Sertoli cells, proximal convoluted tubules in the kidney, and neurons in the brain contain cyclic protein-2.

We analyze by immunocytochemistry the in vivo distribution in rat Sertoli cells of Cyclic Protein-2 (CP-2), which is maximally synthesized and secreted in vitro at stages VI and VII of the cycle of the seminiferous epithelium. This analysis demonstrates that CP-2 staining is strongest in Sertoli cells in stage VI and VII tubules. Additionally, we demonstrate that the staining for CP-2 within a stage VII tubule differs from the staining of another Sertoli cell secretory product, androgen-binding protein. CP-2 is not detected by immunocytochemistry in any other tissues of the reproductive tract, though immunoblot analysis demonstrates the presence of CP-2 in rete testis and epididymal fluids. CP-2 was immunocytochemically detected in only three other organs: the kidney, the brain (with greatest concentration in the supraoptic and paraventricular nuclei), and the posterior pituitary. The presence of CP-2 in the kidney was confirmed by metabolic radiolabeling, immunoprecipitation, and peptide analysis. The presence of CP-2 in the brain was confirmed by immunoblot analysis of radioinert protein immunoprecipitated from the anterior hypothalamus.

Tissue and species specificity of mouse ductus deferens protein.

The ductus deferens of the mouse contains a major protein with a molecular weight of 34.5 kd called mouse vas deferens protein (MVDP). Immunofluorescence histochemistry and immunoblotting with monoclonal antibodies have been used to investigate the localization, tissue, and species distribution, androgen-regulation, and developmental expression of this protein. Consistent positive immunoreaction was achieved in the ductus deferens epithelium, and immunofluorescence revealed that spermatozoa from the deferent duct were coated with MVDP. Western blot analysis showed the organ specificity of MVDP, which could not be detected in several organs in the mouse. Furthermore, MVDP appeared to be species specific since the proteins extracted from the ductus deferens of man, rat, guinea-pig, rabbit, and hamster did not react with the anti-MVDP probe. MVDP, whose expression is regulated by testosterone, was detectable at 20 days of age and its concentration increased rapidly from 20-30 days.

Immunofluorescence localization of vinculin in ectoplasmic ("junctional") specializations of rat Sertoli cells.

We have investigated, using indirect immunofluorescence techniques, the possibility that vinculin is a component of Sertoli cell ectoplasmic specializations. Affinity-purified polyclonal antibodies produced against human platelet vinculin were used to probe fixed frozen sections of rat testis. Specific fluorescence occurs in Sertoli cell regions adjacent to spermatids and to basally situated junctional complexes, sites at which ectoplasmic specializations are known to occur. Staining also occurs in Sertoli cell regions associated with tubulobulbar complexes. The antibody also labels focal contacts in cultured human dermal fibroblasts, apical junctional sites of rat epididymal epithelium, and dense plaques of smooth muscle. Our results are consistent with the prediction that vinculin is likely a component of ectoplasmic specializations and are also consistent with the hypothesis that these structures are a form of actin-associated adhesion complex.

Immunolocalization and concentrations of inhibin alpha in the ovine testis and excurrent duct system.

Inhibin was localized in the ovine testis, excurrent ducts, and accessory sex glands by using a rabbit antiserum against a synthetic polypeptide representing the first 30 amino acids of porcine inhibin alpha-subunit. Concentrations of inhibin in fluids entering and leaving the epididymis also were determined in a radioimmunoassay using the same antibody. In the testis, immunostaining of inhibin was conspicuous in the seminiferous epithelium. Leydig cells occasionally were stained and the tunica media of blood vessels always was stained. Intense staining was observed in the epithelia lining the rete testis and ductuli efferentes. Staining also was intense in the epithelium of the initial segment and proximal caput epididymidis, and became less intense along the length of the epididymis. These observations were consistent with concentrations of inhibin in rete testis fluid (8.2 pmol/ml) entering the ductuli efferentes and in cauda epididymal plasma (0.67 pmol/ml) leaving the epididymis. Epithelia of ampullary and vesicular glands and of some prostatic acini were positively stained, but bulbourethral glands were never stained. Adrenal cortex, some proximal convoluted tubules in the kidney, and transitional epithelium of the urethra also were stained. Based on radioimmunoassay data and fluid flow rates for the ram, it was concluded that almost all of the 328 pmol inhibin that enters the ductuli efferentes daily is endocytosed in the proximal parts of the excurrent duct system. The physiological role(s) for inhibin, or inhibin-like peptides, in the excurrent duct system remains speculative.

Distribution of soltriol [1,25(OH)2-vitamin D3] binding sites in male sex organs of the mouse: an autoradiographic study.

After injection of [3H]-1,25(OH)2-vitamin D3 (soltriol), nuclear labeling is found in Sertoli cells of testes, being highest at the stage of spermiosis, in epithelium of efferent ductules and caput epididymidis and in connective tissue cells of epididymis, in lamina propria and muscular sheath of deferent duct, and in epithelium and muscular sheath of dorsal and ventral prostate of the mouse. This labeling pattern is characteristic for [3H]-soltriol and differs from that for [3H]-dihydrotestosterone and [3H]-estradiol, although with overlap. The nuclear labeling with [3H]-soltriol suggests an action of the hormone on certain processes during spermatogenesis, on sperm maturation, on epididymal fluid resorption, and on secretion and transport of spermatozoa.

Ultrastructural and biochemical changes in epididymis and vas deferens of gossypol treated rats.

Adult male Wister rats when administered with 15 mg/kg body weight/day of gossypol acetic acid proved to be sterile by 10 weeks of treatment. The weight of the whole epididymis did not deviate from the controls but when the caput, corpus and cauda epididymidis were considered separately, the cauda epididymidis weight was significantly reduced. The major changes were observed in the motor apparatus of the sperm. The most common defects in the sperm were the vacuolization and complete degeneration of the midpiece mitochondria and plasma membrane. The total LDH activity of caput and cauda epididymidis were within the range of control values. Sialic acid levels of the epididymis were not affected after the treatment. These results suggest a more proximal site of action of the drug than at the epididymal level.

Age-related changes in the concentration of cytosolic androgen receptors in the epididymis, vas deferens and seminal vesicle of maturing male mice.

In this study, changes in the number of androgen binding sites that occur in cytosols of epididymis, vas deferens and seminal vesicle of mice from 10 to 90 days of age are described. Specific saturable binding of [3H]R-1881 by cytosols of the three organs at all time points studied and age-related differences in the number of binding sites measured were observed. Cytosolic androgen receptor levels in all three organs studied were found to decrease with increasing age, regardless of whether the binding was expressed relative to weight of tissue, cytosolic protein or cellular DNA. The most pronounced change in androgen receptor levels (from 442 to 50 fmol/mg protein) was observed in the epididymis between 10 and 30 days of age. In these three organs there was no significant correlation between androgen (testosterone + dihydrotestosterone) levels and the concentration of androgen binding sites.

Ketanserin reduces a particular monoamine pool in peripheral tissues.

The effect of ketanserin and tetrabenazine treatment on monoamine and metabolite levels in central and peripheral tissues was investigated in young and senescent male Wistar and spontaneously hypertensive Okamota rats. Control animals showed significantly higher brain monoamine levels and 3 and 5.5 times higher dopamine levels in the vas deferens of the senescent and hypertensive rats, as compared with young normotensive rats. Ketanserin (20 mg/kg) produced an average of 20% reduction of brain monoamines without changing metabolite levels. In the vas deferens, dopamine was reduced by 85% and norepinephrine by 30%. In cardiovascular tissues, norepinephrine was 40% to 50% decreased and in the spleen norepinephrine was 60% and 5-hydroxytryptamine 30% reduced. Ketanserin (5 mg/kg) had only a marked effect on dopamine in the vas deferens and on norepinephrine in the portal vein. Tetrabenazine at 20 mg/kg produced complete depletion of the monoamine and 3-methoxytyramine levels in the brain with a concomitant rise in acid metabolites. In peripheral tissues, amine levels were reduced by 55% to 80%; dopamine in the vas deferens was 93% decreased. Tetrabenazine (5 mg/kg) still had marked effects in all tissues. The drug effects were the same in the three types of rats and the effects did not markedly change with chronic treatment up to 20 days. It is hypothesized that at least two different mechanisms are involved in monoamine depletion, 1) the classically proposed inhibition of uptake of monoamines in the storage vesicles, a property of tetrabenazine not shared by ketanserin in vivo and 2) triggering of the release of monoamines from a ketanserin-sensitive pool, which is relatively more important in peripheral tissues than in the brain. The latter process is probably mediated by previously identified ketanserin-binding release sites on nerve terminals and platelets. The ketanserin-sensitive monoamine pools in peripheral tissues may have a role in cardiovascular pathologies.

Effect of progesterone on the stimulation of phosphatidylinositol turnover by epinephrine in guinea pig ductus deferens.

Progesterone enhanced the contractile effect of epinephrine on the ductus deferens of the guinea pig in vitro. In relation to this mechanical phenomenon, we examined the phosphatidylinositol metabolism. In the 3H-myoinositol labeled ductus deferens, radioactivity in phosphatidylinositol bisphosphate was about 2.6 times as high as that in phosphatidylinositol. Phosphatidylinositol and phosphatidylinositol phosphate were not changed by epinephrine (100 microM), but phosphatidylinositol bisphosphate was increased at 10 sec and 1 min after the administration of epinephrine (100 microM). Progesterone (100 microM) added 5 min before the administration of epinephrine increased the stimulatory effect of epinephrine on the phosphatidylinositol bisphosphate metabolism, but had no effect on the phosphatidylinositol and phosphatidylinositol phosphate metabolism. These studies suggest that progesterone expresses its activity not through the cytoplasmic progesterone receptor but through the epinephrine mediated smooth-muscle contractile mechanism.

Influence of long and short photoperiods on the morphology and androgen receptor levels of the epididymis and the ductus deferens of Phodopus sungorus.

The photoperiodic influence (LD 16:8 long photoperiod, LD 8:16 short photoperiod) on the morphology and the androgen receptor level of the epididymis and the ductus deferens of Phodopus sungorus was investigated. Under short day-conditions, the wet weight of the epididymis is reduced to 5-6%, the diameter of the epididymal duct and of its lumen are reduced, the height of the epithelium and the thickness of the smooth muscle layer are increased. Number and size of epithelial and smooth muscle cells are not changed, no atrophy of the smooth muscle cells is found. The loss in wet weight during short photoperiods is discussed in relation to the loss of stored sperm and luminal fluid. The wet weight of the ductus deferens is decreased to about 30% at short photoperiods, the total length of the organ, its diameter and the luminal diameter are decreased. The height of the epithelium is slightly, and the thickness of the smooth muscle layer is strongly, reduced, the latter due to an enormous atrophy of the smooth muscle cells. The androgen receptor content of the epididymis (per pair of organs) is reduced to about 5%, that of the ductus deferens to about 10% of the values found at long photoperiods, indicating a significant loss of androgen receptors with low circulating androgen levels.

Distribution of the murine plasma cell antigen PC-1 in non-lymphoid tissues.

PC-1 is an alloantigen of murine plasma cells. Its close association with secretory function in lymphoid cells previously raised the question of whether PC-1 was part of the secretory apparatus. In addition to its expression on lymphocytes, PC-1 had been known to be present in liver, brain, and kidney, although the data were derived almost entirely from bulk absorption studies of polyclonal alloantisera, and virtually nothing was known about the nature of the cells expressing PC-1 in these organs. If PC-1 was functionally involved in the secretory process. it might be expected to be present at secretory sites within these and other organs. We now report the results of an immunohistochemical survey of the distribution of PC-1 in a variety of non-lymphoid organs, using a mAb. The PC-1 Ag was found in a small number of highly discrete locations that were mostly, but not exclusively, associated with epithelia. Sites of strong expression included the distal convoluted tubule of the kidney, ducts of the salivary glands, epididymis, proximal part of the vas deferens, and chondrocytes. The PC-1 glycoprotein was also found in the capillaries of the brain, but did not appear to be present in capillaries elsewhere, a pattern that is strikingly similar to that of the receptor for the iron transport protein, transferrin. Negative sites included the thyroid, pancreas, choroid plexus, smooth and striated muscle, stomach, small and large intestine, gall bladder, renal glomeruli, testis, and seminal vesicles. These results are not consistent with a generalized role for PC-1 in secretion, but are compatible with a role in a specialized subset of macromolecular transport events.

Testosterone concentrations in blood and seminal plasma of turkeys classified as high or low semen producers.

The interrelationships between concentrations of testosterone in blood plasma, seminal plasma (SP) of ductus (d.) deferens semen, and SP of the ejaculate of mature breeder turkeys were compared. The concentration of testosterone in blood plasma was greater than, and positively correlated with (r = .75, P less than .01), the concentration of testosterone in the ejaculate SP and d. deferens SP. Differences between concentrations of testosterone in blood plasma, ejaculate SP, and d. deferens SP were not significantly different for turkeys classified as high (ejaculate volume greater than .38 mL) or low (ejaculate volume less than .26 mL) volume semen producers. Concentrations of testosterone in blood plasma and SP were not correlated with ejaculate spermatozoal concentration, total number of spermatozoa, d. deferens semen volume, or testicular weights.

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.

The physiological relevance of low agonist affinity binding at opioid mu-receptors.

1. Inhibition constant (Ki) were determined for a range of opioid standards using two binding assays; [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]-GLYOL) binding to guinea-pig brain membranes in HEPES buffer and [3H]-naloxone binding to rat whole brain membranes in Krebs/HEPES buffer. 2. These values were compared with affinity measurements determined by antagonism of GLYOL on the rat isolated vas deferens preparation and by the receptor occlusion technique of Furchgott on the guinea-pig ileum longitudinal muscle, myenteric plexus preparation. 3. Agonists demonstrated markedly reduced binding affinity in the [3H]-naloxone binding assay where binding was conducted in the presence of sodium. 4. A strong correlation was obtained between Ki values from the [3H]-naloxone binding assay and affinity values determined in both isolated tissue preparations. Ki values obtained from [3H]-GLYOL binding did not correlate well with affinity data determined by isolated tissue techniques. 5. These findings suggest that functionally relevant receptors exhibit low agonist affinity.

Endocrine cells in the excurrent duct system of the testis. An immunohistochemical analysis.

A systematic search for endocrine cells in the excurrent duct system of the testis was carried out by means of histochemical and immunohistochemical techniques. A panel of antibodies against amine and polypeptide hormones was used. 80 specimens comprising representative areas of rete testis, ductuli efferentes, ductus epididymis and 30 examples of ductus deferens were investigated. Cells immunoreactive for serotonin were detected in four out of 110 specimens. They were invariably in normal-appearing ductuli efferentes. A salient feature was their rarity and focal distribution. We failed to detect any endocrine cells in other segments of the excurrent duct system and notably not among epididymal epithelium. It seems of interest that serotonin cells are specifically distributed throughout remnants of excretory mesonephric tubules in both males and females.

Structure of the complex basement membrane underlying the epithelium of the vas deferens in the rat.

Underlying the epithelium of the vas deferens there is a complex basement membrane showing a thick lamina densa separated from the plasma membrane of epithelial cells by a lamina lucida. On the connective tissue side of the lamina densa, there are plaques composed of a material that is similar to that of the lamina densa but is more compact and has a greater electron density. This material also forms plaques at a short distance from the lamina densa, where it appears as irregular nodular masses. The plaques are bridged by striated anchoring fibrils (SAF) that are variable in structure. Some SAF are long (0.5-0.6 micron) and bilaterally symmetrical, with a central fusiform segment and, on each side, coarsely banded segments. While the fusiform segment presents 5 or 6 diffuse cross striations, the coarsely banded segments show distinct bands labeled B1-B4. Shorter SAF show a coarsely banded segment alone or a coarsely banded segment plus a fusiform segment. Some SAF also branch at the level of the fusiform segments, in which case they form star-shaped structures with three or more branches that have their extremities inserted into plaques. The plaques, as well as the lamina densa, are immunohistochemically reactive to type IV collagen, laminin, and heparan sulfate proteoglycan, whereas the SAF are not immunoreactive to these substances. SAF and plaques, considered as integral components of this basement membrane, form a series of arches or open tunnels traversed by collagen fibrils. It is thus apparent that these elements contribute to the attachment of the basement membrane and the overlying epithelium to the underlying dense connective tissue of the lamina propria.

General toxicity and peripheral nerve alterations induced by chronic vincristine treatment in the rabbit.

The effects of five 0.3 mg/kg intravenous administrations of vincristine (VCR) at weekly intervals were studied in the rabbit. Body weight gain was impaired starting from the first injection, while gross signs of motor paralysis and hair loss initiated from the third week. At the end of the observation period blood analysis revealed normocytic normochromic anemia, elevated serum creatine kinase, and low serum alkaline phosphatase, whereas all the tested parameters related to liver and kidney functions where within normal limits. The decreased number of red blood cells was the consequence of a complete, although reversible, blockade of staminal hematopoietic activity. Two important indexes of peripheral nerve function were clearly altered at the end of the treatment: (i) the sciatic nerve conduction velocity in vitro was 27% reduced and (ii) the latency between sciatic nerve stimulation and extensor digitorum longus (EDL) twitch in vivo was 34% prolonged. The usefulness of the rabbit as an animal model to study side-effects of VCR treatment is discussed.

A possible crossed histamine-containing pathway adjacent to the sympathetic system of the rat vas deferens.

Studies using nerve degeneration techniques (ganglionectomy, interganglionic section, postganglionic axotomy, uni- or bilateral hypogastric nerve section and right pelvic ganglionectomy) and fluorometric determinations of histamine and norepinephrine have shown the presence of nervous pathways containing histamine adjacent to the sympathetic system of the rat vas deferens. The findings suggest that these pathways cross between the ganglionic clusters located at the angle formed by the seminal vesicle and the vas deferens. They are not structurally related to the central nervous system by way of the hypogastric or pelvic ganglion. The histamine-containing pathways are independent of the noradrenergic pathways as dissociation between norepinephrine depletion and histamine depletion can be shown under nerve degeneration. The time course of nerve degeneration over a long period after sympathectomy shows a biphasic effect on histamine levels of the vas deferens. The early histamine depletion would be indicative of degeneration of histamine-containing pathways, and the delayed histamine increasing phase has been considered as due to accumulation of mast cells in the degenerating nerve sheaths. A possible role for the histamine-containing pathways in the modulation of sympathetic activity is envisaged.

Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes.

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.

Intersex mice composed of androgen insensitive Tfm and wild-type cells analysed by 3H dihydrotestosterone autoradiography.

The X-linked testicular feminization mutation (Tfm) in the mouse is characterized by an androgen receptor defect. Due to random X-chromosome inactivation, XTfm/X+ heterozygotes are mosaics with respect to Tfm. They are composed of androgen receptor deficient XTfm cells and normal X+ wild-type cells. If Tfm heterozygotes are converted to XX males by the sex reversal factor (Sxr) the mosaicism is expressed. Therefore in sex reversed Tfm heterozygotes (XTfm/X+-Sxr) intersexual sex organs develop. In five intersexes with small male accessory glands and hypospadia and one heavily feminized intersex with vagina and caudally dislocated deferent ducts the mosaic is visualized by 3H-DHT-autoradiography. In the epididymis differentiated wild-type cells show nuclear labeling, whereas undifferentiated Tfm cells are unlabeled. Unlabeled Tfm cells are also encountered in the vesicular glands of the heavily feminized animal, demonstrating that Tfm cells can participate in the formation of male sex glands. The urethral glands of the mosaic animals are composed of unlabeled Tfm lobules exhibiting the female phenotype of the glands, and of labeled wild-type lobules exhibiting the male phenotype. Formation of a vagina and deviation of the deferent ducts is correlated with lack of androgen binding sites in the connective tissue.