Sperm degradation after vasectomy follows a sperm chromatin fragmentation-dependent mechanism causing DNA breaks in the toroid linker regions.

Vasectomy is a widely used surgical technique creating an obstructive azoospermia. Although sperm cannot be ejaculated, the testis maintains sperm production in vasectomized males. The continuous accumulation of sperm deposited in the epididymis and the vas deferens fraction necessarily need to be degraded and eliminated. While the elimination process is carried out by granulomas that form after vasectomy, the detailed mechanisms of sperm degradation are still not known. The aim was to assess whether sperm chromatin fragmentation (SCF), a mechanism that degrades the entire sperm genome at the toroid linker regions (TLRs), is activated after vasectomy in sperm cells. We vasectomized mice and evaluated the presence of TLR-specific double-strand breaks through pulsed-field gel electrophoresis and the Comet assay at 1, 2 and 3 weeks after surgery. Results for DNA damage (Olive tail moment) at single-cell level showed an increase of double-strand breaks after vasectomy for vas deferens sperm after 1, 2 and 3 weeks postvasectomy (21.78 ± 2.29; 19.71 ± 1.79 and 32.59 ± 1.81, respectively), compared to mock surgery (7.04 ± 1.03; 10.10 ± 1.29 and 8.64 ± 0.85, respectively; P < 0.001). Similar findings were obtained for cauda epididymis sperm (P < 0.001), but not for caput epididymis (P > 0.05). Pulsed-field gel electrophoresis showed the presence of double-stranded breaks between 15 and 145 kb, indicating that DNA breaks were produced mainly in the sperm TLRs. Results presented here suggest that SCF is a mechanism activated in vas deferens after vasectomy to degrade sperm DNA when they cannot be ejaculated, preventing their function.

Pharmacological characterisation of the CB1 receptor antagonist activity of cannabidiol in the rat vas deferens bioassay.

Cannabidiol is increasingly considered for treatment of a wide range of medical conditions. Binding studies suggest that cannabidiol binds to CB1 receptors. In the rat isolated vas deferens bioassay, a single electrical pulse causes a biphasic contraction from nerve-released ATP and noradrenaline. WIN 55,212-2 acts on prejunctional CB1 receptors to inhibit release of these transmitters. In this bioassay, we tested whether cannabidiol and SR141716 were acting as competitive antagonists of this receptor. Monophasic contractions mediated by ATP or noradrenaline in the presence of prazosin or NF449 (P2X1 inhibitor), respectively, were measured to a single electrical pulse delivered every 30 min. Following treatment with cannabidiol (10-100 µM) or SR141716 (0.003-10 µM), cumulative concentrations of WIN 55,212-2 (0.001-30 µM) were applied followed by a single electrical pulse. The WIN 55,212-2 concentration-contraction curve EC50 values were applied to global regression analysis to determine the pKB. The antagonist potency of cannabidiol at the CB1 receptor in the rat vas deferens bioassay matched the reported receptor binding affinity. Cannabidiol was a competitive antagonist of WIN 55,212-2 with pKB values of 5.90 when ATP was the effector transmitter and 5.29 when it was noradrenaline. Similarly, SR141716 was a competitive antagonist with pKB values of 8.39 for ATP and 7.67 for noradrenaline as the active transmitter. Cannabidiol's low micromolar CB1 antagonist pKB values suggest that at clinical blood levels (1-3 µM) it may act as a CB1 antagonist at prejunctional neuronal sites with more potency when ATP is the effector than for noradrenaline.

Identification of PDGFRα-positive interstitial cells in the distal segment of the murine vas deferens.

Platelet-derived growth factor receptor-α (PDGFRα)-positive interstitial cells (ICs) are widely distributed in various organs and may be involved in the motility of various tubular organs. We, for the first time, aimed to investigate the distribution, immunohistochemical characteristics, and ultrastructure of PDGFRα-positive ICs in murine vas deferens, using confocal laser scanning microscopy, transmission electron microscopy (TEM), and immuno-electron microscopy (immuno-EM). For immunofluorescence, we used antibodies against PDGFRα and other markers of ICs. PDGFRα-positive ICs were distributed widely in the lamina propria, smooth muscles, and serosal layers. Although most PDGFRα-positive ICs labeled CD34, they did not label CD34 in the subepithelial layers. Additionally, PDGFRα-positive ICs were in close proximity to each other, as also to the surrounding cells. TEM and immuno-EM findings revealed that PDGFRα-positive ICs established close physical interactions with adjacent ICs. Extracellular vesicles were also detected around the PDGFRα-positive ICs. Our morphological findings suggest that PDGFRα-positive ICs may have several subpopulations, which can play an important role in intercellular signaling via direct contact with the IC network and the extracellular vesicles in the murine vas deferens. Further investigation on PDGFRα-positive ICs in the vas deferens may lead to understanding the vas deferens mortility.

Mutation analysis of the cystic fibrosis transmembrane conductance regulator gene in Chinese congenital absence of vas deferens patients.

To find the variant spectrum of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and evaluate its frequent variants in Chinese congenital absence of vas deferens (CAVD) patients. A total of 276 patients with azoospermia and CAVD (aged from 21 to 44 years old) were investigated from May 2013 to September 2019 in the Third Affiliated Hospital of Sun Yat-sen University. Additionally, 50 healthy, unrelated volunteers were recruited as controls (aged from 21 to 46 years old). The 5'-UTR, exons and their flanking side of the CFTR gene were sequenced by high-throughput sequencing technology. The results were compared with those retrieved from the Ensembl Genome Browser. In addition, all 13 novel variants were further confirmed independently by Sanger sequencing and evaluated in the bioinformatics web servers. A schematic of the variant spectrum of the CFTR gene, including 13 novel variants (12 in CAVD patients, one in the control group), is shown, and the frequent variants in Chinese CAVD patients were 5 T (27.54%), c.-8G > C (7.25%), p.Q1352H (5.98%), and p.I556V (3.08%). 5 T was found to be the most frequent variant. p.Q1352H had a significantly high allelic frequency in CAVD patients (P < 0.05). c.-8G > C and p.I556V had high allelic frequencies but showed no difference between patients and controls (P > 0.05). p.Q1352H is the most common and important missense variant in Chinese patients with CAVD, while the pathological effects of C.-8G > C and p.I556V may be weak after evaluation.

[E-Cadherine and GATA-3 are useful in the confirmation of the vas deferens in vasectomy specimens]. / Cadherina E y GATA-3 son útiles para confirmar la presencia de conducto deferente en muestras de vasectomía.

INTRODUCTION AND OBJECTIVES: Urologists often submit the resected tissue from vasectomies for histopathological examination in order to confirm the presence of the vas deferens. Microscopy is simple and based on haematoxylin-eosin staining; however, sample artefacts can sometimes cause confusion and immunohistochemistry can be used to identify the vas deferens. MATERIALS AND METHODS: We investigated the utility of immunohistochemical analysis using E-cadherin and GATA-3 to confirm the presence of vas deferens epithelium in 110 vasectomy sections with different artefacts, using monoclonal antibodies and a multimer conjugated with peroxidase based technique; 5 renal arteries and 5 renal veins were stained as negative controls. RESULTS: Membrane staining was observed for E-cadherin, which was moderate (2.7%) or strong (97.3%) in the vas deferens epithelium in all cases: 35 without artefacts, 7 with denuded epithelium, 56 with compressed/distorted epithelium, 8 with detached epithelium and 4 with displaced epithelium. GATA-3 showed moderate (31%) or strong (69%) nuclear staining in all cases, including the 76 with artefacts. In the control group, arteries and veins were negative for both markers in the endothelium, but GATA-3 occasionally stained lymphocytes in the blood vessel wall. CONCLUSIONS: E-cadherin membrane positivity and GATA-3 nuclear expression are useful for the identification of the vas deferens in vasectomy samples containing artefacts. Vascular endothelium is negative for both markers and any possible GATA-3 staining of the lymphocytes in the blood vessel wall should not be misinterpreted.

The Role of the Epididymis and the Contribution of Epididymosomes to Mammalian Reproduction.

It is well-established that testicular spermatozoa are immature and acquire motility and fertilization capabilities during transit throughout the epididymis. The epididymis is a duct-like organ that connects the testis to the vas deferens and is comprised of four anatomical regions: the initial segment, caput, corpus, and cauda. Sperm maturation occurs during epididymal transit by the interaction of sperm cells with the unique luminal environment of each epididymal region. In this review we discuss the epididymis as an essential reproductive organ responsible for sperm concentration, maturation (including sperm motility acquisition and fertilizing ability), protection and storage. Importantly, we also discuss specific characteristics and roles of epididymal-derived exosomes (epididymosomes) in establishing sperm competency within the intricate process of reproduction. This review suggests that an increasing body of evidence is working to develop a complete picture of the role of the epididymis in male reproduction, offspring health, and disease susceptibility.

High-resolution imaging of the actin cytoskeleton and epithelial sodium channel, CFTR, and aquaporin-9 localization in the vas deferens.

Vas deferens is a conduit for sperm and fluid from the epididymis to the urethra. The duct is surrounded by a thick smooth muscle layer. To map the actin cytoskeleton of the duct and its epithelium, we reacted sections of the proximal and distal regions with fluorescent phalloidin. Confocal microscopic imaging showed that the cylinder-shaped epithelium of the proximal region has a thick apical border of actin filaments that form microvilli. The epithelium of the distal region is covered with tall stereocilia (13-18 µm) that extend from the apical border into the lumen. In both regions, the lateral and basal cell borders showed a thin lining of actin cytoskeleton. The vas deferens epithelium contains various channels to regulate the fluid composition in the lumen. We mapped the localization of the epithelial sodium channel (ENaC), aquaporin-9 (AQP9), and cystic fibrosis transmembrane conductance regulator (CFTR) in the rat and mouse vas deferens. ENaC and AQP9 immunofluorescence were localized on the luminal surface and stereocilia and also in the basal and smooth muscle layers. CFTR immunofluorescence appeared only on the luminal surface and in smooth muscle layers. The localization of all three channels on the apical surface of the columnar epithelial cells provides clear evidence that these channels are involved concurrently in the regulation of fluid and electrolyte balance in the lumen of the vas deferens. ENaC allows the flow of Na+ ions from the lumen into the cytoplasm, and the osmotic gradient generated provides the driving force for the passive flow of water through AQP channels.

Resveratrol and quercetin attenuate depressive-like behavior and restore impaired contractility of vas deferens in chronic stress-exposed rats: involvement of oxidative stress and inflammation.

Chronic stress is associated with male sexual problems including ejaculatory dysfunctions. The aim of this study was to determine whether resveratrol (RS) or quercetin (QE) has protective effects on vas deferens (VD) contractility in the unpredictable chronic mild stress (UCMS) rat model of depression. Animals were separated into six groups: control, control + RS and control + QE, stress, stress + RS, and stress + QE. Stress groups were subjected to UCMS procedure for 5 weeks. Animals in treatment groups were injected intraperitoneally with RS (20 mg/kg) or QE (30 mg/kg) for 5 weeks during UCMS period. UCMS caused depressive-like behaviors and enhanced systemic levels of corticosterone. The nerve-evoked contractile responses of VD significantly impaired and, noradrenaline- and ATP-induced contractile responses of VD significantly increased in stressed rats. UCMS exposure also markedly enhanced oxidative stress and inflammation in VD tissues. Treatment with RS or QE significantly ameliorated all the aforementioned parameters. The current study demonstrated that RS or QE protected against chronic stress-induced VD dysfunction by their antioxidant and anti-inflammatory effects on VD, suggesting that oxidative stress and inflammation may be synergistic parts in the development of VD dysfunction associated with chronic stress-induced depression.

Castration Induces Down-Regulation of A-Type K+ Channel in Rat Vas Deferens Smooth Muscle.

A-type K+ channels contribute to regulating the propagation and frequency of action potentials in smooth muscle cells (SMCs). The present study (i) identified the molecular components of A-type K+ channels in rat vas deferens SMs (VDSMs) and (ii) showed the long-term, genomic effects of testosterone on their expression in VDSMs. Transcripts of the A-type K+ channel α subunit, Kv4.3L and its regulatory ß subunits, KChIP3, NCS1, and DPP6-S were predominantly expressed in rat VDSMs over the other related subtypes (Kv4.2, KChIP1, KChIP2, KChIP4, and DPP10). A-type K+ current (IA) density in VDSM cells (VDSMCs) was decreased by castration without changes in IA kinetics, and decreased IA density was compensated for by an oral treatment with 17α-methyltestosterone (MET). Correspondingly, in the VDSMs of castrated rats, Kv4.3L and KChIP3 were down-regulated at both the transcript and protein expression levels. Changes in Kv4.3L and KChIP3 expression levels were compensated for by the treatment with MET. These results suggest that testosterone level changes in testosterone disorders and growth processes control the functional expression of A-type K+ channels in VDSMCs.

Testes and duct deferens of mice during space flight: cytoskeleton structure, sperm-specific proteins and epigenetic events.

To analyze the effect of gravity on the structure of germinal tissues, we examined tissues of the testes and duct deferens of mice that were exposed to space flight conditions for 21-24 days (experiment Rodent Research-4, SpaceX-10 mission, February 2017, USA). We evaluated the levels of cytoskeletal proteins, sperm-specific proteins, and epigenetic events; in particular, we evaluated levels of 5-hydroxymethylcytosine and of enzymes that regulate DNA methylation/demethylation. We did not detect changes in the levels of cytoskeletal proteins, sperm-specific proteins, DNA-methylases, DNA demethylases, DNA acetylases, or histone deacetylases. However, there were changes at the gene expression level. In particular, there was an increase in the demethylase Tet2 and a decrease in the histone deacetylase Hdac1. These gene expression changes may be of key importance during the early period of readaptation since they could lead to an increase in the expression of target genes.

Butylidenephthalide facilitates contractions via nonspecific binding to receptors in isolated guinea-pig vas deferens.

Context: Butylidenephthalide (Bdph) has been reported to inhibit rat uterine contractions, but significantly potentiate the noradrenaline (NA)-induced contractions in guinea-pig vas deferens (GPVDs). Objective: The present study elucidates the binding specificity of Bdph in GPVD to potentiate contractions. Materials and methods: Electrical field stimulation (EFS, supramaximal voltage, 1 ms and 1 Hz) or exogenous NA (50 µM) was applied to the GPVD in Krebs or 1/10 Mg-Tyrode's solution, respectively. After the clonidine (10 nM)-induced twitch inhibition or the exogenous NA-induced contractions reached a constant, Bdph (50 µM) was added 2 min prior to the subsequent addition of NA (50 µM). Three experiments were performed. In the presence of Bdph (100 µM), the release of NA in the medium and remaining NA content in the tissues were determined after EFS-stimulation. Results: Bdph (100 µM) significantly antagonized the clonidine (10 nM)-induced twitch inhibition from 22.5 ± 2.1 to -11.4 ± 1.6% (n = 6) and dibutyryl-cAMP (300 µM) from 25.7 ± 3.2 to 7.9 ± 4.0% (n = 8). Bdph (100 µM) significantly increased the electrically stimulated release of NA from 393.0 ± 109.5 to 1000.0 ± 219.1 ng/g (n = 6). Bdph (50 µM) potentiated the exogenous NA (50 µM)-induced contractions from 3.0 ± 0.06 to 3.9 ± 0.06 g (n = 3), but after washout of Bdph, the response to NA gradually curtailed. Discussion and conclusions: Bdph action may be through the nonspecific binding of the butylidene group to prejunctional α2- and postjunctional α1-adrenoceptors to reversibly block K+ channels, and irreversibly block VDCCs on the smooth muscle cell membrane, respectively.

The expression and localization of Toll-like receptors 2, 4, 5 and 9 in the epididymis and vas deferens of a adult tom cats.

Toll-like receptors (TLRs) are important molecules, which provide protection against infections of the reproductive tract. This study demonstrates for the first time the expression and localization patterns of TLRs in the caput, corpus and cauda segments of the epididymal duct (ED) and the vas deferens (VD) of adult domestic cats using immunohistochemistry and western blotting. While immunoblot analyses revealed relatively similar protein levels for TLRs 2, 4, 5, and 9 in three segments of the ED, the protein levels of TLR2 and TLR4 in the VD were found to be significantly higher than those measured in the ED segments (P < 0.05). On the other hand, immunostaining showed that TLRs exhibited regional- and cell-specific localization patterns. TLR2 and TLR5 were immunolocalized to the nucleus and cytoplasm of the principal cells in all ducts. TLR4 was restricted to the stereocilia, and TLR9 was located in the cytoplasm of the principal cells. Narrow cells displayed positive immunoreactions for TLR4 and TLR5. The basal cells of the different ED segments were positive for all four TLRs. TLR2, TLR5 and TLR9 were detected in the cytoplasmic droplets of the spermatozoa. TLR4 and TLR9 were detected along the entire length of the sperm tail, whilst TLR2 and TLR5 were absent in the midpiece. TLR2 and TLR5 were also detected in the equatorial segment of the sperm head. These results suggest that TLR2, TLR4, TLR5 and TLR9 are important not only for the protection of the ED, VD and spermatozoa but also for the maturation and storage of spermatozoa in the ED and VD, respectively.

Non-obstructive vas deferens and epididymis loss in cystic fibrosis rats.

This study utilizes morphological and mechanistic endpoints to characterize the onset of bilateral atresia of the vas deferens in a recently derived cystic fibrosis (CF) rat model. Embryonic reproductive structures, including Wolffian (mesonephric) duct, Mullerian (paramesonephric) duct, mesonephric tubules, and gonad, were shown to mature normally through late embryogenesis, with involution of the vas deferens and/or epididymis typically occurring between birth and postnatal day 4 (P4), although timing and degree of atresia varied. No evidence of mucus obstruction, which is associated with pathology in other CF-affected tissues, was observed at any embryological or postnatal time point. Reduced epididymal coiling was noted post-partum and appeared to coincide with, or predate, loss of more distal vas deferens structure. Remarkably, α smooth muscle actin expression in cells surrounding duct epithelia was markedly diminished in CF animals by P2.5 when compared to wild type counterparts, indicating reduced muscle development. RNA-seq and immunohistochemical analysis of affected tissues showed disruption of developmental signaling by Wnt and related pathways. The findings have relevance to vas deferens loss in humans with CF, where timing of ductular damage is not well characterized and underlying mechanisms are not understood. If vas deferens atresia in humans begins in late gestation and continues through early postnatal life, emerging modulator therapies given perinatally might preserve and enhance integrity of the reproductive tract, which is otherwise absent or deficient in 97% of males with cystic fibrosis.

Hormonal control of vas deferens fluid volume and aquaporin expression in rats.

Precise regulation of vas deferens fluid volume which is important for sperm survival might be influenced by testosterone. In order to investigate changes in vas deferens fluid volume and aquoporins (AQP) isoforms expression under testosterone influence, orchidectomized Sprague-Dawley rats were given 125 and 250 µg/kg/day testosterone with or without flutamide, an androgen receptor blocker or finasteride, a 5alpha-reductase inhibitor for seven consecutive days. Following treatment completion, vas deferens was perfused and changes in the fluid secretion rate and osmolality were determined in the presence of acetazolamide. Rats were then sacrificed and vas deferens was harvested for histology, tissue expression and distribution analyses of AQP-1, AQP-2, AQP-5, AQP-7 and AQP-9 proteins by Western blotting and immunohistochemistry, respectively. Our findings indicate that testosterone causes vas deferens fluid secretion rate to increase, which was antagonized by acetazolamide. Fluid osmolality increased following testosterone treatment and further increased when acetazolamide was given. Co-administration of flutamide or finasteride with testosterone causing both fluid secretion rate and osmolality to decrease. Histology revealed increased size of vas deferens lumen with increased thickness of vas deferens stroma. Expression of AQP-1, AQP-2 and AQP-9 were detected in vas deferens but not AQP-5 and AQP-7, and the levels of these proteins were increased by testosterone treatment mainly at the apical membrane of vas deferens epithelium. In conclusion, increased in vas deferens fluid secretion rate under testosterone influence mediated via the up-regulation of AQP-1, 2 and 9 might be important for vas deferens fluid homeostasis in order to ensure normal male fertility.

Drug-receptor kinetics and sigma-1 receptor affinity differentiate clinically evaluated histamine H3 receptor antagonists.

The histamine H3 receptor is a G protein-coupled receptor (GPCR) drug target that is highly expressed in the CNS, where it acts as both an auto- and hetero-receptor to regulate neurotransmission. As such, it has been considered as a relevant target in disorders as varied as Alzheimer's disease, schizophrenia, neuropathic pain and attention deficit hyperactivity disorder. A range of competitive antagonists/inverse agonists have progressed into clinical development, with pitolisant approved for the treatment of narcolepsy. Given the breadth of compounds developed and potential therapeutic indications, we assessed the comparative pharmacology of six investigational histamine H3 agents, including pitolisant, using native tissue and recombinant cells. Whilst all of the compounds tested displayed robust histamine H3 receptor inverse agonism and did not differentiate between the main H3 receptor splice variants, they displayed a wide range of affinities and kinetic properties, and included rapidly dissociating (pitolisant, S 38093-2, ABT-239) and slowly dissociating (GSK189254, JNJ-5207852, PF-3654746) agents. S 38093-2 had the lowest histamine H3 receptor affinity (pKB values 5.7-6.2), seemingly at odds with previously reported, potent in vivo activity in models of cognition. We show here that at pro-cognitive and anti-hyperalgesic/anti-allodynic doses, S 38093-2 preferentially occupies the mouse sigma-1 receptor in vivo, only engaging the histamine H3 receptor at doses associated with wakefulness promotion and neurotransmitter (histamine, ACh) release. Furthermore, pitolisant, ABT-239 and PF-3654746 also displayed appreciable sigma-1 receptor affinity, suggesting that this property differentiates clinically evaluated histamine H3 receptor antagonists and may play a role in their efficacy.

Testosterone enhances expression and functional activity of epithelial sodium channel (ENaC), cystic fibrosis transmembrane regulator (CFTR) and sodium hydrogen exchanger (NHE) in vas deferens of sex-steroid deficient male rats.

Effects of testosterone on expression and functional activity of ENaC, CFTR and NHE in vas deferens were investigated. METHODS: Orchidectomized, adult male rats were given 125 and 250 µg/kg/day testosterone subcutaneously, with or without flutamide and finasteride for seven consecutive days. At the end of the treatment, rats were anesthetized and vas deferens were perfused. Changes in vas deferens fluid secretion rate, pH, HCO3-, Cl- and Na+ concentrations were recorded in the presence of amiloride and Cftr inh-172. Rats were then sacrificed and vas deferens were harvested and subjected for molecular biological analysis. RESULTS: Testosterone treatment caused the fluid pH and HCO3- concentrations to decrease but secretion rate, Cl- and Na+ concentrations to increase, where upon amiloride administration, the pH and HCO3- concentration increased but Cl- and Na+ concentrations further increased. In testosterone-treated rats, administration of Cftr inh-172 caused all fluid parameters to decrease. In testosterone-treated rats co-administered with flutamide or finasteride, pH and HCO3- concentration increased but fluid secretion rate, Cl- and Na+ concentrations decreased and these parameters were not affected by amiloride or Cftr inh-172 administration. Under testosterone influence, CFTR and γ-ENaC were highly expressed at the apical membrane while NHE-1 and 4 were highly expressed at the basolateral membrane of vas deferens epithelium. Meanwhile, NHE-2 and 3 were highly expressed at the apical membrane. CONCLUSIONS: Differential expression of ENaC, CFTR and NHE in vas deferens under testosterone influence indicated the important role of these transporters in creating optimal fluid microenvironment that is essential for preserving male fertility.

Direct and indirect effects of ephedrine on heart rate and blood pressure in vehicle-treated and sympathectomised male rats.

We have investigated the cardiac and pressor responses to (±)-ephedrine and (-)-ephedrine in pentobarbitone anaesthetized male wistar rats. The tachycardiac responses to (±)- and (-)-ephedrine were similar, but pressor responses to (-)-ephedrine (10 mg/kg) were significantly greater than those to (±)-ephedrine, and for both, the pressor response was followed by a small depressor response. Sympathectomy did not affect pressor responses, but significantly increased the later depressor response to both compounds. Sympathectomy did not affect tachycardiac or depressor responses to the ß-adrenoceptor agonist isoprenaline, but significantly reduced the tachycardia to (±)-ephedrine. (±)-Ephedrine contracted vas deferens from vehicle treatment animals, but in vas deferens from sympathectomised rats, (±)-ephedrine produced almost no tonic contraction (α1A-adrenoceptor mediated), but the phasic contraction was unaffected (α1D-adrenoceptor mediated). It is concluded, firstly, that (-)-ephedrine is more potent than the racemate mixture at producing pressor responses. Secondly, since the depressor response to isoprenaline was unaffected, sympathectomy presumably reduced a pressor component to the response to (±)- and (-)-ephedrine. Hence, a component of the pressor response to both (±)- and (-)-ephedrine is indirect and may involve actions at α1A-adrenoceptors, at which ephedrine does not have marked direct actions.

Testosterone up-regulates vacuolar ATPase expression and functional activities in vas deferens of orchidectomized rats.

Precise regulation of vas deferens fluid pH is essential for sperm. However, the mechanisms underlying effect of testosterone on vas deferens fluid pH have never been identified, which could involve changes in expression and functional activity of vacoular (V)-ATPase. METHODS: Orchidectomized, adult male Sprague-Dawley rats were treated subcutaneously with 125 µg/kg/day and 250 µg/kg/day testosterone with or without flutamide (androgen receptor blocker) and finasteride (5α-reductase inhibitor) for seven (7) days. Following treatment completion, in vivo perfusion of vas deferens lumen was performed and changes in fluid secretion rate, pH and HCO3- content were measured with and without bafilomycin, a V-ATPase inhibitor. Rats were then sacrificed and vas deferens were harvested and subjected for V-ATPase A1 and B1/2 protein expression and distribution analysis by western blotting and immunohistochemistry, respectively. RESULTS: In sham-operated and testosterone-treated orchidectomized rats, higher fluid secretion rate, which was not antagonized by bafilomycin but lower HCO3- content and pH which were antagonized by bafilomycin were observed when compared to orchidectomized-only and orchidectomized, testosterone-treated rats receiving flutamide or finasteride, respectively. Bafilomycin had no effect on fluid secretion rate, HCO3- content and pH in orchidectomized and testosterone-treated orchidectomized rats receiving flutamide and finasteride. V-ATPase A1 and B1/2 proteins were expressed at high levels in vas deferens and were highly distributed at the apical membrane of luminal epithelium and in muscle layer of this organ, mainly in sham and testosterone-treated orchidectomized rats. CONCLUSIONS: V-ATPase is involved in acidification of vas deferens fluid under testosterone influence.

Reproductive aging in male dogs: The epididymal sperm defects and expression of androgen receptor in reproductive tissues.

The present study aimed at investigating age-related changes in epididymal sperm motility, morphology, DNA integrity and androgen receptor (AR) expression in canine reproductive tissues. Fifty-five healthy medium-sized male dogs were divided into four groups: young (1 - 3 years old, n = 14), adult (>3 - 6 years old, n = 12), old (>6 - 9 years old, n = 14) and senile (>9 years old, n = 15). After routine castration, testes, epididymides (head, body and tail) and vas deferens were collected. Spermatozoa were flushed from epididymal tails and their motility, morphology and DNA integrity were examined. Localization of AR was investigated by immunohistochemistry and the positive immunostaining cells were evaluated using image analysis software (NuclearQuant, 3DHISTECH). We found significantly lower percentages of epididymal sperm motility, sperm vigour and viability in adult, old and senile dogs in comparison with young dogs (p < 0.05). Animal's age negatively correlated with epididymal sperm motility, sperm vigour and viability. The primary, secondary, major and minor epididymal sperm defects were significantly higher in senile dogs compared to young dogs. There were positive correlations between age and epididymal sperm defects (p < 0.01). The percentage of sperm with fragmented DNA did not differ between age groups. Testicular AR was expressed in the nucleus of Sertoli cells, Leydig cells and peritubular myoid cells, except for germ cells. Expression of AR was found in all epithelium, lamina propria and smooth muscle cells of the epididymis and vas deferens. Expression levels of AR in testis, epididymis and vas deferens did not differ between age groups (p > 0.05). In conclusion, the present study clearly demonstrated that senescence in dogs was associated with decreased epididymal sperm quality. An age-related increase in the incidence of poor epididymal sperm quality may promote subfertility, especially in senile dogs.

DPI-289, a novel mixed delta opioid agonist / mu opioid antagonist (DAMA), has L-DOPA-sparing potential in Parkinson's disease.

L-DOPA-induced dyskinesia (LID) remains a significant problem in the management of Parkinson's disease (PD). In rodent and macaque models of PD, delta opioid receptor agonists have anti-parkinsonian actions while mu opioid antagonists can reduce the expression of LID. DPI-289 is a novel molecule with a unique combination of opioid receptor DAMA actions: delta agonist (Ki: 0.73 nM); mu antagonist (Ki: 12 nM). We demonstrated that DPI-289 has oral bioavailability and established its pharmacokinetic profile in both rat and primate. We hypothesised that these combined DAMA actions would provide an enhancement of L-DOPA effect without an associated increase in dyskinesia. In parkinsonian 6-OHDA lesioned rats and MPTP-lesioned macaques, DPI-289 provided anti-parkinsonian actions as monotherapy and an enhancement of L-DOPA benefit. Thus, acute administration of DPI-289 (3 mg/kg, p.o.) to 6-OHDA-lesioned rats produced a significant reduction in forelimb asymmetry (by 48%) that was maintained throughout the fifteen-day repeat-treatment period. Importantly, and in contrast to L-DOPA administration (6 mg/kg, i.p.), these benefits were not compromised by the development of abnormal involuntary movements. In the macaque, as monotherapy, DPI-289 (10 and 20 mg/kg) had significant, though incomplete, anti-parkinsonian actions lasting approximately 4 h. These benefits were not associated with dyskinesia. In fact, over the 6 h period of observation, DPI-289 (20 mg/kg) decreased parkinsonism by 19% and increased activity by 67% compared to vehicle treatment. By contrast, while high-dose L-DOPA (LDh) alone alleviated parkinsonism (for 3 h) this benefit was accompanied by significant dyskinesia that was disabling in nature. LDh provided a 50% reduction in parkinsonism over 6 h and 151% increase in activity. The combination of DPI-289 (20 mg/kg) and a low-dose of L-DOPA (LDl) provided anti-parkinsonian benefits greater than LDl alone without eliciting any significant dyskinesia. Treatment with LDl alone provided only transient statistically significant anti-parkinsonian benefit. However, the combination of LDl and DPI-289 reduced parkinsonism for 6 h (duration of monitoring), with parkinsonism being reduced by 35% and activity increased by 90% but with no increase in dyskinesia over that observed with LDl alone. Thus, DPI-289 has potential to improve the benefits of dopaminergic therapy in Parkinson's disease.