Epididymal Duct Research Articles

Length of seminiferous tubules or epididymal duct estimated with profile counting or diameter measurement: a stereological comparison with transverse and isotropic sections

Transverse testicular sections are often used for histopathological studies and length of seminiferous tubules in the testis is often estimated with a stereological method of tubule diameter measurement, i.e. estimated by the tubule volume divided by the tubules’ cross-sectional area that is estimated with measurement of round or elliptical tubule profiles’ (short axial) diameters. But unbiased stereological length estimation is best based on counting of tubule profiles on isotropic sections. To determine the bias of length estimation with diameter measurement, (i) transverse sections (methacrylate embedded) were obtained from a testis of each normal mature male Sprague-Dawley rat (10 rats in total) and isotropic sections obtained (with the orientator) from the other testis of the rat, and (ii) the tubule length densities were estimated with both methods of profile counting and diameter measurement. The results demonstrated that the method of diameter measurement on transverse or isotropic sections overestimated the length density by 20%-25% compared with profile counting on isotropic sections, probably mainly because of underestimation of the tubules’ cross-sectional area estimated from the short-axial diameters. Length density of the epididymal duct in the epididymis was also estimated with profile counting in the present study, using transverse and isotropic sections for comparison, and significant statistical difference was not found between the results obtained with the transverse and isotropic sections.

EphB2, EphB4, and ephrin-B1 expression and localization in postnatal developing epididymis in mice.

Eph receptors and ephrin ligands, the transmembrane proteins, function as a mechanism of communication between cells. Therefore, we intended to explore the expression array of EphB2 and EphB4 receptors and ephrin-B1 ligand in postnatal developing mouse epididymis during 1 day to 8 weeks using RT-PCR amplification and immunofluorescence staining. RT-PCR analysis indicated that the expression levels of EphB2, EphB4, and ephrin-B1 in the epididymis declined with the advancement of age during the initial phases of postnatal development and stayed relatively near to adult levels until 4 weeks. We discovered that the predominant compartments expressing EphB2/B4 and ephrin-B1 emerged in the excurrent duct epithelia of postnatal developing epididymis until 3 weeks. Consequently, even before spermatozoa reach the excurrent duct in epididymis, at the age of 3 weeks, the epididymal excurrent duct system exhibits characteristics similar to those of an adult in terms of expression of EphB2/B4 and ephrin-B1. Moreover, ephrin-B1 was expressed in epididymal epithelial cells throughout the development and EphB4 was expressed only in early postnatal stages while basal cells expressed EphB4 throughout the postnatal development. The study represents the first expression analysis of ephrin-B1, EphB2, and EphB4 in the normal mouse epididymis during the postnatal development.

Microanatomy of the Testes and Male Genital Ducts of Banded Krait Bungarus fasciatus (Schneider, 1801)

The Banded krait is a species of venomous snakes that ranks among the top 5 in Thailand, with a significance role as a predator for controlling other animals. Despite numerous studies on the ecology, the microanatomy of body systems has still not reported clearly. The present study was to investigate the microanatomy of the testes and male genital ducts of Banded krait that collected from Phatthalung Province, Thailand. The snakes were collected after anesthetized, and their testes and genital ducts were fixed in Bouin’s fixative. The reproductive organs were collected during July to October 2022, and were sectioned of 5-µm-thick pieces for staining with Harris’s hematoxylin and eosin (H&E), periodic acid Schiff’s (PAS), alcian blue (AB) pH 2.5 and pH 1.0, bromophenol blue (BB) and Masson’s trichrome (MT). The testes contained the seminiferous tubules with 7 stages of germ cells: (I) type A spermatogonium, (II) type B spermatogonium, (III) primary spermatocyte, (IV) secondary spermatocyte, (V) round spermatid, (VI) elongated spermatid and (VII) spermatozoa. Besides, Sertoli and Leydig’s cells were appeared in the seminiferous tubules and the interstitial area, respectively. During the sampling period, the spermatozoa were observed in the seminiferous tubules and genital ducts. The extra-testicular rete testis, ductuli efferentes and ductus epididymis were embedded in the epididymal sheath. The epithelial cells of rete testis, ductus epididymis, as well as ductus deferens possessed microvilli, whereas those of the ductuli efferentes composed of cilia. The epithelial cells of all portions showed the positive staining with PAS and BB due to the presence of neutral glycoproteins and proteins, respectively, and the negative staining for AB pH 2.5 and pH 1.0 because of the absence of acidic glycoproteins. HIGHLIGHTS During the sampling period from July to October, the Banded Krait exhibited active spermatogenesis The rete testis could be divided into 2 portions: intra-rete testis and extra-rete testis The ductus epididymis was divided into 3 portions: proximal, middle and distal ductus epididymis All portions of the male genital ducts, the epithelium showed the secretion of neutral glycoproteins and proteins without sulfated and carboxylated glycoproteins GRAPHICAL ABSTRACT

Human efferent ductules and epididymis display unique cell lineages with motile and primary cilia.

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.

Inhibition of Prolactin Affects Epididymal Morphology by Decreasing the Secretion of Estradiol in Cashmere Bucks.

Yanshan Cashmere bucks are seasonal breeding animals and an important national genetic resource. This study aimed to investigate the involvement of prolactin (PRL) in the epididymal function of bucks. Twenty eleven-month-old Cashmere bucks were randomly divided into a control (CON) group and a bromocriptine (BCR, a prolactin inhibitor, 0.06 mg/kg body weight (BW)) treatment group. The experiment was conducted from September to October 2020 in Qinhuangdao City, China, and lasted for 30 days. Blood was collected on the last day before the BCR treatment (day 0) and on the 15th and 30th days after the BCR treatment (days 15 and 30). On the 30th day, all bucks were transported to the local slaughterhouse, where epididymal samples were collected immediately after slaughter. The left epididymis was preserved in 4% paraformaldehyde for histological observation, and the right epididymis was immediately preserved in liquid nitrogen for RNA sequencing (RNA-seq). The results show that the PRL inhibitor reduced the serum PRL and estradiol (E2) concentrations (p < 0.05) and tended to decrease luteinizing hormone (LH) concentrations (p = 0.052) by the 30th day, but no differences (p > 0.05) occurred by either day 0 or 15. There were no differences (p > 0.05) observed in the follicle-stimulating hormone (FSH), testosterone (T), and dihydrotestosterone (DHT) concentrations between the two groups. The PRL receptor (PRLR) protein was mainly located in the cytoplasm and intercellular substance of the epididymal epithelial cells. The PRL inhibitor decreased (p < 0.05) the expression of the PRLR protein in the epididymis. In the BCR group, the height of the epididymal epithelium in the caput and cauda increased, as did the diameter of the epididymal duct in the caput (p < 0.05). However, the diameter of the cauda epididymal duct decreased (p < 0.05). Thereafter, a total of 358 differentially expressed genes (DEGs) were identified in the epididymal tissues, among which 191 were upregulated and 167 were downregulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that ESR2, MAPK10, JUN, ACTL7A, and CALML4 were mainly enriched in the estrogen signaling pathway, steroid binding, calcium ion binding, the GnRH signaling pathway, the cAMP signaling pathway, and the chemical carcinogenesis-reactive oxygen species pathway, which are related to epididymal function. In conclusion, the inhibition of PRL may affect the structure of the epididymis by reducing the expression of the PRLR protein and the secretion of E2. ESR2, MAPK10, JUN, ACTL7A, and CALML4 could be the key genes of PRL in its regulation of epididymal reproductive function.

Exploring the Molecular Characteristics and Role of PDGFB in Testis and Epididymis Development of Tibetan Sheep.

Platelet-derived growth factor B (PDGFB), as an important cellular growth factor, is widely involved in the regulation of cellular events such as cell growth, proliferation, and differentiation. Although important, the expression characteristics and biological functions in the mammalian reproductive system remain poorly understood. In this study, the PDGFB gene of Tibetan sheep was cloned by RT-PCR, and its molecular characteristics were analyzed. Subsequently, the expression of the PDGFB gene in the testes and epididymides (caput, corpus, and cauda) of Tibetan sheep at different developmental stages (3 months, 1 year, and 3 years) was examined by qRT-PCR and immunofluorescence staining. A bioinformatic analysis of the cloned sequences revealed that the CDS region of the Tibetan sheep PDGFB gene is 726 bp in length and encodes 241 amino acids with high homology to other mammals, particularly goats and antelopes. With the increase in age, PDGFB expression showed an overall trend of first decreasing and then increasing in the testis and epididymis tissues of Tibetan sheep, and the PDGFB mRNA expression at 3 months of age was extremely significantly higher than that at 1 and 3 years of age (p < 0.05). The PDGFB protein is mainly distributed in testicular red blood cells and Leydig cells in Tibetan sheep at all stages of development, as well as red blood cells in the blood vessel, principal cells, and the pseudostratified columnar ciliated epithelial cells of each epididymal duct epithelium. In addition, PDGFB protein expression was also detected in the spermatocytes of the 3-month-old group, spermatids of the 1-year-old group, spermatozoa and interstitial cells of the 3-year-old group, and loose connective tissue in the epididymal duct space in each developmental period. The above results suggest that the PDGFB gene, as an evolutionarily conserved gene, may play multiple roles in the development and functional maintenance of testicular cells (such as red blood cells, Leydig cells, and germ cells) and epididymal cells (such as red blood cells, principal cells, and ciliated epithelial cells) during testicular and epididymal development, which lays a foundation for the further exploration of the mechanisms by which the PDGFB gene influences spermatogenesis in Tibetan sheep.

Recent advances in the study of the structure and function of the epididymis

Testicular sperm maturation is critical for establishing male fertility. Spermatozoa undergo remodeling of sperm proteins and changes in lipid and ribonucleic acid composition during transport in the epididymal ducts, which play an important role in sperm maturation. The anatomy, epithelial cell types, physiological functions, and epigenetic inheritance of the epididymis are explored, and recent findings in epididymal research are analyzed. Suggesting possible directions for future research on the epididymis. Using the keywords “epididymis”, “sperm”, and “sperm maturation”, a search of the epididymis was performed through databases and official websites of journals related to reproduction. The epididymis was searched in databases and on the official websites of journals related to reproduction. This review introduces the characteristics of the epididymis, as well as the biological functions of cell types such as principal cells, clear cells, and basal cells, providing a detailed description of the overall physiological functions of the epididymis. It highlights current research hotspots in the field of epididymis, including single-cell analysis, epigenetics, and extracellular vesicles, aiming to offer a comprehensive understanding for beginners. The review emphasizes the importance of the epididymis, its impact on sperm maturation and subsequent embryo development, and how it advances research on epididymal diseases while providing new directions for the study and treatment strategies of infertility.

Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis.

The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl- channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential -60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl- concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.

Morphology of the epididymal duct of the domestic pig (Sus scrofa domesticus).

The study provides a general overview of the morphology of the epididymal duct in pigs. Four epididymides from two sexually mature boars were dissected into 32 segments and examined histologically. Duct lumen and wall thickness were measured and relative surface area of different components was assessed by Chalkley's random hit method. The epithelial lining was characterized at X1000. Lumen diameter and wall thickness of efferent ductules averaged 177 and 30 µm, respectively. Of the epididymal duct from caput to distal corpus the luminal diameter was 332 µm, with a narrower section in the proximal corpus. Wall thickness averaged 70 µm. In the cauda, luminal diameter and wall thickness increased to 717and 751 µm, respectively. The epithelial lining of the efferent ductules consists of a single layer of columnar cells with average height 21 µm. The lining of the epididymal duct consists of ciliated, pseudo-stratified columnar epithelium composed of "basal cells" and "principal cells." Particularly tall principal cells (96 µm) were found in the proximal caput. Height decreased to 40 µm at the distal cauda. Microvilli from principal cells were 14-17 µm long in the distal caput but decreased to 5 µm in the distal cauda. The epithelial lining was folded in the proximal caput and more so in the distal cauda. Secretory granules (epididymosomes) were present in small amounts in efferent ductules and epididymal duct; the largest quantities occurred in the distal cauda. Leukocytes were present throughout the duct, albeit in insignificant numbers. Chalkley's random hit method showed rapid spermatozoan transport through efferent ductules and proximal caput in large amounts of fluid. Sperm concentration increased due to fluid resorption in the proximal caput, was highest from caput flexure to proximal cauda and decreased at the caudal flexure, indicating secretory activity.

Steroidogenic activity and morphological characterization of prenatal testes and epididymis of guinea pig (Cavia porcellus)

The present study aims to establish the morphological, morphometric, and immunostaining patterns of the steroidogenic enzymes 17β-HSD and 5α-reductase and androgen receptors (AR) during the prenatal development of the male gonad and epididymis of Cavia porcellus. Fetuses at 22, 25, 30, 40, 45, 50, and 60 days of gestation (DG) were used. Specimens were dissected and subjected to macroscopic, histological, histomorphometric, and immunohistochemical analyses. Genital and scrotal protrusions were identified in 22 DG embryos. Gonocytes were identified at 25 DG and the formation of primary testicular cords was observed at 30 DG. Through anatomical evaluation, we observed differentiation of the epididymis into the head, body, and tail at 45 DG. During development, there is a progressive decrease in the diameters of the testicular cords and epididymal ducts. 17β-HSD enzyme immunostaining was observed in Leydig cells at all ages, while 5α-reductase was observed in Leydig cell cytoplasm and gonocytes at 40, 50, and 60 DG. AR shows gonocyte labeling at 30 DG. Thus, from the second trimester of pregnancy, it is possible to observe patterns of anatomical development, such as genital and scrotal prominence (22 DG), the appearance of gonocytes in the testicular cords at 25 DG, and the beginning of the organization of primary testicular cords at 30 DG, suggesting sexual differentiation. The 17β-HSD, 5α-reductase, and ARs play an essential role in sexual development and differentiation, presenting immunostaining at different reproductive process times.

Staining patterns of PNA and UEA-I lectins in the postnatal developing male genital excurrent duct epithelium in mice.

The research was aimed at determining the staining patterns of Peanut agglutinin (PNA) lectin and Ulex europaeus agglutinin I (UEA-I) lectin in postnatal developing (day 1 to 8 weeks) male genital excurrent duct epithelium in mice. Lectin staining was performed on testis and epididymis of 1-day-old and 1-, 2-, 3-, 4-, 5-, 6-, and 8-week-old ICR mice. Rete testis epithelia were UEA-I- and PNA-negative at all postnatal ages. PNA lectin unclearly stained the epithelial apical surface in efferent ductules (ED) and ductus epididymis up to 2 weeks, while UEA-I was negative in those epithelia. By contrast, at 3 weeks of age, PNA and/or UEA-I turned to clearly label epithelia in the excurrent duct system. At 5 weeks, efferent ductules epithelium was UEA-I-negative but strongly PNA-positive; segment I was PNA-negative but faintly UEA-I positive; segment II was weakly PNA-positive but strongly UEA-I-positive; segment III was strongly-PNA- and UEA-I-positive; segment IV was weakly UEA-I-positive but strongly PNA-positive; and segment V was faintly UEA-I-positive but strongly PNA-positive. At 5 and 8 weeks (the adult), the staining patterns of PNA and UEA-I lectins were nearly identical. Moreover, an increasing staining intensity was observed in both lectins with the advancement of age until 5 weeks of age. Additionally, it was noted that, with the exception of segment I, the staining intensity of PNA gradually increased while that of UEA-I gradually decreased towards the distal section of the ductus epididymis. Lectin PNA and UEA-I staining, in conclusion, indicated epithelial segments in the male genital duct of mice from the postnatal developing age of 3 weeks, and up until 5 weeks, their staining patterns were nearly identical to those in adults.

Impact of Epididymal Sperm Aspiration on Microsurgical Vasoepididymostomy Outcomes in Patients with Obstructive Azoospermia-Report of 21 Cases Attached

ABSTRACT Background: To explore the impact of epididymal sperm aspiration on microsurgical vasoepididymostomy (MVE) outcomes in patients with obstructive azoospermia (OA). Methods: A retrospective analysis was conducted on 21 patients with OA who underwent epididymal sperm aspiration and received treatment at the department of andrology, Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine from September 2016 to March 2023. Under microscopic scrutiny, the structure of the epididymis and any injuries to its ducts were observed and compared during MVE. Results: Microscopic analysis of 21 patients revealed notable changes in the appearance of the epididymis, showcasing substantial scar contracture and evident adhesion between the epididymal fascia and tunica vaginalis within the sheath, posing challenges in their separation. Among them, 12 patients exhibited fractured, shrunk, fibrotic, and semen-devoid epididymal ducts. Consequently, MVE could not be completed in 15 patients because of these factors, and only six patients underwent MVE. Among the patients who successfully underwent MVE, three patients had sperm in the follow-up examination 3 months after surgery, accounting for 50%. Conclusion: Epididymal sperm aspiration can damage the normal structure of the epididymis and epididymal duct, often leading to surgical termination during operation and a decrease in the long-term patency rate following surgery. Therefore, patients with OA are recommended to undergo MVE as the initial procedure. In case the surgery prove unsuccessful, epididymal or testicular sperm aspiration can be performed for intracytoplasmic sperm injection (ICSI) assisted reproduction. Additionally, patients with OA are advised to avoid diagnostic epididymal sperm aspiration.

Morphohistometric and steroidogenic parameters during testicular and epididymal differentiation in cavy (Galea spixii) fetuses

Sexual differentiation and steroidogenic mechanisms have an important impact on postnatal gonadal phenotypic development. Thus, establishing the activities that lead to male phenotypic development can provide a better understanding of this process. This study examined the prenatal development of cavies to establish morphological and histometric development patterns and protein and enzyme immunolocalization processes that are responsible for androgen synthesis in the testes and epididymis. Histological and histometric analyses of the diameter of the seminiferous cords and epididymal ducts of male fetuses on Days 25, 30, 40, and 50 were performed, as well as immunohistochemistry of the steroidogenic enzymes 5α-reductase and 17β-HSD, the androgen receptor, and the anti-Müllerian hormone (AMH). Our findings showed a cellular grouping of gonocytes from Day 30 onward that was characteristic of the seminiferous cord, which was not present in the lumen at any of the studied dates. From Day 50 onward, the differentiation of the three anatomical regions of the epididymis was evident, the head (caput), body (corpus), and tail (cauda), with tissue distinctions. Furthermore, the diameters of the seminiferous cords and epididymal ducts significantly increased with age. On Day 50, the tail showed the greatest diameter of the three regions. The Sertoli and Leydig cells exhibited AMH immunoreactivity at all dates. In addition, the Leydig cells and epididymal epithelial tissue were immunopositive for 5α-reductase, 17β-HSD, and the androgen receptor; therefore, these factors influenced the development and maintenance of the testis and epididymis during cavy prenatal development.

In vivo dynamic volumetric imaging of mouse testis and epididymis with optical coherence tomography†.

The implementation of live imaging in reproductive research is crucial for studying the physiological dynamics. Sperm transport is a highly dynamic process regulated by tubular contractions and luminal flows within the male reproductive tract. However, due to the lack of imaging techniques to capture these dynamics in vivo, there is little information on the physiological and biomechanical regulation of sperm transport through the male reproductive tract. Here, we present a functional in vivo imaging approach using optical coherence tomography, enabling live, label-free, depth-resolved, three-dimensional, high-resolution visualization of the mouse testis and epididymis. With this approach, we spatiotemporally captured tubular contractility in mouse testis and epididymis, as well as microstructures of these reproductive organs. Our findings demonstrated that the contraction frequency varies significantly depending on the epididymal regions, suggesting the spatial regulation of epididymal contractility. Furthermore, we implemented quantitative measurements of the contraction wave and luminal transport through the epididymal duct, revealing the physiological dynamics within the male reproductive tract. The results show that the contraction wave propagates along the epididymal duct and the wave propagation velocity was estimated in vivo. In conclusion, this is the first study to develop in vivo dynamic volumetric imaging of the male reproductive tract, which allows for quantitative analysis of the dynamics associated with sperm transport. This study sets a platform for various studies investigating normal and abnormal male reproductive physiology as well as the pharmacological and environmental effects on reproductive functions in mouse models, ultimately contributing to a comprehensive understanding of male reproductive disorders.

Seasonal variations of the epididymis in donkeys (Equus asinus) with special reference to blood epididymal barrier.

The epididymis, a key component of the male reproductive system, controls spermatozoa's maturation, fertility, and storage. The objective of this study is to evaluate the histological, ultrastructural, and immunohistochemical variations in the epididymis of donkeys that occur throughout the year. During the breeding season (spring) and nonbreeding seasons (summer, autumn, and winter), 20 epididymis were collected from adult, clinically healthy donkeys. Compared to non-breeding seasons, the epididymal duct displayed a more active lining epithelium and more sperm in the lumen during the breeding season. The epithelial height is the lowest and the lumen is the widest during the breeding season. Furthermore, the epididymal epithelium in the tail region exhibits undulations with polyps-like projections. The epididymal epithelium is composed mainly of the principal, basal, and dark cells. Tight junction between adjacent principal cells is more obvious in the breeding season as compared to the non-breeding seasons. However, intraepithelial lymphocytes, phagocytic, and other immune cells are more frequent in non-breeding seasons. β-catenin, which is a component of the adherent junctions between adjacent PCs, exhibits more immunoreactivity during the spring. On the other hand, iNOS, an indicator of oxidative stress, reacts positively during the summer. Additionally, during non-breeding seasons, autophagy was detected within the epididymal epithelium which may be linked to stress adaptation. In conclusion, our findings suggest that the histological and ultrastructural characteristics of the epididymal epithelium are more active during spring compared to other seasons of the year. RESEARCH HIGHLIGHTS: The study aimed to evaluate the histological, ultrastructural, and immunohistochemical variations in the blood epididymal barrier (BEB) and epididymal epithelium of donkeys that occur throughout the year. In comparison to non-breeding seasons, the epididymal duct displayed a more active lining epithelium and more sperm in the lumen during the breeding season. The epithelial height is the lowest and the lumen is the widest during the breeding season. The epididymal epithelium in the tail region exhibits undulations with polyps-like projections that increase the surface area. β-catenin, which is a component of the adherent junctions between adjacent PCs, exhibits more immunoreactivity during the spring. On the other hand, iNOS, an indicator of oxidative stress, reacts positively during the summer.

New aspects of activin biology in epididymal function and immunopathology.

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.

Genital stones: Radiological, histopathological, ultrastructural, and molecular analysis in rooster

Epididymal lithiasis, characterized by the formation of stones in the epididymis, has been associated with a decline in fertility in roosters. This study aimed to investigate the reproductive performance, ultrastructural characteristics, and expression of aromatase cytochrome P450 (CYP19) and aquaporin 9 (AQP9) in aged broiler breeder roosters affected by epididymal lithiasis. X-ray analysis confirmed the presence of genital stones in both the epididymis and testicular tissue regions. While there was a significant decrease in sperm concentration in the affected roosters compared to non-affected roosters, no significant differences were observed in total and progressive sperm motility between the two groups. Furthermore, the affected roosters exhibited significant abnormalities in semen parameters, except for sperm concentration and morphology. Transmission electron microscopy (TEM) revealed the depletion and deciliation of ciliated cells in the distal efferent ductules of the epididymis in affected roosters. Additionally, the expression of CYP19 and AQP9 was found to be increased in the epididymal region of affected roosters. Notably, we report the presence of testicular stones for the first time in this study, in addition to epididymal stones. Considering the male reproductive tract lesions observed, we propose the term “genital stones” to describe these conditions. Moreover, our findings suggest that the overexpression of AQP9, which is associated with a high copy number of the CYP19 gene in the epididymal region of affected aged roosters, may contribute to the formation of genital stones by promoting increased reabsorption of fluids in the epididymis. The condensation of epididymal duct contents and reduction in the population of ciliated cells further impairs semen movement and can lead to the blockage of extra-testicular ducts, resulting in the low fertility syndrome observed in aged roosters.

Knockout of the polysialyltransferases ST8SiaII and ST8SiaIV leads to a dilatation of rete testis during postnatal development.

Polysialic acid (polySia) is a carbohydrate polymer that modulates several cellular processes, such as migration, proliferation and differentiation processes. In the brain, its essential impact during postnatal development is well known. However, in most other polySia positive organs, only its localization has been described so far. For instance, in the murine epididymis, smooth muscle cells of the epididymal duct are polysialylated during the first 2weeks of postnatal development. To understand the role of polySia during the development of the epididymis, the consequences of its loss were investigated in postnatal polySia knockout mice. As expected, no polysialylation was visible in the absence of the polysialyltransferases ST8SiaII and ST8SiaIV. Interestingly, cGMP-dependent protein kinase I (PGK1), which is essentially involved in smooth muscle cell relaxation, was not detectable in peritubular smooth muscle cells when tissue sections of polySia knockout mice were analyzed by immunohistochemistry. In contrast to this signaling molecule, the structural proteins smooth muscle actin (SMA) and calponin were expressed. As shown before, in the duct system of the testis, even the expression of these structural proteins was impaired due to the loss of polySia. We now found that the rete testis, connecting the duct system of the testis and epididymis, was extensively dilated. The obtained data suggest that less differentiated smooth muscle cells of the testis and epididymis result in disturbed contractility and thus, fluid transport within the duct system visible in the enlarged rete testis.

Immunolocalization of Desmin, Vimentin and S-100 proteins in testis and epididymis of African striped ground squirrel (Xerus erythropus)

The aim of the study was to immunolocalize desmin, vimentin and S-100 proteins in the testes and epididymis of African striped ground squirrel (Xerus erythropus) and establish their spatial distribution in the two organs. Formalin fixed-paraffin-embedded sections of the testis and epididymis obtained from ten apparently healthy adult male African striped ground squirrels were processed routinely for immunohistochemistry, using primary antibodies specific to desmin, vimentin and S-100. S-100The results showed that desmin reacted intensely in the myoid cells of the seminiferous tubules and smooth muscle cells of the epididymal ducts. It showed a moderate positive immunoreaction in the interstitial cells of Leydig, and the epididymal inter-ductal loose connective tissue. However, there was no immunoreaction of desmin in the spermatogonia or any other spermatogenic cell of the seminiferous tubule. Vimentin reacted intensely in the Leydig cells and spermatogonia. It showed moderate positive reaction in the myoid cells and the epididymal inter-ductal loose connective tissue. There was no immunoreaction of vimentin in the other spermatogenic cells (other than the spermatogonia). The S-100 proteins expressed a mild positive immunoreaction at the interstitial cells of Leydig, but negative immunoreaction in all other parts of testis and epididymis. Also, there was intense immunoreaction of desmin and vimentin and moderate immunoreaction of S-100 in the vascular endothelium in the testis and epididymis. In conclusion, the spatial distribution of desmin, vimentin and S-100 proteins in the testes and epididymis in the African striped ground squirrel showed some similarities and contrast with other mammals, giving insight into the functions of the proteins in these organs of the rodent.

P-019 Short ejaculatory abstinence time is associated with higher pregnancy and live birth rates and improved DNA fragmentation index (DFI): A Systematic Review

Abstract Study question Does short ejaculatory abstinence time (EA) improve DNA fragmentation and reproductive outcomes compared with long EA following assisted reproductive technology (ART)? Summary answer Shorter EA is associated with improved pregnancy and live birth rates following ART and lower DFI compared with longer EA at different cut-off points. What is known already When spermatozoa pass the epididymal tract, they are exposed to reactive oxygen species (ROS). ROS is known to cause DNA fragmentation. Testicular sperm has a lower DNA fragmentation when compared to ejaculated sperm, which indicates that DNA fragmentation is increased during the transition and storage in the epididymal duct. There are studies that suggest that a high DFI is associated with poor pregnancy and live birth rates following ART. WHO recommends 2-7 days of EA prior to semen collection; however, several recent studies have reported a correlation between shorter EA and improved reproductive outcomes and DFI. Study design, size, duration A systematic review was conducted according to the PRISMA guidelines and registered in PROSPERO (CRD42022379039). The electronic databases PubMed, Embase and Cochrane were searched for eligible studies by a research librarian in April 2022. Additional articles were manually retrieved after reviewing the reference lists from relevant publications. Participants/materials, setting, methods Two authors independently screened studies based on the eligibility criteria; studies including men referred to fertility treatment undergoing short and long EA reporting data on pregnancy rate, live birth rate and DFI. Study demographics, population characteristics and data on study outcomes including statistical analyses were extracted. Risk of bias in each study was evaluated according to The Scottish Intercollegiate Guidelines Network (SIGN). Any disagreements were resolved by a third author. Main results and the role of chance Out of 1237 studies initially identified, 24 of them met eligibility criteria and were included in the review with a total of 14.173 cases. The studies included in this review were conducted in Asia, North and South America and Europe. The cause of referral to fertility treatment includes both male, female, mixed and unexplained infertility. The EA varied from less than a one hour to as long as 15-20 days. Nine of the 13 studies investigated the influence of varying EA on pregnancy rate, found a significantly higher pregnancy rate with short EA compared with long EA, five with EA as short as one day or less. Three studies evaluated live birth rates and found a significantly higher live birth rate when comparing short EA with long EA. Eleven of the 15 studies that reported DFI found significantly lower DFI with short EA compared with long EA. EA of one day or less were associated with the lowest rates of DFI in the studies. In general, pregnancy rate, live birth rate and DNA fragmentation are likely to be improved with short EA compared to long EA. Limitations, reasons for caution Many of the included studies used different EA intervals, leading to heterogenous data, and therefore, only allowed limited conclusion to be formulated regarding the ideal timeframe of EA. Four studies scored poorly in SIGN methodology checklist due to confounding and population size, suggesting their findings to be treated with caution. Wider implications of the findings Even though it is difficult to make a clear recommendation on the ideal timeframe for EA because of the heterogenous abstinence periods, this review suggest decreasing the EA limits of 2-7 days as recommended by WHO to improve pregnancy rate and decrease DNA fragmentation in semen, followed by ART. Trial registration number not applicable