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Background: As a component of the nucleosome remodeling and deacetylating (NuRD) complex, metastasis‐associated protein 1 (MTA1) has been reported to be abundant in male reproductive system and might participate in spermatogenesis and sperm maturation, whereas the precise functional role of MTA1 in these processes is still undetermined. Objective: To investigate the effect and potential function of MTA1 in male fertility. Materials and methods: Mta1 knockout mice (Mta1−/−) were employed to detect their reproductive phenotype. The pH value of Mta1−/− epididymal luminal fluid was measured, and the potential mechanism of MTA1 involved in regulating luminal acidification was detected in vivo and in vitro. A vasectomy model with abnormal pH of epididymal lumen was established to further detect the effect of MTA1 on epididymal luminal microenvironment. Results: Mta1−/− mice were fertile without any detectable defects in spermatogenesis or sperm motility while the deficiency of MTA1 could acidify the initial segment of epididymis to a certain extent. MTA1 could interact with estrogen receptor alpha (ERα) and inhibit the transcription of ERα target gene, hydrogen exchanger 3 (NHE3), and ultimately affect the epididymal luminal milieu. After vasectomy, the Mta1−/− mice presented a more acidic epididymal lumen which was closer to the normal state compared to the wild‐type model. Discussion and conclusion: MTA1 is dispensable for male fertility in mice, but plays a potentially important function in regulating luminal acidification of the epididymis. [ABSTRACT FROM AUTHOR]

Background: Severe acute syndrome coronavirus 2 can invade a variety of tissues, including the testis. Even though this virus is scarcely found in human semen polymerase chain reaction tests, autopsy studies confirm the viral presence in all testicular cell types, including spermatozoa and spermatids. Objective: To investigate whether the severe acute syndrome coronavirus 2 is present inside the spermatozoa of negative polymerase chain reaction‐infected men up to 3 months after hospital discharge. Materials and methods: This cross‐sectional study included 13 confirmed moderate‐to‐severe COVID‐19 patients enrolled 30–90 days after the diagnosis. Semen samples were obtained and examined with real‐time polymerase chain reaction for RNA detection and by transmission electron microscopy. Results: In moderate‐to‐severe clinical scenarios, we identified the severe acute syndrome coronavirus 2 inside spermatozoa in nine of 13 patients up to 90 days after discharge from the hospital. Moreover, some DNA‐based extracellular traps were reported in all studied specimens. Discussion and conclusion: Although severe acute syndrome coronavirus 2 was not present in the infected men's semen, it was intracellularly present in the spermatozoa till 3 months after hospital discharge. The Electron microscopy (EM) findings also suggest that spermatozoa produce nuclear DNA‐based extracellular traps, probably in a cell‐free DNA‐dependent manner, similar to those previously described in the systemic inflammatory response to COVID‐19. In moderate‐to‐severe cases, the blood–testes barrier grants little defence against different pathogenic viruses, including the severe acute syndrome coronavirus 2. The virus could also use the epididymis as a post‐testicular route to bind and fuse to the mature spermatozoon and possibly accomplish the reverse transcription of the single‐stranded viral RNA into proviral DNA. These mechanisms can elicit extracellular cell‐free DNA formation. The potential implications of our findings for assisted conception must be addressed, and the evolutionary history of DNA‐based extracellular traps as preserved ammunition in animals' innate defence might improve our understanding of the severe acute syndrome coronavirus 2 pathophysiology in the testis and spermatozoa. [ABSTRACT FROM AUTHOR]

Background Objectives Materials and methods Results Discussion and conclusion The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virus caused the coronavirus disease 2019 pandemic, and the prevalence of deaths among men is higher than among women. The epididymis, divided into caput, corpus, and cauda, shows a region‐specific immunity. The K18‐hACE2 mouse expresses human angiotensin‐converting enzyme 2 (hACE2), the receptor that allows SARS‐CoV‐2 infection. However, studies using this transgenic mouse to evaluate the impact of this viral infection in epididymis have not yet been performed.We evaluated the expression of hACE2 in the epididymis of SARS‐CoV‐2‐infected K18‐hACE2 mice, and assessed the epididymal immune response, focusing on F4/80+ mononuclear phagocytes and tumor necrosis factor‐alpha expression.The following analyses were performed in the epididymal sections of infected mice: epithelial height and duct diameter, birefringent collagen, Terminal deoxynucleotidyl Transferase‐mediated dUTP Nick End Labelling, immunoreactions for detection of hACE2, spike, FGF, V‐ATPase, F4/80, tumor necrosis factor‐alpha, and iNOS. Viral particles were identified under electron microscopy. hACE2, Rigi, Tgfb1 and Tnfa expression were also evaluated by real‐time quantitative polymerase chain reaction.All epididymal regions expressed hACE2, which increased in all epididymal regions in the infected mice. However, the caput appeared to be the most infected region. Despite this, the caput region showed minimal changes while the cauda showed significant epithelial changes associated with increased iNOS immunoexpression. The F4/80+ mononuclear phagocyte area increased significantly in both stroma and epithelium. In addition to the epithelial and stromal mononuclear phagocytes, tumor necrosis factor‐alpha was also detected in clear cells, whose cytoplasm showed a significant increase of this cytokine in the infected animals.The K18‐hACE2 mouse is a useful model for evaluating the impact of SARS‐CoV‐2 infection in the epididymis. The infection induced hACE2 upregulation, favoring the virulence in the epididymis. The epididymal regions responded differentially to infection, and the activation of F4/80+ mononuclear phagocytes associated with the increased tumor necrosis factor‐alpha immunolabeling in clear cells indicates a role of clear cells/mononuclear phagocytes immunoregulatory mechanisms in the epididymal immune response to SARS‐CoV‐2 infection. [ABSTRACT FROM AUTHOR]

Background Objective Materials and Methods Results Discussion and Conclusions Previous research has illustrated the role of cilia as mechanical and sensory antennae in various organs within the mammalian male reproductive system across different developmental stages. Despite their significance in both organ development and homeostasis, primary cilia in the human male reproductive excurrent duct have been overlooked due to limited access to human specimens.This study aimed to characterize the unique cellular composition of human efferent and epididymal ducts, with a focus on their association with primary cilia.Human efferent ductules/epididymides from five donors aged 32–47 years, were obtained through our local organ transplant program. Cell lineage specificity and primary cilia features were examined by immunofluorescent staining and confocal microscopy in the efferent ductules and the distinct segments of the epididymis.The epithelium of the human efferent duct exhibited estrogen receptor‐positive cells with primary cilia, FoxJ1‐positive multiciliated cells with numerous motile cilia, and non‐ciliated intraepithelial immune cells. Notably, intraluminal macrophages, identified by CD163/CD68 positivity, were observed to engage in sperm phagocytosis. In all three segments of the human epididymis, primary cilia were found on the surface of principal and basal cells.Our research indicates that the human efferent ductules create a distinct environment, characterized by the presence of two types of ciliated cells that are in contact with immune cells. The discovery of sensory primary cilia exposed on the surface of reabsorptive cells in the efferent ductules, as well as on basal and principal cells in the epididymis, lays the foundation for complementary functional studies. This research uncovers novel characteristics exclusive to human efferent ductules and epididymides, providing a basis for exploring innovative approaches to male contraception and infertility treatment. [ABSTRACT FROM AUTHOR]

Background Objective Methods Results Conclusion Sleep deprivation (SD) can cause damage to the male reproductive system. However, the duration required for such damage and the specific sequence and severity of damage to the testis and epididymis remain unclear.To investigate the effects of different durations of SD on different parts of the testis and epididymis caput, corpus, and cauda.Adult ICR mice were randomly assigned to five groups: the SD group (SD for 18 h/day for 1, 2, 3, or 4 weeks), the SD + Vit E group (supplemented with Vit E 50 mg/kg/d during 4 weeks of SD, the SD+NS group (saline supplementation during 4 weeks of SD), the SD + RS group (5 weeks of recovery sleep after 4 weeks of SD), and a normal sleep control (Ctrl) group. Following the interventions, sperm parameters, testicular and epididymal histopathology, inflammatory response, and oxidative stress markers were compared between the groups.Compared to the Ctrl group, the SD group showed a decrease in sperm motility and concentration from SD 2 W and SD 3 W, respectively. Decreases in sperm concentration and motility were more pronounced in the cauda compared to the caput and corpus. Pathological damage was less severe in the epididymis caput than in the corpus and cauda. After 4 weeks of SD, inflammation and oxidative stress increased in both testes and epididymis. Both sleep recovery and vitamin E supplementation showed significant improvements, though they did not fully reach the level of the Ctrl group.Chronic SD for more than 2 weeks causes varying degrees of damage to the testis, epididymis caput, corpus, and cauda in male mice. This damage is not fully reversible after 5 weeks of sleep recovery and antioxidant stress treatment. These findings help us to identify and prevent SD damage to the male reproduction at an early stage. [ABSTRACT FROM AUTHOR]

Background Objectives Material and methods Results Discussion and conclusion The endoplasmic reticulum (ER) is the central hub for protein quality control, where the protein disulfide isomerases (PDIs), encoded by at least 21 genes, play a pivotal role. These multifunctional proteins contribute to disulfide bond formation, proper folding, and protein modifications, and may act as hormone‐binding proteins (e.g., steroids), influencing hormone biology. The interplay between ER proteostasis, PDIs, and epididymis—a crucial site for sperm maturation—remains largely understudied.This study characterizes transcriptional signatures of Pdi genes in the epididymis.Transcriptional profiles of selected Pdi genes were assessed in adult Wistar rat tissues, and epididymis under different experimental conditions (developmental stages, surgical castration, and efferent ductules ligation [EDL]). In silico bioinformatic analyses identified expression trends of this gene family in human epididymal segments.P4hb, Pdia3, Pdia5, Pdia6, Erp44, Erp29, and Casq1 transcripts were detected in both reproductive and non‐reproductive tissues, while Casq2 exhibited higher abundance in vas deferens, prostate, and heart. Pdilt, highly expressed in testis, and Pdia2, highly expressed in heart, showed minimal mRNA levels in the epididymis. In the mesonephric duct, epididymal embryonic precursor, P4hb, Pdia3, Pdia5, Pdia6, and Erp29 mRNAs were found at gestational day (GD) 17.5. Except for Erp29, which remained stable, these Pdi transcript levels increased from GD17.5 to GD20.5, when epididymal morphogenesis occurs, and were maintained to varying degrees in the epididymis during postnatal development. Surgical castration downregulated P4hb, Pdia3, Pdia5, Pdia6, Pdilt and Erp29 transcripts, an effect reversed by testosterone replacement. Conversely, transcript levels remained unaffected by EDL, except P4hb, which was reduced in caput epididymis. All 21 PDI genes exhibited diverse transcriptional profiles across the human epididymis.The findings lay the foundations to explore Pdi genes in epididymal biology. As a considerable proportion of male infertility cases are idiopathic, targeting hormonal regulation of protein quality control in epididymis represents a route to address male infertility and advance therapeutic interventions in this domain. [ABSTRACT FROM AUTHOR]

Background Objectives Materials and methods Results and discussion Conclusion The epididymis is important for sperm maturation and without its proper development, male infertility will result. Biomechanical properties of tissues/organs play key roles during their morphogenesis, including the Wolffian duct. It is hypothesized that structural/bulk stiffness of the capsule and mesenchyme/extracellular matrix that surround the duct is a major biomechanical property that regulates Wolffian duct morphogenesis. These data will provide key information as to the mechanisms that regulate the development of this important organ.To measure the structural/bulk stiffness in Pascals (force/area) of the capsule and the capsule and mesenchyme together that surrounds the Wolffian duct during the development. To examine the relative membrane tension of mesenchymal cells during the Wolffian duct development. Since Ptk7 was previously shown to regulate ECM integrity and Wolffian duct elongation and coiling, the hypothesis that Ptk7 regulates structural/bulk stiffness and mesenchymal cell membrane tension was tested.Atomic force microscopy and a microsquisher compression apparatus were used to measure the structural stiffness. Biomechanical properties within the membranes of cells within the capsule and mesenchyme were examined using a membrane‐tension fluorescent probe.The structural stiffness (Pascals) of the capsule and underlying mesenchyme was relatively constant during development, with a significant increase in the capsule at the later stages. However, this increase may reflect the ECM and associated mesenchyme being close to the capsule because the coiling of the duct pushed or compressed them into that space. Keeping the capsule and mesenchyme/ECM at constant stiffness would ensure that the duct will continue to coil under similar biomechanical forces throughout the development. Cells within the capsule and mesenchyme at different Wolffian duct regions during the development had varying degrees of membrane lipid tension. It is hypothesized that the dynamic changes ensure the duct is kept at a constant stiffness regardless of any external forces. Loss of Ptk7 resulted in an increase in stiffness at E18.5, which was presumable due to the loss of integrity of the ECM within the mesenchyme.Biomechanical properties of the capsule and the mesenchyme/extracellular matrix that surround the Wolffian duct play an important role toward Wolffian duct morphogenesis, thereby allowing for the proper development of the epididymis and subsequent male fertility. [ABSTRACT FROM AUTHOR]

Background: The epididymis has long been of interest owing to its role in promoting the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue responsible for remodeling of the sperm epigenome, both under physiological conditions and in response to diverse forms of environmental stress. Despite this knowledge, the intricacies of the molecular pathways involved in regulating the adaptation of epididymal tissue to paternal stressors remains to be fully resolved. Objective: The overall objective of this study was to investigate the direct impact of corticosterone challenge on a tractable epididymal epithelial cell line (i.e., mECap18 cells), in terms of driving adaptation of the cellular proteome and phosphoproteome signaling networks. Materials and methods: The newly developed phosphoproteomic platform EasyPhos coupled with sequencing via an Orbitrap Exploris 480 mass spectrometer, was applied to survey global changes in the mECap18 cell (phospho)proteome resulting from sub‐chronic (10‐day) corticosterone challenge. Results: The imposed corticosterone exposure regimen elicited relatively subtle modifications of the global mECap18 proteome (i.e., only 73 out of 4171 [∼1.8%] proteins displayed altered abundance). By contrast, ∼15% of the mECap18 phosphoproteome was substantially altered following corticosterone challenge. In silico analysis of the corresponding parent proteins revealed an activation of pathways linked to DNA damage repair and oxidative stress responses as well as a reciprocal inhibition of pathways associated with organismal death. Corticosterone challenge also induced the phosphorylation of several proteins linked to the biogenesis of microRNAs. Accordingly, orthogonal validation strategies confirmed an increase in DNA damage, which was ameliorated upon selective kinase inhibition, and an altered abundance profile of a subset of microRNAs in corticosterone‐treated cells. Conclusions: Together, these data confirm that epididymal epithelial cells are reactive to corticosterone challenge, and that their response is tightly coupled to the opposing action of cellular kinases and phosphatases. [ABSTRACT FROM AUTHOR]

Background: Region‐specific immune environments in the epididymis influence the immune responses to uropathogenic Escherichia coli (UPEC) infection, a relevant cause of epididymitis in men. Toll‐like receptors (TLRs) are essential to orchestrate immune responses against bacterial infections. The epididymis displays region‐specific inflammatory responses to bacterial‐derived TLR agonists, such as lipopolysaccharide (LPS; TLR4 agonist) and lipoteichoic acid (LTA; TLR2/TLR6 agonist), suggesting that TLR‐associated signaling pathways could influence the magnitude of inflammatory responses in epididymitis. Objectives: To investigate the expression and regulation of key genes associated with TLR4 and TLR2/TLR6 signaling pathways during epididymitis induced by UPEC, LPS, and LTA in mice. Material and methods: Epididymitis was induced in mice using UPEC, ultrapure LPS, or LTA, injected into the interstitial space of the initial segment or the lumen of the vas deferens close to the cauda epididymidis. Samples were harvested after 1, 5, and 10 days for UPEC‐treated animals and 6 and 24 h for LPS‐/LTA‐treated animals. Ex vivo epididymitis was induced by incubating epididymal regions from naive mice with LPS or LTA. RT‐qPCR and Western blot assays were conducted. Results: UPEC infection up‐regulated Tlr2, Tlr4, and Tlr6 transcripts and their associated signaling molecules Cd14, Ticam1, and Traf6 in the cauda epididymidis but not in the initial segment. In these epididymal regions, LPS and LTA differentially modulated Tlr2, Tlr4, Tlr6, Cd14, Myd88, Ticam1, Traf3, and Traf6 expression levels. NFKB and AP1 activation was required for LPS‐ and LTA‐induced up‐regulation of TLR‐associated signaling transcripts in the cauda epididymidis and initial segment, respectively. Conclusion: The dynamic modulation of TLR4 and TLR2/TLR6 signaling pathways gene expression during epididymitis indicates bacterial‐derived antigens elicit an increased tissue sensitivity to combat microbial infection in a spatial manner in the epididymis. Differential activation of TLR‐associated signaling pathways may contribute to fine‐tuning inflammatory responses along the epididymis. [ABSTRACT FROM AUTHOR]

Background: Chronic preconception paternal alcohol use adversely modifies the sperm epigenome, inducing fetoplacental and craniofacial growth defects in the offspring of exposed males. A crucial outstanding question in the field of paternal epigenetic inheritance concerns the resilience of the male germline and its capacity to recover and correct sperm‐inherited epigenetic errors after stressor withdrawal. Objectives: We set out to determine if measures of the sperm‐inherited epigenetic program revert to match the control treatment 1 month after withdrawing the daily alcohol treatments. Materials and methods: Using a voluntary access model, we exposed C57BL/6J males to 6% or 10% alcohol for 10 weeks, withdrew the alcohol treatments for 4 weeks, and used RNA sequencing to examine gene expression patterns in the caput section of the epididymis. We then compared the abundance of sperm small RNA species between treatments. Results: In the caput section of the epididymis, chronic alcohol exposure induced changes in the transcriptional control of genetic pathways related to the mitochondrial function, oxidative phosphorylation, and the generalized stress response (EIF2 signaling). Subsequent analysis identified region‐specific, alcohol‐induced changes in mitochondrial DNA copy number across the epididymis, which correlated with increases in the mitochondrial DNA content of alcohol‐exposed sperm. Notably, in the corpus section of the epididymis, increases in mitochondrial DNA copy number persisted 1 month after alcohol cessation. Analysis of sperm noncoding RNAs between control and alcohol‐exposed males 1 month after alcohol withdrawal revealed a ∼100‐fold increase in mir‐196a, a microRNA induced as part of the nuclear factor erythroid 2‐related factor 2 (Nrf2)‐driven cellular antioxidant response. Discussion and conclusion: Our data reveal that alcohol‐induced epididymal mitochondrial dysfunction and differences in sperm noncoding RNA content persist after alcohol withdrawal. Further, differences in mir‐196a and sperm mitochondrial DNA copy number may serve as viable biomarkers of adverse alterations in the sperm‐inherited epigenetic program. [ABSTRACT FROM AUTHOR]

The activins (A and B) and their binding protein, follistatin, play crucial roles in development, immunoregulation and inflammation throughout the body. In the male reproductive tract of the mouse, activin A and B production is largely confined to the initial segment and proximal caput of the epididymis and the efferent ducts, under normal conditions, with very low expression in the corpus, cauda and vas deferens. However, activin A protein is present throughout the epididymis and vas deferens and is largely associated with the epithelium and interstitial macrophages. Conversely, the activin‐binding protein follistatin is produced in the distal epididymis, with very high expression in the vas deferens. Activin activity in the distal tract is inhibited by follistatin, and the activin–follistatin balance is important for regulating coiling of the duct during epididymal development. In further experiments, as described in this report, in situ hybridisation was used to localise activin A mRNA principally to cells in the periductal zone and interstitium in the efferent ducts and proximal caput. Activin B mRNA, on the other hand, was localised to periductal cells in the efferent ducts and proximal epididymis and, most notably, to epithelial cells in the initial segment. Activin A is implicated in the regulation of mononuclear phagocyte function and immune responses in the caput and stimulates the expression of the key immunoregulatory protein, indoleamine 2,3‐dioxygenase in this region. Activin A production in the corpus and cauda increases dramatically during bacterial epididymitis in mice, promoting inflammation and fibrosis and causing damage to the epithelium and obstruction of the epididymal duct. Consequently, it appears that the activin–follistatin axis is crucial for maintaining normal epididymal structure and function, but disruption of this balance during inflammation has deleterious effects on male fertility. Follistatin has therapeutic potential in ameliorating the proinflammatory and profibrotic effects of activins. [ABSTRACT FROM AUTHOR]

Introduction: One of the most intriguing aspects of male reproductive physiology is the ability of the epididymis to prevent the mounting of immune responses against the onslaught of foreign antigens carried by spermatozoa while initiating very efficient immune responses versus stressors. Epithelial clear cells are strategically positioned to work in a concerted manner with region‐specific heterogeneous subsets of mononuclear phagocytes to survey the epididymal barrier and regulate the balance between inflammation and immune tolerance in the post‐testicular environment. Objective: This review aims to describe how clear cells communicate with mononuclear phagocytes to contribute to the unique immune environment in which sperm mature and are stored in the epididymis. Materials/methods: A comprehensive systematic review was performed. PubMed was searched for articles specific to clear cells, mononuclear phagocytes, and epididymis. Articles that did not specifically address the target material were excluded. Results: In this review, we discuss the unexpected roles of clear cells, including the transfer of new proteins to spermatozoa via extracellular vesicles and nanotubes as they transit along the epididymal tubule; and we summarize the immune phenotype, morphology, and antigen capturing, processing, and presenting abilities of mononuclear phagocytes. Moreover, we present the current knowledge of immunoregulatory mechanisms by which clear cells and mononuclear phagocytes may contribute to the immune‐privileged environment optimal for sperm maturation and storage. Discussion and conclusion: Notably, we provide an in‐depth characterization of clear cell‐mononuclear phagocyte communication networks in the steady‐state epididymis and in the presence of injury. This review highlights crucial concepts of mucosal immunology and cellcell interactions, all of which are critical but understudied facets of human male reproductive health. [ABSTRACT FROM AUTHOR]

There is considerable evidence showing that highly ordered aggregate structures known as amyloids carry out essential biological roles in species ranging from bacteria to humans. Indeed, many antimicrobial peptides/proteins form amyloids to carry out their host defense functions and many amyloids are antimicrobial. The similarity of host defense amyloids from bacterial biofilms to the mammalian epididymal amyloid matrix implies highly conserved host defense structures/functions. With an emphasis on the epididymal amyloid matrix, here we review the common properties of host defense amyloids including unique traits that would allow them to function as powerful biosensors of the immune system. [ABSTRACT FROM AUTHOR]

Background: Spermatozoa become mature and competent for fertilization during transit from the caput epididymis to the cauda epididymis. However, detailed molecular mechanisms of epididymal sperm maturation are still unclear. Here, we focused on multiple epididymis‐enriched genes: lipocalin family genes (Lcn5, Lcn6, Lcn8, Lcn9, and Lcn10) and Ly6 family genes (Ly6g5b and Ly6g5c). These genes are evolutionarily conserved in mammals and form clusters on chromosomes 2 and 17 in the mouse, respectively. Objective: To clarify whether these genes are required for epididymal sperm maturation and acquisition of fertilizing ability, we generated knockout (KO) mice using the CRISPR/Cas9 system and analyzed their phenotype. Materials and methods: We generated four lines of KO mice: Lcn9 single KO, the lipocalin family quadruple KO (Lcn5, Lcn6, Lcn8, and Lcn10), quintuple KO (Lcn5, Lcn6, Lcn8, Lcn10, and Lcn9), and double KO of Ly6 family genes (Ly6g5b and Ly6g5c). Results: Although the Lcn9 single KO did not affect male fertility, the quadruple KO and quintuple KO male mice were subfertile and mostly infertile, respectively, with a reduced amount of ADAM3, an essential protein for sperm binding to the zona pellucida. Further analysis revealed that the quintuple KO spermatozoa lack the CMTM2A/B that are required for ADAM3 maturation. Intriguingly, Ly6g5b and Ly6g5c double KO male mice also showed subfertility with reduced sperm ADAM3. Conclusion: These results suggest epididymal secretory proteins are involved in ADAM3 maturation and acquisition of sperm fertilizing ability. [ABSTRACT FROM AUTHOR]

Background: The mammalian epididymis is a specialized duct system that serves a critical role in sperm maturation and storage. Its distinctive, highly coiled tissue morphology provides a unique opportunity to investigate the link between form and function in reproductive biology. Although recent genetic studies have identified key genes and signaling pathways involved in the development and physiological functions of the epididymis, there has been limited discussion about the underlying dynamic and mechanical processes that govern these phenomena. Aims: In this review, we aim to address this gap by examining two key aspects of the epididymis across its developmental and physiological phases. Results and Discussion: First, we discuss how the complex morphology of the Wolffian/epididymal duct emerges through collective cell dynamics, including duct elongation, cell proliferation, and arrangement during embryonic development. Second, we highlight dynamic aspects of luminal fluid flow in the epididymis, essential for regulating the microenvironment for sperm maturation and motility, and discuss how this phenomenon emerges and interplays with epididymal epithelial cells. Conclusion: This review not only aims to summarize current knowledge but also to provide a starting point for further exploration of mechanobiological aspects related to the cellular and extracellular fluid dynamics in the epididymis. [ABSTRACT FROM AUTHOR]

Background: The human epididymis is poorly studied due to the lack of availability of tissue samples. Our understanding of its structure and function depends upon anatomical and histological observations of archived material. Objectives: Here we used single‐cell RNA sequencing (scRNA‐seq) technologies to elucidate the identity of cells within the human efferent ducts (EDs) and compared them to caput epididymis cells. We also compared the cellularity of primary tissues with those of 2D and 3D (organoid) culture models used for functional studies. Materials and methods: Human epididymis tissue was dissected to separate different anatomical regions and digested to release single cells for processing on the 10X Genomics Chromium platform. Primary human epididymis epithelial (HEE) cells and HEE organoids were grown as described previously and subjected to scRNA‐seq. scRNA‐seq data were processed by standard bioinformatics pipelines and used for comparative analysis. Results: We define the cell types in the EDs which include specialized epithelial cells, connective tissue stromal cells, vascular endothelial cells, smooth muscle cells, and immune cells, but lack basal cells that are seen in the caput epididymis. Furthermore, we identify a sub‐population of epithelial cells which have marker genes found in the bladder and urothelium. Comparative genomics analysis of the 2D and 3D culture models shows cellular identities adapted to the culture environment while still maintaining similarity to the primary tissue. Discussion: Our data suggest that EDs are lined with a transitional epithelium, which like the urothelium is able to stretch and contract depending on luminal volume. This is consistent with its primary role in seminal fluid resorption and sperm concentration. Moreover, we describe the cellularity of models to study the human epididymis epithelium in vitro. Conclusion: Single‐cell RNA‐seq data from the human epididymis make a valuable contribution to our understanding of this highly specialized organ. [ABSTRACT FROM AUTHOR]

Background: Due to the scarcity of studies using human tissues, the limited information is currently available on the gross structure of the caput epididymis in humans, at which efferent ducts connect to the epididymal duct. Objective: The present study investigated the three‐dimensional structures of efferent and caput epididymal ducts in humans, with a focus on junctions between the former and the latter. Materials and methods: We examined three sets of human efferent and caput epididymal ducts in specimens obtained from prostatic carcinoma patients. They were reconstructed from serial paraffin sections using a segmentation model created by a deep learning protocol and high‐performance three‐dimensional reconstruction software. Results: Serial sections and three‐dimensional images of human efferent and caput epididymal ducts were combined to obtain the detailed anatomical information. When a single efferent duct was defined as a duct connecting to both the extra‐testicular rete testis and epididymal duct, there were 14.7 efferent ducts with a total length of 3.0 m per specimen on average. The cranial portion of the efferent ducts joined to a single duct and terminated at the end of the epididymal duct, whereas other efferent ducts terminated independently on the side of the epididymal duct. These two types of junctions between the efferent and epididymal ducts differed in the patterns of the epithelial‐type switch. The epididymal duct consisted of multiple segments, which were separated by a minimal amount of connective tissue septa or even without them. Efferent ducts occupied most of the volume of the caput epididymis. Discussion and conclusions: By utilizing deep learning, we reconstructed human efferent and caput epididymal ducts and revealed their precise three‐dimensional structures, which differed from those of rodents in several aspects. The present results may be useful for analyzing anatomical abnormalities related to some types of male infertility. [ABSTRACT FROM AUTHOR]

This document is an editorial on the special issue of the journal Andrology titled "The clinic and biology of the epididymis." The special issue features a series of original and review articles based on talks presented at the "von Behring‐Röntgen‐Symposium The Epididymis" held in Giessen, Germany in 2022. The symposium, funded by the Behring‐Röntgen Foundation, brought together leading experts and young investigators from around the world to discuss the latest research on the epididymis. The editorial highlights some of the topics covered in the symposium, including clinical characteristics and management of epididymitis, immunobiology and immunopathology of the epididymis, epididymis and sperm maturation, and the physiology and cell biology of the epididymis. The document also mentions the conference's social events and the opportunity for attendees to present their research through oral presentations and poster sessions. The authors of the editorial encourage readers to explore the articles in the special issue for more in-depth information on the topics discussed at the symposium. [Extracted from the article]

Background: Vasectomy causes spermatozoa accumulation in the epididymis, which may cause epididymitis. Inflammation is triggered by alert molecules released following tissue stress or injury. These include uracil‐diphosphate glucose (UDP)‐glucose, which activates the pro‐inflammatory P2Y14 receptor (P2Y14), and induces immune cell recruitment. However, little is known about P2Y14 in the epididymis and its potential activation following vasectomy. Objectives: (i) To localize P2Y14 in the human excurrent duct; and (ii) to examine the effect of vasectomy on P2Y14 protein and P2RY14 mRNA content, the production of selected cytokines and chemokines, and immune cell recruitment in the epididymis. Material and methods: In situ hybridization, qRT‐PCR, western blotting, immunohistochemistry, and immunofluorescence were performed in banked human epididymis samples. Results: P2RY14 mRNA and P2Y14 protein were detected in epithelial cells in the efferent duct, epididymis and vas deferens in non‐vasectomized men. Keratin 5 (KRT5)‐positive basal cells were strongly labeled for P2Y14 in all epididymal segments. A progressive apical localization was detected in principal cells (negative for the proton pump V‐ATPase) from the corpus to the cauda. A subset of V‐ATPase‐positive clear cells also showed strong P2Y14 labeling. Vasectomy induced an increase in P2RY14 mRNA in the corpus and cauda, and stronger apical labeling in principal cells in the corpus. CXCL10 mRNA increased in the cauda and CCL2 mRNA decreased in the corpus of vasectomized versus non‐vasectomized men. No change in IL‐8 and IL‐1β mRNA was detected. Numerous CD45+ leukocytes were detected in the interstitium of the corpus and cauda following vasectomy, while only a few were seen in non‐vasectomized men. Several CD45+ leukocytes, some of which containing spermatozoa, were detected in the corpus lumen following vasectomy. Discussion and conclusion: Our study indicates that vasectomy‐induced spermatozoa congestion may lead to an inflamed‐prone local environment characterized by potential activation of P2Y14 and recruitment of immune cells in the epididymis. [ABSTRACT FROM AUTHOR]

Background: Immunoregulatory genes encoding activin A (Inhba) and B (Inhbb), and indolamine 2,3‐dioxygenase‐1 (Ido1) are highly expressed in the murine caput epididymidis, which also has a network of intraepithelial mononuclear phagocytes. This environment is postulated to promote immunological tolerance to epididymal sperm. The factors regulating the immunoregulatory agents in the epididymal caput are poorly understood. Objectives: This study aimed to investigate the potential role of testicular lumicrine factors in regulating activin and other immune‐related genes in the caput epididymidis. Materials and methods: The efferent ducts in adult C57/Bl6 mice were exposed and ligated bilaterally. Serum and tissues were collected seven days later. Animals with bilateral sham ligation and animals with no ligations (collectively referred to as the "intact" group) were used as controls. Results: Pressure‐induced seminiferous epithelial damage due to intratubular fluid accumulation was observed in all ligated testes. Testicular inhibin was significantly increased and testosterone was elevated in some animals following bilateral ligation, but serum testosterone, serum LH, and serum inhibin were normal. Ligation caused epithelial regression in the initial segment, with similar but less severe effects in other caput segments. Activin A staining by immunohistochemistry in the epithelium was reduced in bilateral ligation, particularly in the initial segment, with moderately reduced staining intensity in the rest of the caput. Inhba expression within the caput was not significantly affected by bilateral ligation, but Inhbb was reduced by more than 60%. Transcripts encoding the macrophage‐specific receptor Cx3cr1 were significantly reduced following bilateral ligation, but other immune cell markers, Ido1, and inflammatory genes were unaffected. Conclusion: These data indicate that testicular lumicrine secretion regulates several genes that are preferentially expressed in the initial segment, but has marginal effects on genes such as those encoding activin A and IDO1, which are expressed more widely in the caput. [ABSTRACT FROM AUTHOR]

Background: Ultrasound (US) is the primary modality for the investigation of scrotal pathology, including both intra‐ and paratesticular abnormalities. Objective: To describe the abnormalities of the paratesticular space. Materials/Methods: The paratesticular space contains the epididymis, spermatic cord and the tunica vaginalis cavity and is affected by a variety of inflammatory or tumoral entities. Differential diagnosis based on US criteria is frequently problematic, as the findings are non‐specific. Results: Some general rules apply: (i) unlike testicular lesions, extra‐testicular entities are usually benign in the adult, (ii) the first steps to accurate diagnosis include careful localization of the lesion and assessment of its consistency (solid or cystic) and (iii) magnetic resonance imaging can be useful for further tissue characterization of lesions suspected to contain fat, but surgical biopsy will often provide the definite diagnosis. Contrast‐enhanced ultrasound (CEUS) has been applied with limited experience indicating a narrow role, primarily for the differential diagnosis of echogenic cystic entities and the delineation of a necrotic abscess from a solid neoplasm. Discussion: The various abnormalities are discussed and illustrated. Conclusion: This manuscript summarizes the literature on paratesticular lesions and the value of US in diagnosis. [ABSTRACT FROM AUTHOR]

Background: Spermatogenesis is an elaborately organized and tightly regulated differentiation process. The spermatogenesis duration is stable within a certain species but highly variable between species of the same family. Objectives: In this study, the spermatogenesis duration of the Roborovski hamster was measured for the first time, and the spermatogenesis duration of the Chinese hamster was re‐assessed. Materials and methods: Stage classification and cycle length measurement were carried out by labeling the dividing cells with bromodeoxyuridine and an antibody‐based chromogen as well as with the periodic acid‐Schiff/hematoxylin stain. Analysis was conducted using reference calculation and linear regression. Morphological measurements completed our set of methods. Results: The mean duration of one seminiferous epithelium cycle was 8.58 ± 0.34 days (mean ± SEM; Phodopus roborovskii) and 16.59 ± 0.47 days (Cricetulus griseus) based on the reference calculation. Slightly higher results were obtained using linear regression analysis: 9.72 ± 0.41 days for P. roborovskii and 17.64 ± 0.61 days for C. griseus. Additionally, a newly developed exemplary flowchart was proposed for the Roborovski hamster to facilitate spermatogenesis stage classification also in other species. The Chinese hamster presented an unexpectedly high paired epididymides weight of 1.701 ± 0.046 g (mean ± SEM) although having a body weight of only 40.5 ± 0.7 g. However, no significant correlation between the relative epididymis weight and spermatogenesis duration in mammals (Spearman rank correlation: r = −0.119, p = 0.607, n = 21) or rodents could be found (r = 0.045, p = 0.903, n = 11). Conclusion: Our data emphasize the stability of the spermatogenesis duration within species and its remarkable variability between species. Further research is needed to identify the principal mechanisms and selection drivers that are responsible for such stability within species and the variability between species. [ABSTRACT FROM AUTHOR]

Background: Varicocoele‐associated stressors, such as hypoxia and heat, can damage cell function and viability, and some exosomal biomarkers released from impaired cells may reflect the cell status in testis. Objectives: To find if seminal exosomal microRNAs can reflect the Sertoli cell function in varicocoele. Materials and methods: Experimental left varicocoele rat model was established (n = 24), and patients with different grades of varicocoele (n = 104) were enrolled. Primary rat Sertoli cells were isolated with enzymatic hydrolysis. Exosomes were isolated from primary rat Sertoli cells, rat epididymis tissue, and human seminal plasma with polymer‐based precipitation method. Exosomal microRNAs were quantified with qPCR. Inhibin‐B was detected with enzyme immunoassay. The correlation analysis between microRNA and inhibin‐B was evaluated with Spearman's correlation. Results: We screened 12 previously reported hypoxia‐responsive microRNAs in the primary rat Sertoli cells and found that 4 exosomal microRNAs increased significantly in response to in vitro hypoxia treatment (P <.05). Of the 4 microRNAs, only miR‐210‐3p was upregulated in the rats with experimental varicocoele (P <.01). In the patients with varicocoele, we found that seminal exosomal miR‐210‐3p significantly increased in patients with grade II and III varicocoele (P <.01), and miR‐210‐3p negatively correlated with sperm count (P <.01) and seminal inhibin‐B expression (r = −0.39, P <.01). For the 30 patients with microsurgical varicocelectomy, the operation notably decreased miR‐210‐3p (P <.01). Discussion and Conclusion: Seminal exosomal miR‐210‐3p may be a novel, sensitive, and non‐invasive biomarker of Sertoli cell damage in varicocoele. [ABSTRACT FROM AUTHOR]

Background: The epididymis is a popular research topic in urology and reproduction. Objectives: To explore and identify the anatomical characteristics of the epididymis based on histology, proteomics, and 3D reconstruction of epididymal tubules. Materials and methods: A 3D reconstruction of epididymal tubules was generated based on 7‐μm‐thick transverse serial sections of an epididymis. The proteins in the subcompartments of the epididymis were obtained and analyzed by non‐labeled sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS). Protein function, signaling pathways, protein expression, and the histology in different subcompartments were analyzed. Results: The caput (Cap), corpus (Cor), and cauda (Cau) of the epididymis were divided into 6, 10, and 4 subcompartments, respectively, and the subcompartment between the Cap and Cor is mixed together. A total of 3411 proteins were identified, and 854 proteins were accurately quantified after screening. When the subcompartment Cap 5 transitioned to Cap 6 and Cap 6 to Cap 7, 87 and 52 proteins were upregulated and 14 and 7 proteins were downregulated, respectively. The Cor 9 transition to Cau 1 was marked by 230 proteins that were downregulated, while 74 proteins were upregulated. At the junction of the cauda and the vas deferens, 57 proteins were downregulated, and 410 proteins were upregulated. Cap 6 histology was consistent with that of Cor 1. Discussion and conclusion: The epididymis contains distinct connective tissue septa that can be identified under a surgical tabletop microscope, enabling it to be divided into 20 subcompartments. [ABSTRACT FROM AUTHOR]

Background: Gene expression patterns along the epididymis are established by specific transcription factor networks that coordinate region‐specific functions. In rodents, the epididymis can be divided in up to 19 segments. Based on gross anatomy, the human epididymis is divided into caput, corpus, and cauda segments together with efferent ducts that occupy the proximal part of this organ. Objectives: To determine to which extent gene expression pattern is segmented along the efferent ducts and the proximal region of the epididymis in men of reproductive age. Materials and methods: Epididymal transcriptome profiling was performed on eight distinct regions from three donors. Microarray analysis was performed on a gene‐chip microarray. Differentially expressed genes (DEGs)>2‐fold change (P <.05) were clustered in relation to their intensity profiles. Overrepresented biological functions from gene ontology were searched using DAVID 6.8. Expression profiles were validated by qRT‐PCR quantification of selected genes. Results: There were no DEGs among segments 1‐3 of efferent ducts region neither among segments 4‐6 of the caput epididymis. 1058 DEGs were identified between efferent ducts and the epididymis, whereas 444 and 846 DEGs distinguished the caput from the corpus (section 7) and cauda (section 8) epididymis, respectively. A total of 131 DEGs were detected between corpus (7) and cauda (8) transcriptomes. Up‐regulated DEGs in the efferent ducts were predominantly related to cilium assembly/movement and cell differentiation. Fertilization, defense, and immune responses were associated with caput epididymis (4‐6), while spermatogenesis and protein binding were found all along the epididymis (4‐8). Discussion: The proximal human epididymis is exclusively occupied by efferent ducts with a distinct DEG profile compared with the downstream epididymal segments. Moreover, gene expression profiling revealed two regions in the human epididymis: the caput and the distal corpus/cauda region. Conclusions: Human epididymal transcriptome reveals limited DEGs, and efferent ducts have a distinct DEGs profile. [ABSTRACT FROM AUTHOR]

Background: Myo‐inositol (cis‐1,2,3,5‐trans‐4,6‐cyclohexanehexol; MI) is the most prominent of nine inositol stereoisomers. MI, its phosphate derivatives, and associated lipids are widely found in vegetables and animal tissues and are known to participate in numerous biological processes. Objectives: To perform a review analysis on MI presence, functions, and impact in male fertility. Materials and Methods: A thorough search of listed publications in PubMed on MI and its derivatives was done. Results: Published information was found and compiled on MI identification, natural dietary sources and absorption, biosynthesis, concentrations, as well as MI as its derivatives (PI, PIP, GPI, IPG) roles in several human tissues and body fluids in health and disease. A section was focused on MI presence, biosynthesis, and functions in the mammalian male genital tract and in spermatozoa, and summarized reports describing the impact of in vivo and in vitro MI supplementation on human semen quality and fertility. Studies reported a discrete improvement in sperm motility in fresh and frozen‐thawed semen, and a better sperm performance in natural and assisted fertility. Discussion and Conclusion: MI was reported as an effective supplement for sperm quality. In any case, several study designs lack appropriate controls or data analysis to confirm the relevance of the findings. While promising, larger prospective randomized controlled studies will be required to confirm the positive effect of MI supplementation in male infertility management. Moreover, further investigations are encouraged to unravel MI roles in sperm physiology and the underlying molecular mechanisms. [ABSTRACT FROM AUTHOR]

Background: The presence of miRNAs in human reproductive tissue is intriguing and suggests the possibility that these important regulatory molecules play a role in reproductive function. However, the regulatory role of miRNAs in reproductive tissue remains poorly understood with a significant amount of controversial and contradicting data. Objectives: To systematically review the high‐quality studies published to date investigating miRNAs associated with male human reproduction in order to describe their roles and relations with infertility and update the knowledge in the field. Materials and methods: A comprehensive systematic review of the published literature in MEDLINE–PubMed and EMBASE databases from the earliest available online indexing year until June 2018 (complimentary search until July 2019) was performed, in accordance with the PRISMA guidelines. We have included descriptive, case–control, cross‐sectional, and observational prospective and retrospective studies in which fertile/infertile men were well‐defined. The primary outcome was the miRNA expression in testis, epididymis, sperm cells, seminal plasma, and extracellular vesicles (i.e., exosomes and microvesicles). Results: We identified 25,204 articles, of which 42 were selected for qualitative analysis. Of the 42 articles included, 15 evaluated the miRNAs in testis, five in epididymis, 13 in spermatozoa, and 11 in seminal plasma and/or extracellular vesicles. Two studies tackled more than one sub‐group. As far as miRNA presence and content, the results of this systematic review indicated that every tissue/cell contains a well‐defined and stable population of miRNAs that could be potentially related to spermatogenesis and embryogenesis. Discussion and conclusion: Our systematic review of descriptive and observational studies shows a consistent relationship between aberrant miRNA expression and infertility. Therefore, it seems reasonable that measuring the expression of particular miRNAs might be useful not only as infertility biomarkers, but also for developing therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Ephrin receptors (Eph) and ligands are membrane‐bound cell–cell communication molecules that regulate the spatial organization of various tissues and organs by repulsive or adhesive signals arising from contact between Eph‐ and ephrin‐bearing cells. However, the expression and functions of Eph receptors in the testis and epididymis are virtually unknown. Objectives: We aimed to investigate the expression of several EphB receptors and ephrin‐B ligands in the testis and epididymis of adult mice. Materials and methods: mRNA and protein expression was detected via reverse transcription‐polymerase chain reaction amplification and immunostaining, respectively. Results: Complementary expression patterns were observed in the epithelia along the excurrent duct system in the testis and epididymis; ephrin‐B1 was strongly expressed in the epithelia of the rete testis and segment I in the ductus epididymis, whereas EphB2 and/or EphB4 were strongly expressed in the epithelia of the straight tubules and efferent ductules. Moreover, ephrin‐B1 was expressed in the spermatogonia, Leydig cells, and peritubular myoid cells in the testis, whereas EphB2 was expressed in elongated spermatids and EphB4 was expressed in the spermatogonia and Leydig cells. Furthermore, these receptors were found to be tyrosine‐phosphorylated in the testis and/or epididymis. Discussion: Receptor localization and phosphorylation patterns suggested that EphB/ephrin‐B signaling might occur in the seminiferous tubules and epithelial junctions among the straight tubules, rete testis, efferent ductules, and ductus epididymis. Therefore, we propose that EphB/ephrin‐B signaling may regulate epithelial boundary formation in the excurrent tubule/ductule/duct system as well as modulate spermatogenesis and spermiation. Conclusion: Overall, this study represents the first analysis of EphB receptor and ephrin‐B ligand expression in the normal adult testis and epididymis. [ABSTRACT FROM AUTHOR]

Background: Studies on epididymal toxicology are scarce. Betamethasone (BM) is a glucocorticoid used in clinical practice for antenatal therapy. We previously reported changes to testicular morphology, altered sperm quality, and fertility in adult rats following intrauterine administration of BM. Objectives: Given that high levels of corticosteroids during gestation lead to fetal androgen depletion, and the essential role of testosterone during epididymal development, here we investigated epididymal morphology and physiology in the F1 and F2 male offspring of female rats treated with BM during gestation. Materials and methods: Pregnant rats were randomly divided into two experimental groups: control (saline vehicle, n = 11) and BM‐treated group (0.1 mg/kg betamethasone 21‐phosphate disodium, n = 13). Rats received an intramuscular injection of vehicle or BM on gestational days 12, 13, 18, and 19. This encompasses the beginning of the critical window of male rat reproductive tract development. A subset of three males from each litter (n = 5 litters/group) was used: One rat per litter was euthanized at puberty, one was euthanized at adulthood, while the others were mated with a non‐treated female to obtain the F2 generation. The same protocol described for the F1 was applied for F2, except for the mating protocol. Results: In both F1 and F2 generations, prenatal BM exposure resulted in delayed differentiation of the cauda epididymal epithelium, characterized by increased cribriform appearance on PND 45, and displayed weaker or non‐detectable Cx43 immunostaining. Furthermore, in the F1 generation only, immunostaining of TP63, a transcription factor expressed in basal cells, appeared more intense with a greater number of TP63‐positive cells observed in the cauda epididymis. In adults, the epithelial area was reduced in the F1 BM rats. The contractile activity of isolated epididymal ducts was comparable between groups. Discussion and Conclusion: Prenatal BM exposure leads to intergenerational impairment in the development and structure of the rat epididymis. [ABSTRACT FROM AUTHOR]

The primary job of the epididymis is to mature and protect the luminally transiting spermatozoa. Mounting evidence is showing that innate immune components [including Toll‐like receptors (TLRs) and antimicrobial proteins, among which are β‐defensins] and inflammatory mediators, under the primary influence of androgens, participate in the cellular and molecular processes that define this tissue. Here, we present an overview of the contributions of these signaling pathway components during epididymal homeostasis and discuss the hypotheses as to their involvement in epididymitis, the most common urological inflammatory condition in men, frequently impairing their fertility. Drawing primarily from rodent models, we also focus on how the distribution and functional expression of innate immune components are differentially regulated in the prenatal developing epididymis, providing new insights into the disruption of these signaling pathways throughout the lifespan. Male infertility is caused by a variety of conditions, such as congenital malformations, genetic and endocrine disorders, exposure to environmental toxicants, and inflammatory/infectious conditions. More than one‐third of infertile men with an idiopathic condition cannot currently be adequately diagnosed. Thinking about the innate immunity and inflammation context of the epididymis may provide new insights and directions as to how these systems contribute to male fertility, as well as also uncover urological and andrological outcomes that may aid clinicians in diagnosing and preventing epididymal pathologies. [ABSTRACT FROM AUTHOR]

Background: One third of infertility cases in couples worldwide has an exclusive male origin and immune disorders, essentially due to repetitive infections, are emerging an cause of male infertility. As the place of sperm maturation, epididymis must be preserved from excessive immune responses that may arise following infections of the male genital tract. At the same time, epididymis must set and maintain a tolerogenic environment in order not to destroy sperm cells that enter the tissue at puberty, long after the immune system has been taught to recognize self pathogens. The immune cells that populate the epididymis have raised growing interest over the last thirty years but they may be not sufficient to understand the immune balance existing in this organ, between immune response to pathogens and tolerance to spermatozoa. Indeed, immune cells are the most motile cells in the organism and need blood and lymphatic vessels to traffic between lymphoid organs and sites of infection to induce efficient responses. Objectives: To review the literature on the blood and lymphatic vessels, and on the immune cells present at steady state in the rodent epididymis (rat and mouse). Materials and methods: PubMed database was searched for studies reporting on the spatial organization of the rodent epididymal vasculature and immune cell types at steady state. This search was combined with recent findings from our team. Results: At steady state, the rodent epididymis presents with dense blood and lymphatic networks, and a large panel of immune cells distributed across the interstitum and epithelium along the organ. Conclusions: The immune system of the rodent epididymis is highly organized. Exploring its functions, especially in an infectious context, is the essential coming step before any transposition to human. [ABSTRACT FROM AUTHOR]

Background: The interface between the epididymis and the immune system is implicated in many male reproductive pathologies. The resident immune cell populations and immune‐environment within the epididymis are significantly different from the testis, which is an immune‐privileged site. Moreover, the immune cell subsets and immunological responses between different regions of the epididymis vary considerably. The cauda epididymis is more susceptible to autoimmune responses than the caput in rodent models of active immunization or suppressed immune tolerance, and in men with congenital or physical damage to the reproductive tract. Activins are members of the transforming growth factor‐β family of cytokines that are crucial for testis and epididymal development; however, they also have complex immunoregulatory properties and may play an essential role in the regulation of immunity in the reproductive tract. Materials and methods: Our recent research and relevant publications by other researchers identified following a PubMed search are reviewed. Results: The caput epididymis displays elevated endogenous expression of activins A and B and the immunoregulatory gene, indoleamine‐2,3‐dioxygenase, co‐existing with an extensive population of intra‐epithelial and interstitial macrophages and dendritic cells, which appear to be involved in regulating tolerance against sperm antigens. The caput is also relatively resistant to inflammatory damage caused by autoimmunity or bacterial infection, but the cauda, which exhibits low activin expression and high levels of the activin‐binding protein, follistatin, is highly susceptible to inflammatory damage. Paradoxically, inflammation in the cauda induces increased activin production, and inhibition of activin activity reduces inflammatory responses. Studies using mouse models with altered levels of activins and follistatin indicate a relationship between the activins and genes involved in inflammation and immunoregulation. Conclusion: The existing data indicate that activins play a complex role in controlling inflammation and immunity in the epididymis and vas deferens. [ABSTRACT FROM AUTHOR]

Background: Our understanding of epididymal physiology and function has been transformed over the three decades in which the International Meeting Series on the Epididymis has been hosted. This transformation has occurred along many fronts, but among the most significant advances has been the unexpected discovery of the diversity of small non‐protein‐coding RNAs (sRNAs) expressed in the epididymal epithelium and differentially accumulated in the luminal population of spermatozoa. Objectives: Here we survey recent literature pertaining to profiling the sRNA landscape of the mammalian epididymis with the goal of demonstrating the contribution that these key regulatory elements, and their associated pathways, make to epididymal physiology and sperm maturation. Results and discussion: High throughput sequencing strategies have fueled an unprecedented advance in our understanding of RNA biology. In the last decade, such high throughput profiling tools have been increasingly applied to study the mammalian epididymis, presaging the discovery of diverse classes of sRNA expressed along the length of the tract. Among the best studied sRNA classes are the microRNAs (miRNA), a sRNA species shown to act in concert with endocrine signals to fine‐tune the segmental patterning of epididymal gene expression. In addition to performing this homeostatic role, epithelial cell‐derived sRNAs also selectively accumulate into the epididymosomes and spermatozoa that occupy the duct lumen. This exciting discovery alludes to a novel form of intracellular communication that contributes to the establishment of the sperm epigenome and its modification under conditions of paternal stress. Conclusion: Compelling literature has identified sRNAs as a crucial regulatory tier that allows the epididymis to fulfill its combined roles of sperm transport, maturation, and storage. Continued research in this emerging field will contribute to our growing understanding of the etiology of male factor infertility and potentially allow for the future design of rational therapeutic options for these individuals. [ABSTRACT FROM AUTHOR]

Background: Small non‐coding RNAs (sncRNAs) accomplish a huge variety of biological functions. Over the past decade, we have witnessed the substantial progress in the epididymal sncRNA studies. In the Epididymis 7, we had the true privilege of having a whole session to share our findings and exchange ideas on the epididymal sncRNA studies. Objectives: This mini‐review attempts to provide an overview of what is known about the sncRNAs in the mammalian epididymis and discuss the future directions in this field. Methods: We surveyed literature regarding the sncRNA studies in the mammalian epididymis, and integrated some of our unpublished findings as well. We focus on the progress in methodology and the advances in our understanding of the expression and functions of epididymal sncRNAs. Results and Discussion: The applications of high‐throughput approaches have made great contributions in the discovery of new sncRNA species and profiling their dynamics in the epithelial cells, the passing spermatozoa, and the luminal environment. The diverse classes of epididymal sncRNAs exert important biological functions from the in situ regulation of epididymal gene expression to the epigenetic inheritance in the offspring. Conclusion: Although still in its infancy, we believe that the research on epididymal sncRNAs will not only lead to a better understanding of their physiological and pathological functions, but also contribute to the whole landscape of the RNA field. [ABSTRACT FROM AUTHOR]

Background: The molecular mechanisms involved in the acquisition of mammalian sperm fertilizing ability are still poorly understood, reflecting the complexity of this process. Objectives: In this review, we describe the role of Cysteine RIch Secretory Proteins (CRISP1–4) in different steps of the sperm journey to the egg as well as their relevance for fertilization and fertility. Materials and Methods: We analyze bibliography reporting the phenotypes of CRISP KO mice models and combine this search with recent findings from our team. Results: Generation of individual KO for CRISP proteins reveals they are key mediators in different stages of the fertilization process. However, in spite of their important functional roles, KO males for each of these proteins remain fertile, supporting the existence of compensatory mechanisms between homologous CRISP family members. The development of mice lacking epididymal CRISP1 and CRISP4 simultaneously (DKO) revealed that mutant males exhibit an impaired fertility due to deficiencies in the sperm ability to fertilize the eggs in vivo, consistent with the proposed roles of the two proteins in fertilization. Interestingly, DKO males show clear defects in both epididymal epithelium differentiation and luminal acidification known to be critical for sperm maturation and storage. Whereas in most of the cases, these epithelium defects seem to specifically affect the sperm fertilizing ability, some animals exhibit a disruption of the characteristic immune tolerance of the organ with clear signs of inflammation and sperm viability defects. Discussion and Conclusion: Altogether, these observations confirm the relevance of CRISP proteins for male fertility and contribute to a better understanding of the fine‐tuning mechanisms underlying sperm maturation and immune tolerance within the epididymis. Moreover, considering the existence of a human epididymal protein functionally equivalent to rodent CRISP1 and CRISP4, DKO mice may represent an excellent model for studying human epididymal physiology and pathology. [ABSTRACT FROM AUTHOR]

Background: The primary cilium is a microtubule‐based organelle that extends transiently from the apical cell surface to act as a sensory antenna. Initially viewed as a cellular appendage of obscure significance, the primary cilium is now acknowledged as a key coordinator of signaling pathways during development and in tissue homeostasis. Objectives: The aim of this review was to present the structure and function of this overlooked organelle,with an emphasis on its epididymal context and contribution to male infertility issues. Materials and Methods: A systematic review has been performed in order to include main references relevant to the aforementioned topic. Results: Increasing evidence demonstrates that primary cilia dysfunctions are associated with impaired male reproductive system development and male infertility issues. Discussion: While a large amount of data exists regarding the role of primary cilia in most organs and tissues, few studies investigated the contribution of these organelles to male reproductive tract development and homeostasis. Conclusion: Functional studies of primary cilia constitute an emergent and exciting new area in reproductive biology research. [ABSTRACT FROM AUTHOR]

Background: Lipid metabolic disorders (dyslipidemia) are constantly increasing in occidental societies and lead to the development of pathologies such as obesity, diabetes, and metabolic syndrome. It has been demonstrated that dyslipidemia can alter the reproductive function. Animal models have recently been used to show that the offspring of dyslipidemic males could also develop such pathologies and that the transgenerational transmission involved post‐testicular sperm maturation. These data targeted the essential role of male gamete epididymal maturation and its importance for the health of the offspring. Objectives: This publication summarizes in the first place experimental data obtained using a mouse model of dyslipidemia‐induced post‐testicular infertility, knockout mice for the two isoforms of the 'Liver X Receptors' (Lxrα;β−/−), the major regulators of cholesterol homeostasis. The impact of a high cholesterol diet (HCD) on the protein YWHAZ (14‐3‐3 ζ or tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein Zeta) was also investigated in our model. Materials and methods: In our mouse model, when young fertile Lxrα;β−/− males aged three months were fed four weeks with a HCD, they developed an epididymal phenotype leading to infertility. The level of sperm YWHAZ was evaluated by Western blot and its tyrosine phosphorylation state by immunoprecipitation followed by Western blot. Results: Our data revealed that sperm lipid composition and structure were altered, leading to defects of the capacitation‐associated signaling pathway. They also showed that both the level and the tyrosine phosphorylation state of YWHAZ were affected by the HCD in sperm cells from Lxrα;β−/− males. Discussion and conclusion: YWHAZ could be a new important regulator of capacitation‐associated tyrosine phosphorylation and a marker of dyslipidemia‐induced infertility. [ABSTRACT FROM AUTHOR]

Summary: Background: The epididymis is the hallmark of all vertebrate species practicing internal fertilization. While the functions of the epididymis are well documented in laboratory rodents and some domestic animals, the structure and functions of the epididymis in humans remain poorly documented. Objectives: Using human tissues obtained with the collaboration of our local organ transplantation program, the histology, cell types, and three‐dimensional organization of the excurrent duct were investigated. Microarrays were performed to determine the gene expression pattern along the human epididymis. Materials and methods: The histology of longitudinal sections of the proximal epididymis was described, and immunohistochemistry using specific antibodies was used to characterize cell types of the efferent duct and caput epididymis epithelia. The epididymis was divided into eight segments permitting gene profiling by microarray and gene ontology analysis. Results: The proximal region of the human epididymis is formed exclusively by efferent ducts. These ducts form a complex histological structure particularly at the junction of the efferent ducts and caput epididymis. The efferent ducts exhibit a specific cellular signature when compared with the adjacent epididymis tubule. Efferent duct gene expression is not segmented and is dedicated to cilium differentiation and movement. The gene expression pattern of the caput segment is homogeneous and specialized in defense and immune responses and fertilization. Discussion: In murine species, the epididymis is segmented into the initial segment, caput, corpus, and cauda regions, whereas in humans, the proximal region is formed by efferent ducts. The caput tubules have their own histological organization with a well‐defined gene expression pattern. The distal corpus and cauda epididymis are distinct by a limited number of differentially expressed genes. Conclusions: Knowledge of epididymis functions and structure obtained using laboratory species should be extrapolated to humans with caution. [ABSTRACT FROM AUTHOR]

Background: It is known that exposure to either arsenic or hyperglycemia can induce male reproductive damages. However, their combined effects on male reproductive organs are still unclear. Therefore, the present study investigated morphological and functional parameters of the testis, epididymis, and spermatozoa in diabetic rats exposed to arsenate. Materials and methods: Diabetes was induced in male rats by intraperitoneal streptozotocin injection. While a set of healthy and diabetic animals received saline solution (negative control and diabetes control, respectively), the other set received 10 mg/L sodium arsenate (arsenic control and diabetes + arsenic groups, respectively) for 40 days in drinking water. Testosterone concentration, daily sperm production, sperm counts in the testis and epididymis, and sperm parameters were evaluated in the groups. Moreover, testis and epididymis were subjected to antioxidant enzymes analysis, micromineral determination, and histopathological evaluation. Results: Arsenate exposure reduced serum testosterone concentration in healthy animals and worsened this reduction in diabetic rats. In addition, the number of spermatozoa in testis and epididymis tissues, as well as the daily sperm production, was decreased in these groups. Sperm parameters such as motility, morphology, and integrity of acrosomal and plasma membranes were impaired in health animals exposed to arsenate. The combination of diabetes and arsenate, in turn, increased only the percentage of spermatozoa with abnormal morphology. Moreover, the proportion of arsenic increased in the testis and epididymis of both groups receiving arsenate. Its bioaccumulation in these organs caused an imbalance in antioxidant enzymes activities and mineral content in healthy animals, enhancing these changes in diabetic rats. Testicular pathologies occurred mainly in animals co‐exposed to diabetes and arsenate. Conclusion: Our results indicate that arsenate exposure enhances several damages to male reproductive functions in diabetic rats, mainly by impairing testosterone levels and inducing nitrosative stress in testis and epididymis. [ABSTRACT FROM AUTHOR]

Background: The mechanisms by which the rete testis joins the efferent ducts, which joins the Wolffian duct during development, are not known. Mouse and chick models have been helpful in identifying genes that are important for the development of each part, but genes have not been identified as to those that play a role in the joining of each part. Clinical implications of the failure of the male reproductive tract to form a fully functional conduit for spermatozoa are not trivial. Epididymal disjunction, the failure of the efferent ducts to join the testis, is one of several epididymal anomalies that have been observed in some boys who were cryptorchid at birth. Objective: A systematic review of studies focusing on the morphogenesis of the mesonephric duct and mesonephric tubules in different species, and identification of clinical issues should there be failure of these tissues to develop. Design: PubMed and GUDMAP databases, and review of books on kidney development were searched for studies reporting on the mechanisms of morphogenesis of the kidney and epididymis. Main outcomes measure(s): Gaps in our knowledge were identified, and hypotheses coupled with suggestions for future experiments were presented. Results: A total of 64 papers were identified as relevant, of which 53 were original research articles and 11 were book chapters and reviews covering morphogenesis and clinical issues. Investigators utilized multiple species including, human, mouse, chick, Xenopus, bovine, and sheep. Conclusion: Fundamental understanding of the morphogenesis of the male reproductive tract is limited, especially the morphogenesis of the rete testis and efferent ducts. Therefore, it is not surprising that we do not understand how each part unites to form a whole. Only one mechanism of joining of one part of the tract to another was identified: the joining of the Wolffian duct to the cloaca via controlled apoptosis. [ABSTRACT FROM AUTHOR]

Background: During the post‐testicular maturation that occurs in the epididymis, spermatozoa need to face biochemical and morphological changes that may make them vulnerable to oxidative damage. During spermatogenesis and the epididymal maturation, the spermatozoon acquires antioxidant enzymes needed to face possible increases of reactive oxygen species (ROS) produced by its own aerobic metabolism but also due to ROS produced in high quantities by abnormal spermatozoa. Objectives: Provide an up‐to‐date review of the enzymatic antioxidant system in the epididymis. Material and methods: A thorough literature review was performed for papers concerning the players of the antioxidant defenses in the epididymis. Results: The antioxidant system in the epididymis is composed by superoxide dismutases, catalase, glutathione peroxidases, peroxiredoxins, glutathione‐S‐transferases, thioredoxins and thioredoxin reductase. They work together to maintain low levels of ROS during the epididymal maturation. Knockout models revealed that the absence of one of the enzyme impact sperm quality affecting a variety of proteins involved in motility, the ability to fertilize oocyte, and promotes oxidative damage to the sperm DNA. Discussion and conclusions: These findings suggest that each enzyme is playing a specific role, and in most of the cases, no compensatory mechanisms are put in place when one enzyme is absent. This review highlights the different antioxidant enzymes in the epididymis and their role during maturation of the spermatozoon. [ABSTRACT FROM AUTHOR]

Epithelial cells line the lumen of tubular organs and are key players in their respective functions. They establish a unique luminal environment by providing a protective barrier and by performing vectorial transport of ions, nutrients, solutes, proteins, and water. Complex intercellular communication networks, specific for each organ, ensure their interaction with adjacent epithelial and non‐epithelial cells, allowing them to respond to and modulate their immediate environment. In the epididymis, several epithelial cell types work in a concerted manner to establish a luminal acidic milieu that is essential for the post‐testicular maturation and storage of spermatozoa. The epididymis also prevents autoimmune responses against auto‐antigenic spermatozoa, while ensuring protection against ascending and blood pathogens. This is achieved by a network of immune cells that are in close contact and interact with epithelial cells. This review highlights the coordinated interactions between spermatozoa, basal cells, principal cells, narrow cells, clear cells, and immune cells that contribute to the maturation, protection, selection, and storage of spermatozoa in the lumen of the epididymis. [ABSTRACT FROM AUTHOR]

Background: We previously demonstrated the normal mouse epididymal lumen contains a non‐pathological amyloid matrix that surrounds spermatozoa and plays important roles in sperm maturation and protection. Objective: The objective herein was to present a review of this work, including studies showing the amyloid structures of four members of the CRES (cystatin‐related epididymal spermatogenic) subgroup are integral and essential components of the amyloid matrix. Methods: We used conformation‐dependent reagents that recognize the cross‐β‐sheet structure characteristic of amyloid, including thioflavin S (ThS), thioflavin T (ThT), anti‐amyloid antibodies, and X‐ray diffraction, as well as negative‐stain transmission electron microscopy (TEM) to visualize amyloid structures in the epididymal lumen. Antibodies that specifically detect each CRES subgroup family member were also used in indirect immunofluorescence analysis. Results and Discussion: The epididymal lumen contains an amyloid matrix that surrounds maturing spermatozoa and represents a functional amyloid. Alterations in the structure of the amyloid matrix by the loss of the CRES subgroup members or the overexpression of cystatin C result in epididymal pathologies, including infertility. Preliminary data suggest the epididymal amyloid matrix is structurally and functionally similar to bacterial biofilms. Conclusion: Together, these results suggest the amyloid matrix serves important roles in epididymal function including sperm maturation and protection. [ABSTRACT FROM AUTHOR]

Background: Spermatozoa become competent for fertilization during transit through the epididymis. As spermatozoa from the proximal caudal epididymis can fertilize eggs, proteins from the caput and corpus epididymis are required for sperm maturation. Objectives: Microarray analysis identified that more than 17,000 genes are expressed in the epididymis; however, few of these genes demonstrate expression restricted to the epididymis. To analyze epididymis‐enriched gene function in vivo, we generated knockout (KO) mutations in nine genes that are abundantly expressed in the caput and corpus region of the epididymis. Materials and methods: KO mice were generated using the CRISPR/Cas9 system. The histology of the epididymis was observed with hematoxylin and eosin staining. KO males were caged with wild‐type females for 3–6 months to check fertility. Results: We generated individual mutant mouse lines having indel mutations in Pate1, Pate2, or Pate3. We also deleted the coding regions of Clpsl2, Epp13, and Rnase13, independently. Finally, the 150 kb region encoding Gm1110, Glb1l2, and Glb1l3 was deleted to generate a triple KO mouse line. Histology of the epididymis and sperm morphology of all KO lines were comparable to control males. The females mated with these KO males delivered pups at comparable numbers as control males. Discussion and conclusion: We revealed that nine genes abundantly expressed in the caput and corpus epididymis are dispensable for sperm function and male fecundity. CRISPR/Cas9‐mediated KO mice generation accelerates the screening of epididymis‐enriched genes for potential functions in reproduction. [ABSTRACT FROM AUTHOR]

Oestrogen targeting in the human genital ducts is still not well-known. In fact, to date, the localization of oestrogen receptors, ESR1 and ESR2, is controversial and the presence of the membrane oestrogen receptor GPER (G protein-coupled oestrogen receptor) is unexplored. This study has investigated the expression of GPER, ESR1, ESR2 in human ductuli efferentes and proximal caput epididymis by immunohistochemistry and Western blot analysis. Furthermore, the presence of PELP1 (proline-glutamic acid-leucine-rich protein 1), a co-regulator of the oestrogen receptors, was also evaluated. In ductuli efferentes, GPER and ESR1 were clearly localized in all epithelial cells, while ESR2 was evidenced only in ciliated cells. Conversely, the epithelial cells of proximal caput epididymis revealed moderate GPER immunoreactivity, the absence of ERS1 and the occasional presence of ESR2. Furthermore, PELP1 was observed in ciliated cells of ductuli efferentes and in principal cells of proximal caput epididymis. Therefore, this study firstly demonstrated the expression of GPER in human male genital ducts, revealing a new mediator of oestrogen action in these anatomical sites. ESR1 and ESR2 were differentially localized in the two genital tracts together with PELP1, but cell sites of ERs and their co-regulator were not homogeneous. So, a different regional/cellular association of GPER with the classical oestrogen receptors was highlighted, suggesting that oestrogen action could be mediated by GPER, ESR1, ESR2 in ductuli efferentes, while by GPER and, occasionally by ESR2, in proximal caput epididymis. This study suggests that the specific oestrogen-mediated functions in human genital ducts might result from the different local interactions of oestrogens with oestrogen receptors and their co-regulators. [ABSTRACT FROM AUTHOR]

Epididymal lumen fluids are directly responsible for sperm maturation. However, very little is known about the molecular details of small molecule metabolites in the epididymal lumen fluids until now. Here we identified and compared the metabolic profiles of mouse caput and cauda epididymal lumen fluids using GC-MS technique. Among 236 metabolites identified in caput and cauda epididymis, 36 were significantly enriched in caput epididymis while 18 were significantly enriched in cauda epididymis. Pathway analysis identified ascorbate and aldarate metabolism and beta-alanine metabolism as most relevant pathways in caput and cauda epididymis, respectively. Ascorbate, dehydroascorbic acid and beta-alanine associated with these two pathways were firstly reported in mouse epididymal lumen fluids and might play important roles in sperm maturation. [ABSTRACT FROM AUTHOR]

Hormonally active agents are released into the environment from industrial and manufacturing activity. Evidence in the literature indicates that impaired reproductive capacity in wildlife and laboratory species is associated with chemical exposures. In particular, bisphenol A (BPA) and di (2-ethylhexyl) phthalate (DEHP) have generated public interest due to their presence in several consumer products. In this study, we determined that expression of steroid hormone receptors (estrogen and androgen receptors), Wnt4, and β-catenin was greater ( p < 0.05) in the rat epididymis at 35 days of age compared to 21 and 90 days. Second, timed-pregnant Long-Evans dams were exposed to the chemicals BPA and DEHP by gavage from gestational days 12-21. The caput epididymis was collected from cohorts of male offspring at 35 and 90 days of age and processed for Western blot analysis. Results showed that prenatal BPA and DEHP exposures affected ( p < 0.05) expression of estrogen and androgen receptor, Wnt4, β-catenin, MAPK, and HOXD4 protein in the epididymis. Data have implications for morphological development of the epididymis, a possibility that would be explored in future studies. [ABSTRACT FROM AUTHOR]