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J. Anim. Sci. 2005. 83:358-365
© 2005 American Society of Animal Science

ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

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 boar1

P. A. Sinclair and E. J. Squires2

Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada, N1G 2W1


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Literature Cited
 
This study examined the relationship between sulfoconjugation and the degree to which 5{alpha}-androstenone can accumulate in fat. Analysis of the unconjugated and sulfoconjugated fractions of peripheral plasma from 25 mature Yorkshire boars and testicular vein plasma from an additional 20 mature Yorkshire boars revealed that the majority of 5{alpha}-androsten-one is present as a sulfoconjugate, reaching levels up to 69 ± 4.3 and 72 ± 6.2%, respectively, relative to its unconjugated form. The presence of this steroid in the sulfoconjugate fraction was confirmed by gas chromatography-mass spectrometry. Plasma concentrations of 5{alpha}-androstenone in the sulfoconjugate fraction were negatively correlated (r = –0.36; P < 0.01) with the concentrations of 5{alpha}-androstenone in fat. High concentrations of 5{alpha}-androstenone in the sulfate fraction were only associated with animals that had fat androstenone concentrations < 0.5 µg/g. In addition, there was a positive correlation (r = 0.31; P < 0.01) between the concentrations of unconjugated 5{alpha}-androstenone in plasma and 5{alpha}-androstenone in fat. These findings indicate that the levels of the sulfoconjugated form present in the peripheral plasma influence the accumulation of 5{alpha}-androstenone in fat. The specific sulfotransferase enzyme involved in sulfoconjugating these steroids was identified by incubating Leydig cells with specific sulfotransferase inhibitors for 8 h. It was discovered that the enzyme responsible for the sulfoconjugation of the 16-androstene steroids is hydroxysteroid sulfotransferase. Hydroxysteroid sulfotransferase may play a significant role in determining the levels of sulfated 16-androstene steroids present in plasma. The results of this study indicate that sulfoconjugation may serve to regulate the quantity of unconjugated 5{alpha}-androstenone present in the circulation and thus available for accumulation. Animals with a decreased ability to sulfoconjugate 5{alpha}-androstenone would have a subsequent increase in the levels of unconjugated 5{alpha}-androstenone in circulation, allowing for the accumulation of high levels in fat and thereby potentially leading to the development of boar taint.

Key Words: Androstenone • Boar Taint • Sulfoconjugation • Sulfotransferase


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Literature Cited
 
The 16-androstene steroids, specifically, 5{alpha}-androst-16-en-3{alpha}-ol (3{alpha}-androstenol), 5{alpha}-androst-16-en-3ß-ol (3ß-androstenol), and 5{alpha}-androst-16-en-3-one (5{alpha}-androstenone), have been determined recently to be sulfoconjugated by porcine Leydig cells (our unpublished results). 5{alpha}-Androstenone is known for its involvement in boar taint (Patterson, 1968Go; Gower, 1972Go), which is the unpleasant odor that is associated with boar fat when it is heated.

In the boar, a large proportion of testicular steroids is present in the sulfoconjugated form (Raeside and Renaud, 1983Go; Raeside et al., 1989Go), due to the high levels of sulfotransferase enzymes present in the Leydig cells (Raeside and Renaud, 1983Go; Hobkirk, 1985Go; Hobkirk et al., 1989Go). These enzymes include hydroxysteroid sulfotransferase (HST), estrogen sulfotransferase (EST), and phenol sulfotransferase (PST) (Hobkirk, 1985Go; Baranczyk-Kuzma and Ciszewska-Pilczynska, 1989Go). The major substrate for HST is dehydroepian-drosterone (DHEA); however, HST can also act on 3ß-, 3{alpha}-, and some 17ß-hydroxy steroids (Falany et al., 1989Go; Strott, 1996Go; Pedersen et al., 2000Go). Estrogen sulfotransferase and PST act on the hydroxyl groups of estrogens (Strott, 1996Go). Estrogen sulfotransferase also is capable of sulfating hydroxysteroids such as DHEA and pregnenolone to a certain extent (Falany et al., 1994Go; Negishi et al., 2001Go).

The addition of a highly charged sulfate group greatly increases the polarity of a steroid (Jakoby et al., 1980Go; Strott, 1996Go). This has important implications with respect to the accumulation of 5{alpha}-androstenone in adipose tissue. The aims of this study were to 1) characterize the influence of 16-androstene sulfoconjugation on the accumulation of 5{alpha}-androstenone in fat, and 2) identify the specific sulfotransferase enzyme involved in conjugating the 16-androstene steroids, which could act as a potential regulatory step involved in the development of boar taint.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Literature Cited
 
Animals and Sampling
Twenty-five Yorkshire boars (Group A), 175 ± 6 d of age, were obtained from the Arkell Swine Research Station at the University of Guelph. In selecting these animals, physiological maturity was estimated by plasma testicular steroid concentrations, specifically estrone sulfate (E1S) > 17 ng/mL, and bulbourethral gland size > 11 cm (Allrich et al., 1982Go; Schwarzenberger et al., 1993Go; Sinclair et al., 2001Go). Mature animals were further screened based on total plasma 5{alpha}-androstenone concentratioins. A cut-off level of 15 ng/mL was used, as it has been demonstrated that below this level, animals do not have the capacity to develop boar taint (Sinclair et al., 2001Go). Blood samples were taken from the orbital sinus, centrifuged at 4°C to collect plasma, and stored at –20°C until extraction and analysis for steroid concentrations.

Testicular vein blood samples were taken from an additional 20 physiologically mature Yorkshire boars (Group B), 200 to 250 d of age, to determine the proportion of sulfoconjugated 5{alpha}-androstenone secreted from the testes. Blood was collected from veins on the surface of the testes as described previously (Raeside and Howells, 1971Go). The samples were centrifuged at 4°C to collect serum and stored at –20°C until extraction and analysis.

After the slaughter of Group A boars, bulbourethral glands were removed and measured for length. A back-fat sample was removed from the midline on the point of the 11th rib and frozen at –20°C until assayed for 5{alpha}-androstenone. Testes samples were obtained from five randomly selected animals from Group A immediately after slaughter. Testes were transported to the laboratory within 5 min of removal from the boar. One testis was dissected free from the epididymis, cut longitudinally, and decapsulated. The tissue was sliced, and 100 g of tissue was incubated for 12 min in a shaking water bath at 37°C with 1 mg/mL of collagenase (type 1A), 50 of µg/mL trypsin inhibitor, and 50 µg/mL of DNase in 250 mL of tissue culture medium 199 containing 1 g/L of BSA and 0.1g/L of L-glutamine. Purified Leydig cells were obtained by layering the collagenase dispersed cells onto discontinuous Percoll gradients consisting of 21, 26, 34, 40, and 60% interfaces. The preparations were then centrifuged at 1,500 x g for 15 min at 5°C and the Leydig cells present at the 40 to 60% interface were collected as outlined previously (Raeside and Renaud, 1983Go). Cell viability was determined with a trypan blue exclusion test. The typical viability of the isolated Leydig cells was 90%.

Inhibition Studies
Purified Leydig cells from each of the five boars were resuspended (50 x 106 cells/incubation) into a final volume of 25 mL of Williams Medium E mixture. Radiolabeled [7-3H(N)]-pregnenolone (3.4 µCi/µmol) was added as a substrate to give a final amount of 0.4 µmol/incubation. Leydig cells were incubated for a total of 8 h at 37°C under 95%:5% CO2 atmosphere in a Dubnoff shaking waterbath in the presence of 0, 5, 10, 50, or 100 µM of various sulfotransferase inhibitors. Each incubation was conducted in triplicate. Triethylamine, was used as an HST-inhibitor (Matsui et al., 1993Go). Pentachloro-phenol (PCP) was used as a phenol-sulfotransferase-inhibitor (Meerman et al., 1983Go; Fayz et al., 1984Go; Boles and Klaassen, 1998Go); however, PCP does not inhibit HST (Singer et al., 1984Go; Okuda et al., 1989Go). Estrone is a preferred substrate for EST and was used as a competitive inhibitor (Falany et al., 1994Go; Negishi et al., 2001Go). The inhibitory effects of estrone on EST were confirmed by measurement of decreasing E1S concentrations in the Leydig cell incubations. All the inhibitors were prepared in ethanol to obtain a final ethanol percentage of 0.5% in each incubation. The control incubations also contained 0.5% ethanol. The sulfotransferase activity towards 5{alpha}-androstenone was determined by HPLC analysis of extracted 5-mL media aliquots.

Steroid Extraction and Purification
Before analysis, conjugated steroids were separated from unconjugated steroids with the use of Sep-Pak C18 solid-phase chromatography cartridges as described previously (Raeside et al., 1997Go). After separation, the conjugate fraction was hydrolyzed overnight in trifluoroacetic acid/ethyl acetate (1:100, vol/vol) at 45°C to liberate the sulfoconjugated steroids (Raeside et al., 1999bGo). The hydrolyzed steroids were then re-extracted by solid-phase chromatography. Enzyme hydrolysis was performed on the remaining conjugated material present after solvolysis with 1,250 U of ß-glucuronidase (type B-1, from bovine liver; Sigma Aldrich Canada, Ltd., Oakville, Ontario), and incubated overnight at 37°C (Raeside et al., 1999aGo,bGo).

The identity of 16-androstene steroids extracted from the plasma was confirmed by HPLC purification followed by identification with gas chromatography-mass spectrometry (GC-MS) as outlined subsequently under biochemical analyses. The unconjugated and hydrolyzed steroids were purified by HPLC using a modification of our previous methods (Bonneau et al., 1992Go). A Phenomenex 5 µm C18 HPLC column (250 mm x 4.6 mm) was used with an 85% acetonitrile:15% H2O mobile phase delivered isocratically at 0.7 mL/min. In this system, the 16-androstene steroids elute between 12 and 18 min, with 5{alpha}-androstenone eluting at 17.7 min. This purified fraction was evaporated to dryness under N at 45°C and prepared for GC-MS.

Biochemical Analyses
Fat and extracted plasma samples were analyzed for 5{alpha}-androstenone with a modified ELISA method (Claus et al., 1988Go), as described previously (Squires and Lundstrom, 1997Go). Radioimmunoassay was used for measurements of E1S in plasma samples (Schwarzenberger et al., 1993Go).

Before GC-MS analysis, the purified 16-androstenes were derivatized as O-methyloxime and trimethylsilyl ethers (Khalil and Lawson, 1983Go), and subsequently purified using Lipidex 5000 (Perkin Elmer, Boston, MA) column chromatography (Khalil et al., 1993Go). The eluates were evaporated to dryness under nitrogen at 45°C and reconstituted in 200 µL of hexane/hexamethyldisilazane (20:1, vol/vol). Steroid derivatives were analyzed using a Hewlett Packard (Mississauga, Ontario, Canada) 6890 gas chromatography system equipped with a HP-1 capillary column linked to a Hewlett-Packard 5973N mass selective detector. To identify the steroids, the ion spectra were compared to those produced by the authentic steroid standards (Kwan et al., 1992Go).

The unconjugated and hydrolyzed steroids from the inhibition studies were analyzed by HPLC using the same protocol as stated above; however; radiolabeled steroids were measured on-line with a Canberra-Packard 500TR flow scintillation analyzer (Canberra-Packard Ltd., Mississauga, Ontario, Canada).

Statistical Analyses
Pearson correlation coefficients were calculated between the following measures: 1) plasma concentrations of sulfoconjugated 5{alpha}-androstenone vs. fat concentrations of 5{alpha}-androstenone; and 2) plasma concentrations of unconjugated 5{alpha}-androstenone vs. fat concentrations of 5{alpha}-androstenone (SAS Inst., Inc., Cary, NC). Regression analysis was performed to evaluate the effects of the various sulfotransferase inhibitors on the sulfoconjugation of the 16-androstene steroids.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Literature Cited
 
Proportions of Sulfoconjugated 5{alpha}-Androstenone in Blood
Analysis of the unconjugated and sulfoconjugated fractions of peripheral plasma revealed that the majority of 5{alpha}-androstenone was present as a sulfoconjugate. The sulfoconjugated form of 5{alpha}-androstenone was found to be present up to 69 ± 4.3% relative to its unconjugated form, with a mean concentration of 18 ± 1.4 ng/ mL. The presence of this steroid in plasma was confirmed by GC-MS. The O-methyloxime and trimethylsilyl derivatives of the HPLC-purified steroids were identical to that of the authentic steroid standards. The gas chromatographic retention time for the steroid standard of 5{alpha}-androstenone was 12.52 min, which corresponded to a peak at 12.56 min in the sulfoconjugate fraction. This peak was confirmed to be 5{alpha}-androstenone, as it produced a molecular ion at a mass/charge ratio (M/Z) of 301, with fragment ions at M/Z 286 and 270, which are identical to that of the 5{alpha}-androstenone standard (Figure 1Go). Gas chromatography-mass spectrometry analysis also confirmed the presence of 3ß-androstenol and 3{alpha}-androstenol as sulfoconjugates in peripheral plasma; however, there were no detectable 16-androstenes present as glucuronide conjugates.



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  		Figure 1. Comparison of mass spectra of the O-methyloxime and trimethylsilyl derivative of the authentic steroid standard 5{alpha}-androstenone (A) to the steroid present in the hydrolyzed sulfoconjugate fraction of peripheral plasma with a gas chromatography retention time of 12.56 (B). M/Z = mass/charge ratio. 5{alpha}-androst-16-en-3-one = 5{alpha}-androstenone.

 
Analysis of testicular vein serum produced similar results to that of the peripheral plasma, with proportions of 5{alpha}-androstenone present up to 72 ± 6.2% in the sulfoconjugate fraction relative to the unconjugated fraction. However, the overall concentration of 5{alpha}-androstenone in testicular vein serum was approximately 10 times greater than that found in the peripheral plasma, with a mean concentration of 195 ± 12 ng/ mL. The presence of this steroid in the sulfoconjugate fraction was confirmed by GC-MS, producing similar results to those reported above for peripheral plasma. As with peripheral plasma, 3ß-androstenol and 3{alpha}-androstenol were present primarily as sulfoconjugates. Additionally, the 16-androstenes were not present within the glucuronide fraction of the testicular vein serum.

Relationships Between Steroid Concentrations
A negative correlation(r = –0.36; P < 0.01) was observed between plasma concentrations of 5{alpha}-androsten-one in the sulfoconjugate fraction and concentrations of 5{alpha}-androstenone in fat. Animals with fat androstenone concentrations < 0.5 µg/g had plasma 5{alpha}-androstenone concentrations that ranged from 9 ng/mL to 56 ng/mL in the sulfoconjugate fraction (Figure 2Go). However, high plasma concentrations in the sulfoconjugate fraction were only present in animals with low fat androstenone. This relationship is further characterized on examining the unconjugated plasma fraction (Figure 3Go). There was a significant positive correlation (r = 0.31; P < 0.01) between unconjugated 5{alpha}-androstenone in plasma and the concentrations of 5{alpha}-androstenone in fat. All the animals with fat androstenone concentrations < 0.5 µg/ g had concentrations of 5{alpha}-androstenone below 9 ng/ mL. High levels of unconjugated 5{alpha}-androstenone in the plasma were only associated with fat concentrations greater than 0.5 µg/g. Animals with high concentrations of unconjugated 5{alpha}-androstenone had corresponding low levels of sulfoconjugated 5{alpha}-androstenone, both of which related to increased concentrations in fat.



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  		Figure 2. Distribution of 5{alpha}-androstenone present in the peripheral plasma sulfoconjugate fraction against the concentration of 5{alpha}-androstenone in fat (n = 25). 5{alpha}-an-drost-16-en-3-one = 5{alpha}-androstenone.

 


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  		Figure 3. Distribution of 5{alpha}-androstenone present in the peripheral plasma unconjugated fraction against the concentration of 5{alpha}-androstenone in fat (n = 25). 5{alpha}-an-drost-16-en-3-one = 5{alpha}-androstenone.

 
Inhibition Studies
The effects of various sulfotransferase inhibitors on the ability of the Leydig cells to produce sulfoconjugated 16-androstene steroids were tested. Figure 4Go illustrates the effect of triethylamine on the production of sulfoconjugated 16-androstene steroids (Figure 4AGo) and the production of 16-androstene steroids in the unconjugated fraction (Figure 4BGo), using pregnenolone as a substrate. Triethylamine caused decreased production (P < 0.01) of sulfoconjugated 16-androstenes from pregnenolone in a dose-dependent manner, with concentrations reaching close to zero at 100 µM triethylamine. Conversely, there was an increased production of the unconjugated forms of these steroids when the cells were treated with triethylamine. The viability of the Leydig cells treated with 100 µM of triethylamine was 85%.



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  		Figure 4. Effects (P < 0.01) of treating Leydig cells with 0, 5, 10, 50, or 100 µM of triethylamine on the synthesis of sulfoconjugated (A) and unconjugated (B) 16-androstene steroids: ({diamondsuit}), 3ß-androstenol; ({blacktriangleup}), 3{alpha}-androstenol; and ({blacksquare}), 5{alpha}-androstenone. Values are plotted mean ± standard error (n = 5, of which each incubation was run in triplicate). 5{alpha}-androst-16-en-3{alpha}-ol = 3{alpha}-androstenol; 5{alpha}-androst-16-en-3-one = 5{alpha}-androstenone.

 
Figure 5Go illustrates the effect of estrone and PCP on the production of the 16-androstene steroids sulfates. Neither estrone nor PCP had an effect on the production of sulfoconjugated 16- androstene steroids. After 8 h of exposure to 100 µM of PCP, a significant toxicity to Leydig cells was evident, with viability decreasing to less than 50%.



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  		Figure 5. Effects of treating Leydig cells with 0, 5, 10, 50, or 100 µM of estrone (A) and pentachlorophenol (B) on the synthesis of sulfoconjugated 16-androstene steroids: ({diamondsuit}), 3ß-androstenol; ({blacktriangleup}), 3{alpha}-androstenol; and ({blacksquare}), 5{alpha}-androstenone. Values are plotted mean ± standard error (n = 5, of which each incubation was run in triplicate). 5{alpha}-androst-16-en-3{alpha}-ol = 3{alpha}-androstenol; 5{alpha}-an-drost-16-en-3-one = 5{alpha}-androstenone.

 
Discussion
A large proportion of 5{alpha}-androstenone in the peripheral plasma is found in its sulfoconjugated form; however, 5{alpha}-androstenone does not contain any hydroxyl groups that would allow for sulfoconjugation. The 3-keto group of 5{alpha}-reduced steroids has been reported to undergo enolization in many species (Kouretas et al., 1996Go; Drmanovic et al., 1999Go). Therefore, conjugation of 5{alpha}-androstenone likely occurs through an initial enolization of the 3-keto group to a 3-enol form (our unpublished results). The high level of sulfoconjugation of this steroid is in agreement with that found for many other steroids produced by the boar (Raeside and Howells, 1971Go; Booth, 1983Go). In fact, DHEA has been reported to be present primarily as a sulfoconjugate, reaching proportions of 90% relative to its unconjugated form (Tan and Raeside, 1980Go). The high concentration of sulfoconjugated 5{alpha}-androstenone present in testicular vein plasma indicates that the testes are of major importance in contributing to concentrations present in peripheral plasma.

Metabolism of testicular steroid hormones into secondary products may alter their biological activity or advance their clearance from the body, thereby affecting plasma concentrations. The process of sulfoconjugation has been classically thought to function as a mechanism to facilitate the metabolic clearance of the steroid; however, the high concentrations of sulfoconjugated steroids present in the plasma of the boar suggest that the biological significance of sulfoconjugation may be more complex. It has been suggested that the sulfoconjugates of testicular steroids may act as regulators of androgen and estrogen synthesis by controlling the levels of unconjugated steroids that are capable of interacting with their respective receptors (Payne and Jaffe, 1970Go; Raeside et al., 1999aGo).

In addition to the potential biological significance of sulfoconjugation, a drastic change in the physiochemical properties of the steroid occurs due to an increase in polarity and thus water-solubility (Bongiovanni and Cohn, 1970Go; Strott, 1996Go). The findings of this study indicate that sulfoconjugation of 5{alpha}-androstenone limits the amount of the nonpolar unconjugated form that is available to accumulate in adipose tissue. Animals with high concentrations of sulfoconjugated 5{alpha}-androstenone in plasma displayed low levels of 5{alpha}-androstenone in fat. In addition, these animals had relatively low levels of unconjugated 5{alpha}-androstenone in plasma, and were therefore unable to accumulate fat levels higher than 0.5 µg/g. The relationship between the level of androstenone in plasma and fat has been investigated in a number of studies, with contradictory results, ranging from no correlation (Malmfors and Andresen, 1975Go; Lundstrom et al., 1978Go; Bonneau et al., 1982Go) to positive correlations (Andresen, 1976Go; Groth and Claus, 1977Go; Sinclair et al., 2001Go). These contradictions are likely the result of confounding factors, such as physiological maturity, as well as the genetic capacity to produce and metabolize the 16-androstene steroids. The results of the present study indicate that the extent to which 5{alpha}-androstenone accumulates in fat is ultimately related to the level of unconjugated steroid present in plasma. These findings suggest that the concentration of unconjugated 5{alpha}-androstenone in plasma is the result of a balance between the capacities for testicular 16-androstene synthesis and subsequent sulfoconjugation.

The ability to produce high levels of sulfoconjugated steroids depends on the levels and enzyme activities of the testicular sulfotransferases. Estrogen sulfotransferase and PST displayed very little to no action towards the 16-androstene steroids, respectively, because both EST and PST are specific for hydroxyl groups on phenolic steroids. Estrogen sulfotransferase has been demonstrated to be capable of sulfating hydroxysteroids; however this sulfation is relatively inefficient (Negishi et al., 2001Go). The results of the inhibition studies indicate that the specific sulfotransferase responsible for conjugating the 16-androstene steroids is HST. Hydroxysteroid sulfotransferase that is expressed in the testicular tissue of the boar has been shown to localize in the Leydig cells, and it has a broad substrate specificity (Hobkirk et al., 1989Go). Hydroxysteroid sulfotransferase prefers steroid substrates with 3ß-hydroxy acceptor sites; however, HST is capable of acting on steroids with 3{alpha} and 17ß-hydroxy acceptor sites (Falany et al., 1989Go; Strott, 1996Go; Pedersen et al., 2000Go). Therefore, the 3ß- and 3{alpha}-hydroxyl groups of 3ß-androstenol and 3{alpha}-androstenol, respectively, serve as potential acceptor sites for sulfoconjugation by HST. It is also likely that the hydroxyl group at the 3 position of the proposed enol form of 5{alpha}-androstenone is sulfoconjugated by HST.

The results of this study suggest that the proportion of the sulfoconjugated form present in the peripheral plasma influences the accumulation of 5{alpha}-androstenone in fat. Hydroxysteroid sulfotransferase was found to be a key enzyme involved in the sulfoconjugation of the 16-androstene steroids and may play a significant role in determining the levels of sulfated steroids present in plasma. An animal with a decreased ability to sulfoconjugate 5{alpha}-androstenone would therefore have an increased potential to accumulate high levels of the unconjugated form in adipose tissue. Based on these findings, HST could potentially be used as a genetic marker for selecting animals with low boar taint.


   Footnotes
 
1 This research was supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Ontario Ministry of Agriculture and Food (OMAF). Back

2 Correspondence—phone: 519-824-4120 (ext. 53928); fax: 519-836-9873; e-mail: jsquires{at}uoguelph.ca.

Received for publication July 20, 2004. Accepted for publication October 25, 2004.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Literature Cited
 


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