The involvement of boar submaxillary salivary gland secretions in boar-induced precocious puberty attainment in the gilt

The involvement of secretions from boar submaxillary salivary glands in mediating the induction of precocious puberty in the gilt was investigated as follows. Forty-eight Large White × (Large White × Landrace) prepubertal gilts from 12 litters were randomly allocated within litters by weight, to four treatment groups of six, in two replicates, at 145 days of age. Treatments commencing at a mean group age of 165 days, were: (1) control (no boar exposure); (2) gilts exposed to a mature sialectomised boar (submaxillary salivary glands were removed at 9 weeks of age); (3) gilts exposed to a mature sham-operated boar; (4) gilts exposed to a mature unoperated boar.Boar exposure occurred for 30 min per day for 75 days, or until pubertal oestrus was observed. Gilts showing pubertal oestrus were removed and slaughtered. Ovaries were examined to confirm reproductive status. Gilts failing to exhibit oestrus by 240 days of age were slaughtered and nominally ascribed a pubertal age of 245 days. Age at puberty was significantly earlier in all three boar-exposed treatments than in the control treatment (P<0.05 for treatments 2 and 3, P<0.001 for treatment 4; median ages at puberty being 227.0, 203.5 , 202.0 and 179.0 days for treatments 1 to 4 respectively). No frothy saliva was ever produced by the sialectomised boar, and chromatographic analysis of saliva produced by the sham-operated boar during mating revealed very low levels of 16-androstene pheromones, while levels in the unoperated boar's saliva were normal. These results provide further evidence for an important role of boar salivary pheromones in the induction of precocious puberty attainment in the gilt.     

Changes with age in the occurrence of C19 steroids in the testis and submaxillary gland of the boar.

After extraction from the testes of boars of different ages, C19 steroids including 16-androstenes were determined by gas-liquid chromatography. Similarly, 16-androstenes were determined in the submaxillary glands of these boars. A high concentration of testosterone was found in the testes of 84-day-old fetuses, and this might be significant in the differentiation of male behaviour. The amount of testosterone exceeded that of androstenedione during postnatal development, and dehydroepiandrosterone and 5-androstenediol as free and sulphates were found in high concentrations particularly in postpubertal boars, suggesting that the 5-ene pathway for the synthesis of testosterone might be important. There was a change in the predominance of individual 16-androstenes in the testis during development, which closely paralleled the sequence for the biosynthesis of these compounds proposed from previous studies in vitro. Whereas the amount of 5alpha-androst-16-en-3beta-ol exceeded that of 5alpha-androst-16-en-3alpha-ol in post-pubertal testes, 5alpha-androst-16-en-3alpha-ol was predominant in the submaxillary glands at all ages. The high concentration of 16-androstenes found in the mature boar, are discussed in relation to their release as pheromones and as factors responsible for taint in boar meat.     

Factors affecting the pheromone composition of voided boar saliva

The pheromone binding protein 'pheromaxein' which binds the pheromonal 16-androstene steroids in the saliva of the male pig (boar), was degraded and lost its binding activity in saliva incubated in air for 72 h at 21 degrees C and 37 degrees C. However, pheromaxein and its binding activity were retained in saliva incubated for 168 h at 4 degrees C. When the 3H-labelled pheromones 5 alpha-androst-16-en-3 alpha-ol (3 alpha-androstenol), 5 alpha-androst-16-en-3-one (5 alpha-androstenone) and 5 alpha-androst-16-en-3 beta-ol (3 beta-androstenol) were incubated with boar saliva for 168 h at 21 degrees C, 3 alpha-androstenol was primarily converted to 5 alpha-androstenone and 5 alpha-androstenone to 3 beta-androstenol; 3 beta-androstenol was unchanged. Evidence was obtained for microorganisms being responsible for these steroid transformations.     

16-ene-steroids in the human testis

Incubation of human testicular homogenates with [4-¹⁴C]pregnenolone gave substantial amounts of an unknown metabolite within 1 min, reaching plateau values of 17–23% of total radioactivity added within 5 min. Mass spectrometry of the metabolite showed it to be identical to the boar sex pheromone precursor androsta-5,16-diene-3β-ol (ADL). In cell cultures the major source of ADL and its dehydrogenated metabolite androsta-4,16-diene-3-one (ADN) was the Leydig cell. In rat and monkey testicular homogenates 16-ene-synthetase activity, a prerequisite for the synthesis of ADL and ADN, was completely lacking, limiting the presence of 16-androstenes to boars and men. In contrast to boars, however, in the human testis no 5-reductase activity was found and consequently no 5-reduced-16-androstenes, e.g. androstenol (AL, musk like) and androstenone (AN, urine like), known sex pheromones in pigs. As both sex pheromones have been identified in urine, plasma, sweat and saliva of men and (especially hirsute) women we hypothesize that AL and AN are synthesized from ADL via ADN peripherically in tissues rich in 5-reductase, i.e. skin, axillary sweat glands and probably also the salivary glands. So far, there is some evidence that both sex pheromones may have similar functions in humans as in boars.     

The occurrence of C19 steroids in testicular tissue and submaxillary glands of intersex pigs in relation to morphological characteristics.

Five true hermaphrodite pigs and two male pseudohermaphrodite pigs were studied. A 38XX sex chromosome constitution was found in peripheral leucocytes of three true hermaphrodites and in one male pseudohermaphrodite; XX/XY mixoploidy was present in the leucocytes of the remaining male pseudohermaphrodite. The occurrence of C19 steroids, including 16-androstenes, in the testicular tissue and submaxillary gland of intersex pigs was of a similar pattern to that found previously in mature boars, and masculinization of the genital tract was related to the amount of testicular tissue present. It is postulated that in the absence of germ cells in the testicular tissue of intersex pigs the Sertoli cells may be involved in the metabolism of dehydroepiandrosterone to 5-androstenediol, a possible testosterone precursor in the pig. The high levels of 16-androstenes found in the submaxillary gland of intersex pigs indicates that these steroids are responsible for 'boar taint' in these animals. In contrast to the boar, no consistent relationship was found between the occurrence of C19 steroids and the degree of masculinization of the submaxillary gland; it is postulated that the predominantly female genetic constitution may have affected the response of the salivary gland to androgen.     

Evolution of the ABPA Subunit of Androgen-Binding Protein Expressed in the Submaxillary Glands in New and Old World Rodent Taxa

The salivary androgen-binding proteins (ABPs) are members of the secretoglobin gene family present in mammals. Each ABP is a heterodimer assembled as an ABPA subunit encoded by an Abpa gene and linked by disulfide bridges to an ABPBG subunit encoded by an Abpbg gene. The ABP dimers are secreted into the saliva of mice and then transferred to the pelage after grooming and subsequently to the environment allowing an animal to mark territory with a biochemical signal. The putative role of the mouse salivary ABPs is that of pheromones mediating mate selection resulting in assortative mating in the Mus musculus species complex. We focused on comparing patterns of molecular evolution between the Abpa genes expressed in the submaxillary glands of species of New World and Old World muroids. We found that in both sets of rodents the Abpa genes expressed in the submaxillary glands appear to be evolving under a similar evolutionary regime, with relatively high nonsynonymous substitution rates, suggesting that ABP might play a similar biological role in both systems. Thus, ABP could be involved with mate recognition and species isolation in New World as well as Old World muroids.     

Boar salivary lipocalin. Three-dimensional X-ray structure and androsterol/androstenone docking simulations.

The X-ray structure of variant A of authentic boar salivary lipocalin (SAL), a pheromone-binding protein specifically expressed in the submaxillary glands of the boar, has been solved and refined at 2.1 A resolution. The structure displays a classical lipocalin fold with a nine-stranded sandwiched beta barrel and an alpha helix. A putative glycosylation site, at position 53, has been found to carry a GlcNAc sugar residue. In contrast with what was expected on the basis of mass spectroscopy reports, the internal cavity was found to be devoid of bound pheromonal compound (androstenone or androstenol). Instead, a small electron density volume could be satisfied by a glycerol molecule, a component of the cryoprotecting liquor. The internal cavity was revealed to be very small for steroid compound accommodation. Therefore, docking and molecular dynamics experiments were performed with both pheromonal compounds. These simulations clearly demonstrate a volume increase of the cavity upon steroid binding and the adaptation of the amino-acid side chains to the steroid molecules. This explains the higher affinity of SAL for both steroid molecules compared to other smaller molecules, although no specific interaction is established with either compound.     

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

Immunohistochemical analysis of secretoglobin SCGB 2A1 expression in human ocular glands and tissues

Human secretoglobin (SCGB) 2A1 (or lipophilin C, lacryglobin, mammaglobin B) is a small protein of unknown function that forms heterodimers with secretoglobin 1D1 (lipophilin A) in tears. SCGB 2A1 is homologous to mammaglobin (mammaglobin A) and the C3 component of prostatein, the major secretory protein of the rat ventral prostate. Androgen-dependent expression of SCGB 2A1 has been observed in the prostate. Besides identification of SCGB 2A1 in the tear proteome only its mRNA had been detected in the lacrimal gland. Here, we report expression of SCGB 2A1 in all ocular glands and in the keratinized stratified squamous epithelium of the eyelid as well as in the stratified epithelium of the conjunctiva and in the orbicularis oculi muscle. Almost all of these tissues are also known to express the androgen receptor. Therefore, we conclude that presence of the androgen signalling machinery could be the main general determinant of SCGB 2A1 expression. Implications of the presence in tear fluid of an androgen-regulated secretoglobin, which most likely binds hydrophobic ligands, for tear film lipid layer formation and function is discussed.     

Novel approach to obtain biologically active recombinant heterodimeric proteins in Escherichia coli

The strategy described in this paper provides a novel approach for recombinant expression of heterodimeric proteins, and is especially suitable for the production of proteins whose characteristics lead to aggregation in E. coli expression systems. Pheromaxein, a steroid-binding protein isolated from boar saliva, is a heterodimeric protein consisting of 10x10(3) rel. mol. mass units (pheromaxein A) and 8x10(3) rel. mol. mass units (pheromaxein C) subunits. Expression of pheromaxein subunits in E. coli resulted in extensive insoluble aggregation. The difficulty faced in obtaining soluble recombinant pheromaxein subunits was clearly evident when native pheromaxein immediately formed aggregates when it was separated into its individual subunits. An increase in soluble pheromaxein expression in E. coli was obtained when the subunits were expressed as fusion proteins with thioredoxin. Pheromaxein genes were inserted separately into pET32a+ vectors at the NcoI site, resulting in thioredoxin, S.Tag and His.Tag coding regions being upstream of the inserted gene. Soluble pheromaxein A-thioredoxin (pheroA/trx) and pheromaxein C-thioredoxin (pheroC/trx) fusions were purified to homogeneity, using a laboratory scale S-protein agarose affinity column. Cleavage of thioredoxin under normal conditions was not feasible due to the extensive aggregation problems experienced when pheromaxein subunits exist separately. PheroA/trx and pheroC/trx were therefore mixed together and cleaved from thioredoxin simultaneously so that pheromaxein subunits were given an instant opportunity to associate under oxido-shuffling conditions. The glutathione oxido-shuffling system allowed the disulphide bridges between pheromaxein A and C to rearrange until the correct native structure was formed. This novel approach combines affinity purification with a coupled fusion protein-cleavage and refolding technique.     

Lipophilins: human peptides homologous to rat prostatein

Lipophilin components A, B and C are human homologues of prostatein, the major secreted protein of rat prostate. This report describes their cDNA sequences, tissue expression and chromosomal localization. Lipophilin gene products were widely expressed in normal tissues, especially in endocrine-responsive organs. The gene for lipophilin C (also called mammaglobin b) is located on chromosome 11q12-q13.1, near the mammaglobin gene, a homologue overexpressed in many breast cancers. The lipophilin B gene resides on chromosome 10q23, a region deleted in many tumors, and the lipophilin A gene is on chromosome 15q12-q13.     

B-mode ultrasonographic examination of the accessory sex glands of boars

Thorough examinations of the reproductive system of boars are generally not performed on normal boars to be used for breeding; only boars with problems undergo a form of a breeding soundness examination. In order for veterinarians to identify pathological conditions, the normal architecture of the accessory sex glands needs to be described. The purpose of this study was to use B-mode ultrasonography to describe the accessory sex glands in the boar and to see if transrectal ultrasonography would be a viable option in which to obtain this data. Initially, cross-sectional saline bath examinations of accessory sex glands were performed on crossbred boar reproductive tracts (n = 4) using B-mode ultrasonography equipped with a 5 MHz dual frequency linear array transducer. In situ examinations were also performed on terminal line crossbred boars (n = 16) ranging in age from 10 to 23 months old using the same ultrasound methodology; four boars were under general anesthesia and the remaining 12 were standing in crates. Eight boars were abstinent for 2 days and the other eight had ejaculates collected 2 h prior to examination. The paired bulbourethral glands are best described as a long oval gland with a uniformly echogenic appearance with a large anechoic space in the center of the gland extending most of its length. The walls of the vesicular glands were found to be thin, with the parenchyma having multiple small echolucent areas that appeared to merge and form a central canal. The prostate gland was best identified as a pecan-sized gland with a uniform echogenic appearance. Visualization of the prostate gland was accomplished with more proficiency using the saline bath ultrasonography as compared to in situ examinations. All of the accessory sex glands could be examined using both methodologies of ultrasonographic examination with a 5 MHz frequency linear array transducer. It was determined that each accessory sex gland could be recognized, and differences between ejaculated and nonejaculated boars could be identified. The results of this study demonstrate that transrectal ultrasonography can be used as a diagnostic aid in assessing the accessory sex glands of boars.     

Characterization and cloning of two isoforms of heteroglobin, a novel heterodimeric glycoprotein of the secretoglobin-uteroglobin family showing tissue-specific and sex differential expression.

Heteroglobin (HGB) is a 39-kDa heterodimeric protein detected under non-reducing conditions in harderian, parotid, and submaxillary glands and saliva of the Syrian hamster with antiserum raised against the carboxyl end deduced from the female harderian gland cDNA FHG22 (Dominguez, P. (1995) FEBS Lett. 376, 257-261). After reduction, only one 5.6-kDa polypeptide, named HGB.A, was immunodetected and identified by sequencing as the mature FHG22 product. Tissue-specific expression of HGB.A and HGB mimics that of FHG22 mRNA, with sex differences in submaxillary and harderian glands. Purification of HGB revealed it consists of HGB.A disulfide bonded to HGB.B, a 33.5-kDa N-glycosylated subunit that yields a 9-kDa core polypeptide after deglycosylation. Two highly homologous (96.2%) cDNA clones (HGB.B1 and HGB.B2) encoding 94 amino acid-long isoforms were identified by screening a female harderian gland library with an HGB.B probe. The corresponding mature polypeptides are 78 amino acids long with 12 differences, but 3 putative N-glycosylation sites are maintained. The expression of HGB.B mRNAs is parallel to that of HGB and HGB.A, but no HGB.B2 mRNA was detected in submaxillary glands. Homology studies indicate that HGB.A and HGB.B1/HGB.B2 belong to different subfamilies of the secretoglobin-uteroglobin family and form heterodimers as previously described.     

Differential regulation of alpha 2u globulin gene expression in liver, lachrymal gland, and salivary gland

The alpha 2u globulins, products of a highly homologous multigene family, are synthesized in the liver and submaxillary salivary glands of the rat. Although their precise function has not been ascertained, they are of interest because of the complex developmental and hormonal regulation of their tissue levels. We now report that alpha 2u globulin is synthesized in a third tissue of the rat, the extraorbital lachrymal gland. Immunocytochemical studies indicate that the distribution of alpha 2u globulin is more homogeneous in the lachrymal gland than in the liver or submaxillary gland. In situ hybridization to alpha 2u globulin RNA reveals specific signal only over the acinar cells of the lachrymal gland. Several different isoelectric forms of alpha 2u globulin are encoded by lachrymal gland mRNA. The major lachrymal and salivary gland isoforms are indistinguishable from one another, but more acidic than the hepatic isoforms. In addition, analysis of double-stranded cDNAs with a diagnostic restriction-enzyme pair detects no differences between the alpha 2u globulin mRNAs of lachrymal and salivary gland, but clearly distinguishes these from their hepatic counterparts. In spite of the similarity between the lachrymal and salivary gland alpha 2u globulin gene products, we find that the hormonal and developmental regulation of alpha 2u globulin expression differs markedly in these two tissues. In the liver, where a different subset of alpha 2u globulin genes is expressed, a third regulatory phenotype is observed.     

Morphofunctional research on the effects of steroid-stimulating and -inhibiting drugs on the major salivary glands of rats

Adult male albino rats underwent chronic treatment with synthetic adrenocorticotropin and Dexamethasone, and were examined for a long time after the administration of these drugs. The major salivary glands were observed by means of histological and histochemical methods. We did not find enzymatic modifications whereas morphological damage was particularly evident in the submaxillary and parotid glands, especially all after Dexamethasone treatment; this damage is to be found up to at least 14 days after the end of treatment.     

Synthesis of free and sulphoconjugated 16-androstene steroids by the Leydig cells of the mature domestic boar

This study examined the involvement of sulphoconjugation in the biosynthesis of the 16-androstene steroids in Leydig cells of the mature boar, since the formation of steroid sulphoconjugates can reduce the levels of these steroids that accumulate in fatty tissue. Leydig cells were purified from testes of mature male pigs and incubated with pregnenolone, or various individual 16-androstene steroids for 10 min, 1, 4 and 8h. Sulphoconjugated steroids were recovered by solid-phase extraction followed by solvolysis. Profiles of unconjugated and sulphoconjugated steroids were analysed by HPLC. Steroids present in the sulphoconjugated fractions were purified, derivatised as O-methoxime/trimethylsilyl ethers (MO-TMS), and subsequently identified using gas chromatography-mass spectrometry (GC-MS). The principal metabolite produced from incubations with pregnenolone, androstadienol, androstadienone and 5alpha-androstenone was 3beta-androstenol. 16-Androstene steroids that were sulphoconjugated included 5alpha-androstenone, 3beta-androstenol and 3alpha-androstenol. Approximately 70% of the total amount of each 16-androstene steroid was in its sulphoconjugated form after incubations for 4h or more. The finding that sulphoconjugated 5alpha-androstenone was present in large amounts suggests that this steroid may be converted from a 3-keto to a 3-enol form which is subsequently sulphoconjugated. These findings emphasise the need to consider the impact of sulphoconjugation of the 16-androstene steroids and their role in contributing to boar taint.     

Development of the rat salivary glands. II. The identification of a trypsinlike protease in the submaxillary gland

Trypsinlike protease activity at pH 9.2 was measured in tissue extracts of adult rat salivary glands by using a fluorometric assay in which β-naphthylamine is released by the hydrolysis of benzylarginine β-naphthylamide. The submaxillary gland contains high levels of this activity, and the parotid and sublingual glands have a maximum of 2000-fold and 200-fold less. After polyacrylamide disc gel electrophoresis at pH 8.3, the protease activity of submaxillary extracts is associated with a major protein band. Neither this protein band nor its protease activity is detectable in extracts of parotid or sublingual glands. Homogenates of newborn submaxillary gland do not have this protease activity at detectable levels, suggesting that its major accumulation is postnatal.     

Isolation of two genomic sequences encoding the Mr = 14,000 subunit of rat prostatein

Complementary DNAs to rat ventral prostate poly(A) RNA were cloned into pBR322 by the "dG-dC tailing" procedure. Clones containing cDNAs to the mRNAs coding for each of the three subunits of a major secretory protein (prostatein) were identified by hybrid-arrested translation. A 457-nucleotide base pair cDNA (E45) and a portion of a 365-base pair cDNA (E85) were analyzed to determine the composite complete DNA coding sequence for the Mr = 14,000 (C3) subunit of prostatein. A sequence of 12-nucleotide bases (TTTGCTGCTATG) in the signal peptide of C3 was noted to be homologous to signal peptide nucleotide sequences reported in cDNAs coding for the other two prostatein subunits, Mr = 6,000 (C1) and 10,000 (C2). Complementary DNA coding for the C3 subunit was used as a hybridization probe to screen an EcoRI rat genomic DNA library. Two unique 12-kilobase genomic clones, each containing mRNA coding sequences within 2.5-3-kilobase fragments, were identified by restriction enzyme mapping and Southern blot analysis. Restriction enzyme sites within the coding regions of both genes were analogous to the cDNA. Differences in restriction enzyme sites in regions of intervening sequences and flanking DNA established the uniqueness of the two genes. It is suggested that both genes may be transcribed in vivo.     

Characterization of primary translation products from ovine and rat salivary gland mRNAs

Ovine and rat salivary gland mRNAs have been prepared and their translation products characterized. A 60 kD translation product from ovine submaxillary and sublingual gland mRNAs is identical in mass to the ovine apomucin. Two additional ovine translation products, 25 and 40 kD, are specific to mucin-producing salivary glands. Four rat mRNA translation products are encoded by mucin-producing salivary glands (38, 44, 67, 69 kD). These polypeptides were not detected in the parotid gland mRNAs, a serous gland. Each of these products has a high level of [3H]serine incorporation, a characteristic of mucins. The nature of these products suggests that they are mucins or mucin-like and that their molecular weights should approximate that of the corresponding apomucins.     

Testicular germ cell development in relation to 5alpha-androstenone levels in pubertal entire male pigs

Androstenone is a 16-androstene steroid pheromone produced in the Leydig cells in the testis, and considered to be one of the major compounds responsible for boar taint. In entire male pigs, progress of sexual maturation has been related to an increase in androstenone levels in fat. Onset of puberty and subsequent reproductive function involves genetic factors affected by the internal and external environment. In this study entire male cross-bred pigs were housed under two different light regimens in order to manipulate the onset of puberty. DNA flow cytometry (FCM) was used to study spermatogenesis and monitor the proportions of haploid (1n), diploid (2n), and tetraploid (4n) testicular cells, with conventional histological evaluation used as the reference technique. Agreement between these two methods was found to be good. The best fit model explained 34% of the variation in the androstenone concentrations. Sexual maturation in boars of 125-146 days of age, as assessed by DNA FCM, was not significantly associated with the variation in androstenone concentrations in adipose tissue when various independent variables (breed, age, light strategy, skatole concentrations in fat, and length of the bulbourethralis glands) were included in this model. These findings support the suggestion that selection against androstenone may be an option in the breeding of entire male pigs.