VCP interacting protein (SVIP) and p97/VCP proteins have not been studied in human testicular tissues from different postnatal periods. Therefore, in our study, we aimed to examine the expression of SVIP and p97/VCP in postnatal human testicular tissues. Our study aimed to contribute to further studies on the use of these proteins as testicular cell biomarkers in cases of unexplained male infertility. Materials and Methods: Immunohistochemical studies with the aim of determining the expression of p97/VCP and SVIP proteins in neonatal, prepubertal, pubertal, adult, and geriatric human testis tissues were performed. Results: In testicular sections obtained from a neonatal group, p97/VCP and SVIP were localized in different testicular and interstitial cells, and the lowest expression was observed in this group. While the expressions of these proteins were low in the neonatal period, they increased gradually in the prepubertal, pubertal and adult periods. The expression of p97/VCP and SVIP, which peaked in adulthood, showed a significant decrease in the geriatric period. Conclusions: As a result, the expression of p97/VCP and SVIP correlated with the increase in age, but it decreased significantly in older groups.

Germ Cell Maintenance and Sustained Testosterone and Precursor Hormone Production in Human Prepubertal Testis Organ Culture with Tissues from Boys 7 Years+ under Conditions from Adult Testicular Tissue.

Human prepubertal testicular tissues are rare, but organ culture conditions to develop a system for human in vitro-spermatogenesis are an essential option for fertility preservation in prepubertal boys subjected to gonadotoxic therapy. To avoid animal testing in line with the 3Rs principle, organ culture conditions initially tested on human adult testis tissue were applied to prepubertal samples (n = 3; patient ages 7, 9, and 12 years). Tissues were investigated by immunostaining and transmission electron microscopy (TEM), and the collected culture medium was profiled for steroid hormones by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Culture conditions proved suitable for prepubertal organ culture since SSCs and germ cell proliferation could be maintained until the end of the 3-week-culture. Leydig cells (LCs) were shown to be competent for steroid hormone production. Three additional testis tissues from boys of the same age were examined for the number of germ cells and undifferentiated spermatogonia (SPG). Using TEM micrographs, eight tissues from patients aged 1.5 to 13 years were examined, with respect to the sizes of mitochondria (MT) in undifferentiated SPG and compared with those from two adult testicular tissues. Mitochondrial sizes were shown to be comparable between adults and prepubertal boys from approximately 7 years of age, which suggests the transition of SSCs from normoxic to hypoxic metabolism at about or before this time period.

Persistence of foetal testicular features in patients with defective androgen signalling.

Congenital defects of androgen synthesis or action in 46, XY individuals can result in impaired virilisation, despite the apparent testicular development. In a recent case report of a young adult with complete androgen insensitivity syndrome (CAIS), tumorous gonadal tissue was shown to express HSD17B3 in Sertoli cells (SCs) and not in Leydig cells (LCs). This expression pattern differs from the typical adult human testis and resembles foetal mouse testis, suggesting an underlying testicular development and function defect. Here, we investigate the effect of altered androgen signalling in gonads from five 46, XY individuals with defects in androgen synthesis or action. Gonadal tissue sections from four patients with CAIS, one with CYP17A1 deficiency, and one control were immunostained for LC developmental and steroidogenic markers. The expression of some of these markers during development was investigated by reanalysing previously published single-cell RNA sequencing (scRNA-seq) data from normal human testicular tissues. All gonadal tissues from the patients show exclusive expression of HSD17B3 in SCs and expression of the foetal/immature LC marker DLK1 in a subset of LCs, suggesting an androgen-dependent differentiation defect of adult SCs and LCs. Furthermore, reanalysis of scRNA-seq data reveals an expression of HSD17B3 in foetal and neonatal SCs that is downregulated in adult SCs. Androgen signalling may affect the differentiation of adult but possibly not foetal SCs or LCs and may induce a shift of testosterone production from the tubular compartment in the foetal phase to the interstitial compartment in the adult phase.

The Acute Exposure of Human Adult Testis Tissue to Cannabinoids THC and CBD Does Not Impact Testosterone Production Nor Germ Cell Lineage.

While an increased risk of developing germ cell tumors has been established in regular cannabis consumers, there is conflicting evidence about an association between cannabis use and testosterone levels in those regular consumers. In this context, we aimed to determine whether Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), the two major and best-studied cannabinoids present in cannabis, also the most used for therapeutic applications, can directly impact the steroidogenic function and germ cell lineage of the human adult testis. We used our well-characterized organotypic culture of human testis, in which adult testis explants were exposed to CBD, THC, or CBD/THC [ratio 1:1] from 10-9 to 10-5 M for up to either 48 hours or 9 days of culture. The testes were obtained from multi-organ donors (n=13; mean age: 55.15±5.62 y). The testosterone production and the spatial distribution of Leydig cells did not change upon CBD and/or THC exposure versus controls after 48 hours or 9 days. The overall tissue morphology of the cannabinoids-exposed testis explants was similar to their control upon 24 hours or 9 days of exposure, a finding confirmed by morphometric analyses on short-term cultures. In line, the number of apoptotic cells was not affected by either 48 hours or 9 days of cannabinoids treatment versus mock. Cannabinoids had no impact on the number of proliferating cells nor on mRNA expression of genes encoding proteins involved in germ cell differentiation, meiosis, or Sertoli and Leydig functions after 24 hours exposure. Altogether, these findings show an absence of acute direct effects of exposure to cannabis-derived cannabinoids THC and CBD on testicular testosterone production and germ cells ex vivo. Further studies are warranted to explore an indirect impact of cannabinoids on testis functions through the hypothalamic-pituitary-testis axis, as well as the potential effects of long-term exposures.

Expression of Intermediate Filaments in the Developing Testis and Testicular Germ Cell Cancer.

Cytokeratin and desmin expression have been associated with Sertoli cell maturity and the development of testicular germ cell cancer (TGCC). Thus, the present study aimed to characterize the expression of these intermediate filaments in normal testis development and TGCC. Cytokeratin and desmin were determined by immunohistochemistry and immunofluorescence in human fetal, and adult testis and tissue from patients with pre-invasive germ cell neoplasia in-situ (GCNIS) or invasive TGCC. Desmin was expressed in Sertoli cells of the human fetal testis, and the proportion of desmin expressing Sertoli cells was significantly reduced in the second trimester, compared with the first trimester (31.14% vs. 6.74%, p = 0.0016). Additionally, Desmin was expressed in the majority of Sertoli cells in the adult testis and TGCC samples. Cytokeratin was detected in Sertoli cells of human fetal testis but was not expressed in Sertoli cells of human adult testis. In patients with TGCC, cytokeratin was not expressed in Sertoli cells in tubules with active spermatogenesis but was detected in Sertoli cells in tubules containing GCNIS cells in patients with both pre-invasive and invasive TGCC. In conclusion, desmin was not associated with Sertoli cell maturation or progression to TGCC. However, cytokeratin appeared to be an indicator of impaired Sertoli cell maturation.

Risk Factors, Hyaluronidase Expression, and Clinical Immunogenicity of Recombinant Human Hyaluronidase PH20, an Enzyme Enabling Subcutaneous Drug Administration

Multiple FDA-approved and clinical-development stage therapeutics include recombinant human hyaluronidase PH20 (rHuPH20) to facilitate subcutaneous administration. As rHuPH20-reactive antibodies potentially interact with endogenous PH20, we investigated rHuPH20 immunogenicity risk through hyaluronidase tissue expression, predicted B cell epitopes, CD4+ T cell stimulation indices and related these to observed clinical immunogenicity profiles from 18 clinical studies. Endogenous hyaluronidase PH20 expression in humans/mice was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative RT-PCR, and deep RNA-Seq. rHuPH20 potential T cell epitopes were evaluated in silico and confirmed in vitro. Potential B cell epitopes were predicted for rHuPH20 sequence in silico, and binding of polyclonal antibodies from various species tested on a rHuPH20 peptide microarray. Clinical immunogenicity data were collected from 2643 subjects. From 57 human adult and fetal tissues previously screened by RT-PCR, 22 tissue types were analyzed by deep RNA-Seq. Hyaluronidase PH20 messenger RNA expression was detected in adult human testes. In silico analyses of the rHuPH20 sequence revealed nine T cell epitope clusters with immunogenic potential, one cluster was homologous to human leukocyte antigen. rHuPH20 induced T cell activation in 6–10% of peripheral blood mononuclear cell donors. Fifteen epitopes in the rHuPH20 sequence had the potential to cross-react with B cells. The cumulative treatment-induced incidence of anti-rHuPH20 antibodies across clinical studies was 8.8%. Hyaluronidase PH20 expression occurs primarily in adult testes. Low CD4+ T cell activation and B cell cross-reactivity by rHuPH20 suggest weak rHuPH20 immunogenicity potential. Restricted expression patterns of endogenous PH20 indicate low immunogenicity risk of subcutaneous rHuPH20.Graphical abstract

Behavior and Functional Roles of CD34+ Mesenchymal Cells in Mammalian Testes.

Mammalian testes consist of seminiferous tubules within which Sertoli cells line up at the periphery and nurse germ cells, and of interstitia that harbor various cells such as peritubular myoid cells (PMCs), Leydig cells (LCs), vascular endothelial cells, immune cells such as macrophages, and mesenchymal (stromal) cells. Morphological studies have recently reported the presence of telocytes with telopodes in the interstitium of adult mouse, rat, and human testes. CD34+PDGFRα+ telocytes with long and moniliform telopodes form reticular networks with various cell types such as LCs, PMCs, and vessels, indicating their potential functions in cell–cell communications and tissue homeostasis. Functional studies have recently been performed on testicular interstitial cells and CD34+ cells, using 3D re-aggregate cultures of dissociated testicular cells, and cell cultures. Direct observation of CD34+ cells and adult LCs (ALCs) revealed that CD34+ cells extend thin cytoplasmic processes (telopodes), move toward the LC–CD34+ cell-re-aggregates, and finally enter into the re-aggregates, indicating the chemotactic behavior of CD34+ telocytes toward ALCs. In mammalian testes, important roles of mesenchymal interstitial cells as stem/progenitors in the differentiation and regeneration of LCs have been reported. Here, reports on testicular telocytes so far obtained are reviewed, and future perspectives on the studies of testicular telocytes are noted.

Mammalian testes utilize multiple modalities to moderate spermatogonial responsiveness to retinoic acid

What is knownThe foundation of mammalian spermatogenesis is provided by undifferentiated spermatogonia that proliferate before responding to retinoic acid (RA). RA converts undifferentiated into differentiating spermatogonia committed to entering meiosis. This RA‐regulated fate transition is essential for maintaining normal ratios of spermatogonia during steady‐state spermatogenesis in adult testes. Imbalances in spermatogonial populations often result in infertility – enhanced differentiation leads to eventual germline depletion, while impaired differentiation increases the likelihood of developing testicular cancer.Study ObjectiveDefine the extent cell autonomous and non‐autonomous mechanisms in adult mammalian spermatogonia regulate their response to RA.HypothesisOnly spermatogonia primed to differentiate will respond to RA and precociously progress through spermatogenesis.MethodologyAdult mice were treated in vivo with vehicle (DMSO), RA, or talarozole (RA catabolism inhibitor) and euthanized 12‐48 hours later. Testes were removed and various assays used to assess populations for responsiveness to RA and capacity to precociously differentiate and enter meiosis. Concurrently, a model of synchronized spermatogenesis was used to define the potential of sequential subsets of differentiating spermatogonia to respond to RA and precociously enter meiosis. In complementary ex vivo experiments, mouse testes and patient‐derived testicular biopsies were cultured with DMSO‐alone, RA, and/or talarozole for 6‐24 hours to evaluate RA responsiveness and changes in spermatogonia fate using immunostaining and qRT‐PCR.Main resultsExogenous RA elicited a rapid (12 hour) and significant increase in expression of the RA‐responsive gene ‘stimulated by retinoic acid 8’ (STRA8) in undifferentiated (ZBTB16+) spermatogonia. STRA8 expression decreased over the next 36 hours, such that numbers of STRA8+/ZBTB16+ spermatogonia were comparable in testes from vehicle‐ and RA‐treated mice. This transient increase in STRA8 affected spermatogonia at all stages of spermatogenesis. Exogenous RA also elicited STRA8 expression in differentiating (KIT+) spermatogonia in adults and synchronized mice. Despite this capacity for responding to exogenous RA, obvious precocious progression was not observed within the testis. Talarozole treatment elicited STRA8 expression in spermatogonia akin to RA treatments. Finally, qRT‐PCR showed adult mouse and human testes regulate responsiveness to RA similarly, based on mRNA expression profiles.ConclusionIn response to exogenous RA, undifferentiated spermatogonia transiently upregulate expression of RA‐responsive genes, but this did not result in precocious progression into meiosis.ImplicationsThe adult mammalian testis is a well‐buffered system in which exogenous RA has little effect on cell fate, thus preserving the normal timing and progression of spermatogenesis.

P–017 The maintenance of testicular architecture and germ cell in adult testis tissue under organ culture condition based on the gas-liquid interface method

Abstract Study question Can the gas-liquid interface organ culture system that achieved in vitro spermatogenesis in mice also support in vitro spermatogenesis in human adult testis? Summary answer Although the progression of spermatogenesis was not observed, germ cells were maintained without the degeneration of the architecture in both fresh and cryopreserved testicular tissues. What is known already Although the research on in vitro spermatogenesis have been conducted for 100 years, only the organ culture system using gas-liquid interface method achieved in vitro spermatogenesis in mice. It has not been verified whether this culture system can be applied to other mammals including humans and induce spermatogenesis. Study design, size, duration Testicular tissue was obtained from the transgender patients receiving sex reassignment surgery. Testicular specimens were either immediately processed for cultivation or cryopreserved, using a vitrification freezing protocol. Organ culture of testicular fragments was performed in three different media for a maximum period of 3 weeks to evaluate the short-term changes in the cultured tissues (viability, proliferation and maintenance of germ and somatic cells). Participants/materials, setting, methods Fresh and cryopreserved-thawed testis fragments (1–2 mm3) were cultured using the organ culture system in alpha-MEM with knock-out serum replacement (K group), alpha-MEM with lipid-rich BSA (A group) and DMEM with FBS (D group). Luteinizing hormone, follicle stimulating hormone and testosterone were supplemented. The number of germ cells (using DDX4), proliferative activity of germ cells (using EdU assay) and intratubular cell apoptosis (by TdT-mediated dUTP Nick End Labeling) were evaluated by immunohistochemical staining weekly. Main results and the role of chance The architecture of the seminiferous tubules was maintained until the second week of culture in both the fresh and the cryopreserved culture group. The number of DDX4-positive germ cells per seminiferous tubule in groups D, K, and A was 49 ± 24, 55 ± 21, 50 ± 26 cells/tubule in 1 day, 32 ± 13, 42 ± 7, 36 ± 21 cells/tubule in 1week, respectively. The numbers gradually decreased to 26 ± 8, 24 ± 6 and 27 ± 18 cells/tubule, in 2 weeks, respectively, with no difference among the groups. The number of intratubular EdU-positive cells of groups D, K, and A was 0.2 ± 0.2, 2.8 ± 2.1, 1.1 ± 0.8 cells/tubule at 1 day, 0.1 ± 0.2, 0.5 ± 0.6, 0.3 ± 0.6 cells/tubule at 1 week, respectively. The values were 0.01, 0.05, and 0.03 at 2 weeks. Thus, EdU-positive cells drastically decreased from the first week of culture. The number of DDX4-positive germ cells and the intratubular EdU-positive cells in the cryopreserved culture group was not different from that in the fresh culture group. Limitations, reasons for caution Current organ culture systems are incomplete, being unable to induce human in vitro spermatogenesis. Further research is needed to improve culture condition with the aim of producing fertile sperm of infertile adult male patients. Wider implications of the findings: Our organ culture system could maintain testis structure and germ cells. By using the testis tissues of the transgender patients, which are available with their consent, we will promote the investigation of the culture condition necessary for germ cell proliferation and differentiation. Trial registration number Grant-in-Aid for Scientific Research on Innovative Areas 18H05546, Grant-in-Aid for Young Scientists (A) 17H05098 and Takeda Science Foundation

Insights into the Regulation on Proliferation and Differentiation of Stem Leydig Cells.

Male hypogonadism is a clinical syndrome caused by testosterone deficiency. Hypogonadism can be caused by testicular disease (primary hypogonadism) or hypothalamic-pituitary dysfunction (secondary hypogonadism). The present strategy for treating hypogonadism is the administration of exogenous testosterone. But exogenous testosterone is reported to have negative side effects including adverse cardiovascular events and disruption of physiological spermatogenesis probably due to its inability to mimic the physiological circadian rhythm of testosterone secretion in vivo. In recent years, a growing number of articles demonstrated that stem Leydig cells (SLCs) can not only differentiate into functional Leydig cells (LCs) in vivo to replace chemically disrupted LCs, but also secrete testosterone in a physiological pattern. The proliferation and differentiation of SLCs are regulated by various factors. However, the mechanisms involved in regulating the development of SLCs remain to be summarized. Factors involved in the regulation of SLCs can be divided into environmental pollutants, growth factors, cytokine and hormones. Environmental pollutants such as Perfluorooctanoic acid (PFOA) and Triphenyltin (TPT) could suppress SLCs proliferation or differentiation. Growth factors including FGF1, FGF16, NGF and activin A are essential for the maintenance of SLCs self-renewal and differentiation. Interleukin 6 family could inhibit differentiation of SLCs. Among hormones, dexamethasone suppresses SLCs differentiation, while aldosterone suppresses their proliferation. The present review focuses on new progress about factors regulating SLC's proliferation and differentiation which will undoubtedly deepen our insights into SLCs and help make better clinical use of them. Different factors affect on the proliferation and differentiation of stem Leydig cells. Firstly, each rat was intraperitoneally injected EDS so as to deplete Leydig cells from the adult testis. Secondly, the CD51+ or CD90+ cells from the testis of rats are SLCs, and the p75+ cells from human adult testes are human SLCs. These SLCs in the testis start to proliferate and some of them differentiate into LCs. Thirdly, during the SLCs regeneration period, researchers could explore different function of those factors (pollutants, growth factors, cytokines and hormones) towards SLCs.

Erratum. WNT signalling in the normal human adult testis and in male germ cell neoplasms.

Erratum. WNT signalling in the normal human adult testis and in male germ cell neoplasms.

Endosialin defines human stem Leydig cells with regenerative potential.

STUDY QUESTIONIs endosialin a specific marker of human stem Leydig cells (SLCs) with the ability to differentiate into testosterone-producing Leydig cells (LCs) in vitro and in vivo?SUMMARY ANSWEREndosialin is a specific marker of human SLCs which differentiate into testosterone-producing LCs in vitro and in vivo.WHAT IS KNOWN ALREADYHuman SLCs have been identified and isolated using the marker platelet-derived growth factor receptor α (PDGFRα) or nerve growth factor receptor (NGFR). However, the specificity was not high; thus, LCs and germ cells could be mistakenly sorted as SLCs if PDGFRα or NGFR was used as a marker for human SLCs isolation.STUDY DESIGN, SIZE, DURATIONFirstly, we re-evaluated the specificity of PDGFRα and NGFR for SLCs in adult human testes. Then we analysed the previously published single-cell sequencing data and found that endosialin may identify human SLCs. Subsequently, we sorted endosialin+ cells from four human donors and characterized their self-renewal and multipotent properties. To assess whether endosialin+ cells have the potential to differentiate into functional LCs in vitro, these cells were stimulated by differentiation-inducing medium. We next assessed the in vivo regenerative potential of human endosialin+ cells after xenotransplantation into the testes of immunodeficient mice.PARTICIPANTS/MATERIALS, SETTING, METHODSSingle-cell sequencing analysis, immunofluorescence and flow cytometry were used to characterize human testis tissues. In vitro colony formation, multipotent differentiation (adipogenic, osteogenic and chondrogenic) and Leydig cell-lineage induction were used to assess stem cell activity. Xenotransplantation into 3-week-old immunodeficient mice was used to determine in vivo regenerative potential. Endpoint measures included testosterone measurements, cell proliferation, immunofluorescence, flow cytometry and quantitative RT–PCR.MAIN RESULTS AND THE ROLE OF CHANCEThe results indicate that endosialin is a specific marker of SLCs compared with PDGFRα and NGFR. Additionally, endosialin+ cells isolated from human testes show extensive proliferation and differentiation potential in vitro: their self-renewal ability was inferred by the formation of spherical clones derived from a single cell. Moreover, these cells could differentiate into functional LCs that secreted testosterone in response to LH in a concentration-dependent manner in vitro. These self-renewal and differentiation properties reinforce the proposal that human testicular endosialin+ cells are SLCs. Furthermore, transplanted human endosialin+ cells appear to colonize the murine host testes, localize to peritubular and perivascular regions, proliferate measurably and differentiate partially into testosterone-producing LCs in vivo.LARGE SCALE DATANA.LIMITATIONS, REASONS FOR CAUTIONOwing to the difficulty in collecting human testis tissue, the sample size was limited. The functions of endosialin on SLCs need to be elucidated in future studies.WIDER IMPLICATIONS OF THE FINDINGSA discriminatory marker, endosialin, for human SLCs purification is a prerequisite to advance research in SLCs and logically promote further clinical translation of SLCs-based therapies for male hypogonadism.STUDY FUNDING/COMPETING INTEREST(S)A.P.X. was supported by the National Key Research and Development Program of China (2017YFA0103802 and 2018YFA0107200). C.D. was supported by the National Natural Science Foundation of China (81971314) and the Natural Science Foundation of Guangdong Province, China (2018B030311039). The authors declare no conflict of interest.

WNT signalling in the normal human adult testis and in male germ cell neoplasms.

Is WNT signalling functional in normal and/or neoplastic human male germ cells? Regulated WNT signalling component synthesis in human testes indicates that WNT pathway function changes during normal spermatogenesis and is active in testicular germ cell tumours (TGCTs), and that WNT pathway blockade may restrict seminoma growth and migration. Regulated WNT signalling governs many developmental processes, including those affecting male fertility during early germ cell development at embryonic and adult (spermatogonial) ages in mice. In addition, although many cancers arise from WNT signalling alterations, the functional relevance and WNT pathway components in TGCT, including germ cell neoplasia in situ (GCNIS), are unknown. The cellular distribution of transcripts and proteins in WNT signalling pathways was assessed in fixed human testis sections with normal spermatogenesis, GCNIS and seminoma (2-16 individuals per condition). Short-term (1-7 h) ligand activation and long-term (1-5 days) functional outcomes were examined using the well-characterised seminoma cell line, TCam-2. Pathway inhibition used siRNA or chemical exposures over 5 days to assess survival and migration. The cellular localisation of WNT signalling components was determined using in situ hybridisation and immunohistochemistry on Bouin's- and formalin-fixed human testis sections with complete spermatogenesis or germ cell neoplasia, and was also assessed in TCam-2 cells. Pathway function tests included exposure of TCam-2 cells to ligands, small molecules and siRNAs. Outcomes were measured by monitoring beta-catenin (CTNNB1) intracellular localisation, cell counting and gap closure measurements. Detection of nuclear-localised beta-catenin (CTNNB1), and key WNT signalling components (including WNT3A, AXIN2, TCF7L1 and TCF7L2) indicate dynamic and cell-specific pathway activity in the adult human testis. Their presence in germ cell neoplasia and functional analyses in TCam-2 cells indicate roles for active canonical WNT signalling in TGCT relating to viability and migration. All data were analysed to determine statistical significance. No large-scale datasets were generated in this study. As TGCTs are rare and morphologically heterogeneous, functional studies in primary cancer cells were not performed. Functional analysis was performed with the only well-characterised, widely accepted seminoma-derived cell line. This study demonstrated the potential sites and involvement of the WNT pathway in human spermatogenesis, revealing similarities with murine testis that suggest the potential for functional conservation during normal spermatogenesis. Evidence that inhibition of canonical WNT signalling leads to loss of viability and migratory activity in seminoma cells suggests that potential treatments using small molecule or siRNA inhibitors may be suitable for patients with metastatic TGCTs. This study was funded by National Health and Medical Research Council of Australia (Project ID 1011340 to K.L.L. and H.E.A., and Fellowship ID 1079646 to K.L.L.) and supported by the Victorian Government's Operational Infrastructure Support Program. None of the authors have any competing interests.

The ACE2 expression in Sertoli cells and germ cells may cause male reproductive disorder after SARS-CoV-2 infection.

The serious coronavirus disease‐2019 (COVID‐19) was first reported in December 2019 in Wuhan, China. COVID‐19 is an infectious disease caused by severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2). Angiotensin converting enzyme 2(ACE2) is the cellular receptor for SARS‐CoV‐2. Considering the critical roles of testicular cells for the transmission of genetic information between generations, we analyzed single‐cell RNA‐sequencing (scRNA‐seq) data of adult human testis. The mRNA expression of ACE2 was expressed in both germ cells and somatic cells. Moreover, the positive rate of ACE2 in testes of infertile men was higher than normal, which indicates that SARS‐CoV‐2 may cause reproductive disorders through pathway activated by ACE2 and the men with reproductive disorder may easily to be infected by SARS‐CoV‐2. The expression level of ACE2 was related to the age, and the mid‐aged with higher positive rate than young men testicular cells. Taken together, this research provides a biological background of the potential route for infection of SARS‐CoV‐2 and may enable rapid deciphering male‐related reproductive disorders induced by COVID‐19.

'Snail factors in testicular germ cell tumours and their regulation by the BMP4 signalling pathway'.

Snail transcription factors mediate key cellular transitions in many developmental processes, including spermatogenesis, and their production can be regulated by TGF-β superfamily signalling. SNAI1 and SNAI2 support many cancers of epithelial origin. Their functional relevance and potential regulation by TGF-β superfamily ligands in germ cell neoplasia are unknown. SNAI1, SNAI2 and importin 5 (IPO5; nuclear transporter that selectively mediates BMP signalling) cellular localization was examined in fixed normal adult human and/or neoplastic testes using in situ hybridization and/or immunohistochemistry. SNAI1 and SNAI2 functions were assessed using the well-characterized human seminoma cell line, TCam-2. Cell migration, adhesion/proliferation and survival were measured by scratch assay, xCELLigence and flow cytometry following siRNA-induced reduction of SNAI1 and SNAI2 in TCam-2 cells. The potential regulation of SNAI1 and SNAI2 in TCam-2 cells by TGF-β signalling ligands, activin A and BMP4 was evaluated following 48hours culture, including with siRNA regulation of IPO5 to selectively restrict BMP4 signalling. In normal testes, SNAI1 transcript was identified in some spermatogonia and in spermatocytes, and SNAI2 protein localized to nuclei of spermatogonia, spermatocytes and round spermatids. In neoplastic testes, both SNAI1 and SNAI2 were detected in GCNIS and in seminoma cells. SNAI1 and SNAI2 reduction in TCam-2 cells by siRNAs significantly inhibited migration and survival, respectively. Exposure to BMP4, but not activin A, significantly increased SNAI2 (~18-fold). IPO5 inhibition by siRNAs decreased BMP4-induced SNAI2 upregulation (~5-fold). Additionally, SNAI2 reduction using siRNAs inhibited BMP4-induced TCam-2 cell survival. This is the first evidence that SNAI1 and SNAI2 are involved in human spermatogenesis, with independent functions. These outcomes demonstrate that SNAI1 and SNAI2 inhibition leads to loss of migratory and viability capacities in seminoma cells. These findings show the potential for therapeutic treatments targeting SNAIL or BMP4 signalling for patients with metastatic testicular germ cell tumours.

ScRNA-seq Profiling of Human Testes Reveals the Presence of the ACE2 Receptor, A Target for SARS-CoV-2 Infection in Spermatogonia, Leydig and Sertoli Cells.

In December 2019, a novel coronavirus (SARS-CoV-2) was identified in COVID-19 patients in Wuhan, Hubei Province, China. SARS-CoV-2 shares both high sequence similarity and the use of the same cell entry receptor, angiotensin-converting enzyme 2 (ACE2), with severe acute respiratory syndrome coronavirus (SARS-CoV). Several studies have provided bioinformatic evidence of potential routes of SARS-CoV-2 infection in respiratory, cardiovascular, digestive and urinary systems. However, whether the reproductive system is a potential target of SARS-CoV-2 infection has not yet been determined. Here, we investigate the expression pattern of ACE2 in adult human testes at the level of single-cell transcriptomes. The results indicate that ACE2 is predominantly enriched in spermatogonia and Leydig and Sertoli cells. Gene Set Enrichment Analysis (GSEA) indicates that Gene Ontology (GO) categories associated with viral reproduction and transmission are highly enriched in ACE2-positive spermatogonia, while male gamete generation related terms are downregulated. Cell–cell junction and immunity-related GO terms are increased in ACE2-positive Leydig and Sertoli cells, but mitochondria and reproduction-related GO terms are decreased. These findings provide evidence that the human testis is a potential target of SARS-CoV-2 infection, which may have significant impact on our understanding of the pathophysiology of this rapidly spreading disease.

Evolutionarily conserved pachytene piRNA loci are highly divergent among modern humans.

In the fetal mouse testis, PIWI Interacting RNAs (piRNAs) guide PIWI proteins to silence transposons, but after birth, most post-pubertal pachytene piRNAs map to the genome uniquely and are thought to regulate genes required for male fertility. In human males, the developmental classes, precise genomic origins, and transcriptional regulation of post-natal piRNAs remain undefined. Here, we demarcate the genes and transcripts that produce post-natal piRNAs in human juvenile and adult testes. As in mouse, human A-MYB drives transcription of both pachytene piRNA precursor transcripts and the mRNAs encoding piRNA biogenesis factors. Although human piRNA genes are syntenic to those in other placental mammals, their sequences are poorly conserved. In fact, pachytene piRNA loci are rapidly diverging even among modern humans. Our findings suggest that during mammalian evolution, pachytene piRNA genes are under few selective constraints. We speculate that pachytene piRNA diversity may provide a hitherto unrecognized driver of reproductive isolation.

Characterisation and localisation of the endocannabinoid system components in the adult human testis

Heavy use of cannabis (marijuana) has been associated with decreased semen quality, which may reflect disruption of the endocannabinoid system (ECS) in the male reproductive tract by exogenous cannabinoids. Components of ECS have been previously described in human spermatozoa and in the rodent testis but there is little information on the ECS expression within the human testis. In this study we characterised the main components of the ECS by immunohistochemistry (IHC) on archived testis tissue samples from 15 patients, and by in silico analysis of existing transcriptome datasets from testicular cell populations. The presence of 2-arachidonoylglycerol (2-AG) in the human testis was confirmed by matrix-assisted laser desorption ionization imaging analysis. Endocannabinoid-synthesising enzymes; diacylglycerol lipase (DAGL) and N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), were detected in germ cells and somatic cells, respectively. The cannabinoid receptors, CNR1 and CNR2 were detected at a low level in post-meiotic germ cells and Leydig- and peritubular cells. Different transcripts encoding distinct receptor isoforms (CB1, CB1A, CB1B and CB2A) were also differentially distributed, mainly in germ cells. The cannabinoid-metabolising enzymes were abundantly present; the α/β-hydrolase domain-containing protein 2 (ABHD2) in all germ cell types, except early spermatocytes, the monoacylglycerol lipase (MGLL) in Sertoli cells, and the fatty acid amide hydrolase (FAAH) in late spermatocytes and post-meiotic germ cells. Our findings are consistent with a direct involvement of the ECS in regulation of human testicular physiology, including spermatogenesis and Leydig cell function. The study provides new evidence supporting observations that recreational cannabis can have possible deleterious effects on human testicular function.

Effects and mechanism of lncRNA serving as ceRNA in non-obstructive azoospermia

Objective: To investigate the mechanisms of lncRNA on the occurrence and development of NOA by constructing ceRNA regulation network of lncRNA, miRNA and mRNA. Methods: Samples of adult human testis were obtained from NOA patients and OA patients with normal spermatogenesis (controls), recruited from the Reproductive Medicine Center of Nanfang Hospital from June 2017 to June 2018. Differentially expressed lncRNAs and mRNAs in testicular tissues from patients with NOA were identified by microarray analysis in previous association study. In this study, differentially expressed lncRNAs and mRNA were used to construct the ceRNA regulatory network in NOA and clarify the interaction relationship among lncRNA, miRNA and mRNA. GeneMANIA database was used to construct Protein-Protein Interaction (PPI) of the mRNAs in ceRNA regulatory network. WebGestalt toolkit was employed to perform gene function and pathway enrichment analyses of those coding genes. Finally, qRT-PCR and dual luciferase reporter system were employed for further experimental validation. Results: The ceRNA regulatory network of lncRNA, miRNA and mRNA consists of 21 nodes and 26 edges, of which 4 lncRNAs, 13 miRNAs and 4 mRNAs. 19 proteins were found to interact with the mRNA coding proteins in ceRNA regulatory network by PPI analysis. Gene oncology and KEGG pathway enrichment analyses indicate these coding genes were significantly enriched in pentose metabolic process and pentose phosphate pathway. Furthermore, lncRNA ANXA2P3 was found binding with miR-613 and miR-206 to inhibit mRNA TKT expression. Conclusion: lncRNAs exert an important role in the occurrence and development of NOA via ceRNA regulatory network, which could be used as new biomarkers for NOA treatment.

Single-Cell Signature Explorer for comprehensive visualization of single cell signatures across scRNA-seq datasets.

The momentum of scRNA-seq datasets prompts for simple and powerful tools exploring their meaningful signatures. Here we present Single-Cell_Signature_Explorer (https://sites.google.com/site/fredsoftwares/products/single-cell-signature-explorer), the first method for qualitative and high-throughput scoring of any gene set-based signature at the single cell level and its visualization using t-SNE or UMAP. By scanning datasets for single or combined signatures, it rapidly maps any multi-gene feature, exemplified here with signatures of cell lineages, biological hallmarks and metabolic pathways in large scRNAseq datasets of human PBMC, melanoma, lung cancer and adult testis.