Efferent Ducts Research Articles

C1q/TNF-Related Proteins 1, 6 and 8 Are Involved in Corneal Epithelial Wound Closure by Targeting Relaxin Receptor RXFP1 In Vitro.

Inadequate wound healing of ocular surface injuries can lead to permanent visual impairment. The relaxin ligand-receptor system has been demonstrated to promote corneal wound healing through increased cell migration and modulation of extracellular matrix formation. Recently, C1q/tumor necrosis factor-related protein (CTRP) 8 was identified as a novel interaction partner of relaxin receptor RXFP1. Additional data also suggest a role for CTRP1 and CTRP6 in RXFP1-mediated cAMP signaling. However, the role of CTRP1, CTRP6 and CTRP8 at the ocular surface remains unclear. In this study, we investigated the effects of CTRP1, CTRP6, and CTRP8 on epithelial ocular surface wound closure and their dependence on the RXFP1 receptor pathway. CTRP1, CTRP6, and CTRP8 expression was analyzed by RT-PCR and immunohistochemistry in human tissues and cell lines derived from the ocular surface and lacrimal apparatus. In vitro ocular surface wound modeling was performed using scratch assays. We analyzed the effects of recombinant CTRP1, CTRP6, and CTRP8 on cell proliferation and migration in human corneal and conjunctival epithelial cell lines. Dependence on RXFP1 signaling was established by inhibiting ligand binding to RXFP1 using a specific anti-RXFP1 antibody. We detected the expression of CTRP1, CTRP6, and CTRP8 in human tissue samples of the cornea, conjunctiva, meibomian gland, efferent tear ducts, and lacrimal gland, as well as in human corneal, conjunctival, and meibomian gland epithelial cell lines. Scratch assays revealed a dose-dependent increase in the closure rate of surface defects in human corneal epithelial cells after treatment with CTRP1, CTRP6, and CTRP8, but not in conjunctival epithelial cells. Inhibition of RXFP1 fully attenuated the effect of CTRP8 on the closure rate of surface defects in human corneal epithelial cells, whereas the CTRP1 and CTRP6 effects were not completely suppressed. Conclusions: Our findings demonstrate a novel role for CTRP1, CTRP6, and CTRP8 in corneal epithelial wound closure and suggest an involvement of the relaxin receptor RXFP1 signaling pathway. This could be a first step toward new approaches for pharmacological and therapeutic intervention.

Electro-osmotic transport of a Williamson fluid within a ciliated microchannel with heat transfer analysis

A novel theoretical model is devised for electro osmotic flow of Williamson fluid model by incorporating heat transfer phenomena in a micro ciliated channel. The physical fluid model is designed by exploiting the renowned Debye–Huckel, long wavelength small Reynolds number approximation. The exact expression for Poisson–Boltzmann equation is obtained to find the analytical solution for axial velocity, pressure gradient, temperature and stream functions comprehensively by using perturbation technique. Whereas, transverse velocity and pressure rise profile are explored numerically through the MATHEMATICA software infrastructure. The variations of paramount significant parameters on the velocity distribution, temperature, pressure gradient and pressure rise per wavelength are demonstrated graphically. It has been investigated that axial velocity profiles escalates with enhancing Weissenberg, electro-osmotic, mobility of medium parameter, while dual behavior is recorded for the case of traverse component of velocity. Cilia plays a crucial role in ductus efferentes and assumed to transfer sperm through the rete testis to the epididymis. Besides above mentioned application cilia like actuators serve in micro-mixers to regulate the flow in bio-sensors and in various drug-delivery mechanism.

The chemical composition of the secretions, their antibacterial activity, and the pygidial gland morphology of selected European Carabini ground beetles (Coleoptera: Carabidae)

The pygidial glands of carabids produce strong-smelling vapours. In this study, we examined the chemical composition of the gland secretions and the structure of the glands in five species of Carabini ground beetles (one species from the subtribe Calosomatina and four species from the subtribe Carabina): Calosoma (Calosoma) maderae (Fabricius, 1775), Carabus (Carabus) granulatus Linnaeus, 1758, C. (Limnocarabus) clathratus Linnaeus, 1760, C. (Carabus) ulrichii Germar, 1823, and C. (Procerus) gigas Creutzer, 1799. Additionally, we tested the antibacterial potential of the pygidial gland secretions of the two latter species against 11 bacterial strains. In order to detect the chemical content of the secretions, we used gas chromatography–mass spectrometry (GC–MS). The secretion extracts were applied against selected strains of medically important bacteria. We used bright-field microscopy to examine the morphology of the glands. We discovered a total of 11 chemical compounds in the pygidial gland extracts of the ground beetles we analysed. Ten of these compounds were identified as seven carboxylic acids, two hydrocarbons, and one aromatic aldehyde, while one chemical remained unidentified. Most of the components were isolated from the secretion of C. (L.) clathratus (nine), while the lowest number of compounds was found in C. (P.) gigas (two). Methacrylic acid was the most dominant compound by percentage in all five species, while angelic acid was also detected in all samples. As expected, salicylaldehyde was exclusively found in the species of the genus Calosoma Weber, 1801. The secretion of C. (P.) gigas was shown to achieve the highest level of antibacterial activity against Pseudomonas aeruginosa, Salmonella enterica, and S. typhimurium (even the same level as the positive control streptomycin), while the secretion of C. (C.) ulrichii achieved the highest antimicrobial potential against Staphylococcus epidermidis, S. aureus, Listeria monocytogenes, and Bacillus cereus. The most noticeable difference in the structure of the glands between the two genera is that the reservoir in Calosoma is more significantly narrowed as it leads into the efferent duct, compared to that of Carabus.

The Structure of the Female Genital System of the Diving Beetle Scarodytes halensis (Fabricius, 1787) (Hydroporinae, Dytiscidae), and the Organization of the Spermatheca and the Spermathecal Gland Complex

The fine structure of the female reproductive organs of the diving beetle Scarodytes halensis has been described, with particular attention to the complex organization of the spermatheca and the spermathecal gland. These organs are fused in a single structure whose epithelium is involved in a quite different activity. The secretory cells of the spermathecal gland have a large extracellular cistern with secretions; duct-forming cells, by their efferent duct, transport the secretions up to the apical cell region where they are discharged into the gland lumen. On the contrary, the spermatheca, filled with sperm, has a quite simple epithelium, apparently not involved in secretory activity. The ultrastructure of the spermatheca is almost identical to that described in a closely related species Stictonectes optatus. Sc. halensis has a long spermathecal duct connecting the bursa copulatrix to the spermatheca-spermathecal gland complex. This duct has a thick outer layer of muscle cells. Through muscle contractions, sperm can be pushed forwarding up to the complex of the two organs. A short fertilization duct allows sperm to reach the common oviduct where eggs will be fertilized. The different organization of the genital systems of Sc. halensis and S. optatus might be related to a different reproductive strategy of the two species.

Heat Shock Proteins Are Differentially Expressed in the Domestic Cat (Felis catus) Testis, Epididymis, and Vas Deferens.

Heat shock proteins (HSPs) play key roles in controlling the morphological transformation of germ cells during spermatogenesis and posttesticular maturation of sperm. This study aims to reveal the presence and localization patterns of large adenosine triphosphate (ATP)-dependent HSPs family members in adult domestic cat testis and excurrent ducts utilizing Western blot, immunohistochemistry, and immunofluorescence techniques. The results indicated that the relative amounts of heat shock protein D1 (HSPD1)/HSP60, heat shock protein C (HSPC)/HSP90, and heat shock protein H (HSPH)/HSP105/110 were highest in the testis, while heat shock protein A (HSPA)/HSP70 was highest in the corpus epididymis. HSPs exhibited spermatogenic stage-dependent localization patterns in germ cells. Sertoli and Leydig cells were positive for other HSPs except for HSPC/HSP90. The tubules rectus and rete testis epithelia showed only HSPD1/HSP60 and HSPA/HSP70 immunoreactivity, while the ciliated cells of efferent ductules were positive for all HSPs. In the epididymis and vas deferens, HSPs localizations were cell and region specific. HSPD1/HSP60 was localized in the midpiece of the immature spermatozoa tail, while HSPA/HSP70 and HSP90 were found only in the proximal cytoplasmic droplet (CD). HSPH/HSP105 was observed in CD and the principal piece but not the midpiece. Overall, the different expression of HSPs throughout the domestic cat testis and excurrent ducts indicates their critical roles in maintaining reproductive functions under physiological conditions.

Pathogenic gene variants in CCDC39, CCDC40, RSPH1, RSPH9, HYDIN, and SPEF2 cause defects of sperm flagella composition and male infertility.

Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder affecting the function of motile cilia in several organ systems. In PCD, male infertility is caused by defective sperm flagella composition or deficient motile cilia function in the efferent ducts of the male reproductive system. Different PCD-associated genes encoding axonemal components involved in the regulation of ciliary and flagellar beating are also reported to cause infertility due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we performed genetic testing by next generation sequencing techniques, PCD diagnostics including immunofluorescence-, transmission electron-, and high-speed video microscopy on sperm flagella and andrological work up including semen analyses. We identified ten infertile male individuals with pathogenic variants in CCDC39 (one) and CCDC40 (two) encoding ruler proteins, RSPH1 (two) and RSPH9 (one) encoding radial spoke head proteins, and HYDIN (two) and SPEF2 (two) encoding CP-associated proteins, respectively. We demonstrate for the first time that pathogenic variants in RSPH1 and RSPH9 cause male infertility due to sperm cell dysmotility and abnormal flagellar RSPH1 and RSPH9 composition. We also provide novel evidence for MMAF in HYDIN- and RSPH1-mutant individuals. We show absence or severe reduction of CCDC39 and SPEF2 in sperm flagella of CCDC39- and CCDC40-mutant individuals and HYDIN- and SPEF2-mutant individuals, respectively. Thereby, we reveal interactions between CCDC39 and CCDC40 as well as HYDIN and SPEF2 in sperm flagella. Our findings demonstrate that immunofluorescence microscopy in sperm cells is a valuable tool to identify flagellar defects related to the axonemal ruler, radial spoke head and the central pair apparatus, thus aiding the diagnosis of male infertility. This is of particular importance to classify the pathogenicity of genetic defects, especially in cases of missense variants of unknown significance, or to interpret HYDIN variants that are confounded by the presence of the almost identical pseudogene HYDIN2.

The impact of primary ciliary dyskinesia on female and male fertility: a narrative review.

Primary ciliary dyskinesia (PCD) is a genetic condition affecting the structure and function of sperm flagellum and motile cilia including those in the male and female reproductive tracts. Infertility is a commonly reported feature of PCD, but there is uncertainty as to how best to counsel patients on their fertility prognosis. This review aimed to summarize the prevalence of subfertility, possible underlying mechanisms, and the success of ART in men and women with PCD. The efficacy of ART in this patient group is relatively unknown and, hence, the management of infertility in PCD patients remains a challenge. There are no previous published or registered systematic reviews of fertility outcomes in PCD. Systematic literature searches were performed in Medline, Embase, Cochrane Library, and PubMed electronic databases to identify publications between 1964 and 2022 reporting fertility outcomes in men and women with PCD. Publications were excluded if they reported only animal studies, where gender was not specified or where subjects had a medical co-morbidity also known to impact fertility. Quality of evidence was assessed by critical appraisal and application of an appraisal tool for cross-sectional studies. The primary outcomes were natural conception in men and women with PCD, and conception following ART in men and women with PCD. A total of 1565 publications were identified, and 108 publications were included after screening by two independent researchers. The quality of available evidence was low. The exact prevalence of subfertility in PCD is unclear but appears to be higher in men (up to 83% affected) compared to women (up to 61% affected). Variation in the prevalence of subfertility was observed between geographic populations which may be explained by differences in underlying genotype and cilia function. Limited evidence suggests subfertility in affected individuals is likely caused by abnormal cilia motion in the fallopian tubes, endometrium and efferent ductules, and dysmotile sperm. Some men and women with PCD benefited from ART, which suggests its use should be considered in the management of subfertility in this patient group. Further epidemiological and controlled studies are needed to determine the predictors of fertility and optimal management in this patient group. It is important that patients with PCD receive evidence-based counselling about the potential impact of their condition on their fertility prognosis and what management options may be available to them if affected. Understanding the pathophysiology and optimal management of subfertility in PCD will increase our understanding of the role of cilia and the impact of wider secondary ciliopathies on reproduction.

Development and application of a novel cervical lymph collection method to assess lymphatic transport in rats.

Background: Fluids, solutes and immune cells have been demonstrated to drain from the brain and surrounding structures to the cervical lymph vessels and nodes in the neck via meningeal lymphatics, nasal lymphatics and/or lymphatic vessels associated with cranial nerves. A method to cannulate the efferent cervical lymph duct for continuous cervical lymph fluid collection in rodents has not been described previously and would assist in evaluating the transport of molecules and immune cells from the head and brain via the lymphatics, as well as changes in lymphatic transport and lymph composition with different physiological challenges or diseases. Aim: To develop a novel method to cannulate and continuously collect lymph fluid from the cervical lymph duct in rats and to analyze the protein, lipid and immune cell composition of the collected cervical lymph fluid. Methods: Male Sprague-Dawley rats were cannulated at the carotid artery with or without cannulation or ligation at the cervical lymph duct. Samples of blood, whole lymph and isolated lipoprotein fractions of lymph were collected and analyzed for lipid and protein composition using commercial kits. Whole lymph samples were centrifuged and isolated pellets were stained and processed for flow cytometry analysis of CD3+, CD4+, CD8a+, CD45R+ (B220) and viable cell populations. Results: Flow rate, phospholipid, triglyceride, cholesterol ester, free cholesterol and protein concentrations in cervical lymph were 0.094 ± 0.014mL/h, 0.34 ± 0.10, 0.30 ± 0.04, 0.07 ± 0.02, 0.02 ± 0.01 and 16.78 ± 2.06mg/mL, respectively. Protein was mostly contained within the non-lipoprotein fraction but all lipoprotein types were also present. Flow cytometry analysis of cervical lymph showed that 67.1 ± 7.4% of cells were CD3+/CD4+ T lymphocytes, 5.8 ± 1.6% of cells were CD3+/CD8+ T lymphocytes, and 10.8 ± 4.6% of cells were CD3-/CD45R+ B lymphocytes. The remaining 16.3 ± 4.6% cells were CD3-/CD45- and identified as non-lymphocytes. Conclusion: Our novel cervical lymph cannulation method enables quantitative analysis of the lymphatic transport of immune cells and molecules in the cervical lymph of rats for the first time. This valuable tool will enable more detailed quantitative analysis of changes to cervical lymph composition and transport in health and disease, and could be a valuable resource for discovery of biomarkers or therapeutic targets in future studies.

Unilateral testicular atrophy and epididymal lithiasis in a Gramasree rooster

Testicular atrophy is a condition impairing the reproductive health of male chicken. Lithiasis or stone formation in testicles or epididymis adds to the gravity of the problem. The present study describes a case of unilateral testicular atrophy with epididymal lithiasis in a one-year-old male Gramasree cock. The carcass, was brought to the Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Pookode, for postmortem examination. The lesions were recorded and tissues for histopathology were collected in 10% neutral buffered formalin, sectioned, and stained using routine hematoxylin and eosin staining procedure. Grossly, there was diffused congestion of the testicular tunic with atrophy of right testis. The epididymis was congested with multifocal white spots. Histologically, seminiferous tubules were severely degenerated. The efferent ductules of epididymis were dilated with basophilic amorphous and granular liths in multiple locations. Sperm stasis was evident in a few ductules and rete testes. The lith formation might have caused sperm stasis and degeneration and atrophy of the testis.

Photoperiodic-dependent histomorphological changes in the excurrent duct system of helmeted guinea fowl subjected to short day (8L:16D), long-day (16L:8D) light/dark cycles and exogenous melatonin

In the present study, the influence of varying photoperiods [short day light 8L:16D, long day light 16L;8D] and exogeneous melatonin on the excurrent duct system of male helmeted guinea fowl was investigated using histo-morphological and histometric approaches. A total of twenty-eight (28) guinea fowl birds were randomly divided into Group I: Short daylight (SD; 8 HL), Group II: (SD +1mg/kg melatonin; 8 HL+ Mel), Group III: Long daylight (LD; 16 HL) and Group IV: (LD +1mg/kg; 16 HL + Mel)] and comprises of seven birds (n=7) per group. At the end of the 8 weeks of experimentation, the excurrent ducts were excised and processed for routine histological examination and the variations in histo-morphometrical parameters were determined using the GIMP2 software. Histologically, apart from the moderate cellular degeneration observed in efferent duct epithelia of the SD subgroups: (8 HL and 8 HL + Mel), there was remarkable spermatozoa presence in the lumens of the epididymal duct and ductus deferens of both 16 HL and 16 HL + Mel groups. The histo-morphometric data (luminal, ductal diameters and epithelial heights) were significantly increased (p <0.05) in the excurrent ducts of guinea fowl exposed to 16 HL and 16 HL + Mel, as compared to other groups. There was significant decrease (p <0.05) in stereocilia height (SH) in 16 HL compared to 8 HL sub-groups of lower segments. Although, a non-significant (p >0.05) increase in SH was observed in melatonin-treated groups, regardless of photoperiod. Taken together, these sets of data from this study indicate the importance of artificial light and exogenous melatonin in the control of seasonality of reproduction and which could be used to influence the reproductive cycle of the guinea fowl.

Improving the process of spermatogenesis in azoospermic mice using spermatogonial stem cells co-cultured with epididymosomes in three-dimensional culture system

AimsThis study aimed to explore the effect of epididymosomes on the proliferative efficiency of spermatogonial stem cells (SSCs) in vitro and the resumption of spermatogenesis in the azoospermic mice. Main methodsThe epididymosomes were extracted from the epididymis and characterized. SSCs were cultured in 2D (two-dimensional) and hydrogel-based 3D culture in the presence of 20 μg/mL epididymosome or 10 ng/mL GDNF. After two weeks of culture, the proliferation and purity of the separated SSCs were evaluated using the MTT test and flow cytometry, respectively. qRT-PCR was used to analyze PLZF, caspase-3, TGF-β, miR-10b, and miR-21 expression levels. Then, SSCs grown in the 3D culture system were labeled by DiI and transplanted into azoospermic mice via the efferent duct. After 2 weeks, tracing of DiI and cell homing were evaluated. Subsequently, histomorphometric studies and immunohistochemistry analysis were performed in testes after eight weeks of transplantation. Key findingsThe expression of PLZF, TGF-β, miR-10b, and miR-21 increased significantly (*p < 0.05) in the 3D + GDNF and 3D + epididymosomes groups than in the 2D group. Transplanted SSCs migrated into the seminiferous tubules of recipient mice and the number of spermatogenic cells and protein expression of PLZF, SCP3 and ACRBP in the 3D + GDNF and 3D + epididymosomes groups were considerably higher (∗ ∗ ∗ p < 0.001) compared to the azoospermic group. SignificanceThis finding indicates that culturing SSCs on decellularized testicular matrix (DTM) hydrogel with 10 ng/mL GDNF or 20 μg/mL epididymosomes could lead to an increase in SSCs proliferation which provides a sufficient number of SSCs for successful transplantation in azoospermic mice.

An investigation on cauda storage of sperm in hamster epididymis

Abstract Immature testicular sperm of mammals acquire the potential to move in a forward direction during their journey through excurrent ducts, efferent ductules and the epididymis. The present study aimed to determine the sperm storage time using the hamster cauda epididymis. Ligation of the left epididymal tubule at the junction between the distal corpus and the proximal cauda was carried out to determine the storage time. The right epididymis was left unaltered as the control. On days 3, 12, 15, 24, 28, 32, and 40 after ligation of the cauda, experiments were carried out and repeated five times. Sperm total count and mortality were determined using the haemocytometer counting method and sperm viability was assessed with the live/dead viability kit. The morphology of cauda sperm was observed by preparing sperm smears from each animal. Data were analyzed using SPSS and all values were expressed as mean ± SEM. On day 40, the total number of cauda sperms was reduced remarkably. In the experimental groups and in the control, 3–6% of sperm motility was maintained until day 40. By day 3, the percentage of live sperm was 50% and by the 40th day, it was decreased up to 10%. In the control group, the live sperm percentage was 24% by the 40th day (). By day 32, 76% of the cauda spermatozoa appeared abnormal with head defects, mid piece and neck defects and multiple defects. Findings of the present study indicate that cauda sperm storage time is more than 40 days. Motility, viability and morphology of these spermatozoa were decreased remarkably during this storage time.

The murine male reproductive organ at a glance: Three-dimensional insights and virtual histology using label-free light sheet microcopy.

The unique anatomy of the male reproductive organ reflects its complex function from sperm maturation to their storage for months until emission. Since light microscopy in two dimensions (2d) cannot sufficiently demonstrate its complex morphology, a comprehensive visualization is required to identify pathologic alterations in its entire anatomical context. Aim of this study was to use three-dimensional (3d) light sheet fluorescence microscopy (LSFM) to visualize entire murine testes in 3d, label-free and at subcellular resolution, and to assign local autofluorescence to testicular and deferent structures. Murine testes were fixed with four different fixatives and subsequently cleared with benzoic acid/benzyl benzoate. Hereafter, complete murine testes were scanned with LSFM with different fluorescence filter sets and subsequently embedded in paraffin for further conventional planar histology. Autofluorescence signals of the murine reproductive organ allowed the unambiguous identification of the testicular anatomy from the seminiferous tubules to the vas deferens with their specific stratification independent of the used fixative. Blood vessels were visualized from the pampiniform plexus to the small capillaries of single tubules. Moreover, due to the specific intrinsic fluorescence properties of the efferent ducts and the epididymis, luminal caliber, the epithelial stratification and retronuclear cytoplasmic inclusions gave a unique insight into the interface of both morphological structures. Subsequent 2d histology confirmed the identified morphological structures. LSFM analysis of the murine reproductive organ allows due to its intrinsic fluorescence a simple, label-free 3d assessment of its entire duct morphology, the epithelial composition, and the associated blood supply in its anatomical relation. LSFM provides the technical basis for comprehensive analyses of pathologically altered murine testes in its entirety by depicting specific autofluorescence. Thereby it facilitates mouse studies of testicular disease or their drug-related alterations in more detail potentially for clinical translation assessing human testicular biopsies.

The roles of plasma accessible and cytosolic carbonic anhydrases in bicarbonate (HCO3-) excretion in Pacific hagfish (Eptatretus stoutii).

Pacific hagfish (Eptatretus stoutii) are marine scavengers and feed on decaying animal carrion by burrowing their bodies inside rotten carcasses where they are exposed to several threatening environmental stressors, including hypercapnia (high partial pressures of CO2). Hagfish possess a remarkable capacity to tolerate hypercapnia, and their ability to recover from acid-base disturbances is well known. To deal with the metabolic acidosis resulting from exposure to high CO2, hagfish can mount a rapid elevation of plasma HCO3- concentration (hypercarbia). Once PCO2 is restored, hagfish quickly excrete their HCO3- load, a process that likely involves the enzyme carbonic anhydrase (CA), which catalyzes HCO3- dehydration into CO2 at the hagfish gills. We aimed to characterize the role of branchial CA in CO2/HCO3- clearance from the plasma at the gills of E. stoutii, under control and high PCO2 (hypercapnic) exposure conditions. We assessed the relative contributions of plasma accessible versus intracellular (cytosolic) CA to gill HCO3- excretion by measuring in situ [14C]-HCO3- fluxes. To accomplish this, we employed a novel surgical technique of individual gill pouch arterial perfusion combined with perifusion of the gill afferent to efferent water ducts. [14C]-HCO3- efflux was measured at the gills of fish exposed to control, hypercapnic (48h) and recovery from hypercapnia conditions (6h), in the presence of two well-known pharmacological inhibitors of CA, the membrane impermeant C18 (targets membrane bound, plasma accessible CA) and membrane-permeant acetazolamide, which targets all forms of CA, including extracellular and intracellular cytosolic CAs. C18 did not affect HCO3- flux in control fish, whereas acetazolamide resulted in a significant reduction of 72%. In hypercapnic fish, HCO3- fluxes were much higher and perfusion with acetazolamide caused a reduction of HCO3- flux by 38%. The same pattern was observed for fish in recovery, where in all three experimental conditions, there was no significant inhibition of plasma-accessible CA. We also observed no change in CA enzyme activity (measured in vitro) in any of the experimental PCO2 conditions. In summary, our data suggests that there are additional pathways for HCO3- excretion at the gills of hagfish that are independent of plasma-accessible CA.

Expression of the pro-inflammatory P2Y14 receptor in the non-vasectomized and vasectomized human epididymis.

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

OP-12 - Three-dimensional and immunohistochemical analysis of sex steroid receptors in the developing murine mesonephros

Sex steroids are essential for the development of the reproductive tracts, and their function is mediated by activating their receptors. The expression patterns of the receptors have been investigated in previous studies, but the results differ. One of the reasons is a lack of a method to distinguish each region of the reproductive tracts. To distinguish the cranial and caudal Wolffian duct (WD), which contribute to the common efferent duct and epididymal duct, respectively, it is necessary to check the connection with the mesonephric tubules (MTs) that differentiate into the efferent ductules. In this study, we examined the expression patterns of sex steroid receptors in detail to combine the three-dimensional analysis. Serial paraffin sections of the developing murine mesonephros were prepared, and immunohistochemistry for sex steroid receptors, such as estrogen receptor (ESR1), androgen receptor (AR), and progesterone receptor (PR), was performed. Additionally, the sections in which Collagen Type IV was detected were used to reconstruct the MTs and WD. ESR1 was found in the mesenchymal cells around the WD, and the MTs and cranial WD also became positive from the middle period of the development, but the caudal WD was negative for ESR1. AR was first found at the distal end (gonadal side) of the MTs and weakly in the mesenchymal cells. The epithelial expression became strong in the whole length of the MTs and cranial portion of the WD in the middle period of the development. Finally, the caudal part of the WD also showed strong positive just before birth. PR was weakly positive in the MTs and cranial WD, but the distal end of the MTs where the rete cells were adherent was negative for PR. Androgen secreted from the gonad probably acts on the distal end of the MTs to maintain and develop the efferent tubules. ESR1 and PR may also contribute to the development of the efferent duct. Additionally, estrogen treatment in the neonatal male mice induces epididymal inflammation after puberty, but its mechanisms are still unclear. This study found the ESR1 expression in not epithelial cells but mesenchymal cells around the caudal WD. These results suggest that neonatal estrogen treatment probably affects the mesenchymal cells around the epididymal duct, resulting in the inability to suppress the autoimmune response to spermatozoa after puberty.

Male reproductive cycle in a population of the endemic butterfly lizard, Leiolepis ocellata Peters, 1971 (Squamata: Agamidae) from northern Thailand

BackgroundFundamental knowledge on the seasonal reproductive microanatomy and endocrinology of reptiles has been collected from several studies of various species. The present study was to determine annual changes in hormonal profiles, and detailed histomorphometric and histochemical characteristics of the entire male reproductive system of the tropical agamid lizard, Leiolepis ocellata.ResultsMale L. ocellata individuals (n = 75) collected from the territory of two provinces (Lampang and Tak) in northern Thailand exhibited annual variation in sex hormonal, histomorphometric, and histochemical characteristics of the male reproductive system. The reproductive cycle was subdivided into eight reproductive periods (early first active, first active, resting, second recrudescent, second active, regressive, quiescent, and first recrudescent), thus displaying a bimodal pattern with two actively reproductive periods. Circulating sex hormones (testosterone, estradiol, and progesterone) peaked in the first active (February) and the second active (June–July) periods. Likewise, gonadosomatic index (GSI) and histomorphometric variables of the testes and of the genital ducts (rete testis, ductuli efferentes, ductus epididymis, and ductus deferens) revealed their highest values in the first active period. Marked increase in protein and carbohydrate production was detectable in the ductuli efferentes during the active periods.ConclusionsThe male reproductive cycle of L. ocellata showed a biannual pattern of the hormonal profile, and detailed histomorphometric and histochemical characteristics of the entire reproductive system. Hence, the present study provides improved basic knowledge on the reptilian reproductive biology with comparative viewpoints to other reptiles.

The appendage of the testis: morphogenesis, structural and functional characteristics in physiological and pathological conditions

The article focuses on the data from Russian and foreign literature on the morphofunctional characteristics of the appendage of the testis in humans and mammalian animals. In the appendage of the testis, the head, body and tail are isolated. The head is formed by the efferent ductules, then they pass into the duct of the appendage forming the body and tail of the appendage. The epithelial cell classification in the appendage is debatable, which is due to both different methodological approaches and species differences in the epithelial lining of the testis appendage in different species. Species differences in the morphofunctional organization of the epithelium of the appendage of the testis are insignificant, this manifests the principle of parallelism in the evolutionary dynamics of the appendage epithelium. The appendage of the testis is a hormone-dependent organ, androgens play a leading role in regulating the development and functioning of the appendage; in addition, estrogens and prolactin take a significant part in regulating the appendage activity. Morphological equivalents of the negative influence of various damaging factors are basically similar, which evidences the presence of a nonspecific mechanism of reaction to the action of adverse factors formed during evolution. Despite a significant number of studies related to the morphology of the testis appendage, many aspects of morphofunctional, molecular and biochemical transformations occurring in the testis appendage still remain poorly understood and require further in-depth study. There are also insufficient number of comparative studies, and studies investigating the appendage of the testis of animals of natural biocenosis.

The Infection Route of Tomato Zonate Spot Virus in the Digestive System of Its Insect Vector Frankliniella occidentalis.

Tomato zonate spot virus (TZSV) is a phytopathogen of the genus Orthotospovirus (Bunyaviridae) that is widespread in many areas of Southwest China. TZSV is mainly transmitted by Frankliniella occidentalis, but its exact infection route remains unclear. To explore this issue, we detected the nucleocapsid protein of TZSV in the digestive systems of first-instar F. occidentalis nymphs fed with TZSV-infected pepper leaves. TZSV infection in the F. occidentalis digestive system begins within 4 h post-first access to diseased plants: The foregut is likely the primary site of infection, and primary salivary glands (PSGs) are the destination. There are three potential routes for TZSV transmission from the alimentary canal to the PSGs: (1) virus dissemination from the midgut to hemocoel followed by movement to the PSGs; (2) accumulation in midgut epithelial cells and arrival at PSGs via tubular salivary glands and efferent ducts; and (3) arrival at epitheliomuscular cells of the forepart of the midgut and movement along the ligament to the PSGs. We tested the transmission efficiency of F. occidentalis in second-instar nymphs and female and male adults. TZSV was transmitted in a persistent-propagative mode by both nymphs and adults, with adults appearing to show slightly higher transmission efficiency than nymphs. We confirmed the presence of all three routes for TZSV transmission in F. occidentalis and determined that like other Orthotospoviruses, TZSV is transmitted in a persistent-propagative manner. These results should facilitate the control of TZSV-related diseases and further our understanding of the transmission biology of Orthotospoviruses in general.

Dynamic volumetric imaging and cilia beat mapping in the mouse male reproductive tract with optical coherence tomography.

Spermatozoa transport within the male reproductive tract is a highly dynamic and biologically important reproductive event. However, due to the lack of live volumetric imaging technologies and quantitative measurements, there is little information on the dynamic aspect and regulation of this process. Here, we presented ex vivo dynamic volumetric imaging of the mouse testis, efferent duct, epididymis, and vas deferens at a micro-scale spatial resolution with optical coherence tomography (OCT). Micro computed tomography imaging is presented as a reference for the proposed OCT imaging. Application of functional OCT analysis allowed for 3D mapping of the cilia beat frequency in the efferent duct, which volumetrically visualized the spatial distribution of the ciliated cells and corresponding ciliary activities. Potentially these analyses could be expanded to in vivo settings through intravital approach. In summary, this study demonstrated that OCT has a great potential to investigate the microstructure and dynamics, such as cilia beating, muscle contractions, and sperm transport, within the male reproductive tract.