Epididymal Epithelium Research Articles

Functional genomics of the human epididymis: Further characterization of efferent ducts and model systems by single-cell RNA sequencing analysis.

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. 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. 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. 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. 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. Single-cell RNA-seq data from the human epididymis make a valuable contribution to our understanding of this highly specialized organ.

Conditioned medium and secretome from epididymal epithelial cell cultures improve sperm kinetics and capacitation.

Sperm maturation occurs in the epididymis through interactions with existing molecules inside the lumen. However, the mechanism of epididymis molecular transfer is currently unclear. This study was aimed to determine the necessity of the epididymal epithelial cells (EECs) in the process of sperm maturation in terms of sperm kinetics and tyrosine phosphorylation. A true experimental research design was used in this study. The medium tested was a primary culture of mice caput epididymal cells (cells and culture medium), conditioned medium (CM) (supernatant of EECs), and secretome (CM filtered at 0.22 μm). Sperm was cocultured in EEC culture, CM, and secretome for 1, 2, 3, or 4 h. The original culture medium was used as the control. Sperm kinetic analysis was performed after the indicated times using computer-assisted sperm analysis, and tyrosine phosphorylation was detected using the Western blot technique. A primary culture of caput EECs was successfully generated. The results showed increased sperm motility and progressive movement after 3 h of incubation (p < 0.05). There was a significant decrease in the average path velocity (VAP) values after 4 h of incubation (p < 0.05), but there was no significant change in the 1, 2, and 3 h incubation groups. The EEC culture-CM and secretome groups showed a significant increased progressivity and VAP percentage values compared with the control medium (p < 0.05). In terms of percentage motility, the culture and CM groups were significantly different from the control medium, but the secretome group was not. The sperm kinetics of sperm cultured in CM, secretome, and EEC were significantly increased after 3 h of incubation, suggesting that CM and secretome can be used to replace EECs, especially when analyzing molecules secreted by the epididymal epithelium during sperm maturation. The results of this study highlight the potential of CM and secretome as therapy mediums for sperm kinetic abnormalities.

Aerobic exercises regulate the epididymal anion homeostasis of high-fat diet-induced obese rats through TRPA1-mediated Cl- and HCO3- secretion†.

Aerobic exercises could improve the sperm motility of obese individuals. However, the underlying mechanism has not been fully elucidated, especially the possible involvement of the epididymis in which sperm acquire their fertilizing capacity. This study aims to investigate the benefit effect of aerobic exercises on the epididymal luminal milieu of obese rats. Sprague-Dawley male rats were fed on a normal or high-fat diet (HFD) for 10 weeks and then subjected to aerobic exercises for 12weeks. We verified that TRPA1 was located in the epididymal epithelium. Notably, aerobic exercises reversed the downregulated TRPA1 in the epididymis of HFD-induced obese rats, thus improving sperm fertilizing capacity and Cl- concentration in epididymal milieu. Ussing chamber experiments showed that cinnamaldehyd (CIN), agonist of TRPA1, stimulated an increase of the short-circuit current (ISC) in rat cauda epididymal epithelium, which was subsequently abolished by removing the ambient Cl- and HCO3-. In vivo data revealed that aerobic exercises increased the CIN-stimulated Cl- secretion rate of epididymal epithelium in obese rats. Pharmacological experiments revealed that blocking cystic fibrosis transmembrane regulator (CFTR) and Ca2+-activated Cl- channel (CaCC) suppressed the CIN-stimulated anion secretion. Moreover, CIN application in rat cauda epididymal epithelial cells elevated intracellular Ca2+ level, and thus activate CACC. Interfering with the PGHS2-PGE2-EP2/EP4-cAMP pathway suppressed CFTR-mediated anion secretion. This study demonstrates that TRPA1 activation can stimulate anion secretion via CFTR and CaCC, which potentially forming an appropriate microenvironment essential for sperm maturation, and aerobic exercises can reverse the downregulation of TRPA1 in the epididymal epithelium of obese rats.

Morphological and histochemical identification of telocytes in adult yak epididymis

Telocytes (TCs) are a newly discovered type of mesenchymal cell that are closely related to the tissue’s internal environment. The study aimed to investigate the morphological identification of TCs in the epididymis of adult yak and their role in the local microenvironment. In this study, transmission electron microscopy (TEM), scanning electron microscopy, immunofluorescence, qRT-PCR, and western blotting were used to analyze the cell morphology of TCs. The results showed that there are two types of TCs in the epididymal stroma of yak by TEM; one type is distributed around the capillaries with full cell bodies, longer TPs, and a large number of secretory vesicles; the other is distributed outside the basement membrane with irregularly long, striped, large nuclei and short telopodes (TPs). In addition, these TCs formed complex TC cell networks through TPs with epididymal interstitial capillaries and basal fibroblasts. TCs often appear near the capillaries and basement membrane by special staining. The surface markers of TCs (CD34, vimentin, and CD117) were positively expressed in the epididymal stroma and epithelium by immunohistochemistry, and immunofluorescence co-expression of vimentin + CD34 and CD117 + CD34 was observed on the surface of TCs. The trends in the mRNA and protein expression of TCs surface markers revealed expression was highest in the caput epididymis. In summary, this is first report of TCs in the epididymis of yak, and two phenotypes of TCs were observed. The existence and distribution characteristics of TCs in the epididymis of plateau yaks provide important clues for further study of the adaptation to reproductive function in the plateau.

Transfer of Galectin-3-Binding Protein via Epididymal Extracellular Vesicles Promotes Sperm Fertilizing Ability and Developmental Potential in the Domestic Cat Model.

Key proteins transferred by epididymal extracellular vesicles (EVs) to the transiting sperm cells contribute to their centrosomal maturation and developmental potential. Although not reported in sperm cells yet, galectin-3-binding protein (LGALS3BP) is known to regulate centrosomal functions in somatic cells. Using the domestic cat model, the objectives of this study were to (1) detect the presence and characterize the transfer of LGALS3BP via EVs between the epididymis and the maturing sperm cells and (2) demonstrate the impact of LGALS3BP transfer on sperm fertilizing ability and developmental potential. Testicular tissues, epididymides, EVs, and spermatozoa were isolated from adult individuals. For the first time, this protein was detected in EVs secreted by the epididymal epithelium. The percentage of spermatozoa with LGALS3BP in the centrosome region increased as cells progressively incorporated EVs during the epididymal transit. When LGALS3BP was inhibited during in vitro fertilization with mature sperm cells, less fertilized oocytes and slower first cell cycles were observed. When the protein was inhibited in epididymal EVs prior to incubation with sperm cells, poor fertilization success further demonstrated the role of EVs in the transfer of LGALS3BP to the spermatozoa. The key roles of this protein could lead to new approaches to enhance or control fertility in clinical settings.

Seasonal spermatogenesis, epididymal storage, and creatine kinase expression in Pelodiscus sinensis

The soft-shelled turtle, Pelodiscus sinensis, is an important economic aquaculture species. Its reproduction exhibits seasonality; however, there is a lack of systematic studies focused on sperm maturation and epididymal storage. The testes and epididymides of P. sinensis were sampled from March to December. The seasonal reproduction and maturation of the spermatozoa were examined by anatomy, hematoxylin and eosin staining, AB-PAS staining, and immunohistochemistry. Spermatogenesis exhibited obvious seasonality in P. sinensis. It was found that the spermatogenic epithelium was most active during June to September, whereas the diameter of the epididymal tubules was smallest during June to October. As key enzymes of ATP metabolism, creatine kinases were highly expressed in the epididymal tubule epithelium during the breeding season, which may be important for the regulation of sperm maturation. In addition, the epididymal tubule epithelium changed with the season in June to September, the epididymal tubule epithelium proliferated to form villous structures, and secreted a large number of glycoproteins, which may be related to the rapid maturation of sperm during the breeding season. In conclusion, this study provided insights into the spermatogenesis of P. sinensis through histological analysis and enriched our understanding of reproduction in reptiles.

Pharmacological inhibition of endoplasmic reticulum stress mitigates testicular pathology in a mouse model of simulated microgravity

BackgroundSimulated microgravity during hindlimb unloaded (HU) induces multi-organ pathologies in mice, including testicular dysfunction. However, a detailed characterization of testicular histology and its driving molecular mechanisms remains elusive. We investigated the potential contribution of elevated endoplasmic reticulum (ER) stress to testicular atrophy in HU mice. MethodsWe divided male c57BL/6j mice into a control group (C) or unloaded mice without treatment (U), or treated with 4PBA (UP), an ER stress inhibitor, for three weeks. At the end of the experiment, mice were euthanized, and testes tissues were dissected for histopathology and mRNA sequencing. ResultsHU was associated with significant testicular atrophy (p = 0.03) and several histopathological alterations, including a reduction in the diameter of seminiferous tubules (p < 0.001), epithelial thinning (p < 0.001), reduced sperm density (p < 0.001), and thickening of the epididymal epithelium (p < 0.001). mRNA sequencing revealed alterations in several pathways associated with oxidative stress, protein catabolism, and inflammation induction (all p < 0.05). Three weeks of treatment with 4PBA prevented testicular atrophy (p = 0.214), partly restored testicular microarchitecture, and reversed changes in the transcriptomic profiling of HU mice. ConclusionOur novel findings indicate a mechanistic role of elevated ER stress in causing testicular pathology during HU conditions. Based on findings, we report a therapeutic potential of 4PBA in reversing testicular pathology in conditions mimicking prolonged bed rest and spaceflight.

Immunolocalisation of aquaporins 3, 7, 9 and 10 in the epididymis of three wild ruminant species (Iberian ibex, mouflon and chamois) and sperm cryoresistance.

In the epididymis, epithelial cells manage changes in the luminal environment for proper sperm maturation. Moreover, aquaglyceroporins, a subgroup of aquaporins (AQP), modulate the transport of water, glycerol and other small molecules in epithelial cells. We aim to characterise the lining epithelium, quantify its cell composition and immunolocalise the aquaglyceroporins AQP3, AQP7, AQP9 and AQP10 alongside the epididymal ductus of three wild ruminant species, and to determine if species-specific differences could be associated with cauda sperm cryoresistance variations. Epididymides from Iberian ibex (n =5), mouflon (n =5) and chamois (n =6) were obtained. Cauda spermatozoa were collected and sperm parameters were analysed before and after freezing. Histology and immunohistochemistry of AQP3, 7, 9, 10 and T-CD3 were performed in the caput, corpus and cauda epididymal regions. This work first describes the lining epithelium in Iberian ibex, mouflon and chamois epididymis along the three anatomical regions, consisting of principal, basal, apical, clear and halo cells. However, the percentage of each cell type differed in ibex compared to mouflon and chamois. The positive T-CD3 immunolabeling of all the halo cells confirmed their T-lymphocyte nature. Aquaglyceroporin expression patterns were similar among species, except for differences in AQP7 and AQP10 immunolocalisation in ibex. Species-specific differences in epididymal sperm cryoresistance were confirmed. The epididymal epithelium of the three wild ruminants differ in their relative number of cell types and AQP immunolocalisation, which ultimately appears to affect cauda epidydimal spermatozoa cryoresistance. Our study provides information on the relevance of the quantitative composition and AQP pattern expression in epididymal lining epithelium on sperm cryoresistance.

Cathepsin B plays a role in spermatogenesis and sperm maturation through regulating autophagy and apoptosis in mice.

Spermatogenesis and sperm maturation are complex and highly ordered biological processes. Any failure or disorder in these processes can cause defects in sperm morphology, motility, and fertilization ability. Cathepsin B (CTSB) is involved in the regulation of a variety of pathological processes. In the present study, we found that CTSB was abundantly expressed in the male reproductive system, however, the specific role of CTSB in regulating spermatogenesis and sperm maturation remained elusive. Hence, we generated Ctsb -/- mice using CRISPR/Cas9 technology. In Ctsb -/- mice, sperm count was significantly decreased while the level of morphologically abnormal sperm was markedly increased. Additionally, these mice had significantly lower levels of progressive motility sperm and elevated levels of immobilized sperm. Histological analysis showed slight vacuolization in the testis epithelium, as well as the loss of epididymal epithelium cells. Further investigation showed that autophagic activity was inhibited and apoptotic activity was increased in both the testis and epididymis of Ctsb -/- mice. Together, our findings demonstrate that CTSB plays an important role in spermatogenesis and sperm maturation in mice.

Identification of some calcium binding proteins and neural cell markers in rat testis and epididymis during postnatal development.

Calcium-binding proteins (CaBPs) have an essential role in male reproduction by modulating calcium ion metabolism. Although the brain and testis are structurally and functionally different, they show a high degree of transcriptomic and proteomic similarities. The purpose of the present study was to explore some CaBPs (Iba-1, Calbindin, Calretinin and Parvalbumin) and neural cell markers (CNPase, NG2 and Drebrin) expression in rat testis and epididymis during postnatal periods by using immunohistochemistry. Iba-1, calbindin, calretinin, parvalbumin, CNPase, NG2 and drebrin were expressed in the epididymal epithelium in each postnatal period. Iba-1 and calbindin expression showed stage-dependent expression in spermatids. CaBPs and neural cell markers showed a positive reaction in Leydig cells in the postpubertal and mature periods. Sertoli cells, gonocytes, spermatogonium, and peritubular myoid cells showed heterogeneity in the expression of CaBPs and nerve markers throughout postnatal development. Interestingly, CNPase, NG2 and drebrin were positive in spermatocytes, spermatids, and sperm. Expression dynamics of calcium-binding proteins and nerve markers showed differences in germ cells and somatic cells during postnatal development. The expression of these proteins in the testis and epididymis supports that they may have important roles in reproductive physiology.

Arl13b controls basal cell stemness properties and Hedgehog signaling in the mouse epididymis.

Epithelial cells orchestrate a series of intercellular signaling events in response to tissue damage. While the epididymis is composed of a pseudostratified epithelium that controls the acquisition of male fertility, the maintenance of its integrity in the context of tissue damage or inflammation remains largely unknown. Basal cells of the epididymis contain a primary cilium, an organelle that controls cellular differentiation in response to Hedgehog signaling cues. Hypothesizing its contribution to epithelial homeostasis, we knocked out the ciliary component ARL13B in keratin 5-positive basal cells. In this model, the reduced size of basal cell primary cilia was associated with impaired Hedgehog signaling and the loss of KRT5, KRT14, and P63 basal cell markers. When subjected to tissue injury, the epididymal epithelium from knock-out mice displayed imbalanced rates of cell proliferation/apoptosis and failed to properly regenerate in vivo. This response was associated with changes in the transcriptomic landscape related to immune response, cell differentiation, cell adhesion, and triggered severe hypoplasia of the epithelium. Together our results indicate that the ciliary GTPase, ARL13B, participates in the transduction of the Hedgehog signaling pathway to maintain basal cell stemness needed for tissue regeneration. These findings provide new insights into the role of basal cell primary cilia as safeguards of pseudostratified epithelia.

Hindlimb unloading induces time-dependent disruption of testicular histology in mice

Mechanical unloading of the body in the hindlimb unloaded (HU) mice induces pathology in multiple organs, but the effects on testes are poorly characterized. We investigated the histology and Raman spectroscopy of the mouse testes following HU condition. We divided male, c57BL/6j mice into ground-based controls or experimental groups for two and four weeks of HU. The testes tissues were dissected after euthanasia to investigate histological and Raman spectroscopic analysis. We found an HU-induced atrophy of testes irrespective of the time duration (p < 0.05). Histological analysis revealed that the HU induced epithelial thinning, luminal widening, and spermatozoa decline in the seminiferous tubules of the mouse testes. In addition, we found a thickening of the epididymal epithelia and tunica albuginea. These changes were accompanied by a generalized depression in the Raman spectra, indicating an altered concentration and/or orientation of several molecules. We also report reduced signal intensities of hydroxyproline and tryptophan, potentially contributing to testicular pathology during HU. Taken together, our findings indicate that the two or four weeks of HU induce disruption of testicular architecture and molecular phenotypes. Our results may have implications for understanding and/or treating male infertility associated with prolonged bed rest and spaceflight.

The Role of the Epididymis in Linking the Legacy of Paternal Exposures to Alteration of the Sperm Epigenome

In what has become a well-established paradigm, paternal exposure to a range of environmental stressors is known to elicit distinct changes to the sperm small noncoding RNA (sncRNA) profile; modifications that can have significant post-fertilization consequences. Despite this knowledge, there remains limited mechanistic understanding of how paternal exposures modify the sperm sncRNA landscape. To address this question, our laboratory has exploited a tractable exposure model in which male mice were subjected to acute administration of the reproductive toxicant, acrylamide; a challenge that elicited robust changes to their sperm sncRNA profile. Further, we traced the differential accumulation of acrylamide-responsive sncRNAs to coincide with sperm transit of the proximal (caput) segment of the epididymis, wherein acrylamide exposure altered the expression of several transcription factors implicated in the expression of acrylamide-sensitive sncRNAs. We have also identified extracellular vesicles (epididymosomes) secreted from the caput epididymal epithelium in relaying altered sncRNA profiles to maturing spermatozoa, the implications of which manifest in the form of dysregulated gene expression during early embryonic development following fertilization by acrylamide-exposed sperm. Overall, these data provide mechanistic links to account for how environmental insults can alter the sperm epigenome and compromise the transcriptomic profile of early embryos.

Zika virus persistence in the male macaque reproductive tract.

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in that it is also vertically and sexually transmitted by humans. The male reproductive tract is thought to be a ZIKV reservoir; however, the reported magnitude and duration of viral persistence in male genital tissues vary widely in humans and non-human primate models. ZIKV tissue and cellular tropism and potential effects on male fertility also remain unclear. The objective of this study was to resolve these questions by analyzing archived genital tissues from 51 ZIKV-inoculated male macaques and correlating data on plasma viral kinetics, tissue tropism, and ZIKV-induced pathological changes in the reproductive tract. We hypothesized that ZIKV would persist in the male macaque genital tract for longer than there was detectable viremia, where it would localize to germ and epithelial cells and associate with lesions. We detected ZIKV RNA and infectious virus in testis, epididymis, seminal vesicle, and prostate gland. In contrast to prepubertal males, sexually mature macaques were significantly more likely to harbor persistent ZIKV RNA or infectious virus somewhere in the genital tract, with detection as late as 60 days post-inoculation. ZIKV RNA localized primarily to testicular stem cells/sperm precursors and epithelial cells, including Sertoli cells, epididymal duct epithelium, and glandular epithelia of the seminal vesicle and prostate gland. ZIKV infection was associated with microscopic evidence of inflammation in the epididymis and prostate gland of sexually mature males, pathologies that were absent in uninfected controls, which could have significant effects on male fertility. The findings from this study increase our understanding of persistent ZIKV infection which can inform risk of sexual transmission during assisted reproductive therapies as well as potential impacts on male fertility.

Histological study and immunohistochemical location of cytoskeletal proteins in the testis and epididymis of the three species of lizards of the family Leiosauridae (Reptilia: Squamata)

AbstractThe objective of the study was to histologically describe the structure of the testis and epididymis, identifying the GAGs in them. Furthermore, the distribution of smooth muscle alpha‐actin, desmin, and vimentin is described of E. bilineatus, E. perditus, and U. vautieri, through immunohistochemical method. Two adult males of each species of the lizards were collected in southeastern Brazil. In the testicular albuginea, the presence of neutral glycoproteins was detected. In E. bilineatus, the epididymal duct epithelium is simple, composed of secretory cells, cylindrical in shape and acidophilic cytoplasm. In the testis of the three lizards there was a positive immunolocation accentuated for α‐SMA in the dense connective tissue of the tunica albuginea. A moderate reaction was observed in the connective tissue albuginous and in the layer of smooth muscle fibers in a circumferential arrangement of the epididymal duct. The immunolocation for desmin was accentuated in the fibers of the testicular albuginea of E. bilineatus, while in U. vautier and E. perditus was moderate. In the epididymis, only in E. perditus and E. bilineatus positive immunoreaction was verified. In the three lizards there were no observations of immunostaining for vimentin with the antibody utilized.

Cell function and identity revealed by comparative scRNA-seq analysis in human nasal, bronchial and epididymis epithelia

The evolutionary relationship of cells within tissues having a similar function but located in different anatomical sites is of considerable biological interest. The development of single-cell RNA sequencing (scRNA-seq) protocols has greatly enhanced opportunities to address this topic. Here we focus on cells in the epithelium which lines two regions of the human respiratory tract and the male genital ducts to delineate the shared, differentiated functions of the different cell populations. Transcriptomic data were used to assess the gene expression profiles of human bronchial, nasal, and epididymal epithelium (HBE, HNE, and HEE). Bulk RNA-seq showed many shared genes expressed in cells from the nasal and bronchial epithelium and highlighted their divergence from the epididymal epithelium. ScRNA-seq in HBE and HNE cells demonstrated overlapping gene expression patterns within basal and secretory cell populations. Moreover, the distribution of cell types was altered in HNE cells derived from donors with cystic fibrosis (CF) when compared to cells from healthy donors. Next, the HBE and HNE datasets were merged and confirmed intersection of cell type gene expression profiles from the two sites. However, secretory and ciliated cells were the most abundant types in the HBE samples, while more basal cells were seen in the HNE populations. We then merged single-cell data from the epididymis to determine if overlapping functions of these cells corresponded to those in the airway. Of note, only the pulmonary ionocytes/epididymis clear cells showed a strongly conserved identity, which was confirmed by imputation in bulk RNA-seq datasets from the same cells.

Integrative Analysis of Small RNA and mRNA Expression Profiles Identifies Signatures Associated With Chronic Epididymitis.

Chronic epididymitis (CE) refers to a long-lasting inflammatory condition of the epididymis, which is considered the most common site of intrascrotal inflammation and an important aetiological factor of male infertility. Recent studies demonstrate that small RNAs secreted from epididymal epithelium modulate embryo development and offspring phenotypes via sperm transmission, and the resulting modifications may lead to transgenerational inheritance. However, to date, the genome-wide analysis of small RNA together with the transcriptomic expression profiles of human epididymis and CE is still lacking. In this study, we facilitated next-generation sequencing and bioinformatics to comprehensively analyze the small RNA and mRNA in an integrative way and identified signatures associated with CE. Both of the small RNA and mRNA expression data demonstrated relatively larger molecular differences among the segmental region of the epididymides, including caput, corpus, and cauda, than that of the inflammatory conditions. By comparing the inflamed caputs to the controls, a total of 1727 genes (1220 upregulated and 507 downregulated; 42 most significant genes, adjusted P <0.05) and 34 miRNAs (23 upregulated and 11 downregulated) were identified as differentially expressed. In silico functional enrichment analysis showed their roles in regulating different biological activities, including leukocyte chemotaxis, extracellular milieu reconstruction, ion channel and transporter-related processes, and nervous system development. Integrative analysis of miRNA and mRNA identified a regulatory network consisting of 22 miRNAs and 31 genes (miRNA-mRNA) which are strong candidates for CE. In addition, analysis about other species of small RNA, including (miRNA), piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), Y RNA, and rsRNA identified the distinct expression pattern of tsRNA in CE. In summary, our study performed small RNA and miRNA profiling and integrative analysis in human CE. The findings will help to understand the role of miRNA-mRNA in the pathogenesis of CE and provide molecular candidates for the development of potential biomarkers for human CE.

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.

Freezability biomarkers in the epididymal spermatozoa of swamp buffalo

As an alternative to ejaculated semen, epididymal spermatozoa (ES) can be recovered and cryopreserved for use in artificial insemination and other assisted reproductive technologies. However, variabilities in the sperm response to freeze-thaw procedures challenge its inherent value. Therefore, the present study aims to clarify the freezability phenomena in the swamp buffalo ES, where pieces of literature do not abound. Here, we isolated ES from swamp buffaloes using abattoir-derived, post-mortem caudal epididymis by slicing-flushing technique. Following cryopreservation by slow-freezing, ES samples were classified into high (HF) and low freezability (LF) based on their post-thaw total and progressive motilities. Conventional sperm parameters and proteins of interest, such as glutathione S-transferase Mu 3 (GSTM3) and ATP synthase beta subunit (ATP1B1), were assessed and compared between HF and LF. Computer-assisted sperm analysis revealed that nearly all motion and kinematic parameters significantly differed among freezability groups except for wobble, linearity, and straightness. Moreover, intracellular reactive oxygen species production was evident in both HF and LF after fluorescence staining, with the latter having considerably greater malondialdehyde levels than the former. Immunohistochemical labeling demonstrated that both GSTM3 and ATP1B1 proteins were present in the ES and the epididymal tubular epithelium. Although the GSTM3 relative amounts, as analyzed through Western blot, were significantly higher in LF than HF in association with lipid peroxidation, no significant differences were observed in the case of ATP1B1. Such variations in motility, motion and kinematics, oxidative stress status, and specific sperm proteins suggest their potential utility in distinguishing freezability phenotypes in swamp buffalo ES.

Maternal Protein Restriction Alters the Expression of Proteins Related to the Structure and Functioning of the Rat Offspring Epididymis in an Age-Dependent Manner.

Nutrition is an environmental factor able to activate physiological interactions between fetus and mother. Maternal protein restriction is able to alter sperm parameters associated with epididymal functions. Since correct development and functioning of the epididymides are fundamental for mammalian reproductive success, this study investigated the effects of maternal protein restriction on epididymal morphology and morphometry in rat offspring as well as on the expression of Src, Cldn-1, AR, ER, aromatase p450, and 5α-reductase in different stages of postnatal epididymal development. For this purpose, pregnant females were allocated to normal-protein (NP—17% protein) and low-protein (LP—6% protein) groups that received specific diets during gestation and lactation. After weaning, male offspring was provided only normal-protein diet until the ages of 21, 44, and 120 days, when they were euthanized and their epididymides collected. Maternal protein restriction decreased genital organs weight as well as crown-rump length and anogenital distance at all ages. Although the low-protein diet did not change the integrity of the epididymal epithelium, we observed decreases in tubular diameter, epithelial height and luminal diameter of the epididymal duct in 21-day-old LP animals. The maternal low-protein diet changed AR, ERα, ERβ, Src 416, and Src 527 expression in offspring epididymides in an age-dependent manner. Finally, maternal protein restriction increased Cldn-1 expression throughout the epididymides at all analyzed ages. Although some of these changes did not remain until adulthood, the insufficient supply of proteins in early life altered the structure and functioning of the epididymis in important periods of postnatal development.