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Anterior gradient-2 (AGR2) is highly expressed in several tumors and plays an important role in tumor development. However, the biological function of AGR2 in teratomas has not yet been thoroughly studied. In this study, AGR2 was found to be upregulated in teratoma tissues and in human testicular teratoma cell lines by Western blotting and qRT-PCR assays. A DNA Methylation-Specific PCR assay demonstrated that AGR2 upregulation resulted from hypomethylated AGR2 in teratoma cells. NCC-IT and NT2-D1 cells were transfected with pcDNA-AGR2 or sh-AGR2 to obtain AGR2-overexpressed or -silenced cells, and cell proliferation, invasion and glycolysis were determined using CCK-8, 5-ethynyl-2'-deoxyuridine (EdU), Transwell assays, and commercial kits. The results revealed that overexpression of AGR2 promoted teratoma cell proliferation and invasion and elevated glycolysis levels evidencing by the increase in lactate secretion, glucose consumption, ATP levels and the expression of glycolysis-related proteins, while knockdown of AGR2 showed the opposite results. The interactions between AGR2 and annexin A2 (AnXA2), as well as between AnXA2 and epidermal growth factor receptor (EGFR) were verified by co-immunoprecipitation assay. Mechanistic studies revealed that AGR2 interacts with AnXA2 and increases the level of AnXA2 to recruit more AnXA2 to EGFR, there by promoting EGFR expression. A series of rescue experiments showed that knockdown of AnXA2 or EGFR weakened the promotional effects of AGR2 overexpression on the proliferation, invasion, and glycolysis of teratoma cells. Finally, tumorigenicity assays were performed using NT2-D1 cells stably transfected with either LV-NC-shRNA or LV-shAGR2. The results showed that AGR2 knockdown significantly inhibited teratoma tumor growth in vivo. In conclusion, our data suggested that AGR2 facilitates glycolysis in teratomas through promoting EGFR expression by interacting with AnXA2, thereby promoting teratoma cells proliferation and invasion., Competing Interests: Declaration of competing interest There is no conflict of interest to be declared by the authors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Germ cell tumors (GCT) are the most common solid tumors in young men of age 15 - 40. In previous studies, we profiled the interaction of GCT cells with cells of the tumor microenvironment (TM), which showed that especially the 3D interaction of fibroblasts (FB) or macrophages with GCT cells influenced the growth behavior and cisplatin response as well as the transcriptome and secretome of the tumor cells, suggesting that the crosstalk of these cells with GCT cells is crucial for tumor progression and therapy outcome. In this study, we shed light on the mechanisms of activation of cancer-associated fibroblasts (CAF) in the GCT setting and their effects on GCT cells lines and the monocyte cell line THP-1. Ex vivo cultures of GCT-derived CAF were established and characterized molecularly and epigenetically by performing DNA methylation arrays, RNA sequencing, and mass spectrometry-based secretome analysis. We demonstrated that the activation state of CAF is influenced by their former prevailing tumor environment in which they have resided. Hereby, we postulate that seminoma (SE) and embryonal carcinoma (EC) activate CAF, while teratoma (TER) play only a minor role in CAF formation. In turn, CAF influence proliferation and the expression of cisplatin sensitivity-related factors in GCT cells lines as well as polarization of in vitro-induced macrophages by the identified effector molecules IGFBP1, LGALS3BP, LYVE1, and PTX3. Our data suggests that the vital interaction of CAF with GCT cells and with macrophages has a huge influence on shaping the extracellular matrix as well as on recruitment of immune cells to the TM. In conclusion, therapeutically interfering with CAF and / or macrophages in addition to the standard therapy might slow-down progression of GCT and re-shaping of the TM to a tumor-promoting environment. Significance: The interaction of CAF with GCT and macrophages considerably influences the microenvironment. Thus, therapeutically interfering with CAF might slow-down progression of GCT and re-shaping of the microenvironment to a tumor-promoting environment., Competing Interests: Conflict of interest statement All authors declare to have no conflict of interest, (Copyright © 2024. Published by Elsevier B.V.)

Estrogen drives the growth of some cancers, such as breast cancer, via estrogen receptor alpha (ERα). Estrogen also activates ERβ, but whether ERβ is expressed and has a role in different cancers is debated. The use of nonspecific antibodies has contributed to the confusion, and this review delves into ERβ's controversial role in cancer and focuses on tumor expression that can be supported by non-antibody-dependent assays. We discuss its expression at the transcript level and focus on its potential role in lymphoma, granulosa cell tumors, testicular, and adrenal cancers, emphasizing recent findings and the complexities that necessitate further research., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Embryonic-type neuroectodermal elements are often intimately mixed with primitive endodermal-type glands, like those of yolk sac tumors, in germ cell neoplasia in situ (GCNIS)-derived germ cell tumors of the testis. Because the primitive glands mimic tubules or rosettes of embryonic-type neuroectodermal elements, these embryonic-type neuroectodermal/glandular complexes may be misinterpreted as pure lesions of embryonic-type neuroectodermal elements, which, if of sufficient size, may lead to a diagnosis of embryonic-type neuroectodermal tumor, despite that the criteria of the World Health Organization for a "somatic-type malignancy" are not met. A diagnosis of embryonic-type neuroectodermal tumor in the testis may lead to retroperitoneal lymphadenectomy even in clinical stage I patients, and in postchemotherapy resections indicates a poor prognosis. The distinction of the neuroectodermal and glandular elements is not always straightforward based on morphology alone. We, therefore, studied 34 testis-derived germ cell tumors with embryonic-type neuroectodermal/glandular complexes and 2 purely glandular yolk sac tumors to characterize the immunophenotypes and determine an efficient immunohistochemical panel to aid in this differential. We found that GFAP, synaptophysin, and paired-like homeobox 2B (PHOX2B) expression was specific to embryonic-type neuroectodermal elements, although PHOX2B had poor sensitivity. In contrast, positive reactions with antibodies directed against AFP, villin, and CDX2 were specific for the glandular elements, although CDX2 had poor sensitivity. Other markers, including AE1/AE3 cytokeratin, SALL4, glypican 3, SOX2, SOX11, CD56, INSM1, and neurofilament, proved less helpful because of their nonspecificity and/or poor sensitivity. We conclude that the optimal immunohistochemical panel for distinguishing the components of embryonic-type neuroectodermal/glandular complexes includes stains for synaptophysin, GFAP, villin, and AFP., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Testicular Leydig cell tumours (LCTs) are the most common type of sex cord-stromal tumour in men, representing 1%-3% of all testicular neoplasms. Among testicular sex cord-stromal tumours, CTNNB1 mutations and nuclear expression of β-catenin have been typically associated with Sertoli cell tumour. Recent genomic analyses have shown that CTNNB1 variants are also identified in a subset of LCTs; however, the frequency and clinicopathologic associations of β-catenin alterations remain incompletely understood in this tumour type., Methods: In this study, we evaluated 32 LCTs (five malignant/metastasizing, 27 nonmetastasizing) using β-catenin immunohistochemistry and DNA sequencing., Results: Immunohistochemistry revealed focal or multifocal nuclear β-catenin expression in 47% of the tumours. Diffuse nuclear β-catenin expression (in >50% of the tumour cells) was not detected in any of the cases analysed herein. Comparison of β-catenin-positive and β-catenin-negative cases did not show significant differences in the frequency of adverse histopathologic findings or malignant clinical behaviour. DNA sequencing performed de novo on a subset of seven cases revealed the presence of exon 3 CTNNB1 variants in four of them (4/7, 57%), with variant allele frequencies (VAF) ranging from 7 to 33%. Two additional β-catenin-positive cases that had been sequenced as part of a previous study harboured exon 3 CTNNB1 variants at VAF of 28% and 7%, respectively., Conclusion: These results demonstrate that β-catenin alterations are relatively common in LCT, most likely occurring as subclonal events that are not enriched in cases with aggressive features. Further studies are needed to clarify the oncogenic role of β-catenin in this tumour type., (© 2024 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Background: The transcription factor SALL4 is associated with embryonic pluripotency and has proposed as a novel immunohistochemistry (IHC) marker for diagnosing germ cell tumours. SALL4 comprises three isoforms, and SALL4-A being the full-length isoform. Studying its isoforms could revolutionize testicular cancer prognosis and subtype differentiation., Methods: The expression and clinical significance of isoform 'A' of SALL4 was evaluated in 124 testicular germ cell tumours (TGCTs) subtypes, adjacent 67 normal tissues and 22 benign tumours, using immunohistochemistry on tissue microarrays (TMA)., Results: A statistically significant higher expression of nuclear and cytoplasmic SALL4-A was detected in TGCTs histological subtypes and benign tumours compared to the normal tissues. Seminoma and yolk sac tumours had the highest nuclear and cytoplasmic expression of SALL4-A. A significant correlation was detected between the higher nuclear expression of SALL4-A and increased pT stages ( P  = 0.026) in seminomas. Whereas in embryonal carcinomas, cytoplasmic expression of SALL4-A was associated with the tumour recurrence ( P  = 0.04) and invasion of the epididymis ( P  = 0.011)., Conclusions: SALL4-A isoform expression in the cytoplasm and nucleus of TGCTs may be associated with histological differentiation. In the seminoma subtype of TGCTs, higher expression of SALL4-A may be used as a predictive indicator of poorer outcomes and prognosis.

SummaryWe previously demonstrated that among various histological types of human testicular germinal cell tumors (GCTs), embryonal carcinoma (EC) preferentially expresses low-sulfated keratan sulfate (KS) consisting of repeating N -acetyllactosamine (LacNAc) disaccharide units composed of galactose and 6- O -sulfated N -acetylglucosamine (GlcNAc), which is recognized by the R-10G antibody. Recently, we generated another anti-low-sulfated KS monoclonal antibody, 294-1B1. Immunohistochemical analysis of testicular GCTs ( n =83) revealed that the low-sulfated KS recognized by 294-1B1 is also preferentially expressed in EC but minimally in other GCT histological types. Moreover, immunolabeling with R-10G and 294-1B1 antibodies was resistant to peptide- N -glycosidase F digestion, and EC was not stained with the MECA-79 antibody, indicating that low-sulfated KS expressed in EC contains mucin-type core 2 O -glycans carrying GlcNAc-6- O -sulfated oligo-LacNAc. Double immunofluorescence staining showed that R-10G and 294-1B1 antibody signals colocalized with those for podocalyxin (PODXL). Furthermore, western blot analysis of recombinant human PODXL•IgG fusion proteins secreted from low-sulfated KS-expressing human embryonic kidney 293T cells revealed that PODXL functions as a core protein for low-sulfated KS. Taken together, these findings strongly suggest that the PODXL glycoform decorated with low-sulfated KS is preferentially expressed in human testicular EC and may therefore serve as a diagnostic marker for this malignancy., Competing Interests: Competing InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

This comprehensive review examines the complex interplay between endocrine disrupting chemicals (EDCs) and the development of testicular germ cell tumors (TGCTs). Despite the high cure rates of TGCTs, challenges in diagnosis and treatment remain, necessitating a deeper understanding of the etiology of the disease. Here, we emphasize current knowledge on the role of EDCs as potential risk factors for TGCTs, focusing on pesticides and perfluorinated and polyfluoroalkyl substances (PFAs/PFCs). Evidence suggests that EDCs disrupt endocrine pathways and induce epigenetic changes that contribute to the development of TGCTs. However, the direct link between EDCs and TGCTs remains elusive and requires further investigation of the molecular mechanisms. We also highlighted the importance of studying nuclear receptors as potential targets for understanding TGCT etiology. In addition, recent evidence implicates PFAs/PFCs in TGCT incidence, highlighting the need for further research into their impact on human health. Overall, this review provides valuable insights into the potential role of EDCs in TGCT development and suggests avenues for future research, while also highlighting how understanding their influence may pave the way for novel therapeutic approaches to improve disease management., Competing Interests: Declaration of competing interest The authors declare to have no conflict of interests., (Copyright © 2024. Published by Elsevier Ltd.)

Aberrant male germline development can lead to the formation of seminoma, a testicular germ cell tumor. Seminomas are biologically similar to primordial germ cells (PGCs) and many bear an isochromosome 12p [i(12p)] with two additional copies of the short arm of chromosome 12. By mapping seminoma transcriptomes and open chromatin landscape onto a normal human male germline trajectory, we find that seminoma resembles premigratory/migratory PGCs; however, it exhibits enhanced germline and pluripotency programs and upregulation of genes involved in apoptosis, angiogenesis, and MAPK/ERK pathways. Using pluripotent stem cell-derived PGCs from Pallister-Killian syndrome patients mosaic for i(12p), we model seminoma and identify gene dosage effects that may contribute to transformation. As murine seminoma models do not exist, our analyses provide critical insights into genetic, cellular, and signaling programs driving seminoma transformation, and the in vitro platform developed herein permits evaluation of additional signals required for seminoma tumorigenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Abstract: Although abnormal 68 Ga-PSMA uptake in the prostate and its metastases can be seen in a variety of diseases, it is rare to see in the testis. In these 2 cases, 68 Ga-PSMA PET/CT revealed unilateral 68 Ga-PSMA uptake in the testis of 2 patients. One of these patients was diagnosed with testis metastases from prostate cancer after an orchiectomy. The other patient was diagnosed with an orchitis. 68 Ga-PSMA uptake should be considered as an infection, as well as a malignancy in the initial differential diagnosis., Competing Interests: Conflicts of interest and sources of funding: none declared., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Recently we reported expressional alterations in 219 genes and their transcripts in Leydig cell tumors but nowadays there is still a lack of full basic biochemical characteristics of these tumors. The discovery of potential biochemical markers for tumor management from early detection, treatments, and control of therapy results may markedly supplement genetic data. Leydig cell micronodules were obtained from patients with azoospermia who were qualified for testicular biopsy. The biochemistry of Leydig cell tumors was analyzed using histological staining and spectrophotometric measurements of total proteins, carbohydrates, lipids, and nucleic acids. In addition, the levels of calcium (Ca2 +), copper (Cu2 +), zinc (Zn2 +), and selenium (Se2 +) ions were measured. When compared to healthy testis we revealed, for the first time, that in the interstitial tissue with Leydig cell tumors, great amounts of proteins, carbohydrates, lipids, and acids were dislocated from the seminiferous tubules. Measurements of organic compounds showed a decrease (P < 0.05) only in the Cu2 + content in Leydig cell tumors which may be related to their altered biochemical structure. This specific result may be promising for designing further approaches to manage this tumor based on combining morphological and molecular data., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Purpose: Testicular Germ Cell Tumors (TGCTs) are the most frequent solid malignancies in young adult men. Regardless of differences in their cell of origin, all TGCTs are considered highly curable malignancies. However, approximately 3-5% of all TGCTs do not respond to platinum-based chemotherapies. The purpose of our paper is to investigate whether immunohistochemical expression of MLH1 and REV-7 can be used as predictive tissue markers for TGCTs., Material and Methods: The main demographic and clinicopathological characteristics of 64 male patients with TGCTs who underwent orchiectomy from 2007 to 2022 were retrospectively obtained from two large Oncology Clinics in Greece. Both patients with chemosensitive and chemoresistant disease were included. Immunohistochemical staining for MLH1 and REV-7 proteins was applied in specimens of these patients., Results: 31 seminomas and 33 non-seminomas were included. 48 patients had chemosensitive disease, while 16 had chemoresistant disease. 53 specimens showed preserved MLH1 expression, while 11 specimens had lost MLH1 expression. Expression of MLH1 was only significantly associated with patients' age. 16 specimens showed positive REV-7 expression, while 48 specimens were REV-7 negative. Interestingly, 50% of patients with chemoresistant disease and 16,7% of patients with chemosensitive disease were REV-7 positive. This difference was statistically significant. Moreover, REV-7 positivity was significantly associated with chemoresistance, various clinicopathological parameters and patients' prognosis and survival., Conclusion: Loss of MLH1 expression was only found to be significantly associated with lower patients' age. Positive immunohistochemical REV-7 expression was significantly associated with various clinicopathological parameters, while it was also associated with significantly lower survival and greater hazard. REV-7 positive percentages were significantly higher in patients with chemoresistant disease. Our findings imply that immunohistochemical staining for REV-7 could potentially be used as a predictive tissue marker for TGCT tumors. Moreover, targeting of REV-7 protein, could represent a potential therapeutic strategy for chemoresistant TGCT cases. The implementation of well-designed studies on a larger scale is of utmost importance, in order to draw safer conclusions. Additional studies are needed so as to draw safer conclusions., (© 2024. The Author(s).)

microRNAs (miRNAs) are small endogenous noncoding RNAs, and alterations in their expression may contribute to oncogenesis. Discovering a unique miRNA pattern holds the potential for early detection and novel treatment possibilities in cancer. This study aimed to evaluate miRNA expression in pediatric patients with gonadal germ cell tumors (GCTs), focusing on characterizing the miRNA profiles of each histological subtype and identifying a distinct histological miRNA signature for a total of 42 samples of pediatric gonadal GCTs. The analysis revealed distinct miRNA expression profiles for all histological types, regardless of the primary site. We identified specific miRNA expression signatures for each histological type, including 34 miRNAs for dysgerminomas, 13 for embryonal carcinomas, 25 for yolk sac tumors, and one for immature teratoma, compared to healthy controls. Furthermore, we identified 26 miRNAs that were commonly expressed in malignant tumors, with six miRNAs (miR-302a-3p, miR-302b-3p, miR-371a-5p, miR-372-3p, miR-373-3p, and miR-367-3p) showing significant overexpression. Notably, miR-302b-3p exhibited a significant association with all the evaluated clinical features. Our findings suggest that miRNAs have the potential to aid in the diagnosis, prognosis, and management of patients with malignant GCTs., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Testicular germ cell tumors (TGCTs) exhibit diverse biological and pathological features and are divided in two main types, seminomas and nonseminomatous germ cell tumors (NSGCTs). CD44 is a cell surface receptor, which is highly expressed in malignancies and is implicated in tumorigenesis affecting cell-matrix interactions and cell signaling., Methods and Results: Here, we examined the expression of CD44 in tumor cell lines and in patients' material. We found that CD44 is over-expressed in TGCTs compared to normal tissues. Immunohistochemical staining in 71 tissue specimens demonstrated increased expression of CD44 in some patients, whereas CD44 was absent in normal tissue. In seminomas, a high percentage of tumor and stromal cells showed cytoplasmic and/or cell surface staining for CD44 as well as increased staining for CD44 in the tumor stroma was found in some cases. The increased expression of CD44 either in tumor cells or in stromal components was associated with tumor size, nodal metastasis, vascular/lymphatic invasion, and disease stage only in seminomas. The increased stromal expression of CD44 in TGCTs was positively associated with angiogenesis., Conclusions: CD44 may exhibit diverse biological functions in seminomas and NSGCTs. The expression of CD44 in tumor cells as well as in tumor stroma fosters an aggressive phenotype in seminomas and should be considered in disease treatment., (© 2024. The Author(s).)

Background: Acetaminophen and ibuprofen are widely administered to babies due to their presumed safety as over-the-counter drugs. However, no reports exist on the effects of cyclooxygenase inhibitors on undifferentiated spermatogonia and spermatogonial stem cells. Infancy represents a critical period for spermatogonial stem cell formation and disrupting spermatogonial stem cells or their precursors may be associated with infertility and testicular cancer formation., Objectives: The goal of this study was to examine the molecular and functional impact of cyclooxygenase inhibition and silencing on early steps of undifferentiated spermatogonia (u spg) and spermatogonial stem cell development, to assess the potential reproductive risk of pharmaceutical cyclooxygenase inhibitors., Methods: The effects of cyclooxygenase inhibition were assessed using the mouse C18-4 undifferentiated juvenile spermatogonial cell line model, previously shown to include cells with spermatogonial stem cell features, by measuring prostaglandins, cell proliferation, and differentiation, using cyclooxygenase 1- and cyclooxygenase 2-selective inhibitors NS398, celecoxib, and FR122047, acetaminophen, and ibuprofen. Cyclooxygenase 1 gene silencing was achieved using a stable short-hairpin RNA approach and clone selection, then assessing gene and protein expression in RNA sequencing, quantitative real-time polymerase chain reaction, and immunofluorescence studies., Results: Cyclooxygenase 2 inhibitors NS398 and celecoxib, as well as acetaminophen, but not ibuprofen, dose-dependently decreased retinoic acid-induced expression of the spg differentiation gene Stra8, while NS398 decreased the spg differentiation marker Kit, suggesting that cyclooxygenase 2 is positively associated with spg differentiation. In contrast, short-hairpin RNA-based cyclooxygenase 1 silencing in C18-4 cells altered cellular morphology and upregulated Stra8 and Kit, implying that cyclooxygenase 1 prevented spg differentiation. Furthermore, RNA sequencing analysis of cyclooxygenase 1 knockdown cells indicated the activation of several signaling pathways including the TGFb, Wnt, and Notch pathways, compared to control C18-4 cells. Notch pathway genes were upregulated by selective cyclooxygenase inhibitors, acetaminophen and ibuprofen., Conclusion: We report that cyclooxygenase 1 and 2 differentially regulate undifferentiated spermatogonia/spermatogonial stem cell differentiation. Cyclooxygenases regulate Notch3 expression, with the Notch pathway targeted by PGD2. These data suggest an interaction between the eicosanoid and Notch signaling pathways that may be critical for the development of spermatogonial stem cells and subsequent spermatogenesis, cautioning about using cyclooxygenase inhibitors in infants., (© 2023 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Testicular germ cell tumours (TGCTs) derived from immature (type I) and pluripotent germ cell neoplasia in situ (GCNIS, type II) are characterised by remarkable phenotypic heterogeneity and plasticity. In contrast, the rare spermatocytic tumour (SpT, type III), derived from mature spermatogonia, is considered a homogenous and benign tumour but may occasionally present as an anaplastic or an aggressive sarcomatoid tumour. While various oncogenic processes had been proposed, the precise mechanism driving malignant progression remained elusive until the molecular characterisation of a series of atypical SpTs described in a recent issue of The Journal of Pathology. The emerging picture suggests the presence of two distinct trajectories for SpTs, involving either RAS/mitogen-activated protein kinase pathway mutations or a ploidy shift with secondary TP53 mutations and/or gain of chromosome 12p, the latter known as pathognomonic for type II GCNIS-derived TGCTs. Here, we discuss the implications of these findings, seen from the perspective of germ cell biology and the unique features of different TGCTs. The evolving phenotype of SpTs, induced by genomic and epigenetic changes, illustrates that the concept of plasticity applies to all germ cell tumours, making them inherently heterogenous and capable of significant transformation during progression. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Background: Testicular germ cell tumours (TGCTs) represent a clinical challenge; they are most prevalent in young individuals and are triggered by molecular mechanisms that are not fully understood. The origin of TGCTs can be traced back to primordial germ cells that fail to mature during embryonic development. These cells express high levels of pluripotency factors, including the transcription factor NANOG which is highly expressed in TGCTs. Gain or amplification of the NANOG locus is common in advanced tumours, suggesting a key role for this master regulator of pluripotency in TGCT stemness and malignancy., Methods: In this study, we analysed the expression of microRNAs (miRNAs) that are regulated by NANOG in TGCTs via integrated bioinformatic analyses of data from The Cancer Genome Atlas and NANOG chromatin immunoprecipitation in human embryonic stem cells. Through gain-of-function experiments, MIR9-2 was further investigated as a novel tumour suppressor regulated by NANOG. After transfection with MIR9-2 mimics, TGCT cells were analysed for cell proliferation, invasion, sensitivity to cisplatin, and gene expression signatures by RNA sequencing., Results: For the first time, we identified 86 miRNAs regulated by NANOG in TGCTs. Among these, 37 miRNAs were differentially expressed in NANOG-high tumours, and they clustered TGCTs according to their subtypes. Binding of NANOG within 2 kb upstream of the MIR9-2 locus was associated with a negative regulation. Low expression of MIR9-2 was associated with tumour progression and MIR9-2-5p was found to play a role in the control of tumour stemness. A gain of function of MIR9-2-5p was associated with reduced proliferation, invasion, and sensitivity to cisplatin in both embryonal carcinoma and seminoma tumours. MIR9-2-5p expression in TGCT cells significantly reduced the expression of genes regulating pluripotency and cell division, consistent with its functional effect on reducing cancer stemness., Conclusions: This study provides new molecular insights into the role of NANOG as a key determinant of pluripotency in TGCTs through the regulation of MIR9-2-5p, a novel epigenetic modulator of cancer stemness. Our data also highlight the potential negative feedback mediated by MIR9-2-5p on NANOG expression, which could be exploited as a therapeutic strategy for the treatment of TGCTs., (© 2024. The Author(s).)

In the post-chemotherapy setting, germ cell tumors of the testis (GCTT) that resemble non-specific sarcomas and co-express cytokeratins and glypican-3 (GPC3) are diagnosed as "sarcomatoid yolk sac tumor postpubertal-type (YSTpt)". The diagnosis of sarcomatoid YSTpt is clinically relevant but challenging due to its rarity, non-specific histology, and negative α-fetoprotein (AFP) staining. Recently, FOXA2 has emerged as a key-gene in the reprogramming of GCTT (activating the transcription of several genes, among which GATA3), and immunohistochemical studies showed that GATA3 and FOXA2 have a higher sensitivity for non-sarcomatoid YSTpt than GPC3 and AFP. We found that sarcomatoid YSTpt did not express FOXA2 [0: 14/14 (100%)] and showed focal expression of GATA3 [0: 12/14 (85.7%), 1 + : 2/14 (14.3%)], thus suggesting that these markers are not useful in diagnosing this tumor. Furthermore, we proposed a potential mechanism of sarcomatoid transformation in the post-chemotherapy setting of GCTT, mediated by the downregulation of FOXA2 and GATA3., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Testicular cancer is the most common form of cancer in young men of reproductive age and its incidence is increasing globally. With the currently successful treatment and 95% survival rate, there is a need for deeper understanding of testicular cancer-related infertility. Most patients with testicular cancer experience semen abnormalities prior to cancer therapy. However, the exact mechanism of the effect of testicular cancer on sperm anomalies is not known. Mitochondria are organelles that play a crucial role in both tumorigenesis and spermatogenesis and their malfunction may be an important factor resulting in sperm abnormalities in testicular cancer patients. Within the scope of this review, we will discuss current knowledge of testicular cancer-related alterations in the ATP production pathway, a possible pathophysiological switch from oxidative phosphorylation (OXPHOS) to glycolysis, as well as the role of oxidative stress promoting sperm dysfunction. In this regard, the review provides a summary of the impact of testicular cancer on sperm quality as a possible consequence of impaired mitochondrial function including the energy metabolic pathways that are known to be altered in the sperm of testicular cancer patients., (© 2023 The Authors. European Journal of Clinical Investigation published by John Wiley & Sons Ltd on behalf of Stichting European Society for Clinical Investigation Journal Foundation.)

Serpinb9 is an inhibitor of granzyme B and is potentially involved in the immune escape of tumor cells. In the present study, bioinformatics analysis using open databases suggested that SerpinB9 is overexpressed in testicular embryonal carcinoma. Immunohistological analysis was performed on 28 cases of testicular germ cell tumors to investigate the relationship between SerpinB9 expression in testicular germ cell tumors and the tumor immune environment. SerpinB9 was significantly upregulated in the non-seminoma group and inversely correlated with the number of tumor-infiltrating CD8-positive cells. In addition, yolk sac tumors were characterized by the loss of human leukocyte antigen-class I expression. These findings suggest that SerpinB9 contributes to the immune escape of testicular germ cell tumors. Targeting therapy for SerpinB9 might therefore be useful in immunotherapy for testicular germ cell tumors resistant to immune checkpoint inhibitors., (© 2023. The Author(s) under exclusive licence to The Japanese Society for Clinical Molecular Morphology.)

Background: Seminoma and dysgerminoma are rare testicular and ovarian germ cell tumors characterized by a significant infiltration of immune cells in the tumor microenvironment. According to the failure of conventional treatments in some patients, it is crucial to identify novel prognostic and therapeutic biomarkers for these patients. The objectives of this study were to evaluate the expression of CD45RO and PD-1/PD-L1 and investigate their association with the clinicopathological characteristics of the patients., Methods: Immunohistochemistry was performed to assess the expression of CD45RO, PD-1, and PD-L1 in tumor-infiltrated lymphocytes (TILs), and tumor cells in 33 seminoma and 31 dysgerminoma patients. The expression levels were evaluated using a semiquantitative approach, weighted histoscore, which considers both the intensity and extent of staining., Results: All seminoma and dysgerminoma patients exhibited CD45RO expression in TILs, with 66.7 % and 90.3 % displaying high levels of expression, respectively. PD-1 expression in TILs was observed at low levels in 81.8 % and 77.4 % and at high levels in 18.2 % and 19.4 % of seminoma and dysgerminoma patients, respectively. Likewise, low expression of PD-L1 in tumor cells was detected in 63.6 % of seminoma and 61.3 % of dysgerminoma patients, while none of the patients exhibited high expression of PD-L1. In seminoma patients, a positive correlation was observed between PD-1 expression in TILs and CD45RO expression and between PD-L1 expression in tumor cells and TILs score., Conclusion: The frequent infiltration of CD45RO, along with variable expression of PD-1 and PD-L1 on TILs and tumor cells, could impact the effectiveness of anti-tumor responses and immunotherapy., Competing Interests: Declaration of Competing Interest There was no conflict of interest among the authors., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Spermatocytic tumor (ST) is a rare type of germ cell tumor that occurs exclusively in the postpubertal testis and typically affects elderly men. Most STs are benign, but rare cases exhibit aggressive clinical behavior, often in association with transition to sarcomatoid histology. Limited molecular analyses have been performed on STs; therefore, their genomic and epigenomic features remain incompletely described. Twenty-seven samples from 25 individual patients were analyzed with a combination of DNA sequencing panels, genomic methylation profiling, SNP array, isochromosome (12p) [i(12p)] FISH, and immunohistochemistry. The series included five metastasizing tumors (three with sarcomatoid transformation, one anaplastic, and one conventional) and 20 non-metastasizing tumors (14 anaplastic and six conventional). Anaplastic tumors comprised a monomorphic population of intermediate-sized neoplastic cells, as previously described. Multiomic analyses demonstrated that there were two genomic subgroups of STs: one with diploid genomes and hotspot RAS/RAF variants and the other with global ploidy shift and absence of recurrent mutations. Relative gain of chromosome 9 was a consistent finding in both subgroups. A comparison of metastasizing and non-metastasizing cases demonstrated that aggressive behavior was associated with the acquisition of pathogenic TP53 mutations and/or relative gains of 12p/i(12p). In cases with sarcomatoid transformation, TP53 mutations seem to underlie the transition to sarcomatoid histology. Genomic methylation analysis demonstrated that aggressive cases with gains of 12p cluster closer to pure seminomas than to STs without gains of 12p. In conclusion, STs include two genomic subgroups, characterized by global ploidy shifts without recurrent mutations and diploid genomes with RAS/RAF hotspot mutations, respectively. Biologic progression was associated with relative gains of 12p and TP53 mutations. The findings in STs with relative gains of 12p suggest that they may exhibit biologic characteristics akin to those seen in germ cell neoplasia in situ-related germ cell tumors rather than non-germ cell neoplasia in situ-derived STs. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Seminoma is the most common malignant solid tumor in 14 to 44 year-old men. However, its molecular features and tumor microenvironment (TME) is largely unexplored. Here, we perform a series of studies via genomics profiling (single cell multi-omics and spatial transcriptomics) and functional examination using seminoma samples and a seminoma cell line. We identify key gene expression programs share between seminoma and primordial germ cells, and further characterize the functions of TFAP2C in promoting tumor invasion and migration. We also identify 15 immune cell subtypes in TME, and find that subtypes with exhaustion features were located closer to the tumor region through combined spatial transcriptome analysis. Furthermore, we identify key pathways and genes that may facilitate seminoma disseminating beyond the seminiferous tubules. These findings advance our knowledge of seminoma tumorigenesis and produce a multi-omics atlas of in situ human seminoma microenvironment, which could help discover potential therapy targets for seminoma., (© 2023. The Author(s).)

The myoid gonadal stromal tumor is a very rare testicular spindle cell tumor. Few cases have been reported in the literature from 1977 to 2022. It was an emerging entity in the fourth edition of the WHO Classification of Tumors of the Urinary System and Male Genital Organs and has been instated as a full benign entity in the new edition of 2022. We report herein an additional case of a myoid gonadal stromal tumor. It was a 27-year-old man who presented with an asymptomatic mass in his left testicle for 2 months. The mass was confirmed by ultrasound as a 3 cm solid well-circumscribed lesion for which orchiectomy has been performed. The histological analysis of the specimen and the immunohistochemistry staining was consistent with a myoid gonadal stromal tumor. The characteristics of this tumor are summarized in well-circumscribed, nonencapsulated masses of uniform spindle cells, and immunohistochemically, tumor cells coexpress SMA and S-100 protein. The benign behavior of this tumor is demonstrated by our results and those presented in the previous cases reported in the literature., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Glypican-3 (GPC3) is highly expressed in testicular yolk sac tumor (TYST). GPC3 has been evaluated as a cancer vaccine for some types of tumors, but little is known on the effects of GPC3 peptide-based therapy on TYST. Here, we evaluated the antitumor effect of GPC3 144-152 on TYST and its potential mechanisms., Methods: GPC3 144-152 -specific CD8 + T cells were induced by vaccine immunization and examined by ELISPOT. The CD8 + T cells were purified for testing their cytotoxicity in vitro against TYST cells by CCK-8 and TUNEL assays and in vivo against tumor growth. The influence of GPC3 144-152 loading and/or cGAS silencing on the tumor growth, apoptosis and cGAS/STING signaling was tested by immunohistochemistry, immunofluorescence, flow cytometry, and Western blot., Results: Vaccination with GPC3 144-152 induced tumor-specific CD8 + T cells that secreted high levels of IFN-γ and granzyme B, and had potent cytotoxicity against TYST in a dose-dependent manner. Adoptive transfer of CD8 + T cells and treatment with GPC3 144-152 significantly inhibited the growth of TYST tumors, but less effective for cGAS-silenced TYST tumors in vivo. Treatment with GPC3 144-152 enhanced the infiltration of CD8 + T cells into the tumor environment and their cytotoxicity against TYST tumors in vivo by up-regulating granzyme B and IFN-β expression, but down-regulating GPC3 expression in the tumors. Co-culture of CD8 + T cells with TYST in the presence of exogenous GPC3 144-152 enhanced peptide-specific CD8 + T-cell cytotoxicity in vitro, accompanied by enhancing cGAS, γH2AX, TBK1, and IRF3 phosphorylation in TYST cells, but less effective in cGAS-silenced TYST cells., Conclusions: These data indicated that GPC3 peptide-specific CD8 + T cells had potent antitumor activity against TYST tumor, particularly for combined treatment with the peptide, which was partially dependent on the intratumoral cGAS/STNG signaling. GPC3 peptide vaccine may be valuable for the combination treatment of TYST., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The transforming growth factor β (TGFβ) superfamily, consisting of protein ligands, receptors, and intracellular SMAD transducers, regulates fundamental biological processes and cancer development. Our previous study has shown that sustained activation of TGFβ receptor 1 (TGFBR1) driven by anti-Mullerian hormone receptor type 2 ( Amhr2 )-Cre in the mouse testis induces the formation of testicular granulosa cell tumors (TGCTs). As Amhr2 -Cre is expressed in both Sertoli cells and Leydig cells, it remains unclear whether the activation of TGFBR1 in Sertoli cells alone is sufficient to induce TGCT formation. Therefore, the objective of this study was to determine whether Sertoli cell-activation of TGFBR1 drives oncogenesis in the testis. Our hypothesis was that overactivation of TGFBR1 in Sertoli cells would promote their transdifferentiation into granulosa-like cells and the formation of TGCTs. To test this hypothesis, we generated mice harboring constitutive activation of TGFBR1 in Sertoli cells using anti-Mullerian hormone ( Amh )-Cre. Disorganized seminiferous tubules and tumor nodules were found in TGFBR1 CA ; Amh -Cre mice. A histological analysis showed that Sertoli cell-specific activation of TGFBR1 led to the development of neoplasms resembling granulosa cell tumors, which derailed spermatogenesis. Moreover, TGCTs expressed granulosa cell markers including FOXL2, FOXO1, and INHA. Using a dual fluorescence reporter line, the membrane-targeted tdTomato (mT)/membrane-targeted EGFP (mG) mouse, we provided evidence that Sertoli cells transdifferentiated toward a granulosa cell fate during tumorigenesis. Thus, our findings indicate that Sertoli cell-specific activation of TGFBR1 leads to the formation of TGCTs, supporting a key contribution of Sertoli cell reprogramming to the development of this testicular malignancy in our model.

Testicular adrenal rest tumors (TARTs), commonly occurring in males with congenital adrenal hyperplasia, may arise from chronic stimulation of adrenocorticotropic hormone (ACTH)-sensitive cells in the testes. It is not yet established whether the human fetal testis (HFT) is responsive to ACTH. To investigate this, we cultured HFT tissue with and without ACTH for up to 5 days, and quantified adrenal steroid hormones and expression of adrenal steroidogenic enzymes. Fetal testis and adrenal tissue produced high levels of testosterone and cortisol, respectively, indicating viability. In contrast to fetal adrenal tissues, the expression of ACTH receptor MC2R was either absent or expressed at extremely low levels in ex vivo HFT tissue and no clear response to ACTH in gene expression or steroid hormone production was observed. Altogether, this study suggests that the HFT is unresponsive to ACTH, which would indicate that a TART does not arise from fetal testicular cells chronically exposed to ACTH in utero., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Maturation arrest (MA) is a subtype of non-obstructive azoospermia, and male infertility is a known risk factor for testicular tumors. However, the genetic basis for many affected individuals remains unknown. Here, we identified a deleterious hemizygous variant of X-linked retinoblastoma-binding protein 7 (RBBP7) as a potential key cause of MA, which was also found to be associated with the development of Leydig cell tumors. This mutation resulted in premature protein translation termination, affecting the sixth WD40 domain of the RBBP7 and the interaction of the mutated RBBP7 with histone H4. Decreased BRCA1 and increased γH2AX were observed in the proband. In mouse spermatogonial and pachytene spermatocyte-derived cells, deprivation of rbbp7 led to cell cycle arrest and apoptosis. In Drosophila, knockdown of RBBP7/Caf1-55 in germ cells resulted in complete absence of germ cells and reduced testis size, whereas knockdown of RBBP7/Caf1-55 in cyst cells resulted in hyperproliferative testicular cells. Interestingly, male infertility caused by Caf1-55 deficiency was rescued by ectopic expression of wild-type human RBBP7 but not mutant variants, suggesting the importance of RBBP7 in spermatogenesis. Our study provides insights into the mechanisms underlying the co-occurrence of MA and testicular tumors and may pave the way for innovative genetic diagnostics of these 2 diseases.

Autophagy is a lysosomal degradation process known as autophagic flux, involving the engulfment of damaged proteins and organelles by double-membrane autophagosomes. It comprises microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. Macroautophagy consists of three stages: induction, autophagosome formation, and autolysosome formation. Atg8-family proteins are valuable for tracking autophagic structures and have been widely utilized for monitoring autophagy. The conversion of LC3 to its lipidated form, LC3-II, served as an indicator of autophagy. Autophagy is implicated in human pathophysiology, such as neurodegeneration, cancer, and immune disorders. Moreover, autophagy impacts urological diseases, such as interstitial cystitis /bladder pain syndrome (IC/BPS), ketamine-induced ulcerative cystitis (KIC), chemotherapy-induced cystitis (CIC), radiation cystitis (RC), erectile dysfunction (ED), bladder outlet obstruction (BOO), prostate cancer, bladder cancer, renal cancer, testicular cancer, and penile cancer. Autophagy plays a dual role in the management of urologic diseases, and the identification of potential biomarkers associated with autophagy is a crucial step towards a deeper understanding of its role in these diseases. Methods for monitoring autophagy include TEM, Western blot, immunofluorescence, flow cytometry, and genetic tools. Autophagosome and autolysosome structures are discerned via TEM. Western blot, immunofluorescence, northern blot, and RT-PCR assess protein/mRNA levels. Luciferase assay tracks flux; GFP-LC3 transgenic mice aid study. Knockdown methods (miRNA and RNAi) offer insights. This article extensively examines autophagy's molecular mechanism, pharmacological regulation, and therapeutic application involvement in urological diseases.

Thus far, several monoclonal antibodies directed against cell-surface carbohydrate antigens have been generated. Among them, R-10G reportedly reacts selectively with human embryonic stem and induced pluripotent stem cells, but not with embryonal carcinoma (EC) cells. However, EC cells derived from patients' EC tumors may exhibit varying levels of R-10G-reactive antigen expression. Thus, we asked whether human EC tissues or germ cell tumor (GCT) tissues other than EC express R-10G-reactive antigen. To do so, we quantitatively analyzed R-10G-reactive antigen expression in 83 testicular GCT surgical specimens containing a total of 125 various GCT components. Accordingly, in all EC components examined, the EC cell plasma membrane was immunolabeled with R-10G, while most seminoma components were R-10G-negative. In non-seminomatous GCT (NSGCT) other than EC (non-EC NSGCT), R-10G-reactive antigen expression was variable, but signal distribution was focal, and the average intensity was weaker than that seen in EC. The percentages of R-10G-positive cells in these three groups varied with high statistical significance ( p <0.001 for all combinations). These findings indicate that the R-10G-reactive antigen is preferentially expressed in human testicular EC tissues and, thus, could be used as a diagnostic marker for this malignancy.

Cryptorchidism, the failed descent of one or both testes into the scrotum, is a common developmental disorder in male dogs. Cryptorchidism may affect canine fertility, reducing the quality of the semen, and may promote spermatic cord torsion and onset of neoplasia. MicroRNAs (miRNAs) are epigenetic regulators of gene expression and their dysregulation is associated with disorders of spermatogenesis and testis neoplasia. The present study aimed at investigating the expression of miRNAs in formalin-fixed, paraffin-embedded (FFPE) canine retained testes and testes affected by seminoma, and at integrating miRNAs to their target genes. Forty testicular FFPE specimens from 30 dogs were included - 10 scrotal and 10 contralateral retained from 10 unilateral cryptorchid dogs; 10 tumoral testes affected by seminoma from non-cryptorchid dogs; 10 scrotal normal testes from non-cryptorchid dogs included as the control. The expression level of three miRNAs, namely miR-302c-3p, miR-302a-3p, and miR-371-3p, associated with testicular disorders, were quantified using RT-qPCR. The comparative analysis demonstrated that the level of miR-302a-3p and miR-371a-3p were quantifiable exclusively in control testes. The expression level of miR-302c-3p was higher in the control than in the other groups; its expression decreased in retained testes compared to scrotal testes and testes with seminoma. Gene Ontology analysis pointed out that these miRNAs may be involved in the modulation of estrogen and thyroid hormone signaling pathways. In conclusion, this study demonstrated that miRNAs are dysregulated in canine cryptorchid and seminoma-affected testes compared to control tissues, confirming the pivotal role of miRNAs in cryptorchidism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cryptorchidism is a congenital abnormality resulting in increased rates of infertility and testicular cancer. We used cryptorchidism model mice that presented with the translocation of the left testis from the scrotum to the abdominal cavity. Mice underwent the surgical procedure of the left testis at day 0 and were sacrificed at days 3, 5, 7, 14, 21, and 28 post-operatively. The weight of the left cryptorchid testis decreased significantly at days 21 and 28. The morphological changes were observed after 5 days and showed detached spermatogenic cells and abnormal formation of acrosome at day 5, multinucleated giant cells at day 7, and atrophy of seminiferous tubules at days 21 and 28. The high abdominal temperature disrupted the normal expression of cell adhesion molecule-1, Nectin-2, and Nectin-3 which are essential for spermatogenesis. In addition, the pattern and alignment of acetylated tubulin in cryptorchid testes were also changed at days 5, 7, 14, 21, and 28. Ultrastructure of cryptorchid testes revealed giant cells that had been formed by spermatogonia, spermatocytes, and round and elongating spermatids. The study's findings reveal that cryptorchidism's duration is linked to abnormal changes in the testis, impacting protein marker expression in spermatogenic and Sertoli cells. These changes stem from the induction of high abdominal temperature.

Large cell calcifying Sertoli cell tumour (LCCSCT) is a type of testicular sex cord-stromal tumour that may occur sporadically or in the context of Carney complex and other genetic syndromes. A subset is clinically malignant, and the molecular mechanisms that drive such aggressive behaviour remain unknown. METHODS AND RESULTS: We analysed 21 samples from 20 patients with LCCSCT (12 non-metastasising and eight metastasising) using PRKAR1A immunohistochemistry (IHC) and next-generation sequencing. All tumours except two (cases 17 and 20, both metastasising) demonstrated loss of PRKAR1A expression. Among 11 cases with interpretable sequencing results, all harboured pathogenic single nucleotide variants of PRKAR1A. Evidence of loss of heterozygosity (LOH) of PRKAR1A was present in all tumours with interpretable zygosity data, but the mechanisms of LOH were different for non-metastasising and metastasising tumours. Non-metastasising tumours demonstrated only copy-neutral LOH, while metastasising tumours demonstrated a spectrum of mechanisms of LOH, including copy-loss LOH, two concurrent mutations or copy-neutral LOH. Relevant molecular findings in non-metastasising LCCSCT were limited to PRKAR1A variants. In contrast, all metastasising LCCSCTs with interpretable data harboured additional pathogenic variants, including (but not restricted to) BRCA2 mutations with evidence of LOH and bi-allelic CDKN2A/B deletions. Three patients harboured PRKAR1A variants of inferred germline origin, including one with Carney complex and two without known syndromic features. CONCLUSIONS: This study further confirms that PRKAR1A IHC is a useful diagnostic tool for both non-metastasising and metastasising tumours and suggests that molecular analyses can be helpful to identify non-metastasising tumours with malignant potential in selected patients. Importantly, these results highlight that germline assessment could be beneficial for all patients presenting with LCCSCT., (© 2023 John Wiley & Sons Ltd.)

Background: It has been estimated that microorganisms are involved in the pathogenesis of approximately 20% of all cancers. Testicular germ cell tumours (TGCTs) are the most common type of malignancy in young men and arise from the precursor cell, germ cell neoplasia in situ (GCNIS). The microbiome of seminal plasma and testicular tissue has not been thoroughly investigated in regard to TGCTs., Objectives: To investigate the differences in the seminal plasma microbiome between men with TGCT or GCNIS-only compared with controls., Materials and Methods: The study population consisted of patients with GCNIS-only (n = 5), TGCT (n = 18), and controls (n = 25) with different levels of sperm counts in the ejaculate. RNA was isolated from the seminal plasma and sequenced. Reads not mapping to the human genome were aligned against a set of 2784 bacterial/archaeal and 4336 viral genomes using the Kraken pipeline., Results: We identified reads from 2172 species and most counts were from Alteromonas mediterranea, Falconid herpesvirus 1, and Stigmatella aurantiaca. Six species (Acaryochloris marina, Halovirus HGTV-1, Thermaerobacter marianensis, Thioalkalivibrio sp. K90mix, Burkholderia sp. YI23, and Desulfurivibrio alkaliphilus) were found in significantly (q-value < 0.05) higher levels in the seminal plasma of TGCT and GCNIS-only patients compared with controls. In contrast, Streptomyces phage VWB was found at significantly higher levels among controls compared with TGCT and GCNIS-only patients combined., Discussion: Often the microbiome is analysed by shotgun or 16S ribosomal sequencing whereas our present data build on small RNA sequencing. This allowed us to identify more viruses and phages compared with previous studies but also makes the results difficult to directly compare., Conclusion: Our study is the first to report identification of the microbiome species in seminal plasma of men with TGCT and GCNIS-only, which potentially could be involved in the pathogenesis of TGCTs. Further studies are, however, needed to confirm our findings., (© 2022 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Testicular teratomas are the major histologic type of testicular germ cell tumors and their incidence continues to grow. Moreover, teratomas can develop from undifferentiated cells in induced pluripotent stem (iPS) cell transplantation therapy, seriously hampering the progress of regenerative medicine. Germinal center-associated nuclear protein (GANP) is thought to be important to the biogenetic control of primordial germ cells and is among the genes susceptible to testicular germ cell tumors. Thus, we analyzed the expression of GANP in human testicular postpubertal-type teratomas and established a novel mouse model to reveal the association between GANP and teratomagenesis. We analyzed 31 cases of human testicular postpubertal-type teratomas and, in all cases, GANP was overexpressed. The aberrant expression was also detected in germ cell neoplasia in situ accompanied by the teratoma. GANP expression was particularly high in the epithelia of the epidermis, cutaneous appendages, and trachea-like ciliated epithelium. To further clarify the association between GANP and teratomagenesis, we established a novel teratomagenesis mouse model (CAG-ganp Tg mice). In the GANP-teratoma mice, GANP-overexpressing teratomas were more frequent at the testes and the middle portion of the uterus than has been seen in the previously established mouse models. In conclusion, GANP is overexpressed in testicular postpubertal-type teratomas and is an essential teratomagenic factor. We also found that CAG-ganp Tg mice are useful mouse models of teratomagenesis that mimics human midline teratomas and that teratomas may originate from the overexpression of GANP in primordial germ cells., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-β, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-β, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-β superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-β signalling pathway outcomes in TGCTs.

Environmental cyanotoxin exposure may be a trigger of testicular cancer. Activation of PI3K/AKT/mTOR signaling pathway is the critical molecular event in testicular carcinogenesis. As a widespread cyanotoxin, microcystin-leucine arginine (MC-LR) is known to induce cell malignant transformation and tumorigenesis. However, the effects of MC-LR on the regulatory mechanism of PI3K/AKT/mTOR pathway in seminoma, the most common testicular tumor, are unknown. In this study, mouse spermatogonia cell line (GC-1) and nude mice were used to investigate the effects and mechanisms of MC-LR on the malignant transformation of spermatogonia by nude mouse tumorigenesis assay, cell migration invasion assay, western blot, and cell cycle assay, and so forth. The results showed that, after continuous exposure to environmentally relevant concentrations of MC-LR (20 nM) for 35 generations, the proliferation, migration, and invasion abilities of GC-1 cells were increased by 120%, 340%, and 370%, respectively. In nude mice, MC-LR-treated GC-1 cells formed tumors with significantly greater volume (0.998 ± 0.768 cm 3 ) and weight (0.637 ± 0.406 g) than the control group (0.067 ± 0.039 cm 3 ; 0.094 ± 0.087 g) (P < .05). Furthermore, PI3K inhibitor Wortmannin inhibited the PI3K/AKT/mTOR pathway and its downstream proteins (c-MYC, CDK4, CCND1, and MMP14) activated by MC-LR. Blocking PI3K alleviated MC-LR-induced cell cycle disorder and malignant proliferation, migration and invasive of GC-1 cells. Altogether, our findings suggest that MC-LR can induce malignant transformation of mouse spermatogonia, and the PI3K/AKT/mTOR pathway-mediated cell cycle dysregulation may be an important target for malignant proliferation. This study provides clues to further reveal the etiology and pathogenesis of seminoma., (© 2022 Wiley Periodicals LLC.)

Seminoma is the most common type of testicular germ cell tumour and is highly sensitive to cisplatin therapy, which has not been fully elucidated. In this study, we comprehensively monitored dynamic changes of TCam-2 cells after cisplatin treatment. At an early stage, we found that both low and high concentrations of cisplatin induced the S-phase arrest in TCam-2 cells. By contrast, the G0G1 arrest was caused by cisplatin in teratoma NTERA-2 cells. Afterwards, high concentrations of cisplatin promoted the extrinsic apoptosis and high expressions of related genes (Fas/FasL-caspase-8/-3) in TCam-2 cells. However, when decreasing the cisplatin, the apoptotic cells were significantly reduced, and accompanied by cells showing senescence-like morphology, positive SA-β-gal staining and up-regulation of senescence-related genes (p53, p21 and p16). Furthermore, the cell cycle analysis revealed that most of the senescent TCam-2 cells were irreversibly arrested in the G2M phase. G2M arrest was also observed in NTERA-2 cells treated with a low concentration of cisplatin, while no senescence-related phenotype was discovered. In addition, we detected the γ-H2AX, a DNA damage marker protein, and reactive oxygen species (ROS) and found both of which were elevated with the increase of cisplatin in TCam-2 cells. In conclusion, the extrinsic apoptosis and senescence are involved in the growth kinetics of TCam-2 cells to cisplatin, which provide some implications for studies on cisplatin and seminoma., (© 2022 John Wiley & Sons Australia, Ltd.)

Background: Testis-specific genes encoding for long non-coding RNA (lncRNA) have been detected in several cancers; many produce proteins with restricted or aberrant expression patterns in normal or cancer tissues., Objective: To characterize new lncRNA involved in normal and/or pathological differentiation of testicular cells., Methods: Using bioinformatics analysis, we found that lncRNA LOC100130460 (CAND1.11) is expressed in normal and tumor testis; its expression was assessed in several human cell lines by qRT-PCR. CAND1.11 protein, produced by a single nucleotide mutation, was studied by western blot and immunofluorescence analysis on normal, classic seminoma, and Leydig cell tumor testicular tissues., Results: CAND1.11 gene is primate-specific; its expression was low in SH-SY5Y cells and increased when differentiated with retinoic acid treatment. CAND1.11 expression in PC3 cells was higher than in PNT2 cells. CAND1.11 protein is present in the human testis and overexpressed in testicular cancer tissues., Conclusions: This report is one of the few providing evidence that a lncRNA produces a protein expressed in normal human tissues and overexpressed in several testicular cancers, suggesting its involvement in regulating cell proliferation and differentiation. Although further studies are needed to validate the results, our data indicate that CAND1.11 could be a potential new prognostic biomarker to use in proliferation and cancer.

Recent reports indicate an increase in Leydig cell tumor (LCT) incidence. Radical orchiectomy is the standard therapy in children and adults, although it entails physical and psychosocial side effects. Testis-sparing surgery can be a consideration for benign LCT of 2.5 cm or less in size. Malignant LCTs respond poorly to conventional chemotherapy, so new treatment modalities are needed. In this study, we observed increased histidine decarboxylase expression and pro-angiogenic potential in LCT surgically resected from pediatric patients (fetal to pubertal) vs control samples from patients without endocrine or metabolic disorders which were collected at necropsy. We, therefore, evaluated for the first time the antitumor efficacy of two histidine decarboxylase inhibitors (α-methyl-dl-histidine dihydrochloride (α-MHD) and epigallocatechin gallate (EGCG)), alone and combined with carboplatin, in two preclinical models of LCT. MA-10 and R2C Leydig tumor cells, representing two different LCT subtypes, were used to generate syngeneic and xenograft mouse LCT models, respectively. In the syngeneic model, monotherapy with α-MHD effectively reduced tumor growth and angiogenesis. In the xenografts, which showed co-expression of histidine decarboxylase and CYP19, the combination of EGCG plus carboplatin was the most effective therapy, leading to LCT growth arrest and undetectable levels of plasmatic estradiol. Testicular and body weights remained unaltered. On the basis of this study, histidine decarboxylase may emerge as a novel pharmacological target for LCT treatment.

Sertoli cells play critical roles in regulating spermatogenesis and testis development by providing structural and nutritional support. This study aimed to develop a standard protocol for canine Sertoli cell isolation and culture; and characterize its biological features, functionality, and application of compound toxicity testing. Canine testicles were received from the neuter clinic, and three-step of enzymatic digestion was applied to isolate Sertoli cells. We characterized the growth and purity of Sertoli cells with the expression of SOX9, GATA4, and Clusterin. In addition, we selected cadmium as a model toxicant to evaluate the toxic responses in the newly established Sertoli cells using High-content Analysis (HCA). With our optimized protocol, the purity of isolated Sertoli cells was above 95%, as determined with Sertoli cell-specific protein markers of SOX9 and GATA4. More importantly, primary Sertoli cell populations could be expanded rapidly in vitro, passaged (up to seven), and cryopreserved. The HCA-based assay revealed that cadmium at 1 μM induced both disruptions of cytoskeletal and DNA damage responses. Furthermore, we established an HCA assay with the newly isolated and optimized culture of canine Sertoli cells to evaluate the epigenetic markers of histone modification. We found cadmium-induced differential changes in histone modifications H3Me3K9, H3Me3K36, H4Me3K20, and H4acK5. In summary, we have established the standardized protocol to produce canine Sertoli cells with Sertoli cell-specific phenotype. The isolation and expansion of large quantities of canine Sertoli cells will provide broad applications in studying male infertility, reproductive toxicology, testicular cancer, and cell therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Some pediatric patients with cryptorchidism preserve cells with gonocyte characteristics beyond their differentiation period, which could support the theory of the gonocyte as a target for malignancy in the development of testicular neoplasia. One of the key molecules in gonocyte malignancy is represented by microRNAs (miRNAs). The goal of this review is to give an overview of miRNAs, a class of small non-coding RNAs that participate in the regulation of gene expression. We also aim to review the crucial role of several miRNAs that have been further described in the regulation of gonocyte differentiation to spermatogonia, which, when transformed, could give rise to germ cell neoplasia in situ, a precursor lesion to testicular germ cell tumors. Finally, the potential use of miRNAs as diagnostic and prognostic biomarkers in testicular neoplasia is addressed, due to their specificity and sensitivity compared to conventional markers, as well as their applications in therapeutics.

Cadmium and lead are widespread, nonbiodegradable heavy metals of perpetual environmental concerns. The present study aimed to evaluate whether sub-chronic exposure to cadmium chloride (CdCl 2 ) and lead acetate [Pb(CH 3 COO) 2 ] induces reproductive toxicity and development of testicular germ cell neoplasia in situ (GCNIS) in swiss albino mice. The effects of resveratrol to reverse the metal-induced toxicity were also analyzed. The mice were randomly divided into four groups for metal treatments and two groups received two different doses of each metal, CdCl 2 (0.25 and 0.5 mg/kg) and Pb(CH 3 COO) 2 (3 and 6 mg/kg). The fourth group received oral doses of 20 mg/kg resveratrol in combination with 0.5 mg/kg CdCl 2 or 6 mg/kg Pb(CH 3 COO) 2 for 16 weeks. Toxic effects of both metals were estimated qualitatively and quantitatively by the alterations in sperm parameters, oxidative stress markers, testicular histology, and protein expressions of the treated mice. Pronounced perturbation of sperm parameters, cellular redox balance were observed with severe distortion of testicular histo-architecture in metal exposed mice. Significant overexpression of Akt cascade and testicular GCNIS marker proteins were recorded in tissues treated with CdCl 2 . Notable improvements were observed in all the evaluated parameters of resveratrol cotreated mice groups. Taken together, the findings of this study showed that long-term exposure to Cd and Pb compounds, induced acute reproductive toxicity and initiation of GCNIS development in mice. Conversely, resveratrol consumption abrogated metal-induced perturbation of spermatogenesis, testicular morphology, and the upregulation of Akt cascade proteins along with GCNIS markers, which could have induced the development of testicular cancer., (© 2022 Wiley Periodicals LLC.)

Male reproductive development starts early in the embryogenesis with somatic and germ cell differentiation in the testis. The LIN28 family of RNA-binding proteins promoting pluripotency has two members-LIN28A and LIN28B. Their function in the testis has been investigated but many questions about their exact role based on the expression patterns remain unclear. LIN28 expression is detected in the gonocytes and the migrating, mitotically active germ cells of the fetal testis. Postnatal expression of LIN28 A and B showed differential expression, with LIN28A expressed in the undifferentiated spermatogonia and LIN28B in the elongating spermatids and Leydig cells. LIN28 interferes with many signaling pathways, leading to cell proliferation, and it is involved in important testicular physiological processes, such as cell renewal, maturation, fertility, and aging. In addition, aberrant LIN28 expression is associated with testicular cancer and testicular disorders, such as hypogonadotropic hypogonadism and Klinefelter's syndrome. This comprehensive review encompasses current knowledge of the function of LIN28 paralogs in testis and other tissues and cells because many studies suggest LIN28AB as a promising target for developing novel therapeutic agents.

Background: Incidence of type II germ cell tumors (T2GCT) has increased in young men possibly due to fetal/perinatal exposure to estrogenic compounds. Three-fold increased incidence of T2GCT was reported in men exposed in utero to diethylstilbestrol (DES). T2GCT is a development-related disease arising due to blocked differentiation of gonocytes into spermatogonia in fetal testes which survive as germ cell neoplasia in situ (GCNIS) and initiate T2GCT. In our earlier study, T2GCT-like features were observed in 9 out of 10 adult, 100-day-old mice testes upon neonatal exposure to DES (2 μg/pup/day on days 1-5). Neonatal DES exposure affected testicular very small embryonic-like stem cells (VSELs) and spermatogonial stem cells and resulted in infertility, reduced sperm counts and tumor-like changes leading to our postulate that testicular dysgenesis syndrome possibly has a stem cell basis. The present study was undertaken to further characterize testicular tumor in mice testes., Methods: DES-exposed mice pups (n = 70) were studied on D100 and after 12 months to understand how T2GCT progresses. Besides histological studies, a carefully selected panel of markers were studied by immuno-fluorescence and qRT-PCR., Results: DES resulted in either atrophied or highly vascularized, big-sized testes and extra-testicular growth was also observed. GCNIS-like cells with big, vacuolated cytoplasm and increased expression of OCT-4, SSEA-1, SCA-1 and CD166 (cancer stem cells marker) along with reduced c-KIT, MVH and PTEN were evident. Global hypomethylation was found associated with altered expression of Dnmts, Igf2-H19 and Dlk-Meg3 imprinted genes along with reduced expression of Ezh2, cell cycle regulator p57KIP2 and Meg3; however, Pten remained unaltered. Increased expression of PCNA and Ki67 was observed in concert with complete lack of SOX-9 suggesting Sertoli cells independent proliferation., Conclusions: Mouse model for T2GCT is described which will have immense potential to understand cancer initiation, cancer stem cells and also to develop effective therapies in future. T2GCT initiates from tissue-resident, pluripotent VSELs due to their altered epigenome. Neonatal exposure to DES blocks differentiation (spermatogenesis) and VSELs get transformed into CD166 positive cancer stem cells that undergo excessive self-renewal and initiate cancer in adult life challenging existing concept of fetal origin of T2GCT., (© 2022. The Author(s).)

The accurate identification of different components in testicular germ cell tumors (GCT) is essential for tailoring treatment and informing the clinical prognosis. PRAME (preferentially expressed antigen in melanoma), a member in the family of cancer testis antigens, plays critical roles in regulating pluripotency and suppressing somatic/germ cell differentiation in seminomas (SEM). To investigate the potential diagnostic value of PRAME in testicular GCT, here we comparatively examined the expression patterns of PRAME and SOX17 by immunohistochemistry in both pure and mixed GCT. Tissue microarrays constructed from 66 pure or mixed GCT were examined, including 25 seminomas (13 pure and 12 mixed), 35 embryonal carcinomas (EC; 7 pure and 28 mixed), 23 teratomas (TER; 10 pure and 13 mixed), 15 yolk sac tumors (YST; 1 pure and 14 mixed), and 5 choriocarcinomas (CC; 1 pure and 4 mixed), with 11 germ cell neoplasia in situ (GCNIS) and 6 normal testicular tissue as controls. The expression levels of PRAME or SOX17 were evaluated by a scoring system counting for intensity and extent of staining. PRAME nuclear expression was present in 92% (23/25) of SEM, including all 13 pure SEM, and 10 out of 12 seminomatous component of mixed GCT. In contrast, all EC and TER were completely negative for PRAME, and focal expression was demonstrated in 33.3% of YST and 20% of CC. As for SOX17, 96% of SEM and 73% of YST stained positively, whereas EC and CC were negative. Focal nuclear positivity was identified in the epithelial cell component of 17.4% (4/23) of TER. We found the sensitivity of PRAME to detect SEM to be comparable to SOX17, although SOX17 staining is more diffuse and stronger in the majority of cases. The specificity of PRAME for SEM appeared to be superior to that of SOX17 (92% versus 81%). In conclusion, PRAME is preferentially expressed in SEM or within the seminomatous component of mixed GCT with only focal variable expression in YST and CC, but shows no expression in EC and TER. These findings suggest that PRAME can be explored as a diagnostic marker for SEM., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The frequencies of adverse outcomes associated with male reproductive health, including infertility and testicular cancer, are increasing. These adverse trends are partially attributed to increased exposure to environmental agents such as endocrine-disrupting chemicals (EDCs). This study addresses effects on EDCs on adjacent prepubertal Sertoli TM4 cells, specifically on 1) testicular gap junctional intercellular communication (GJIC), one of the hallmarks of non-genotoxic carcinogenicity, 2) GJIC building blocks connexins (Cx), and 3) mitogen-activated protein kinases MAPKs. We selected eight representatives of EDCs: organochlorine chemicals such as pesticides dichlorodiphenyltrichloroethane, lindane, methoxychlor, and vinclozolin, industrial chemicals bisphenol A and 2,2',4,4',5,5'-hexachlorobiphenyl, and components of personal care products, triclocarban and triclosan. EDCs rapidly dysregulated GJIC in Sertoli TM4 cells mainly via MAPK p38 and/or Erk1/2 pathways by the intermediate hyper- or de-phosphorylation of Cx43 (Ser368, Ser282) and translocation of Cx43 from the plasma membrane, suggesting disturbed intracellular trafficking of Cx43 protein. Surprisingly, EDCs did not rapidly activate MAPK Erk1/2 or p38; on the contrary, TCC and TCS decreased their activity (phosphorylation). Our results indicate that EDCs might disrupt testicular homeostasis and development via testicular GJIC, junctional and non-junctional functions of Cx43 and MAPK-signaling pathways in Sertoli cells., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Testicular seminoma is a relatively rare tumor which is mostly detected in male population aged from 15 to 35 years old. Although several molecular biomarkers have been identified to be associated with testicular seminoma pathogenesis, the exact mechanism for testicular seminoma progression remains largely unknown. CDKN2A interacting protein (CDKN2AIP) has previously been identified as a tumor suppressor in multiple malignant diseases. In this study, we aimed to further explore its role in testicular seminoma as well as the underlying molecular mechanisms. Retrospective testicular seminoma clinical samples, normal tissues, NTERA-2 cell line, and mouse xenograft models were used in this study. RT-qPCR, western blot analysis, immunofluorescence microscopy, Co-IP and IP-MS experiments were performed to detect the expression of CDKN2AIP and its interaction with CARM1 and eIF4β. SA-β-gal staining assay and H3K9me3 activity experiments were used to subsequently evaluate the cell senescence and apoptosis. Mouse xenograft animal model was used for in vivo study. The results showed that CDKN2AIP is highly expressed in normal testis samples, and is significantly suppressed in testicular seminoma clinical samples and cell line model. Up-regulation of CDKN2AIP is significantly associated with the inhibition of testicular seminoma tumor growth and the increase of cell senescence and apoptosis. CDKN2AIP exhibits anti-tumor activity by interacting with CARM1 and eIF4β. CDKN2AIP induces testicular seminoma cell senescence by suppressing CARM1 expression and eIF4β phosphorylation. The CDKN2AIP-CARM1 and CDKN2AIP-eIF4β interactions, which induce tumor cell senescence and apoptosis, may be the potential druggable molecular pathways in testicular seminoma tumor pathogenesis and progression.