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a Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Dihydrotestosterone was the principal metabolite, but the amounts formed depended on the source, with little from the epididymal tissues and seminal vesicles, but greater quantities from the vas deferens (> 25%) and prostate (> 30%). The most noteworthy feature of estrogen metabolism was the extent of conjugation by all tissues. Almost all radioactivity in the conjugate fractions for the epididymis and vas was present as sulfates. Glucuronidates were seen for the prostate and were the dominant form of conjugation (about 60%) for the seminal vesicles. A striking parallel existed for the profiles of estrogen metabolites from all tissues for unconjugated and hydrolyzed fractions. Only in quantitative terms were some distinctions noted. These overall findings underscore a need to consider local metabolism of steroid hormones in target tissues of the male reproductive system.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dihydrotestosterone [1]. Further metabolism may result in products that either contribute to additional biological activation or represent a pathway for reduction in the stimulation by testosterone [2]. This latter objective would include the possibility of formation of water-soluble conjugates, such as those from glucuronosyltransferase activity with C19 steroids in the human prostate [3, 4]. To this end also, it may be noted that steroid sulfotransferase activity has been recorded for the reproductive tract of the male hamster [5]; however, its relevance for testosterone metabolism is in some doubt since 
5-3ß-hydroxysteroids are the preferred substrates. In addition to conjugation, it is likely that metabolism of testosterone to such polar compounds as androstanetriols would give rise to hydrophilic products that could be removed easily as water-soluble substances and thus contribute to down-regulation in the response.
A role for estrogens in the male reproductive system has become the focus of increased attention [6]. Recently, a specific physiological endpoint of estrogen action in the male has been demonstrated for the first time, in the stimulation of reabsorption of luminal fluid by the efferent ductules of rat testes [7]. Reports on the presence of the estrogen receptors (ER and ERß) at many sites in the male reproductive system [8–10] suggest that estrogens may have an extensive role in the regulation of reproductive functions in the male. In this context, our earlier work revealed that estrogens act synergistically with testosterone on the accessory sex glands and libido in boars castrated after reaching maturity [11]. It may be noted here that the boar is remarkable for its high levels of testicular estrogen secretion [12–14] as well as for its exceedingly large volume of semen (200–500 ml) at ejaculation [15]. Estrogens also appear to be important along with androgens as regulators of epididymal development and function in the rabbit [16, 17] and rat [18]. Moreover, the early investigations on the induction of prostatic hypertrophy in the dog [19] were followed by many studies confirming that estrogens synergize with 5-reduced androgens in the dog prostate gland [2]. Enhanced prostate growth may have resulted from a synergistic effect of estrogens and androgens leading to an elevation of 5-reductase activity as seen in the rat [20].
Estrogen metabolism in the body occurs mainly in the liver. However, evidence is accumulating that metabolism of estrogens in target cells may contribute to the overall response to the hormone [21]. A hypothesis is emerging that the biological effects of an estrogen will depend on the profile of multiple metabolites formed and the biological activities of each of the metabolites. This view was expressed earlier for testosterone [22] and is now well recognized. The aim of the present study was to investigate the potential for metabolism of testosterone, as well as estrogens, by tissues from several components of the reproductive system in the mature boar.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Radiolabeled [1,2,6,7-3H(N)]testosterone (96.5 Ci/mmol) was obtained from NEN Life Science Products Inc. (Boston, MA); both [2,4,6,7-3H]estrone (101 Ci/mmol) and [2,4,6,7-3H]estradiol (83 Ci/mmol) were from Amersham Canada Ltd. (Oakville, ON, Canada). Nonradioactive steroids were purchased from Steraloids Inc. (Wilton, NH). Solvents were glass-distilled, reagent-, or HPLC-grade from Caledon Laboratories Ltd. (Georgetown, ON, Canada). Sep-Pak C18 cartridges were purchased from Waters Scientific (Mississauga, ON, Canada). ß-Glucuronidase (Type B-1, from beef liver) was supplied by Sigma Chemical Co. (St. Louis, MO). All other chemicals were analytical grade from Sigma or from Fisher Scientific (Toronto, ON, Canada).
Preparation and Incubation of Tissues
Seminal vesicles were removed at slaughter from 4 pubertal Yorkshire male pigs (6 mo old) in experiment 1. For experiments 2 and 3, an adult male animal (10 mo old) and a mature boar (2 yr old) were used, respectively. In the latter two experiments, the reproductive tracts and accessory sex glands were recovered at slaughter (experiment 2) or after intravenous injection of pentobarbital (experiment 3). All tissues were placed on ice and taken to the laboratory for incubation within 2 h. The glands were dissected free of extraneous tissues and minced finely by hand, or with a McIlwain tissue chopper (Gomshall, Surrey, UK), before being divided into equal portions. After the vas deferens was stripped, it was minced and transferred to a test tube containing physiological saline. Portions of the midregions of the caput, corpus, and caudal epididymides were dissected out and placed separately in saline after mincing. Three washes of all tissues from the reproductive tract were done to remove spermatozoa by suspension and gravity sedimentation in saline solution.
Tissues were incubated for 2 h in 5 ml of culture medium (TC-199) in 25-ml Erlenmeyer flasks at 34°C under 5% CO2 and 95% air in a shaking water bath, with 3H-labeled steroids (about 2 x 106 cpm, in duplicate or quadruplicate). The amount of tissue dispensed was approximately the same for both glands and various regions of the tract in each experiment, and was approximately 0.5 g (wet weight) per flask. After incubation, the contents of the flasks were transferred to tubes for centrifugation to recover the media. The tissues were washed twice by resuspension and centrifugation in 1 ml of TC-199, and the washings were pooled with the spent media for storage in glass vials at -20°C until processed as follows. The thawed contents of each vial were vortexed, and an aliquot (1/100th) was taken to measure radioactivity by liquid scintillation counting (LSC).
Analytical Procedures
Steroids in the media were recovered by solid-phase extraction (Waters C18 Sep-Pak cartridges) as described previously [23]. Unconjugated and conjugated steroids were eluted from the primed cartridges with 5 ml diethyl ether and 5 ml methanol, successively. The ether and methanol eluates were evaporated separately under nitrogen at < 45°C. The conjugated material underwent two hydrolytic processes, in series; in each case, the recovery of free steroids from Sep-Pak cartridges was as described above. These steps were briefly as follows: the dried methanol (conjugate) fraction from the first cartridge was acid-solvolysed overnight at 45°C with trifluoroacetic acid:ethyl acetate (1:100; v:v) to obtain a "sulfate" fraction, as free steroids; and the conjugated material (methanol eluate) from the second cartridge was submitted to enzyme hydrolysis. The dried methanol fraction was reconstituted in 0.5 ml of 0.5 M sodium acetate buffer (pH 5.0), and 1250 units of ß-glucuronidase (type B-1, from beef liver) were added in 25 µl of the buffer for incubation at 37°C overnight. Free steroids obtained at this stage (third cartridge) were designated as the "glucuronidate" fraction; and the nonhydrolysed steroids eluted with methanol were considered to be "conjugated" in an unknown form(s).
The amount of radioactive material recovered from each incubation was determined by LSC in a 5-ml cocktail (Ecolite; ICN, Costa Mesa, CA), by taking an aliquot after thawing but before solid-phase extraction on Sep-Pak C18 cartridges. Similarly, the dried eluates of subsequent separations on C18 cartridges were dissolved in methanol, and aliquots were removed for counting radioactivity. This was done at each stage in a series and served also to ensure that sufficient radioactive material was taken for a reliable estimation of its distribution at the next step.
Unconjugated and hydrolyzed steroids in the fractions obtained serially from Sep-Pak cartridges were examined by HPLC. Profiles for the steroids were generated by a method developed in our laboratory (unpublished results) and described briefly in a recent report [23]. A binary solvent gradient of acetonitrile-water was used with a Waters HPLC (Waters Corp., Milford, MA) at a flow rate of 2 ml/min, and absorbance was monitored at 254 and 280 nm. Fractions (1 ml) were collected automatically (LKB Redi-Frac; Pharmacia, Kalamazoo, MI) into vials, and 5 ml Ecolite cocktail was added for LSC.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most of the radioactivity was recovered in the media after incubation regardless of the source of the tissue (mean% ± SD = 74.7 ± 5.0, n = 20). The data that follow relate only to work done on the media samples. Radioactivity was present mainly (88.0% ± 3.2, n = 4) in the unconjugated fraction in experiment 1, in which only the seminal vesicle glands from peripubertal animals were used. For the older, single boars, the amounts of unconjugated ("free") steroids were predominant again from incubations of the seminal vesicles (91.8% and 90.2%) as well as from all other sources of tissues (ranging from 82.8% to 95.2%, Table 1).
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Distribution of Estrogen Metabolites
Metabolism of the estrogens was markedly different from that of testosterone and was notable for the extent to which conjugation had occurred (Table 2). Data were obtained from the two older animals only. With few exceptions the extent of conjugation was about the same for both estrogens. The highest percentage of radioactivity recovered in the conjugated fraction was found with incubations of estradiol in the caput epididymidis (80.7%). Conjugated material from both estrogens was present in greater quantities for tissues from the reproductive tract as compared to those from the two accessory sex glands. In fact, the prostate gland seemed least able to form conjugates. Separation of the conjugated material into its components revealed that most of the radioactivity was in the sulfate fraction in the case of tissues from the tract, but not from the glands (Table 3). Much lesser amounts were found either as glucuronidates or in the nonhydrolyzed fractions for all segments of the tract. In contrast, the glucuronidate fraction was the major one for the seminal vesicles; and a more even distribution resulted for the smaller quantities of conjugates formed in incubations with prostatic tissues. No marked differences were seen between the two estrogens upon comparison of the distribution of radioactivity after incubation with the various tissues.
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Profiles of Unconjugated and Hydrolyzed Steroids on HPLC
No major peaks were formed from [3H]testosterone by the tissues of the seminal vesicles from all of the peripubertal animals (data not shown). However, one lesser peak having the same retention time (25 min) as a reference standard of [3H]5-dihydrotestosterone (5-DHT) was seen with the seminal vesicles of the 10-mo-old animal (Fig. 1). Profiles for other tissues from this boar showed marked variation in the amounts of radioactivity corresponding to 5-DHT, with low activity in the epididymis and much higher levels in the vas deferens and prostate (Fig. 1). In general the patterns differed only in quantitative terms. No profiles were run for the minor amounts of conjugated material from testosterone metabolism in the seminal vesicles and in all of the other tissues examined from the older boars.
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A remarkable similarity was seen in the profiles of the unconjugated metabolites of the estrogens for all tissues. In each case the substrate was the major peak, and differences were largely restricted to some variation in the distribution in quantitative terms. Some examples are presented for [3H]estrone (Fig. 2), where it is clear that little or no estradiol was formed. The same held true for the sulfate fractions, in which the substrate was dominant in the profiles and the metabolic differences among the tissue sources again were mainly quantitative (Fig. 3). The same situation applied for the glucuronidate fractions. Only the sex glands produced significant amounts of glucuronidates, but all profiles suggested formation of some estrone glucuronidate (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-DHT was the principal metabolite, but the relative amounts formed varied markedly among the tissue sources. Its identification was based on coincident elution with a reference standard on HPLC and on drastic reduction in the size of the peak when an inhibitor of 5-reductase was used in studies with tissues from the accessory sex glands of 6-wk-old males (unpublished results). The profiles of metabolites were practically identical for the various regions of the epididymis, with only 6–7% occurring as 5-DHT. This finding was unlike the distribution of the enzyme in the rat epididymis [24, 25], where it is regulated differentially with respect to region. However, if the lower part of the tract in the pig is included, one sees that regional differences in expression of the enzyme exist, with about 4-fold higher levels of 5-DHT produced by the vas deferens. Because of the key role of the epididymis in sperm transport, maturation, and storage, further studies including the use of molecular tools [25] are needed for a better understanding of the significance of local androgen metabolism in the pig.
The most extensive metabolism of testosterone was noted with tissues from the prostate, where the amount of 5-DHT formed was twice as great as that from the seminal vesicles. This result was the inverse of that in an earlier report on incubation of minced tissues from these glands in mature boars [26]. Direct comparison between the two studies is difficult because we used fresh tissues with no cofactors added, rather than thawed tissues with cofactors in the media. The formation of other metabolites by the prostate was also evident in the HPLC profile. At much lower levels than 5-DHT, some radioactivity appeared to be present as 5-androstane-3,17ß-diol, which might be expected from the earlier report [26]. In other respects the profiles for all tissues bore a striking resemblance to one another, with all minor peaks being coincident and differing only slightly in quantitative terms. How these profiles translate to the overall biological responses to testosterone has yet to be addressed.
Both estrone and estradiol-17ß are secreted as sulfoconjugated steroids by the testes of the boar [27, 28] and, as such, are present in high concentrations in peripheral blood in early postnatal life as well as in adult males [29].The most noteworthy feature of estrogen metabolism was the relatively high level of conjugation. Although no definitive identification of the conjugate forms has yet been made, the data reveal strong support for the presence of both sulfotransferase and glucuronosyltransferase activities. Such enzyme activity involving estrogens has not been recorded for the reproductive tract and associated glands in males of any mammalian species to our knowledge [30, 31]. This contrasts with the numerous reports on glucuronidation of 5-reduced C19 steroids by the prostate in several species [32]. Since conjugation of a steroid may abolish its interaction with its receptor and facilitate its clearance from the cell, the activity expressed in the boar could be an important component in the regulation of the levels of active hormone present in the various target tissues. The secretion of characteristically large amounts of estrogens by the boar testes [12–14] makes it likely that conjugating enzymes have a significant role to play in this species. We have recently reported that even in the young male pig (6 wk old), the accessory sex glands are active in conjugating estrogens [33]. On the other hand, an earlier study with the prostate and seminal vesicles of mature boars did not show conjugated material when tissues that had been stored at -70°C were incubated with [3H]estrone and estradiol [26]. Comparison with our positive findings using fresh tissues is clearly limited by differences in experimental conditions.
A striking difference is seen in the apparent sulfotransferase activity between the tissues from the reproductive tract and those of the sex glands. While quantitative comparisons were not made, the caput epididymidis seemed to be the site of highest estrogen sulfate formation. In contrast, estrogen glucuronidates were clearly most prominent in the seminal vesicles. The outstanding feature for the prostate was the extent to which estrogen metabolites could not be liberated by the hydrolysis steps. No explanation can be offered for the obvious specificity in conjugate formation among the tissues; however, the end result would be a reduction in exposure of all of the tissues to the biologically active form of the estrogen. In this context, mRNA encoding the estrogen receptor (ERß) has been detected in the reproductive tract of the adult rat, from the efferent ductules to the vas deferens and in the prostate [10]. Similar studies in the pig are warranted, especially in view of the in vivo responses to estrogens recorded in our earlier work [11].
Oxidative metabolism of estrogens takes place mainly in the liver, but many examples of extrahepatic metabolism have been reported since the early demonstration of 2-hydroxylation of estradiol by the rat brain [34]. In an extensive review of the subject, no reports on the male reproductive system were cited [21]. Our study shows that estradiol (data not shown) and estrone may undergo oxidative metabolism. Although the substrates were predominant in the HPLC profiles, several other peaks were present from both unconjugated and conjugated fractions. No identification has yet been made, but most peaks were more polar than that for the substrate, which suggests oxidative metabolism to more hydroxylated forms. Whether any of these metabolites function as secondary hormones with unique functions and receptors, or compete for the known estrogen receptors, remains to be determined. In this regard, it has been recently proposed that the biological effects of an estrogen will depend on the profile of multiple metabolites formed and on the biological activities of each of the metabolites [21].
In conclusion, our data demonstrate an extensive metabolism of estrogens and, to a lesser degree, of testosterone in the reproductive tract and accessory sex glands in the mature male pig. An outstanding feature of estrogen metabolism was the high level of conjugation expressed in most tissues. Thus, further attention has been drawn to the need to consider the functional role of steroid metabolism in target tissues of the male reproductive system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 519 767 1450; jraeside{at}ovcnet.uoguelph.ca
Accepted: June 17, 1999.
Received: April 21, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß) expression in the excurrent ducts of the adult male rat reproductive tract. J Androl 1997; 18:602–611.) within the testis and excurrent ducts of the rat and marmoset monkey from perinatal life to adulthood. J Endocrinol 1997; 153:485–495.[CrossRef][Medline]This article has been cited by other articles:
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  E.E. At-Taras, A.J. Conley, T. Berger, and J.F. Roser Reducing Estrogen Synthesis Does Not Affect Gonadotropin Secretion in the Developing Boar Biol Reprod, January 1, 2006; 74(1): 58 - 66. [Abstract] [Full Text] [PDF]
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 P. A. Sinclair and E. J. Squires Testicular sulfoconjugation of the 16-androstene steroids by hydroxysteroid sulfotransferase: Its effect on the concentrations of 5{alpha}-androstenone in plasma and fat of the mature domestic boar J Anim Sci, February 1, 2005; 83(2): 358 - 365. [Abstract] [Full Text] [PDF]
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J. I. Raeside, H. L. Christie, R. L. Renaud, R. O. Waelchli, and K. J. Betteridge Estrogen Metabolism in the Equine Conceptus and Endometrium During Early Pregnancy in Relation to Estrogen Concentrations in Yolk-Sac Fluid Biol Reprod, October 1, 2004; 71(4): 1120 - 1127. [Abstract] [Full Text] [PDF]
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 M. H. Laughlin, W. V. Welshons, M. Sturek, J. W. E. Rush, J. R. Turk, J. A. Taylor, B. M. Judy, K. K. Henderson, and V. K. Ganjam Gender, exercise training, and eNOS expression in porcine skeletal muscle arteries J Appl Physiol, July 1, 2003; 95(1): 250 - 264. [Abstract] [Full Text] [PDF]
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