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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
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* Norsvin, PO Box 504, 2304 Hamar, Norway;
and
Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, PO Box 5003, 1432 Ås, Norway; and
Division of Farm Animal Science, School of Clinical Veterinary Science, University of Bristol, Langford, Bristol, BS40 5DU, United Kingdom
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: androstenone • breed • cytochrome P450-c17 • 3ß-hydroxysteroid dehydrogenase • pig • sulfotransferase 2B1
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-androst-16-en-3-one) in adipose tissue (Bonneau, 1987
; Zamaratskaia et al., 2005). Androstenone is produced in the Leydig cells of testis (Gower, 1984). The main enzymes involved in this process are 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and andien-ß synthase, which consists of cytochrome P450-c17 (CYP17) and cytochrome b5. In rats, CYP17 was reported to be involved in control of steroidogenesis not only in testis, but also in liver (Katagiri et al., 1998). Androstenone degradation takes place mainly in liver and includes 2 stages: 1) oxidative, which is catalyzed by 3ß-HSD (Doran et al., 2004; Sinclair et al., 2005), and 2) conjugative, which involves a number of enzymes, including sulfotransferases (SULT; Sinclair and Squires, 2005).
It has previously been reported that the elevated adipose tissue androstenone concentration is related to a low activity and expression of the enzymes controlling androstenone metabolism (Doran et al., 2004; Nicolau-Solano et al., 2006; Sinclair et al., 2006). Moreover, cDNA microarray studies have established that the genes encoding porcine CYP17 and sulfotransferase 2B1 (SULT2B1) are differentially expressed in animals with high and low androstenone concentrations [M. Moe, T. Meuwissen (Norwegian University of Life Sciences, Norway), S. Lien (Norwegian University of Life Sciences, Norway), C. Bendixen (University of Aarhus, Denmark), X. Wang (University of Aarhus, Denmark), L. Conley (University of Aarhus, Denmark), I. Berget (Norwegian University of Life Sciences, Norway), H. Tajet (Norsvin and Norwegian University of Life Sciences, Norway), and E. Grindflek; unpublished data].
The above-mentioned studies have generally been conducted within one breed. It is currently unknown whether there are breed differences in the mechanisms regulating androstenone concentration in pig adipose tissue. The aim of the current study was to investigate the possible effect of breed [Norwegian Landrace (N. Landrace) and Duroc] on expression of the enzymes involved in androstenone biosynthesis and degradation (3ß-HSD, CYP17, and SULT2B1) in animals that vary in adipose tissue androstenone concentrations.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The study was conducted in compliance with Norwegian regulations for humane care and slaughter.
The study was conducted on nucleus boars of 2 breeds: N. Landrace and Duroc. The animals were raised at the boar testing stations of the Norwegian Pig Breeder’s organization Norsvin (Hamar, Norway) and were slaughtered at the end of the test. These animals were not used in the breeding program, because they were not the top boars for traits included in the breeding scheme. The animals were reared on a standard commercial diet containing 14.9 MJ of DE, 17.8% CP, 5.6% fiber, 6% raw fat, 6% raw ash, and 1.12% Lys (as fed-basis), without food or water restrictions.
The pigs were reared and slaughtered at the age of 143 to 156 d (100 kg of live BW). The slaughtering for each breed was carried out once per week over a period of 26 mo. Samples from the left lateral lobe of the liver (approximately 50 g) were collected directly on the slaughter line and frozen immediately in liquid N2. One of the testicles was also removed immediately after slaughter and a sample of testicle (approximately 20 g) was frozen in liquid N2. All samples were stored at –80°C until used. Samples of subcutaneous adipose tissue for androstenone measurement (approximately 100 g) were collected from the neck of each boar and stored at –20°C until analyzed.
Analyses of Androstenone in Subcutaneous Adipose Tissue
Androstenone concentration in subcutaneous adipose tissue was analyzed by a modified time-resolved fluoroimmunoassay (Tuomola et al., 1997) by using antiserum produced at the Norwegian School of Veterinary Science (Andresen, 1974). Androstenone concentration was analyzed in 1,533 N. Landrace boars and 1,027 Duroc boars. The mean androstenone values with SEM for all the animals within each breed were 1.17 ± 0.029 and 3.22 ± 0.083 µg/g for N. Landrace and Duroc boars, respectively. Androstenone values were log normal distributed. For further analyses of enzyme expression, the 20 most extreme animals from each breed were selected, out of which 10 animals had low concentrations of androstenone and 10 animals had increased concentrations of androstenone, further referred to as the low-androstenone and high-androstenone groups. The animals were selected from the "tails" of the distribution within each breed. The mean androstenone value and SEM for means for the low-androstenone and high-androstenone N. Landrace pigs were 0.1 ± 0.01 and 7.58 ± 0.68 µg/g of adipose tissue, respectively. The androstenone values for the low-androstenone and high-androstenone Duroc pigs were 0.22 ± 0.04 and 13.5 ± 1.14 µg/g of adipose tissue, respectively. Assay sensitivity was 0.04 µg/g.
Isolation of Microsomal and Cytosolic Fractions
3ß-Hydroxysteroid dehydrogenase and CYP17 are membrane-bound enzymes and their expression was analyzed in isolated microsomes. Expression of SULT2B1 protein was analyzed in the cytosolic fraction. Microsomal and cytosolic fractions from pig liver and testis were isolated by differential centrifugation. Approximately 1 g of frozen tissue was homogenized in 20 mL of cold sucrose buffer (10 mM Tris-HCl, 250 mM sucrose, pH 7.4) followed by centrifugation at 12,000 x g for 10 min. The supernatant was collected and 8 mM CaCl2 was added to the supernatant to facilitate sedimentation of the microsomes. The microsomes were separated from the cytosolic fraction by centrifugation at 25,000 x g for 50 min. The resulting pellet (microsomal fraction) was resuspended in a buffer containing 50 mM Tris-HCl, 10 mM KH2PO4, 0.1 mM EDTA, 20% glycerol, and 0.1 mM protein inhibitors antipain hydrochloride, pepstatin, and leupeptin hydrochloride (Sigma Aldrich, St. Louis, MO) to a final protein concentration of approximately 25 mg/mL. Microsomal and cytosolic proteins were determined by the Bradford method, with BSA used as the standard (Bradford, 1976).
Western Blotting
Microsomal and cytosolic proteins were separated by SDS gel electrophoresis and electroblotted onto a nitrocellulose membrane (Sigma, Dorset, UK) at 100 V for 1 h. The membrane with separated proteins was probed with one of the following primary antibodies: rabbit anti-porcine 3ß-HSD, goat anti-human CYP17, or goat anti-human SULT2B1 for 1.5 h. The anti-porcine 3ß-HSD were produced against a synthetic peptide, GSLVKQHKEAKTK, corresponding to AA 357 to 370 of the porcine 3ß-HSD protein (GenBank accession no. NP 001004049) as described previously (Nicolau-Solano et al., 2006). The CYP17 and SULT2B1 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The CYP17 antibody was raised against a peptide mapping at the C-terminus of human CYP17A1, which is 75% identical to the corresponding porcine CYP17 AA sequence. The CYP17 antibody produced an immunoreactive band with a porcine protein of approximately 50 kDa, which corresponds to the molecular weight of the pig CYP17 (Moran et al., 2002). The SULT2B1 antibody was raised against a peptide mapping at the C-terminus of human SULT2B1 and produced an immunoreactive band of approximately 45 kDa with a porcine protein, which corresponds to the SULT2B1 molecular weight reported for humans (Meloche and Falany, 2001).
After probing with the primary antibody, the membrane was washed in PBS Tween for 25 min and reprobed with the appropriate commercial secondary antibody [horseradish peroxidase linked donkey anti-rabbit IgG (Amersham, Buckinghamshire, UK) or horseradish peroxidase linked donkey anti-goat IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA); diluted 1:10,000 in PBS Tween] for 1 h. The protein bands were visualized with an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK) and the intensity of the signals was quantified by using the ImageQuant program (Molecular Dynamics, GE HealthCare UK Ltd., Buckinghamshire, UK).
The optimal amount of protein (6 µg per well) required for analyses of 3ß-HSD expression was detected in our previous work (Nicolau-Solano et al., 2006). To determine the optimal amount of protein required for analyses of CYP17 and SULT2B1 expression, the relationships between the amount of protein loaded on the SDS gel and the intensity of the CYP17 and SULT2B1 signals were investigated. A positive linear relationship was observed between the amount of protein and the intensity of both SULT2B1 and CYP17 signals while loading up to 25 µg of the protein (Figure 1A and 1B, respectively). Therefore, in the subsequent blots, 10 µg of microsomal or cytosolic protein was used for analyses of CYP17 and SULT2B1. This would allow detection of a decrease or increase in protein expression by at least 50%. One sample of microsomes or cytosol from a particular pig was present on every blot, and the intensity of the signal from this sample was taken as 100 arbitrary units throughout (reference sample). The amount of 3ß-HSD, CYP17, and SULT2B1 in other samples on the blot was expressed as a percentage of this reference sample.
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Data were analyzed by using GLM with breed (N. Landrace vs. Duroc) and androstenone concentration (high androstenone vs. low androstenone) as factors and including the interaction term (Minitab release 14, Minitab Inc., State College, PA). Paired comparisons between means in the event of a significant breed x androstenone level interaction were assessed post hoc at P 
0.05 with Tukey’s test (balanced case) or the Tukey-Kramer test (unbalanced case).
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The results of analyses of 3ß-HSD protein expression in microsomes isolated from liver and testis are presented in Figure 2A and 2B, respectively. A significant interaction was found between breed and androstenone concentration for hepatic 3ß-HSD (P < 0.05). In N. Landrace pigs, the level of hepatic 3ß-HSD protein was significantly less (P < 0.01) in the high-androstenone group compared with the low-androstenone group (Figure 2A). In contrast to N. Landrace pigs, there were no significant differences in the expression of hepatic 3ß-HSD between the high-androstenone and low-androstenone Duroc pigs. The low-androstenone Duroc group also had approximately 60% less 3ß-HSD expression compared with the low-androstenone N. Landrace pigs.
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show that in testis, 3ß-HSD protein expression did not differ significantly between breeds (P = 0.27), nor did it differ between the low-androstenone and high-androstenone groups (P = 0.65). CYP17 Protein Expression
Expression of hepatic and testicular CYP17 is presented in Figure 3A and 3B. The protein concentration did not differ significantly in the microsomes isolated from liver of N. Landrace and Duroc pigs (P = 0.80). No differences were found between the high-androstenone and low-androstenone groups (P = 0.20). Similar results were observed for testis, in which expression of CYP17 protein did not differ significantly between breeds (P = 0.55) or between groups (P = 0.87).
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Expression of SULT2B1 has not previously been reported for pig tissues. Figure 4 shows the presence of an immunoreactive band of approximately 45 kDa in liver and testis of both the N. Landrace and Duroc breeds. The size of the immunoreactive band was similar to the molecular weight of the SUL2B1 protein reported for humans, which is approximately 43 kDa (Meloche and Falany, 2001).
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shows that both the N. Landrace and Duroc animals had less expression of SULT2B1 protein in the high-androstenone groups compared with the respective low-androstenone groups. However, in the case of Durocs, the differences between the high-and low-androstenone animals were more pronounced (63.7%, P < 0.001) than in the N. Landrace pigs (36.7%, P < 0.05). |
DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Nicolau-Solano et al., 2006; Sinclair et al., 2006).
In the current study, we established that the relationship between expression of some of the androstenone-metabolizing enzymes and androstenone deposition in subcutaneous adipose tissue is breed specific. Elevated androstenone concentrations in N. Landrace boars were accompanied by reduced expression of 3ß-HSD, the enzyme involved in the first stage of hepatic androstenone degradation, without significant changes in the expression of SULT2B1, the enzyme controlling the conjugative stage of steroid metabolism. The results were opposite in Duroc pigs, in which the expression of hepatic SULT2B1, but not 3ß-HSD, was inhibited in animals with increased androstenone concentrations. These data suggest that the mechanisms regulating androstenone concentrations in pigs might be breed specific. This is in agreement with our previous report on breed-specific mechanisms regulating accumulation of another boar taint compound, skatole (Doran et al., 2002). In that study, we demonstrated that accumulation of skatole was negatively correlated with the activity and expression of the hepatic skatole-metabolizing enzyme CYP2E1 in Large White pigs. However, in Meishan crossbreed, CYP2E1 expression was generally low, and an additional factor, a multidrug-resistant protein, was suggested to be involved in skatole deposition in those pigs (Doran et al., 2002).
The groups of high- and low-androstenone animals selected for this research were more extreme compared with those in other studies that have been done on this trait (Doran et al., 2004; Zamaratskaia et al., 2005; Nicolau-Solano et al., 2006). Selecting animals with extremely high and low androstenone concentrations was possible because of the availability of a large number of animals from each breed. Using pigs with extremely high and low androstenone concentrations provides a better model for studying possible variations in enzyme expression. The mean values for the low- and high-androstenone N. Landrace groups were approximately 50% of those for pigs within the low- and high-androstenone Duroc groups. This is consistent with data showing that the mean androstenone values in the whole population of N. Landrace pigs (1.17 µg/g) were approximately 65% less than those in the whole population of Duroc animals (3.32 µg/g).
Hepatic androstenone metabolism consists of 2 stages: oxidative and conjugative. The oxidative stage is catalyzed by 3ß-HSD, which reduces a keto group at the 3 position to a hydroxyl group in the ß conformation. The main product of the oxidative stage of the pig hepatic androstenone metabolism is 3ß-androstenol (Doran et al., 2004; Sinclair et al., 2005). The enzyme 3ß-HSD is also present in testis, where it is involved in androstenone biosynthesis (Gower, 1984; Conley and Bird, 1997). In the current study, down-regulation of 3ß-HSD protein expression was observed in liver, but not in testis, of high-androstenone N. Landrace pigs. This finding is consistent with the results of our previous work on Large White x Landrace and Meishan x Large White x Landrace crossbreeds, which demonstrated a significant negative relationship between adipose tissue androstenone concentrations and expression of the hepatic, but not the testicular, 3ß-HSD protein (Nicolau-Solano et al., 2006).
Interestingly, in this study the concentration of 3ß-HSD protein in the liver of low-androstenone Duroc pigs was more than 50% lower than in low-androstenone N. Landrace animals. The reason for these differences is not clear. One possible explanation could be the existence of breed-specific mechanisms regulating pig hepatic 3ß-HSD expression. Our previous research on primary cultured pig hepatocytes established that sex steroids play a key role in regulation of 3ß-HSD protein expression (Nicolau-Solano and Doran, 2007). Therefore, it might be possible that the breed-specific regulation of enzyme expression is due to breed-related hormonal homeostasis.
The main enzymes involved in the second, conjugative stage of steroid metabolism are cytosolic SULT, which catalyze the transfer of the sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate to a variety of compounds containing a hydroxyl or amine group to form sulfates or sulfamates (see Strott, 1996, for a review). Sulfoconjugation increases the water solubility of compounds, and hence facilitates their excretion (Jakoby et al., 1980; Strott, 2002). Glatt and Meinl (2004) have also proposed that sulfoconjugative enzymes are important for regulating the amount of active steroids in tissues. Among the main members of the SULT family are SULT2A and SULT2B (Weinshilboum et al., 1997; Geese and Raftogianis, 2001). The presence of SULT2A1 has been reported for a number of species and tissues, including pig liver and testis (Alnouti and Klaassen, 2006; Sinclair et al., 2006; Urquhart et al., 2007). The enzyme SULT2B1 has also been identified in a number of species (He et al., 2005; Falany et al., 2006; Kohjitani et al., 2006), but no information is available regarding the presence of the SULT2B isoforms in pig tissues. An interesting point is that the human SULT2A1 has a broad substrate specificity, whereas SULT2B1 is selective and stereospecific for sulfation of 3ß-hydroxysteroids (Meloche and Falany, 2001), the process which plays the major role in inactivation of steroids in pig tissue (Sinclair and Squires, 2005). In this study, we detected the presence of an approximately 45-kDa SULT2B1 immunoreactive band in pig liver and testis. The molecular weight of the immunoreactive band in our study is consistent with the size of the human SULT2B1 protein (Meloche and Falany, 2001). Expression of the hepatic SULT2B1 protein in the current study was negatively related to the androstenone concentration in Duroc, but not in N. Landrace pigs, whereas expression of the testicular SULT2B1 was negatively related to the androstenone concentration in both breeds investigated. These results suggest that the degree of the enzyme contribution in the conjugative stage of steroid catabolism in different tissues (testis and liver) might be breed specific.
The finding in the current study that expression of SULT2B1 protein in pig tissues is related to androstenone concentrations is in agreement with the results of Sinclair and Squires (2005), who established the relationship between androstenone concentrations and expression of another SULT isoform, SULT2A1, in pig testis. Those authors also suggested that sulfoconjugation is important not only for elimination of the products of steroid catabolism, but also for binding "free" steroids (including androstenone), and thus eliminating the steroids from circulation in plasma. This was associated with a reduction of androstenone in adipose tissue.
The microsomal enzyme CYP17 plays a key role in steroid metabolism and has been reported to be present in both steroidogenic and nonsteroidogenic tissue (Katagiri et al., 1998; Niwa et al., 2002; Valle et al., 2002). In the pig, particular attention has been paid to testicular CYP17 in relation to androstenone biosynthesis. Lin et al. (2005) cloned and sequenced the coding region of the porcine CYP17 gene and detected a polymorphism, which affected an AA sequence but did not have any effect on CYP17 activity. Therefore, the authors suggested that synthesis of androstenone in pig testis is not directly affected by a polymorphism in the CYP17 coding region. Further, Davis and Squires (1999) investigated whether an increased concentration of 16-androstene steroids in fat in Yorkshire boars is related to expression of testicular CYP17 protein. The authors did not find any significant correlation between these 2 variables in the breed studied. Because there are indications in the literature that mechanisms controlling accumulation of boar taint compounds might be breed specific (Xue et al., 1996; Doran et al., 2002), it was of interest to extend the study on expression of CYP17 to other breeds and to investigate expression of the testicular and hepatic CYP17 protein. In the current study, we investigated the expression of hepatic and testicular CYP17 in 2 breeds of pigs and found no differences in the expression of CYP17 between the high-androstenone and low-androstenone groups in either Duroc or N. Landrace pigs. Therefore, although CYP17 is important for androstenone metabolism in pigs (Meadus et al., 1993; Davis and Squires, 1999), there is no evidence regarding the contribution of this enzyme to within-breed or between-breed variations in the content of androstenone in adipose tissue.
In conclusion, in the current study, we report breed differences in expression of the androstenone-metabolizing enzymes 3ß-HSD and SULT2B1 in the liver of pigs with high and low androstenone concentrations. We suggest that the excessive accumulation of androstenone in adipose tissue of N. Landrace pigs might be related to a low rate of hepatic androstenone degradation in metabolic stage I, whereas the high androstenone concentration in pigs of the Duroc breed might be related to a low rate of androstenone metabolism in metabolic stage II. In addition, we suggest that the mechanisms regulating androstenone concentrations in pig adipose tissue are breed specific.
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Footnotes |
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2 Corresponding author: o.doran{at}bristol.ac.uk
Received for publication May 18, 2007. Accepted for publication July 25, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-adrost-16-en-3-one in pig peripheral plasma. Acta Endocrinol. (Copenh.) 76:377–387.-androstenone levels in young boars fed two planes of nutrition. Reprod. Nutr. Dev. 27:413–422.[CrossRef][Medline]-hydroxylase/17,20-lyase P450c17. J. Steroid Biochem. Mol. Biol. 64:121–128.[CrossRef][Medline]-androstenone in plasma and fat of the mature domestic boar. J. Anim. Sci. 83:358–365.
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