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.

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.

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.

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.

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.

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.

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.

Expression of oestrogen receptors (GPER, ESR1, ESR2) in human ductuli efferentes and proximal epididymis.

Oestrogen targeting in the human genital ducts is still not well-known. In fact, to date, the localization of oestrogen receptors, ESR1 and ESR2, is controversial and the presence of the membrane oestrogen receptor GPER (G protein-coupled oestrogen receptor) is unexplored. This study has investigated the expression of GPER, ESR1, ESR2 in human ductuli efferentes and proximal caput epididymis by immunohistochemistry and Western blot analysis. Furthermore, the presence of PELP1 (proline-glutamic acid-leucine-rich protein 1), a co-regulator of the oestrogen receptors, was also evaluated. In ductuli efferentes, GPER and ESR1 were clearly localized in all epithelial cells, while ESR2 was evidenced only in ciliated cells. Conversely, the epithelial cells of proximal caput epididymis revealed moderate GPER immunoreactivity, the absence of ERS1 and the occasional presence of ESR2. Furthermore, PELP1 was observed in ciliated cells of ductuli efferentes and in principal cells of proximal caput epididymis. Therefore, this study firstly demonstrated the expression of GPER in human male genital ducts, revealing a new mediator of oestrogen action in these anatomical sites. ESR1 and ESR2 were differentially localized in the two genital tracts together with PELP1, but cell sites of ERs and their co-regulator were not homogeneous. So, a different regional/cellular association of GPER with the classical oestrogen receptors was highlighted, suggesting that oestrogen action could be mediated by GPER, ESR1, ESR2 in ductuli efferentes, while by GPER and, occasionally by ESR2, in proximal caput epididymis. This study suggests that the specific oestrogen-mediated functions in human genital ducts might result from the different local interactions of oestrogens with oestrogen receptors and their co-regulators.

Comparative analysis of activins A and B in the adult mouse epididymis and vas deferens.

Activin A regulates testicular and epididymal development, but the role of activin B in the epididymis and vas deferens is unknown. Mouse models with reduced activin A (Inhba+/- and InhbaBK/+), or its complete absence (InhbaBK/BK), were investigated to identify specific roles of activins in the male reproductive tract. In 8-week-old Inhba+/- mice, serum activin A decreased by 70%, with a 50% reduction of gene expression and protein in the testis, epididymis and vas deferens. Activin B and the activin-binding protein, follistatin, were similar to wild-type. Testis weights were slightly reduced in Inhba+/- mice, but the epididymis and vas deferens were normal, while the mice were fertile. Activin A was decreased by 70% in the serum, testis, epididymis and vas deferens of InhbaBK/+ mice and was undetectable in InhbaBK/BK mice, but activin B and follistatin levels were similar to wild-type. In 6-week-old InhbaBK/BK mice, testis weights were 60% lower and epididymal weights were 50% lower than in either InhbaBK/+ or wild-type mice. The cauda epididymal epithelium showed infoldings and less intra-luminal sperm, similar to 3.5-week-old wild-type mice, but at 8 weeks, no structural differences in the testis or epididymis were noted between InhbaBK/BK and wild-type mice. Thus, Inhbb can compensate for Inhba in regulating epididymal morphology, although testis and epididymal maturation is delayed in mice lacking Inhba Crucially, reduction or absence of activin A, at least in the presence of normal activin B levels, does not lead to major defects in the adult epididymis or vas deferens.

Synthesis of Gly-ψ[(Z)CF═CH]-Phe, a Fluoroalkene Dipeptide Isostere, and Its Incorporation into a Leu-enkephalin Peptidomimetic.

A new Leu-enkephalin peptidomimetic designed to explore the hydrogen bond acceptor ability of the third peptide bond has been prepared and studied. This new analog is produced by replacing the third amide of Leu-enkephalin with a fluoroalkene. An efficient and innovative synthesis of the corresponding dipeptide surrogate Fmoc-Gly-ψ[(Z)CF═CH]-Phe-OH is described. The key step involves the alkylation of a tin dienolate from the less hindered face of its chiral sulfonamide auxiliary derived from camphor. Once its synthesis was complete, its incorporation into the peptidomimetic sequence was achieved on a solid support with chlorotrityl resin following the Fmoc strategy. The peptidomimetic was characterized using competition binding with [125I]-deltorphin I on membrane extracts of HEK293 cells expressing the mouse delta opioid receptor (DOPr) and based on its abilities to inhibit the electrically induced contractions of the mouse vas deferens and to activate the ERK1/2 signaling pathway in DRGF11/DOPr-GFP cells. Together with our previous observations, our findings strongly suggest that the third amide bond of Leu-enkephalin primarily acts as a hydrogen bond acceptor in DOPr. Consequently, this amide bond can be successfully replaced by an ester, a thioamide, or a fluoroalkene without greatly impacting the binding or biological activity of the corresponding analogs. The lipophilicity (LogD7.4) of the active analog was also measured. It appears that fluoroalkenes are almost as efficient at increasing the lipophilicity as normal alkenes.

Novel mutations and polymorphisms in the CFTR gene associated with three subtypes of congenital absence of vas deferens.

OBJECTIVE: To study the new genotypes in congenital absence of vas deferens (CAVD) and the correlation with different phenotypes, and to investigate the pathogenesis of the disease based on bioinformatics analysis. DESIGN: Case-control study. SETTING: University-affiliated tertiary teaching hospital. PATIENT(S): Nineteen patients with CAVD and azoospermia. The time period of the study was from May 2013 to April 2014. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sanger sequencing was performed in the coding regions and intron-exon boundaries of the cystic fibrosis transmembrane regulator CFTR gene on the polymerase chain reaction (PCR) products. Mutations/variations were identified and compared with the control subjects, and bioinformatics analysis searched in the dbSNP and 1000 Genomes Project. Functional effects of the novel mutations were predicted. Structural modeling of the wild and mutant proteins was also performed. RESULT(S): A total of 8 mutations were identified in 12 patients, 4 of which were novel (c.4433C>G, c.3469-3C>A, c.1357delT, and c.3407C>T). The mutation c.4433C>G occurred in the PSD-95/DLG/ZO-1 (PDZ)-binding motif in the CFTR protein, which was predicted to disrupt the interaction between CFTR and CFTR-associated ligand (CAL). Another missense mutation, c.3407C>T, was predicted to damage and destroy the transmembrane adenosine triphosphate (ATP)-binding cassette domain. The splicing mutation, c.3469-3C>A, was predicted to truncate exon 22 by Human Splicing Finder. The frameshift mutation, c.1357delT, was predicted to introduce a premature stop codon at position 453 and lead to 1,012 amino acids truncation at the carboxyl terminus of the CFTR protein. CONCLUSION(S): This study illustrates the significance of whole exon sequencing of the CFTR gene in patients with CAVD. It is essential for determining the pathogenesis of novel mutations using bioinformatics analysis and to identify correlation between new genotypes and phenotypes.

Evaluation of the reproductive toxicity of fungicide propiconazole in male rats.

The propiconazole (Prop) is a fungicide extensively used in agriculture. There are evidences that this compound may cause endocrine disrupting effects. In vitro studies have demonstrated that Prop inhibits the activity of CYP 19 (aromatase), responsible for converting androgens into estrogens and also is an androgen and estrogen receptor antagonist. Therefore, this study evaluated the reproductive toxicity of Prop treatment in male rats. The Wistar rats were divided in three groups and were treated daily, by gavage, with corn oil (control group), propiconazole 4 mg/kg (Prop 4) and 20 mg/kg (Prop 20), from post-natal day 50 to 120. The following were observed: the body weight gain, sexual behavior, testosterone and estradiol plasmatic levels, organs weight, sperm count and morphology and testicular histomorphology. There was an increase in abnormal tail morphology sperm, seminal vesicle and vas deferens weight, and a decrease in estradiol levels in Prop 4 group. Sexual behavior was affected only in the Prop 20 group. These results suggest that Prop treatment induced alterations in some reproductive parameters, what could be related with an endocrine disruption.

Effect of resveratrol on restoring spermatogenesis in experimental cryptorchid mice and analysis of related differentially expressed proteins.

The present study aimed to evaluate the effect of trans-Resveratrol on spermatogenesis. Male Kunming suckling mice (10 days old) were surgically rendered cryptorchid and subcutaneously injected with trans-Resveratrol at doses of 5, 10, 20, and 40 µg/g/day as groups I, II, III, and IV, respectively, for 35 days. Animals in the control group received 10 µL/mouse/day of olive oil. Serum estradiol, testosterone, FSH, and LH levels were measured on day 45. Tissue analysis and sperm morphological abnormalities analysis were done. Results showed that in the control group and group I only spermatogonia and primary spermatocytes were present, whereas spermatogenesis was totally restored in groups II, III, and IV. Sperm counts in groups III and IV were remarkably higher than the control group (P<0.05). The morphological abnormalities in resveratrol-treated groups were higher than the mature mice. Serum estradiol levels in the resveratrol-treated groups were not significantly different from the control group, but were lower than the mature mice (P<0.05). There was no significant difference in serum testosterone levels between the resveratrol-treated groups and mature mice, but the levels in the resveratrol-treated groups was significantly lower than the control group (P<0.05). No significant influence of trans-Resveratrol was observed on serum FSH levels in all cryptorchid mice. Serum LH levels in groups I, II, and III were higher than the control group. These results indicate that trans-Resveratrol restores spermatogenesis in cryptorchid mice. In addition, proteomic analysis between the 20 µg/g/day resveratrol-treated group and the control group was carried out, and five kinds of proteins (BAF250, ZFP261, CHD1L, RBBP9, and SOHLH2) were identified. The expression of SOHLH2 increased, while that of BAF250, ZFP261, CHD1L, and RBBP9 decreased in the 20 µg/g/day resveratrol-treated group, indicating that SOHLH2 may contribute to testicular germ cell differentiation.

Heterogeneous spectrum of mutations in CFTR gene from Indian patients with congenital absence of the vas deferens and their association with cystic fibrosis genetic modifiers.

Cystic fibrosis (CF) is usually considered a rare disease in the Indian population. Two studies have reported on the frequency of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Indian males with congenital absence of the vas deferens (CAVD), however, data on the spectrum of CFTR gene mutations are still lacking. Therefore, the present study was designed to identify the spectrum of CFTR gene mutations as well as to investigate an association of CF genetic modifiers in the penetrance of CAVD in infertile Indian men. A total of 60 consecutive infertile males with a diagnosis of CAVD were subjected to CFTR gene analysis which revealed 13 different CFTR gene mutations and 1 intronic variant that led to aberrant splicing. p.Phe508del (n = 16) and p.Arg117His (n = 4) were among the most common severe forms of CFTR mutations identified. The IVS8-T5 allele, which is considered as a mild form of CFTR mutation, was found with an allelic frequency of 28.3%. Eight novel mutations were also identified in the CFTR gene from our patient cohort. It is noteworthy that the spectrum of CFTR gene mutation is heterogeneous, with exon 4 and exon 11 as hot spot regions. Moreover, we also found an association of the CF genetic modifiers, viz., transforming growth factor (TGF)-ß1 and endothelial receptor type-A (EDNRA) genes with the CAVD phenotype. The findings are of considerable clinical significance because men suffering from infertility due to CAVD can decide to use artificial reproduction technology. The children of men with CAVD are at risk of carrying CFTR mutations; therefore, genetic counseling is a crucial step for such patients. With special reference to developing countries, such as India, where whole gene sequencing is not feasible, the outcome of our study will make the screening procedure for CFTR gene simpler and more cost-effective as we have identified hot spot regions of the CFTR gene which are more prone to mutation in Indian males with CAVD. Moreover, this is the first study from the Indian population to investigate the association of CF genetic modifiers with penetrance of the CAVD phenotype. The observed association of the genetic modifiers TGF-ß1 and EDNRA in the penetrance of CAVD further supports their involvement in genesis of the vas deferens.

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.

P2X1 receptors localized in lipid rafts mediate ATP motor responses in the human vas deferens longitudinal muscles.

To assess the role of the P2X1 receptors (P2X1R) in the longitudinal and circular layers of the human vas deferens, ex vivo-isolated strips or rings were prepared from tissue biopsies to record isometric contractions. To ascertain its membrane distribution, tissue extracts were analyzed by immunoblotting following sucrose gradient ultracentrifugation. ATP, alpha,beta-methylene ATP, or electrical field stimulation elicited robust contractions of the longitudinal layer but not of the circular layer which demonstrated inconsistent responses. Alpha,beta-methylene ATP generated stronger and more robust contractions than ATP. In parallel, prostatic segments of the rat vas deferens were examined. The motor responses in both species were not sustained but decayed within the first minute, showing desensitization to additional applications. Cross-desensitization was established between alpha,beta-methylene ATP or ATP-evoked contractions and electrical field stimulation-induced contractions. Full recovery of the desensitized motor responses required more than 30 min and showed a similar pattern in human and rat tissues. Immunoblot analysis of the human vas deferens extracts revealed a P2X1R oligomer of approximately 200 kDa under nonreducing conditions, whereas dithiothreitol-treated extracts showed a single band of approximately 70 kDa. The P2X1R was identified in ultracentrifugation fractions containing 15%-29% sucrose; the receptor localized in the same fractions as flotillin-1, indicating that it regionalized into smooth muscle lipid rafts. In conclusion, ATP plays a key role in human vas deferens contractile responses of the longitudinal smooth muscle layer, an effect mediated through P2X1Rs.

Undernutrition affects cell survival, oxidative stress, Ca2+ handling and signaling pathways in vas deferens, crippling reproductive capacity.

BACKGROUND: The aim of this work was to investigate the mechanisms by which chronic malnutrition (CM) affects vas deferens function, leading to compromised reproductive capacity. Previous studies have shown that maternal malnutrition affects the reproductive tracts of adult male offspring. However, little is known about the effects of CM, a widespread life-long condition that persists from conception throughout growth to adult life. METHODOLOGY/PRINCIPAL FINDINGS: Young adult male rats, which were chronically malnourished from weaning, presented decreased total and haploid cells in the vas deferens, hypertrophy of the muscle layer in the epididymal portion of the vas deferens and intense atrophy of the muscular coat in its prostatic portion. At a molecular level, the vas deferens tissue of CM rats exhibited a huge rise in lipid peroxidation and protein carbonylation, evidence of an accentuated increase in local reactive oxygen species levels. The kinetics of plasma membrane Ca(2+)-ATPase activity and its kinase-mediated phosphorylation by PKA and PKC in the vas deferens revealed malnutrition-induced modifications in velocity, Ca(2+) affinity and regulation of Ca(2+) handling proteins. The severely crippled content of the 12-kDa FK506 binding protein, which controls passive Ca(2+) release from the sarco(endo) plasmic reticulum, revealed another target of malnutrition related to intracellular Ca(2+) handling, with a potential effect on forward propulsion of sperm cells. As a possible compensatory response, malnutrition led to enhanced sarco(endo) plasmic reticulum Ca(2+)-ATPase activity, possibly caused by stimulatory PKA-mediated phosphorylation. CONCLUSIONS/SIGNIFICANCE: The functional correlates of these cellular and molecular hallmarks of chronic malnutrition on the vas deferens were an accentuated reduction in fertility and fecundity.

Molecular basis of bicarbonate membrane transport in the male reproductive tract.

Bicarbonate (HCO3⁻) membrane transport systems are crucial players in the physiology of several tissues. The molecular basis of HCO3⁻ membrane transport is of major physiological relevance since this ion is involved in the establishment of intracellular and extracellular ionic composition, osmolariy and pH. The membrane HCO3⁻ transporters are divided in two main families: solute carrier 4 (SLC4) and solute carrier 26 (SLC26), although HCO3⁻ concentration can also be regulated by the cystic fibrosis transmembrane regulator (CFTR). In most tissues the SLC4 family represents the majority of HCO3⁻ transporters members, which can be divided in two subgroups: the Na⁺-dependent and the Na⁺-independent transporters. The SLC26 family consists of ten members that can transport diverse ions besides HCO3⁻. In the male reproductive tract, HCO3⁻ transport occurs in several processes in order to assure a correct pursuance of the spermatogenetic event and spermatozoa capacitation, being also necessary for egg fertilization. Indeed, the formation of competent spermatozoa, the maintenance of an adequate ductal luminal milieu and spermatozoa capacitation are highly dependent of ionic balance and pH. Perturbations in these processes result in reduced male reproductive health and consequently male subfertility and/or infertility. Thus, it is imperative to understand HCO3⁻ transport dynamics in order to identify and counteract possible alterations related with reduced male fertility caused by pathological conditions. Herein, we will review the major families and subfamilies of HCO3⁻ membrane transport, discussing the molecular basis of HCO3⁻ transport in the male reproductive tract and its role in male-associated subfertility and/or infertility.

Transforming growth factor-ß1 impairs CFTR-mediated anion secretion across cultured porcine vas deferens epithelial monolayer via the p38 MAPK pathway.

The goal of this study was to determine whether transforming growth factor-ß1 (TGF-ß1) affects epithelial cells lining the vas deferens, an organ that is universally affected in cystic fibrosis male patients. In PVD9902 cells, which are derived from porcine vas deferens epithelium, TGF-ß1 exposure significantly reduced short-circuit current (Isc) stimulated by forskolin or a cell membrane-permeant cAMP analog, 8-pCPT-cAMP, suggesting that TGF-ß1 affects targets of the cAMP signaling pathway. Electrophysiological results indicated that TGF-ß1 reduces the magnitude of current inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) channel blockers. Real-time RT-PCR revealed that TGF-ß1 downregulates the abundance of mRNA coding for CFTR, while biotinylation and Western blot showed that TGF-ß1 reduces both total CFTR and apical cell surface CFTR abundance. These results suggest that TGF-ß1 causes a reduction in CFTR expression, which limits CFTR-mediated anion secretion. TGF-ß1-associated attenuation of anion secretion was abrogated by SB431542, a TGF-ß1 receptor I inhibitor. Signaling pathway studies showed that the effect of TGF-ß1 on Isc was reduced by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). TGF-ß1 exposure also increased the amount of phospho-p38 MAPK substantially. In addition, anisomycin, a p38 MAPK activator, mimicked the effect of TGF-ß1, which further suggests that TGF-ß1 affects PVD9902 cells through a p38 MAPK pathway. These observations suggest that TGF-ß1, via TGF-ß1 receptor I and p38 MAPK signaling, reduces CFTR expression to impair CFTR-mediated anion secretion, which would likely compound the effects associated with mild CFTR mutations and ultimately would compromise male fertility.