Preliminary characterization, androgen-dependence and ontogeny of an abundant protein from mouse vas deferens

Polyacrylamide gel electrophoresis analysis revealed that the vas deferens of adult mouse contains a major protein. Mouse vas deferens protein is a basic glycoprotein with a molecular weight of 34,800 +/- 300. The protein represents 17 +/- 0.7% and 42 +/- 2.4% of soluble proteins from homogenate and luminal fluid respectively, an estimate based on densitometric scanning of polyacrylamide gels. The protein originated from the vas deferens since it was not detected in blood plasma or in sexual organs and it was still present after ligation of the epididymis. Changes in androgen status of the animal markedly affected the vas deferens protein. After castration a progressive decrease in the protein was observed and its relative percentage dropped to 2 +/- 0.4% after 45 days. The concentration of the protein returned to precastration levels after 2 weeks of testosterone treatment but oestradiol, progesterone and corticosterone were ineffective in this respect. The vas deferens protein was not synthesized in significant amounts until animals were 20 days old and its concentration increased rapidly from 20 to 30 days in concert with the pubertal increase of androgens in the vas deferens.     

NORADRENALINE METABOLIZING ENZYMES IN NORMAL AND SYMPATHETICALLY DENERVATED VAS DEFERENS

—A surgical technique for sympathetically denervating the vas deferens has been evaluated biochemically. A slight fall in soluble muscle protein content and no significant change in DNA content of the operated vas deferens were found. This indicates that the surgical procedure causes only a slight degree of tissue damage and may be useful for investigating the cellular localization and properties of noradrenaline metabolizing enzymes. In three species examined (rat, guinea pig and rabbit), monoamine oxidase activity of the vas deferens fell by approximately 50 per cent after denervation. The time course of the fall in monoamine oxidase activity of rat vas deferens was parallel to that of the disappearance of noradrenaline suggesting that this proportion of the total enzyme activity had a neuronal localization. The remaining enzyme activity is presumably located extraneuronally. Significant falls in catechol-O-methyl transferase activity were found in rat and rabbit vas deferens after denervation but not in guinea pig. The rabbit and rat vas deferens had respectively approximately 60 and 30 per cent of the catechol-O-methyl transferase activity associated with the sympathetic nerves. A complete loss of DOPA decarboxylase and tyrosine hydroxylase activities occurred in rat vas deferens after denervation, suggesting that these noradrenaline synthesizing enzymes have an entirely neuronal localization.     

Effects of streptozotocin-induced diabetes and insulin on calcium responsiveness of the rat vas deferens

In the present study, effects of streptozotocin-induced diabetes and insulin treatment on the reactivity of rat vas deferens to KCl and calmidazolium, a calmodulin antagonist, were evaluated and calmodulin levels in vas deferens tissue from diabetic and insulin-treated rats were determined. Diabetes was induced in rats by a single injection of streptozotocin. Five weeks after the induction of diabetes, one group of diabetic rats was injected with insulin for 3 weeks. After 8 weeks, vas deferens tissues on one side of diabetic and insulin-treated diabetic rats and their controls were mounted in organ bath to measure isometric tension, while the tissues on the other side of rats were homogenized to determine calmodulin levels by radioimmunoassay. Concentration-response curves to KCl were obtained in vas deferens tissues in the absence and presence of calmidazolium. The effects of KCl and calmidazolium on vas deferens isolated from 8-weeks diabetic rats were decreased. Calmodulin levels were also found to be decreased in vas deferens from diabetic rats. Decreased calmodulin levels in diabetic rat vas deferens were not corrected by insulin treatment. Only a partial correction following insulin treatment was observed in contractile effect of KCl on diabetic rat vas deferens, whereas insulin treatment increases the affinity of calmodulin in this muscle. Experimental diabetes causes an impairment in calcium/calmodulin-dependent contractile process of vas deferens, which is correctable partially following insulin therapy. The changes in the function of rat vas deferens due to streptozotocin diabetes seem to be related to impaired sexual functions in human diabetes.     

Effect of L-prolyl-L-leucyl-glycinamide (MIF-I) on some neutrotransmitter-receptor interactions

Some neurotransmitter-receptor interactions have been studied in an attempt to determine how L-prolyl-L-leucyl-glycinamide (MIF-I) exerts its antiparkinson effect. MIF-I affected neither the contractile responses of isolated mouse vas deferens and guinea pig ileum to noradrenaline, acetylcholine, substance P and histamine, nor the inhibitory effects of dopamine and GABA on the rat vas deferens and guinea pig ileum. MIF-I, as well as L-leucine and Pro-Leu, antagonized the contractile response of the ileum to 5-hydroxytryptamine (5-HT). Behavioural tests were used to examine the action of MIF-I on CNS transmitter-receptor interactions. MIF-I did not modify the circling produced by either dopamine agonists in nigro-striatal lesioned rats of 5-HT agonists in rats with a lesion of the medial raphe nucleus. MIF-I affected neither 5-hydroxytryptophan-induced head twitches in mice, which is a measure of 5-HT receptor stimulation, nor striatally-evoked head turning in the rat, which is a model for brain GABA function. It is concluded that MIF-I, at the doses used, does not directly modify the function of any of the CNS transmitter examined. Other possibilities to explain its antiparkinson action are discussed.     

A Review of Transplantation Practice of the Urologic Organs: Is It Only Achievable for the Kidney?

Transplantation is a viable treatment option for failure of most major organs. Within urology, transplantation of the kidney and ureter are well documented; however, evidence supporting transplantation of other urologic organs is limited. Failure of these organs carries significant morbidity, and transplantation may have a role in management. This article reviews the knowledge, research, and literature surrounding transplantation of each of the urologic organs. Transplantation of the penis, testicle, urethra, vas deferens, and bladder is discussed. Transplantation attempts have been made individually with each of these organs. Penile transplantation has only been performed once in a human. Testicular transplantation research was intertwined with unethical lucrative pursuits. Interest in urethra, bladder, and vas deferens transplantation has decreased as a result of successful surgical reconstructive techniques. Despite years of effort, transplantations of the penis, testicle, urethra, vas deferens, and bladder are not established in current practice. Recent research has shifted toward techniques of reconstruction, tissue engineering, and regenerative medicine.Key words: Urology, Transplantation, Reconstruction, Tissue engineeringOrgan transplantation is still a relatively novel treatment modality; its practice only developed in the latter half of the 20th century, in part due to the advent of immunosuppressive drugs. Key questions driving progress in transplant surgery is how far this science can be pushed, and if any part of the human body can be transplanted.In urology, transplantation of the kidney was previously inconceivable, but is now well established, following the first successful renal transplant performed in 1950. However, there is minimal evidence in the scientific literature of the successful transplantation of other urologic organs.There has, in the past, been great interest in exploring this lesser-known area of urologic transplantation, as the morbidity for patients with failure or damage to urologic organs such as the penis, testicle, or bladder is considerable. There is renewed interest in recent years due to the increased prevalence of devastating injuries to the genitalia caused by improvised explosive devices among soldiers stationed in Afghanistan.¹ There is minimal evidence in the literature addressing how these injuries should be best managed or whether transplantation may have a role in helping such patients.Remarkable progress in transplantation surgery has allowed development of new strategies of treatment for many urologic patients. Greater insight into the practice of transplantation may lead to improved management of other urologic pathologies. We comprehensively reviewed the historical evidence for transplantation of those urologic structures for which the practice is not well established. To our knowledge, no such review exists in the literature.     

3H-noradrenaline metabolism in the isolated epididymal and prostatic portions of the rat vas deferens

The spontaneous ³H-noradrenaline efflux from the isolated epididymal and prostatic portions of the rat vas deferens, was investigated. The spontaneous tritium efflux was higher in the epididymal portion than in the prostatic one from normal animals. Such differences were abolished after castration. On the other hand, acetylsalicylic acid enhanced the total tritium efflux only in the epididymal portion, whereas phentolamine and phenoxybenzamine increased the total tritium efflux from the two portions of the rat vas deferens in both experimental groups. The prostatic portion released a similar amount of ³H normetanephrine than the apididymal end, whereas other metabolic fractions (3,4-dihydroxyphenylglycol; 3,4-dihydroxymandelic acid and O-methylated deaminated metabolites) were smaller in the prostatic portion in comparison with the epididymal portion from control vas deferens. The results presented in the isolated rat vas deferens suggest the existence of a prostaglandin as well as an alpha adrenoceptive modulation of the spontaneous total tritium efflux.     

Do endogenous prostaglandins modulate noradrenergic transmission in the rat isolated perfused vas deferens?

The effects of the prostaglandin synthetase inhibitors aspirin, indomethacin and meclofenamic acid have been studied on the response of the rat isolated perfused vas deferens. None of these drugs, up to a concentration of 5 x 10(-5) M affected either phase of the biphasic constrictor response to 30 s periods of field stimulation. In the same preparations an inhibition of the response to field stimulation was seen in the presence of prostaglandin E1 at concentrations of 1 to 5 ng ml-1. All three prostaglandin synthetase inhibitors, at 5 x 10(-5) M, caused significant reduction of prostaglandin biosynthesis by homogenates of rat vas deferens. It is, therefore, suggested that stimuli which activate directly the noradrenergic nerves in the rat vas do not activate simultaneously a release of endogenous prostaglandins.     

Purification and characterization of the inositol 1,4,5-trisphosphate receptor protein from rat vas deferens

Among rat peripheral tissues examined, Ins(1,4,5)P(3) receptor binding is highest in the vas deferens, with levels about 25% of those of the cerebellum. We have purified the InsP(3) receptor binding protein from rat vas deferens membranes 600-fold. The purified protein displays a single 260 kDa band on SDS/PAGE, and the native protein has an apparent molecular mass of 1000 kDa, the same as in cerebellum. The inositol phosphate specificity, pH-dependence and influence of various reagents are the same for purified vas deferens and cerebellar receptors. Whereas particulate InsP(3) binding in cerebellum is potently inhibited by Ca(2+), particulate and purified vas deferens receptor binding of InsP(3) is not influenced by Ca(2+). Vas deferens appears to lack calmedin activity, but the InsP(3) receptor is sensitive to Ca(2+) inhibition conferred by brain calmedin. The vas deferens may prove to be a valuable tissue for characterizing functional aspects of InsP(3) receptors.     

Species-specific expression of microsomal prostaglandin E synthase-1 and cyclooxygenases in male monkey reproductive organs

We investigated the tissue distribution and cellular localization of microsomal PGE synthase-1 (mPGES-1) and cyclooxygenase (COX)-1 and -2 in male monkey reproductive organs. Western blotting revealed that monkey mPGES-1 was expressed most intensely in the seminal vesicles, moderately in the testis, and weakly in the epididymis and vas deferens. The tissue distribution profile was quite different from those profiles for rats, rabbits, and pigs, e.g., rat mPGES-1 was the most abundant in the vas deferens, and the rabbit and pig enzymes, in the testis. Immunohistochemical staining with mouse monoclonal anti-human mPGES-1 antibody revealed that monkey mPGES-1 was localized in spermatogonia, Sertoli cells, and primary spermatocytes of testis and in epithelial cells of the epididymis, vas deferens, and seminal vesicles. In monkeys, COX-1 was localized in epithelial cells of the epididymis and vas deferens, whereas COX-2 was dominantly found in epithelial cells of the seminal vesicles.     

Differential contractile response of normal vas deferens of rodents in correlation to their calcium and electrolyte levels

The contractile pattern of the vas deferens in three different rodents, rat, guinea pig and mouse was studied in response to adrenaline and noradrenaline. The left vas deferens of rat was more responsive to the graded doses of adrenaline and noradrenaline than the right. The same was also true for guinea pig and mouse vas deferens. This differential response has been correlated with the greater concentrations of calcium and sodium in the right vas deferens in rats and guinea pigs and it might also be related to the levels of membrane-bound and intracellular calmodulin-bound calcium. It is suggested that the left vas deferens might possess more calmodulin-bound calcium than the right, which might have instead, more membrane-bound calcium.     

Effect of mating on sperm distribution in the reproductive tract of the male rat

Extragonadal sperm reserves in male rats were measured in different regions of the genital tract before and subsequent to normal ejaculation. In sexually rested rats, the sperm count (million spermatozoa for the paired organs) in different regions was: distal vas, 18; proximal vas, 9.8; cauda epididymidis, 229; caput + corpus epididymidis, 154. Following mating, the sperm count was reduced in the proximal and distal vas deferens and in the cauda epididymidis. The reproductive tract of mated females was found to contain 29% (no copulatory plug) or 59% (with copulatory plug) of the estimated mean ejaculate, which was estimated from the difference between the sperm counts in the sexually rested rat and following ejaculation. It is concluded that in the rat the immediate source of spermatozoa for ejaculation is the cauda epididymidis, with a smaller contribution arising from the vas deferens.     

Carbonyl reductases from rat testis and vas deferens. Purification, properties and localization

Three enzyme forms (T1, T2, T3) from rat testis and two from rat vas deferens (V1, V2) of carbonyl reductase have been highly purified to apparent homogeneity. These carbonyl reductases from rat reproductive organs have several similarities in terms of molecular mass (32-33 kDa), isoelectric point (pI 5.9-6.4), immunochemical properties, cofactor requirement (NADPH dependency) and sensitivity to sulfhydryl reagents. The isoenzymes from the vas deferens (V1, V2) have similar catalytic activities, whereas those from the testis (T1, T2, T3) showed different catalytic activities from each other. All enzymes, however, reduced quinones, aromatic aldehydes and ketones, while T3, V1 and V2 were characterized as possessing high affinity towards prostaglandins. An immunoinhibition study using a specific antibody indicated that these enzymes were solely responsible for the overall catalytic activities of 13, 14-dihydro-15-oxo-prostaglandin F2 alpha, 4-benzoylpyridine, and 4-nitroacetophenone reduction and prostaglandin F2 alpha oxidation in both testis and vas deferens cytosol. The immunohistochemical staining revealed a positive immunoreactivity to antibody only in the Leydig cells of the testis, but neither the germ cells nor Sertoli cells in the seminiferous tubule. The staining also showed that the enzymes in the vas deferens were primarily localized in mucosal epithelium cells.     

An endogenous brain substance, CDS (clonidine-displacing-substance), inhibits the twitch response of rat vas deferens

The effect of CDS, an endogenous brain substance that specifically displaces bound [3H]clonidine and [3H]rauwolscine in rat brain membranes and human platelets, has been tested in isolated, field-stimulated rat vas deferens. CDS, obtained after an extensive purification procedure as a single peak from an HPLC sizing column, inhibited the electrically stimulated rat vas deferens similarly to the inhibitory action of clonidine, an alpha 2-agonist. The effective dose of CDS as an inhibitor of the vas deferens is equivalent to its effective dose in displacing specifically bound [3H]-clonidine in rat brain membranes. Furthermore, the CDS inhibition of the twitch response is reversed by two alpha 2-adrenergic antagonists, yohimbine and phentolamine. From these results, it is suggested that CDS extracted from brain, with affinity for clonidine sites, may be involved in the nonadrenergic fast response of the sympathetic transmission of the vas deferens.     

Morphological and biochemical changes in supersensitive smooth muscle.

The effect of postganglionic denervation on the incidence of nexal contacts in the smooth muscle of the rat vas deferens was investigated. The chronically denervated tissue exhibited twice as many nexuses as control. This increase in the incidence of cell contacts may contribute to the supersensitivity and/or the increase in maximum response of the denervated vas deferens. The effects of denervation, decentralization, and pretreatment with reserpine on the concentration of adenosine triphosphate (ATP) in vasa deferentia of rats and guinea pigs were also investigated. One day after denervation there was a substantial decrease in the endogenous norepinephrine and ATP concentrations. The norepinephrine concentration remained low (less than 10% of control) throughout subsequent days (up to 14 days) whereas the ATP concentration, after the first postoperative day, rose significantly. The rise in ATP concentration was temporally correlated with the development of postjunctional supersensitivity. Decentralization and pretreatment with reserpine both resulted in a significant increase in ATP concentration which preceded by 2 to 3 days a significant increase in sensitivity of the vas deferens. It appears that a change in the tissue concentration of ATP may be one of the initial events that occurs following interruption of the neural contact to the smooth muscle of the vas deferens.     

Testosterone stimulates prostanoid production by rat vas deferens.

The rat isolated vas deferens produces and releases prostanoids into an incubation medium. Production of these substances from the exogenous precursor 14C arachidonic acid was studied in prepubertal, pubertal and adult animals. Synthesis of prostaglandin F, prostaglandin E, prostaglandin D and thromboxane B2 is lower in prepubertals arid increases significantly in pubertals, with no further modifications in adults. Castration of pubertals and adults dramatically reduces the production of all measured arachidonic acid metabolites but does not modify it in prepubertals. Replacement therapy with testosterone propionate significantly enhances prostanoid production in pubertal and adult castrated rats. Similar treatment on normal prepubertals also increases synthesis, indicating that androgens could be modulators of prostanoid synthesis in vas deferens. The lower effects obtained treating castrated adults with progesterone and with 17-beta estradiol suggest an action, at least partially specific for androgenic steroids. It is concluded that prostanoid production by the rat vas deferens from an exogenous precursor is closely related to the presence of androgens.     

Effect of NO donors on protein phosphorylation in intact vascular and nonvascular smooth muscles

It is generally well accepted that nitrovasodilator-induced relaxation of vascular smooth muscle involves elevation of cGMP and activation of a specific cGMP-dependent protein kinase [protein kinase G (PKG)]. However, the protein targets of PKG and the underlying mechanisms by which this kinase leads to a relaxant response have not been elucidated. Several types of smooth muscle, including rat myometrium and vas deferens, are not relaxed by sodium nitroprusside, even at concentrations that produce marked elevation of cGMP and activation of PKG. The main objective of our studies was to compare PKG-mediated protein phosphorylation in intact rat aorta, rat myometrium, and rat vas deferens using two-dimensional gel electrophoresis. In intact rat aorta, seven PKG substrates were detected during relaxation of the tissue. None of the PKG substrates identified in the rat aorta appeared to be phosphorylated in the myometrium or vas deferens after administration of various cGMP-elevating agents. Thus the failure of the rat myometrium and rat vas deferens to relax in the face of cGMP elevation and PKG activation may be due to a lack of PKG substrate phosphorylation.     

Beta-endorphin: peripheral opioid activity of homologues from six species

The peripheral opioid activity of six homologous beta-endorphins (beta-EPs) were assayed on the guinea pig ileum and the vas deferens of the mouse, the rat and the rabbit. In the guinea pig ileum assay, human beta-EP (beta h-EP) was less potent than camel, turkey, and ostrich beta-EPs, of the same potency as equine beta-EP and more active than des-acetyl salmon beta-EP. In the rat vas deferens, mammalian beta-EPs showed higher activity than those from the bird and the fish, whereas in the mouse vas deferens assay, beta h-EP is more active than those from other species. In the rabbit vas deferens, however, all homologous beta-EPs show very weak activity. The relative potency of beta-EP homologues obtained from rat vas deferens assay is in good correlation with the analgesic potency, while the receptor binding activity does not correlate with any of the four bioassays, but appears to be related to the charge properties of the peptides.