Blood-epididymis Barrier Research Articles

Hypoxia-sensitive cells trigger NK cell activation via the KLF4-ASH1L-ICAM-1 axis, contributing to impairment in the rat epididymis

Male infertility is a global health problem especially prevalent in high-altitude regions. The epididymis is essential for sperm maturation, but the influence of environmental cues on its reshaping remains poorly understood. Here, we use single-cell transcriptomics to track the cellular profiles of epidydimal cells in rats raised under normoxia or extended hypoxia. The results show that hypoxia impairs epididymal function, evident in reduced epithelial cells, compromised blood-epididymis barrier integrity, and increased natural killer cells. Through combined analysis of gene-regulatory networks and cell-cell interaction maps, we identify epididymal hypoxia-sensitive cells that communicate with natural killer (NK) cells via increased intercellular adhesion molecule 1 (ICAM-1) driven by KLF4 recruitment of the histone methyltransferase ASL1L to the Icam1 promoter. Taken together, our study offers a detailed blueprint of epididymal changes during hypoxia and defines a KLF4-ALSH1L-ICAM-1 axis contributing to NK cell activation, yielding a potential treatment targeting hypoxia-induced infertility.

CX3CR1 deficiency leads to impairment of immune surveillance in the epididymis.

Mononuclear phagocytes (MPs) play an active role in the immunological homeostasis of the urogenital tract. In the epididymis, a finely tuned balance between tolerance to antigenic sperm and immune activation is required to maintain epididymal function while protecting sperm against pathogens and stressors. We previously characterized a subset of resident MPs that express the CX3CR1 receptor, emphasizing their role in antigen sampling and processing during sperm maturation and storage in the murine epididymis. Bacteria-associated epididymitis is the most common cause of intrascrotal inflammation and frequently leads to reproductive complications. Here, we examined whether the lack of functional CX3CR1 in homozygous mice (CX3CR1EGFP/EGFP, KO) alters the ability of MPs to initiate immune responses during epididymitis induced by LPS intravasal-epididymal injection. Confocal microscopy revealed that CX3CR1-deficient MPs located in the initial segments of the epididymis displayed fewer luminal-reaching membrane projections and impaired antigen capture activity. Moreover, flow cytometry showed a reduction of epididymal KO MPs with a monocytic phenotype under physiological conditions. In contrast, flow cytometry revealed an increase in the abundance of MPs with a monocytic signature in the distal epididymal segments after an LPS challenge. This was accompanied by the accumulation of CD103+ cells in the interstitium, and the prevention or attenuation of epithelial damage in the KO epididymis during epididymitis. Additionally, CX3CR1 deletion induced downregulation of Gja1 (connexin 43) expression in KO MPs. Together, our study provides evidence that MPs are gatekeepers of the immunological blood-epididymis barrier and reveal the role of the CX3CR1 receptor in epididymal mucosal homeostasis by inducing MP luminal protrusions and by regulating the monocyte population in the epididymis at steady state as well as upon infection. We also uncover the interaction between MPs and CD103+ dendritic cells, presumably through connexin 43, that enhance immune responses during epididymitis. Our study may lead to new diagnostics and therapies for male infertility and epididymitis by identifying immune mechanisms in the epididymis.

Differential expression and localization of tight junction proteins in the goat epididymis.

The blood-epididymis barrier (BEB) forms a unique microenvironment that is crucial for the maturation, protection, transport, and storage of spermatozoa in the epididymis. To characterize the function of tight junctions (TJs), which are constitutive components of the BEB, we determined the expression and localization of TJ proteins such as zonula occludens (ZO)-1, 2, and 3, occludin, and claudin3 (Cldn3) during postnatal development in the goat epididymis. To assess the expression patterns of TJ proteins in immature (3 months of age) and mature (14 months of age) goat epididymides, two different experimental methods were used including immunofluorescence labeling and western blotting. We show that, ZO-1, 2, and 3, and occludin, were strictly expressed and localized to the TJs of the goat epididymis, whereas Cldn3 was present in basolateral membranes as well as TJs. All TJ proteins examined were more highly expressed in the immature epididymis compared to levels in mature tissue. In conclusion, our study indicates that at least five TJ proteins, namely ZO-1, ZO-2, ZO-3, occludin, and Cldn3, are present in TJs, and the expression strength and pattern of TJ proteins tend to be age dependent in the goat epididymis. Together, these data suggest that the distinct expression patterns of TJ proteins are essential for regulating components of the luminal contents in the epididymal epithelium and for forming adequate luminal conditions that are necessary for the maturation, protection, transport, and storage of spermatozoa in the goat epididymis.

Testis and blood-testis barrier in Covid-19 infestation: role of angiotensin-converting enzyme 2 in male infertility.

Severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) that causes COVID-19 infections penetrates body cells by binding to angiotensin-converting enzyme-2 (ACE2) receptors. Evidence shows that SARS-CoV-2 can also affect the urogenital tract. Hence, it should be given serious attention when treating COVID-19-infected male patients of reproductive age group. Other viruses like HIV, mumps, papilloma and Epstein-Barr can induce viral orchitis, germ cell apoptosis, inflammation and germ cell destruction with attending infertility and tumors. The blood-testis barrier (BTB) and blood-epididymis barrier (BEB) are essential physical barricades in the male reproductive tract located between the blood vessel and seminiferous tubules in the testes. Despite the significant role of these barriers in male reproductive function, studies have shown that a wide range of viruses can still penetrate the barriers and induce testicular dysfunctions. Therefore, this mini-review highlights the role of ACE2 receptors in promoting SARS-CoV-2-induced blood-testis/epididymal barrier infiltration and testicular dysfunction.

Distribution pattern of ZO-1 and claudins in the epididymis of vampire bats

ABSTRACT Epithelial cells connect with each other by tight junctions (TJs) in several tissues. In epididymides, TJs proteins form the blood-epididymis barrier (BEB), which is crucial for male fertility. However, little is known about BEB morphological and physiological aspects in wild animals. This study examines the region-specific distribution pattern of TJs proteins in D. rotundus’ epididymis, assessing their regulation in rainy and dry season. The expression of zonula occludens-1 (ZO-1), and claudins (Cldn)-1, −3, and −4 were evaluated by confocal immunofluorescence and ELISA analysis. Herein, ZO-1 was strictly expressed in TJs, whereas Cldns were expressed in TJs and basolateral membranes of epithelial cells. Their co-localization and intensity of expression varied in the epididymal regions examined. The effect of season on protein expression was detected mainly in TJ proteins located in the proximal regions. As such, in the initial segment (IS), Cldn-3 and −4 were detected at low levels in basolateral membranes in the rainy season compared to the dry season. Furthermore, in the distal IS, Cldn-1 expression was lower in TJs of epithelial cells during the rainy season than the dry season. ZO-1 expression was higher in the cauda region than the corpus region by ELISA analysis. Additionally, in the corpus region, ZO-1 expression was higher in TJs during dry season compared to the rainy season. Our study sheds light on the understanding of BEB in D. rotundus, improving the knowledge of their reproductive biology.

Chronic sleep loss disrupts blood-testis and blood-epididymis barriers, and reduces male fertility.

Sleep loss increases blood-brain barrier permeability. As the blood-brain barrier and the blood-tissue barriers in the reproductive tract (blood-testis and blood-epididymis barriers) share common characteristics, we hypothesized that sleep restriction may also modify their barrier function. Previous reports showed that sleep loss decreased sperm viability and progressive fast mobility, which may be a consequence of altered blood-testis and blood-epididymis barrier. Therefore, we quantified changes in blood-testis and blood-epididymis barrier after sleep loss and related them to male fertility. Adult male Wistar rats were sleep restricted using the multiple-platform technique in a protocol of 20hr daily sleep deprivation plus 4hr of sleep recovery in the home-cage. At the 10th day, barrier permeability assays were performed with Na-fluorescein, 10kDa Cascade blue-dextrans and Evans blue, and the expression of tight junction proteins, actin and androgen receptor was quantified. At the 10th day of sleep restriction and after sleep recovery days 1-7, males were placed with sexually receptive females, sexual behaviour was tested, and the percentage of pregnancies was calculated. Sleep restriction increased the barrier permeability to low- and high-molecular-weight tracers, and decreased the expression of tight junction proteins, actin and androgen receptor. Concomitantly, sleep restriction reduced the percentage of ejaculating males and the number of pregnancies. Sleep recovery for 2-3days progressively re-established fertility, as indicated by a higher percentage of ejaculating males and impregnated females. In conclusion, chronic sleep loss alters fertility concomitantly with the disruption of the blood-tissue barriers at the reproductive tract, the mechanism involves androgen signalling.

Identification of a missense variant in CLDN2 in obstructive azoospermia.

Obstructive azoospermia (OA), defined as an obstruction in any region of the male genital tract, accounts for 40% of all azoospermia cases. Of all OA cases, ~30% are thought to have a genetic origin, however, hitherto, the underlying genetic etiology of the majority of these cases remain unknown. To address this, we took a family-based whole-exome sequencing approach to identify causal variants of OA in a multiplex family with epidydimal obstruction. A novel gain-of-function missense variant in CLDN2 (c.481G>C; p.Gly161Arg) was found to co-segregate with the phenotype, consistent with the X-linked inheritance pattern observed in the pedigree. To assess the pathogenicity of this variant, the wild and mutant protein structures were modeled and their potential for strand formation in multimeric form was assessed and compared. The results showed that dimeric and tetrameric arrangements of Claudin-2 were not only reduced, but were also significantly altered by this single residue change. We, therefore, envisage that this amino acid change likely forms a polymeric discontinuous strand, which may lead to the disruption of tight junctions among epithelial cells. This missense variant is thus likely to be responsible for the disruption of the blood-epididymis barrier, causing dislodged epithelial cells to clog the genital tract, hence causing OA. This study not only sheds light on the underlying pathobiology of OA, but also provides a basis for more efficient diagnosis in the clinical setting.

Busulfan administration produces toxic effects on epididymal morphology and inhibits the expression of ZO-1 and vimentin in the mouse epididymis.

Busulfan is an alkane sulphonate currently used as an anticancer drug and to prepare azoospermic animal models, because it selectively destroys differentiated spermatogonia in the testes. However, few studies have focussed on the exact effects of busulfan treatment on the epididymis currently. The present study assessed the effect of busulfan on epididymal morphology and the blood–epididymis barrier in mice. We treated mice with a single injection of busulfan and detected the effect at different time points. We showed that busulfan was toxic to the morphological structure and function of the epididymis. Furthermore, busulfan treatment down-regulated the epididymal expression of vimentin and zonula occludens-1 (ZO-1) at the mRNA and protein levels. In addition, there was an increase in total androgen receptor (AR) levels, whereas the estrogen receptor-α (ER-α) levels were reduced, both in the caput and cauda regions after busulfan treatment, which may be secondary to the testicular damage. In conclusion, our study describes the effects of busulfan administration on the mouse epididymis and also provides a potential understanding of male infertility arising from chemotherapy-related defects in the epididymis.

Two fibrinogen-like proteins, FGL1 and FGL2 are disulfide-linked subunits of oligomers that specifically bind nonviable spermatozoa

Nevertheless, a nonviable sperm population is present in the cauda epididymidis of many species. Degenerating spermatozoa release enzymes that could have detrimental effects on the viability of neighboring cells, and they are source of autoantigens that induce an autoimmune response if they escape the blood-epididymis barrier. Does the epididymis have specialized protective mechanism(s) to segregate the viable sperm population from defective spermatozoa? Previously, we identified a fibrinogen-like protein-2 (fgl2) that specifically binds to and polymerizes into a cocoon-like complex coating defective spermatozoa and sperm fragments. The objective of the present study is to identify the subunit composition of the fgl2-containing oligomers both in the soluble and cocoon-like complex. Our proteomic studies indicate that the 260/280kDa oligomers (termed eFGL) contain two distinct disulfide-linked subunits; 64kDa fgl2 and 33kDa fgl1. Utilizing a PCR-based cloning strategy, the 33kDa polypeptide has been identified as fibrinogen-like protein-1 (fgl1). Immunocytochemical studies revealed that fgl1 selectively binds to defective spermatozoa in the cauda epididymidis. Northern blot analysis and in situ hybridization demonstrated the high expression of fgl1 in the principal cells of the proximal cauda epididymidis. Co-immunoprecipitation analyses of cauda epididymal fluid, using anti-fgl2, demonstrate that both fgl1 and fgl2 are present in the soluble eFGL. Our study is the first to show an association of fgl1 and fgl2 both in the soluble and in the sperm-associated eFGL. We conclude that our results provide new insights into the mechanisms by which the potentially unique epididymal protein functions in the recognition and elimination of defective spermatozoa.

The MAPK/ERK-Signaling Pathway Regulates the Expression and Distribution of Tight Junction Proteins in the Mouse Proximal Epididymis.

The initial segment (IS) in rodents is functionally and structurally distinct from other epididymal segments and plays an important role in sperm maturation. The MAPK/ERK1/2 pathway is maintained active in the IS by testicular luminal factors and plays crucial roles in the maintenance and differentiation of the IS epithelium. Tight junctions (TJs) are constituents of the blood-epididymis barrier, which mediates the paracellular transport of ions, solutes, and water and controls epithelial cell differentiation, thereby contributing to the establishment of a unique luminal environment. We examine here the role of the MAPK/ERK1/2 pathway in the regulation of TJ proteins in the IS. Inhibition of mitogen activated protein kinase kinase (MAPKK or MEK1/2) with PD325901, followed by reduction of ERK1/2 phosphorylation (pERK), decreased zonula occludens (ZO)-2 expression and increased ZO-3 expression in TJs but had no effect on ZO-1 expression. In control mice, in addition to being located in TJs, claudin (Cldn)-1, Cldn-3, and Cldn-4 were detected in the basolateral membrane of epithelial cells, with enriched expression of Cldn-1 and Cldn-4 in basal cells. PD325901 reduced the expression of Cldn-1 and Cldn-4 at all locations without affecting Cldn-3. Occludin was undetectable in the IS of control mice, but PD325901 triggered its expression in TJs. No effect was observed for any of the proteins examined in the other epididymal regions. Our results indicate the participation of the MAPK/ERK1/2 pathway in the regulation of cell-cell events that control the formation and maintenance of the blood-epididymis barrier.

Isolated Rat Epididymal Basal Cells Share Common Properties with Adult Stem Cells.

There is little information on the function of epididymal basal cells. These cells secrete prostaglandins, can metabolize radical oxygen species, and have apical projections that are components of the blood-epididymis barrier. The objective of this study was to develop a reproducible protocol to isolate rat epididymal basal cells and to characterize their function by gene expression profiling. Integrin-alpha6 was used to isolate a highly purified population of basal cells. Microarray analysis indicated that expression levels of 552 genes were enriched in basal cells relative to other cell types. Among these genes, 45 were expressed at levels of 5-fold or greater. These highly expressed genes coded for proteins implicated in cell adhesion, cytoskeletal function, ion transport, cellular signaling, and epidermal function, and included proteases and antiproteases, signal transduction, and transcription factors. Several highly expressed genes have been reported in adult stem cells, suggesting that basal cells may represent an epididymal stem cell population. A basal cell culture was established that showed that these basal cells can differentiate in vitro from keratin (KRT) 5-positive cells to cells that express KRT8 and connexin 26, a marker of columnar cells. These data provide novel information on epididymal basal cell gene expression and suggest that these cells can act as adult stem cells.

Tricellulin and its role in the epididymal epithelium of the rat.

Tricellulin is a tight-junction protein present at tricellular tight junctions. It has been suggested that basal cells are implicated in the blood-epididymis barrier. Basal cells express claudins, a component of tight junctions; however, there is no information regarding the potential architecture or regulation of basal cell-principal cell interactions. The present objectives were to determine the expression and localization of tricellulin in rat epididymis in relation to occludin, basal cell-principal cell interactions, and other junctional proteins. Tricellulin levels were similar in all segments of the adult epididymis, and the protein was localized to the apical region of the epithelium. Postnatal development showed that tricellulin levels increased with age and localization changed from cytoplasmic to membrane-bound as a function of age. Colocalization with occludin indicated that both proteins are in the region of the tight junction. In the initial segment, the proteins did not colocalize compared to the epididymis where they were both colocalized. Tricellulin did not colocalize with cytokeratin 5, a marker of basal cells, in any region of the epididymis, including the corpus and cauda epididymidis, where apical projections of basal cells were apparent. Tricellulin knockdown studies using small interfering RNA in rat caput epididymal principal cells resulted in decreased transepithelial resistance and was correlated with decreased levels of Cldn3, Cldn1, and occludin. Tight-junction protein1, also known as ZO-1, and cadherin1 levels were unchanged. This is the first report of tricellulin in the epididymis and on the interaction between tricellulin and other tight-junction proteins.

Mononuclear phagocytes rapidly clear apoptotic epithelial cells in the proximal epididymis.

We have shown previously that a network of mononuclear phagocytes (MPs) expressing macrophage and dendritic cell markers such as CD11c, F4/80 and CX3CR1, lines the base of the epididymal tubule. However, in the initial segment (IS) and only in that particular segment, epididymal MPs establish extremely close interactions with the epithelium by projecting slender dendrites between most epithelial cells. We undertook the present study to determine how epididymal phagocytes respond to the transient wave of apoptosis initiated by unilateral efferent duct ligation (EDL) in the epididymal epithelium. We show profound morphological and phenotypical changes restricted to the MPs populating the proximal epididymis following EDL. Within 48 h, a large subset of IS epithelial cells had entered an apoptotic state, visualized by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and CD11c(+) and CX3CR1(+) MPs readily engulfed TUNEL-positive cells and other debris. Despite the high levels of apoptosis and the rapid clearance of apoptotic cells occurring after EDL, the epithelium preserved its overall architecture and maintained tight junctions of the blood-epididymis barrier (BEB). The discovery of a functional population of MPs in the epididymal epithelium responsible for maintaining the integrity of the BEB raises further questions regarding the role of these cells in clearing defective epithelial cells in the steady-state epididymis, as well as pathogens and abnormal spermatozoa in the lumen.

TGFβs Modulate Permeability of the Blood-Epididymis Barrier in an In Vitro Model

The blood-epididymis barrier (BEB) is formed by epithelial tight junctions mediating selective permeability of the epididymal epithelium. Defective barrier function can disturb the balance of the epididymal milieu, which may result in infertility. The stroma of the epididymis contains high amounts of cytokines of the TGFβ family of unknown function. We screened possible effects of all three TGFβ isoforms on paracellular tightness in a BEB in vitro model based on the strongly polarized mouse epididymal epithelial MEPC5 cells in the transwell system. In this model we found a robust transepithelial electrical resistance (TER) of about 840 Ω x cm2. Effects on the paracellular permeability were evaluated by two methods, TER and FITC-Dextran-based tracer diffusion assays. Both assays add up to corresponding results indicating a time-dependent disturbance of the BEB differentially for the three TGFβ isoforms (TGFβ3>TGFβ1>TGFβ2) in a TGFβ-recetor-1 kinase- and Smad-dependent manner. The tight junction protein claudin-1 was found to be reduced by the treatment with TGFβs, whereas occludin was not influenced. Epididymal epithelial cells are predominantly responsive to TGFβs from the basolateral side, suggesting that TGFβ may have an impact on the epididymal epithelium from the stroma in vivo. Our data show for the first time that TGFβs decrease paracellular tightness in epididymal epithelial cells, thus establishing a novel mechanism of regulation of BEB permeability, which is elementary for sperm maturation and male fertility.

Are the basal cells of the mammalian epididymis still an enigma?

Basal cells are present in the columnar pseudostratified epithelium covering the epididymis of all mammalian species, which regulates the microenvironment where the functionally incompetent germ cells produced by the testis are matured and stored. Striking novelties have come from investigations on epididymal basal cells in the past 30-40 years. In addition to an earlier hypothesised scavenger role for basal cells, linked to their proven extratubular origin and the expression of macrophage antigens, basal cells have been shown to be involved in cell-cell cross-talk, as well as functioning as luminal sensors to regulate the activity of principal and clear cells. Involvement of basal cells in the regulation of electrolyte and water transport by principal cells was hypothesised. This control is suggested to be mediated by the local formation of prostaglandins. Members of the aquaporin (AQP) and/or aquaglyceroporin family (AQP3, AQP7 and AQP8) are also specifically expressed in the rat epididymal basal cells. Transport of glycerol and glycerylphosphorylcholine from the epithelium of the epididymis to the lumen in relation to sperm maturation may be mediated by AQP. Most probably basal cells collaborate to the building up of the blood-epididymis barrier through cell adhesion molecules, implying an involvement in immune control exerted towards sperm cells, which are foreigners in the environment in which they were produced.

A Novel Effect of Polychlorinated Biphenyls: Impairment of the Tight Junctions in the Mouse Epididymis

Polychlorinated biphenyls (PCBs) exhibit a broad range of adverse biological effects, including reproductive toxicity. However, the mechanisms by which PCBs disrupt the epididymis remain obscure. We analyzed the gene expression profile in mice epididymis exposed to PCBs (Aroclor 1254) at doses comparable to human exposure using a cDNA microarray. Differentially expressed genes were involved in a variety of function categories and biological pathways, including GTP binding, nucleosome assembly, and ribosome and protein disulfide isomerase. The differentially expressed genes related to GTP binding were highly enriched. The abundance of GTP binding proteins related to tight junctions being reduced and the phosphorylation level of their downstream effectors were impaired after exposure to PCBs. The results of tracer studies demonstrated that the permeability of blood-epididymis barrier was increased by PCB exposure. In addition, PCB exposure also disrupted the expression of the tight junction proteins, zonula occludens-1 and occludin. We demonstrated for the first time that exposure to PCBs at doses relevant for the general population was able to affect the blood-epididymis barrier in mice through altering GTP binding and tight junction proteins. Our results provided a novel insight into the molecular mechanisms linking PCB exposure to sperm maturation.

The Blood-Testis and Blood-Epididymis Barriers Are More than Just Their Tight Junctions1

The terms blood-testis barrier (BTB) or blood-epididymis barrier (BEB), are often described as Sertoli cell-Sertoli cell tight junctions (TJs) or TJs between the epithelial cells in the epididymis, respectively. However, in reality, the BTB and BEB are much more complex than just the TJ. The focus of this minireview is to remind readers that the complete BTB and BEB are comprised of three components: anatomical, physiological, and immunological. The TJs form the anatomical (physical) barrier that restricts passage of molecules and cells from entering or exiting the lumen. The physiological barrier is comprised of transporters that regulate movement of substances in or out of the lumen, thus creating a microenvironment, which is critical for the proper development and maturation of germ cells. The immunological barrier limits access by the immune system and sequesters the majority of the autoantigenic germ cells. Combined with the overall immune-privilege of the testis, this suppresses detrimental immune responses against the autoantigenic germ cells. These three components on their own do not create a complete functional barrier; instead, it is the interaction between all three components that create a barrier of maximal competence.

Alterations in the Human Blood-Epididymis Barrier in Obstructive Azoospermia and the Development of Novel Epididymal Cell Lines from Infertile Men1

Post-testicular sperm maturation requires a specific luminal environment in the epididymis that is created, in part, by the blood-epididymis barrier. There is limited information on gene expression in the epididymis of infertile obstructive azoospermia (OA) patients due to the difficulty in obtaining tissues. The objectives of this study were to determine if epididymal tight junction proteins are altered in OA and to develop cell lines that could serve to elucidate alterations in the epididymis of infertile men. Epididymal claudin (CLDN) 1, CLDN4, and CLDN10 mRNA levels were altered in OA downstream from the obstruction site. Epithelial cell lines derived from the caput epididymidis of one OA patient were developed (infertile human caput epididymal cell line [IHCE]). IHCEs were composed of homogenous populations of diploid cells that ultrastructurally resembled in vivo principal cells. The cells expressed cytokeratin, SPAG11B, CLDN2, CLDN3, desmoplakin, and vimentin. However, the cells did not express several other epididymal markers (CRISP1, SPINLW1, NPC2, CD52, or DCXR) or junctional proteins (CDH1, CDH2, CLDN1, CLDN4, CLDN7, or CLDN8). Further studies using IHCE1 and transepithelial resistance indicated that the cells were unable to form tight junctions. Microarray analyses comparing gene expression in IHCE1 and a recently developed fertile human caput epididymal cell line revealed differential expression of genes encoding junctional proteins, cell junction regulators, and epididymal proteins. Together, these data indicate that epididymal cellular junctions appear to be altered in OA.

Assessing the Role of Claudins in Maintaining the Integrity of Epididymal Tight Junctions Using Novel Human Epididymal Cell Lines1

The epididymis is responsible for posttesticular sperm maturation. Sperm maturation is dependent on the luminal microenvironments along the epididymis. Though the role of the epididymis is well established, the molecular and cellular mechanisms responsible for sperm maturation remain to be elucidated, particularly in the human, as limited biological tools exist. We have established the first stable epithelial cell lines transformed with SV40 large T antigen (LTAg) from two regions of the human adult epididymis. The cell lines are composed of homogenous populations of diploid principal cells that possess ultrastructural characteristics similar to those of human principal cells in vivo. These cells express transcripts for adherens (cadherins CDH1 and CDH2) and tight (claudins CLDN1, CLDN2, CLDN3, CLDN4, CLDN7, and CLDN8) junctions as well as desmosomes (desmoplakin, DSP). Transepithelial resistance (TER) measurements in fertile human caput epididymal cell line 1 (FHCE1) as well as the immunolocalization of tight junctional protein 1 (TJP1), occludin, and CLDN1 indicate that these cells form functional tight junctions. Furthermore, knockdown of CLDN1, CLDN3, CLDN4, or CLDN7 using specific siRNAs resulted in significant decreases in TER, suggesting that these CLDNs are essential for the barrier function of the blood-epididymis barrier. Disruption of CLDN1, CLDN3, CLDN4, and CLDN7 could, therefore, lead to epididymal dysfunction, resulting in male infertility.

Investigation of epididymal immunology

Immunology is the study of the structure and function of the immune system. The immune system consists of an earlier-stage innate immunity and a later-stage adaptive immunity. The task of the immune system is to efficiently respond to non-self antigens and the invasion of pathogens, thereby protecting the host's homeostasis. This review article discusses the structure and function of the epididymis, including the composition of the epithelial cells of the epididymis and their relationship to the immune system, through the assessment of alterations in the immune cells of the epididymis. The review also shows the anti-inflammatory properties of rat epididymal defensin and the description of the blood-epididymis barrier, immune barrier, epididymitis and pathological mechanisms of infertility in males. Taken together, we see that the epididymis possesses a close link with immunology. Finally, this review discusses the future of studies involving epididymal immunology.