Identification of glandular (preputial and clitoral) proteins in house rat (Rattus rattus) involved in pheromonal communication

Proteins (18-20 kDa) belonging to lipocalin family have been reported to act as carriers for ligands binding to pheromones in mouse urine, pig saliva, hamster vaginal fluid and human sweat, that are involved in pheromonal communication. As the preputial gland is a major pheromonal source, the present study was aimed to detect the specific protein bands (around 18-20 kDa) in the preputial and clitoral glands of the house rat, R. rattus. The amount of protein was higher in preputial gland of the male than that of female (clitoral) gland. A 20 kDa protein was noted in male and female glands; however, the intensity of the band was much higher in male than in female. In addition, 70, 60, 35 kDa bands, identified in male preputial gland, were absent in females. The presence of higher concentration of glandular proteins in the male preputial gland suggests that male rats may depend more on these glandular proteins for the maintenance of reproductive and dominance behaviours. The results further suggest that these glandular proteins (20 kDa) may act as a carrier for ligand binding.     

Identification of sex-associated protein in the preputial gland of house rat (a new insight in rodent pest management)

Our recent findings revealed that the preputial gland of male house rat contains 20 kDa protein, however, the role of androgen in the production of this protein is not known. Hence, the present study was carried out to evaluate the androgen dependency of 20 kDa protein in the preputial gland of house rat (Rattus rattus) and to compare its presence in female clitoral gland. Further, on castration the amount of glandular protein in male was significantly decreased to a certain extent, while testosterone treatment on castrated males showed an increasing trend. The electrophorogram of male house rat showed six different protein fractions with molecular weights of 90, 70, 60, 50, 35 and 20 kDa. However, the 70, 60, 50 and 35 kDa were absent in female. Among the different fractions, 90 and 20 kDa proteins were prominent. On castration, the 20 kDa protein was disappeared; while on testosterone treatment the protein reappeared. Thus, the present study concludes that the 20 kDa protein is a testosterone dependent sex-associated protein. Since urinary protein is found to act as carrier for volatile substances in pheromonal communication. The present study suggests that the glandular protein may bind with the volatile compounds produced from preputial gland. Identification of this carrier protein in the preputial gland explores the possibility of developing pheromonal trap for rodent pest management (RPM).     

Immunohistochemical localization of physiologic urinary proteins in Wistar rats.

Rat physiologic urinary proteins were immunohistochemically localized. In male Wistar rats, urinary protein antigens were present in both hepatic and epithelial cells of the salivary ducts, the coagulating gland, and the prostate gland. In female rats, urinary protein antigens were present in the same proportion of the salivary glands as in males and in the uterine glands, but to a lesser extent than in salivary glands. The results of this study indicate multiple origins of rat urinary proteins. It remains to be determined if female uterine glands contribute to urinary proteins.     

Immunocytochemical localization of seminal proteins in salivary and lacrimal glands of the rat

Antibodies against 10 different secretory proteins from the accessory sex glands of the male rat were used for immunohistochemical studies of salivary and lacrimal glands from intact and castrated rats, at the light- and electron-microscopic levels. In the parotid gland, secretory acinar cells showed immunoreactivity with antibodies against prostatic binding protein, cystatin-related peptide and acid phosphatase (isoenzyme pI 8.0; 5.6) typical of ventral prostate, and seminal vesicle secretion VI. Western blotting analysis indicated that immunoreactivity against prostatic binding protein was attributable to a subunit, presumably C3. Acid phosphatase pI 5.6 showed a molecular weight of 66 kDa, which is at variance with the prostatic form. Immunoreactivity for secretory transglutaminase, derived from the coagulating gland, was restricted to myoepithelial and stromal cells. In castrated animals, the immunoreactivity of acinar cells was reduced to the background level, whereas stromal transglutaminase immunoreactivity was unaltered. The distribution pattern of immunoreactivity for the proteins mentioned was almost identical in the lacrimal gland. Significant differences were however observed in the immunoreactivity of the inframandibular gland, where serous glandular cells were non-immunoreactive for seminal proteins, with the exception of acid phosphatase isoenzyme pI 8.0. Granules present in the convoluted granular ducts were immunoreactive particularly for acid phosphatase (isoenzyme pI 5.6)but much less for cystatin-related peptide; immunoreactivity was reduced after castration. The straight portion of the inframandibular duct system was immunoreactive for transglutaminase, but no influence of castration was visible. The distribution of immunoreactivity for seminal proteins present in the salivary and lacrimal glands and the pronounced androgen-dependence of their expression point to functional relationships of the respective proteins at both glandular sites.     

Immunological characterization of soluble peroxidases from rat tissues including preputial gland

A highly active soluble peroxidase has been identified in the preputial gland of rats and characterized immunologically along with other soluble peroxidases of a number of rat tissues such as submaxillary gland, exorbital lacrimal gland and also of the uterine fluid of the estrogen treated rats. All these peroxidases have the native molecular weight around 73K as determined by gel filtration on Sephadex G-150. An antiserum raised against the pure bovine lactoperoxidase interacts with all these soluble peroxidases and immunoprecipitates the enzyme activity in a similar fashion when titrated against varied concentration of the antiserum. Following electrophoretic transfer to nitrocellulose by Western blotting, the antiserum crossreacts with the preputial, submaxillary and lacrimal gland protein of molecular weight around 73K and with the uterine fluid protein of molecular weight of 80K. An additional crossreacting protein of molecular weight of 80K is also evident in the lacrimal gland. All these enzyme preparations, however, contain another immunoreactive protein of molecular weight of about 64K. While 73–80K molecular weight interacting proteins may represent different forms of peroxidase, presumably with varied carbohydrate moieties, 64K molecular weight protein may be a precursor of the peroxidase which after posttranslational modification such as heme conjugation and glycosylation leads to formation of native enzyme. Rat harderian gland, unlike bovine origin, does not contain any detectable peroxidase activity. The immunoblot does not show the presence of any immunoreactive protein around 73K except the 64K molecular weight protein indicating that this gland can not synthesize the native peroxidase from this precursor probably due to some block in posttranslational modification.     

Sex pheromone production by preputial gland: the regulatory role of estrogen

The present contribution reviews progress in the identificationof molecular and metabolic events that underlie the synthesisand secretion of putative sex attractants by preputial glands.The participation of androgens and peptide hormones, alone andtogether, in modification of glandular structure and functionis briefly summarized. Special emphasis is given to recent findingswhich define the interaction of estrogens with specific receptorsof preputial acinar and ductal cells from the female rat. Changesin the basal metabolic and biochemical characteristics of thelatter cells as a consequence of estradiol-receptor bindingappear to contribute to hormone-induced secretion of sex pheromone(s).Evidence for the preferential attraction of male rats to thehormone-dependent, odoriferous secretion of female preputialglands is reviewed. Although the attractant substance in femalesecretions remains to be purified and characterized, speculationon its probable nature is considered. The combined data suggestthat further investigation of the suspected preputial gland-olfacto-hypothalamo-pituitarygonadalcircuit in the rat may provide an important model to examinethe complexities of endocrineexocrine interrelations in sexualcommunication among mammals.     

Alpha 2u-globulin in modified sebaceous glands with pheromonal functions: localization of the protein and its mRNA in preputial, meibomian, and perianal glands

alpha 2u-Globulin, the principal urinary protein of the male rat, has extensive sequence homology with many lipid binding proteins. The highest concentration of alpha 2u-globulin is found in the preputial gland, a holocrine secretory organ with pheromonal function. Meibomian and perianal glands are two other modified sebaceous glands with holocrine secretory cycles and pleiomorphic peroxisomes capable of synthesizing pheromonal lipids. Immunocytochemical examination shows the presence of alpha 2u-globulin in the acinar cells of all three of these modified sebaceous glands. Whereas in the preputial gland all of the acinar cells exhibit immunoreactivity, in the meibomian and perianal glands only selective cells contain alpha 2u-globulin. In the case of the preputial gland, in addition to the acinar cells some stratified epithelial cells also were immunoreactive. In the perianal and meibomian glands, keratinocytes lining nearby hair shafts and select cells of accessory oil glands stained for alpha 2u-globulin. In situ hybridization with a cloned cRNA probe confirmed the immunocytochemical data. Presence of the alpha 2u-globulin mRNA in these glands was also established by Northern blot analysis. Immunoelectron microscopic examination of preputial alpha 2u-globulin showed the presence of this protein in secretory granules of various maturational stages. Immunolabeled alpha 2u was also found in attached vesicles containing protein and lipid inclusions. The lytic cells were not only loaded with alpha 2u-globulin but also contained sharp-edged, irregularly shaped electron-dense granules which stained heavily for this protein. Specific localization of alpha 2u-globulin and its mRNA in three pheromone-producing sebaceous glands and its structural homology with known lipid binding proteins indicate a pheromone carrier role of alpha 2u-globulin.     

Identification of alpha-2u globulin in the rat preputial gland by MALDI-TOF analysis

The low molecular mass proteins found in the pheromonal sources such as urine, saliva, glandular secretion etc have been reported as ligand carriers for the processes of chemocommunication in mammals. The preputial gland plays an important role in the production of olfactory signals for pheromonal communication. Thus, in the present study, alpha-2u globulin having molecular mass of 18 kDa has been identified in the preputial gland of Norway rat (Rattus norvegicus) by in-gel trypsin digestion and analyzing the resulting peptides by MALDI-TOF. Since preputial gland is one of the major pheromonal sources in rat, the results suggest that alpha-2u globulin might act as a carrier for hydrophobic odorants of preputial gland.     

Characterization of a 22 kDa protein with widespread tissue distribution but which is uniquely present in secretions of the testis and epididymis and on the surface of spermatozoa

The purification is reported of a 22 kDa protein which was first identified as one of the major components of the luminal secretion of the rat testis and epididymis. Antibodies against the 22 kDa protein cross-reacted with a protein of the same molecular weight in cytosolic extracts of other tissues from both male and female rats. However, since the protein could not be detected in blood, peritoneal fluid, saliva, milk, uterine fluid, seminal vesicle secretion, coagulating gland secretion or prostatic secretion, it would appear that the testis and epididymis may be unique in containing the protein in a soluble form within their luminal secretions. Proteins with slightly lower molecular weight were detected by the antibodies in cytosolic extracts of tissues from other animals (mice, rabbits, sheep, pigs, cattle), indicating that the protein may be conserved in a variety of species. However, in contrast to the rat, the protein was apparently not present in the testicular and epididymal secretions of these species. In addition to the occurrence of the 22 kDa protein as a soluble moiety in rat testicular and epididymal fluids, the protein was also located on sperm plasma membranes where its distribution was restricted to the surface of the flagellum. Amongst sperm surface proteins, the 22 kDa protein was the major protein containing sulphydryl groups and one of the major entities containing disulphide bonds. These properties may be of importance in the maintenance of sperm viability.     

Tissue specific gene activities and proteins in the Chironomus salivary gland

Summary The secretory proteins of the larval salivary gland of some Chironomus species were analysed according to a method developed by Grossbach (1969). After reduction of the disulphide bonds and alkylation the secretion of Chironomus thummi was separated by disc electrophoresis at acrylamide concentrations of 4.8 to 9.5% into seven main and some minor fractions. By increasing the acrylamide concentration up to 20% nine main fractions could be resolved. In addition to these high molecular weight structural proteins a number of proteins soluble in simple buffer solutions and separable at pH 8.8 in a 15% acrylamide gel are present in the secretion but only to a very small amount.The electrophoretic pattern of the secretory proteins of Chironomus strenzkei, Ch. luridus and Ch. obtusidens are qualitatively the same as the Ch. thummi pattern. Some quantitative differences may exist. In the secretion of Chironomus plumosus at least eleven main fractions were detected in 20% acrylamide gels. A comparison between the number of Balbiani rings of the salivary gland chromosomes and the number of main protein fractions in the salivary gland secretion yielded no direct correlation.The results are discussed in respect to the question wether the major puffs of the gland, the Balbiani rings, encode the major products of the gland, the secretory proteins.Abbreviations used BR Balbiani ring - bis N,N-Methylenebisacrylamide     

The induction of liver microsomal proteins by 3-methylcholanthrene in rats of different age,sex and strain

The treatment of male and female adult and male and female immature rats with 3-methylcholanthrene results in increases in the intensity of two liver microsomal protein bands separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The induced protein species have estimated molecular weights of about 55 000 and 52 500. The time course of the induction of these species has been followed in a semi-quantitative manner. In immature rats an additional band, corresponding to a protein species with a molecular weight of about 43 000, is also increased by 3-methylcholanthrene treatment. In male adults rats exclusively, treatment with 3-methylcholanthrene also results in a decrease in the intensity of a band corresponding to a protein species of about 51 000. The appearance of this band in the liver microsomal fraction of adult male rats occurs following sexual maturation and its presence represents a significant difference in the microsomal proteins between male and female rats, this protein is not detected in younger male rats or in female rats at any age. These findings are discussed in terms of their possible relevance to the cytochrome P-450 content of the endoplasmic reticulum of rat hepatocytes.     

白背飞虱若虫和雄雌成虫唾液腺蛋白鉴定

【目的】对白背飞虱Sogatella furcifera唾液腺蛋白进行鉴定及功能注释分类,探究不同发育阶段和不同性别的白背飞虱唾液腺蛋白之间的区别与联系。【方法】麻醉白背飞虱若虫、雄成虫和雌成虫,解剖收集唾液腺组织,提取蛋白,还原烷基化和酶解,利用液相色谱串联质谱技术鉴定蛋白。与Unigene蛋白数据库进行比对,并通过KOG分析,对唾液腺蛋白进行功能注释分类。【结果】白背飞虱若虫、雄成虫和雌成虫特有的唾液腺蛋白分别为385, 168和82个,若虫与雄成虫、若虫与雌成虫及雄成虫与雌成虫共有的唾液腺蛋白分别为319, 60和60个。KOG功能注释显示与细胞过程和信号传导相关的蛋白数量最多,若虫、雄成虫和雌成虫特有以及若虫与雄成虫、若虫与雌成虫及雄成虫与雌成虫共有的这类蛋白数量分别为81, 22, 70, 19, 21和12个,这些蛋白主要发挥着翻译后修饰、蛋白质更新、伴侣蛋白,细胞内运输、分泌和囊泡运输及信号转导作用。【结论】白背飞虱唾液腺蛋白在参与信号转导机制方面表现较为活跃,这可能与其刺吸危害有一定的关系。     

Detection of gender differences in rat lens proteins using 2-D-DIGE

The glass-like transparency of the human eye lens is achieved by the tight packing of abundant crystallin proteins. However, the precise role of the accessory non-crystallin proteins is not well understood. We have carried out 2-DE mapping of these proteins in rat lens. This showed the presence of the high molecular weight filamentous structural proteins spectrin, filensin, tubulin, vimentin, actin and phakinin as well as several forms of potential crystallin oligomers comprised of alphaA, betaB1, betaA1 and betaA4 chains. Other proteins that were present include, heat shock protein 71, WD repeat protein 1, and several enzymes including alpha-enolase, pyruvate kinase, transketolase and aldose reductase. 2-D-DIGE analysis revealed several expression differences between the lens proteomes of male and female rats. Female rat lenses contained lower levels of aldose reductase, increased proteolyic fragments of the structural proteins filensin, vimentin and phakinin and higher levels of potential alphaA, betaB1 and betaA1 crystallin oligomers. Taken together these findings suggest that there are potential differences in oxidative stress regulation between male and female rat lenses, which may have implications on susceptibility to cataract formation. Future studies aimed at elucidating pre-cataractic changes in the non-crystallin proteins described here may facilitate identification of novel markers involved in cataractogenesis.     

Accessory gland secretory proteins in relation to fitness parameters of Drosophila ananassae and D. varians

Developmental morphometry, qualitative and quantitative analysis of the accessory gland secretory proteins, fecundity and productivity in relation to protein ejected during subsequent (first to fourth-time) matings have been studied in Drosophila ananassae Doleschall and Drosophila varians Bock. In both species, size and secretion of accessory glands increases from 1 to 8 days and the stored secretion ejected from males to the female genital tract during subsequent mating varies. The maximum number of eggs and flies are produced from the females mated with bachelor males and it is a minimum when virgin females are mated with fourth-time mated males. Sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis of accessory gland secretory protein patterns and their glycosylation differs in both the species. Correlation coefficient analysis between gland size and quantity of secretion, percentage of secretory protein transferred per mating, and eggs and flies that emerged showed a highly significant, positive relationship. Among different matings, the number of eggs laid and flies that emerged per female between subsequent (first to fourth-time) matings of males was found to be highly significant and the difference between fecundity and productivity between the two species was highly significant.     

Interaction of proteins RSV IV and RSV V in rat seminal vesicle secretion

The RSV IV polypeptide, molecular weight ratio (Mr = 10,000), which is produced by the rat seminal vesicle, has previously been suggested to be associated with another polypeptide in the gland secretion (Higgins et al., '76). This study provides that RSV IV is a component of a protein shown by immunoassays, electrophoresis, and amino acid composition analysis to contain, together with RSV IV, the seminal vesicle secretory RSV V polypeptide (Mr = 13,000). This RSV IV-RSV V complex (namely CFS protein) had an isoelectric point at pH 7.2 and an approximate molecular weight of 22,000 daltons. This complex inhibits the previously reported in vitro binding of the isolated RSV IV to epididymal sperm cells, thus suggesting a functional role for the RSV IV-RSV V interaction.     

Primary structure and cellular distribution of two fatty acid-binding proteins in adult rat kidneys

Fatty acid-binding proteins (FABPs) were purified from the kidneys of female and male rats and characterized by primary structure and histological distribution in the kidney. Two FABPs (14 and 15.5 kDa) were found in male rat kidney cytosol whereas only 14-kDa FABP could be recognized in female rat kidneys throughout the purification steps. The amino acid sequence of the 14-kDa FABP was identical to that of rat heart FABP deduced from the cDNA sequence (Heuckeroth, R. O., Birkenmeier, E. H., Levin, M. S., and Gordon, J. I. (1987) J. Biol. Chem. 262, 9709-9717). Structural analysis of the male-specific 15.5-kDa FABP identified this second FABP as a proteolytically modified form of alpha 2u-globulin, an 18.7-kDa major urinary protein of adult male rats (Unterman, R. D., Lynch, K. R., Nakhasi, H. L., dolan, K. P., Hamilton, J. W., Cohn, D. V., and Feigelson, P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3478-3482) which shares a common ancestry with a number of hydrophobic ligand-binding proteins such as serum retinol-binding proteins. Immunohistochemical investigation disclosed that heart-type FABP (14-kDa FABP) is localized in the cytoplasm of the epithelia of the distal tubules in both male and female rat kidneys whereas 15.5-kDa FABP immunostaining was observed predominantly in the endosomes or lysosomes of proximal tubules in male rat kidneys. These results suggest strongly the functional divergence of two FABPs in the rat kidney.     

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.     

Occurrence of autoimmune antibodies to liver microsomal proteins in association with fulminant hepatitis in the LEC strain of rats

The Long Evans Cinnamon (LEC) rat, which has been established as a strain showing hereditary hepatitis and hepatic carcinoma, was found to possess autoimmune antibodies to liver microsomal proteins, particularly to a protein with the molecular weight of 56kD. The antibodies also recognized a protein(s) in liver microsomes from Long Evans Agouti and Sprague-Dawley rats. About 42 and 15 percent of respective female and male LEC rats died within a week after acute hepatitis; sera from all of the animals contained the antibodies. About 43 and 0 percent of the surviving female and male LEC rats possessed the antibodies, respectively. These results suggest that the autoantibodies occur in association with acute lethal hepatitis in the LEC rats.     

Cell-specific synthesis and glycosylation of secretory proteins in larval salivary glands of Chironomus thummi

Synthesis and glycosylation of larval salivary gland secretory proteins of Chironomus thummi were analyzed with respect to cell specific differences in the Balbiani ring (BR) pattern and glycoprotein composition of secretion formerly detected by histochemical staining procedures. In the secretion of a special cell type in salivary glands, which is characterized by the appearance of an additional BR, an additional polypeptide with a relative molecular weight (M_r) of 160 kD was found differing in its antigenic properties and tryptic fingerprint pattern from main cell secretion proteins. This so-called ssp-160 component is preferentially synthesized and glycosylated in the special cells. In the same cells, both the synthesis and glycosylation of all other major secretory proteins was found to be diminished or even repressed. In contrast to the conspicuous cell-specific differences at the level of protein synthesis, RNA analyses show the prominent synthesis of 75 S RNA in both main and special cells and gave no clear indication of the synthesis of a smaller RNA fraction as expected from the size of ssp-160 component. — These and further data on synthesis and properties of secretory proteins as well as expression of BR DNA are discussed with regard to the assumption that at least some of the eight major secretory polypeptides are coded for by BR DNA. The BR gene(s) might have originated by manifold duplications and modifications of short repetitive prototype DNA sequences, which are coordinatively expressed.On the occasion of the 60th anniversary of his birth-day we wish to dedicate this paper to Professor Wolfgang Beermann who was the first to detect, by the discovery of cell specific expression of BR 4 of Chironomus pallidivittatus salivary gland chromosomes and the concomitant occurrence of cell specific secretion granules, a causual relationship between the activity of a Balbiani ring and the appearance of a secretion component (Beermann, 1961)addressee for reprint requests