Monocytes expressing activin A and CCR2 exacerbate chronic testicular inflammation by promoting immune cell infiltration.

STUDY QUESTION: Does the chemokine/chemokine receptor axis, involved in immune cell trafficking, contribute to the pathology of testicular inflammation and how does activin A modulate this network? SUMMARY ANSWER: Testicular chemokines and their receptors (especially those essential for trafficking of monocytes) are elevated in orchitis, and activin A modulates the expression of the chemokine/chemokine receptor network to promote monocyte/macrophage and T cell infiltration into the testes, causing extensive tissue damage. WHAT IS KNOWN ALREADY: The levels of CC motif chemokine receptor (CCR)2 and its ligand CC motif chemokine ligand (CCL)2 are increased in experimental autoimmune orchitis (EAO) compared with healthy testes, and mice deficient in CCR2 are protected from EAO-induced tissue damage. Activin A induces CCR2 expression in macrophages, promoting their migration. Moreover, there is a positive correlation between testicular activin A concentration and the severity of autoimmune orchitis. Inhibition of activin A activity by overexpression of follistatin (FST) reduces EAO-induced testicular damage. STUDY DESIGN, SIZE, DURATION: EAO was induced in 10-12-week-old male C57BL/6J (wild-type; WT) and B6.129P2-Ccr2tm1Mae/tm1Mae (Ccr2-/-) mice (n = 6). Adjuvant (n = 6) and untreated (n = 6) age-matched control mice were also included. Testes were collected at 50 days after the first immunization with testicular homogenate in complete Freund's adjuvant. In another experimental setup, WT mice were injected with a non-replicative recombinant adeno-associated viral vector carrying a FST315-expressing gene cassette (rAAV-FST315; n = 7-9) or an empty control vector (n = 5) 30 days prior to EAO induction. Appropriate adjuvant (n = 4-5) and untreated (n = 4-6) controls were also examined. Furthermore, human testicular biopsies exhibiting focal leukocytic infiltration and impaired spermatogenesis (n = 17) were investigated. Biopsies showing intact spermatogenesis were included as controls (n = 9). Bone-marrow-derived macrophages (BMDMs) generated from WT mice were treated with activin A (50 ng/ml) for 6 days. Activin-A-treated or untreated BMDMs were then co-cultured with purified mouse splenic T cells for two days to assess chemokine and cytokine production. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of chemokines in total testicular RNA collected from mice. Immunofluorescence staining was used to detect activin A, F4/80, and CD3 expression in mouse testes. The expression of chemokine/chemokine-receptor-encoding genes was examined in human testicular biopsies by qRT-PCR. Correlations between chemokine expression levels and either the immune cell infiltration density or the mean spermatogenesis score were analyzed. Immunofluorescence staining was used to evaluate the expression of CD68 and CCR2 in human testicular biopsies. RNA isolated from murine BMDMs was used to characterize these cells in terms of their chemokine/chemokine receptor expression levels. Conditioned media from co-cultures of BMDMs and T cells were collected to determine chemokine levels and the production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interferon (IFN)-γ by T cells. MAIN RESULTS AND THE ROLE OF CHANCE: Induction of EAO in the testes of WT mice increased the expression of chemokine receptors such as Ccr1 (P < 0.001), Ccr2 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.0001), CXC motif chemokine receptor (Cxcr)3 (P < 0.01), and CX3C motif chemokine receptor (Cx3cr)1 (P < 0.001), as well as that of most of their ligands. Ccr2 deficiency reversed some of the changes associated with EAO by reducing the expression of Ccr1 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.01), Cxcr3 (P < 0.001), and Cx3cr1 (P < 0.0001). Importantly, the biopsies showing impaired spermatogenesis and concomitant focal leukocytic infiltration exhibited higher expression of CCL2 (P < 0.01), CCR1 (P < 0.05), CCR2 (P < 0.001), and CCR5 (P < 0.001) than control biopsies with no signs of inflammation and intact spermatogenesis. The gene expression of CCR2 and its ligand CCL2 correlated positively with the immune cell infiltration density (P < 0.05) and negatively with the mean spermatogenesis score (P < 0.001). Moreover, CD68+ macrophages expressing CCR2 were present in human testes with leukocytic infiltration with evidence of tubular damage. Treatment of BMDMs, as surrogates for testicular macrophages, with activin A increased their expression of Ccr1, Ccr2, and Ccr5 while reducing their expression of Ccl2, Ccl3, Ccl4, Ccl6, Ccl7 Ccl8, and Ccl12. These findings were validated in vivo, by showing that inhibiting activin A activity by overexpressing FST in EAO mice decreased the expression of Ccr2 (P < 0.05) and Ccr5 (P < 0.001) in the testes. Interestingly, co-culturing activin-A-treated BMDMs and T cells reduced the levels of CCL2 (P < 0.05), CCL3/4 (P < 0.01), and CCL12 (P < 0.05) in the medium and attenuated the production of TNF (P < 0.05) by T cells. The majority of cells secreting activin A in EAO testes were identified as macrophages. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: BMDMs were used as surrogates for testicular macrophages. Hence, results obtained from the in vitro experiments might not be fully representative of the situation in the testes in vivo. Moreover, since total RNA was extracted from the testicular tissue to examine chemokine expression, the contributions of individual cell types as producers of specific chemokines may have been overlooked. WIDER IMPLICATIONS OF THE FINDINGS: Our data indicate that macrophages are implicated in the development and progression of testicular inflammation by expressing CCR2 and activin A, which ultimately remodel the chemokine/chemokine receptor network and recruit other immune cells to the site of inflammation. Consequently, inhibition of CCR2 or activin A could serve as a potential therapeutic strategy for reducing testicular inflammation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the International Research Training Group in 'Molecular pathogenesis on male reproductive disorders', a collaboration between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK1871/1-2) funded by the Deutsche Forschungsgemeinschaft and Monash University, a National Health and Medical Research Council of Australia Ideas Grant (1184867), and the Victorian Government's Operational Infrastructure Support Programme. The authors declare no competing financial interests.

T cells in testicular germ cell tumors: new evidence of fundamental contributions by rare subsets.

BACKGROUND: Immune cell infiltration is heterogeneous but common in testicular germ cell tumors (TGCT) and pre-invasive germ cell neoplasia in situ (GCNIS). Tumor-infiltrating T cells including regulatory T (Treg) and follicular helper T (Tfh) cells are found in other cancer entities, but their contributions to TGCT are unknown. METHODS: Human testis specimens from independent patient cohorts were analyzed using immunohistochemistry, flow cytometry and single-cell RNA sequencing (scRNA-seq) with special emphasis on delineating T cell subtypes. RESULTS: Profound changes in immune cell composition within TGCT, shifting from macrophages in normal testes to T cells plus B and dendritic cells in TGCT, were documented. In most samples (96%), the CD4+ T cell frequency exceeded that of CD8+ cells, with decreasing numbers from central to peripheral tumor areas, and to tumor-free, contralateral testes. T cells including Treg and Tfh were most abundant in seminoma compared to mixed tumors and embryonal carcinoma. CONCLUSION: Despite considerable heterogeneity between patients, T cell subtypes form a key part of the TGCT microenvironment. The novel finding of rare Treg and Tfh cells in human testis suggests their involvement in TGCT pathobiology, with implications for understanding tumor progression, to assess patients' prognosis, and as putative targets for personalized immunotherapy.

Proteomic biomarkers in seminal plasma as predictors of reproductive potential in azoospermic men.

Introduction: Azoospermia, characterized by an absence of sperm in the ejaculate, represents the most severe form of male infertility. While surgical sperm retrieval in obstructive azoospermia (OA) is successful in the majority of cases, patients with non-obstructive azoospermia (NOA) show retrieval rates of only about 50% and thus frequently have unnecessary surgery. Surgical intervention could be avoided if patients without preserved spermatogenesis are identified preoperatively. This prospective study aimed to discover biomarkers in seminal plasma that could be employed for a non-invasive differential diagnosis of OA/NOA in order to rationalize surgery recommendations and improve success rates. Methods: All patients signed written informed consent, underwent comprehensive andrological evaluation, received human genetics to exclude relevant pathologies, and patients with azoospermia underwent surgical sperm retrieval. Using label-free LC-MS/MS, we compared the proteomes of seminal plasma samples from fertile men (healthy controls (HC), n=8) and infertile men diagnosed with 1) OA (n=7), 2) NOA with successful sperm retrieval (mixed testicular atrophy (MTA), n=8), and 3) NOA without sperm retrieval (Sertoli cell-only phenotype (SCO), n=7). Relative abundance changes of two candidate markers of sperm retrieval, HSPA2 and LDHC, were confirmed by Western Blot. Results: We found the protein expression levels of 42 proteins to be significantly down-regulated (p ≤ 0.05) in seminal plasma from SCO NOA patients relative to HC whereas only one protein was down-regulated in seminal plasma from MTA patients. Analysis of tissue and cell expression suggested that the testis-specific proteins LDHC, PGK2, DPEP3, and germ-cell enriched heat-shock proteins HSPA2 and HSPA4L are promising biomarkers of spermatogenic function. Western blotting revealed a significantly lower abundance of LDHC and HSPA2 in the seminal plasma of men with NOA (SCO and MTA) compared to controls. Discussion: The results indicate that certain testis-specific proteins when measured in seminal plasma, could serve as indicators of the presence of sperm in the testis and predict the success of sperm retrieval. Used in conjunction with conventional clinical assessments, these proteomic biomarkers may assist in the non-invasive diagnosis of idiopathic male infertility.

Rescue of p53 functions by in vitro-transcribed mRNA impedes the growth of high-grade serous ovarian cancer.

BACKGROUND: The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the very aggressive high-grade serous ovarian carcinoma (HGSOC) exhibits the highest prevalence of TP53 mutations, present in >96% of cases. Despite intensive efforts to reactivate p53, no clinical drug has been approved to rescue p53 function. In this study, our primary objective was to administer in vitro-transcribed (IVT) wild-type (WT) p53-mRNA to HGSOC cell lines, primary cells, and orthotopic mouse models, with the aim of exploring its impact on inhibiting tumor growth and dissemination, both in vitro and in vivo. METHODS: To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient-derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models. RESULTS: Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA-based methods. The introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent long-lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner. CONCLUSIONS: The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.

Effects of a Sertoli cell-specific knockout of Connexin43 on maturation and proliferation of postnatal Sertoli cells.

Adult male Sertoli cell-specific Connexin43 knockout mice (SCCx43KO) exhibit higher Sertoli cell (SC) numbers per seminiferous tubule compared to their wild type (WT) littermates. Thus, deletion of this testicular gap junction protein seems to affect the proliferative potential and differentiation of "younger" SC. Although SC have so far mostly been characterised as postmitotic cells that cease to divide and become an adult, terminally differentiated cell population at around puberty, there is rising evidence that there exist exceptions from this for a very long time accepted paradigm. Aim of this study was to investigate postnatal SC development and to figure out underlying causes for observed higher SC numbers in adult KO mice. Therefore, the amount of SC mitotic figures was compared, resulting in slightly more and prolonged detection of SC mitotic figures in KO mice compared to WT. SC counting per tubular cross section revealed significantly different time curves, and comparing proliferation rates using Bromodesoxyuridine and Sox9 showed higher proliferation rates in 8-day old KO mice. SC proliferation was further investigated by Ki67 immunohistochemistry. SC in KO mice displayed a delayed initiation of cell-cycle-inhibitor p27Kip1 synthesis and prolonged synthesis of the phosphorylated tumour suppressor pRb and proliferation marker Ki67. Thus, the higher SC numbers in adult male SCCx43KO mice may arise due to two different reasons: Firstly, in prepubertal KO mice, the proliferation rate of SC was higher. Secondly, there were differences in their ability to cease proliferation as shown by the delayed initiation of p27Kip1 synthesis and the prolonged production of phosphorylated pRb and Ki67. Immunohistochemical results indicating a prolonged period of SC proliferation in SCCx43KO were confirmed by detection of proliferating SC in 17-days-old KO mice. In conclusion, deletion of the testicular gap junction protein Cx43 might prevent normal SC maturation and might even alter also the proliferation potential of adult SC.

Sertoli cell-enriched proteins in mouse and human testicular interstitial fluid.

Sertoli cells support the development of sperm and the function of various somatic cells in the interstitium between the tubules. Sertoli cells regulate the function of the testicular vasculature and the development and function of the Leydig cells that produce testosterone for fertility and virility. However, the Sertoli cell-derived factors that regulate these cells are largely unknown. To define potential mechanisms by which Sertoli cells could support testicular somatic cell function, we aimed to identify Sertoli cell-enriched proteins in the testicular interstitial fluid (TIF) between the tubules. We previously resolved the proteome of TIF in mice and humans and have shown it to be a rich source of seminiferous tubule-derived proteins. In the current study, we designed bioinformatic strategies to interrogate relevant proteomic and genomic datasets to identify Sertoli cell-enriched proteins in mouse and human TIF. We analysed proteins in mouse TIF that were significantly reduced after one week of acute Sertoli cell ablation in vivo and validated which of these are likely to arise primarily from Sertoli cells based on relevant mouse testis RNASeq datasets. We used a different, but complementary, approach to identify Sertoli cell-enriched proteins in human TIF, taking advantage of high-quality human testis genomic, proteomic and immunohistochemical datasets. We identified a total of 47 and 40 Sertoli cell-enriched proteins in mouse and human TIF, respectively, including 15 proteins that are conserved in both species. Proteins with potential roles in angiogenesis, the regulation of Leydig cells or steroidogenesis, and immune cell regulation were identified. The data suggests that some of these proteins are secreted, but that Sertoli cells also deposit specific proteins into TIF via the release of extracellular vesicles. In conclusion, we have identified novel Sertoli cell-enriched proteins in TIF that are candidates for regulating somatic cell-cell communication and testis function.

Bi-allelic variants in INSL3 and RXFP2 cause bilateral cryptorchidism and male infertility.

STUDY QUESTION: What is the impact of variants in the genes INSL3 (Insulin Like 3) and RXFP2 (Relaxin Family Peptide Receptor 2), respectively, on cryptorchidism and male infertility? SUMMARY ANSWER: Bi-allelic loss-of-function (LoF) variants in INSL3 and RXFP2 result in bilateral cryptorchidism and male infertility, whereas heterozygous variant carriers are phenotypically unaffected. WHAT IS KNOWN ALREADY: The small heterodimeric peptide INSL3 and its G protein-coupled receptor RXFP2 play a major role in the first step of the biphasic descent of the testes, and variants in the INSL3 and RXFP2 genes have long been implicated in inherited cryptorchidism. However, only one single homozygous missense variant in RXFP2 has clearly been linked to familial bilateral cryptorchidism, so the effects of bi-allelic variants in INSL3 and heterozygous variants in both genes on cryptorchidism and male infertility remain unclear. STUDY DESIGN, SIZE, DURATION: Exome data of 2412 men from the MERGE (Male Reproductive Genomics) study cohort including 1902 infertile men with crypto-/azoospermia, of whom 450 men had a history of cryptorchidism, were screened for high-impact variants in INSL3 and RXFP2. PARTICIPANTS/MATERIALS, SETTING, METHODS: For patients with rare, high-impact variants in INSL3 and RXFP2, detailed clinical data were collected and the testicular phenotype was determined. Genotyping of family members was performed to analyse the co-segregation of candidate variants with the condition. Immunohistochemical staining for INSL3 in patient testicular tissue and measuring serum INSL3 concentration was performed to analyse the functional impact of a homozygous loss-of-function variant in INSL3. For a homozygous missense variant in RXFP2, its impact on the protein's cell surface expression and ability to respond to INSL3 in CRE reporter gene assay was determined. MAIN RESULTS AND THE ROLE OF CHANCE: This study presents homozygous high-impact variants in INSL3 and RXFP2 and clearly correlates these to bilateral cryptorchidism. Functional impact of the identified INSL3 variant was demonstrated by absence of INSL3-specific staining in patients' testicular Leydig cells as well as undetectable blood serum levels. The identified missense variant in RXFP2 was demonstrated to lead to reduced RXFP2 surface expression and INSL3 mediated receptor activation. LIMITATIONS, REASONS FOR CAUTION: Further investigations are needed to explore a potential direct impact of bi-allelic INSL3 and RXFP2 variants on spermatogenesis. With our data, we cannot determine whether the infertility observed in our patients is a direct consequence of the disruption of a possible function of these genes on spermatogenesis or whether it occurs secondarily due to cryptorchidism. WIDER IMPLICATIONS OF THE FINDINGS: In contrast to previous assumptions, this study supports an autosomal recessive inheritance of INSL3- and RXFP2-related bilateral cryptorchidism while heterozygous LoF variants in either gene can at most be regarded as a risk factor for developing cryptorchidism. Our findings have diagnostic value for patients with familial/bilateral cryptorchidism and additionally shed light on the importance of INSL3 and RXFP2 in testicular descent and fertility. STUDY FUNDING/COMPETING INTEREST(S): This study was carried out within the frame of the German Research Foundation (DFG) funded by Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG, CRU326). Research at the Florey was supported by an NHMRC grant (2001027) and the Victorian Government Operational Infrastructure Support Program. A.S.B. is funded by the DFG ('Emmy Noether Programme' project number 464240267). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies.

BACKGROUND: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression. METHODS: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines' representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling. RESULTS: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells. CONCLUSION: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression.

DDX3Y is likely the key spermatogenic factor in the AZFa region that contributes to human non-obstructive azoospermia.

Non-obstructive azoospermia, the absence of sperm in the ejaculate due to disturbed spermatogenesis, represents the most severe form of male infertility. De novo microdeletions of the Y-chromosomal AZFa region are one of few well-established genetic causes for NOA and are routinely analysed in the diagnostic workup of affected men. So far, it is unclear which of the three genes located in the AZFa chromosomal region is indispensible for germ cell maturation. Here we present four different likely pathogenic loss-of-function variants in the AZFa gene DDX3Y identified by analysing exome sequencing data of more than 1,600 infertile men. Three of the patients underwent testicular sperm extraction and revealed the typical AZFa testicular Sertoli cell-only phenotype. One of the variants was proven to be de novo. Consequently, DDX3Y represents the AZFa key spermatogenic factor and screening for variants in DDX3Y should be included in the diagnostic workflow.

Linking human Dead end 1 (DND1) variants to male infertility employing zebrafish embryos.

STUDY QUESTION: Is the vertebrate protein Dead end (DND1) a causative factor for human infertility and can novel in vivo assays in zebrafish help in evaluating this? SUMMARY ANSWER: Combining patient genetic data with functional in vivo assays in zebrafish reveals a possible role for DND1 in human male fertility. WHAT IS KNOWN ALREADY: About 7% of the male population is affected by infertility but linking specific gene variants to the disease is challenging. The function of the DND1 protein was shown to be critical for germ cell development in several model organisms but a reliable and cost-effective method for evaluating the activity of the protein in the context of human male infertility is still missing. STUDY DESIGN, SIZE, DURATION: Exome data from 1305 men included in the Male Reproductive Genomics cohort were examined in this study. A total of 1114 of the patients showed severely impaired spermatogenesis but were otherwise healthy. Eighty-five men with intact spermatogenesis were included in the study as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: We screened the human exome data for rare, stop-gain, frameshift, splice site, as well as missense variants in DND1. The results were validated by Sanger sequencing. Immunohistochemical techniques and, when possible, segregation analyses were performed for patients with identified DND1 variants. The amino acid exchange in the human variant was mimicked at the corresponding site of the zebrafish protein. Using different aspects of germline development in live zebrafish embryos as biological assays, we examined the activity level of these DND1 protein variants. MAIN RESULTS AND THE ROLE OF CHANCE: In human exome sequencing data, we identified four heterozygous variants in DND1 (three missense and one frameshift variant) in five unrelated patients. The function of all of the variants was examined in the zebrafish and one of those was studied in more depth in this model. We demonstrate the use of zebrafish assays as a rapid and effective biological readout for evaluating the possible impact of multiple gene variants on male fertility. This in vivo approach allowed us to assess the direct impact of the variants on germ cell function in the context of the native germline. Focusing on the DND1 gene, we find that zebrafish germ cells, expressing orthologs of DND1 variants identified in infertile men, failed to arrive correctly at the position where the gonad develops and exhibited defects in cell fate maintenance. Importantly, our analysis facilitated the evaluation of single nucleotide variants, whose impact on protein function is difficult to predict, and allowed us to distinguish variants that do not affect the protein's activity from those that strongly reduce it and could thus potentially be the primary cause for the pathological condition. These aberrations in germline development resemble the testicular phenotype of azoospermic patients. LIMITATIONS, REASONS FOR CAUTION: The pipeline we present requires access to zebrafish embryos and to basic imaging equipment. The notion that the activity of the protein in the zebrafish-based assays is relevant for the human homolog is well supported by previous knowledge. Nevertheless, the human protein may differ in some respects from its homologue in zebrafish. Thus, the assay should be considered only one of the parameters used in defining DND1 variants as causative or non-causative for infertility. WIDER IMPLICATIONS OF THE FINDINGS: Using DND1 as an example, we have shown that the approach described in this study, relying on bridging between clinical findings and fundamental cell biology, can help to establish links between novel human disease candidate genes and fertility. In particular, the power of the approach we developed is manifested by the fact that it allows the identification of DND1 variants that arose de novo. The strategy presented here can be applied to different genes in other disease contexts. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the German Research Foundation, Clinical Research Unit, CRU326 'Male Germ Cells'. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Functional assessment of DMRT1 variants and their pathogenicity for isolated male infertility.

OBJECTIVE: To study the impact of Doublesex and mab-3-related transcription factor 1 (DMRT1) gene variants on the encoded protein's function and the variants' pathogenic relevance for isolated male infertility caused by azoospermia. DESIGN: This study established a novel luciferase assay for DMRT1 missense variants using 2 different target promotors and validated the assay by analyzing previously published variants associated with differences in sex development. SETTING: University genetics research institute and tertiary referral center for couples' infertility. PATIENT(S): Eleven infertile men with severely impaired spermatogenesis resulting in crypto- or azoospermia and carrying rare heterozygous missense variants in DMRT1 were identified within the Male Reproductive Genomics study. MAIN OUTCOME MEASURE(S): Luciferase assays with human DMRT1 variants to test functional effects on the CYP19A1 and Stra8 target promoters. RESULT(S): We first developed and refined luciferase assays to reliably test the functional impact of DMRT1 missense variants. Next, the assay was validated by analyzing 2 DMRT1 variants associated with differences in sex development, of which c.240G>C p.(Arg80Ser) displayed highly significant effects on both target promoters compared with the wild-type protein (-40% and +100%, respectively) and c.331A>G p.(Arg111Gly) had a significant effect on the Stra8 promoter (-76%). We then systematically characterized 11 DMRT1 variants identified in infertile men. The de novo variant c.344T>A p.(Met115Lys) showed a pronounced loss of function in both DMRT1 target promoters (-100% and -86%, respectively). Variants c.308A>G p.(Lys103Arg) and c.991G>C p.(Asp331His) showed a significant gain of function exclusively for the CYP19A1 promoter (+15% and +19%, respectively). Based on these results, 3 variants were reclassified according to clinical guidelines. CONCLUSION(S): The present study highlights the importance of functionally characterizing DMRT1 variants of uncertain clinical significance. Using luciferase assays for diagnostic purposes enables an improved causal diagnosis for isolated male infertility.

Activin A and CCR2 regulate macrophage function in testicular fibrosis caused by experimental autoimmune orchitis.

Experimental autoimmune-orchitis (EAO), a rodent model of chronic testicular inflammation and fibrosis, replicates pathogenic changes seen in some cases of human spermatogenic disturbances. During EAO, increased levels of pro-inflammatory and pro-fibrotic mediators such as TNF, CCL2, and activin A are accompanied by infiltration of leukocytes into the testicular parenchyma. Activin A levels correlate with EAO severity, while elevated CCL2 acting through its receptor CCR2 mediates leukocyte trafficking and recruits macrophages. CCR2 + CXCR4 + macrophages producing extracellular matrix proteins contribute widely to fibrogenesis. Furthermore, testicular macrophages (TMs) play a critical role in organ homeostasis. Therefore, we aimed to investigate the role of the activin A/CCL2-CCR2/macrophage axis in the development of testicular fibrosis. Following EAO induction, we observed lower levels of organ damage, collagen deposition, and leukocyte infiltration (including fibronectin+, collagen I+ and CXCR4+ TMs) in Ccr2-/- mice than in WT mice. Furthermore, levels of Il-10, Ccl2, and the activin A subunit Inhba mRNAs were lower in Ccr2-/- EAO testes. Notably, fibronectin+ TMs were also present in biopsies from patients with impaired spermatogenesis and fibrotic alterations. Overexpression of the activin A antagonist follistatin reduced tissue damage and collagen I+ TM accumulation in WT EAO testes, while treating macrophages with activin A in vitro increased the expression of Ccr2, Fn1, Cxcr4, and Mmp2 and enhanced migration along a CCL2 gradient; these effects were abolished by follistatin. Taken together, our data indicate that CCR2 and activin A promote fibrosis during testicular inflammation by regulating macrophage function. Inhibition of CCR2 or activin A protects against damage progression, offering a promising avenue for therapeutic intervention.

The changing landscape of immune cells in the fetal mouse testis.

Fetal testis growth involves cell influx and extensive remodeling. Immediately after sex determination in mouse, macrophages enable normal cord formation and removal of inappropriately positioned cells. This study provides new information about macrophages and other immune cells after cord formation in fetal testes, including their density, distribution, and close cellular contacts. C57BL6J mouse testes from embryonic day (E) 13.5 to birth (post-natal day 0; PND0), were examined using immunofluorescence, immunohistochemistry, and RT-qPCR to identify macrophages (F4/80, CD206, MHCII), T cells (CD3), granulocytes/neutrophils (Ly6G), and germ cells (DDX4). F4/80+ cells were the most abundant, comprising 90% of CD45+ cells at E13.5 and declining to 65% at PND0. Changes in size, shape, and markers (CD206 and MHCII) documented during this interval align with the understanding that F4/80+ cells have different origins during embryonic life. CD3+ cells and F4/80-/MHCII+ were absent to rare until PND0. Ly6G+ cells were scarce at E13.5 but increased robustly by PND0 to represent half of the CD45+ cells. These immunofluorescence data were in accord with transcript analysis, which showed that immune marker mRNAs increased with testis age. F4/80+ and Ly6G+ cells were frequently inside cords adjacent to germ cells at E13.5 and E15.5. F4/80+ cells were often in clusters next to other immune cells. Macrophages inside cords at E13.5 and E15.5 (F4/80Hi/CD206+) were different from macrophages at PND0 (F4/80Dim/CD206-), indicating that they have distinct origins. This histological quantification coupled with transcript information identifies new cellular interactions for immune cells in fetal testis morphogenesis, and highlights new avenues for studies of their functional significance.

In Situ Hybridization of Estrogen Receptors α and ß in the Human Testis.

In situ hybridization (ISH) is an excellent method for detecting RNA in histological sections, both to detect gene expression and to assign gene expression to a distinct cell population. Therefore, ISH may be used in basic cell biology to detect the expression of certain genes within a tissue containing various cell populations. Here, we describe the detection and cellular localization of two estrogen receptors, both isoforms of the genomic estrogen receptor (ERα and ERß) in the human testis.

Task force veterinary anatomy: joint efforts of the five German veterinary schools to ensure education during the COVID-19 pandemic.

At the start of the COVID-pandemic in March 2020, the Institutes of Veterinary Anatomy of the five German educational institutions were confronted with the challenge of digitalising all lectures for the second and fourth semesters of veterinary students. After an online kick-off event and a preliminary status quo meeting, available digital teaching material was exchanged for students to stream from learning platforms. Lectures were either synchronized or made available as audio recordings and connotated slides on the learning platforms. Fortunately, digital microscopic slides had already been in use, which made it easy for students to access them. Dissection exercises mostly consisted of self-study, using instructive videos and interactive exercises. In the second half of the semester, four of the educational institutions were able to offer a restricted number of in-person gross anatomy classes under reinforced conditions. Success monitoring took place online through different formats, and partially on a voluntary basis, via the learning platforms. Although the past two semesters had to almost exclusively take place online due to the unprecedented circumstances, and joint efforts of the five veterinary institutions, there is a general consensus that the practical education in anatomy, histology and embryology is essential to veterinary students. In fact, it is the only way they can obtain the necessary skills to successfully complete the rest of their degree.

Modelling the Functions of Polo-Like Kinases in Mice and Their Applications as Cancer Targets with a Special Focus on Ovarian Cancer.

Polo-like kinases (PLKs) belong to a five-membered family of highly conserved serine/threonine kinases (PLK1-5) that play differentiated and essential roles as key mitotic kinases and cell cycle regulators and with this in proliferation and cellular growth. Besides, evidence is accumulating for complex and vital non-mitotic functions of PLKs. Dysregulation of PLKs is widely associated with tumorigenesis and by this, PLKs have gained increasing significance as attractive targets in cancer with diagnostic, prognostic and therapeutic potential. PLK1 has proved to have strong clinical relevance as it was found to be over-expressed in different cancer types and linked to poor patient prognosis. Targeting the diverse functions of PLKs (tumor suppressor, oncogenic) are currently at the center of numerous investigations in particular with the inhibition of PLK1 and PLK4, respectively in multiple cancer trials. Functions of PLKs and the effects of their inhibition have been extensively studied in cancer cell culture models but information is rare on how these drugs affect benign tissues and organs. As a step further towards clinical application as cancer targets, mouse models therefore play a central role. Modelling PLK function in animal models, e.g., by gene disruption or by treatment with small molecule PLK inhibitors offers promising possibilities to unveil the biological significance of PLKs in cancer maintenance and progression and give important information on PLKs' applicability as cancer targets. In this review we aim at summarizing the approaches of modelling PLK function in mice so far with a special glimpse on the significance of PLKs in ovarian cancer and of orthotopic cancer models used in this fatal malignancy.

TRIM71 Deficiency Causes Germ Cell Loss During Mouse Embryogenesis and Is Associated With Human Male Infertility.

Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling germ cell development. The RNA-binding protein and E3 ubiquitin ligase TRIM71 is essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of germ cells in the seminiferous tubules. Infertility in male Trim71 cKO mice originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, TCam-2 cells, a human GCT-derived seminoma cell line which was used as an in vitro model for PGCs, showed proliferation defects upon TRIM71 knockdown. Additionally, in vitro growth competition assays, as well as proliferation assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells showed that TRIM71 also promotes proliferation in this malignant GCT-derived non-seminoma cell line. Importantly, the PGC-specific markers BLIMP1 and NANOS3 were consistently downregulated in Trim71 KO PGCLCs, TRIM71 knockdown TCam-2 cells and TRIM71 mutant NCCIT cells. These data collectively support a role for TRIM71 in PGC development. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with an SCO phenotype. Altogether, our work reveals for the first time an association of TRIM71 deficiency with human male infertility, and uncovers further developmental roles for TRIM71 in the germline during mouse embryogenesis.

Sperm proteins and cancer-testis antigens are released by the seminiferous tubules in mice and men.

Sperm develop from puberty in the seminiferous tubules, inside the blood-testis barrier to prevent their recognition as "non-self" by the immune system, and it is widely assumed that human sperm-specific proteins cannot access the circulatory or immune systems. Sperm-specific proteins aberrantly expressed in cancer, known as cancer-testis antigens (CTAs), are often pursued as cancer biomarkers and therapeutic targets based on the assumption they are neoantigens absent from the circulation in healthy men. Here, we identify a wide range of germ cell-derived and sperm-specific proteins, including multiple CTAs, that are selectively deposited by the Sertoli cells of the adult mouse and human seminiferous tubules into testicular interstitial fluid (TIF) that is "outside" the blood-testis barrier. From TIF, the proteins can access the circulatory- and immune systems. Disruption of spermatogenesis decreases the abundance of these proteins in mouse TIF, and a sperm-specific CTA is significantly decreased in TIF from infertile men, suggesting that exposure of certain CTAs to the immune system could depend on fertility status. The results provide a rationale for the development of blood-based tests useful in the management of male infertility and indicate CTA candidates for cancer immunotherapy and biomarker development that could show sex-specific and male-fertility-related responses.

Excessive unilateral proliferation of spermatogonia in a patient with non-obstructive azoospermia - adverse effect of clomiphene citrate pre-treatment?

BACKGROUND: Clomiphene citrate has been proposed as pre-treatment for infertile men with non-obstructive, testicular azoospermia (NOA) before surgery for testicular sperm extraction (TESE), especially when serum testosterone is low. CASE PRESENTATION: Here, we report on a 33-year old azoospermic patient with a previous history of repeated "fresh" TESE and clomiphene citrate therapy (50 mg/day over 6 months) before undergoing microscopically assisted, bilateral testicular biopsy. Comprehensive histological and immunohistochemical work-up revealed a heterogeneous spermatogenic arrest at the level of spermatogonia or primary spermatocytes, with focally preserved spermatogenesis up to elongated spermatids in the right testis. In the left testis, the majority of tubules (> 70%) showed no tubular lumen or regular seminiferous epithelium but a great number of spermatogonia-like cells. These cells proved to be normally differentiated spermatogonia (positive for melanoma associated antigen 4 (MAGEA4), negative for placental alkaline phosphatase (PlAP)) with increased proliferative activity (positive for proliferating cell nuclear antigen (PCNA)) and a slightly higher rate of apoptotic cells. When compared to a tissue control with normal spermatogenesis, expression of sex hormone receptors androgen receptor (AR), estrogen receptor (ER) alpha, and G-protein coupled estrogen receptor 1 (GPER1) was not altered in patient samples. Sertoli cells appeared to be mature (positive for vimentin, negative for cytokeratin 18), whereas the expression of zona occludens protein 1 (ZO-1), claudin 11, and connexin 43 was absent or dislocated in the tubules with abundance of spermatogonia. CONCLUSION: This result suggests that formation of the blood-testis barrier is disturbed in affected tubules. To our knowledge this is the first observation of excessive, non-malignant proliferation of spermatogonia in a NOA patient. Although underlying molecular mechanisms remain to be elucidated, we hypothesize that the unusual pathology was triggered by the high-dose clomiphene citrate treatment preceding testicular biopsy.

CONTEXTE: Chez les hommes infertiles qui présentent une azoospermie non obstructive (NOA), le citrate de clomifène a été proposé comme pré-traitement avant la chirurgie pour extraction testiculaire de spermatozoïdes (TESE), surtout quand la testostérone sérique est basse. PRÉSENTATION DU CAS: Nous rendons compte, ici, d'un patient azoospermique de 33 ans avec antécédent de traitements répétés par TESE « frais ¼ et par citrate de clomifène (50 mg/jour sur 6 mois) avant de subir une biopsie testiculaire bilatérale assistée microscopiquement.L'étude histologique et immunohistochimique a révélé un arrêt hétérogène de la spermatogénèse au stade de spermatogonies ou de spermatocytes primaires, avec des foyers de spermatogenèse préservée jusqu'au stade de spermatides allongées dans le testicule droit.Dans le testicule gauche, la majorité des tubules (>70%) ne présentaient ni lumière tubulaire ni épithélium séminifère régulier mais un grand nombre de cellules spermatogonies-like. Ces cellules se sont avérées être des spermatogonies normalement différenciées (positives pour l'antigène 4 associé au mélanome (MAGEA4), négatives pour la phosphatase alcaline placentaire (PlAP)) avec une activité proliférative accrue (positives pour l'antigène nucléaire de prolifération cellulaire (PCNA)) et un taux un peu plus élevé de cellules apoptotiques. Comparée à celle d'un tissu témoin avec spermatogenèse normale, l'expression des récepteurs aux hormones sexuelles, récepteur aux androgènes (AR), récepteur aux estrogènes (ER) alpha et récepteur 1 à la protéine G couplée aux estrogènes (GPER1), n'était pas modifiée dans les échantillons du patient. Les cellules de Sertoli semblaient matures (positives à la vimentine, négatives cytokératine 18), tandis que l'expression de la protéine 1 de la zone occludens (ZO-1), de la claudine 11, et de la connexine 43 était absente ou délocalisée dans les tubules présentant une abondance de spermatogonies. CONCLUSION: Ces résultats suggèrent que la formation de la barrière hémato-testiculaire est perturbée dans les tubules affectés. À notre connaissance, il s'agit de la première observation d'une prolifération excessive et non maligne de spermatogonies chez un patient avec NOA. Bien que les mécanismes moléculaires sous-jacents restent à élucider, nous supposons que cette pathologie inhabituelle a été déclenchée par le traitement au citrate de clomifène à haute dose précédant la biopsie testiculaire.

Bi-allelic Mutations in M1AP Are a Frequent Cause of Meiotic Arrest and Severely Impaired Spermatogenesis Leading to Male Infertility.

Male infertility affects ∼7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported. To address this gap, we performed whole-exome sequencing in 58 men with unexplained meiotic arrest and identified the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 associated protein, in three unrelated men. This variant most likely results in a truncated protein as shown in vitro by heterologous expression of mutant M1AP. Next, we screened four large cohorts of infertile men and identified three additional individuals carrying homozygous c.676dup and three carrying combinations of this and other likely causal variants in M1AP. Moreover, a homozygous missense variant, c.1166C>T (p.Pro389Leu), segregated with infertility in five men from a consanguineous Turkish family. The common phenotype between all affected men was NOA, but occasionally spermatids and rarely a few spermatozoa in the semen were observed. A similar phenotype has been described for mice with disruption of M1ap. Collectively, these findings demonstrate that mutations in M1AP are a relatively frequent cause of autosomal recessive severe spermatogenic failure and male infertility with strong clinical validity.