Maxi K+ channels on human vas deferens epithelial cells

The vas deferens forms part of the male reproductive tract and extends from the cauda epididymis to the prostate. Using the patch clamp technique, we have identified a Ca²⁺-activated, voltage-dependent, maxi K⁺ channel on the apical membrane of epithelial cells cultured from human fetal vas deferens. The channel had a conductance of 250 pS in symmetrical 140 mm K⁺ solutions, and was highly selective for K⁺ over Na⁺. Channel activity was increased by depolarization and by an elevation of bath (cytoplasmic) Ca²⁺ concentration, and reduced by cytoplasmic Ba²⁺ (5 mm) but not by cytoplasmic TEA (10 mm). Channel activity was also dependent on the cation bathing the cytoplasmic face of the membrane, being higher in a Na⁺-rich compared to a K⁺-rich solution. We estimated that up to 600 maxi K⁺ channels were present on the apical membrane of a vas cell, and that their density was 1–2 per ² of membrane. Activity of the channel was low on intact cells, suggesting that it does not contribute to a resting K⁺ conductance. However, fluid in the lumen of the human vas deferens has a high K⁺ concentration and we speculate that the maxi K⁺ channel could play a role in transepithelial K⁺ secretion.Funded by grants from the Cystic Fibrosis Trust and the Medical Research Council (UK). We thank Mr. David Stephenson for excellent technical assistance.     

Contractile responses of the rat vas deferens after epithelium removal

The purpose of the present investigation was to verify the role of the epithelium in the functional response of the rat vas deferens. Our results showed that the contractile effect of cumulative doses of clonidine (3.10(-5)-3.10(-3)) was increased after the removal of the epithelium. The effect of clonidine in epithelium-free vas deferens returned to normal values when an isolated epithelium from another vas deferens was added to the organ bath, showing that the epithelium is responsible for this increase of maximum effect for clonidine. Drugs functionally or structurally related to clonidine, such as oxymetazoline, alpha-methylnorepinephrine and moxonidine, did not have their dose-response curves altered. The curves for other contractile agents, such as noradrenaline, acetylcholine, ATP, 5HT, bradykinin and histamine, or the relaxation induced by isoprenaline and forskolin were also not modified. Electrically-induced contractions at frequencies from 0.1 to 20 Hz and the mechanism of negative feed-back, brought about by clonidine (10(-10)-10(-8) M) through pre-synaptic alpha2-adrenoceptors, were not changed after the removal of epithelium. In conclusion, a significant function of the epithelium in the contractility of the rat vas deferens was demonstrated for clonidine, but not for other agonists.     

Opioid effects in developing rat vas deferens

The inhibitory effects of various opiates on developing rat vas deferens were studied by determining the degree of depression of mechanical responses elicited by electrical field stimulation. All agonists showed decreased effects with maturation, but the decrease occurred at different times. With normorphine the loss of agonist activity was greatest at days 12–16, while withd-Met²,Pro⁵-enkephalinamide it was greatest at days 16–30. -Endorphin also was less effective in adult than 30-day preparations, but methionine enkephalin was ineffective at all ages. Morphine and normorphine were weak antagonists of opiate agonists in the adult preparations. These results indicate that the nature and pharmacologic sensitivity of opiate actions change with development.     

Light microscopic features of the rete testis,the vas efferens,the epididymis and the vas deferens in the adult rhesus monkey

The present study was carried out to determine the detailed histological and cytological features of the excurrent ducts of the male reproductive system in the rhesus monkey. The excurrent ducts show a regional difference in their histological features. The use of some of these features as histological markers and their possible functional significance are discussed. The epithelial cells in the different components of the excurrent duct system possess cytological features which suggest their involvement in absorption and the secretion of different products into the lumen.     

Effects of veratrine and paeoniflorin on isolated mouse vas deferens

In this study, we attempted to identify the interactions and mechanisms between veratrine and paeoniflorin on isolated mouse vas deferens. Paeoniflorin had no effect on isolated mouse vas deferens. Veratrine (1 x 10(-5) approximately 1 x 10(-3) g/ml) could directly induce contraction of isolated rat and mouse vas deferens. The concentration induced by veratrine (1 x 10(-5) g/ml) was completely inhibited by Ca2+-free solution and verapamil (1 x 10(-5) M), in both the epididymal and the prostatic portions of isolated mouse vas deferens. Naloxone (1 x 10(-5) M) did not alter the contraction induced by veratrine (1 x 10(-5) g/ml) in either the epididymal or the prostatic portions of isolated mouse vas deferens. Paeoniflorin (4.8 x 10(-5) g/ml) inhibited the contraction induced by veratrine (1 x 10(-5) g/ml) in both the epididymal and the prostatic portions of isolated mouse vas deferens. Paeoniflorin (4.8 x 10(-5) g/ml) potentiated norepinephrine (1 x 10(-5) M)-induced phasic contraction in the epididymal portion, but decreased contractions in the prostatic portion. Paeoniflorin (4.8 x 10(-5) g/ml) increased KCI (56 mM)-induced phasic contraction in the epididymal portion, but decreased the tonic contraction in either the epididymal or the prostatic portion. Veratrine (1 x 10(-5) g/ml)-induced contractions could be decreased by pretreatment with ryanodine (1 x 10(-5) M) in both the epididymal and the prostatic portions. Pretreatment with the combination of paeoniflorin (4.8 x 10(-5) g/ml) and ryanodine (1 x 10(-5) M) did not potentiate the inhibition of paeoniflorin in the veratrine-induced contraction in both the epididymal and the prostatic portions of isolated mouse vas deferens.     

Galanin potentiates electrical stimulation and exogenous norepinephrine-induced contractions in the rat vas deferens

In order to evaluate the mode of action of galanin (GAL) on the neuroeffector mechanism of peripheral sympathetic nerve fibers, the effects of this peptide were tested on the electrical stimulated and the unstimulated preparations of the isolated rat vas deferens in the presence of 10(-7) M atropine. The contractile responses, which were mediated predominantly by activation of postganglionic noradrenergic nerve fibers were dose-dependently potentiated by GAL in concentrations ranging from 1 to 50 nM. The facilitatory action induced by GAL in high concentrations (greater than 10 nM) usually returned to the control level at 2-3 min and were tachyphylactic. The potentiating action of GAL was not modified by pretreatment with 10(-7) M propranolol. Contractions produced by exogenous norepinephrine (NE) in the unstimulated preparations were not affected by pretreatment with low concentrations (less than 5 nM) of GAL. On the other hand, the contractions were dose-dependently potentiated 1 min after pretreatment with higher concentrations (greater than 10 nM) of GAL, which recovered 15 min after constant flow washout. Contractions developed by exogenous 5-hydroxytryptamine were not affected, or slightly inhibited, by GAL (1-50 nM). In some preparations without electrical stimulation, high concentrations of GAL caused a slight contraction, which was not blocked by pretreatment with 10(-6) M phentolamine and 10(-6) M tetrodotoxin. These results suggest that GAL receptors exist presynaptically in the rat vas deferens and that stimulation of the receptors by GAL potentiates the release of NE from the nerve terminals during postganglionic sympathetic nerve stimulation. Other mechanisms for GAL action, such as influence on neuronal uptake and catecholamine metabolism, cannot be ruled out.     

Regional distribution of dopamine, 5-hydroxytryptamine,and noradrenaline in the rat vas deferens

The concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT) and noradrenaline (NA) in the rat vas deferens divided in eight or four sections were determined by high performance liquid chromatography with electrochemical detection. Dopamine and NA had the same regional distribution; their concentrations were maximal near the prostatic end and decreased towards the epididymis. The concentration of 5-HT also decreased from the prostatic to the epididimal end, but 5-HT did not follow the same regional distribution as DA and NA. Reserpine (0.02 or 0.2 mg/kg, i.p., 24 hr) and 6-hydroxydopamine (2×80 mg/kg, i.v., 6 days) decreased the contents of DA and NA; the concentrations of both amines were modified to a similar extent. Reserpine also diminished the content of 5-HT. Pargyline (200 mg/kg, i.p., 2 hr) increased the concentration of 5-HT whilep-chlorophenylalanine (300 mg/kg, oral, 3 days) decreased the contents of the amine in some sections of the vas deferens. This study suggests that DA and NA co-exist in the same sympathetic neurons. Some of the 5-HT could be stored in mast cells as previously proposed, but the finding that tissue content of 5-HT changes after inhibiting the deamination or synthesis of the amine suggests that other source(s) of 5-HT distinct from mast cells exist in the rat vas deferens.     

Postmortem recovery and cryopreservation of spermatozoa from the vas deferens of rhesus macaques (Macaca mulatta)

To determine whether sperm derived from the vas deferens could be retrieved and successfully cryopreserved, testes were collected from 20 rhesus macaques (Macaca mulatta). The males ranged in age from 3 to 19 yr with an average age of 8.5 yr. No sperm was obtained from three animals that were younger than 4 yr. The remaining 17 samples contained sperm with an average sperm cell number of 421.8 ± 88.7 × 10⁶ and an average motility of 72.8 ± 4.4%. After 24 h of culture in TALP medium at 37 °C in 5% CO₂ and 95% air, the overall motility decreased significantly in all samples regardless of treatment. Freezing in TEST (TES-Tris buffer)-yolk buffer containing 6% (vol/vol) glycerol had a significant effect on sperm, reducing the immediate postthaw motility to 42.4% in nontreated samples. Treatment with dibutyryl-cAMP and caffeine further reduced sperm motility after 4 h in fresh sperm (72.8% vs. 50.4%) but increased motility in sperm that had been frozen (14.0% vs. 23.2%). The age of the male did not influence sperm concentration or grade but proved to be a significant factor in determining motility of frozen-thawed treated sperm, with lower motility found in samples from older males. Overall, the study demonstrates that motile sperm can be obtained from postmortem males, although subsequent studies will be needed to determine whether the quality is sufficient to facilitate its use in assisted reproduction.     

Foreign DNA introduced into the vas deferens is gained by mammalian spermatozoa

The uptake of exogenous DNA by mouse and rat spermatozoa was analyzed using in vitro and in vivo methods. Two DNA constructs were used, one containing the Growth hormone (GH) gene and the other the c-myc oncogene linked to the αA-crystallin promoter (CPV-1 plasmid). For the in vitro approach, washed epididymal spermatozoa were incubated for 2 hr in the presence of linearized DNA. For in vivo experiments, DNA was injected into the proximal region of the vas deferens, and spermatozoa were recovered 6 hr later. In situ hybridization employing fluorescent markers and electron microscopy were used to localize the exogenous genes in spermatozoa. The precise localization of the foreign DNA in spermatozoa was visualized by tridimensional reconstructions using a confocal laser microscopy. Uptake of exogenous DNA occurred in 60–70% of the spermatozoa after in vitro or in vivo treatments. A positive signal was detected in the sperm nucleus and was not affected by DNase treatments. Incorporation of exogenous DNA was also evaluated by slot blot and PCR techniques using the DNA isolated from the sperm nuclei and the corresponding labelled probes. Comparison of a nucleotide sequence between the DNA isolated from in vivo treated spermatozoa and CPV-1 plasmid showed a 98.6% identity. These results show the in vivo capacity of spermatozoa to incorporate exogenous DNA, the ability of this DNA to reach the nucleus, and also demonstrate that epididymal and vas deferens secretions do not block these capacities. Mol. Reprod. Dev. 51:42–52, 1998. © 1998 Wiley-Liss, Inc.     

Sympathetic histamine exerts different pre- and post-synaptic functions according to the frequencies of nerve stimulation in guinea pig vas deferens

Histamine (HA) was found to be present in the sympathetic nerve terminals of guinea pig hearts and vasa deferentia in our previous study; however, little is known about the functions of this neurogenic HA. In this study, we used guinea pig vasa deferentia to investigate the pre- and post-synaptic functions of HA evoked by different frequencies of sympathetic nerve stimulation. We found that sympathetic nerve stimulation could evoke HA release, which was independent to mast cell degranulator compound 48/80 and mast cell stabilizer cromolyn, but was highly sensitive to Na⁺ channel blocker tetrodotoxin and chemical sympathectomy with 6-hydroxydopamine. The neurogenically released HA evoked by 12.5 Hz of nerve stimulation activated only pre-synaptic H₃ receptors and mediated pre-synaptic inhibitory effects, while under 25 or 50 Hz stimulation condition, HA simultaneously activated both pre-synaptic H₃ receptors and post-synaptic H₁ receptors. However, the direct contractile responses evoked by sympathetic HA via H₁ receptors were observed at 50 Hz. HA release and HA-mediated contractile responses upon sympathetic nerve stimulation were significantly inhibited by pre-treatment of histidine decarboxylase inhibitor α-fluoromethylhistidine. Furthermore, application of exogenous HA could mimic these pre- and post-synaptic effects. Our findings indicate that HA in sympathetic neurons acts as a neurotransmitter and its functions vary from pre-synaptic inhibition, to post-synaptic facilitation, to direct post-synaptic contractile responses according to sympathetic nerve stimulation frequencies.     

一氧化氮和K+通道参与乙酰胆碱引起的大鼠离体输精管平滑肌细胞超极化

本文旨在探讨大鼠新鲜离体输精管平滑肌细胞中乙酰胆碱（acetylcholine,ACh）引起超极化反应的机制,采用细胞内微电极记录技术和细胞内荧光标记技术研究ACh对大鼠输精管不同走行方向平滑肌细胞的作用。用尖端含0．1％碘化吡啶（propidium iodide,PI）的记录电极标记电生理记录后的平滑肌细胞,其中37个为外层纵行细胞,17个为内层环行细胞。它们的平均静息膜电位分别为（-53．56±3．88）mV和（-51．62±4．27）mV,膜输入阻抗分别为（2245．60±372．50）MQ和（2101．50±513．50）MQ。ACh引起的膜超极化反应是浓度依赖性的,EC50为36 μmol／L。ACh引起的超极化反应可被非选择性的毒草碱（muscarinic receptor,M）受体阻断剂阿托品（atropine,1 μmol／L）和选择性的M3受体阻断剂diphenylacetoxy-N-methylpiperidine-methiodide（DAMP,100nmol/L）阻断。ACh引起的超极化还能被一氧化氮合酶抑制剂L-硝基-精氨酸甲酯（N-nitro-L-arginine methylester,L．NAME,300μmol／L）阻断,并可被ATP敏感的钾通道阻断剂glipizide（5μmol／L）或内向整流钾通道阻断剂钡离子（50μmol／L）部分阻断。Glipizide和钡离子联合使用可完全阻断ACh引起的超极化反应。上述结果表明：ACh通过作用于大鼠输精管平滑肌细胞膜上的M3受体引起超极化反应,一氧化氮、ATP敏感性钾通道和内向整流钾通道参与了ACh引起的超极化反应。     

Effect of calcitonin gene-related peptide on the neuroeffector mechanism of sympathetic nerve terminals in rat vas deferens

In order to evaluate the mode of action of calcitonin gene-related peptide (CGRP) on the neuroeffector mechanism of peripheral sympathetic nerve fibers, the effects of CGRP were tested on the electrical stimulated and the non-stimulated preparations of the isolated rat vas deferens. The contractile responses, which were mediated predominantly by activation of postganglionic noradrenergic nerve fibers, were dose-dependently inhibited by CGRP in concentrations ranging from 0.1 to 10 nM. The inhibitory response produced by CGRP in high concentrations (greater than 2 nM) usually returned to the control level at 20-30 min and were rarely tachyphylactic. The inhibitory action of CGRP was not modified by pretreatment with 10(-7) M propranolol or 10(-7) M atropine. Contractions produced by exogenous norepinephrine (NE) and 5-hydroxytryptamine (5-HT) in unstimulated preparations were not affected by pretreatment with CGRP in a low concentration (less than 2 nM). On the other hand, the contractions were slightly reduced 1 min after pretreatment with CGRP in high concentrations (greater than 5 nM), which recovered in 15 min after constant flow washout. High concentrations of CGRP also caused a concentration-dependent relaxation on the precontracted preparations produced by high potassium (60 mM K+) solution. These results suggest that CGRP in high concentrations (greater than 5 nM) may have a non-specific inhibitory action on the postsynaptic plasma membrane of the smooth muscle cell and a postulated CGRP receptor exists presynaptically in the rat vas deferens and that CGRP may inhibit the release of NE during adrenergic nerve stimulation.     

Light and electron microscopic studies on the seminal secretions and the vas deferens of the penaeiodean shrimp,Sicyonia ingentis

Unlike the other penaeiodean shrimp, the ridge back shrimp, Sicyoniaingentis does not produce a spermatophore, but transfers sperm suspended in seminal plasm. This paper reports on the histomorphology and ultrastructure of the vas deferens with reference to its functional role in secreting the sperm bearing materials. The vas deferens is divisible into proximal secretory, mid storage and distal ejaculatory regions. The epithelial cells lining the proximal vas deferens are comprised of secretory and absorptive cell types. The loose sperm cells found in the lumen of this region are in an immature condition, and are agglutinated into a compact mass with signs of spermiogenesis in the mid vas deferens. The epithelial cells lining the mid vas deferens are short flattened cells. The distal vas deferens is ensheathed by muscular fibres. The inner epithelial cells are highly secretory and contain numerous microvilli at the luminal end. The sperm cord gets liquefied in this region facilitating the transfer of sperm in liquid form to the female during mating.     

Structure and physiology of mammalian vas deferens in relation to fertility regulation

The structural and functional integrity of the vas deferens and its role in ensuring the fertilizing ability, viability of spermatozoa and their survival in the vas deferens, are elucidated. The regulation of the function of the vas deferens and the differential androgen dependency of its two regions have been discussed in relation to its secretory and absorptive activities as well as its contractility. The importance of ascorbic acid in maintaining its functions has also been investigated. The potentiality of the use of an androgen antagonist, steroids (testosterone, estradiol benzoate), prostaglandins, copper devices, vasectomy, vasocclusive agents, effects of nutr itional deficiencies, human chorionic gonadotropin-antiserum and plant products as antifertility agents have been discussed.     

L-NAME pre-treatment partially inhibits the agmatine-evoked depression of the electrically induced twitch contraction of isolated rat vas deferens

The effect of the putative endogenous ligand for alpha(2)-adrenoceptors and imidazoline receptors agmatine was studied in sympathetic neurotransmission in the rat epididymal vas deferens. Tissues were obtained from N(varpi)-nitro-l-arginine methyl ester (l-NAME)-treated or normal animals and were contracted by electrical stimulation or by exogenous adenosine 5'-triphosphate (ATP). In the electrically stimulated epididymal end, agmatine produced an inhibitory effect on twitch contraction that was partially reversed in l-NAME-treated animals, whereas the inhibition produced by clonidine was not affected by l-NAME treatment. The nitric oxide (NO)-donor S-nitroso-N-acetyl-penicillamine (SNAP) also inhibited twitch contraction. Neither agmatine nor SNAP interfered with the responses induced by exogenous ATP in the epididymal end. Removal of the epithelium of the preparation did not modify the agmatine response. We conclude that a nitrergic pathway activated by agmatine plays a role in its inhibitory effect in rat vas deferens, but it remains to be investigated whether it results from a direct action on the enzyme NO-synthase or a receptor-mediated mechanism.     

The effects of pancreatic polypeptides and neuropeptide Y on the rat vas deferens

In order to study the physiological significance of the coexistence of pancreatic polypeptide and norepinephrine (NE) in peripheral noradrenergic nerves, the effects of pancreatic polypeptides of several species were tested on the isolated rat vas deferens. Neuropeptide Y (NPY) was also studied because of its sequence homology to the pancreatic polypeptides. The contractile responses, which were mediated predominantly by activation of noradrenergic nerves following electrical stimulation, were inhibited by bovine pancreatic polypeptide (BPP), human pancreatic polypeptide (HPP), avian pancreatic polypeptide (APP) and NPY in a dose-dependent manner using a constant flow bath. The decreasing order of the inhibitory responses was as follows: BPP = HPP greater than NPY greater than APP. The inhibitory responses produced by BPP and HPP lasted more than 1 hr and displayed a marked tachyphylaxis. In contrast, the inhibitory effects induced by NPY and APP usually returned to the control level after 20-30 min and had minimal tachyphylaxis. The inhibitory action of NPY was still present during alpha-adrenergic blockade. Contractions produced by a single submaximal dose of exogenous NE or serotonin (5-HT) in unstimulated preparations were not affected by pretreatment with NPY. The amplitude of contractions was partially reduced 1 min after pretreatment with BPP or HPP; recovery occurred about 15 min after peptide pretreatment in a constant flow bath. These results suggest that an NPY receptor exists presynaptically in the rat vas deferens and that stimulation of the receptor by NPY inhibits the release of NE from noradrenergic nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ascaris suum: Free amino acids and proteins in the pseudocoelom,seminal vesicle and glandular vas deferens

The free amino acids and proteins of the seminal vesicle and pseudocoelomic fluids in the male Ascaris suum were examined and compared. The seminal fluid contained a high concentration of lysine (lysine: glutamic acid ratio of 5:1) while the pseudocoelomic fluid contained more glutamic acid than lysine with alanine, serine, glycine and proline being the most abundant free amino acids. The proteins present in the seminal fluid differed from those in the pseudocoelomic fluid in both number and molecular weight. The sperm activating substance (SAS) present in homogenates of the glandular vas deferens of male worms is nondialyzable, heat-sensitive and can be precipitated using 45% saturated ammonium sulfate. Active moieties can be recovered following passage of the ammonium sulfate precipitates through ultrafiltration membranes or by applying gland homogenates to an ion exchange column. When subjected to SDS-polyacrylamide gel electrophoresis, the active fractions revealed both low and high molecular weight substances. During attempts to purify a single activating substance, it was noted that the more heterogeneous fractions contained the highest activating capacity. Thus, no precise relationship between the biological activity and the purity of the various fractions was determined.     

E-2078, a potent, selective and stable dynorphin analog with preferential activity for the kappa-opioid receptor subtype on the mouse vas deferens neuroeffector junction

The profile of opioid activity of E-2078, a synthetic stable dynorphin analog, was examined in the mouse vas deferens bioassay and compared to that of methionine enkephalin and nonpeptide kappa agonists in the absence and in the presence of selective antagonists for the mu-, kappa- and delta-opioid receptor subtypes. The inhibitory action of E-2078 and related kappa agonists was specifically and potently antagonized only by norbinaltorphimine, revealing the presence of kappa receptors in this tissue and the predominant kappa activity of E-2078.