THE OCCURRENCE OF TESTOSTERONE AND 5\g=a\-DIHYDROTESTOSTERONEIN THE SUBMAXILLARY SALIVARY GLAND OF THE BOAR W. D. BOOTH A.R.C. Unit of Reproductive Physiology and Biochemistry, University of Cambridge (Received 14 February 1972) SUMMARY Testosterone and 5\g=a\-dihydrotestosteronewere 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\p=m-\liquidchromatography and combined gas\p=m-\liquidchromatography\p=m-\massspectrometry. In the submaxillary gland of the mature boar, a high concentration of androgen (> l \g=m\/g) was found, and the concentration of 5\g=a\-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. INTRODUCTION There is growing evidence that the two musk-smelling 16-unsaturated C19 steroids, 5a-androst-16-en-3a-ol and 5a-androst-16-en-3-one, are the prime contributors to the pheromone complex of the boar (Melrose, Reed & Patterson, 1971). Patterson (1968) has identified these two steroids in the submaxillary glands of the boar but could find only traces in the castrated male and none in the female. These results show that there is a sexual dimorphism in the occurrence of 16-unsaturated C19 steroids in the submaxillary gland of the pig which is related to the presence of the testis in the male (Rhodes & Patterson, 1971). The purpose of this present study was to investigate the possibility that other C19 steroids such as testosterone and its immediate precursors or metabolites might accumulate in the submaxillary gland of the boar, and thus indicate that the sexual dimorphism observed in the submaxil lary salivary gland of the pig could be related to the presence of androgens. The following trivial names and abbreviations are used: 5a-dihydrotestosterone, 17/?- hydroxy-5a-androstan-3-one; 5-androstenediol, androst-5-ene-3/?,17/?-diol; dehydro epiandrosterone, (DHA); androstenedione, androst-4-ene-3,17-dione. * Postal address: Animal Research Station, 307 Huntingdon Road, Cambridge CB3 OJQ.
20 MATERIALS AND METHODS Authentic testosterone was obtained from the M.R.C. Steroid Reference Collection by courtesy of Professor W. Klyne and 5a-dihydrotestosterone was purchased from the Sigma Chemical Co. Ltd., Lettice Street, London S.W. 6. [7a-3H]Testosterone (85 mci/mmol) and [la,2a-3h]5a-dihydrotestosterone (49 Ci/mmol), for use as in ternal recovery standards, were purchased from the Radiochemical Centre, Amer sham, Bucks. Radioactive and non-radioactive steroids were checked for purity by thin-layer chromatography (t.l.c). All solvents (Analar grade) were obtained from B.D.H. Ltd., Poole, Dorset, and redistilled before use. A 0 g batch of MN-Kieselgel G/UV254 (Camlab, Cambridge) for t.l.c. was washed with 10 ml redistilled methanol and dried before coating on glass plates to a thickness of 0-5 mm. This procedure removed substances from the Kieselgel which would otherwise have interfered with gas-liquid Chromatographie (g.l.c.) analyses. Paper chromatography was carried out on Whatman No. 2 paper which had been washed with redistilled methanol for at least 48 h. Gas-liquid chromatography was performed using a Series 104, Model 64, dual-flame ionization Chromatograph (Pye-Unicam, Cambridge). Combined gasliquid chromatography-mass spectrometry (g.l.c.-m.s.) was carried out with an LKB 9000 instrument. Reference 4-en-3-oxo steroids were located on thin-layer and paper chromatograms under u.v. light (254 nm), 5-ene-3/?-hydroxy steroids on paper chromatograms by antimony trichloride in chloroform and all reference steroids on t.l.c. by the Allen's reagent (Allen, Hayward & Pinto, 19). Submaxillary glands Pigs were slaughtered either by electric stunning or the captive-bolt method before being bled, and the submaxillary glands removed. (The male genital tract was also removed for further studies and to check the reproductive status of the animal.) After pieces of tissue had been taken for histological studies, the remaining tissue was stored over solid C02 until analysed for steroids. Extraction of submaxillary gland The submaxillary gland tissue (25 g) from a 4-yr-old boar was homogenized in ml 2-5 % NaOH solution using a Waring Blendor and the homogenate extracted with diethyl ether (2 300 ml). The pooled ether extracts were washed with water (3 60 ml), dried over anhydrous Na2S04, and the extract evaporated to dryness under reduced pressure. The extract was partially defatted by 'freezing-out' the lipid at C overnight from its solution in a small volume (3 ml) of light petroleum (40-60 C). After centrifugation the supernatant was made up to 20 ml with light petroleum and extracted with 83% aqueous methanol (6 20 ml). The petroleum phase was retained for the analysis of 16-unsaturated C19 steroids (as part of another investigation), and the pooled aqueous methanol extract evaporated to dryness under reduced pressure. Thin-layer and paper chromatography In order to separate C19 steroids from 16-unsaturated C19 steroids not extracted by light petroleum, the aqueous methanol extract was subjected to t.l.c. and the plate developed twice with toluene:ethyl acetate (9:1, v/v) and once with benzene:
ether (9:1, v/v) (Gower, 1964). The areas on the plate corresponding to the non polar 16-unsaturated C19 steroids were eluted and kept for future investigation. The moderately polar region corresponding to C19 steroids was eluted and further purified by t.l.c. benzene:acetone (4:1, v/v). The area corresponding to the positions of 5-androstenediol, testosterone, DHA, 5a-dihydrotestosterone and androstenedione was eluted, and any oestrogen which might have been present, removed by the method of Brown (1955). The elutions from silica so far described were carried out with chloroform:methanol (2:1, v/v; 2 5 ml). The resulting neutral fraction was chromatographed on Whatman No. 2 paper in the Bush A system (Bush, 1952) giving three fractions corresponding to: (a) 5-androstenediol and testosterone, (b) DHA and (c) 5a-dihydrotestosterone and androstenedione. Each fraction was acetylated over night with acetic anhydride in pyridine (0-1 ml of each), and the products were submitted to t.l.c. using benzene : ether (4:1, v/v) in order to separate 5-androstene diol diacetate from testosterone acetate, and 5a-dihydrotestosterone acetate from androstenedione. The steroids were eluted on this occasion with ethyl acetate (2x5 ml), since it has been observed that this solvent gives rise to fewer spurious peaks on subsequent g.l.c. than water-miscible solvents. Gas-liquid chromatography After acetylation, steroids were tentatively identified by g.l.c. A 1 cm glass column was used containing 1 % XE-60 stationary phase on diatomite CQ (-200 mesh). The oven temperature was maintained at 215 C, FID detector at 2 C, injection port heater at 2 C and the argon carrier gas flow rate at 60 ml/min. Authentic testosterone acetate was used as the internal standard for 5-androstenediol diacetate, DHA acetate and 5a-dihydrotestosterone acetate fractions, and DHA acetate as the internal standard for the testosterone acetate and androstenedione fractions. RESULTS Gas-liquid chromatography revealed two peaks which had the same retention times as authentic testosterone acetate and 5a-dihydrotestosterone acetate respec tively (Fig. 1). However, there was no evidence for the presence of 5-androstenediol diacetate, DHA acetate or androstenedione. The remaining testosterone acetate and 5a-dihydrotestosterone acetate samples were hydrolysed (Bush & Willoughby, 1957) and rechromatographed on t.l.c. before subjecting them to g.l.c. Peaks with the same retention times as authentic testosterone and 5a-dihydrotestosterone were observed. The possibility that the 5a-dihydrotestosterone fraction might also have contained the 5/?-epimer was considered. However, under the g.l.c. conditions used, authentic 5a and 5/?-dihydrotestosterone acetates had retention times relative to testosterone acetate of 0-64 and 0-59 respectively and the corresponding free steroids 0-58 and 0-53 respectively: in the submaxillary gland extracts, no peaks corresponding to the authentic 5/?-epimer were observed. A further 25 g of submaxillary gland tissue from the same boar were extracted to provide more material for a definite identification of the acetates of testosterone and 5a-dihydrotestosterone by means of g.l.c.-m.s. The LKB 9000 (g.l.c.-m.s.) was equipped with a 1 cm glass column containing 1-5% QF-1 stationary phase. The
10 Time (min) 20 10 Time (min) Fig. 1. (a) Gas-liquid chromatography (g.l.c.) of the testosterone acetate fraction after acetyla tion of material from the boar submaxillary gland; (b) g.l.c. of authentic testosterone acetate (peak 2). Dehydroepiandrosterone acetate (peak 1) was added as internal standard; (c) g.l.c. of the 5a-dihydrotestosterone acetate fraction after acetylation of material from the boar sub maxillary gland; (d) g.l.c. of authentic 5a-dihydrotestosterone acetate (peak 1). Testosterone acetate (peak 2) was added as internal standard. Column: 1 % XE-60 at 215 C with carrier gas flow rate 60 ml/min.
75 («) " " 124 147 3 S 25 o S 75 o Oí 25 ) 124 147 228 228 JuJL 288 288 330 (m+) 330 (m + ) 0 -^ -1 1 200 2 300 3 75 W " - CO o 25 -o g3 -S o Cl> > JS 75 cu eí 25 0 ( 1149 li M- 149 1 200 m/e 231 _1_ 231 2 )57 257 272-1 300 332 (m*). 332 (m+) Fig. 2. Mass spectra of (a) testosterone acetate prepared from testosterone isolated from the boar submaxillary gland; (6) authentic testosterone acetate; (c) 5œ-dihydrotestosterone acetate prepared from 5œ-dihydrotestosterone isolated from the boar submaxillary gland; (d) authentic 5a-dihydrotestosterone acetate. column temperature was maintained at 238 CC, injection port heater at 240 C, mole cular separator at 230 C and the helium gas-flow rate at 30 ml/min. The results are presented in (Fig. 2). Subsequent investigations on the occurrence of testosterone and 5a-dihydrotestosterone in the boar submaxillary gland have included observations on the submaxil lary gland of female pigs and the parotid gland of boars. Both testosterone and 5a-dihydrotestosterone were isolated from the submaxillary gland of mature boars (Table 1), but neither was found in the submaxillary gland of the female pig, or in the parotid gland of the boar. Evidence for losses of steroids during analysis is limited. 3
However, in the case of the Large White Landrace boar (see Table 1), recoveries of [7a-3H]testosterone and [la,2a-3h]5a-dihydrotestosterone were 41-6 and 27-7% respectively. Table 1. Testosterone and 5ct-dihydrotestosterone in the submaxillary salivary glands of boars at different ages Breed LW Esx LW Esx LW Ldr LW Age* 12 wk 36 wk 2yr 4yr /tg/ g Trace 11-2 2-80 Testosterone /ig/submaxillary glands Trace 0-70 36-0 4-87 5a-Dihydrotestosterone / g/ g 0-74 2-10 42-8 4-48 /tg/submaxillary glands 013 1-42 137 7-76 * 12 wk = mean of four animals, 36 wk = mean of two animals. All steroid values in the Table are given uncorrected for losses during extraction. LW Esx = Essex, Ldr = Landrace. Large White, DISCUSSION The high level of 5a-dihydrotestosterone in relation to testosterone found in the boar submaxillary gland supports the current concept that testosterone is readily metabolized to 5a-dihydrotestosterone in androgen target organs (Baulieu, 1970; Wilson & Gloyna, 1970). The amounts of testosterone and 5a-dihydrotestosterone which have been isolated from the submaxillary glands of mature boars are consider able, and yet there was no evidence for the presence of intermediary steroids which are associated with testosterone synthesis in the boar testis (Booth, 1970; Raeside & Howells, 1971). These observations differ from those made in the dog. In recent experiments Weiner, Ofner & Sweeney (1970) infused radioactive testosterone into the canine submaxillary gland in vivo, where it was primarily converted to the less potent androgen, androstenedione. However, there is no evidence to suggest that the submaxillary gland of the dog is in any way a significant target organ for androgen action. The high concentration of 16-unsaturated C19 steroids in the submaxillary gland of a mature boar (W. D. Booth, unpublished observations), and their disappearance after castration (Rhodes & Patterson, 1971), suggests that testosterone plays a role in the occurrence of the musk-smelling steroids, which are either of testicular origin (Booth, 1970; Katkov & Gower, 1970) or newly formed in the salivary gland. Lacassagne (1940a, b, c) observed that a sexual dimorphism occurs in the submaxil lary glands of mice and rats. He observed that in the male rodent the serous tubules of the submaxillary gland were hypertrophied, and that this condition was related to testosterone. The sexual dimorphism observed in the present study and in that of Patterson (1968) indicates that of the three types of salivary gland, the submaxillary gland displays a marked degree of sexual dimorphism; in this respect the pig is similar to the mouse (Berkman & Kronman, 1970). Current observations on the histology and histochemistry of the pig salivary glands, support these observations.
I am grateful to Dr R. L. S. Patterson of the Meat Research Institute, Langford, Bristol, who carried out the combined gas-liquid chromatography-mass spectro metry. REFERENCES Allen, W. M., Hayward, S. J. & Pinto, A. (19). A colour test for dehydroisoandrosterone and closely related steroids of use in the diagnosis of adrenocortical tumours. J. clin. Endocr. Metab. 10, 54 70. Baulieu, E. E. (1970). The action of hormone metabolites: a new concept in endocrinology (on the metabolism and the activity of testosterone). Revue europ. Étud. clin. biol. 15, 723-726. Berkman, M. D. & Kronman, J. H. (1970). A histochemical study of the effects of castration and testo sterone administration on the major salivary glands of Swiss mice. Ada anat. 76, 200 219. Booth, W. D. (1970). The occurrence of some C19-steroids and vitamin A in boar testis. J. Reprod. Fert. 23, 533-534. Brown, J. B. (1955). A chemical method for the determination of oestriol, oestrone and oestradiol in human urine. Biochem. J. 60, 185 193. Bush, I.E. (1952). Methods of paper chromatography of steroids in mammalian blood and tissues. Biochem. J., 370-378. Bush, I. E. <& Willoughby, M. (1957). The extraction of allotetrahydrocortisol in human urine. Biochem. J. 67, 689-700. Gower, D. B. (1964). Chromatographie separation of C19-16-dehydrosteroids. J. Chromât. 14, 423 1. Katkov, T. «fe Gower, D. B. (1970). The biosynthesis of androst-16-enes in boar testis tissue. Biochem. J. 117, 533-538. Lacassagne,. (1940a). Dimorphisme sexual de la gland sous-maxillaire chez la souris. C. r. Séanc. Soc. Biol. 133, 180-181. Lacassagne, A. (19406). Mesure de l'action des hormones sexuelles sur la glande sous-maxillaire de la souris. C. r. Séanc. Soc. Biol. 133, 227-229. Lacassagne, A. (1940 c). Reaction de la glande sous-maxillaire a l'hormone maie chez la souris et le rat. C. r. Séanc. Soc. Biol. 133, 539-542. Melrose, D. R., Reed, H. C. B. «fe Patterson, R. L. S. (1971). Androgen steroids associated with boar odour as an aid to the detection of oestrus in pig artificial insemination. Br. vet. J. 127, 497-2. Patterson, R. L. S. (1968). Identification of 3cx-hydroxy-5a-androst-16-ene as the musk odour component of boar submaxillary salivary gland and its relationship to the sex odour taint in pork meat. J. Sci. Fd Agrie. 19, 4-8. Raeside, J. I. «fe Howells, G. A. (1971). The isolation and identification of androstenediol sulfate from spermatic vein blood and testes of the boar. Can. J. Biochem. 49, 80-84. Rhodes, D. N. «fe Patterson, R. L. S. (1971). Effects of partial castration on growth and the incidence of boar taint in the pig. J. Sci. Fd Agrie. 22, 320-324. Weiner, A. L., Ofner, P. «fe Sweeney,.. (1970). Metabolism of testosterone-4-14c by the canine sub maxillary gland in vivo. Endocrinology 87, 406-409. Wilson, J. D. «fe Gloyna, R. E. (1970). The intranuclear metabolism of testosterone in the accessory organs of reproduction. Recent Prog. Horm. Res. 26, 309-336.