Rat Preputial Gland Research Articles

Detection of α2u-globulin in rat pup preputial gland by MALDI-TOF mass spectrometry

Abstract The α2u-globulin, a soluble protein identified in the urine and preputial gland of adult male rat is reported to be pheromone carrier. The pup preputial gland plays a significant role in chemical communication for mother-young interaction; however, the presence of a pheromone-carrying protein in the pup preputial gland has not been confirmed. Therefore, the present study was carried out to identify the α2u-globulin in the pup preputial gland by Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF). The preputial glands of prepubertal rats were subjected to one-dimensional SDS-PAGE. In-gel trypsin digestion of a 18 kDa band was carried out and analyzed by MALDI-TOF. The results of a MASCOT search showed the presence of α2u-globulin in the 18 kDa band. In contrast to the report of the synthesis of this protein only in adult rats, the identification of this protein in pup preputial gland is significant. The results suggest that synthesis of α2u-globulin in the rat preputial gland starts in the prepubertal stage itself. In prepubertal rats, the preputial gland is a source of pheromone for performing anogenital licking behaviour by the mother rat. Since α2u-globulin belongs to the lipocalin (ligand carrier) family, it might carry the volatile for processing pheromonal communication in mother-pup bonding in rat.

Sex- and Gonad-Affecting Scent Compounds and 3 Male Pheromones in the Rat

This study was aimed at identifying sex pheromones of the rat (Rattus norvegicus). We characterized the volatiles and semivolatiles of rat preputial gland and voided urine by using gas chromatography–mass spectrometry (GC–MS) and quantified them by their GC areas (abundances) and percentage of GC areas (relative abundances). Although all the compounds other than 4-heptanone and phenol detected were shared by males and females, the quantities for some of these sex-common compounds exhibited sexual dimorphism and decreased with gonadectomy. Thus, these compounds might be sex pheromones. Among them, squalene from preputial glands and 2-heptanone and 4-ethyl phenol from urine were 3 major compounds. They were richer in males and could be suppressed by castration. Adding any of the 3 compounds (at a concentration higher than its physiological level in male urine) to castrated male urine (CMU) increased the attractiveness of CMU to sex-naive females. Adding the 3 together (at the levels in normal male urine) to CMU significantly increased the attractiveness of CMU to females. However, such combination did not fully restore females' preference for urine from intact males, suggesting that some other trace compounds such as 4-heptanone and phenol might also play some roles in sex attractiveness. Thus, squalene, 2-heptanone, and 4-ethyl phenol were indeed male pheromone molecules in rats. Our study also indicates that E,E-β-farnesene and E-α-farnesene, both richer in females than males, might be putative female pheromones.

LXRα Enhances Lipid Synthesis in SZ95 Sebocytes

Differentiation of sebocytes is strongly associated with enhanced lipid synthesis and accumulation in the cells. Liver X receptors (LXRs) are members of the nuclear receptor superfamily, which play a critical role in cholesterol homeostasis and lipid metabolism. We examined whether LXRalpha regulated lipid synthesis in the immortalized human sebaceous gland cell line SZ95. When the SZ95 sebocytes were treated with the ligand of LXR such as TO901317 or 22(R)-hydroxycholesterol, lipid droplets were accumulated in the majority of cells when examined by Oil Red O staining. The expression of the known LXR targets, such as fatty acid synthase and sterol regulatory-binding protein-1, was induced by TO901317. TO901317 treatment increased expression of LXRalpha but not that of LXRbeta. Transfection of antisense LXRalpha significantly decreased TO901317-induced target gene expression and lipid droplet accumulation, suggesting a major role of LXRalpha in differentiation of sebocytes. Further, TO901317 decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase that was induced by lipopolysaccharide treatment. Together, these results indicate that important roles of LXRalpha in differentiation and inflammatory signaling in sebaceous glands. Thus, we suggest that LXR ligands could provide a new class of therapeutic agents for sebaceous gland-associated disorders such as seborrhea and acne.

The Melanocortin 5 Receptor is Expressed in Human Sebaceous Glands and Rat Preputial Cells

Melanocortins regulate pigmentation, adrenal hormone secretion, immune functions, lipid metabolism, and feeding behaviors in rodents. These peptides include adrenocorticotrophic hormone, melanocyte stimulating hormone, beta-lipotrophin, and the endorphins. Lipid metabolism in sebaceous glands and preputial glands of rodents is regulated by alpha-melanocyte stimulating hormone, the major agonist for melanocortin receptors. Five melanocortin receptor subtypes have been identified that differ in their tissue localization and affinities for melanocortin ligands. Targeted disruption of the melanocortin 5 receptor in transgenic mice results in widespread dysfunction of exocrine glands, including a marked decrease in sebum production. A role for melanocortins in the modulation of human sebum production has not been established. The goal of this study is to determine which melanocortin receptors are expressed in human sebaceous glands. Messenger RNA was isolated from human sebaceous glands and the reverse transcriptase polymerase chain reaction was performed using primers specific for each of the melanocortin receptor subtypes. Transcripts were detected for the melanocortin 5 receptor. A polyclonal chicken antihuman antibody to the melanocortin 5 receptor localized to sebaceous glands, eccrine glands, hair follicles, and epidermis in human skin, rat skin, cultured human sebocytes, and rat preputial cells. Presence of the melanocortin 5 receptor protein in human sebaceous glands and rat preputial glands was further verified by Western blotting. These data support further investigation of the role of melanocortins in the regulation of human sebum production and support the use of the rat preputial system as an experimental model in sebaceous gland physiology.

Phosphomannosyl receptors on the surface of spermatozoa from the cauda epididymis of the rat

This study demonstrates that beta-glucuronidase from rat preputial glands binds with high affinity to spermatozoa from the cauda epididymis. The binding was calcium-independent and was inhibited by mannose-6-phosphate, but not by other phosphorylated or non-phosphorylated sugars. Binding was also inhibited by alpha-mannosidase from Dictyostelium discoideum, an enzyme known to have mannose-6-phosphate as the ligand. From solubilized sperm membranes, a protein of > 200 kDa and one of 45 kDa, were absorbed to a column of D. discoideum enzyme and to a phosphomannan column respectively, and eluted with mannose-6-phosphate. According to histochemical observations at the light and the electron microscopic level, gold particles coated with the enzyme became bound to the external surface of the plasmalemma in the acrosomal region of caudal spermatozoa. Similar labelling was observed using gold particles coated with antibodies against the rat 300 kDa phosphomannosyl receptor. The existence of phosphomannosyl receptors on the sperm plasma membrane, and our previous demonstration of the presence of affinity sites for epididymal beta-galactosidase on these gametes which is inhibited by phosphofructosyl derivatives, suggest strongly that maturing spermatozoa could be a target for glycosidases secreted into the lumen of the cauda epididymis, which then become bound to these cells via different ligand-receptor systems.

Purification and characterization of a soluble peroxidase of rat preputial gland: comparison with lactoperoxidase.

A highly active soluble peroxidase (donor: H2O2 oxidoreductase EC 1.11.1.7) has been purified from the preputial gland of the rat by hydroxylapatite chromatography, ammonium sulfate fractionation, Sephadex gel filtration and affinity chromatography on con A-Sepharose. The enzyme shows apparent homogeneity when analysed by acid and alkaline-PAGE. Its molecular, spectral, kinetic and catalytic properties were compared with those of bovine lactoperoxidase (LPO). Preputial gland peroxidase (PPO) is a glycoprotein of molecular weight of 70-73 kDa slightly lower (78 kDa) than that of LPO. Using isoelectric focussing, PPO was resolved into eight different closely spaced protein species spanning a pI range of 5.4 to 6.4, while LPO focuses into several closely spaced protein bands in the pI range 8.5-9.3. PPO is similar to LPO in its spectral (Soret) and some kinetic properties, but it differs significantly from LPO in substrate (H2O2) tolerance and substrate inactivation. PPO also differs from LPO in showing differential inactivation by SDS. Both enzymes are glycoproteins and although concanavalin A (con A) showed a variable interaction with both enzymes, wheat germ agglutinin interacted specifically with LPO only. We suggest that PPO, the secretory peroxidase of the preputial gland, differs significantly from LPO in its molecular and catalytic properties.

Cyclic AMP-Receptor Protein Activity in Rat Preputial Cells

Cells from the rat preputial gland--a type of sebaceous gland--exhibited specific responsiveness of cyclic 3',5'-adenosine monophosphate (AMP) dependent protein kinase to stimulation by agents that elevate intracellular cyclic AMP. Electron microscopy shows that the rat preputial gland resembles the human sebaceous gland, not only in terms of containing a sebocyte-like population of cells in an acinar arrangement at different maturational stages, but also in the morphology of its organelles such as abundant and sometimes atypical mitochondria, many perinuclear lysosomes with crystalline inclusions, lipid droplets of various sizes, and peroxisomes. Other cell types, among them duct and inflammatory cells, were evident in the tissue sections, but constituted a minor component. Responses to stimulation of the adenylate cyclase-protein kinase pathways were determined using preputial cells that had been both freshly dispersed and grown in monolayer culture. Stimulation with isoproterenol (IPR) or forskolin (FS) resulted in both cases in an increase of cyclic AMP binding of the regulatory (R) subunits of cyclic AMP-dependent protein kinase, as determined by photoaffinity labeling of R subunits with an azido analog of cyclic AMP ([32P]-8-azido cyclic AMP). Cells from the epidermis under comparable conditions responded to a lesser degree and with a different distribution of R subunit isoforms. There are, therefore, differences in receptor activity as well as in the transduction pathways between the two types of epithelial cell populations. These results indicate that the preputial gland contains precursor cells that differentiate in culture to retain specific molecular mechanisms of action mediated via cyclic AMP.

Dodecyl propionate, attractant from rat pup preputial gland: Characterization and identification

A chemical agent contained in the rat pup preputial gland was found to regulate anogenital licking, a behavioral pattern crucial to pup survival. Combined gas chromatography and mass spectrometric analysis led to identification of four components in addition to high molecular alkanes (waxes). Independent synthesis added to behavioral observations were used to quantify the dam's response to these components. Among them, dodecyl propionate (DP), a pheromone-like compound, appears to be the only one efficient enough to induce, in the dam, behavior patterns similar to those observed with crude lipidic extract from the preputial gland secretion. Various esters previously were predicted in the adult rat preputial gland. DP is the first ester identified from mammalian infant tissue. These results, in combination with previous findings, allow discussion of the DP pheromonal role.

Anti-5α-Reductase Autoantibodies in the Serum of Patients with Prostatic Cancer*

Human sera were tested for their ability to inhibit 5 alpha-reductase binding of a potent inhibitor of the enzyme. Thirty one of 227 serum samples from patients diagnosed or suspected of prostatic cancer had a significant inhibitory activity, whereas 128 serum samples from other patients were inactive. The majority of the inhibitory activity was in the IgG fraction purified by chromatography on a protein A-Sepharose affinity column and an anti-human IgG-agarose column. IgG fractions from non-inhibitory sera were inactive. Inhibitory IgG also inhibited the enzymatic activity of microsomal 5 alpha-reductase from liver, ventral prostate and preputial gland of rat, and liver, prostate, and facial skin of human. The inhibitory IgG had no effect on NADH-menadione reductase or 17 beta-hydroxysteroid dehydrogenase. These results suggest that 5 alpha-reductase autoantibodies are present in the blood of some prostatic cancer patients.

The Macrophage Mannose Receptor: Current Status

Sugar-specific recognition of oligosaccharide chains present on macromolecules and particles has emerged as an important mechanism by which macrophages carry out a variety offunctions including host defense, molecular scavenging, and cell-cell recognition. Among the oligosaccharide determinants that are recognized by macrophages, terminal mannose residues are the most thoroughly studied. More than a decade ago, in vivo experiments showed that a variety of glycoproteins bearing high mannose chains, including the lysosomal enzyme {j-glucuronidase, were rapidly and specifically cleared from the bloodstream after intravenous infusion (1). It is now widely appreciated that in vivo clearance of mannose glycoconjugates is mediated by a receptor found in liver Kupffer and endothelial cells and in mononuclear phagocytes (2). With the exception of lysosomal hydrolases, which possess high mannose chains as a remnant of the mannose-phosphate targeting marker, glycoproteins and membrane glycoconjugates of most higher organisms bear processed or complex N-linked oligosaccharide chains (3). As such, mannose is nearly always relegated to a penultimate or more inferior position in the carbohydrate structure. Lower organisms, however, including many infectious agents, have little facility to process N-linked oligosaccharide chains and, consequently, express abundant mannose, glucose, and other sugars on their surfaces. It is not surprising, then, that higher organisms have evolved sugar-specific recognition molecules as part oftheir host-defense system. Macrophages recognize, in addition to mannose and L-fucose, molecules and particles bearing galactose, glucose, and N-acetylneuraminic acid residues (2). The mannose receptor has served as an excellent model for studies of receptor recycling in the macrophage. The availability of several high affinity ligands and anti-receptor antibodies have permitted a detailed analysis of receptor movement in macrophages. {j-Glucuronidase, a lysosomal glycosidase that can be easily purified from rat preputial glands, and mannose-derivitized bovine serum albumin (mannoseBSA) , bind the mannose receptor with high affinity. Many other glycoconjugates have been used as ligands as well, including ricin A-chain, yeast mannan and invertase, ovalbumin, and ribonuclease B. Binding and internalization studies have shown that the mannose receptor is both phagocytic (4)

Relationship of sebaceous cell stage to growth in culture.

The maturational stage at which sebaceous epithelial cells are irreversibly committed to autolysis has not been determined in a biologic system. The purpose of these experiments was to determine the relationship of sebocyte maturation to their ability to grow in culture. Single cell suspensions from adult rat preputial glands were prepared, and sebocyte stages were classified using lipid staining as one criterion of maturation. Cells were subjected to one-step, isokinetic discontinuous density gradient centrifugation in Percoll. Cells were cultured on 3T3 feeder layers in an epithelial cell medium containing 20% fetal calf serum, growth factors, and antibiotics. Growth in culture occurred from sebocytes in all gradient fractions. The least growth was found from cells in the lightest density fraction (1.020), which contained sebocytes with the most lipid, i.e., the most mature. Over fivefold more growth occurred from cells in the most dense fraction (1.080), which contained undifferentiated and immature sebocytes. Cell growth in culture was then correlated with the type of sebocyte in each fraction plated. Although cell growth in culture correlated significantly with the number of undifferentiated cells (r2 = 0.460), epithelial colonies were found in the absence of discernible undifferentiated cells in most fractions. Growth in culture correlated much better with the number of early plus mid-differentiated sebocytes (r2 = 0.702). These data suggest that, whereas mature sebocytes are not capable of attachment/proliferation, early differentiation of sebocytes is compatible with retention of the capacity to proliferate. If this property is shared by human sebocytes, it would contribute to the intransigence and diversity of acne-form lesions.

Relationship of sebaceous cell stage to growth in culture

The maturational stage at which sebaceous epithelial cells are irreversibly committed to autolysis has not been determined in a biologic system. The purpose of these experiments was to determine the relationship of sebocyte maturation to their ability to grow in culture.Single cell suspensions from adult rat preputial glands were prepared, and sebocyte stages were classified using lipid staining as one criterion of maturation. Cells were subjected to one-step, isokinetic discontinuous density gradient centrifugation in Percoll. Cells were cultured on 3T3 feeder layers in an epithelial cell medium containing 20% fetal calf serum, growth factors, and antibiotics.Growth in culture occurred from sebocytes in all gradient fractions. The least growth was found from cells in the lightest density fraction (1.020), which contained sebocytes with the most lipid, i.e., the most mature. Over fivefold more growth occurred from cells in the most dense fraction (1.080), which contained undifferentiated and immature sebocytes. Cell growth in culture was then correlated with the type of sebocyte in each fraction plated. Although cell growth in culture correlated significantly with the number of undifferentiated cells (r2= 0.460), epithelial colonies were found in the absence of discernible undifferentiated cells in most fractions. Growth in culture correlated much better with the number of early plus mid-differentiated sebocytes (r2=0.702).These data suggest that, whereas mature sebocytes are not capable of attachment/proliferation, early differentiation of sebocytes is compatible with retention of the capacity to proliferate. If this property is shared by human sebocytes, it would contribute to the intransigence and diversity of acne form lesions.

Rat liver β-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells

A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.

Monoclonal antibody toward lysosomal cathepsin B cross-reacts preferentially with distinct histone classes

1. 1. A set of monoclonal antibodies (Mab) was prepared against cathepsin B (CB) from rat preputial-gland, an organ characterized by rapidly-renewing cell populations, which is a uniquely enriched source of lysosomal enzymes, including CB. Minute amounts of CB are known to be transferred abruptly to the nuclear compartment in a variety of activated cells. 2. 2. Since, on the basis of its stringent substrate requirements, CB was expected to function at limited protein loci in chromatin, Mab Line II-B4 was used to probe Western blots of chromatin fractions and selected proteins. 3. 3. The Mab, which was not directed against the active site of CB, cross-reacted preferentially with histones 3 and 4 (H3 and H4) in acid-soluble fractions of chromatin from rat preputial-gland. Line II-B4 also recognized H3 and H4 selectively in calf thymus histones and among histones purified from a wide range of sources from yeast to man. HMG 1 was minimally immunoreactive among preputial gland constituents and carbonic anhydrase (CA) was also sensitive to the Mab. 4. 4. The common determinants were not shared by any of the H1 series, nor by H2A, H2B, protein A24 or a wide range of natural and synthetic products. 5. 5. Origin of the antigenicity was traced by chemical modifications of H3, H4 and CA to the critical contribution of arginine and hydrophobic amino acid residues in its immediate environment, indicating that Line II-B4 may be directed against an epitope comprising the specific binding-site of CB and its selective substrate(s). 6. 6. These data suggest that certain highly conserved cellular constituents may be uniquely vulnerable to limited proteolysis in preproliferative cells responding to mitotic signals.

Sex-Independent Synthesis of a2u-Globulin and Its Messenger Ribonucleic Acid in the Rat Preputial Gland: Biochemical and Immunocytochemical Analyses*

alpha 2u-Globulin is the principal urinary protein of the mature male rat. The major urinary source of this protein is the liver where it is synthesized and secreted by hepatocytes under hormonal regulation. High levels of alpha 2u-globulin and its messenger RNA (mRNA) are also present in the preputial gland of both male and female rats, and neither castration nor ovariectomy significantly alters the preputial concentration of this protein and its mRNA. Per unit mass of RNA and protein, the preputial gland as compared to liver contains about 3-fold higher level of alpha 2u-globulin mRNA and about 300-fold higher level of alpha 2u-globulin. Despite a 3-fold (300%) difference in the content of alpha 2u-globulin mRNA, nuclear run-off experiments show only a 30% higher rate of alpha 2u-globulin gene transcription in the preputial gland than in the liver. Immunocytochemical analyses reveal that the liver possesses two alpha 2u-globulin cell populations, one showing higher immunoreactivity than the other. In contrast, the preputial gland contains only one type of alpha 2u-globulin containing acinar cells, and a large amount of alpha 2u-globulin accumulates in the ductal lumen. From these results we conclude that the 300% higher level of alpha 2u-globulin mRNA in the preputial gland is not due to a corresponding difference in the rate of transcription of alpha 2u-globulin gene. Such a difference may represent tissue-specific regulation at a posttranscriptional level of mRNA metabolism. Furthermore, the huge difference in the alpha 2u-globulin content of the preputial gland and the liver is primarily due to the cellular and ductal accumulation of this protein in the preputial gland vs. its rapid secretion by the liver.

Identification, properties and effect of hormones on rat preputial gland peroxidase.

A highly active soluble peroxidase has been identified in the preputial glands of the rat. The enzyme was detectable in the sebaceous secretion of the glands and showed catalytic properties characteristic of true peroxidase. It had a native molecular weight of around 73,000 as determined by gel-permeation studies. Immunologically the enzyme cross-reacted with an antiserum against bovine lactoperoxidase. Administration of progesterone resulted in a significant increase in the total activity of the enzyme, while testosterone and oestradiol had no such effect. The enzyme had a similar molecular weight and similar catalytic and immunological properties to rat uterine fluid peroxidase but differed markedly in respect to sensitivity to oestradiol.

Nucleotide sequence of rat preputial gland beta-glucuronidase cDNA and in vitro insertion of its encoded polypeptide into microsomal membranes.

We have selected the rat preputial gland beta-glucuronidase as a model protein to study the sorting of newly synthesized lysosomal hydrolases to the lysosome. The complete coding sequence of beta-glucuronidase messenger RNA was determined from the sequences of a group of overlapping cDNA clones isolated from preputial gland cDNA libraries. The beta-glucuronidase mRNA primary translation product contains 648 amino acids, including an amino-terminal signal sequence of 22 residues. The polypeptide has four potential sites for the addition of asparagine-linked core oligosaccharides. A 376-residue segment of beta-glucuronidase shows extensive homology (23% sequence identity) to a portion of Escherichia coli beta-galactosidase. This homology most likely reflects an evolutionary relationship between the bacterial and eukaryotic enzymes and the conservation of structural features necessary for the glycosidase activity of both proteins. Translation of mRNA synthesized in vitro by transcription of a cDNA containing the entire beta-glucuronidase coding region yielded a polypeptide that was immunoprecipitated with anti-beta-glucuronidase antiserum and had the same electrophoretic mobility as the primary translation product of natural beta-glucuronidase mRNA. In the presence of microsomal membranes, the in vitro-synthesized beta-glucuronidase underwent cotranslational incorporation into the microsomes, as indicated by removal of the signal sequence and the addition of several oligosaccharide chains. The beta-glucuronidase cDNA will provide a useful tool to study the mechanism of mannose phosphorylation and other aspects of the sorting of lysosomal enzymes to lysosomes.

Characterization of the secretory proteins of rat preputial gland in relation to urinary proteins.

The water-soluble proteins of the rat preputial gland secretion were characterized in native and SDS-treated form on polyacrylamide gel electrophoresis. Nine major proteins were present in the secretion. One protein was a glycoprotein of molecular weight greater than 200,000 with beta-glucuronidase activity, and the other eight proteins had a molecular weight of 17,000, but with different charges. Acid phosphatase and arylsulphatase activities were present in the secretion in minor amounts. The isoelectric points of the secretory proteins ranged from 8.5 to 5.3; none of the proteins were lipoproteins, and there were no sex differences. The male and female rat urinary proteins were also characterized electrophoretically. The male rats had two different protein patterns, probably genetically determined. The female rats showed basically one urinary protein pattern, but their urines were frequently mixed with the preputial gland secretory proteins, which most likely played a part in the chemical communication. The mixing could not be correlated to daytime or estrous cycle.

The In Vitro Metabolism of 13-cis-Retinoic Acid in a Model Sebaceous Structure, the Rat Preputial Gland

In order to study the metabolism of 13-cis-retinoic acid (13-cis-RA) in animal sebaceous glands and analogues, preputial glands from normal and vitamin A-deficient male rats were incubated with [3H]13-cis-RA for up to 24 h; vitamin A-normal hamster costovertebral glands (flank organs) were incubated for 24 h as well. High-performance liquid chromatography was used to identify the metabolites. [3H]13-cis-RA was rapidly converted to a less polar compound, [3H]all-trans-retinoic acid, by the preputial glands from both normal and deficient rats. In normal preputial glands, the level of [3H]all-trans-RA decreases and two more polar compounds, metabolite I and [3H]4-keto-13-cis-RA appear. In contrast, [3H]all-trans-RA is not metabolized further by the preputial glands from deficient rats, while [3H]13-cis-RA in the hamster costovertebral glands remains intact for up to 24 h. The major metabolite of [3H]13-cis-RA in rat preputial glands is [3H]4-keto-13-cis-RA. Initially, [3H]13-cis-RA is converted to [3H]all-trans-RA. In vitamin A-deficient rats the preputial glands fail to further metabolize [3H]13-cis-RA to the more polar [3H]13-cis-RA derivatives. This may be due to the reduced level of P-450 enzyme in vitamin A-deficient rat preputial glands.

Rat ?2u-globulin mRNA expression in the preputial gland

Rat alpha 2u-globulin and the mouse major urinary proteins (MUP) are encoded by homologous multigene families whose members exhibit diverse tissue-specific, developmental, and hormonal controls of expression. Although their patterns of expression and hormonal control appear to be very similar in many respects, we have found high levels of alpha 2u-globulin mRNA in rat preputial glands, whereas MUP mRNA could not be detected in the male mouse preputial gland. Male and female rat preputial have similar concentrations of alpha 2u-globulin mRNA, suggesting an absence of endocrine regulation as occurs in the liver and lachrymal glands. Two-dimensional polyacrylamide gel electrophoresis of proteins encoded by hybrid-selected alpha 2u-globulin mRNA indicates that the liver and lachrymal translation products have different mobilities. However, many of the preputial gland products comigrate with most or all of the liver and lachrymal products. Among the possibilities suggested by these results is that alpha 2u-globulin genes expressed in liver and lachrymal glands under endocrine control are also expressed constitutively in the preputial gland.