Rat Preputial Gland Research Articles

Properties of rat and mouse β-glucuronidase mRNA and cDNA, including evidence for sequence polymorphism and genetic regulation of mRNA level

cDNA clones containing partial sequences for β-glucuronidase (βG) were constructed from rat preputial gland RNA and identified by their ability to selectively hybridize βG mRNA. One such rat clone was used to isolate several cross-hybridizing clones from a mouse-cDNA library prepared from kidney RNA from androgen-treated animals. Together, the set of mouse clones spans about 2.0 kb of the 2.6-kb βG mRNA. Using these cDNA clones as probes, a genomic polymorphism for DNA restriction fragment size was found that proved to be genetically linked to the βG gene complex. A fragment of βG cDNA was subcloned into a vector carrying an SP6 polymerase promoter to provide a template for the in vitro synthesis of single-stranded RNA complementary to βG mRNA. This provided an extremely sensitive probe for the assay of βG mRNA sequences. Using either nick-translated cDNA or transcribed RNA as a hybridization probe, we found that mouse βG RNA levels are strongly induced by testosterone, and that induction by testosterone is pituitarydependent. During the lag period preceding induction, during the induction period itself, and during deinduction following removal of testosterone, βG mRNA levels paralleled rates of βG synthesis previously measured by in vivo pulse-labelling experiments. Genetic variation in the extent of induction affected either the level of βG mRNA or its efficiency of translation depending on the strain of mice tested.

Transformation of dehydroepiandrosterone into dihydrotestosterone by isolated cells from rat preputial gland

After the rat preputial gland was treated with collagenase and trypsin, five bands of cells were isolated by centrifugation in Ficoll gradients. Homogenates of the heavier cells (Bands IV and V) which contained less lipids, were more active than the homogenates of the lighter cells (Bands I, II and III) in transforming [1,2- 3H]-dehydroepiandrosterone ([l,2- 3H]-DHA) into [ 3H]-androstenedione and [ 3H]-testosterone and the latter into [ 3H]-dihydrotestosterone (DHT). In the presence of NAD, NADH and NADPH-generating system, [1,2- 3H]-DHA was transformed into [ 3H]-DHT in 50–60% yield by homogenates of cells in Bands IV and V. DHT levels in the preputial gland were measured by radio-immunoassay. The levels in female rats reduced by 77% from 3.14 ± 0.27 to 0.72 ± 0.10pg/mg tissue after adrenalectomy, and by 45% to 1.71 ± 0.10 pg/mg tissue after ovariectomy. In male rats, the level reduced by 15% from 4.58 ± 0.55 to 3.88 ± 0.62pg/mg tissue after adrenalectomy and by 40% to 2.74 ± 0.21 pg/mg tissue after orchidectomy. These results demonstrated the transformation of DHA into DHT in the preputial gland of the rat, and that the adrenal is an important source of precursor steroid (DHA) for DHT formation in the preputial gland.

Receptor-mediated entry of beta-glucuronidase into the parasitophorous vacuoles of macrophages infected with Leishmania mexicana amazonensis.

125I-labeled rat preputial gland beta-glucuronidase was shown by light and electron microscopic radioautography to accumulate within the parasitophorous vacuoles of in vitro derived bone marrow macrophages infected with Leishmania mexicana amazonensis. beta-glucuronidase uptake was mediated by the mannose receptor, since the penetration of the ligand was inhibited by mannan. Uptake was detected as soon as 4 h after incubation of infected cells with the ligand, and increased at 24 and 48 h. The label persisted in the vacuoles for at least 24 h after a 24-h pulse with the ligand, a finding compatible with the relatively long half-life of labeled beta-glucuronidase in normal macrophages. Parasitophorous vacuoles were also labeled in macrophages exposed to the ligand only before infection, indicating that secondary lysosomes containing the ligand fused with the parasitophorous vacuoles. Another mannosylated ligand, mannose-BSA, which, in contrast to beta-glucuronidase, is rapidly degraded in macrophage lysosomes, did not detectably accumulate in the vacuoles. The results support and extend information previously obtained with electron opaque tracers that emphasizes the phagolysosomal nature of Leishmania parasitophorous vacuoles. In addition, the results suggest that appropriate mannosylated molecules may be used as carriers for targeting of leishmanicidal drugs to the parasitophorous vacuoles of infected macrophages.

129 Antimitotic effect of the hormonal contraceptive on the preputial gland of rat

129 Antimitotic effect of the hormonal contraceptive on the preputial gland of rat

Mechanism of action of alpha-melanocyte-stimulating hormone in rat preputial glands: the role of androgen metabolism.

The metabolism of testosterone and 5 alpha-dihydrotestosterone has been studied in vitro in preputial glands of posterior hypophysectomized, totally hypophysectomized and control sham-operated rats. The level of C19 steroid 5 alpha-reductase activity/unit of preputial gland DNA did not fall after removal of the neurointermediate lobe and rose after total hypophysectomy. It was concluded from this that the androgen unresponsiveness of the preputial glands of hypophysectomized rats was not due to a near-total lack of 5 alpha-reductase and hence that the combined synergistic action of testosterone and alpha-melanocyte-stimulating hormone (alpha-MSH) on preputial gland activity was unlikely to be due to an alpha-MSH-mediated restoration of 5 alpha-reductase levels in hypophysectomized rats. Levels of 3 alpha and 3 beta-hydroxysteroid dehydrogenase but not of 17 beta-hydroxysteroid dehydrogenase appeared to be altered by hypophysectomy.

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution.

By in vitro translation of mRNA's isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, beta-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA's for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.

Cloning of a cDNA complementary to rat preputial gland β-glucuronidase mRNA

Messenger RNA was isolated from rat preputial glands by guanidine HCl extraction, ethanol and salt precipitation, followed by chromatography on oligo(dT) cellulose. Double-stranded cDNA was synthesized from the mRNA and inserted into the Pst 1 site of the plasmid pBR322 by the poly(dG)·poly(dC) tailing and annealing procedure. The hybrid plasmids were used to transform E. coli HB101. Recombinant clones were screened for those containing cDNA inserts complementary to β-glucuronidase mRNA by a hybridization-selection procedure. One clone, containing an insert of about 1.2 kilobases, hybridized to preputial gland mRNA which, when translated in vitro , gave a product that migrated with the β-glucuronidase subunit on polyacrylamide gels.

Isolation and partial characterization of the oligosaccharide moieties of rat preputial β-glucuronidase

The oligosaccharide alditols of rat preputial gland β-glucuronidase have been isolated by reductive alkaline hydrolysis of the enzyme followed by gel filtration. Since the product was about decasaccharide in size, and as β-glucuronidase contains about 20 sugar residues per subunit, the enzyme must contain two oligosaccharides per subunit. Two major subfractions were obtained. Chemical and enzymatic studies on the initial oligosaccharide alditol fraction and on the separated subfractions indicated the presence of a mannosyl-chitobiosyl core and permitted formulation of the following structures for the two different oligosaccharide alditols: ▪ ▪

The effect of sexual hormones on the lipid and proteinaceous secretion of the rat preputial sebaceous gland

The diameters of Sudan Black B stained sebum vesicles and acid hematein stained perinuclear granules, and the numerical density of the latter, were determined in mature sebaceous preputial cells from normal male and female rats, testosterone-treated female rats and estradiol-treated male rats. Statistical analysis by Student's t-test showed that the diameter of lipid droplets was significantly higher in male than in female controls and that testosterone increased female values up to make control levels. Estradiol treatment decreased male values to levels below those of normal male and female controls and of testosterone-treated female rats. Diameters of perinuclear granules did not vary among animal groups but their numerical density was larger in testosterone treated than in normal female rats or estradiol treated male rats. Lipid droplet sizes and perinuclear granule numbers are thus increased by androgens and decreased by estrogens, which was interpreted an meaning that sexual hormones do not only act on sebaceous cell multiplication or turnover time as previously known, but also on the production of lipid and the output of beta-glucuronidase containing granules in this gland.

Lysosomal cathepsin B-like activity: mobilization in prereplicative and neoplastic epithelial cells.

Extracellular release of acid thiol proteinase activity by prereplicative and neoplastic epithelial cells was studied in serum-free, chemically defined media (CDM) in vitro. Cells isolated from urinary bladder of male bullfrogs and endometrium of ovariectomized rats each showed preferential secretion of cathepsin B-like (CB) activity within 30 min after exposure to carcinogenic nitrosamines (5 X 10(-4) M) or to mitogenic estrogen 1 X 10(-9) M), respectively. In contrast, release of such proteinase, and stimulation of cell proliferation were far less extensive in rat preputial gland cells treated with estradiol-17 beta. Striking secretion of CB was characteristic of neoplastic, but not noncancerous, epithelial cells from human ectocervix. Neoplastic cells with divergent rates of cell-to-cell aggregation were separated by a filtration method. Those cells with high rates of intercellular aggregation also exhibited higher rates of cell proliferation in CDM, as well as in soft gels, and a greater level of CB release than corresponding cancer cells with a relatively low degree of intercellular adhesion. Brief treatment of neoplastic cervical epithelial cells with liposomes containing entrapped leupeptin, a potent inhibitor of CB activity, elicited a sharp reduction in both cellular thiol proteinase activity and cell growth as compared to appropriate controls. These data indicate that mobilization of lysosomal CB activity in prereplicative and malignant cells may play a significant role in the promotion of cell proliferation.

Innervation of the rat preputial gland.

Acetylcholinesterase, butyrylcholinesterase and monoamine oxidase activities were observed in the neuronal and non-neuronal components of the preputial gland. Histological, histochemical and experimental studies clearly indicate that the preputial gland of the rat is innervated by cholinergic as well as adrenergic nerves.

Immunocytochemistry on acid hydrolases in preputial gland cells

Present knowledge of the in situ localization of acid hydrolases is mainly based on enzyme-cytochemical observations on acid phosphatase and aryl sulfatase. In rat preputial gland cells, β-glucuronidase and acid phosphatase were demon- strated this way in secretory granule-like lysosomes. we have applied immunocytochemistry, which in principle allows the detection of proteins at intracellular sites where they are not (yet) enzymatically active.

Studies on the αL-Iduronidase Activity of β-Glucuronidase Preparations from Bovine Liver, Rat Liver, and Rat Preputial Gland<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

A commercial preparation of bovine liver beta-glucuronidase contained two distinct enzyme species, both of which catalyze the hydrolysis of 4-methylumbelliferyl alpha-L-iduronide. The species with a molecular weight of about 290,000 was devoid of phenyl alpha-L-iduronidase activity and exhibited 4-methylumbelliferyl beta-D-glucuronidase activity. The species with a molecular weight of about 78,000 was active towards phenyl alpha-L-iduronide but lacked the latter activity. Studies of the kinetics of inhibition and heat inactivation suggested that the hydrolysis of 4-methylumbelliferyl alpha-L-iduronide is due to the beta-glucuronidase in the case of the 290,000-dalton species. The highly purified beta-glucuronidase preparations derived from rat preputial gland and liver lysosomes also exhibited 4-methylumbelliferyl alpha-L-iduronidase activity. These findings support the view that beta-glucuronidase can hydrolyze certain alpha-L-iduronide bonds and raise the possibility that beta-glucuronidase may play a role in the catabolism of iduronic acid-containing glycosaminoglycans.

A preliminary crystallographic study of β-glucuronidase from rat preputial gland

Large single crystals of rat preputial gland β-glucuronidase have been grown by dialysis against 2-methyl-2,4-pentanediol. The crystals are tetragonal, space group P4 12 12 or P4 32 12 with cell dimensions a = 103.5 A ̊ and c = 279.8 A ̊ . Each asymmetric unit contains one half of a tetramer composed of subunits of 69,000 molecular weight.

In vitro characterization of adrenergic receptors mediating extrusion of preformed sebum from preputial gland of rat.

Extrusion of preformed sebum from the preputial glands of rat after phenylephrine and adrenaline treatment, and its inhibition by alpha-receptor blocking agents under in vitro conditions, shows that secretory response of the glands is influenced by alpha-adrenergic receptors, and isoproterenol--a beta-agonist--is not effective in elicitation of exudation of secretion from the preputial gland.

Multiple kinetic forms of beta-glucuronidase.

Partially purified chick embryo liver beta-glucuronidase and highly purified beta-glucuronidase from human placenta and rat preputial gland exhibit multiple kinetic forms which appear to exist in an equilibrium which can be shifted by varying the assay conditions. All three enzymes exist in a low Km form, which predominates at pH 3 and is stabilized by bovine serum albumin, and a high Km form, which predominates at pH 5.5 to 6.0 in the absence of serum albumin. At intermediate pH values both forms are present. Addition of 0.2 M NaCl shifts the equilibrium toward the high Km form. Both forms of these enzymes are active on 4-methyl umbelliferyl-beta-D-glucuronide and on the hexasaccharides of chondroitin-6-SO4, chondroitin, and hyaluronic acid, with the low Km forms showing 2- to 20-fold more activity on the oligosaccharide substrates than the high Km forms.

An electron-microscope study of β-glucuronidase crystals

beta-Glucuronidase from rat preputial glands was crystallized as thin sheets having p6 symmetry in projection with a equal 20.2nm. A filtered image was produced by Fourier methods to a resolution of 2.2 nm by averaging information from six areas. This suggests an approximately triangular molecular outline in projection, and this is taken to indicate a probable tetrahedral arrangement of the four subunits of the beta-glucuronidase molecule.

Chick embryo liver beta-glucuronidase. Comparison of activity on natural and artificial substrates.

The beta-glucuronidase in homogenates of 12-day chick embryo livers catalyzed the release of glucuronic acid from 4-methylumbelliferyl-beta-D-glucuronide and from the nonreducing terminals of the hexasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4 at rates of 143, 114, and 108 nmol of glucuronic acid/h/mg of protein, respectively, when assayed at pH 3.5 in 0.05 M sodium acetate buffer. During a 60-fold purification of the enzyme, the ratios of the activities on these substrates did not change. When 4-methylumbelliferyl-beta-D-glucuronide was used as substrate the enzyme was active at pH values from 3.0 to 5.5, with maximal activity between pH values 4.0 and 4.5. Concentrations of NaCl from 0.15 to 0.3 M inhibited the activity at low pH values but activated the enzyme between pH 4.0 and 5.5. The enzyme was active on the chondroitin-6-SO4 hexasaccharide from pH 3.0 to 5.5, with a broad optimum between 3.0 and 4.5. NaCl inhibited the activity on the oligosaccharide substrate at all pH values. Eadie-Scatchard plots of rates of 4-methylumbelliferyl-beta-D-glucuronide hydrolysis at substrate concentrations ranging from 2 to 1000 microM showed multiple kinetic forms of the enzyme, a form with a Km of approximately 11 microM, and a second form with a Km of approximately 225 microM. The pH optimum of the low Km form was 3.5 to 4.0; that of the high Km form was pH 4.5. NaCl inhibited the activity of the low Km form, but activated the high Km form of the enzyme. Chondroitin SO4 oligosaccharides competed with 4-methylumbelliferyl-beta-D-glucuronide for the low Km form of the enzyme but had little effect on the hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide by the high Km form of the enzyme. The activities of the beta-glucuronidase on tetra-, hexa-, octa-, and decasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4, measured using a new assay procedure which can detect the formation of 1 nmol of product, were similar, although rates were somewhat lower for the higher oligosaccharides. With the exception of the chondroitin-4-SO4 tetrasaccharide, all of the oligosaccharide substrates saturated the enzyme at concentrations of 20 to 30 microM, indicating Km values of less than 10 to 15 microM for the oligosaccharides. Highly purified beta-glcuronidases from human placenta and from rat preputial gland also showed multiple kinetic forms when assayed using 4-methylumbelliferyl-beta-D-glucuronide as substrate.

Attraction of rats to sulfur compounds

Adult male and female Sprague—Dawley rats were assayed for olfactory preference of various sulfur-containing compounds. Both sexes preferred dimethyl disulfide. Male rats were also interested in dimethyl sulfite and hexyl mercaptan odor, while females preferred methyl isothiocyanate. The three latter compounds were found to be present in rat preputial gland volatiles and may aid in sex recognition.

Rat olfactory response to aliphatic acetates

Volatile compounds of the male rat preputial gland extracts were found to containn-aliphatic acetates, by gas chromatography and mass spectroscopy. Female rats preferred the odor of various saturated and unsaturatedn-aliphatic acetates while the male rats were repelled by or indifferent to, most of these compounds. A bioassay apparatus recorded the number of approaches and the investigation time for each animal offered the test compounds. The possibility of aliphatic acetates' involvement as sex attractants produced by the male rat is considered.