Androgen dependence during development of the mouse vas deferens protein mRNA.

The mRNA encoding a major protein of the mouse vas deferens (MVDP) was first detected in 10-day-old males and its concentration increased sharply between 10 and 20 days, reaching adult levels at 40 days. This increase was not associated with an increase in tissular androgen concentrations. In 30-day-old mice castrated at birth or treated with cyproterone acetate over 29 days, MVDP mRNA levels were not abolished and were similar to those measured in 10- and 20-day-old controls. These results suggest that the neonatal expression of MVDP gene is independent of androgens. In addition, precocious accumulation of MVDP mRNA could be induced by injection of excess amounts of androgens in 20- but not in 10-day-old animals. The prepubertal increase in MVDP mRNA levels is androgen-dependent but other factors may be necessary for MVDP expression.     

Binding of [3H]dihydroazapetine to alpha-adrenoreceptor-related proteins from rat vas deferens.

The potent alpha-adrenoreceptor blocking agent, azapetine, has been catalytically reduced with tritium gas to form [3H]dihydroazapetine. [3H]Dihydroazapetine retains significant ability to block alpha-adrenoreceptors and has been used as a ligand to study the receptor in a subcellular fraction containing membrane fragments from rat vas deferens. Specific binding of [3H]dihydroazapetine rapidly reaches equilibrium and is also reversible and saturable with a dissociation constant similar to that determined pharmacologically. The binding capacity is approximately 40 pmol/mg of protein. All alpha-adrenergic blockers tested were able to inhibit specific binding. High concentrations of alprenolol, atropine, or chlorpheniramine had no effect. In addition, all alpha-adrenergic agonists of the imidazoline class inhibit binding in low concentrations, whereas soterenol or carbamylcholine did not. There is good correlation (r=0.84) between blockade or stimulation of the receptor in intact tissues and inhibition of binding of [3H]dihydroazapetine to the subcellular fraction. These findings suggest that the fraction contains alpha-adrenoreceptor-related proteins. Alpha-adrenergic agonists structurally related to norepinephrine caused a stereoselective increase in binding in favor of the (-)-isomer, possibly reflecting an allosteric interaction at a different binding site on the receptor protein. The possibility of two different modes of binding for structurally dissimilar agonists is suggested.     

Proteomic analysis of androgen-regulated protein expression in a mouse fetal vas deferens cell line

During sex differentiation, androgens are essential for development of the male genital tract. The Wolffian duct is an androgen-sensitive target tissue that develops into the epididymis, vas deferens, and seminal vesicle. The present study aimed to identify androgen-regulated proteins that are involved in development of Wolffian duct-derived structures. We have used male mouse embryos transgenic for temperature-sensitive simian virus 40 large tumor antigen at 18 d of gestation, to generate immortalized mouse fetal vas deferens (MFVD) parental and clonal cell lines. The MFVD parental and clonal cell lines express androgen receptor protein and show features of Wolffian duct mesenchymal cells. Clonal cell line MFVD A6 was selected for proteomic analysis and cultured in the absence or presence of androgens. Subsequently, two-dimensional gel electrophoresis was performed on total cell lysates. Differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and two androgen-regulated proteins were identified as mElfin and CArG-binding factor-A (CBF-A). CBF-A and mElfin are known to bind to cytoskeletal F-actin. Both proteins appeared to be regulated by androgens at the posttranslational level, possibly involving phosphorylation. Posttranslational modification of mElfin and CBF-A by androgens may be associated with a cytoskeletal change that is involved in androgen-regulated gene expression.     

Androgen-estrogen synergy in the regulation of energy metabolism in epididymis and vas deferens of rhesus monkey.

The possible physiological role of estrogen in the regulation of energy metabolism of epididymis and vas deferens of rhesus monkey was investigated. A few selected key enzymes of glycolysis (hexokinase, phosphofructokinase and pyruvate kinase) and TCA cycle (succinate dehydrogenase and malate dehydrogenase) were measured in these two organs of (a) castrated estrogen treated, (b) castrated estrogen + dihydrotestosterone (DHT) treated animals and compared with those in castrated and castrated + DHT treated animals. Results reveal that DHT stimulated the activities of all these enzymes whereas estrogen failed to stimulate any of the enzymes in castrated animals. However, estrogen in combination with DHT caused a marked stimulation of the enzymes and the response of the epididymis and vas deferens to combination treatment was significantly more than that caused by DHT alone. The results suggest that circulating estrogen in male has a physiological role and acts synergistically with androgen in regulating accessory sex organ function.     

Testicular control of prostaglandin E2 production in rat vas deferens

Orchidectomy decreased and testosterone (T) replacement restored prostaglandin E2 (PGE2) concentrations in adult rat vas deferens. To explain this finding, phospholipase A (PLase-A) and PG synthetic activity were studied in vas tissue from 9-week-old rats orchidectomized with or without T replacement as well as in rats in which the cauda epididymidis was ligated. PG synthetic activity fell to 3% of intact levels in 14-day castrate rats and was restored to normal by T replacement. Although vas PLase-A activity was also significantly (P less than 0.01) reduced to 38% of the control level in 14-day castrate rats, this change appears in part to reflect a castration-related increase in endogenous phospholipid concentrations. Further, T replacement only partially restored PLase-A activity to 59% of intact levels. Ligation of the cauda epididymidis in intact rats reduced vas PLase-A activity to castrate levels without altering vas T concentration. These results demonstrate both a direct effect of T on the biosynthesis of PGs in rat vas deferens as well as a paracrine effect, which appears to be mediated by a factor(s) other than T. These data suggest the existence of a new mechanism through which testicular products contribute to the function of the vas deferens.     

Testicular regulation and sub-cellular distribution of zinc in the epididymis and vas deferens of rhesus monkey (Macaca mulatta)

The zinc concentration in the epididymis (caput, corpus and cauda regions), vas deferens and caudal lobe of prostate of adult rhesus monkeys was determined by atomic absorption spectrophotometry. Zinc content (microgram/g wet weight) was found to be maximum in the prostate (709 micrograms) followed by epididymis and vas deferens. The three segments of the epididymis did not differ from one another in their zinc content (165-177 micrograms). On a protein basis maximum concentration of zinc was present in the nuclear fraction followed by microsomal, cytosolic and mitochondrial fractions in that order. Ligation of testicular efferent ducts or castration 90 days prior to autopsy caused a marked reduction in zinc concentration in different sub-cellular fractions of the organs examined; castration was relatively more effective in this regard. The importance of androgen and other testicular products in controlling zinc content and the possible physiological role of zinc in the male genital tract are discussed.     

Age-, cell- and region-specific immunoexpression of estrogen receptor alpha (but not estrogen receptor beta) during postnatal development of the epididymis and vas deferens of the rat and disruption of this pattern by neonatal treatment with diethylstilbestrol

This study in rats sought to 1) characterize immunoexpression of estrogen receptor alpha (ERalpha) and ERss in the efferent ducts, epididymis, and vas deferens during postnatal development; 2) establish whether ER expression changed after neonatal treatment with diethylstilbestrol (DES); and 3) determine whether ER changes coincided with abnormal epididymal/vas development. Rats were administered 10 microg DES or vehicle on days 2, 4, 6, 8, 10, and 12 and were sampled on days 10, 18, 25, 35, and 90+. At all ages, ERalpha was immunoexpressed intensely in the efferent ducts. On day 10, immunoexpression of ERalpha was absent from the epididymis and vas, but was detectable on day 18 in epithelial cells in the caput, corpus, and proximal cauda. Epithelial expression of ERalpha was absent from the distal cauda and in the proximal and distal vas was confined to a band of periductal stromal cells. Thus, on day 18, the site of ERalpha expression delineated the epididymis-vas boundary. On days 25-35, epithelial expression of ERalpha was absent, but stromal expression persisted in the vas and distal cauda. In adults, immunoexpression of ERalpha in the epididymis and vas was absent. In contrast, ERbeta was immunoexpressed in epithelial cells and some stromal cells in the efferent ducts, epididymis, and vas at all ages. In the vas, stromal expression of ERalpha and ERbeta was in different layers. DES treatment caused 1) underdevelopment of the epididymal duct and reduced epithelial height in epididymis and vas; 2) coiling of the extraepididymal vas; 3) thickening of the periductal actin-free stromal layer in the distal cauda and vas; and 4) reduced cell proliferation on day 18 in the epididymis and vas, based on incorporation of bromodeoxyuridine, especially in the epithelium. These changes coincided with abnormalities in cell- and region-specific immunoexpression of ERalpha, but not ERbeta. Thus, in DES-treated rats on day 18, epithelial expression of ERalpha occurred in all regions of the epididymis and vas instead of being confined to the caput, corpus, and proximal cauda as in controls. Similarly, stromal ERalpha expression in the vas of DES-treated rats was not confined to a periductal layer as in controls, but occurred diffusely in the muscle layer. It is suggested that 1) estrogens play a role in peripubertal development of the epididymis and vas; 2) the cellular site of expression of ERalpha either plays a role in or reflects demarcation of the epididymal/vas boundary; and 3) blurring of this boundary in DES-treated rats coincides with altered ERalpha immunoexpression.     

Catenins in the rat epididymis: their expression and regulation in adulthood and during postnatal development

Tight and adhering junctions are important in maintaining the integrity of the epididymal epithelium and formation of the blood epididymal barrier, which are crucial for sperm maturation and storage. The composition of the catenin-adhering junctional family of proteins and their relationship with tight junctions remain to be established in the epididymis. In the normal adult rat epididymis, immunostaining for three anticatenin antibodies (alpha, beta-, and p120ctn) was noted along the lateral plasma membranes (LPM) between adjacent epithelial cells. Although alpha-catenin and beta-catenin were maximally expressed in the corpus and cauda epididymis, p120 expression was intense and similar in all epididymal regions. Bilateral orchidectomy of adult rats indicated that the expression of p120 at the LPM was not altered compared with that in control animals. On the other hand, staining at the LPM for alpha- and beta-catenin was markedly reduced, concomitant with an increased cytoplasmic reaction in each epididymal region. As the staining pattern for alpha- and beta-catenin returned to that seen in control animals after testosterone supplementation, it is suggested that their localization and targeting to the LPM are regulated by androgens. This is confirmed by postnatal studies in which maximal expression at the LPM for each catenin occurs by d 49, when androgen levels are adult-like. Immunolocalization of zona occludens-1 along with immunoprecipitation of epididymal homogenates of the initial segment/caput region of the epididymis revealed that zona occludens-1 is an integral part of the adhering junctional complex in young rats and coprecipitates with beta-catenin at the level of the apical tight junctions.     

Identification and characterization of two classes of receptors for oxytocin and vasopressin in porcine tunica albuginea, epididymis, and vas deferens

The neurohypophysial hormones oxytocin (OT) and vasopressin (VP) are involved in the regulation of the contractility of the male genital tract in several animal species. We investigated the presence of specific binding sites for [3H]OT and [3H]arginine VP (AVP) in membranes prepared from tunica albuginea, epididymis, and vas deferens from prepubertal pigs 2-16 weeks of age. Membranes were incubated with [3H]OT and [3H]AVP in the presence or absence of the corresponding unlabeled peptides. Binding equilibrium was reached in 60 min at 22 C. Millimolar concentrations of Mg2+ increased the specific binding of both ligands. Analysis of families of self- and cross-displacement curves using the computer program LIGAND clearly demonstrated that two classes of binding sites were present in all tissues investigated. The first class of sites, designated the OT site, shows high affinity for OT, AVP, lysine vasopressin, arginine vasotocin, the selective OT agonists [Thr4,Gly7]OT and [Asu1,6]OT, and the OT antagonists derived from ornithine vasotocin (OVT), namely d(CH2)5Tyr(Et)OVT and dEt2OVT. The second class of sites, designated the VP site, shows high affinity for AVP, lysine vasopressin, arginine vasotocin, and the selective V1 antagonist d(CH2)5Tyr(Me)AVP. The V2 agonist [1-deamino,4-valine]8-D-AVP shows low affinity for both sites. Isotocin, desglycinamide [Arg-8]AVP and tocinoic acid were ineffective in displacing [3H]AVP or [3H]OT. The highest density of OT receptors was found in tunica albuginea and epididymis, whereas the highest density of AVP receptors was found in vas deferens. Adenylate cyclase was not activated in any of the tissues studied by concentrations of AVP or OT up to 100-fold greater than their Kd values. This is the first demonstration and pharmacological characterization of specific OT and V1 VP receptors in the tunica albuginea, epididymis, and vas deferens. The recent demonstration of high local concentration of neurohypophysial hormones in the gonads of several mammals support a physiological role of these OT and VP receptors in regulation of the motility of the male genital tract.     

Androgen-controlled synthesis of specific proteins in the rat epididymis

Previous investigations have established the production of specific epididymal proteins (SEP) in the rat which become attached to the sperm surface as these cells pass along the duct. The present study is concerned with the regulation of SEP synthesis by androgens. For this purpose, we determined the concentrations of SEP and protein DE, one of its main components (40%), during sexual maturation, after castration with and without androgen administration, and after ligation of the efferent ductuli in rats. SEP were first detectable at 25 days of age and attained adult values at 60-90 days of age. Protein DE behaved similarly. Castration of the adult rat led to a decrease in SEP and DE concentrations. The fall was more rapid and marked in the caput than in the caudal segments. SEP synthesis seemed to stop promptly after castration; the different rates of decrease of SEP in caput and cauda may reflect different rates of exit of spermatozoa from those segments. SEP and DE concentrations in castrated rats were increased by the administration of testosterone (100 micrograms/day). The SEP concentration was increased after 4 days and restored to control values after 11 days of treatment. Testosterone and 5 alpha-dihydrotestosterone were equipotent in inducing SEP and DE synthesis, while 5 alpha-androstandiols were less potent. The effects of androgens were significantly reduced by the simultaneous administration of cyproterone acetate. We propose that SEP is a suitable marker for following the action of androgens in the epididymis.     

Protein kinases in divergent eukaryotes: identification of protein kinase activities regulated during trypanosome development.

The role of protein kinases in organisms that diverged early in the eukaryotic lineage is relatively unexplored. In this study, we determined that primitive parasitic protozoa possess multiple protein-serine kinases and inferred the presence of protein-tyrosine kinases through sensitive immunoblotting techniques. To further explore the role of protein kinases in parasite development, we examined the activity of eight renaturable protein kinases during the life cycle of the protozoan parasite Trypanosoma brucei. The activities of six protein-serine/threonine kinases were regulated during development, with several distinct patterns of regulation. In addition, an 89-kDa protein kinase was detected in dividing cells but not in nondividing cells. Our data indicate that even the most primitive eukaryotes possess a large complement of protein kinases, including protein-tyrosine kinases as well as protein-serine/threonine kinases. The data further suggest that protein kinases may play a pivotal role in regulation of proliferation and differentiation in protozoa.     

Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation and several other cytogenetic aberrations, including heterozygous loss of chromosomal arms 1p, 6q, 11q and 13q and/or gains of 3q and 8q. The common intervals of chromosomal imbalance have been narrowed down using array-comparative genomic hybridization (CGH). However, the chromosomal intervals still contain many genes potentially involved in MCL pathogeny. Combined analysis of tiling-resolution array-CGH with gene expression profiling on 11 MCL tumours enabled the identification of genomic alterations and their corresponding gene expression profiles. Only subsets of genes located within given cytogenetic anomaly-intervals showed a concomitant change in mRNA expression level. The genes that showed consistent correlation between DNA copy number and RNA expression levels are likely to be important in MCL pathology. Besides several 'anonymous genes', we also identified various fully annotated genes, whose gene products are involved in cyclic adenosine monophosphate-regulated pathways (PRKACB), DNA damage repair, maintenance of chromosome stability and prevention of rereplication (ATM, ERCC5, FBXO5), energy metabolism (such as genes that are involved in the synthesis of proteins encoded by the mitochondrial genome) and signal transduction (ARHGAP29). Deregulation of these gene products may interfere with the signalling pathways that are involved in MCL tumour development and maintenance.     

Kinetics of alpha-adrenoreceptor blockade by phentolamine in the normal and denervated rabbit aorta and rat vas deferens.

The rates of onset and offset of alpha-adrenoreceptor blockade by phentolamine (5 X 10(-7) M) have been studied on the normal and denervated rabbit aorta and rat vas deferens. Removal of the adventitia with its accompanying sympathetic nerve component results in an approximately threefold increase in the rate of onset of alpha-adrenoreceptor blockade by phentolamine in the rabbit aorta. The rate of offset of blockade by the antagonist on the aorta is likewise faster after the adventitia has been removed (approximately threefold faster for loss of 90% of the antagonist from the region of the receptors). This effect is not likely to be attributed to the absence of sympathetic nerve terminals since surgical denervation of the rat vas deferens has no effect on the kinetics of receptor blockade in this densely innervated organ. These data suggest that the adventitia of the rabbit aorta may serve as a diffusion barrier which limits the antagonist's access to, and efflux from, the region of the receptors. The dissociation constant of phentolamine (calculated from the equilibrium dose ratio) on the alpha-adrenoreceptor is unaltered by denervation in either tissue and is identical in both tissues. It is suggested that alpha-adrenoreceptors in the rabbit aorta and rat vas deferens are of a single type and are not significantly affected, with respect to antagonist affinity, by denervation.     

The effect of efferent duct ligation on the uptake of 65-ZN by the epididymis and vas deferens of rhesus monkeys (Macaca mulatta)

A study dealing with the uptake of zinc-65 by different segments of the epididymis and vas deferens of the rhesus monkey (Macaca mulatta) in the presence or absence of spermatozoa is reported. It was determined that zinc-65 uptake by the vas deferens of the control side was significantly higher than that of the epididymal segments (vas deferens vs. caput, proximal and distal corpus, or cauda, p less than .01). Amon g the epididymal segments themselves there was no significant difference . Efferent duct ligation consistently reduced the uptake of zinc-65 in all portions of the epididymis and vas deferens (control vs. ligated: caput, corpus, cauda, and vas, p less than .01). The percentage reduction in the uptake by different segments of the epididymis and vas remained virtually constant in the ligated side (caput, 57%; proximal corpus, 53%; distal corpus, 64%; cauda, 55%; vas deferens, 55%). Nevertheless, zinc-65 uptake by vas continued to be significantly greater than that of the epididymis. It is noted that the reduced zinc-65 accumulated on the ligated side could at least be partly due to absence of spermatozoa. It is further suggested that the lowered uptake of zinc-65 by the epididymis and vas deferens following efferent duct ligation in the monkey may be also due to a local androgen deficiency.     

Diverse expression of ErbB receptor proteins during rat liver development and regeneration

BACKGROUND & AIMS: The protein expression and interactions of the ErbB receptors were examined in different liver proliferation models in vivo and in vitro, including ontogeny and regeneration following partial hepatectomy. METHODS: Expression and tyrosine phosphorylation status of specific ErbB proteins were studied by immunologic methods. RESULTS: The epidermal growth factor receptor, ErbB2, and ErbB3 were the only ErbB proteins detected in the liver parenchyma on embryonic day 19. ErbB2 disappeared by the third week after birth and could not be appreciably induced in the adult animal by partial hepatectomy. ErbB2 was also detected in multipotent stem (RLE) and hepatoma (H4IIe) cell lines as well as in fetal, but not adult, hepatocyte cultures. Only epidermal growth factor receptor and ErbB3 were detected in adult liver, and both showed circadian variation in protein expression. ErbB4 was not detected in any model. Patterns of ligand-induced ErbB phosphorylation differed between fetal and adult hepatocytes. CONCLUSIONS: Complex and independent programs regulate the ErbB receptors, with implications for differential cell signaling in hepatic development and regeneration.     

Proteomic identification of phosphatidylinositol (3,4,5) triphosphate-binding proteins in Dictyostelium discoideum

Phosphatidylinositol (3,4,5)-triphosphate (PtdInsP₃) mediates intracellular signaling for directional sensing and pseudopod extension at the leading edge of migrating cells during chemotaxis. How this PtdInsP₃ signal is translated into remodeling of the actin cytoskeleton is poorly understood. Here, using a proteomics approach, we identified multiple PtdInsP₃-binding proteins in Dictyostelium discoideum, including five pleckstrin homology (PH) domain-containing proteins. Two of these, the serine/threonine kinase Akt/protein kinase B and the PH domain-containing protein PhdA, were previously characterized as PtdInsP₃-binding proteins. In addition, PhdB, PhdG, and PhdI were identified as previously undescribed PH domain-containing proteins. Specific PtdInsP₃ interactions with PhdB, PhdG, and PhdI were confirmed using an in vitro lipid-binding assay. In cells, PhdI associated with the plasma membrane in a manner dependent on both the PH domain and PtdInsP₃. Consistent with this finding, PhdI located to the leading edge in migrating cells. In contrast, PhdG was found in the cytosol in WT cells. However, when PtdInsP₃ was overproduced in pten⁻ cells, PhdG located to the plasma membrane, suggesting its weak affinity for PtdInsP₃. PhdB was found to bind to the plasma membrane via both PtdInsP₃-dependent and -independent mechanisms. The PtdInsP₃-independent interaction was mediated by the middle domain, independent of the PH domain. In migrating cells, the majority of PhdB was found at the lagging edge. Finally, we deleted the genes encoding PhdB and PhdG and demonstrated that both proteins are required for efficient chemotaxis. Thus, this study advances our understanding of the PtdInsP₃-mediated signaling mechanisms that control directed cell migration in chemotaxis.