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ABSTRACT
Submaxillary glands of mature Göttingen miniature pigs were examined for the presence of a sexual dimorphism. Gland weights, serous cell hypertrophy and total protein in the glands were much greater in male than female pigs. High concentrations of the pheromonal 16-androstene steroids were present in the glands of males and exceeded 2 mmol/g in some animals; this was primarily due to 5α-androst-16-en-3α-ol. The high concentration of 16-androstene steroids in boar glands was correlated with the presence of large amounts of binding protein for these steroids in the glands; smaller amounts of the binding protein were detected in female glands. These findings are similar to those found in domestic pigs, but the degree of sexual dimorphism assessed from these findings is more extreme in the miniature pig.
J. Endocr. (1984) 100, 195-202
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SUMMARY
Testosterone and 5α-dihydrotestosterone were extracted from the submaxillary salivary glands of boars at different ages, but were not detected in these glands of the female pig. After purification by thin-layer and paper chromatography the steroids were identified by gas—liquid chromatography and combined gas—liquid chromatography—mass spectrometry. In the submaxillary gland of the mature boar, a high concentration of androgen (> l μg/100 g) was found, and the concentration of 5α-dihydrotestosterone was two to four times higher than testosterone. Immediate steroid precursors of testosterone which are found in boar testis were not isolated. The isolation of the two potent androgens, and the known occurrence of 16-unsaturated C19 steroids in the boar submaxillary gland, shows that there is a biochemical sexual dimorphism in this gland of the pig, which is primarily under the influence of testicular hormones.
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Homogenates of the submaxillary glands of mature domestic boars were incubated with radioactively labelled C19 steroids. The metabolism of [7α-3H]dehydroepiandrosterone and [7α-3H]androstenedione was primarily to the end products 5α-androstane-3,17-dione and androsterone, but some testosterone, 5α-dihydrotestosterone and 5α-androstane-3α,17β-diol were also formed. When [7α-3H]5-androstene-3β,17β-diol was used as a substrate, the major metabolite was testosterone. Both [7α-3H]5-androstene-3β,17β-diol and [1α,2α-3H]-testosterone were converted to 5α-dihydrotestosterone and high yields of 5α-androstane-3α,17β-diol, but only small yields of 5α-androstane-3β,17β-diol and androsterone were obtained. High yields of 5α-androstane-3α,17β-diol and to a lesser extent 5α-androstane-3β,17β-diol were formed from the substrate [1α,2α-3H]5α-dihydrotestosterone. Conversely, when both 3H-labelled androstanediols were used as substrates, 5α-dihydrotestosterone was formed in equal amounts together with a large yield of 5α-androstane-3α,17β-diol from the 3β-epimer. There was no evidence for steroid sulphatase or aromatase activity in these incubations. These findings support the prohormone concept that precursor steroids of testosterone, known to be present in boar testis, could act as an additional source of potent androgens in peripheral target organs to androgens in this species.
In a number of incubations with [1α,2α-3H]testosterone, free oestradiol-17β and oestrone, but not oestrone sulphate enhanced the formation of androstenedione, but reduced the formation of 5α-dihydrotestosterone. However, both free and conjugated oestrogen reduced the formation of 5α-androstane-3α,17β-diol. These findings are discussed in relation to the high levels of oestrogen found in male pigs.
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Homogenates of the submaxillary glands of immature and mature pigs were incubated with 3H-labelled C19 steroids which have been shown previously to be metabolized in vitro by the submaxillary gland of mature boars. Dehydroepiandrosterone was metabolized largely to androstenedione, 5α-androstane-3,17-dione and androsterone, and to small amounts of testosterone, 5α-dihydrotestosterone and 5α-androstanediols. Testosterone yielded predominantly 5α-androstane-3α,17β-diol with smaller amounts of other 5α-reduced products, i.e. 5α-dihydrotestosterone, 5α-androstane-3β,17β-diol, 5α-androstane-3,17-dione and androsterone; 5α-dihydrotestosterone and the two epimeric 5α-androstane-3α/3β,17β-diols were interconverted. These and earlier results show that the porcine submaxillary gland has the capacity in vitro to metabolize selected C19 steroids in a way which is not related to either sexual maturity or sex of the animal; in this respect the findings support certain aspects of previous histochemical studies.
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Minces of the prostate and seminal vesicles of the mature boar were incubated with the following major testicular steroids of the pig: [3H]dehydroepiandrosterone, [3H]5-androstene-3β,17β-diol, [3H]oestrone, [3H]oestradiol-17β and their respective sulphate conjugates (excluding oestradiol-17β). Incubations were also carried out with [3H]testosterone, [3H]5α-dihydrotestosterone and [3H]5α-androstanediols. Minces of the epididymides were incubated with [3H]dehydroepiandrosterone and [3H]oestrone sulphates. The prostate and seminal vesicles converted dehydroepiandrosterone predominantly to weak androgens, whereas 5-androstene-3β,17β-diol was primarily converted to testosterone; testosterone and its 5α-reduced metabolites were metabolized in a manner typical of androgen end target organs. Unconjugated oestrone and oestradiol-17β were interconverted by the prostate and seminal vesicles. The metabolism of C19 steroid sulphates was < 1% in all incubations; some oestrone sulphate, however, was converted to unconjugated oestrone and oestradiol, particularly by the caput epididymidis. The significance of these results is discussed in relation to recent studies in vivo.
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20α-Hydroxypregn-4-en-3-one (20α-dihydroprogesterone) has been isolated from porcine ovaries under in vitro conditions (Bjersing & Carstensen, 1967; Cook, Kaltenbach, Norton & Nalbandov, 1967; Schomberg, 1967). No 20β-epimer has hitherto been demonstrated in the pig.
While the progesterone concentration was being investigated in the corpora lutea of the pregnant gilt using a method similar to that of Rowlands & Short (1959), two u.v. absorbing substances were found on the paper chromatograms with Rf values identical to authentic 20α- and 20β-hydroxypregn-4-en-3-one (20α-ol and 20β-ol) These substances were eluted, and gave the same response as the authentic steroids when examined by spectrophotometry, namely an absorption maximum in ethanol at 240 mμ, an absorption maximum at 285 mμ on heating with cone. H2SO4, and 20α-ol gave an absorption maximum at 485 mμ when treated with sulphuric acid:80% ethanol (Wiest, 1959). Both substances were acetylated with 0·1 ml. pyridine and acetic anhydride
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ABSTRACT
Pheromaxein, the 16-androstene steroid-binding protein with a relative molecular mass of 15 000 was isolated in sub-milligram quantities from the submaxillary gland and saliva of the Gottingen miniature boar, after a fourfold purification involving the following methods: ultrafiltration for submaxillary gland cytosols and ethanol precipitation for saliva, Concanavalin-A-Sepharose affinity chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, 'Extractigel-D' affinity chromatography (to remove sodium dodecyl sulphate) and fast protein-liquid chromatography. Yields of purified pheromaxein obtained after fast protein-liquid chromatography represented 10–20% of total protein present in an ultrafiltrate of a submaxillary gland cytosol. Fast protein-liquid chromatography separated the α- and β-charge isomers of pheromaxein which were shown to have isoelectric points of 4·78 and 5·35 respectively on flat-bed isoelectric focusing. Some data are provided for the variable occurrence of the isomeric forms of pheromaxein in relation to different breeds of pig. Five 16-unsaturated steroids showed the highest binding to pheromaxein. Other steroids of the 5α- and 5β-androstane series also showed some binding to pheromaxein, i.e. 17β-hydroxy-5α-androstan-3-one (19·2%), with 5α-androstan-3-one, which has a similar urinous odour to 5α-androst-16-en-3-one, showing the greatest binding (42·6%) relative to 5α-androst-16-en-3-one (100%).
J. Endocr. (1988) 118, 47–57
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SUMMARY
The metabolism of radioactively labelled testosterone, 5α-dihydrotestosterone, 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-diol by the rabbit epididymis and prostate has been investigated in vitro and in vivo. In vitro, the rate of conversion of testosterone to 5α-reduced androgens was low in both glands. Varying the nature of the tissue preparation, age of animal or incubation medium did not improve the situation substantially. However, the rabbit prostate and epididymis metabolized 5α-reduced androgens readily. The prostate was particularly efficient at interconverting 5α-androstane-3α,17β-diol and 5α-dihydrotestosterone, whereas the capacity of the epididymis to carry out this step was much lower. Small amounts of 5α-androstane-3,17-dione and androsterone were also identified. Both glands interconverted 5α-dihydrotestosterone and 5α-androstane-3β,17β-diol to a comparable degree.
Following the intravenous injection of 3H-labelled testosterone, significant levels of 3H-labelled 5α-dihydrotestosterone were found in the prostate and epididymis within 30 min. Furthermore, 5α-androstane-3α,17β-diol was detected in both glands. In blood plasma, the ratio of radioactively labelled testosterone: 5α-dihydrotestosterone was 2: 1, i.e. similar to that for endogenous steroids. The intravenous injection of 3H-labelled 5α-androstane-3α-17β-diol gave rise to much higher amounts of 5α-dihydrotestosterone in the prostate than in the epididymis, whereas the reverse was found for the levels of unmetabolized diol.
The results indicate that the prostate and epididymis of the adult rabbit differ in their capacity to metabolize 5α-reduced androgens and that both glands depend to a large extent on the relatively high levels of 5α-dihydrotestosterone and 5α-androstanediols present in the peripheral circulation, rather than the metabolism of testosterone in situ.
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ABSTRACT
Submaxillary salivary gland tissue from large White, Göttingen miniature and Meishan (Chinese) breeds of pig, and European wild boars, was incubated with [35S]methionine. The radiolabelled amino acid was incorporated into protein in all incubations as demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Specifically [35S]methionine was predominantly incorporated into the α- and β-charge isomers of pheromaxein, a 16-androstene steroid-binding protein, as shown by SDS-PAGE in combination with vertical isoelectric focusing on polyacrylamide slab gels. The synthesis of pheromaxein occurred in submaxillary gland tissue from both sexes, including tissues stored frozen at −70 °C for long periods. There was little evidence for pheromaxein synthesis in parotid gland tissue or skeletal muscle. Total protein, pheromaxein and total 16-androstenes were determined in the submaxillary gland cytosols of six mature Göttingen miniature boars and a positive correlation was found between these glandular constituents. The amounts of endogenous pheromaxein relative to total protein in the submaxillary gland cytosols (range 10·3–18·0%), together with the predominant synthesis of this protein in vitro, indicate that pheromaxein is a major protein produced in porcine submaxillary glands, particularly in those of the male.
Journal of Endocrinology (1991) 128, 205–212
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ABSTRACT
An enzyme-linked immunosorbent assay has been developed for the direct assay of cortisol in the saliva of man, sheep, calf and pig. The assay is sensitive (∼1 pg), highly specific and reproducible, and has the advantage over radioimmunosassay in being cheaper and quicker to carry out. The relatively-non invasive method of collecting saliva on cotton buds coupled with simple assay equipment provide a good potential for assessing cortisol status in studies on stress and welfare in farm animals as well as the possibility of monitoring cortisol status in mammals generally.
