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.     

两种兔尾鼠的化学信息及其在群体中的作用

黄兔尾鼠(Lagurus lutens)及草原兔尾鼠(Lagurus lagurus)是新疆某些地区主要害鼠之一,它们的繁殖能力很强,数量高峰时,对草场危害极大。此外,它们还能传染某些地方性流行病,所以对这两种害鼠的防治是十分必要的。 近年来,我们在研究动物化学通讯的过程中,了解到外激素(信息素)在动物间传递信     

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.     

四川短尾鼩包皮腺的挥发性化学成分分析

哺乳动物的包皮腺分泌物对个体间性引诱及繁殖行为的信息交流和种群调节有着重要的作用。本研究以四川短尾鼩（Anourosorex squamipes）为研究对象对劳亚食虫目动物包皮腺挥发性化学成分和化学通讯功能进行报道。采用顶空固相微萃取—气质联用（HS-SPME-GC/MS）的方法,分析四川短尾鼩雄体包皮腺中挥发性物质的化学组成。结果表明：（1）四川短尾鼩雄体包皮腺的挥发性化学成分主要含烷烃类、醇类、酮类、醛类、醚类、酯类、酸类、芳香烃类等45种化合物;（2）成年雄性四川短尾鼩的包皮腺中挥发性成分有39种,幼年雄性含有28种,说明四川短尾鼩成年雄体的包皮腺中挥发性化学成分多于幼年雄体;（3）成年雄体的包皮腺分泌物中含有4种特有的挥发性化学成分,幼年雄体中特有成分仅为1种,表明四川短尾鼩包皮腺中的挥发性化学成分存在年龄差异;（4）四川短尾鼩包皮腺分泌物中不同化学成分相对含量不同,同种化学成分在不同个体间的相对含量存在差异。四川短尾鼩雄性包皮腺挥发性化合物种类丰富,个体、年龄差异明显。推测挥发性化合物中丁酸（Butanoicacid）、乙酸乙酯（Ethyl acetate）、苯酚（Phenol）为四川短尾鼩的信息素。本研究为进一步验证该物种的信息素成分及其传递机制提供了基础数据。     

Role of male scent glands in improving poison bait acceptance in female rats,Rattus norvegicus

The poison (Zn3P2) bait mixed with preputial gland and cheek gland extract was highly effective as compared to poison bait mixed without the scent gland extract, in increasing the food consumption and mortality rate in rats. Among these two scent glands, preputial gland extract was more effective than cheek gland extract in increasing the bait acceptance. The scent gland extract mixed with poison bait was capable of improving the poison bait acceptance. A 10% concentration of scent gland extract was more effective than 5% concentration in acceptance of poison bait. The results suggest efficacy of scent gland in improving the poison bait acceptance.     

The rodent preputial gland

Researches published in the last 2 years have advanced direct evidence for regarding the rodent preputial gland as a scent organ, confirming a speculation made at least as long ago as 1725. The gland is the source of olfactory stimuli which mediate behavioural responses in social and sexual interactions between conspecifics. Although the gland has received little direct attention from investigators, a considerable amount of information on it is available. Most of this was gained ancilliary to other, and very diverse, research objectives so that the literature is very scattered. These references are collated to present a comprehensive review of knowledge of the biology of the rodent preputial gland. Theoretical concepts of population structure and regulation, involving density-dependent factors, require a sensory mechanism to activate the physiological effects attributed to social interactions. The preputial gland, as a source of olfactory cues, has all the biological characteristics required of such a sensory modulator.     

Hormonal regulation of preputial gland function in male Microtus montanus, the montane vole

1. Preputial gland function in male Microtus montanus is androgen-dependent, both in terms of preputial weights and in the production of a series of lipids which are present in M. montanus and absent from Microtus pennsylvanicus. 2. Production of these species-typical lipids is decreased but not eliminated in castrates, as well as in adrenalectomized castrates treated with corticosterone. Therefore, in the total absence of androgens, a low level of these lipids is still produced. 3. 5 alpha-dihydrotestosterone and 17 beta-estradiol enanthate have limited effects on maintenance of preputial weight, suggesting that testosterone itself is the active steroid in the preputial gland of this species.     

Aggression and preputial gland of male mice affected by the presence of other males

The level of aggressiveness and the weight of preputial gland and testis in male mice (Mus musculus) were influenced by housing condition, especially by the presence of cohabitant males. In this study, the relation between aggressiveness and the preputial gland and testis weight was studied for various housing conditions. The mouse individually housed in a cage that was linked to another cage containing another male separated by wire net was more aggressive than isolated or paired mice. The preputial gland weight also showed the same tendency, suggesting that the odor from other males promotes pituitary-gonadal activity in males, and that long-term cohabitance inhibits it.     

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).     

Der Einfluss verschiedener Fixierungsmittel auf das Strukturbild der Präputialdrüsen der Ratte

Summary The influence of various methods of fixation on the preputial gland of the rat was investigated. In addition to the lipids, the cells contain numerous granules which are dissolved by alcoholic and certain acid fixatives. In future investigation concerning either the histochemistry of the gland or the effect on the gland of various experimental conditions, its peculiar ability to secrete two different substances must be considered. In any event the preputial gland of the rat is more than a simple sebaceous gland.     

Catalase-positive particles (“microperoxisomes”) from rat preputial gland and bovine adrenal cortex

Catalase activity was detected histochemically within membrane-bound cell organelles in epithelial cells of rat preputial gland and bovine adrenal cortex. These particles are oval to worm-like in rat preputial gland, 0.08 – 0.15 μm thick and up to 1.0 μm long. In bovine adrenal cortex the shape of catalase-positive particles is rather spherical (diameter 0.1 to 0.3 μm). Particles of both organs lack crystalline or dense cores.Biochemical examination of cell fractions prepared from tissue homogenates by differential centrifugation revealed the presence of two typical peroxisomal oxidases, viz. α-hydroxy acid and -amino acid oxidase, with maximal relative specific activities in the ‘microsomal’ fraction (preputial gland) and in the ‘lysosomal’ fraction (adrenal cortex), respectively. Urate oxidase is absent in both tissues.The concomitant occurrence of catalase and hydrogen peroxide producing oxidases in the particles described characterizes them as true peroxisomal systems (‘microperoxisomes’).     

The metabolism of androgens in rat preputial glands

The metabolism of testosterone, androstenedione and dehydroepiandrosterone in the rat preputial gland has been studied. A high activity of 5α-reductase is present as shown by the formation of 17β hydroxy-5α-androstan-3-one and 5α-androstan-3, 17-dione as the major products from testosterone and androstenedione respectively. Other enzyme activities are present including 17β-hydroxy steroid dehydrogenase, but the amounts of testosterone and 17β-hydroxy-5α-androstan-3-one formed from androstenedione and dehydroepiandrosterone are low. The main product of dehydroepiandrosterone metabolism was androstenedione indicating a high level of 3β-hydroxy steroid dehydrogenase 4-5 isomerase activity. The metabolism was compared with that in rat skin where it was found that the extent of metabolism was much less. The possible significance of the various products formed and of differences between skin and preputial gland metabolism is discussed. Some differences were noted between the metabolism of androgens by rat skin and preputial gland and the metabolism of androgens by human skin.     

在自然状态下黄毛鼠包皮腺的引诱力及季节变化

黄毛鼠(Rattus rattoides Hodgson)包皮腺成对位于下腹部的皮下,腺体开口于尿道口外包皮内的两侧。腺体形态,雌雄各异。雌性呈豆瓣形;雄性呈瓜籽形。体长150毫米以上的成年鼠,其包皮腺的长和宽约18和9毫米。包皮腺是动物的一个外激素释放源,对于种群的调节有着重要的作用(Christiansen ＆ Dving 1975)。关于它的性引诱作用见诸于Bronson ＆ Caroom(1971)、刘炳谦、刘素霞(1979)等。但在自然情况下包皮腺分泌及其引诱作用尚少报道。本文通过野外试验,初步研究了黄毛鼠包皮腺的分泌及引诱力的变化规律,探讨利用此特性的可能性。无疑,这对进一步研究外激素对种群调节作用以及鼠害防治方面是有意义的。     

Effects of prolactin, progesterone, and 17beta-hydroxy-5alpha-androstan-3-one on squalene production by the preputial gland of the immature female rat

To examine further the previously demonstrated synergism between prolactin and progesterone on preputial glands of hypophysectomized, ovariectomized, immature rats, their effects on squalene production were determined and compared with the ability of 17beta-hydroxy-5alpha-androstan-3-one (DHT) and prolactin to increase the amount of squalene in the preputial glands. Glands from progesterone-treated rats incubated in vitro with [14C]mevalonic acid incorporated radioactivity into squalene (identified by chromatographic mobility) more rapidly than glands from controls or prolactin-treated rats. Using the same in vitro system, glands from prolactin-treated rats incorporated more [14C]acetate into squalene than those from progesterone-treated animals. In addition, results showed that prolactin and DHT increased nonradioactive squalene (identified by mass spectral analysis) content in the gland while progesterone had no effect. It is proposed that prolactin increases preputial gland squalene content by enhancing synthesis of mevalonic acid, while progesterone increases incorporation of mevalonic acid into squalene.     

Reproductive biology of the greater bandicoot rat Bandicota indica （Rodentia： Muridae） in the rice fields of southern Thailand

This study investigated some aspects of the reproductive biology of male and female greater bandicoot rats,Bandicota indica,in southern Thailand from September 2004 to September 2006. In females,body,uterine and preputial gland weights,occurrences of pregnancies and placental scars,and in males,testicalar weights and histology,and sizes of accessory sex glands,were recorded. Pregnancies occurred predominantly,but not exclusively,in the wet season,with a higher incidence pregnancies in the second,than in the first,dry season. Uterine and preputial gland weights tended to be lower in the first,but not the second dry season,with placental scars occurring at all times of year. Males tended to have heavier testes in the wet season but some seminiferous tubules contained sperm even in the dry season. Seminal vesicles,but not prostates and preputial glands,tended to be heavier in animals in the wet season. We conclude that the greater bandieoot rat in southern Thailand shows maximal reproductive activity in the wet season with some reproductive activity,albeit variable from year to year,occurring in the dry season depending upon environmental conditions. This study has also shown that females,as well as males,have large preputial glands,and that males invariably have small testes regardless of the time of year. These observations suggest a similar timing of reproduction,but a different breeding biology and perhaps social organisation,from that of the sympatric ricefield rat,Rattus argentiventer.     

Stimulation of growth by testosterone via the mesenchyme

Summary To establish testosterone-dependent growth in organ-culture, anlagen of preputial glands from normal wild-type and from androgeninsensitive mouse embryos carrying the testicular feminization mutation (Tfm) were explanted and cultured in the presence of testosterone. Within six days a size difference developed between Tfm and wild-type explants involving length of hair follicle, amount of preputial gland tissue, and overall size.Anlagen from Tfm and wild-type preputial glands were then separated into epithelial bud and mesenchyme. Reciprocal recombinants were prepared and cultured with testosterone. In the recombinants development of hair follicles and gland tissue was inconsistent. Nevertheless, the effect of testosterone was expressed in the overall size of the explants. The size correlated with the type of mesenchyme used, but not with the type of epithelium: Androgen-insensitive Tfm epithelium combined with wild-type mesenchyme reached the same size as whole wild-type glands and wild-type/wild-type recombinants. Wild-type epithelium with Tfm mesenchyme resulted in small explants, which were in the range of the whole androgen-insensitive Tfm glands. Tfm/Tfm recombinants showed very poor growth, probably related to the fact that in this group no hair or gland structures developed.     

Tissue distribution of constitutive and induced soluble peroxidase in rat. Purification and characterization from lacrimal gland.

A thorough search for a soluble peroxidase in 31 different tissues of rat indicated the presence of a constitutive activity only in lacrimal, preputial and submaxillary gland. An induced soluble peroxidase activity was also detected in the lactating mammary gland and in the estrogen-induced uterine secretory fluid. The lacrimal gland was the richest source of the enzyme. No peroxidase activity was detected in the lactating mammary gland of mouse and hamster nor in the preputial gland of mouse and uterine fluid of hamster. The three constitutive and two induced soluble peroxidases of rat had a native molecular mass of 73 kDa by gel filtration and they showed a similar mobility in native PAGE. Lactoperoxidase of cow's milk and solubilized rat membrane-bound peroxidases of uterus, intestine and bone marrow showed in native PAGE a mobility which was distinctly different from that of rat soluble peroxidases. As the lacrimal gland of rat was the richest source of soluble peroxidase, the enzyme was purified from this gland to apparent homogeneity; SDS/PAGE then showed a single band of molecular mass 75 kDa which was similar to that obtained by gel filtration. Peroxidase also purified from preputial and submaxillary gland, as well as commercial lactoperoxidase, had a similar molecular mass on SDS/PAGE to purified lacrimal peroxidase. The visible spectrum of lacrimal peroxidase was similar to that of lactoperoxidase but different from membrane-bound peroxidase of rat neutrophils. On isoelectric focussing, purified lacrimal peroxidase resolved into about 14 multiple forms spanning a pI range of 6.5-3.5 while lactoperoxidase focussed at the cathode. Evidence presented suggests that the multiple forms are possibly due to differences in glycosylation. Immunodiffusion, immunoprecipitation and Western blot using antilacrimal peroxidase serum showed a similar interacting species for all five soluble peroxidases of rat while membrane-bound peroxidases showed no interaction. Although in immunodiffusion, the antiserum failed to cross-react with lactoperoxidase it did interact with lactoperoxidase on Western blot. The results indicate that the various constitutive and induced soluble peroxidases of rat tissues are similar to lacrimal peroxidase but are distinctly different from the known membrane-bound peroxidases of rat. However the lacrimal peroxidase shows both similarities as well as dissimilarities with bovine lactoperoxidase. This soluble peroxidase system of rat could be useful to study tissue-specific regulation of gene expression at the molecular level.     

Regulation of acyl coenzyme A reductase by a heat-stable cytosolic protein during preputial gland development

A low-molecular-weight protein located in the cytosol of mouse preputial glands has been shown to stimulate the activity of a microsomal acyl coenzyme A (CoA) reductase in the gland. This cytoplasmic protein was stable to heating and lyophilization, but was destroyed by trypsin digestion. It was able to bind palmitoyl-CoA and gel elution behavior indicated it had a molecular weight of 10,000–12,000. The level of this stimulatory cytosolic protein and the activity of acyl-CoA reductase were shown to correlate with differentiation of the preputial gland during development of puberty in male mice; the acyl-CoA reductase activity first appeared at 4 weeks of age and increased dramatically up to 6 weeks of age. By 8 weeks, when sexual maturity was attained, the reductase activity decreased to that level found in mature male mice. The cytosol from the preputial glands of the youngest mice (3 weeks) contained sufficient heat-stable acyl-CoA binding protein to stimulate acyl-CoA reduction; however, the 3-week-old preputial gland microsomes had little or no acyl-CoA reductase activity. As the animal matured, the stimulatory capacity in the heat-treated cytosol increased, reaching a maximum at 6 weeks; by 8 weeks, the stimulatory capacity of the soluble fraction had decreased to that found in mature male mouse. Results of this study suggest that the concentration of acyl-CoA, cytoplasmic acyl-CoA binding protein, and acyl-CoA reductase activity regulate the level of fatty alcohols in vivo and that the reductase activity and binding protein have similar patterns of development during puberty.     

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.