We studied a unique case of osteosarcoma of the testis. The tumor either originated from indifferent mesenchymal cells, or it represents a one-sided monomorphic development of a teratoma.

The identification of cancer testis (CT) antigens has been an important advance in determining potential targets for cancer immunotherapy. Multiple previous studies have shown that CT antigen vaccines, using both peptides and dendritic cell vaccines, can elicit clinical and immunologic responses in several different tumors. This review details the expression of melanoma antigen family A, 1 (MAGE-A1), melanoma antigen family A, 3 (MAGE-A3), and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) in various malignancies, and presents our current understanding of CT antigen based immunotherapy.

The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier "leaky." Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons.

Throughout spermatogenesis, two important processes occur at late stage VIII of the seminiferous epithelial cycle in the rat testis: preleptotene spermatocytes commence entry into the adluminal compartment and step 19 spermatids release from the seminiferous epithelium. Presently, it is not clear how these processes, which involve extensive restructuring of unique Sertoli-Sertoli and Sertoli-germ cell junctions, are mediated. We aimed to determine whether annexin A2 (ANXA2), a Ca2+-dependent and phospholipid-binding protein, participates in cell junction dynamics. To address this, in vitro and in vivo RNA interference studies were performed on prepubertal Sertoli cells and adult rat testes. The endpoints of Anxa2 knockdown were determined by immunoblotting, morphological analyses, fluorescent immunostaining, and barrier integrity assays. In the testis, ANXA2 localized to the Sertoli cell stalk, with specific staining at the blood-testis barrier and the concave (ventral) surface of elongated spermatids. ANXA2 also bound actin when testis lysates were used for immunoprecipitation. Anxa2 knockdown was found to disrupt the Sertoli cell/blood-testis barrier in vitro and in vivo. The disruption in barrier function was substantiated by changes in the localization of claudin-11, zona occludens-1, N-cadherin, and β-catenin. Furthermore, Anxa2 knockdown resulted in spermiation defects caused by a dysfunction of tubulobulbar complexes, testis-specific actin-rich ultrastructures that internalize remnant cell junction components prior to spermiation. Additionally, there were changes in the localization of several tubulobulbar complex component proteins, including actin-related protein 3, cortactin, and dynamin I/II. Our results indicate that ANXA2 is critical for the integrity of the blood-testis barrier and the timely release of spermatids.

In December 2019, a new type of pneumonia caused by SARS-Cov-2 (COVID-19) occurred in Wuhan and has been discovered in many countries around the world. ACE2 (angiotensin-converting enzyme 2) has been shown to be one of the major receptors that mediate the entry of SARS-Cov-2 into human cells. Here in this study, we used the online datasets to analyze ACE2 expression in different human organs. The results indicated that ACE2 highly expresses in renal tubular cells, Sertoli cells, Leydig cells, and cells in seminiferous ducts in testis. Recombinant SARS-CoV-2 spike protein (RBD) domain and ACE2 of RPTEC/SerC cell-binding assays confirmed that SARS-Cov-2 can bind to ACE2 on the surface of these cells. Our results suggest that ACE2 expression could contribute to kidney and testis infection after COVID-19 infection. Renal function evaluation and special care should be performed during clinical work. Clinicians should also pay attention to the risk of testicular lesions in patients during hospitalization and later clinical follow-up, especially the assessment and appropriate intervention in young patients' fertility.

the aim of this study was to investigate the effects of apocynin (APO) on hormone levels, the blood-testis barrier, and oxidative biomarkers in monosodium glutamate (MSG) induced testicular degeneration.

Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed.

The formation of matured and individual sperm involves a series of molecular and spectacular morphological changes of the developing cysts in Drosophila melanogaster testis. Recent advances in RNA Sequencing (RNA-Seq) technology help us to understand the complexity of eukaryotic transcriptomes by dissecting different tissues and developmental stages of organisms. To gain a better understanding of cellular differentiation of spermatogenesis, we applied RNA-Seq to analyse the testis-specific transcriptome, including coding and non-coding genes.

Two important events that occur during mammalian spermatogenesis are the release of elongated spermatids at late stage VIII of the seminiferous epithelial cycle and the restructuring of the blood-testis barrier (BTB) during stages VIII-XI. Still, it is not completely understood how these cellular events are accomplished within the seminiferous epithelium. In the present study, we investigate how sertolin, a protein that was initially identified, cloned, and partially characterized by our laboratory, functions in these critical events. Sertolin was found at the BTB, as well as at the apical ectoplasmic specialization and apical tubulobulbar complex, where it colocalized with epidermal growth factor receptor kinase substrate 8 and actin-related protein 3, two actin-regulatory proteins. Knockdown of sertolin by RNA interference showed Sertoli cell barrier function to be enhanced when assessed by transepithelial electrical resistance measurements and immunolocalization experiments. By contrast, the integrity of the BTB was disrupted when sertolin was overexpressed in vitro and in vivo. Sertolin overexpression also prompted germ cell loss from the seminiferous epithelium. Taken collectively, these results suggest that sertolin may be involved in coordinating spermatid release and BTB restructuring during spermatogenesis in the rat.

The testis is responsible for sperm production and androgen synthesis. Abnormalities in testis development and function lead to disorders of sex development and male infertility. Currently, no in vitro system exists for modelling the testis. Here, we generated testis organoids from neonatal mouse primary testicular cells using transwell inserts and show that these organoids generate tubule-like structures and cellular organization resembling that of the in vivo testis. Gene expression analysis of organoids demonstrates a profile that recapitulates that observed in in vivo testis. Embryonic testicular cells, but not adult testicular cells are also capable of forming organoids. These organoids can be maintained in culture for 8-9 weeks and shows signs of entry into meiosis. We further developed defined media compositions that promote the immature versus mature Sertoli cell and Leydig cell states, enabling organoid maturation in vitro. These testis organoids are a promising model system for basic research of testes development and function, with translational applications for elucidation and treatment of developmental sex disorders and infertility.

Abnormal fetal testis development can result in disorders of sex development (DSDs) and predispose to later testicular dysgenesis syndrome (TDS) disorders such as testicular germ cell tumours. Studies of human fetal testis development are hampered by the lack of appropriate model, and intervention systems. We hypothesized that human fetal testis xenografts can recapitulate normal development.

The rete testis (RT) is a region of the mammalian testis that plays an important role in testicular physiology. The RT epithelium consists of cells sharing some well-known gene markers with supporting Sertoli cells (SCs). However, little is known about the differences in gene expression between these two cell populations. Here, we used fluorescence-activated cell sorting (FACS) to obtain pure cultures of neonatal RT cells and SCs and identified differentially expressed genes (DEGs) between these cell types using RNA sequencing (RNA-seq). We then compared our data with the RNA-seq data of other studies that examined RT cells and SCs of mice of different ages and generated a list of DEGs permanently upregulated in RT cells throughout testis development and in culture, which included 86 genes, and a list of 79 DEGs permanently upregulated in SCs. The analysis of studies on DMRT1 function revealed that nearly half of the permanent DEGs could be regulated by this SC upregulated transcription factor. We suggest that useful cell lineage markers and candidate genes for the specification of both RT cells and SCs may be present among these permanent DEGs.

Zika virus (ZIKV) is a unique flavivirus with high tropism to the testes. ZIKV can persist in human semen for months and can cause testicular damage in male mice. However, the mechanisms through which ZIKV enters the testes remain unclear. In this study, we revealed that matrix metalloproteinase 9 (MMP9) was upregulated by ZIKV infection in cell culture and in A129 mice. Furthermore, using an in vitro Sertoli cell barrier model and MMP9-/- mice, we found that ZIKV infection directly affected the permeability of the blood-testis barrier (BTB), and knockout or inhibition of MMP9 reduced the effects of ZIKV on the Sertoli cell BTB, highlighting its role in ZIKV-induced disruption of the BTB. Interestingly, the protein levels of MMP9 were elevated by ZIKV nonstructural protein 1 (NS1) in primary mouse Sertoli cells (mSCs) and other cell lines. Moreover, the interaction between NS1 and MMP9 induced the K63-linked polyubiquitination of MMP9, which enhanced the stability of MMP9. The upregulated MMP9 level led to the degradation of essential proteins involved in the maintenance of the BTB, such as tight junction proteins (TJPs) and type Ⅳ collagens. Collectively, we concluded that ZIKV infection promoted the expression of MMP9 which was further stabilized by NS1 induced K63-linked polyubiquitination to affect the TJPs/ type Ⅳ collagen network, thereby disrupting the BTB and facilitating ZIKV entry into the testes.

While the molecular cues initiating testis determination have been identified in mammals, the cellular interactions involved in generating a functional testis with cord and interstitial compartments remain poorly understood. Previous studies have shown that testis cord formation relies on cell migration from the adjacent mesonephros, and have implicated immigrant peritubular myoid cells in this process. Here, we used recombinant organ culture experiments to show that immigrant cells are endothelial, not peritubular myoid or other interstitial cells. Inhibition of endothelial cell migration and vascular organisation using a blocking antibody to VE-cadherin, also disrupted the development of testis cords. Our data reveal that migration of endothelial cells is required for testis cord formation, consistent with increasing evidence of a broader role for endothelial cells in establishing tissue architecture during organogenesis.

The synthesis and release of testosterone (T) depends both on circulating luteinizing hormone (LH) and on an array of testicular factors whose role remains incompletely understood. Corticotropin-releasing factor (CRF) had been reported in the rat testes, where it was thought to inhibit T secretion. However, the discovery that the CRF-related peptides urocortins (Ucns), of which there are currently three subtypes (Ucn 1, 2 and 3), cross-react with many reagents previously used to detect CRF, has cast doubt on this concept. Here we show that while CRF was readily measurable in rat hypothalami (which served as controls), signals for this peptide were barely detectable in total RNA extracted from the testes. On the other hand, microarray, RT-PCR and real-time quantitative RT-PCR (qRT-PCR) analyses all indicated strong signals for Ucn 1 in the male gonads, with weaker levels of Ucn 2 and 3 mRNA gene expression. Results obtained for Ucn 1 gene expression were corroborated by immunohistochemical detection, which appeared restricted to Leydig cells. Finally, to investigate possible changes in testicular Ucn 1 levels induced by homeostatic challenges, we measured them in rats exposed to alcohol. We observed that indeed, the intragastric injection of this drug significantly increased testicular Ucn 1, but not Ucn 2, Ucn 3, CRF, CRFR1 or CRFR2 mRNA levels. Collectively, these results provide novel information regarding the presence of CRF-like peptides in the adult male rat testis.

FlyTED, the Drosophila Testis Gene Expression Database, is a biological research database for gene expression images from the testis of the fruit fly Drosophila melanogaster. It currently contains 2762 mRNA in situ hybridization images and ancillary metadata revealing the patterns of gene expression of 817 Drosophila genes in testes of wild type flies and of seven meiotic arrest mutant strains in which spermatogenesis is defective. This database has been built by adapting a widely used digital library repository software system, EPrints (http://eprints.org/software/), and provides both web-based search and browse interfaces, and programmatic access via an SQL dump, OAI-PMH and SPARQL. FlyTED is available at http://www.fly-ted.org/.

Synovial sarcoma (SS) is a rare cancer that disproportionately affects children and young adults. Cancer testis antigens (CTAs) are proteins that are expressed early in embryonic development, but generally not expressed in normal tissue. They are aberrantly expressed in many different cancer types and are an attractive therapeutic target for immunotherapies. CTAs are expressed at high levels in SS. This high level of CTA expression makes SS an ideal cancer for treatment strategies aimed at harnessing the immune system to recognize aberrant CTA expression and fight against the cancer. Pivotal clinical trials are now underway, with the potential to dramatically alter the landscape of SS management and treatment from current standards of care. In this review, we describe the rationale for targeting CTAs in SS with a focus on NY-ESO-1 and MAGE-A4, the current state of vaccine and T-cell receptor-based therapies, and consider emerging opportunities for future development.

Mammalian sex determination depends on a complex interplay of signals that promote the bipotential fetal gonad to develop as either a testis or an ovary, but the details are incompletely understood. Here, we investigated whether removal of the signaling molecule retinoic acid (RA) by the degradative enzyme CYP26B1 is necessary for proper development of somatic cells of the testes. Gonadal organ culture experiments suggested that RA promotes expression of some ovarian markers and suppresses expression of some testicular markers, acting downstream of Sox9. XY Cyp26b1-null embryos, in which endogenous RA is not degraded, develop mild ovotestes, but more important, steroidogenesis is impaired and the reproductive tract feminized. Experiments involving purified gonadal cells showed that these effects are independent of germ cells and suggest the direct involvement of the orphan nuclear receptor DAX1. Our results reveal that active removal of endogenous RA is required for normal testis development in the mouse.

As a result of decades of effort by many investigators we now have an advanced level of understanding about several molecular systems involved in the control of gene expression. Examples include CpG islands, promoters, mRNA splicing and epigenetic signals. It is less clear, however, how such systems work together to integrate the functions of a living organism. Here I describe the results of a study to test the idea that a contribution might be made by focusing on genes specifically expressed in a particular tissue, the human testis.

Human adult spermatogenesis balances spermatogonial stem cell (SSC) self-renewal and differentiation, alongside complex germ cell-niche interactions, to ensure long-term fertility and faithful genome propagation. Here, we performed single-cell RNA sequencing of ~6500 testicular cells from young adults. We found five niche/somatic cell types (Leydig, myoid, Sertoli, endothelial, macrophage), and observed germline-niche interactions and key human-mouse differences. Spermatogenesis, including meiosis, was reconstructed computationally, revealing sequential coding, non-coding, and repeat-element transcriptional signatures. Interestingly, we identified five discrete transcriptional/developmental spermatogonial states, including a novel early SSC state, termed State 0. Epigenetic features and nascent transcription analyses suggested developmental plasticity within spermatogonial States. To understand the origin of State 0, we profiled testicular cells from infants, and identified distinct similarities between adult State 0 and infant SSCs. Overall, our datasets describe key transcriptional and epigenetic signatures of the normal adult human testis, and provide new insights into germ cell developmental transitions and plasticity.