Efferent Ducts Research Articles

ARL13B controls male reproductive tract physiology through primary and Motile Cilia

ARL13B is a small regulatory GTPase that controls ciliary membrane composition in both motile cilia and non-motile primary cilia. In this study, we investigated the role of ARL13B in the efferent ductules, tubules of the male reproductive tract essential to male fertility in which primary and motile cilia co-exist. We used a genetically engineered mouse model to delete Arl13b in efferent ductule epithelial cells, resulting in compromised primary and motile cilia architecture and functions. This deletion led to disturbances in reabsorptive/secretory processes and triggered an inflammatory response. The observed male reproductive phenotype showed significant variability linked to partial infertility, highlighting the importance of ARL13B in maintaining a proper physiological balance in these small ducts. These results emphasize the dual role of both motile and primary cilia functions in regulating efferent duct homeostasis, offering deeper insights into how cilia related diseases affect the male reproductive system.

A computation analysis with heat and mass transfer for micropolar nanofluid in ciliated microchannel: With application in the ductus efferentes

Inherited heat enhancement capabilities and their significance in the field of medical sciences and industry make nanofluids the focus of research nowadays. Furthermore, due to the remarkable advancements in bionanotechnology and its significance in biomedical fields such as drug delivery systems, cancer tumor therapy, bioimaging, and many others, it has emerged as a key research area. Contribution of cilia for the flow in ductus efferentes of human male reproductive tract is elaborated. A novel mathematical scheme is presented for the heat and mass transfer of MHD micropolar nanofluid transport in an asymmetric channel lined with cilia. The pertinent equations of nanofluid transport are exposed to lubrication approximation theory and solution for the physical problem is examined with efficient bvp4c technique in MATLAB. Fluid rheology is explored with the variations of different transport parameters like Hartmann number, Grashof number, Brownian motion, buoyancy, thermophoresis and Darcy number. It is reported that nanofluid transport is affected with rise in the Lorentz force and show reverse behavior with rising permeability. The temperature of the nanofluid in ciliated microchannel is raised with enhanced value of Hartmann number, Grashof number, Prandtl number, and Darcy number while diffusion phenomenon of nanofluid is slowed down with these parameters. Spinning motion of the nanofluid is enhanced with Grashof number and slow down with nanoparticle Grashof number and different behavior is recorded for Darcy and viscosity parameters in different flow regime. Reported investigation presents crucial findings for ciliary transport of micropolar nanofluid and tackled with appropriate selection of micropolar parameter, Brownian motion parameter, thermophoresis and Grashof number. Moreover, this investigation will be handful for cilia-based actuators which work as micro-mixers in controlling the flow in minute bio-sensors and may prove their worth in micro-pumps employed in various drug-delivery systems.

Impact of Sexual Maturity and Seasonality on Immunolocalization of S100 and Alpha Smooth Muscle Actin in the Efferent Ductules of the Dromedary Camel (Camelus dromedarius)

Impact of Sexual Maturity and Seasonality on Immunolocalization of S100 and Alpha Smooth Muscle Actin in the Efferent Ductules of the Dromedary Camel (Camelus dromedarius)

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

Histological Study of Development Structure of Testis and Epididymis in Gazelle Gazelle Subgutturosa

Objective: The aim of this study for focused the light on the development structure of the testis of gazelle. Materials and Method: 10 healthy male gazelle were used to study the histology structure of testis (5 pre- Puberty and 5 post- Puberty). Results: The testis is surrounded by a thick, irregular connective tissue capsule. Each lobule of the testis is composed of seminiferous tubules and interstitial tissue, and trabecular and interlobular septa divide the lobules apart. Neighboring the seminiferous tubules are Sertoli cells and spermatogenic cells. From spermatogenic cells, spermatozoa are produced. The first spermatogonia are small, spherical cells with black, spherical nuclei that reside on the basement membrane. Spermatogonia were earliest stages of the spermatogenic cells. Larger cells with frequently distinct chromatin are called primary spermatocytes. Secondary spermatocytes are rare because of their quick second meiotic division and the haploid spermatids that follow. Spermatids are round, pale-nucleated cells that are grouped close to the lumen of the seminiferous tubule. Prostatogenic cells outnumber Sertoli cells in the body. Oval or triangular in shape, the nucleus has a light coloration. In the connective tissue separating adjacent tubules, Leydig cells are present. Lines the epididymis are pseudostratified epithelium. Extensions of the cytoplasm into the lumen are called stereocilia. In different proportions, there is circular smooth muscle, and a connective tissue lamina propria supports the epithelium. The rete testis is connected to the ductus epididymidis by the efferent ductules, which are convolutions of tubules. Numerous valved veins were visible in the tunica albuginea, which covered the initial ascending portion of the epididymal head. Cuboidal epithelium was lining extratesticular rete testis. Three different cell types; migrating, basal, and columnar had epithelial membranes lining the efferent ductules. Many columnar, non-ciliated cells with many cytoplasmic vacuoles and tiny granules lined the epithelium of the first segment of the efferent ductules. Conclusion: In post-puberty age, the structure of the gazelle's testis exhibited a pattern resembling that of other ruminants; the seminiferous tubules contain adult sperm and a broad lumen.

Histological observations and transcriptome analyses reveal the dynamic changes in the gonads of the blotched snakehead (Channa maculata) during sex differentiation and gametogenesis

BackgroundBlotched snakehead (Channa maculata) displays significant sexual dimorphism, with males exhibiting faster growth rates and larger body sizes compared to females. The cultivation of the all-male population of snakeheads holds substantial economic and ecological value. Nonetheless, the intricate processes governing the development of bipotential gonads into either testis or ovary in C. maculata remain inadequately elucidated. Therefore, it is necessary to determine the critical time window of sex differentiation in C. maculata, providing a theoretical basis for sex control in production practices.MethodsThe body length and weight of male and female C. maculata were measured at different developmental stages to reveal when sexual dimorphism in growth initially appears. Histological observations and spatiotemporal comparative transcriptome analyses were performed on ovaries and testes across various developmental stages to determine the crucial time windows for sex differentiation in each sex and the sex-related genes. Additionally, qPCR and MG2C were utilized to validate and locate sex-related genes, and levels of E2 and T were quantified to understand sex steroid synthesis.ResultsSexual dimorphism in growth became evident starting from 90 dpf. Histological observations revealed that morphological sex differentiation in females and males occurred between 20 and 25 dpf or earlier and 30–35 dpf or earlier, respectively, corresponding to the appearance of the ovarian cavity or efferent duct anlage. Transcriptome analyses revealed divergent gene expression patterns in testes and ovaries after 30 dpf. The periods of 40–60 dpf and 60–90 dpf marked the initiation of molecular sex differentiation in females and males, respectively. Male-biased genes (Sox11a, Dmrt1, Amh, Amhr2, Gsdf, Ar, Cyp17a2) likely play crucial roles in male sex differentiation and spermatogenesis, while female-biased genes (Foxl2, Cyp19a1a, Bmp15, Figla, Er) could be pivotal in ovarian differentiation and development. Numerous biological pathways linked to sex differentiation and gametogenesis were also identified. Additionally, E2 and T exhibited sexual dimorphism during sex differentiation and gonadal development. Based on these results, it is hypothesized that in C. maculata, the potential male sex differentiation pathway, Sox11a–Dmrt1–Sox9b, activates downstream sex-related genes (Amh, Amhr2, Gsdf, Ar, Cyp17a2) for testicular development, while the antagonistic pathway, Foxl2/Cyp19a1a, activates downstream sex-related genes (Bmp15, Figla, Er) for ovarian development.ConclusionsThis study provides a comprehensive overview of gonadal dynamic changes during sex differentiation and gametogenesis in C. maculata, establishing a scientific foundation for sex control in this species.

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.

Localization of β-Nerve Growth Factor in the Stallion Reproductive Tract.

β-Nerve growth factor (β-NGF) is a protein produced in the reproductive tract of camelids (camels, llamas, and alpacas) that has been identified as the ovulation inducing factor in seminal plasma. β-NGF from seminal plasma deposited into the reproductive tract of the female camelid acts systemically to stimulate the secretion of luteinizing hormone (LH) from the anterior pituitary, which in turn induces follicle maturation and ovulation. The objectives of the present study were to determine if β-NGF is present in the reproductive tract of the stallion and identify the specific site(s) of production. The hypotheses were that β-NGF would be present in the stallion reproductive tract and would primarily be localized in Sertoli cells of the testes and the prostate gland. Immunohistochemistry on paraffin-embedded paraformaldehyde-fixed tissues was performed using a rabbit polyclonal anti-β-NGF antibody on a total of six male equine reproductive tracts, including a one-day old colt, a one-year-old colt, and four adult stallion tracts. Strong immunostaining was observed in the efferent ducts of the testes and the epithelial cells of the prostate, seminal vesicles, bulbourethral glands, and ampullae. Weaker β-NGF staining was noted in Leydig cells, Sertoli cells, and spermatogonia within the testes and in epithelial cells of the epididymis. In conclusion, immunohistochemistry revealed that β-NGF is present in the stallion reproductive tract, and the protein is primarily present in the efferent ducts of the testes and in all accessory sex glands.

Epididymal mRNA expression profiles for the protein disulfide isomerase gene family: Modulation by development and androgens.

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

Electro osmotic flow of nanofluids within a porous symmetric tapered ciliated channel

AbstractIn this investigation, a comprehensive study has been made to reveal electro osmosis flow through a tapered ciliated symmetric porous channel. The flow is initiated due to metachronal dynamics of cilia. Axial electric field is deployed and thermal radiation phenomenon is scrutinised by applying convective conditions. The equations tackling the flow are non dimensionalized and simplified by capitalizing the low Reynolds number and long wave length approximations. Analytical solution is presented for well reputed Poissson equation and the axial velocity. Whereas, traverse velocity, temperature and nanofluids concentration profiles are examined numerically in MATHEMATICA. Variation of emerging crucial parameters on the velocity profile, temperature and concentration profiles, pressure gradient, pressure rise per wavelength, and on the velocity distribution inside the micro ciliated are exhibited with the aid of graphical deliberations. It worth to mention in this work that in case of tapered channel transverse velocity also has significant contribution in the flow, which is observed trivial in symmetric and non‐symmetric channel flows. Temperature of the nanofluid in the ciliated tapered channel is raised with permeability and thermal radiations phenomena and can be controlled with Helmholtz Smoluchowski velocity, and electroosmotic parameter. Pumping phenomena is affected with increase in Helmholtz Smoluchowski velocity and permeability. Reported investigation cover a informative insight about biological fluid system, may be beneficial for the understanding the flow through ductus efferentes of human reproductive tract since it assumed that cilia are responsible for the transport of sperm from rete testis to the epididymis, also have worth in cilia designed bio‐sensors and in certain drug delivery systems.

A new type of spermiogenesis in teleost fish: Formation of the aflagellate sperm in Campylomormyrus compressirostris (Osteoglossomorpha: Mormyridae)

Osteoglossomorpha, the bony tongue fishes, show great variation in morphology, behavioural strategies, reproductive biology and gamete ultrastructure. The order Osteoglossiformes is the only vertebrate taxon, in which four types of sperm (monoflagellate, biflagellate and aflagellate aquasperm and the complex introsperm) have been described. It is also the only vertebrate lineage in which aflagellate spermatozoa exist. The aim of this study was to analyse the structure of the testis and the process of spermiogenesis in the mormyrid Campylomormyrus compressirostris during the breeding season using light and electron microscopy (transmission and scanning). Males of this species have a single testis of the anastomosing tubular type. The tubules of the anterior part of the testis contain cysts with developing germ cells, and this region is much wider than the posterior part, which consists of efferent ducts filled with sperm cells. The cysts are filled with single or mitotic spermatogonia, primary and secondary spermatocytes and early spermatids. At the stage of spermatids with fine granular chromatin, the cysts rupture and successive stages of spermatid differentiation take place in the testicular lumen; we therefore characterise this process as ‘extracystic spermiogenesis’. Sperm development in C. compressirostris is extremely simple and involves chromatin condensation in the central region of the nucleus, a slight decrease in nuclear volume, the appearance of numerous vesicles in the cytoplasm that form a tubular-vesicular system at the base of the nucleus. Both centrioles and mitochondria are translocated to the peripheral region of the midpiece, which forms the opposite pole to the nucleus. There are many differences between the types of spermiogenesis described so far in teleosts and that found in C. compressirostris, including the loss of flagellum formation. This unique type of spermiogenesis is restricted to species of the families Mormyridae and Gymnarchidae, all of which possess aflagellate spermatozoa. Our data demonstrate that the spermatid differentiation and existence of the aflagellate spermatozoon are a unique phenomena not only among teleosts but also in the whole vertebrate lineage.

PH Homeodynamics and Male Fertility: A Coordinated Regulation of Acid-Based Balance during Sperm Journey to Fertilization.

pH homeostasis is crucial for spermatogenesis, sperm maturation, sperm physiological function, and fertilization in mammals. HCO3- and H+ are the most significant factors involved in regulating pH homeostasis in the male reproductive system. Multiple pH-regulating transporters and ion channels localize in the testis, epididymis, and spermatozoa, such as HCO3- transporters (solute carrier family 4 and solute carrier family 26 transporters), carbonic anhydrases, and H+-transport channels and enzymes (e.g., Na+-H+ exchangers, monocarboxylate transporters, H+-ATPases, and voltage-gated proton channels). Hormone-mediated signals impose an influence on the production of some HCO3- or H+ transporters, such as NBCe1, SLC4A2, MCT4, etc. Additionally, ion channels including sperm-specific cationic channels for Ca2+ (CatSper) and K+ (SLO3) are directly or indirectly regulated by pH, exerting specific actions on spermatozoa. The slightly alkaline testicular pH is conducive to spermatogenesis, whereas the epididymis's low HCO3- concentration and acidic lumen are favorable for sperm maturation and storage. Spermatozoa pH increases substantially after being fused with seminal fluid to enhance motility. In the female reproductive tract, sperm are subjected to increasing concentrations of HCO3- in the uterine and fallopian tube, causing a rise in the intracellular pH (pHi) of spermatozoa, leading to hyperpolarization of sperm plasma membranes, capacitation, hyperactivation, acrosome reaction, and ultimately fertilization. The physiological regulation initiated by SLC26A3, SLC26A8, NHA1, sNHE, and CFTR localized in sperm is proven for certain to be involved in male fertility. This review intends to present the key factors and characteristics of pHi regulation in the testes, efferent duct, epididymis, seminal fluid, and female reproductive tract, as well as the associated mechanisms during the sperm journey to fertilization, proposing insights into outstanding subjects and future research trends.

The effect of epididymosomes on the development of frozen-thawed mouse spermatogonial stem cells after culture in a decellularized testicular scaffold and transplantation into azoospermic mice.

This study examined SSC proliferation on an epididymosome-enriched decellularized testicular matrix (DTM) hydrogel and spermatogenesis induction in azoospermic mice. Epididymosomes were extracted and characterized using SEM and western blotting. After cryopreservation, thawed SSCs were cultured in a hydrogel-based three-dimensional (3D) culture containing 10 ng/mL GDNF or 20µg/mL epididymosomes. SSCs were assessed using the MTT assay, flow cytometry, and qRT-PCR after two weeks of culture. The isolated SSCs were microinjected into the efferent ducts of busulfan-treated mice. DiI-labeled SSCs were followed, and cell homing was assessed after two weeks. After 8 weeks, the testes were evaluated using morphometric studies and immunohistochemistry. The expression of PLZF, TGF-β, and miR-10b did not increase statistically significantly in the 3D + GDNF and 3D + epididymosome groups compared to the 3D group. Among the groups, the GDNF-treated group exhibited the highest expression of miR-21 (*P < 0.05). Caspase-3 expression was lower in the epididymosome-treated group than in the other groups (***P < 0.001). Compared to the 3D and negative control groups, the 3D + epididymosomes and 3D + GDNF groups showed an increase in spermatogenic cells. Immunohistochemical results confirmed the growth and differentiation of spermatogonial cells into spermatids in the treatment groups. The DTM hydrogel containing 20µg/mL epididymosomes or 10 ng/mL GDNF is a novel and safe culture system that can support SSC proliferation in vitro to obtain adequate SSCs for transplantation success. It could be a novel therapeutic agent that could recover deregulated SSCs in azoospermic patients.

Male genital system of Ameiva ameiva (Squamata: Teiidae).

Understanding squamate reproductive morphology is crucial for investigating ecological, behavioral, and evolutionary questions. Here, we describe the anatomy and histology of the male genital system of Ameiva ameiva from southeastern Brazil. Ten adult males were dissected to characterize genital macroscopy and collect fragments of the testes, gonadoducts, and kidneys for histological examination. We examined 10 transverse histological sections per individual and measured the epithelial height of the epididymis and ductus deferens. The male reproductive system consists of a pair of yellowish oval testes, the rete testis, ductuli efferentes, epididymis, ductus deferens, ampulla ductus deferentis, sexual segment of the kidney (SSK), cloaca, and hemipenis. The hemipenis is elongated, cylindrical, and unilobed, with a sulcate face and an asulcate face, which has continuous fringes throughout its length. Seminiferous tubules exhibited germ cells at various stages. The epididymis is wider and more coiled than the ductus deferens. The rete testis has a simple squamous epithelium with long stereocilia, while the narrower ductuli efferentes are lined by a simple ciliated cuboidal epithelium. The epididymal epithelium is pseudostratified columnar, with basal and ciliated principal cells, whereas the ductus deferens epithelium is pseudostratified to simple cuboidal. The epididymal epithelium is 1.5 times taller than the ductus deferens epithelium. Here, we observed the SSK present in the cortex of the ventral region of the kidneys due to the hypertrophy of the distal convoluted tubules, as well as its secretory activity. Our findings will contribute to future research into the evolution of squamate reproductive morphology.

Symptomatic Intraparenchymal Epididymal Cysts: Description of 11 cases.

Epididymal cysts are benign cystic formations of the epididymis that usually appear in adolescence or early adulthood. Their frequency doubles after the age of 14-15. Obstruction in the epididymal efferent ductules with subsequent prostenotic dilatation of them, as well as dysgenesis due to hormonal disorders during fetal or postnatal life, are possible. At the 1st Department of Pediatric Surgery of A.U.Th. we treated 11 cases of boys at the age of 11-16 who presented with acute scrotum because of an epididymal cyst. The diagnosis was confirmed by ultrasound scanning . Due to persistent symptomatology, patients underwent surgical exploration and removal of the cyst. The postoperative care of the patients was uncomplicated with immediate remission of symptoms. In one case, ipsilateral acute epididymitis occurred after 10 days, which was successfully treated with antibiotic therapy. It is reported that approximately 50% of epididymal cysts involute within an average of 17 months. In conclusion, using the data obtained from the review, of the small in number of international bibliography studies, it is proposed conservative treatment of asymptomatic cysts with diameter smaller than 1 cm and surgical excision [1] of large asymptomatic cysts with diameter greater than 1 cm, which do not regress after a follow-up of 24-48 months, cysts, regardless of their diameter, responsible for persistent symptoms and in the manifestation of acute scrotal symptoms due to inflammation, intravesical bleeding or secondarily torsion of the epididymis.

The antiapoptotic effects of conditioned medium from bone marrow-derived mesenchymal stromal stem cells on cyclophosphamide-induced testicular damage in rat: An experimental study.

Cyclophosphamide (CP) has some negative effects on the reproductive system. Stem cells and their metabolites are being utilized to enhance fertility after chemotherapy. This study aimed to investigate the impact of conditioned medium (CM) derived from bone marrow mesenchymal stromal stem cells (BM-MSCs) on the toxic effects of CP on testicles. BM-MSCs were isolated, a CM was collected and 25-fold concentrated. 24 male Wistar rats (8 wk, 200-250 gr) were randomly divided into following groups: control, CP, CP+ Dulbecco's Modified Eagle Medium (DMEM), CP+CM. CP was given at a single dose of 100 mg/kg. 2 wk after the CP administration, CM was injected into the testicular efferent duct. Sperm parameters, testicular histopathology, and the level of testosterone were analyzed 2 months after treatment. The expression of B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) genes were evaluated by real-time polymerase chain reaction. CP had a negative effect on testis histology (p 0.001) and sperm quality (p 0.001). It changed the expression of genes associated with apoptosis (p 0.001). Treatment with CM reduced the expression of Bax (p 0.001), while significantly increasing the expression of Bcl2 (p = 0.01). It improved sperm count (p = 0.03), viability (p 0.001), motility (p 0.001), spermatogonial count (p 0.001), and epithelial thickness of testicular tubules (p = 0.02). These findings suggest that CM produced from BM-MSCs may be valuable for therapeutic approaches in reproductive medicine and may lessen the side effects of CP.

(221) Anatomical Transcriptome Atlas of the Male Mouse Reproductive System During Aging

Abstract Introduction The elderly males undergo degenerative fertility and testicular endocrine function that jeopardize the reproductive health and well-being. However, the mechanisms underlying reproductive aging are unclear. Objective This study aims to investigate the phenotypes and transcriptomes of seven regions of the male mouse reproductive tract: the testis, efferent ductules, initial segment, caput, corpus and cauda epididymidis, and vas deferens, in adult (3 months) and aged (21 months) mice. Methods Quantitative PCR, immunohistochemistry, immunofluorescent staining, and enzyme-linked immunosorbent assay were performed for the analysis of gene expression in mice, human tissues, and semen samples. Aged male mice showed both systematic and reproductive changes, and remarkable histological changes were detected in the testis and proximal epididymis. Transcriptomes of the male reproductive tract were mapped, and a series of region-specific genes were identified and validated in mouse and/or human tissues, including Protamine 1 (Prm2), ADAM metallopeptidase domain 28 (Adam28), Ribonuclease A family member 13 (Rnase13), WAP four-disulfide core domain 13 (Wfdc13), and Wfdc9. Meanwhile, age-related transcriptome changes of different regions of the male reproductive tract were characterized. Results Notably, increased immune response was functionally related to the male reproductive aging, especially the T cell activation. An immune response-associated factor, phospholipase A2 group IID (Pla2g2d), was identified as a potential biomarker for reproductive aging in mice. And the PLA2G2D level in human seminal plasma surged at approximately 35 years of age. Furthermore, we highlighted Protein tyrosine phosphatase receptor type C (Ptprc), Lymphocyte protein tyrosine kinase (Lck), Microtubule associated protein tau (Mapt), and Interferon induced protein with tetratricopeptide repeats 3 (Ifit3) as critical molecules in the aging of initial segment, caput, caput, and cauda epididymidis, respectively. Conclusions This study provides an RNA-seq resource for the male reproductive system during aging in mice, and is expected to improve our understanding of male reproductive aging and infertility. Disclosure No.

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.

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.

Adeno-associated-virus-mediated delivery of CRISPR-CasRx induces efficient RNA knockdown in the mouse testis.

Rationale: In male mammals, many developmental-stage-specific RNA transcripts (both coding and noncoding) are preferentially or exclusively expressed in the testis, where they play important roles in spermatogenesis and male fertility. However, a reliable platform for efficiently depleting various types of RNA transcripts to study their biological functions during spermatogenesis in vivo has not been developed. Methods: We used an adeno-associated virus serotype nine (AAV9)-mediated CRISPR-CasRx system to knock down the expression of exogenous and endogenous RNA transcripts in the testis. Virus particles were injected into the seminiferous tubules via the efferent duct. Using an autophagy inhibitor, 3-methyladenine (3-MA), we optimized the AAV9 transduction efficiency in germ cells in vivo. Results: AAV9-mediated delivery of CRISPR-CasRx effectively and specifically induces RNA transcripts (both coding and noncoding) knockdown in the testis in vivo. In addition, we showed that the co-microinjection of AAV9 and 3-MA into the seminiferous tubules enabled long-term transgene expression in the testis. Finally, we found that a promoter of Sycp1 gene induced CRISPR-CasRx-mediated RNA transcript knockdown in a germ-cell-type-specific manner. Conclusion: Our results demonstrate the efficacy and versatility of the AAV9-mediated CRISPR-CasRx system as a flexible knockdown platform for studying gene function during spermatogenesis in vivo. This approach may advance the development of RNA-targeting therapies for conditions affecting reproductive health.