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

INTRODUCCIÓN Y OBJETIVOS: Con frecuencia los urólogos remiten el tejido resecado durante las vasectomías para estudio anatomopatológico con el fin de confirmar la presencia de conducto deferente. El estudio microscópico es sencillo y se suele hacer con hematoxilina-eosina. En ocasiones, se ve dificultado por artefactos de la muestra y la inmunohistoquímica puede ayudar a reconocer la presencia de deferente. MATERIALES Y MÉTODOS: Hemos investigado la utilidad de la determinación inmunohistoquímica de cadherina E y GATA-3 para confirmar epitelio del deferente en 110 secciones de vasectomías con diferentes artefactos, utilizando anticuerpos monoclonales y técnica de multímero conjugado con peroxidasa; 5 arterias y 5 venas renales fueron controles negativos. RESULTADOS: Con cadherina E se observó tinción de membrana moderada (2,7%) o intensa (97,3%) en el epitelio del deferente en todos los casos: 35 sin artefacto, 7 con epitelio denudado, 56 con epitelio comprimido o distorsionado, 8 con epitelio desprendido y 4 con epitelio desplazado. GATA-3 mostró positividad nuclear moderada (31%) o intensa (69%) en todos los casos, incluyendo los 76 con los artefactos señalados. Las arterias y venas fueron negativas para ambos marcadores en el endotelio, con positividad para GATA-3 en ocasionales linfocitos de la pared. CONCLUSIONES: La inmunohistoquímica puede ayudar a reconocer la presencia de epitelio del deferente en vasectomías artefactadas con positividad de membrana para cadherina E y expresión nuclear de GATA-3. El endotelio vascular, por el contrario, es negativo para ambos marcadores. No se debe malinterpretar como positividad la posible tinción para GATA-3 de linfocitos de la pared

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

Ultrastructure and histochemistry of the male reproductive system of the genus Callinectes Stimpson, 1860 (Brachyura: Portunidae).

We described the ultrastructure and histochemistry of the reproductive system of five Callinectes species, and evaluate the seasonal variation in weight of the reproductive system and hepatopancreas by comparing annual changes of somatic indices. The somatic indices changed little throughout the year. In Callinectes, spermatogenesis occurs inside the lobular testes and, within each lobule, the cells are at the same developmental stage. Spermatogenesis and spermiogenesis follow the same development pattern in all Callinectes studied. Mature spermatozoa are released into the seminiferous ducts through the collecting ducts. Cells of the vas deferens are secretory as evidenced by rough endoplasmic reticulum, Golgi complex, and secretory vesicles that produce the seminal fluid. The anterior vas deferens shows two portions: proximal and distal. In proximal portion (AVDp), spermatozoa are clustered and embedded in an electron-dense, basophilic glycoproteinaceous secretion Type I. In the distal portion (AVDd), the spermatophore wall is formed by incorporation of a less electron-dense glycoproteinaceous secretion Type II. The secretion Type I change to an acid polysaccharide-rich matrix that separates the spermatophores from each other. The median vas deferens (MVD) stores the spermatophores and produces the granular glycoproteinaceous seminal fluid. The posterior vas deferens (PVD) has few spermatophores. Its epithelium has many mitochondria and the PVD seminal fluid changes into a liquid and homogeneous glycoprotein. Many outpocketings in the PVD and MVD help to increase the fluid production. Overall, the reproductive pattern of Callinectes is similar to other species that produce sperm plugs. The secretions of AVD, MVD, and PVD are responsible for the polymerization that forms the solid, waxy plug in the seminal receptacle. The traits identified here are common to all Portunidae species studied so far.

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.

An atlas of cell types in the mouse epididymis and vas deferens.

Following testicular spermatogenesis, mammalian sperm continue to mature in a long epithelial tube known as the epididymis, which plays key roles in remodeling sperm protein, lipid, and RNA composition. To understand the roles for the epididymis in reproductive biology, we generated a single-cell atlas of the murine epididymis and vas deferens. We recovered key epithelial cell types including principal cells, clear cells, and basal cells, along with associated support cells that include fibroblasts, smooth muscle, macrophages and other immune cells. Moreover, our data illuminate extensive regional specialization of principal cell populations across the length of the epididymis. In addition to region-specific specialization of principal cells, we find evidence for functionally specialized subpopulations of stromal cells, and, most notably, two distinct populations of clear cells. Our dataset extends on existing knowledge of epididymal biology, and provides a wealth of information on potential regulatory and signaling factors that bear future investigation.

Functional anatomy of the male reproductive system of the American lobster (Homarus americanus).

Despite supporting a valuable fishery, the reproductive system of the male American lobster (Homarus americanus) is poorly understood. The elongated H-shaped testis is responsible for spermatogenesis and is composed of follicles, a common collecting duct with interlaced scattered striated muscles, and a serosa as an external wall. Sertoli cells are associated with the spermatogenesis that produces spermatozoa, which are transferred to the collecting duct through a temporary passageway. Spermatogenesis is asynchronous between follicles and occurs on a continuous basis. The anterior and posterior lobes of the testes are independent and connect to the vasa deferentia through the Y-shaped collecting tubules that have a different cell anatomy and function than the two organs they connect. The vas deferens is divided into four regions. Spermatophores, produced in the proximal vas deferens, are packets of spermatozoa encapsulated in a single layer-the spermatophoric wall, which is composed of mucopolysaccharide acid. Large dense ovoid granules and the seminal fluid, composed of acidic sulfated mucosubstances, are secreted in the median vas deferens. Spermatophores within these secreted substances (i.e., semen) are stored in the distal vas deferens that, with the spermiduct (last region of the vas deferens), is responsible for the extrusion of the semen by striated muscle contractions. Smooth muscles suggest a peristaltic movement of the spermatophores within the vas deferens. Finally, the gonopores and the first pair of pleopods (i.e., gonopod) move the semen to the female seminal receptacle during copulation.

Morphological and histological structure of the male reproductive system of the water strider Gerris lacustris (Linnaeus 1758) (Gerridae, Heteroptera).

This work presents the male reproductive system morphology and histology of the water strider Gerris lacustris (Linnaeus 1758) (Gerridae, Heteroptera) using light microscopy and scanning electron microscopy. The male reproductive system of G. lacustris comprise of a pair of testes, two vasa deferentia, two seminal vesicles, an ejaculatory duct. There is no bulbus ejaculatorius and the long vas deferantia uniting to form a simple ductus ejaculatorius which is connected to the aedeagus. The testes are white colored and this cylindiric-shaped structure lies along genital abdominal segment. The testicular follicles have three different development zones (growth zone, maturation zone, differentiation zone). Each testis has two follicles, which are not lined by a common peritoneal sheath and involving many cysts arranged in a progressive order of maturation from the distal to the proximal region; spermiogenesis occurs in mature males, finishing with the organization of sperm bundles. The testes are connected to the seminal vesicles, specialized sperm storage places, by the vas deferentia.

Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na+ channels of mouse vas deferens myocytes and recombinant NaV1.6 channels.

Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na+ channel ß-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other ß-subunit genes (Scn2b - Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na+ current peak amplitude in myocytes (i.e., native INa) were compared with its inhibitory potency on recombinant NaV1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INa and recombinant NaV1.6 expressed in HEK293 cells (i.e., recombinant INa). The current decay of native INa was similar to the recombinant NaV1.6 current co-expressed with ß1-subunits. The current-voltage (I-V) relationships of native INa were similar to those of recombinant NaV1.6 currents co-expressed with ß1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native INa (K i = 510 nM), recombinant INa (K i = 112 nM), and recombinant INa co-expressed with ß1-subunits (K i = 92 nM). The half-maximal (Vhalf) activation and inactivation of native INa values were similar to those observed in recombinant INa co-expressed with ß1-subunits. These results suggest that ß1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native INa but also recombinant INa and recombinant INa co-expressed with ß1-subunits in a concentration-dependent manner.

Deciphering the mechanisms involving cenexin, ninein and centriolin in sperm maturation during epididymal transit in the domestic cat.

The sperm centrosome is an essential organelle with a key role in organizing the sperm aster for proper syngamy and formation of the first mitotic spindle. The sperm cell acquires the functional capability during epididymal transit by incorporation of key factors. The objective of the study was to identify these key maturation proteins, such as ninein and centriolin as well as cenexin-a scaffold protein that serves to bind ninein and centriolin. Epididymal samples were dissected from 17 adult cat testes (>1 year old) and spermatozoa were extracted from the different regions, including rete testis, caput, corpus, cauda and vas deferens. Tissue samples and sperm cells were fixed separately in 4% paraformaldehyde before immunostaining with anticenexin, ninein or centriolin antibodies. Results showed that the proportion of sperm cells with cenexin localized at the centrosome progressively increased along the tract with the lowest percentage of stained cells in the testis (mean = 45%) and highest in the cauda (mean = 81%). Although not significant, the intensity of cenexin immunofluorescence in positive cells increased twofold from the testis to vas deferens. There was no significant difference in the proportion of sperm labelled with centriolin or ninein (ranges of 21%-26% and 33%-48% between segments, respectively) or the intensity (±58% and ±63% change as compared to testis, respectively). Cenexin may serve as a scaffold protein for centriolin and ninein, as the vast majority of spermatozoa only displayed colocalization of these proteins when cenexin was also present (mean = 85% and 91% colocalization, respectively). In summary, these results could be applied to future efforts to create an in vitro culture system capable of rescuing the impaired centrosome of an infertile male, with particular potential for wild felid conservation.

Stereoselectivity of butylidenephthalide on non-adrenergic prejunctional voltage-dependent Ca(2+) channels in prostatic portion of rat vas deferens.

The naturally occurring and synthetic butylinenephthalide (Bdph) has two geometric isomers. Z- and E-Bdph were reported to have geometric stereoselectivity for voltage-dependent calcium channels (VDCCs) in guinea-pig ileum. The aim of this study was to investigate whether the binding of Z- and E-Bdph on prejunctional VDCCs of rat vas deferens (RVD) is stereoselective. The twitch responses to electrical field stimulation (EFS, supramaximal voltage, 1 ms, 0.2Hz) were recorded on a polygraph. Z- and E-Bdph concentration-dependently inhibited the twitch responses to EFS in full tissue, prostatic portion and epididymal portion of RVD. The pIC50 value of Z-Bdph was greater than that of E-Bdph in the electrically stimulated prostatic portion of RVD, suggesting that the binding of Bdph on the non-adrenergic prejunctional VDCCs of cell membrane is stereoselective. In the prostatic portion, exogenous Ca(2+) only partially reversed the twitch inhibition by Z-Bdph, but effectively reversed those by Ca(2+) channel blockers, such as verapamil, diltiazem and aspaminol, suggesting that the action mechanisms may be different from those of Ca(2+) channel blockers. K(+) channel blockers, such as tetraethylammonium (TEA) and 4-aminopyridine (4-AP), may prolong duration of action potential to allow greater Ca(2+) entry and induced more release of transmitters. Therefore both blockers via their prejunctional actions reversed the twitch inhibition induced by Z-Bdph in all preparations of RVD by a non-specific antagonism.

Morphological evidence for a permeability barrier in the testis and spermatic duct of Gymnotus carapo (Teleostei: Gymnotidae).

Cell-cell interactions play essential roles in the regulation of gametogenesis. The involvement of junctional complexes in permeability barriers, for example, provides structural and physiological support for male germ-cell development. This study describes morphological characteristics of the reproductive system of Gymnotus carapo, a neo-tropical freshwater fish widely distributed in South and Central America, focusing on the detection of permeability barriers using morphological and biochemical approaches. Ultrastructural analysis of testes treated with the lanthanum nitrate exclusion technique showed that the tracer penetrated the interstitial compartment of the testis, surrounding and appearing within cysts containing spermatogonia and spermatocytes in early stages of meiosis, but was not detected in the spermatid cysts or inside the lumen of spermatogenic tubules. These results suggest the presence of a permeability barrier that is stabilized after meiosis is completed and serves to protect the haploid cells from the vascular system. In the spermatic-duct region, the tracer was obstructed near the lumen of the duct. Junctional complexes and focal tight junctions between adjacent cells were observed in the testis and spermatic duct. Freeze-fracture methods indeed confirmed the presence of tight junctions, which were visualized as parallel rows of individual particles between adjacent cells. More evidence supporting the existence of a permeability barrier was gathered from differences observed in the electrophoretic protein profiles of testis and spermatic-duct fluids compared to blood plasma. Together, these observations demonstrate the existence of a permeability barrier formed by tight junctions in the testis and spermatic duct of G. carapo.

Seminal fluid promotes in vitro sperm-oviduct binding in the domestic cat (Felis catus).

From many endangered or threatened species which are expected to profit from assisted reproduction techniques, mainly epididymal sperm of dead or freshly castrated males are available. These sperm had contact to epididymal secretion products but not to seminal fluid components. Notably, products of accessory sex glands have been shown in domestic animals to condition sperm for fertilization, in particular by mediating sperm-oviduct interaction. We report for the first time that motile epididymal sperm from domestic cats are able to bind to fresh oviduct epithelial cell explants from preovulatory females (median [min, max] of 10 [8, 16] and 10 [8, 17] sperm per 0.01 mm(2) explant surface from both isthmic and ampullar regions, respectively). More sperm attach to the explants when epididymal sperm were preincubated for 30 minutes with seminal fluid separated from electroejaculates of mature tomcats (median [min, max] of 17 [13, 25] and 16 [12, 21] sperm per 0.01 mm(2) explant surface from isthmus and ampulla, respectively). The proportion of bound sperm increased from a median of 54% to 62% by seminal fluid treatment. Sperm-oviduct binding could be facilitated by the decelerated sperm motion which was observed in seminal fluid-treated samples or supported by seminal fluid proteins newly attached to the sperm surface. Seminal fluid had no effect on the proportion of sperm with active mitochondria. Extent and pattern of sperm interaction in vitro were independent of explant origin from isthmus or ampulla. Sperm were attached to both cilia and microvilli of the main epithelial cell types present in all explants. In contrast to published sperm-binding studies with porcine and bovine oviduct explants where predominantly the anterior head region of sperm was attached to ciliated cells, the tails of some cat sperm were firmly stuck to the oviduct cell surfaces, whereas the heads were wobbling. Whether this response is a preliminary step toward phagocytosis or a precondition to capacitation and fertilization remains to be determined. In conclusion, treatment of epididymal sperm with seminal fluid or particular protein components should be considered in future investigations for its potential to improve the outcome of artificial insemination in felids.

Molecular analysis of ATP-sensitive K⁺ channel subunits expressed in mouse vas deferens myocytes.

BACKGROUND AND PURPOSE: ATP-sensitive K(+)(K(ATP)) channels, which are composed of K(IR)6.x associated with sulphonylurea receptor (SUR) subunits, have been detected in native smooth muscle cells, but it is currently not known which of these is expressed in mouse vas deferens myocytes. EXPERIMENTAL APPROACH: Pharmacological and electrophysiological properties of K(ATP) channels in mouse vas deferens myocytes were investigated using patch clamp techniques. Molecular biological analyses were performed to examine the properties of these K(ATP) channels. KEY RESULTS: During conventional whole-cell recording, pinacidil elicited an inward current that was suppressed by glibenclamide, a sulfonylurea agent, and by U-37883A, a selective K(IR)6.1 blocker. When 0.3 mM ATP was added to the pipette solution, the peak amplitude of the pinacidil-induced current was much smaller than that recorded in its absence. When 3 mM UDP, GDP or ADP was included in the pipette solution, an inward current was elicited after establishment of the conventional whole-cell configuration, with potency order being UDP > GDP > ADP. These nucleoside diphosphate-induced inward currents were suppressed by glibenclamide. MCC-134, a SUR modulator, induced glibenclamide-sensitive K(ATP) currents that were similar to those induced by 100 µM pinacidil. In the cell-attached configuration, pinacidil activated channels with a conductance similar to that of K(IR)6.1. Reverse transcription PCR analysis revealed the expression of K(IR)6.1 and SUR2B transcripts and immunohistochemical studies indicated the presence of K(IR)6.1 and SUR2B proteins in the myocytes. CONCLUSIONS AND IMPLICATIONS: Our results indicate that native K(ATP) channels in mouse vas deferens myocytes are a heterocomplex of K(IR)6.1 channels and SUR2B subunits.

Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance.

We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the broken DNA ends remain attached to the nuclear matrix, causing the DNA to remain associated with the dispersion halo, and the Comet tails to be weak. Therefore, we term these hidden dsDNA breaks. When the Comet assay was modified to include an additional incubation with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) after the conventional lysis, thereby solubilizing the nuclear matrix, the broken DNA was released from the matrix, which resulted in a reduction of the sperm head halo and an increase in the Comet tail length, exposing the hidden dsDNA breaks. Conversely, SCF-induced vas deferens sperm had small halos and long tails with the conventional neutral Comet assay, suggesting that the broken DNA ends were not tethered to the nuclear matrix. These results suggest that the attachment to the nuclear matrix is crucial for the religation of SCF-induced DNA breaks in sperm. Our data suggest that the neutral Comet assay identifies only dsDNA breaks that are released from the nuclear matrix and that the addition of an SDS treatment can reveal these hidden dsDNA breaks.

Newly developed techniques in andrology: endoscopy of the vas deference and a new imaging technique for in situ localization of vital spermatozoa.

During the past decade, endourology represents the most advanced technology in urology which has provided minimal invasive diagnostic and therapeutic tool for patients with urology diseases. However, because of the much smaller lumen of genital tract, endoscopic andrology remained a dream for andrologists until the first clinical application of vesiculoscopy in laser lithotripsy of seminal vesicle stones in 2006. Lately, Dr Trottmann at Ludwig-Maximilians University, Germany, for the first time established vasoscopy in the seminal duct using a new prototype of a microendoscope and also applied a new imaging technique for in situ localization of vital spermatozoa. These newly developed techniques will greatly speed up the clinical practice of endoandrology.

Plasticity of basal cells during postnatal development in the rat epididymis.

Our previous study has shown that basal cells sense luminal factors by forming a narrow body projection that can cross epithelial tight junctions. As a first step toward characterizing the structural plasticity of basal cells, in this study, we followed their appearance and morphology in the rat epididymis and vas deferens (VD) during postnatal development and examined their modulation by androgens in adulthood. Immunofluorescence labeling for cytokeratin 5 showed that basal cells are absent at birth. They progressively appear in a retrograde manner from the VD and cauda epididymis to the initial segments during the postnatal weeks PNW1-3. At the onset of differentiation, basal cells are in contact with the lumen and their nucleus is located at the same level as that of adjacent epithelial cells. Basal cells then position their nucleus to the base of the epithelium, and while some are still in contact with the lumen, others have a 'dome-shaped' appearance. At PNW5-6, basal cells form a loose network at the base of the epithelium, and luminal-reaching basal cells are rarely detected. The arrival of spermatozoa during PNW7-8 did not trigger the development of projections in basal cells. However, cells with a narrow luminal-reaching projection began to reappear between PNW8 and PNW12 in the corpus and the cauda. Treatment with flutamide from PNW10 to PNW12 significantly reduced the number of luminal-reaching basal cell projections. In summary, basal cells exhibit significant structural plasticity during differentiation. Fewer apical-reaching projections were detected after flutamide treatment in adulthood, indicating the role of androgens in the luminal-sensing function of basal cells.

The reproductive cycle of the male sleep snake Sibynomorphus mikanii (Schlegel, 1837) from southeastern Brazil.

This study describes the male reproductive cycle of Sibynomorphus mikanii from southeastern Brazil considering macroscopic and microscopic variables. Spermatogenesis occurs during spring-summer (September-December) and spermiogenesis or maturation occurs in summer (December-February). The length and width of the kidney, the tubular diameter, and the epithelium height of the sexual segment of the kidney (SSK) are larger in summer-autumn (December-May). Histochemical reaction of the SSK [periodic acid-Schiff (PAS) and bromophenol blue (BB)] shows stronger results during summer-autumn, indicating an increase in the secretory activity of the granules. Testicular regression is observed in autumn and early winter (March-June) when a peak in the width of the ductus deferens occurs. The distal ductus deferens as well as the ampulla ductus deferentis exhibit secretory activities with positive reaction for PAS and BB. These results suggest that this secretion may nourish the spermatozoa while they are being stored in the ductus deferens. The increase in the Leydig cell nuclear diameter in association with SSK hypertrophy and the presence of sperm in the female indicate that the mating season occurs in autumn when testes begin to decrease their activity. The peak activity of Leydig cells and SSK exhibits an associated pattern with the mating season. However, spermatogenesis is dissociated of the copulation characterizing a complex reproductive cycle. At the individual level, S. mikanii males present a continuous cyclical reproductive pattern in the testes and kidneys (SSK), whereas at the populational level the reproductive pattern may be classified as seasonal semisynchronous.

Distribution of vitamin D receptor and 1α-hydroxylase in male mouse reproductive tract.

Vitamin D has been introduced as one of the main regulators of spermatogenesis. Here, for the first time, we evaluated the expression of vitamin D receptor (VDR) and 1α-hydroxylase in all organs of male mice reproductive tract by immunohistochemistry and Western blotting. Epithelial cells of epididymis, seminal vesicle, coagulating gland, ductus deferens, preputial gland, and prostate were the prominent cell types that concomitantly expressed VDR and 1α-hydroxylase. Nearly all cell types in testis expressed both proteins. Interestingly, VDR intensity in epididymis epithelial cells was reduced toward cauda, in which only strong staining of stereocilia was observed. Although been positive in caput epididymis, sperms lost their VDR expression in cauda region. In all organs, sperms failed to express 1α-hydroxylase. Specific bands of the VDR and 1α-hydroxylase were determined in all tissues, except testis in which novel unprecedented isoforms of 1α-hydroxylase were observed. Our findings could provide further convincing evidence of pivotal role of this hormone in male reproductive biology.

Resurgent-like currents in mouse vas deferens myocytes are mediated by NaV1.6 voltage-gated sodium channels.

Patch-clamp experiments were performed to investigate the molecular properties of resurgent-like currents in single smooth muscle cells dispersed from mouse vas deferens, utilizing both Na(V)1.6-null mice (Na(V)1.6(-/-)), lacking the expression of the Scn8a Na(+) channel gene, and their wild-type littermates (Na(V)1.6(+/+)). Na(V)1.6 immunoreactivity was clearly visible in dispersed smooth muscle cells obtained from Na(V)1.6(+/+), but not Na(V)1.6(-/-), vas deferens. Following a depolarization to +30 mV from a holding potential of -70 mV (to produce maximal inactivation of the Na(+) current), repolarization to voltages between -60 and +20 mV elicited a tetrodotoxin (TTX)-sensitive inward current in Na(V)1.6(+/+), but not Na(V)1.6(-/-), vas deferens myocytes. The resurgent-like current in Na(V)1.6(+/+) vas deferens myocytes peaked at approximately -20 mV in the current-voltage relationship. The peak amplitude of the resurgent-like current remained at a constant level when the membrane potential was repolarized to -20 mV following the application of depolarizing rectangular pulses to more positive potentials than +20 mV. 4,9-Anhydrotetrodotoxin (4,9-anhydroTTX), a selective Na(V)1.6 blocking toxin, purified from a crude mixture of TTX analogues by LC-FLD techniques, reversibly suppressed the resurgent-like currents. ß-Pompilidotoxin, a voltage-gated Na(+) channel activator, evoked sustained resurgent-like currents in Na(V)1.6(+/+) but not Na(V)1.6(-/-) murine vas deferens myocytes. These results strongly indicate that, primarily, resurgent-like currents are generated as a result of Na(V)1.6 channel activity.

Changes of cytosolic calcium and contractility of young rat vas deferens by acute treatment with amphetamine, fluoxetine or sibutramine.

Previous studies conducted in our laboratory indicated that administration of amphetamine, fluoxetine or sibutramine affects the sympathetic nervous system of the rat vas deferens. Therefore, our goal was to verify the role of calcium in vasa deferentia from young rats pretreated with a single dose of these drugs. Young 40-day-old male Wistar rats were pretreated with amphetamine 3 mg/kg, fluoxetine 10 mg/kg or sibutramine 6 mg/kg for 4 h before the experiments. CaCl(2) (10 mM) was used to induce contraction through time-effect curves in calcium-free solution to measure phasic and tonic components. We also evaluated the calcium-induced fluorescence of vas deferens cut into thin slices. In rats pretreated with amphetamine, we found an increase of the tonic contraction component which was reduced by verapamil. The phasic and tonic responses were increased in the group treated with fluoxetine, but only the tonic response was more sensitive to the antagonism by verapamil. The group treated with sibutramine showed an increase of phasic response whereas the tonic component was decreased. In this group an increase of the affinity for verapamil antagonism was found. In the calcium fluorescence study it was observed that the group treated with amphetamine, fluoxetine or sibutramine showed higher basal Ca(2+) fluorescence after stimulus with KCl (70 mM), noradrenaline (10(-4)M) or acetylcholine (10(-4)M). In all pretreated groups the calcium fluorescence was diminished by nifedipine 10(-7)M. Therefore, the pretreatment with amphetamine, fluoxetine or sibutramine seems to affect the calcium contractility and homeostasis in young rat vas deferens.

Cellular mechanism underlying the facilitation of contractile response of vas deferens smooth muscle by sodium orthovanadate.

In the earlier study, sodium orthovanadate (SOV) has been reported to be a powerful inhibitor of (Na(+), K(+)) adenosine triphosphatase, exhibit widespread actions on the renal and cardiovascular systems, induces smooth muscle contraction by inhibiting the phosphorylation of the protein tyrosine phosphatases. In the current study, we aimed to investigate the cellular mechanisms by which SOV facilitated contractile response of vas deferens smooth muscle and its potential therapeutic advantage. Exogenous application of ATP and NA-caused contraction was strengthened by pretreatment with SOV. This facilitation was inhibited not by bath with the inhibitor of P2 receptor, PPADS, or the inhibitor of α1 receptor, Prazosin, but by bath with the protein tyrosine kinase inhibitor, Genistein. SOV induced a sustained increase in intracellular Ca(2+) of smooth muscle cells, which was abolished by 100 µM Genistein or Ca(2+)-free solution. The facilitation of SOV could also be inhibited by the selective inhibitors of TRP channel, 2-APB and non-selective cation channel, Gd(3+), Ni(+). The in vivo study showed that peritoneal injection of SOV in dystrophic mice (mdx mice) enhanced the contraction of vas deferens smooth muscle stimulated by electrical field stimulation, ATP, noradrenaline, or KCl. The above results suggest that SOV facilitates the concentration of vas deferens smooth muscle through the tyrosine phosphorylation activated the non-selective cation channels, which has potential use in the therapy for muscle dysfunction.