Localization of the endogenous benzodiazepine ligand octadecaneuropeptide in the rat testis.

Diazepam binding inhibitor (DBI) is the precursor of a family of peptides, including an octadecaneuropeptide (ODN), which share with DBI the ability to specifically displace benzodiazepines (BZD) from their receptors. BZD receptors have been found not only in the brain, but also in a variety of peripheral tissues, including the testis. To clarify the role of ODN in the testis, we have investigated the localization of ODN in the rat testis using two different cytochemical approaches: immunocytochemistry and in situ hybridization. Immunocytochemical localization was achieved using rabbit antibodies developed against rat ODN. At the light microscopic level, immunostaining was exclusively located in interstitial cells; the seminiferous tubules were totally unlabeled. In the developing rat, immunostaining in the interstitial cells was first detected in an 18-day-old fetus. The immunolabeling increased as a function of age to reach a plateau at 40 days of age. The ultrastructural localization of ODN was achieved by immunogold staining. The gold particles were exclusively found in the cytoplasm of Leydig cells. HPLC analysis performed in adult rat testicular extracts revealed that immunoreactive material was detected in a peak eluted later than synthetic rat ODN. The cellular distribution of ODN was also studied by in situ hybridization using a 35S-labeled single stranded RNA probe complementary to DBI mRNA. Hybridization signal obtained at the light microscopic level was only detected over interstitial cells. The data obtained clearly indicate that in the rat, Leydig cells synthesize ODN and accumulate ODN-like immunoreactivity. Since Leydig cells have been shown to contain BZD receptors, it might be hypothesized that ODN and/or other DBI-related peptides can play a role in Leydig cell regulation.

Measurement of circulating parathyroid hormone-related protein in rats with humoral hypercalcemia of malignancy using a two-site immunoradiometric assay.

The Rice H500 rat Leydig cell tumor is a well characterized model of humoral hypercalcemia of malignancy (HHM). Circulating concentrations of PTH-related protein (PTHRP) have not been reported in this or any other animal model of HHM. Taking advantage of the marked N-terminal amino acid homology between rodent and human PTHRPs, we have adapted a sensitive two-site immunoradiometric assay developed for measurement of human PTHRP for use in measuring rat PTHRP. Circulating calcium and PTHRP concentrations were serially measured after sc passage of the Leydig cell tumor in rats. Significant hypercalcemia and elevation of PTHRP occurred on day 9 after tumor inoculation. When grouped by tumor size, both plasma calcium and PTHRP levels were significantly elevated in animals with tumor burdens greater than 10 cc. The PTHRP concentration was strongly correlated with both serum calcium (r = 0.88) and tumor size (r = 0.80). Circulating rat PTHRP averaged 12.8 pM on day 9 and 27.5 pM on day 10 or 11. PTHRP was undetectable in the plasma of 19 control rats. In 3 rats, plasma calcium returned to normal, and PTHRP became undetectable within 24 h after tumor excision. Rat milk displayed a PTHRP concentration of 2000 pM, while acid-urea extract of the rat tumor contained 0.32 pmol/mg protein. Dilutions of rat plasma, milk, and tumor extract displayed response curves that were parallel to the human PTHRP-(1-74) standard in the assay. This two-site immunoradiometric assay is a sensitive and easily performed means of measuring rat PTHRP. It should be useful in studying this animal model of HHM and the function of PTHRP in normal tissues.

Escherichia coli DNA polymerase I: inherent exonuclease activities differentiate between monofunctional and bifunctional adducts of DNA and cis- or trans-diamminedichloroplatinum(II). An exonuclease investigation of the kinetics of the adduct formation.

[3H]dGMP-3'-labelled, activated salmon testis DNA and [32P]dGMP-5'-labelled open circular M13 DNA were reacted with cis-diamminedichloroplatinum(II), cis-diamminechloroaquaplatinum(II), cis-diamminediaquaplatinum(II) or trans-diamminechloroaquaplatinum(II). The reaction was arrested after arbitrary times by adjustment to slightly alkaline solution conditions. The platinum-containing DNA was digested with Escherichia coli DNA polymerase I. The progress of nucleotide release was measured by acid precipitation of undigested DNA. Solubilized nucleotides and adducts were analyzed by HPLC. The 3'-5'-exonuclease activity liberated single-coordinated dGMP-platinum(II) adducts from both cis- and trans-platinum(II) treated salmon testis DNA and a small fraction of adducts of cis-platinum(II) that coordinated two molecules of dGMP. The bisadduct was derived from non-neighboring guanine residues probably located at or close to 3'-termini. This nuclease activity neither cut between nor after neighboring guanine residues crosslinked by cis-platinum(II). No bisadduct was liberated for trans-platinum(II). The 5'-3'-exonuclease activity did not liberate any nucleotide adducts from cis-platinum(II)-treated DNa. However, it removed single-coordinated guanine adducts of trans-diamminedichloroplatinum(II). From the kinetics of the appearance of dGMP monoadducts and the inhibition of digestion, a reaction scheme is formulated for the reaction of platinum(II) complexes with DNA that confirms and extends the previously published one [W. Schaller, H. Reisner & E. Holler (1987) Biochemistry 26, 943-950]. The longevity of the dGMP monoadduct intermediate is discussed in the context of the efficiency of cis-diamminedichloroplatinum(II) as an antitumor drug.

The flow cytometric analysis of undescended testes in children.

Flow cytometric analysis was performed on the testicular aspirates of 45 consecutive children with unilateral cryptorchidism undergoing elective orchiopexy or orchiectomy. Concomitant histological analysis was performed on the testicular tissue obtained from either biopsy or orchiectomy specimens. In all cases deoxyribonucleic acid histograms appeared to correspond with microscopic appearance. Histograms from prepubescent patients demonstrated 85 to 95% of cells in the diploid (2c) peak and less than 10% of cells in the tetraploid peak (4c), representing prepubertal testes without active spermatogenesis. Three distinct patterns of ploidy were identified in postpubertal children corresponding to the histological appearances of normal spermatogenesis, maturation arrest and the Sertoli-cell-only syndrome, respectively. In addition, we identified an aneuploid cell population in the specimen from 1 patient, suggesting that this testis may be at risk for future malignant degeneration. We conclude that flow cytometry of testicular aspirates is an easy and effective means of testicular evaluation, which may permit predictions regarding the fertility and malignant potential of undescended testes in postpubertal children.

Efecto del ejercicio físico agudo sobre la distribución y el metabolismo de la vitamina A / Effect of acute physical exercise on the distribution and metabolism of vitamin A

Se evaluó el efecto del ejercio físico agudo sobre la distribución y el metabolismo de la vitamina A en un modelo experimental animal. Se estudiaron las concentraciones plasmáticas, hepaticas, renales, testiculares y de glándulas suprarrenales de la vitamina A y de sus principales formas moleculares: retinol y ésteres de retinol, en animales sometidos a ejercicio físico durante 2 h sin previo entrenamiento. Se observó una disminución en la concentración hepática y testicular de la vitamina A y un incremento de ésta en los riñones de los animales ejercitados. Se demostró, además, el incremento del catabolismo hepático del retinol bajo las condiones experimentales

Identification of interleukin-1 receptors in mouse testis.

The cytokine interleukin-1 (IL-1) has been reported to alter reproductive functions through both effects in brain and direct actions at the level of the gonads. To further define the role of IL-1 at the gonads, we have used 125I-labeled recombinant human IL-1 to identify and characterize IL-1 receptors in crude membrane preparations of mouse (C57BL/6) testis and to study the distribution of IL-1-binding sites using autoradiography. In preliminary homogenate binding and autoradiographic studies, [125I]IL-1 alpha showed significantly higher specific binding than [125I]IL-1 beta. Thus, [125I]IL-1 alpha was used in all subsequent assays. The binding of [125I]IL-1 alpha was linear over a broad range of membrane protein concentrations, saturable, and reversible, and on Scatchard analysis revealed an equilibrium dissociation constant (Kd) of 82 +/- 4 pM and a maximum number of binding sites (Bmax) of 10.8 +/- 1.5 fmol/mg protein. In competition studies, IL-1 alpha, IL-1 beta, and a weak IL-1 beta analog IL-1 beta+ inhibited [125I]IL-1 alpha binding to mouse testis in parallel with their relative bioactivities in immune assays, with inhibitory binding affinity constant (Ki) values of 14.2 +/- 1.7, 88.8 +/- 5.7, and 7183.3 +/- 603 pM, respectively; rat/human CRF and human tumor necrosis factor showed no effect on [125I]IL-1 alpha binding. In autoradiographic studies, IL-1 receptors were heterogeneously distributed, with highest densities present in the luminal border of the epididymis and interstitial areas of the testis. After hypophysectomy, the testes were significantly atrophied, and homogenate binding and autoradiographic studies showed that while the total number of binding sites per testis was significantly decreased in hypophysectomized mice in proportion to the reduction in testicular mass, there was no apparent change in the relative density of IL-1 receptors. These data provide the first identification of IL-1 receptors in testis and provide further support for a physiological role for IL-1 to alter reproductive functions through a direct effect at the gonads.

In the absence of a downstream element, the apolipoprotein E gene is expressed at high levels in kidneys of transgenic mice.

Human apolipoprotein (apo) E gene constructs with 30 or 5 kilobases of 5'-flanking and 1.5 kilobases of 3'-flanking regions were used to create transgenic mice. High levels of human apoE mRNA were present in the transgenic kidney, but none was detected in the liver, which is normally the major source of apoE. When a construct with 5 kilobases of 5'- and 23 kilobases of 3'-flanking regions was used, only trace levels of human apoE mRNA were detected in the kidney, whereas high levels were found in the liver. These results indicated that regulatory elements downstream of the human apoE gene interacted with the transcription initiation complex to stimulate gene expression in the liver while suppressing expression in the kidney. In each case, human apoE was secreted into the plasma. The source of human apoE in the transgenic kidney was the epithelial cells lining the proximal tubule and Bowman's capsule.

Identification and characterization of a testis-specific isoform of a chaperonin in a moth, Heliothis virescens.

Two relatively abundant proteins having subunit molecular weights of 60,000 and 63,000 (p60 and p63, respectively) have been purified as a 16 to 18S complex from sperm mitochondria of a moth. Heliothis virescens. Although the function of these proteins had heretofore not been established, interest in the p63 polypeptide stemmed from its sperm-specific expression and its striking occurrence as a net charge variant among several insect species surveyed, using two-dimensional gel electrophoresis. Genomic and cDNA clones corresponding to the p63 protein have now been isolated and their sequencing has revealed extensive amino acid sequence identity with both the Escherichia coli GroEL protein and its eukaryotic homologues, the chaperonins. Immunoblot studies with a Tetrahymena chaperonin antiserum demonstrated that the p60 protein, which is expressed in all cell types, is structurally related to p63 and is itself a chaperonin subunit. While the chaperonin complex from Heliothis sperm shares certain properties with GroEL, including the ability to hydrolyze ATP and organization of its subunits into a seven-member ring, electron microscopic analysis revealed that its higher-order structure differed from GroEL (and other lower eukaryotic chaperonins) in that the native particle comprises one such ring rather than a doublet. It is not yet known whether the two chaperonin isoforms coexpressed in moth sperm assemble separately or give rise to hybrid particles. In either case, the existence of multiple chaperonin subunits in sperm leaves open the possibility that some aspect of mitochondrial biogenesis that is dependent upon the activity of these proteins is qualitatively or quantitatively different in this cell type.

Similarities between metallothionein and low molecular weight testicular cadmium-binding protein.

The incorporation of 35S-cysteine and 3H-glutamic acid was studied in mouse hepatic and renal metallothionein and in testicular cadmium-binding protein of similar molecular weight. Preferential incorporation of 35S-cysteine over 3H-glutamic acid was observed not only in hepatic and renal metallothionein, but also in testicular cadmium-binding protein. When the antigenic reactivity of these proteins was compared, all three proteins reacted with the metallothionein antibody. These similarities suggest that the low molecular weight testicular cadmium-binding protein is apparently metallothionein.

Changes in glycolipid expression in human testicular tumor.

Glycolipids were extracted from testicular tumor tissues of 13 patients, and their pattern of expression compared with that of normal testicular tissue. The most conspicuous and consistent change in the tumor extracts was marked accumulation of CTH (ceramide trihexoside). Structural analysis by enzyme cleavage showed that CTH which accumulated in the tumor tissue was Gb3 (Gal alpha 1-4Gal beta 1-4Glc beta 1-Cer). Immunohistochemistry using anti-Gb3 monoclonal antibody (MAb) (1A4) also indicated massive accumulation of Gb3 in the tumor tissue. Gb3 may be a new marker of testicular tumors, especially seminomas, for which useful markers are so far lacking.

Localization and characterization of epidermal growth factor receptors on human testicular tissue by biochemical and immunohistochemical techniques.

In the present study attempts were made to characterize the epidermal growth factor (EGF) receptor on human testicular tissue. A radioligand exchange assay with 125I-labelled EGF was used to detect a high affinity, low capacity, single binding site in the 105,000 g particulate fraction of human testicular tissue. Binding was optimal at 32 degrees C following a 40-min incubation with a mean (+/- S.D.) dissociation constant of 327 +/- 59 pmol/l (d.f.9). The number of binding sites ranged from 0.07 to 0.21 pmol/mg protein. Competition studies with other peptide hormones including LH, FSH, prolactin, insulin-like growth factor-I, fibroblast growth factor and nerve growth factor have confirmed the specificity of EGF for its receptor. The receptor was also found to be heat-labile and sensitive to trypsinization. Cross-linking experiments using disuccinimidyl suberate revealed major binding species at the 125 kDa region and this is thought to represent a proteolysed form of the receptor. Immunohistochemical localization of the receptors demonstrated their presence in the interstitial tissue and not within the seminiferous tubules. The presence of specific EGF binding in the interstitial tissue suggests that EGF may play some role in testicular steroidogenesis.

Interaction between aluminum and zinc or copper and its effects on the pituitary-testicular axis. II. Testicular enzyme and serum gonadotropin assay.

Sprague-Dawley male rats were divided into groups and maintained on a semipurified diet containing either 5 or 40 ppm of zinc or 2 or 8 ppm of copper. Half of the rats in each group received 1000 ppm aluminum in the diet. It was found that aluminum accumulated in the pituitary glands and testes when dietary copper levels were suboptimal. The ALP activity in testes was depressed by the added aluminum when the intake of zinc or copper was suboptimal. SDH, LDH, and LDH-X activities were inhibited and GRS and GGTP activities were elevated in rats fed either the suboptimal zinc or copper diet. However, the added aluminum in the diet reversed the changes to normal levels. The testosterone levels in plasma changed very little when the zinc or copper intake was suboptimal. An increase in plasma FSH was demonstrated in groups of both suboptimal zinc and copper intake. But the plasma LH was elevated only in the group receiving the suboptimal copper diet, and the added aluminum reversed plasma LH to control levels. A lower level of testosterone was demonstrated in the group given suboptimal copper with aluminum. It was concluded that dietary aluminum influenced the pituitary-testicular axis by interacting with certain essential trace metals, particularly zinc.

[Chemical composition of the penis and testes of deer before and after processing].

Compositional analysis has been made of Penis et testis Cervi before and after processing, taking extracts of EtOH (50%) and chloroform, nitrogen contents, ash contents, 18 amino-acids and 20 trace elements as chemical indexes. The result shows no remarkable difference between the raw and processed products. The processing procedure is only helpful in eliminating the fish mell and making grinding and extracting easier.

Intracellular cadmium mobilization sequelae.

The consequences of the mobilization of aged intracellular cadmium from its in vivo deposits in mice by chelating agents were examined. The chelating agents used were BAL, sodium N-benzyl-D-glucamine dithiocarbamate (NaB), Diisopropyl meso-2,3-dimercaptosuccinate(Di-PDMS) and sodium N-(4-methoxybenzyl)-D-glucamine dithiocarbamate(4-Me0), all previously shown capable of causing statistically significant decreases in either renal or hepatic cadmium burdens in rodents. They were given at a level of 400 mumol/kg (i.p.) daily for 10 days to mice previously loaded with a total of 10 mg CdCl2.2.5 H2O/kg. Under these conditions a significant decrease in the renal cadmium level occurred following treatment with BAL, NaB, and 4-MeO; hepatic cadmium levels decreased significantly following treatment with NaB and 4-MeO. Pathological examination of the kidneys, liver, and testes in these animals showed that chelate mobilization of the cadmium produced no noticeable changes in the histopathology of these organs in comparison with that observed for the animals which had been given only cadmium and had undergone no chelate treatment. The results suggest that the mobilization of such aged cadmium from in vivo deposits need not result in any deleterious changes in the kidneys, liver or testes.

Immunohistochemical detection of transforming growth factor-alpha in Leydig cells during the development of the rat testis.

In this paper the localization of transforming growth factor alpha (TGF-alpha) is described in the rat testis at various stages throughout development, e.g. neonatal, prepubertal, and adult, in order to examine somatic cells and germinal cells at different stages of differentiation. This was done by immunoperoxidase staining using a monoclonal antibody that does not cross-react with epidermal growth factor (EGF). In sections of testes from neonatal rats, intense staining was present in Leydig cells. In the cells of the seminiferous tubules the staining was faint or undetectable. At the time when many mesenchymal cells differentiate into Leydig cells in the 21-day-old rat, TGF-alpha was visualized in most but not all of the identifiable Leydig cells. In interstitial cell cultures derived from 21-day-old rats, the majority of the Leydig cells contained TGF-alpha, but in a proportion of the Leydig cells TGF-alpha was undetectable. No staining was apparent in Sertoli cells and germ cells in seminiferous tubules or in Sertoli cell cultures derived from 21-day-old rats. Under these in vitro conditions it was found that peritubular-myoid cells also possessed TGF-alpha immunoreactivity. In the adult testis all Leydig cells stained positively for TFG-alpha, whereas no staining was found in the cells of the seminiferous tubules. Treatment of adult rats with ethylene-1,2-dimethane-sulfonate (EDS) resulted in the destruction of Leydig cells and the loss of all positively stained for TGF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic ethanol diet on pituitary-testicular function of the rat.

We studied the effects of a 6% ethanol liquid diet administered for 5 wk on the pituitary-gonadal function of adult male rats. Because ethanol is known to reduce body weight, we used sucrose-fed animals as controls. No significant differences in body, testis, or prostate weights were found between the rats exposed to ethanol and their sucrose-fed controls at the end of the 5-week treatment. Seminal vesicle weights decreased significantly (p less than 0.05) in the ethanol-treated group. Serum and testicular testosterone concentrations were significantly reduced in the ethanol-treated group, to 43.6% and 48.3% of levels in the sucrose-fed controls, respectively (p less than 0.05). Serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels of the ethanol-treated rats were 37.9% and 41.3%, respectively, of those of the sucrose-fed controls (p less than 0.01-0.05). The pituitary levels of these hormones were similar to those of controls, but the ratios of pituitary LH and FSH to their serum levels were clearly increased after ethanol exposure, to 492% and 206.1%, respectively (p less than 0.05). In contrast, pituitary prolactin (PRL) of the ethanol-treated rats was decreased to 40.2% (p less than 0.01) of sucrose-fed controls. Testicular content of LH receptors was significantly reduced (to 77.0% of controls; p less than 0.01), but content of FSH receptors was slightly increased by the ethanol diet (to 121.5% of sucrose-fed controls; p less than 0.05). No ethanol-associated changes were apparent in testicular PRL and gonadotropin-releasing hormone (GnRH) receptors or in pituitary GnRH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Combination of the peroxidase anti-peroxidase (PAP)- and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining.

A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step--Incubation of the tissue sections with the monoclonal primary antibodies; Second step--biotinylated anti-rat or anti-mouse IgG; Third step--monoclonal PAP complex; Fourth step--ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step--the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4- and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical detection of prolactin or prolactin-like immunoreactivity in epididymis of mature male mouse.

Prolactin (PRL) binds to the testis of mice and rats where it increases the number of luteinizing hormone receptors, increases the binding of human chorionic gonadotropin (hCG) to LH receptors, and enhances testosterone synthesis and secretion. PRL also binds to the prostate and seminal vesicles of rats and humans where it increases organ weight and stimulates growth and uptake of testosterone. PRL binds to the epididymis of rats but the effect of PRL on this organ is unknown. In the present study, a standard immunoperoxidase (PAP) technique was used to detect the binding of endogenous and exogenous PRL or PRL-like peptides to the epididymis of the mature mouse. Throughout the epididymal duct, a positive reaction for peroxidase, suggesting PRL or PRL-like binding, occurred in the Golgi area of principal cells. In segment 1, positive reactions were also visualized in the perinuclear area and in the region located between the Golgi area and the apical surface of the principal cells (supra-Golgi area). In the corpus and cauda epididymidis, scattered entire principal cells were also positive. Throughout the epididymal duct, the reactions indicating the binding of exogenous PRL were slightly stronger than those testing for binding of endogenous peptides. The significance of such binding to the epididymis is uncertain but PRL may perform the same functions in epididymal principal cells as it does in the testis, prostate, and seminal vesicles.

Detection of sex-specific proteins in chick embryo gonads and mesonephros: effects of estradiol benzoate or tamoxifen on their expression.

Gonadal and mesonephric protein patterns from 19 day old normal chick embryos were investigated by two-dimensional polyacrylamide gel electrophoresis. Under these conditions, several sex-specific polypeptides were detected. As concerns gonadal extracts, four sex-specific polypeptides, all restricted to the cytosol, were present in the testis, whereas three sex-specific polypeptides, two localized in the cytosol, the other being membrane-bound, were identified in the ovary. Among the ovary-specific polypeptides two proved to be estrogen-dependent. They appeared in the left testis of embryos after early estradiol benzoate treatment and their expression was reduced in the ovary after early exposure to the antiestrogen, tamoxifen. Mesonephros extracts of both sexes also differed in their protein composition since three additional polypeptides (one in both the cytosolic and membrane fractions, the others in the cytosol) not found in females were found to be present in males. None appeared to be affected after either estradiol or tamoxifen treatment.

Purification and identification of ADP-ribosylated proteins from bull testis intact nuclei.

Isolated, intact bull testis nuclei were incubated with [14C] NAD. A large amount of radioactivity was associated to loosely bound chromosomal proteins extracted with 0.35M NaCl and fractionated with trichloroacetic acid. The labelled nuclear proteins included essentially a number of components belonging to the low mobility group. Mg2(+)-catalyzed alkali digestion of radioactive proteins and further analysis demonstrated that the final products were 5'-AMP and phospho-ribosyl-AMP, which arise from the hydrolysis of poly(ADP-ribose).