Nucleoside triphosphate diphosphohydrolase-1 ectonucleotidase is required for normal vas deferens contraction and male fertility through maintaining P2X1 receptor function.

In this work, we report that Entpd1(-/-) mice, deficient for the ectonucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), produce smaller litters (27% reduction) compared with wild-type C57BL6 animals. This deficit is linked to reduced in vivo oocyte fertilization by Entpd1(-/-) males (61 ± 11% versus 88 ± 7% for Entpd1(+/+)). Normal epididymal sperm count, spermatozoa morphology, capacitation, and motility and reduced ejaculated sperm number (2.4 ± 0.5 versus 3.7 ± 0.4 million for Entpd1(+/+)) pointed to vas deferens dysfunction. NTPDase1 was localized by immunofluorescence in the tunica muscularis of the vas deferens. Its absence resulted in a major ATP hydrolysis deficiency, as observed in situ by histochemistry and in primary smooth muscle cell cultures. In vitro, Entpd1(-/-) vas deferens displayed an exacerbated contraction to ATP, a diminished response to its non-hydrolysable analog αßMeATP, and a reduced contraction to electrical field stimulation, suggesting altered P2X1 receptor function with a propensity to desensitize. This functional alteration was accompanied by a 3-fold decrease in P2X1 protein expression in Entpd1(-/-) vas deferens with no variation in mRNA levels. Accordingly, exogenous nucleotidase activity was required to fully preserve P2X1 receptor activation by ATP in vitro. Our study demonstrates that NTPDase1 is required to maintain normal P2X1 receptor functionality in the vas deferens and that its absence leads to impaired peristalsis, reduced spermatozoa concentration in the semen, and, eventually, reduced fertility. This suggests that alteration of NTPDase1 activity affects ejaculation efficacy and male fertility. This work may contribute to unveil a cause of infertility and open new therapeutic potentials.

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.

High expression and activity of ecto-5'-nucleotidase/CD73 in the male murine reproductive tract.

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, steroidogenesis, and maintenance of fluid composition. Interestingly, adenosine might act as a key capacitative effector for mammalian spermatozoa to acquire the capacity for fertilisation. Extracellular nucleotide levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family regroups the most abundant and effective enzymes to hydrolyse ATP and ADP to AMP in physiological conditions. In the male reproductive tract three members of this family have been indentified: NTPDase1, NTPDase2 and NTPDase3 (Martín-Satué et al. in Histochem Cell Biol 131:615-628, 2009). The purpose of the present study was to characterize in the male reproductive tract the expression profile of the main enzyme responsible for the generation of adenosine from AMP, namely the ecto-5'-nucleotidase (CD73). The enzyme was identified by immunological techniques and by in situ enzymatic assays, including inhibition experiments with alpha,beta-methylene-ADP, a specific CD73 inhibitor. High levels of ecto-5'-nucleotidase were detected in testes in association with both germinal and somatic cells, in smooth muscle cells throughout the tract, in secretory epithelia from exocrine glands, and remarkably, in principal cells of epididymis, where co-localization with NTPDase3 was found. The relevance of this co-expression on nucleotide hydrolysis in these cells directly involved in the control of sperm fluid composition was addressed biochemically. This study suggests close regulation of extracellular nucleoside and nucleotide levels in the genital tract by ecto-5'-nucleotidase that, in concurrence with NTPDases, may impact male fertility.

Localization of cyclooxygenase-2 in mice vas deferens and its effects on fertility upon suppression using nimesulide: a preferential cyclooxygenase-2 inhibitor.

Accumulating evidence on constitutive expression of cyclooxygenase-2 (COX-2), one of the isoforms of enzyme cyclooxygenase (COX) the other isoform being cyclooxygenase-1 (COX-1), questions the safety profile of non-steroidal anti-inflammatory drugs (NSAIDs). This COX-2 isoform which is induced not only during inflammation but also by factors such as cytokines, steroid hormones and mitogenic stimuli is constitutively expressed in brain, kidney and reproductive organs. Present NSAIDs, particularly COX-2 inhibitors is no longer considered safe since suppression of COX-2 in tissues which it is constitutively expressed may lead to adverse effects. Though intense expression of COX-2 in vas deferens is proved, lack of information with respect to its function has attracted a wide scope for research as to whether COX-2 in vas deferens contributes to male fertility. In the present study, the authors investigated the localization of COX-2 as well as COX-1 in mice vas deferens and also assessed the activity of COX-2 and total prostaglandin (PG) levels in vas deferens. Further they suppressed the expression of COX-2 using a preferential COX-2 inhibitor nimesulide and analyzed the sperm from vas deferens for any defects. COX-2 was intensely expressed in the epithelial cells of mice vas deferens and nimesulide was able to effectively suppress most of COX-2 expression. A decrease in PG levels was observed initially but interestingly, the levels tend to rise on sustained suppression of COX-2. The motility of sperm was affected severely after 6h of nimesulide administration that suggested a crucial role of COX-2 towards fertility of mice sperm.

Carbonic anhydrase activity in the vas deferens of the cotton leafworm - Spodoptera littoralis (Lepidoptera: Noctuidae) controlled by circadian clock.

The male reproductive tract of Lepidoptera is an ideal model for the study of the physiological role of peripheral clocks in insects. The latter are significant in the generation and coordination of rhythmic phenomena which facilitate the initial stages of sperm capacitation. This process requires the maintenance of pH in the upper vas deferens (UVD) aided by, among others, H+-ATPase. Our aim was to determine the potential involvement of carbonic anhydrase (CA) in this process, an enzyme tasked with generating protons subsequently utilized by H+-ATPase to acidify the UVD milieu in S. littoralis, during the time when the lumen of this organ is filled with sperm. We attempted to answer the question whether CA activity can be controlled by the biological oscillator present in the male reproductive tract of the cotton leafworm. Using PAGE zymography, the presence of CA was demonstrated in the UVD wall, but not in the luminal fluid nor in the sperm. Using histochemistry, it was shown that CA is active in the UVD epithelium, and that this activity varies throughout the day and is most likely controlled by an endogenous biological clock. Conversely, the application of CA inhibitors, acetazolamide and sodium thiocyanate, in conjunction with an analysis of H+-ATPase activity in the acidification the UVD environment shows that CA most likely does not play a direct role in the regulation of the pH in this organ.

Adaptive expression pattern of different proteins involved in cellular calcium homeostasis in denervated rat vas deferens.

The activity and protein expression of plasma membrane and sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPases and ryanodine receptors were investigated in surgically denervated rat vas deferens. The function of thapsigargin-sensitive but not thapsigargin-resistant (Ca2+-Mg2+)ATPase (from sarco(endo)plasmic reticulum and plasma membrane, respectively), evidenced by enzyme activity and Ca2+ uptake experiments, was significantly depressed by 30-50% when compared to innervated vas. Western blots showed that such reduction in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase performance was accompanied by a decrement of similar magnitude in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase type 2 protein expression, without any significant change in plasma membrane (Ca2+-Mg2+)ATPase expression. Finally, [3H]ryanodine binding revealed that the density of ryanodine binding sites was reduced by 45% after denervation without modification in affinity. The present findings demonstrate that sarco(endo)plasmic reticulum proteins involved in intracellular calcium homeostasis are clearly down-regulated and brings further evidence of a modified calcium translocation in denervated rat vas deferens.

The in vitro and in vivo pharmacological activity of Boiga dendrophila (mangrove catsnake) venom.

The great taxonomic and prey base diversity of colubrids (non-front-fanged snakes) suggests that their venoms may represent a 'literal gold mine' for scientists eager to find novel pharmacological probes. While pharmacological characterization is lacking for most of these venoms, this is even more so with regard to activity of colubrid venoms on the mammalian autonomic nervous system. This study characterizes the activity of venom from the colubrid, Boiga dendrophila using in vitro smooth muscle preparations and the anaesthetized rat. In the prostatic segment of the rat vas deferens, cumulative additions of venom (1-150 microg ml(-1)) induced concentration-dependent inhibition of electrically evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches. The inhibitory effect of venom (100 microg ml(-1)) was attenuated by 8-phenyltheophylline (8-PT) (20 microM) and 8-cyclopentyl-1, 3-dipropylxanthine (20 microM) but not idazoxan (1 microM), or a combination of ranitidine (0.2 microM) and thioperamide (10 microM). The inhibitory effect of venom (100 microg ml(-1)) was augmented by dipyridamole (10 microM) but abolished by pretreatment with adenosine deaminase (7.5 units/100 microl) suggesting that it contains components with adenosine A(1) receptor activity, most likely adenosine. In isolated segments of guinea-pig ileum, venom (10-100 microg ml(-1)) caused concentration-dependent contractions which were inhibited by the muscarinic receptor antagonist atropine (0.1 microM) but not by the histamine receptor antagonist mepyramine (0.5 microM). In the anaesthetized rat, venom (5-7.5 mg kg(-1), i.v.) caused a hypotensive effect. Our data suggest that the venom contains components with purinergic and muscarinic receptor activity.

Effects of cooling and ARL 67156 on synaptic ecto-ATPase activity in guinea pig and mouse vas deferens.

We have studied the influence of temperature and ARL 67156 on ATP hydrolysis in mouse and guinea pig vas deferens in order to explore the properties of the enzymatic inactivation mechanism proposed to regulate purinergic neurotransmission at the sympathetic neuromuscular junction of smooth muscle. The ectonucleotidase activity was determined by using the malachite green method to measure the inorganic phosphate (Pi) liberated with ATP used as a substrate. ATP hydrolysis in both species was found to be insensitive to ouabain (100 microM), sodium azide (1 mM), sodium vanadate (100 microM) and beta-glycerophosphate (10 mM) and was also found to depend on Ca2+ and Mg2+. V(MAX) of the ectonucleotidase activity for guinea pig and mouse vas deferens was 958.4+/-66.3 and 79.7+/-8.5 pmol/min/mg, while K(M) was 625.1+/-45.2 and 406.0+/-29.0 microM, respectively. Cooling the tissues from 35 to 25 degrees C reduced the enzyme activity significantly (P<0.01) by 52.7+/-9.2% in guinea pig vas deferens and 34.9+/-5.3% in mouse vas deferens. ARL 67156 (100 microM), the specific ecto-ATPase inhibitor, caused a reduction in enzyme activity in guinea pig and mouse vas of 54.1+/-16.4% and 53.0+/-7.6%, respectively (P<0.01). The degree of inhibition of ATP hydrolysis by lowered temperature and 100 microM ARL 67156 correlates well with the reported potentiation and prolongation of junction potentials under these conditions. It is concluded that ecto-ATPase or a closely related ectonucleotidase plays an important role in the physiological regulation of purinergic neurotransmission.

Transgenic mouse with high Cre recombinase activity in all prostate lobes, seminal vesicle, and ductus deferens.

BACKGROUND: Prostate-specific gene ablation provides a powerful tool for functional characterization of genes that have impact on embryonic development or on other organs, specifically in the prostate. Uniform expression of Cre with high recombinase activity in the prostate is needed for prostate-specific gene ablation based on Cre-loxP recombinations. Currently, available strains of Cre transgenic mice only express Cre recombinase adequately in certain lobes of the prostate. In other lobes, the expression is low and mosaic. Additional strains of transgenic mice expressing high levels of prostate-specific Cre in all prostate lobes would be useful to study the impact of genome manipulation in all prostate lobes. METHODS: The ARR2PB composite promoter with improved capacity to drive androgen-responsive gene expression was used to initiate expression of a transgene bearing the cDNA encoding a recently modified Cre recombinase with improved recombination activity. In addition, an insulator element from the chicken globin locus that minimized negative effect on transcription of the transgene imposed by chromosome structure was employed. The derived transgenic founders were crossed with the Z/AP reporter mouse and Fgfr2(f/f) mice bearing loxP flanking the FGFR2 locus. Immunochemical and mRNA analyses were employed to test expression and efficacy of the Cre recombinase in the prostate and other tissues. RESULTS: The ARR2PBi-Cre transgenic mouse specifically and uniformly expressed Cre recombinase in the dorsal, lateral, ventral, and anterior lobes of the prostate, seminal vesicles, and ductus deferens. The Cre recombinase in these tissues effectively excised loxP flanked DNA fragments in the Z/AP reporter that triggered expression of beta-galactosidase, and the loxP-flanked FGFR2(f/f) locus resulting in specific ablation of FGFR2 in the prostate. CONCLUSIONS: Compared with the currently available prostate-specific Cre strains, the new ARR2PBi-Cre strain exhibited higher and more uniform expression of Cre recombinase in the prostate as well as in seminal vesicles and ductus deferens. This provides an additional tool for efficient hormone-dependent gene targeting in epithelial cells of all lobes of the adult prostate, seminal vesicle, and ductus deferens.

Evidence for trichloroethylene bioactivation and adduct formation in the rat epididymis and efferent ducts.

Recent studies indicate that trichloroethylene (TCE) may be a male reproductive toxicant. It is metabolized by conjugation with glutathione and cytochrome p450-dependent oxidation. Reactive metabolites produced along both pathways are capable of forming protein adducts and are thought to be involved in TCE-induced liver and kidney damage. Similarly, in situ bioactivation of TCE and subsequent binding of metabolites may be one mechanism by which TCE acts as a reproductive toxicant. Cysteine-conjugate beta-lyase (beta-lyase) bioactivates the TCE metabolite dichlorovinyl cysteine (DCVC) to a reactive intermediate that is capable of binding cellular macromolecules. In the present study, Western blot analysis indicated that the soluble form of beta-lyase, but not the mitochondrial form, was present in the epididymis and efferent ducts. Both forms of beta-lyase were detected in the kidney. When rats were dosed with DCVC, no protein adducts were detected in the epididymis or efferent ducts, although adducts were present in the proximal tubule of the kidney. Trichloroethylene can also be metabolized and form protein adducts through a cytochrome p450-mediated pathway. Western blot analysis detected the presence of cytochrome p450 2E1 (CYP2E1) in the efferent ducts. Immunoreactive proteins were localized to efferent duct and corpus epididymis epithelia. Metabolism of TCE was demonstrated in vitro using microsomes prepared from untreated rats. Metabolism was inhibited 77% when efferent duct microsomes were preincubated with an antibody to CYP2E1. Dichloroacetyl adducts were detected in epididymal and efferent duct microsomes exposed in vitro to TCE. Results from the present study indicate that the cytochrome p450-dependent formation of reactive intermediates and the subsequent covalent binding of cellular proteins may be involved in the male reproductive toxicity of TCE.

Ultrastructural localization of phosphoglycerate kinase in adult Clonorchis sinensis.

Phosphoglycerate kinase (PGK) is an enzyme that produces one ATP molecule in the glycolytic pathway. Clonorchis sinensis is largely dependent on glycolysis for energy production. We performed immunoelectron microscopy on adult C. sinensis by using mouse immune serum raised against recombinant C. sinensis PGK. A high density of gold particles was found in the microvilli of the intestinal epithelium and in lamellae of the sperm duct. PGK was common in the somatic cells of intra-uterine eggs and in excreted products. It was localized with moderate intensity in muscular fibers of the subtegumental muscle layer, and in the myoepithelia of the intestine and excretory bladder. We suggest that PGK plays an essential role in C. sinensis energy production for movement via muscle contraction.

Enzyme kinetics and pharmacological characterization of nucleotidases released from the guinea pig isolated vas deferens during nerve stimulation: evidence for a soluble ecto-nucleoside triphosphate diphosphohydrolase-like ATPase and a soluble ecto-5'-nucleotidase-like AMPase.

Previously, we have demonstrated that stimulation of the sympathetic nerves of the guinea pig vas deferens evokes release not only of the cotransmitters ATP and norepinephrine but also of soluble nucleotidases that break down extracellular ATP, ADP, and AMP into adenosine. In this study we show that the apparent K(m) values of the releasable enzyme activity vary depending on which of these adenine nucleotides is used as initial substrate. The K(m) value for ATP was 33.6 +/- 2.3 microM, 21.0 +/- 2.3 microM for ADP, and 10.0 +/- 1.1 microM for AMP. The ratios of the V(max) values for each enzyme reaction were 4:2:3. We have also found a different sensitivity of the metabolism of ATP and AMP by releasable nucleotidases to known nucleotidase inhibitors. Suramin inhibited the breakdown of ATP by releasable nucleotidases in a noncompetitive manner and with a K(i) value of 53 microM, but had no effect on the breakdown of AMP. The 5'-nucleotidase inhibitor alpha,beta-methylene ADP inhibited the breakdown of AMP but not that of ATP. Concanavalin A inhibited the breakdown of AMP but had neither inhibitory nor facilitatory effects on the breakdown of ATP. 6-N,N-Diethyl-beta,gamma-dibromomethylene-D-ATP (ARL67156), an ecto-ATPase inhibitor, suppressed ATPase and AMPase activities, whereas NaN(3) (10 mM) affected neither reaction, but inhibited the ADP metabolism. Phosphatase- and phosphodiesterase inhibitors did not affect the activity of the releasable nucleotidases. This evidence suggests that the soluble nucleotidases released during neurogenic stimulation of the guinea pig vas deferens combine an ecto-5'-nucleotidase-like and an ecto-nucleoside triphosphate diphosphohydrolase-like activity.

Estrogen sulfotransferase: discrete and androgen-dependent expression in the male reproductive tract and demonstration of an in vivo function in the mouse epididymis.

Estrogen sulfotransferase (EST) catalyzes the sulfoconjugation and inactivation of the steroid hormone estrogen. It is known previously that EST is expressed abundantly in Leydig cells of the testis. We recently have shown that male mice with targeted EST gene disruption developed age related Leydig cell and seminiferous tubule abnormalities as a consequence of increased local estrogen stimulation. In the same study, we also found that epididymal sperm isolated from the mutant mice had significantly reduced motility, but whether this reflected impaired epididymal function or was secondary to the testicular lesions was not known. The purpose of the current study was to investigate if EST is normally present in the mouse epididymis and/or other parts of the male reproductive tract where, as in testis, it may play a role in regulating local estrogen homeostasis. We describe here that EST is expressed in the epithelium of corpus and cauda but not caput regions of the mouse epididymis. It is also expressed in the luminal epithelium and smooth muscle cells of the vas deferens but was present at very low levels, if at all, in the prostate or seminal vesicle/ coagulating gland. Hypophysectomy, castration, and epididymal ligation experiments, together with the use of an androgen receptor antagonist, established that EST expression in the epididymis and vas deferens is critically dependent on pituitary hormone(s) and androgen but not on other factors in the testicular fluid. Administration of exogenous estradiol to mice with surgically ligated epididymis resulted in a more pronounced reduction in sperm motility in EST mutant mice than in wild-type mice. We conclude that EST is discretely expressed and regulated in the male reproductive tract and plays a physiological role in maintaining the functional integrity of the epididymis by regulating luminal estrogen homeostasis.

Localization of a proteolytic enzyme within the efferent and deferent duct epithelial cells of the turkey (Meleagris gallopavo) using immunohistochemistry.

Turkey seminal plasma contains a serine protease found to be distinct from the spermatozoal acrosin. However, the origin and biological roles of this enzyme are unknown. Our experimental objective was to identify the cellular source of this protease within the male reproductive tract. The enzyme was isolated from seminal plasma using benzamidine-Sepharose 6B chromatography. A synthetic substrate, Nalpha-benzoyl-DL-arginine p-nitroanilide, was used to detect fractions containing the enzyme. The affinity chromatography technique yielded a 150-fold increase in amidase activity. Analysis of the protease by SDS-PAGE revealed two protein bands with relative molecular masses of 37 000 and 61 000. Proteolytic activity was detected within the smaller band as evidenced by casein digestion. Further analysis of the purified protein revealed that the smaller protein band was glycosylated. To determine the cellular source of the protease, a panel of mouse monoclonal antibodies was then developed against the purified protease, and used in immunohistochemistry. Frozen tissue sections from the liver, testis, epididymal region, and deferent duct were fixed in 4% (w/v) paraformaldehyde, permeabilized with 0.2% (v/v) (octylphenoxy)polyethoxyethanol followed by routine immunohistochemistry procedures. Monoclonal antibodies did not bind to tissue sections from either the liver or testis, or to blood plasma proteins. Both the distal portion of the efferent duct and the deferent duct were immunoreactive. We concluded that the protease found in turkey seminal plasma is concentrated to the distal efferent duct and the deferent duct epithelial cells.

Urodilatin attenuates sympathetic neurotransmission in the rabbit isolated vas deferens without activating guanylyl cyclase.

Natriuretic peptides are produced in cardiovascular, renal and neural tissues and are believed to reduce arterial blood pressure by augmenting sodium and water loss in the urine. Another potential antihypertensive action of these peptides involves a suppression of adrenergic neurotransmission. Atrial, brain and C-type natriuretic peptides suppress sympathetic neurotransmission but no data are available on neuromodulatory actions of urodilatin. This study investigates the hypothesis that urodilatin and brain natriuretic peptide inhibit sympathetic neurotransmission by elevating guanylyl cyclase activity. Both brain natriuretic peptide and urodilatin suppressed force generation in response to electrical stimulation of the vas deferens. Brain natriuretic peptide accelerated the production of cyclic guanosine monophosphate equipotently with its effects on neurotransmission. However, urodilatin failed to increase guanylyl cyclase activity, thus dissociating its effects on neurotransmission from guanylyl cyclase stimulation. None of the natriuretic peptides altered contractile effects of either adenosine triphosphate or norepinephrine, the two putative neurotransmitters secreted from adrenergic nerves in the vas deferens. These data are consistent with the following conclusions: 1) all of the known endogenous natriuretic peptides suppress adrenergic neurotransmission; 2) guanylyl cyclase activation is not required for the inhibition of sympathetic neurotransmission by natriuretic peptides; and 3) inhibitory effects of the natriuretic peptides on neurotransmission result from a suppression of neurotransmitter exocytosis. The novel findings of this study include both the suppression of sympathetic neurotransmission by urodilatin and its biological activity in the absence of guanylyl cyclase activation.

Expression of CPI-17 and myosin phosphatase correlates with Ca(2+) sensitivity of protein kinase C-induced contraction in rabbit smooth muscle.

1. Various smooth muscles have unique contractile characteristics, such as the degree of Ca(2+) sensitivity induced by physiological and pharmacological agents. Here we evaluated six different rabbit smooth muscle tissues for protein kinase C (PKC)-induced Ca(2+) sensitization. We also examined the expression levels of myosin light chain phosphatase (MLCP), the MLCP inhibitor phosphoprotein CPI-17, and the thin filament regulator h-calponin. 2. Immunohistochemical and Western blot analyses indicated that CPI-17 was found primarily in smooth muscle, although expression varied among different tissues. Vascular muscles contained more CPI-17 than visceral muscles, with further distinction existing between tonic and phasic subtypes. For example, the tonic femoral artery possessed approximately 8 times the cellular CPI-17 concentration of the phasic vas deferens. 3. In contrast to CPI-17 expression patterns, phasic muscles contained more MLCP myosin-targeting subunit than tonic tissues. Calponin expression was not statistically different. 4. Addition of phorbol ester to alpha-toxin-permeabilized smooth muscle caused an increase in contraction and phosphorylation of both CPI-17 and myosin light chain (MLC) at submaximal [Ca(2+)]i. These responses were several-fold greater in femoral artery as compared to vas deferens. 5. We conclude that the expression ratio of CPI-17 to MLCP correlates with the Ca(2+) sensitivities of contraction induced by a PKC activator. PKC stimulation of arterial smooth muscle with a high CPI-17 and low MLCP expression generated greater force and MLC phosphorylation than stimulation of visceral muscle with a relatively low CPI-17 and high MLCP content. This implicates CPI-17 inhibition of MLCP as an important component in modulating vascular muscle tone.

Sperm disposal system in spermatic granuloma: a link with superoxide radicals.

Spermatic granulomas are believed to maintain "physiological harmony" in the male reproductive tract by maintaining a balance of hydrostatic pressure post-vasectomy. The mechanism for the disposal of deposited spermatozoa in the granuloma core is not clear. A fourfold rise in the production of superoxide along with ascorbyl and dienyl radicals and a 50% drop in the production of nitric oxide (NO) radicals by granuloma tissue hints that a reaction between NO and superoxide radicals could lead to the formation of peroxynitrite species which may contribute to the disposal of spermatozoa in the granuloma core. A higher protease activity and low hypoxanthine content in the granuloma indicates that a free radical driven sperm disposal system is active in granulomas.

Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts.

Testicular fluid is concentrated and acidified during its passage through the excurrent ducts. These processes involve bicarbonate absorption, in which carbonic anhydrases are implicated. In this study, the distribution of two transmembrane carbonic anhydrase isozymes (CA IX and CA XII) in the human excurrent ducts was investigated using isozyme-specific antibodies in conjunction with immunohistochemical and immunoblotting techniques. Specific staining for CA XII was present in the basolateral plasma membrane of the epithelial cells in the efferent ducts, predominantly in the non-ciliated cells. In the epididymal duct, CA XII was detected only in sporadic cells, which also contained CA II, thus suggesting that they are apical mitochondria-rich cells. CA IX was also localized to the basolateral plasma membrane of the epithelium in the efferent ducts, but its staining was weaker and less uniform compared to CA XII. No signal for CA IX was detected in the epididymal duct. Western blot analysis from efferent duct samples revealed specific bands for CA IX and CA XII, confirming that the immunohistochemical stainings represent these isozymes. The expression of CA XII and CA IX in the excurrent duct system and co-expression of CA XII with Aquaporin-1 in the same efferent duct epithelial cells suggest their functional involvement in ion transport and concentration processes of testicular fluid.

Constant expression of cyclooxygenase-2 gene in prostate and the lower urinary tract of estrogen-treated male rats.

Expression of cyclooxygenase-2 (E. C. 1.14.99.1) in prostate and the lower urinary tract (LUT) of the neonatally estrogenized male rat has been studied by using a COX-2's PCR fragment of 724 nt spanning 3 introns and a 478nt internal standard for quantitative RT-PCR. The same fragment of 724 nt was used for RNA probe in Northern hybridization. Neonatal estrogenization (10 microg/day of diethylstilbestrol on days 1-5) had no effect on COX-2 expression in prostatic urethra, prostatic lobes, or bladder. Acute estrogen treatment of castrated animals did not induce COX-2 expression, either. In addition the differential expression of basal level of COX-2 in the different lobes of prostate in normal rat was demonstrated. Our results suggest a constant expression of COX-2 gene in prostate and the lower urinary tract of the neonatally estrogenized (neoDES) rats. The present study indicates that the increased expression of COX-2 is probably not essential for the estrogen-driven development of stromal inflammation or hyperplastic and dysplastic alterations in the prostate of neoDES rats.

Distribution of the vacuolar H+ atpase along the rat and human male reproductive tract.

Luminal acidification in parts of the male reproductive tract generates an appropriate pH environment in which spermatozoa mature and are stored. The cellular mechanisms of proton (H+) secretion in the epididymis and the proximal vas deferens involve the activity of an apical vacuolar H+ ATPase in specialized cell types, as well as an apical Na+/H+ exchanger in some tubule segments. In this study we used Western blotting and immunocytochemistry to localize the H+ ATPase in various segments of the male reproductive tract in rat and man as a first step toward a more complete understanding of luminal acidification processes in this complex system of tissues. Immunoblotting of isolated total cell membranes indicated a variable amount of H+ ATPase in various segments of the rat reproductive tract. In addition to its known expression in distinct cell types in the epididymis and vas deferens, the H+ ATPase was also localized at the apical pole and in the cytoplasm of epithelial cells in the efferent duct (nonciliated cells), the ampulla of the vas deferens and the ventral prostate (scattered individual cells), the dorsal and lateral prostate, the ampullary gland, the coagulating gland, and all epithelial cells of the prostatic and penile urethra. Both apical and basolateral localization of the protein were found in epithelial cells of the prostatic ducts in the lateral prostate and in periurethral tissue. Only cytoplasmic, mostly perinuclear localization of the H+ ATPase was found in all epithelial cells of the seminal vesicles and in most cells of the ventral prostate and coagulating gland. No staining was detected in the seminiferous tubules, rete testis, and bulbourethral gland. In human tissue, H+ ATPase-rich cells were detected in the epididymis, prostate, and prostatic urethra. We conclude that the vacuolar H+ ATPase is highly expressed in epithelial cells of most segments of the male reproductive tract in rat and man, where it may be involved in H+ secretion and/or intracellular processing of the material endocytosed from the luminal fluid or destined to be secreted by exocytosis.