Effects of the expression of bovine growth hormone on the testes and male accessory reproductive glands in transgenic mice

The effects of physiological and excessive levels of growth hormone (GH) on reproductive functions are poorly understood, and impairment of fertility is frequently observed in transgenic animals overexpressing GH genes. The present study was undertaken to determine the effects of chronic exposure to heterologous bovine GH (bGH) on the testes and accessory reproductive glands in transgenic mice. Endocrine function of the testes was evaluated by measuring the activities of two steroidogenic enzymes, 5-3-hydroxysteroid dehydrogenase (5-3-HSD) and 17-hydroxysteroid dehydrogenase (17-HSD). The activities of acid phosphatase, alkaline phosphatase and -glucuronidase, important hydrolytic enzymes of lysosomal origin, were measured in testes, seminal vesicles and ventral prostates in normal and transgenic mice. Testicular 5-3-HSD activity was higher in transgenic than in normal mice, while testicular 17-HSD activity in transgenic mice was not altered. Acid phosphatase activity was elevated in both seminal vesicles and ventral prostates of transgenic mice, while alkaline phosphatase activity was increased only in the prostate. The activity of -glucuronidase was elevated in the testes, seminal vesicles and ventral prostate gland of transgenic mice. These results suggest that chronic exposure to bGH is associated with significant stimulation of some hydrolytic enzymes in the testes and in the accessory reproductive glands of transgenic mice.     

Steroid-induced regulation of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase activity in wild type and mutants of Streptomyces hydrogenans

The effect of estradiol, hydrocortisone and progesterone on 3,20-and 3,17-hydroxysteroid dehydrogenase (HSD) in mutants of Streptomyces hydrogenans was compared to the steroid response of the wild type. Mutants were defective in arginine biosynthesis and/or aerial mycelial formation and lacked both enzymes or only 17-HSD. Some 17-HSD mutants had lost the ability to be induced by estradiol, by progesterone or by both. Some 20-HSD mutants had lost the ability to be induced by hydrocortisone, by progesterone or by both. Non-inducibility of 17-and 20-HSD by progesterone was not co-ordinate. An additional study of the growth phase-dependent enzyme activity of the wild type after induction with estradiol, hydrocortisone and progesterone was performed.Non-standard abbreviations 17-HSD 3,17-Hydroxysteroid dehydrogenase (EC 1.1.1.51) - 20-HSD 3,20-hydroxysteroid dehydrogenase (EC 1.1.1.53) - AO acridine orange - EBr ethidium bromide - EMS ethyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Histochemical study of enzyme patterns in the human submaxillary gland

Summary The histochemical distribution of various enzymes, such as alkaline phosphatase, acid phosphatase, esterase, -glycosidase, aminopeptidase, succinic dehydrogenese and TPN diaphorase, in human submaxillary glands has been determined.Acini and ducts of human submaxillary gland were devoid of alkaline phosphatase activity, but this enzyme was observed in capillaries and somewhat in myoepithelium.Activities of acid phosphatase, esterase, -glucuronidase and -galactosidase were generally observed in the entire cytoplasm of serous acini; but the cytoplasm of mucous acini was either negative or showed only trace amounts.Aminopeptidase reaction of both acini and ducts was generally negative.Succinic dehydrogenase and TPN diaphorase activities were strongly active in intralobuler ducts. Serous acini exhibited less activity with these enzymes; and mucous cells showed still less and were almost negative. In serous acini, there was much greater activity of TPN diaphorase than of succinic dehydrogenase.With 7 Figures in the Text     

The ultrastructural localization of the enzymes related to steroid hormone metabolism in the guinea-pig testis

Synopsis A study of the ultrastructural localization of 3-hydroxysteroid dehydrogenase (3-HSD), 11-hydroxysteroid dehydrogenase (11-HSD), glucose-6-phosphate dehydrogenase (G-6-PD), -hydroxybutyrate dehydrogenase (-HBD), NADH diaphorase (NADH-D) and NADPH diaphorase (NADPH-D) in the guinea-pig testis is reported.The procedures employed included short immersion or perfusion fixation with aldehydes followed by incubation of small blocks in a tetrazolium salt or a ferricyanide medium. The effects of incubation conditions were investigated, and a reaction medium for the ultracytochemical demonstration of 11-HSD is described. Using suitable controls, evidence for the specificity of the cytochemical reactions is presented.It was found that all the enzymes studied were present in both the Leydig and Sertoli cells of the guinea-pig testis and that the intracellular distribution pattern for each enzyme was independent of the cell type. Using tetrazolium salt techniques, both 3-HSD and 11-HSD activities were localized on or in membranes of smooth endoplasmic reticulum and within the mitochondria. With the ferricyanide techniques, G-6-PD activity was found to be associated mainly with the smooth endoplasmic reticulum membranes, while -HBD activity was limited to mitochondria. With both the tetrazolium salt and ferricyanide techniques, the reaction products for NADH-D and NADPH-D activities showed localizations which were similar to those observed for the steroid dehydrogenases.     

Histochemical evidence for granulosa steroids in follicle maturation in the African catfish,Clarias lazera

Summary The effects of carp pituitary suspension (CPS) and 11-desoxycorticosterone-acetate (DOCA) on 3-hydroxysteroid dehydrogenase (3-HSD) and glucose-6-phosphate dehydrogenase (G6PD) activity in the ovary of Clarias lazera are described. Strong 3-HSD and G6PD activities are localized in the stroma, of both control and treated fish. A single CPS injection stimulates 3-HSD activity in the granulosa of postvitellogenic, maturing and postovulatory follicles, but DOCA has no such effect on the postvitellogenic and maturing follicles, and only stimulates a weak response in the postovulatory ones.     

Light and electron microscopic immunocytochemistry on the localization of 3β-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

Summary The localization of 3-hydroxysteroid dehydrogenase/isomerase (3-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.Supported by grants from the Ministry of Education Science and Culture, Japan     

Histochemical localization of 3 beta-hydroxysteroid dehydrogenase in the testes of chicken-pheasant hybrids.

Synopsis Histochemical studies on the activity of 3-hydroxysteroid dehydrogenase (3-HSD) in the testes of adult chicken-pheasant hybrids and domestic fowl of similar age were carried out using dehydroepiandrosterone as the substrate. The reaction for 3-HSD was positive in the interstitial tissue and negative within seminiferous tubules of domestic fowl. In chicken-pheasant hybrids, a strong positive reaction for 3-HSD was observed in the interstitial tissue and within the seminiferous tubules suggesting that, in hybrid testes, both Leydig cells and cells of seminiferous tubules may be capable of steroid biosynthesis. Since the plasma testosterone levels in these birds were found to be extremely low, it is hypothesized that either the chicken-pheasant hybrids do not release testosterone in sufficient amounts in the circulation or the type of steroid produced by the 3HSD-positive cells may be different from testosterone that is required for the maintenance of normal fertility and the development of secondary sexual characteristics.     

Histochemistry of 11-beta-hydroxysteroid dehydrogenase in rat submandibular gland. Effect of cortisol stimulation.

Synopsis The activity pattern of NAD/NADP-linked 11-hydroxysteroid dehydrogenase (HSD) in the submandibular gland of the rat was re-evaluated using several control experiments. The incubation time needed for the initial appearance of red and blue formazans was used to investigate the activity of NAD-dependent 11-HSD in control and cortisol-treated rats. The following results were obtained. (1) Prefixation of small tissue blocks with 1% w/v methanol-free formaldehyde (pH 7.2) for up to 20 min preserved morphological integrity and maximal enzyme activity. The substantivity of formazans was enhanced. (2) The substantivity of Nitro BT was highly variable. The implication of this forin situ localization of enzymes was analysed. (3) Pretreatment with acetone and application of phenanthroline was necessary to avoid a false positive reaction caused by alcohol dehydrogenase. (4) No diffusion of 11-HSD was noticed within 30 min of incubation, nor was rediffusion of reduced intermediates seen. (5) With either NAD or NADP as coenzyme, 11-HSD was localized in the striated, intralobular, interlobular, interlobar, and main duct. (6) 11-HSD was found to be primarily NAD-dependent. (7) With DMF or DMSO as solvent, the rate of utilization of substrates was as follows: Cortisol=11-hydroxyandrostendione>11-hydroxyprogesterone. Aldosterone was utilized very poorly, if at all. (8) After injection (i.p.) of a single pharmacological dose of cortisol, the activity of NAD-linked 11-HSD was significantly increased 24 h later. (9) NADH-tetrazolium reductase was not inhibited by levamisole. (10) Distinct NADPH-tetrazolium reductase activity was localized in the apical part of cells (or cell membranes) of the interlobar ducts and the main duct.     

Fine structural localization of 3beta-hydroxysteroid dehydrogenase in rat corpus luteum

Synopsis A procedure for the ultracytochemical demonstration of 3-hydroxysteroid dehydrogenase (3-HSD) is described and the results for the localization of this enzyme in the lutein cells of the rat corpus luteum are presented.The procedure involved pre-fixation with aldehydes and incubation of small blocks. Short fixation (maximum 30 min) in 0.1% glutaraldehyde, 2% depolymerized paraformaldehyde or in 6.25% hydroxyadipaldehyde was found to be the best compromise for the preservation of both 3-HSD activity and fine structure. The method utilizes 3-hydroxy-5-androstan-17-one as substrate, tetranitro blue tetrazolium as a final electron acceptor, and phenazine methosulphate or menadione as an intermediate electron carrier to bypass the NADH₂-diaphorase. Control and inhibitor (cyano-ketone) experiments provide evidence for the specificity of the cytochemical reaction.Results showed that 3-HSD activity is localized on or in membranes of smooth endoplasmic reticulum, in outer compartments of mitochondria, and possibly within smooth endoplasmic reticulum cisternae and intracristal spaces of mitochondria.Localization of NADH₂-diaphorase activity in the same tissue was also examined. With the ferricyanide techniques, the reaction product was found to be associated mainly with the mitochondria.     

Penicillinase activity in three strains ofClostridium butyricum

Three strains ofClostridium butyricum exhibited elevated minimal inhibitory concentrations (MICs) to penicillin (64–1,024 g/ml), ampicillin (32–256 g/ml), carbenicillin (128–1,024 g/ml), and oxacillin (32–64 g/ml). Cephalosporin/cephamycin agents were more active than penicillin drugs. All isolates were found to possess a -lactamase. The -lactamases were primarily cell associated during the logarithmic phase of growth. Stationary-phase cells released most of the enzyme into the culture medium. Cephalothin supplementation of broth cultures with concentrations equivalent to one-eighth of the MIC significantly increased the quantity of -lactamase synthesized. The -lactamases produced by these three isolates exhibited greatest activity with penicillin followed by ampicillin>cephaloridine>carbenicillin and oxacillin. No enzymatic activity was observed using cephalothin, cephalexin, cefazolin, cefoxitin, or cephamandole substrates. The -lactamases were inhibited by clavulanic acid and para-chloromurcuribenzoate and not inhibited by cloxacillin. Each enzyme exhibited an isoelectric point of 4.2.     

Cellular composition and steroidogenic capacity of the ovary of Macrotus californicus (Chiroptera: Phyllostomatidae) during and after delayed embryonic development

Summary In the leaf-nosed bat, Macrotus californicus, a 4,5-month period of delayed early embryogenesis (October–March) precedes a 3.5-month period of normal embryogenesis (March–June). This prolonged gestation provides a unique opportunity to correlate ovarian changes with the events following implantation. The present study investigated luteal cell development and follicular biology during gestation. Circulating progesterone (P) levels following implantation were unchanged before transition to normal development, and were maximal at the start of active gestation. Luteal cell diameters increased during this period. Serum P levels declined prior to parturition, when cells staining positive for 3-hydroxy-5-steroid dehydrogenase-5,4-isomerase (3-HSD) activity were reduced in number and diameter, and enzyme staining was less intense in tissue slices. Subcellular steroidogenic organelles were present during delayed development, but smooth endoplasmic reticulum (SER) was markedly increased after the resumption of normal development at which time also luteal cells reacted positively to staining for 17-HSD. Before parturition, lipid droplet accumulation and reduced SER suggested a reduction in steroid secretion. Large multilaminar follicles stained positive for 3-HSD activity throughout gestation and for 17-HSD except in late delayed development. Thus, the delay in embryogenesis may be due to an inadequately developed corpus luteum or to the steroidogenic activity of the multilaminar follicles.     

Contrasting effects of ACTH and cyanoketone on Δ5-3β-hydroxysteroid dehydrogenase activity in interrenal glands of tadpoles of Rana catesbeiana in vitro

Summary The present communication describes an investigation of stimulation and inhibition of ⁵-3-hydroxysteroid dehydrogenase in interrenal glands of tadpoles of Rana catesbeiana. Frozen sections of interrenal glands, together with kidneys, were prepared histochemically for assay of ⁵-3-HSD activity. Concentrations of 0.01, 0.1, 1, and 10 IU/ml of ACTH or of 0.01, 0.1, 1, and 10 g/ml of cyanoketone were added to the incubation media. The reaction products of the histochemically prepared slides, in terms of absorbance, were scanned at a defined area with a computerized microscope spectrophotometer. The results indicate that ACTH causes a significant dose-response stimulation of ⁵-3-HSD activity in tadpole interrenals; cyanoketone, on the other hand, causes significant dose-dependent inhibition.     

Changes in the distribution of integrins and their basement membrane ligands during development of human thyroid follicular epithelium

Interactions between cells and basement membrane components are crucial for the regulation of epithelial cell differentiation and polarization. We have studied by immunohistochemical methods the distribution of integrin adhesion proteins and some of their basement membrane ligands in foetal (16--19 weeks) and adult thyroid follicular epithelia. A diffuse immunoreactivity for only 3, v and 1 integrins was found in foetal follicular epithelium, whereas in adult follicular epithelium these integrins were expressed basally in a polarized manner. Additionally, 3 integrin was seen in a more basolaterally confined manner in adult follicular epithelium. Among basement membrane components, laminin 1, 1, 1 and 2 chains were found in epithelial basement membranes of the foetal thyroid gland, suggestive of the presence of laminins-1 and -3. In contrast, the basement membranes of adult follicular epithelium presented a much weaker immunoreactivity for the laminin 2 chain. Furthermore, immunoreactivity for the laminin 2 chain was occasionally seen in adult thyroid glands, apparently confined to myofibroblasts. Immunoreactivity for type IV collagen 1 and 2 (IV) chains was found in follicular basement membranes of foetal as well as adult thyroid gland. The results suggest that during maturation of foetal thyroid follicular epithelium a distinct polarization of integrins takes place. In mature thyroid follicular epithelium, the presumable adhesion-mediating integrin complexes are 31, v1 and/or v3 mediating adhesion to laminin-1 (1-1- 1) and type IV collagen trimer 12 (IV)     

Morphologische Untersuchungen an den Glandulae inguinales des Kaninchens

Zusammenfassung In den Glandulae inguinales 4 geschlechtsreifer Kaninchen (2, 2) wurden folgende Enzyme mit histochemischen Methoden nachgewiesen: Alkalische Phosphatase, Adenosintriphosphatase, Glucose-6-phosphatase, 5-Nukleotidase, unspezifische Esterase, -D-Glucuronidase und -D-Galactosidase. Die Histotopik dieser Enzyme in der braunen und weißen Inguinaldrüse des Kaninchens zeigt in zahlreichen Punkten eine weitgehende Übereinstimmung mit dem Enzymverteilungsbild in den Schweiß- und Talgdrüsen der Haut. Die Befunde werden diskutiert.

---

Summary In the glandulae inguinales of 4 adult rabbits (2 male, 2 female) the distribution of alkaline phosphatase, adenosintriphosphatase, glucose-6-phosphatase, 5-nucleotidase, unspecific esterase, -D-glucuronidase and -D-galactosidase was studied by histochemical means. The site of enzymatic activity in the white and in the brown inguinal gland of the rabbit shows a condition very similar to that found in the sweat glands and the sebaceous glands of the skin. The findings obtained being discussed.

     

Immunocytochemical localization of 17β-hydroxysteroid dehydrogenase in porcine testis

Summary The immunocytochemical localization of 17-hydroxysteroid dehydrogenase (17-HSD) in porcine testes was examined by applying an indirect-immunofluorescence method using an antiporcine testicular 17-HSD antibody. Only the Leydig cells located in the interstitial tissue exhibited a positive immunoreaction for 17-HSD: the germ cells and Sertoli cells located in the seminiferous tubules were entirely negative. These results suggest that, in porcine testis, the biosynthesis of testicular testosterone, the final step of which is the conversion of androstenedione to testosterone, takes place in the Leydig cells.Supported by grants from the Ministry of Education, Science, and Culture, Japan     

Evidence that the same structural gene encodes testicular and adrenal 3β-hydroxysteroid dehydrogenase-isomerase

Thermostability of 3-hydroxysteroid dehydrogenase-isomerase (3HSD) activity was examined in testes and adrenal glands from several inbred lines and feral mice. A thermolabile varant of 3HSD was detected in the feral Brno mice. The thermostability (t _1/2) of 3HSD was approximately 7 min for both testes and adrenal glands from C57BL/6J mice, compared with 4 min for both tissues from Brno mice. Comparison of testicular and adrenal 3HSD thermostability in six kinds of mice indicated that the t _1/2of 3HSD was correlated in the two tissues and could be classified into two distinct types, thermolabile and thermostable. In contrast, quantitative variants in 3HSD activity were not correlated in the two tissues. These data are consistent with the hypothesis that testicular and adrenal 3HSD is encoded by the same structural gene but that expression of 3HSD activity is independently controlled in testes and adrenal glands.This work was supported by NICHHD National Research Service Award HD-06392 to J.R.D.S. and Grant HD-17916 to A.H.P.     

Localization of type 5 17β-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization

The mouse enzyme type 5 17-hydroxysteroid dehydrogenase (17-HSD) catalyzes the conversion of androstenedione to testosterone and, to a lesser degree, the conversion of estrone to estradiol. In order to determine the exact sites of action of type 5 17-HSD, we studied the cellular localization of the mRNA of the enzyme in mouse tissues by using in situ hybridization. Specific hybridization signal was found in the liver, ovary, adrenal cortex, and kidney. In the liver of mice of both sexes, a strong signal was observed in all hepatocytes. In the ovary, specific labeling was detected in the granulosa and theca interna cells in growing follicles and in luteal cells. In the female adrenal cortex, intense labeling was restricted to the zona reticularis, whereas no type 5 17-HSD mRNA expression could be found in the male adrenal cortex. In the kidney of mice of both sexes, type 5 17-HSD mRNA was expressed in epithelial cells in both the proximal and distal convoluted tubules. The data indicate that androgens and estrogens are formed via the action of type 5 17-HSD in specific cell types in the liver, ovary, adrenal cortex, and kidney.This work was supported by Genome Canada and Genome Québec.     

Correlation of plasma estradiol-17β and progesterone levels with ultrastructure and histochemistry of ovarian follicles in the white-spotted char,Salvelinus leucomaenis

Summary Plasma estradiol-17 and progesterone profiles were correlated with morphological changes in ovarian follicles during the preovulatory and postovulatory periods in the white-spotted char, Salvelinus leucomaenis. Plasma estradiol levels were highest in September, and were followed by a sharp drop in October; they remained very low throughout the postovulatory period. There was a good correlation between plasma estradiol levels and the gonadosomatic index, thus suggesting that estradiol is responsible for the synthesis of vitellogenic proteins. Plasma progesterone levels were very low in August, began to rise in September and reached a peak soon after ovulation; progesterone remained high for several days after ovulation. A preovulatory rise in plasma progesterone levels was recorded, and this is discussed in relation to the induction of oocyte maturation.In the preovulatory follicles, neither granulosa cells nor special thecal cells (ST cells) showed ⁵, 3-hydroxysteroid dehydrogenase (3-HSD) activity. In the young postovulatory follicles, in contrast, the ST cells showed intense 3-HSD activity with extensive agranular endoplasmic reticulum and numerous large mitochondria, while granulosa cells did not show 3-HSD activity. These results strongly suggest that the ST cells are the major sites of progesterone synthesis during the postovulatory period.Nanae Fish Culture Experimental Station Contribution No 14     

Exposure to high fluoride concentration in drinking water will affect spermatogenesis and steroidogenesis in male albino rats

Sodium fluoride (NaF) administered orally to adult male rats at a dose level of 4.5 ppm and 9.0 ppm for 75 days caused significant decrease in the body weight, brain index and testicular index. A significant decrease in sperm count, sperm motility, sperm viability and sperm function (HOS positive) with increased sperm abnormalities was also observed in NaF-exposed male rats. The activity levels of testicular steroidogenic marker enzymes 3-hydroxysteroid dehydrogenase (3-HSD) and 17-hydroxysteroid dehydrogenase (17-HSD) were significantly decreased in NaF-treated rats indicating decreased steroidogenesis and in turn spermatogenesis in rats exposed to NaF.