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ANIMAL PRODUCTS |
* Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol, BS40 5DU, UK;
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Meat and Livestock Commission, PO Box 44, Winterhill House, Snowdon Drive, Milton Keynes, MK6 1AX, UK
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Abstract |
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Key Words: androstenone • boar taint • 3ß-hydroxysteroid dehydrogenase • pig
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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; Bonneau, 1982; Lundstrom and Bonneau, 1996). A positive relationship between fat androstenone level and sensory perception of boar taint is well established (Patterson, 1968; Fuchs, 1972; Malmfors and Andresen, 1975; Hansson et al., 1980; Xue et al., 1996). In particular, our previous work reported that abnormal odor of pork depends on the concentration of backfat androstenone or on the combined effect of androstenone, skatole, and indole concentrations (Annor-Frempong et al., 1997). Desmoulin and Bonneau (1982) demonstrated that consumers could detect boar taint in pork at the fat androstenone level 0.5 µg/g. For processed pork products this level was 1 µg/g. Accumulation of androstenone in adipose tissue could be due to at least 2 factors: overproduction of androstenone in the testis, a low rate of androstenone metabolism in the liver, or both.
The enzyme 3ß-hydroxysteroid dehydrogenase (3ß-HSD) is involved in both processes (Von Teichman et al., 2001; Doran et al., 2004). This enzyme catalyzes 2 types of reactions: 1) dehydrogenation and isomerization of 3ß-hydroxysteroids to 3-ketosteroids, and 2) reduction of 3-ketosteroids to 3ß-hydroxysteroids (Simard et al., 2005). The involvement of 3ß-HSD in the 2 different types of reactions is often linked to the existence of different 3ß-HSD isoforms (Zhao et al., 1991; Rogerson et al., 1995). The 3ß-HSD isoform spectrum for pig liver and testis remains unclear. So far only one 3ß-HSD isoform has been characterized in pig adipose tissue (Von Teichman, 2001).
Our previous research established that pigs with high androstenone levels exhibit low hepatic 3ß-HSD activity (Doran et al., 2004). Therefore it was suggested that a low rate of hepatic androstenone metabolism via 3ß-HSD might contribute to high levels of androstenone in adipose tissue. However, the reason for the low hepatic 3ß-HSD activity remains unclear. One of the possibilities could be a low expression of the hepatic 3ß-HSD gene/protein. The relationship between expression of the testicular 3ß-HSD and androstenone deposition in adipose tissue has not yet been investigated.
The aims of the present research were 1) to investigate the relationship between the expression of 3ß-HSD protein in liver and testis and androstenone deposition in backfat and 2) to clone and sequence the coding regions of pig hepatic and testicular 3ß-HSD in order to compare isoform spectrums in these 2 tissues.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Animals
Thirteen intact (uncastrated) male pigs of Large White (50%) x Landrace (50%) and Meishan (25%) x Large White (25%) x Landrace (50%) cross-breeds, which differed in the level of backfat androstenone, were used in the study, hereafter referred to as Large White and Meishan pigs. Animals were allocated to groups depending on the breed and backfat androstenone level. An androstenone level over 1 µg/g of adipose tissue was considered as the threshold to differentiate between low and high androstenone pigs. Three experimental groups were defined: 1) Large White with low adipose tissue androstenone levels (n = 5); 2) Meishan with low adipose tissue androstenone levels (n = 4); and 3) Meishan with high adipose tissue androstenone levels (n = 4). The androstenone levels in these groups were: 0.72 ± 0.07, 0.49 ± 0.19, and 1.32 ± 0.1 µg/g of adipose tissue, respectively.
The animals were reared on the same commercial standard pelleted diets (BOCM Pauls Ltd., Portishead, UK), which consisted of growing and finishing diets. The growing diet was fed ad libitum to pigs up to 12 wk of age. This diet contained 5% vegetable oil, 18.5% CP, 1.20% lysine, and 3.3% crude fiber. After 12 wk, the pigs were transferred to the finishing diet, which was fed until harvest. The finishing diet contained 3.5% vegetable oil, 16.7% CP, 1.25% lysine, and 3.8% crude fiber.
After slaughter, carcass weights were recorded as 70 to 85 kg, which is in agreement with the mean for pig carcass weight in the UK (74 kg; Scientific Report on the Scientific Panel for Animal Health and Welfare, 2004). Samples of liver, testis, and subcutaneous fat were collected within 5 to 10 min after exsanguination in the EU-approved abattoir of the Department of Clinical Veterinary Science, University of Bristol. Subcutaneous fat samples were removed from the region of the last cervical vertebra, one of the areas commonly used for analysis of androstenone content in backfat (Walstra et al., 1999; Zeng et al., 2002; Quintanilla et al., 2003). Liver samples were obtained from the left lateral lobe. To obtain testicular samples, the testes were decapsulated to remove connective tissues, fasciae, and the main blood vessels. The inner part of the testicular tissue, containing Leydig cells, was collected and subsequently used for isolation of testicular microsomes. All the samples were immediately frozen in liquid nitrogen and stored at –80°C until analyzed.
Producing 3ß-HSD Antibody
A synthetic peptide, GSLVKQHKEAKTK, corresponding to amino acids 357 to 370 of the porcine 3ß-HSD protein (GenBank accession No. NP 001004049) was linked to Keyhole Limpet Haemocyanin via its N-terminal cysteine and was injected into rabbits using a standard protocol. The unfractionated serum was used as a source of anti-3ß-HSD.
Isolation of Microsomes
In pig tissues, 3ß-HSD was shown to be mainly or only associated with the microsomal fraction (Hebert and Cooke, 1992; Furster, 1999). Microsomes were isolated by differential centrifugation (Schenkman and Cinti, 1978) with minor modifications. In brief, 10 g of frozen tissue was homogenized in 20 mL of ice-cold Tris-sucrose buffer (10 mM Tris-HCl, 250 mM sucrose, pH 7.4). The mitochondria were separated from the postmitochondrial fraction (containing microsomes) by centrifugation at 12,000 x g for 10 min. The microsomes were obtained by centrifugation of the postmitochondrial fraction at 25,000 x g for 30 min in the presence of 8 mM CaCl2. The microsomal pellet was washed with Tris-KCl buffer (10 mM Tris-HCl, 150 mM KCl, pH 7.4) and centrifuged at 25,000 x g for 30 min. The resulting pellet was resuspended at a protein concentration of about 20 mg/mL in a medium containing 50 mM Tris-HCl, 10 mM KH2PO4, 0.1 mM EDTA, 20% glycerol, and inhibitors of proteolytic enzymes antipain+pepstatin+leupeptin (1 µg/mL; Sigma-Aldrich, St. Louis, MO). Protein was determined by the Bradford method using bovine serum albumin as a standard (Bradford, 1976).
Measurements of 3ß-HSD Activity
In the current study, the microsomal 3ß-HSD activity [with androstenone (Sigma-Aldrich, Dorset, UK) as a substrate] was determined by recording the rate of ß-androstenol formation, as described previously (Doran et al., 2004). In brief, isolated microsomes (1 mg of protein) were incubated in 3 mL (total volume) of medium containing 50 mM Tris-HCl, 10 mM KH2PO4, 0.1 mM EDTA, and 20% glycerol (pH 7.4) in a shaking waterbath at 37°C for 20, 40, or 60 min in the presence of 0.5 mM of androstenone (if not otherwise stated) and 1 mM NADH (Sigma-Aldrich) as a cofactor.
To extract the product of the reaction, 200 µL of each sample was mixed with 200 µL of chloroform/isopropanol (1:1, vol/vol), vortexed for 1 min, and centrifuged for 10 min at 2,000 x g. The lower (chloroform) fraction was taken for quantification of ß-androstenol by high resolution gas chromatography (Doran et al., 2004) using androstanedione as an internal standard. The linearity of the detector response was checked by using a "calibration cocktail" containing known amounts of ß-androstenol, androstenone, and androstanedione. It was reported in our previous study that the rates of ß-androstenol formation in microsomes isolated from fresh and frozen tissues were similar (Doran et al., 2004).
In the inhibition study, trilostane was used as a specific inhibitor of 3ß-HSD (Tueni et al., 1987; Ukena et al., 1999; Sakamoto et al., 2001). Isolated hepatic and testicular microsomes were incubated in the absence (control) or presence of various concentrations of trilostane (0.5, 1, 2.5, 5, or 25 µM), 0.5 mM androstenone, and 1 mM NADH at 37°C for 20 min. Trilostane was kindly donated by Stegram Pharmaceuticals (Northumberland, UK). The 3ß-HSD activity was determined by measuring the rate of ß-androstenol formation, as described above.
Analysis of Androstenone in Adipose Tissue
Androstenone level in adipose tissue was measured by high resolution gas chromatography using a modification of the method of De Brabander and Verbeke (1986). In this procedure, 0.4 g of fat was saponified for 1 h at 60°C in toluene and KOH in methanol, with 5
-androstane-3,17-dione (Sigma-Aldrich, Dorset, UK) as an internal standard. After cooling, aqueous methanol was added and the samples were extracted 4 times with petroleum ether (boiling point 40 to 60°C):diethyl ether (1:1, vol/vol). The pooled upper layers were reduced in volume under nitrogen before derivatization using TriSil reagent (Pierce, Cheshire, UK). The samples as trimethylsilyl-derivatives were analyzed using a Fisons 8000 series gas chromatograph (Fisons Instruments, Crawley, Sussex, UK) in hot splitless/split mode (20:1), equipped with a Sil8 capillary column (25 m x 0.25 mm i.d.; Chrompack Ltd., London, UK), with helium as the carrier gas and a flame ionization detector.
Western Blotting
To determine the optimum amount of microsomal protein required for analysis of 3ß-HSD expression, preliminary experiments were performed. The relationship between the intensity of 3ß-HSD signal and the amount of the protein used was investigated. In these experiments microsomal proteins (2, 4, 6, 10, 12, 15, or 20 µg per well) were separated by SDS-PAGE, electroblotted onto a nitrocellulose membrane (pore size 0.45 µm, BioRad, Herts, UK) at a constant 100 V for 1 h. The membrane with transferred proteins was probed with rabbit antiporcine 3ß-HSD antibody for 1.5 h (for details of antibody production, see above). The antibody was diluted 1:300 in phosphate buffered saline Tween (PBST).
After probing with primary antibody, the nitrocellulose membranes were washed with PBST for 15 min and incubated with a commercial secondary antibody [horseradish peroxidase-linked, donkey anti-rabbit Ig (Amersham, Bucks, UK)] for 1 h. The secondary antibody was diluted 1:10,000 in PBST. After the incubation, the membrane with the complex of 3ß-HSD-primary antibody-secondary antibody was washed for 15 min with PBST. The protein bands were visualized using an enhanced chemiluminescence detection system (Amersham, Bucks, UK). The films were scanned, and the intensity of the corresponding bands was quantified using the ImageQuant program (Molecular Dynamics, GE HealthCare UK Ltd., Buckinghamshire, UK).
Figure 1A shows an example of a blot for 3ß-HSD when varying amount of microsomal protein was used. Quantification of the 3ß-HSD signals is presented on Figure 1B. A linear relationship was observed between the amount of the microsomal protein and the intensity of 3ß-HSD signal, when up to 12 µg of the protein was loaded (r2 = 0.95; P < 0.01). Therefore, 6 µg of microsomal protein was used for all of the subsequent blots. This allowed detection of a decrease or increase in 3ß-HSD protein expression by at least 2 times. One sample from a particular pig (the reference sample) was present on every blot, and the 3ß-HSD content of this sample was taken as 100 arbitrary units throughout. The amount of 3ß-HSD in other samples on the blot was expressed as a percentage of this reference sample.
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). The intensity of the calreticulin signals was measured for microsomal preparations from testis and liver of 4 different pigs. The averages of the signals were similar for the hepatic and testicular microsomal preparations: 83.9 ± 5.8 and 91.7 ± 8.5 arbitrary units, respectively (P > 0.05).
Northern Blotting
Total RNA was isolated from pig liver using TriReagent (Sigma, Dorset, UK). Thirty micrograms of RNA were denatured, separated on a denaturing 0.9% (w/ vol) agarose gel, and transferred by capillary action onto nylon membrane (Hybond-N, Amersham, Buckinghamshire, UK) as described by Sambrook et al. (1989). A specific cDNA probe (305 bp), corresponding to bases 722 to 1,027 of the pig 3ß-HSD sequence (Gen-Bank accession No. AF 232699), was produced by PCR with pig liver cDNA as a template. The PCR reaction was performed at the annealing temperature of 54°C for 35 cycles. The identity of the probe was confirmed by DNA sequencing after cloning the PCR product into pGem-T-easy vector (Promega, Southampton, UK). The probe was labeled with -32P-dCTP using a Rediprime kit (Amersham, Buckinghamshire, UK).
After prehybridization, the blot was hybridized for 3 h at 68°C in Perfecthyb buffer (Sigma), washed twice at 42°C in buffer containing 0.3 M NaCl, 20 mM Na2PO4, and 2 mM EDTA (pH 7.4), followed by 2 washes in the same buffer but with addition of 0.1% (wt/vol) SDS. The blot was exposed to film, and the signals were scanned using ImageQuant program (Molecular Dynamics). The blot was then reprobed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe as a control to ensure that an equivalent amount of RNA was used for each sample. The probe (353 bp) corresponding to bases 939–1,290 of the pig GAPDH sequence (GenBank accession No. AF 017079), was produced by PCR reaction using pig liver cDNA as a template. The reaction was performed at the annealing temperature 50°C for 35 cycles. The ratio of the signals 3ß-HSD:GAPDH was calculated for each sample.
Cloning and Sequencing of 3ß-HSD
The coding region (1,008 bp) of the hepatic and testicular 3ß-HSD was generated by PCR using corresponding cDNA as the templates and the following primers: 5'-GAGGATCGTCCACTTGTTGC-3' (forward) and 5'-GTTTTCTGCTTGGCTTCCTCC-3' (reverse). The primers corresponded to the bases 223 to 242 and 1,211 to 1,231, respectively, of the 3ß-HSD cDNA sequence from pig adipose tissue (GenBank accession No. AF 232699). These regions are largely conserved in different 3ß-HSD isoforms. The PCR reaction was performed at the annealing temperature of 55°C for 35 cycles. The PCR products were ligated into pGem-T-easy vector (Promega, Southampton, UK), amplified in E-coli XL-1 Blue, and the inserts were sequenced.
Statistics
The significance of differences between groups of experimental data was assessed by SPSS (Chicago, IL) univariate ANOVA and the Tukey honestly significant difference post hoc test (P 
0.05) for multiple comparisons. Simple linear regression analysis was performed to investigate the relationship between parameters.
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RESULTS |
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). To compare the activities of the hepatic and testicular 3ß-HSD with androstenone as a substrate, the rates of ß-androstenol formation per milligram of protein were measured in microsomes isolated from these 2 tissues. The time-course of ß-androstenol formation is presented in Figure 2. For microsomes from both tissues there was a linear increase in ß-androstenol level for up to 40 min of incubation (r2 = 0.9, P < 0.05) The 3ß-HSD activity at the time-point 40 min was almost 4-fold greater (P = 0.05) in the hepatic microsomes compared with the testicular microsomes.
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). At 5 µM of trilostane the formation of the hepatic and testicular ß-androstenol were inhibited by 81.7% (P < 0.001) and 67.4% (P < 0.001), respectively. These results provide direct proof that androstenone is metabolized via 3ß-HSD in isolated microsomes from pig liver and testis.
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) could occur for at least 2 reasons: 1) 3ß-HSD is expressed at a higher level in liver than in testis or 2) there is a polymorphism in the testicular 3ß-HSD cDNA, which affects AA composition and the activity of the enzyme. Both possibilities have been explored.
3ß-HSD Protein and mRNA Expression
Protein expression was estimated by Western blotting. Figure 4A shows a representative blot with 3ß-HSD antibody for liver and testis. A single 50-kDa band, which corresponds to the molecular weight of 3ß-HSD, was obtained in both tissues. To confirm the specificity of the antibody, the blots were repeated in the presence of excess of the peptide, to which the antibody was produced. Figure 4B shows that the presence of the peptide prevents binding of the primary antibody to 3ß-HSD protein for hepatic and testicular microsomal preparations.
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shows that in liver, 3ß-HSD protein expression was more than 3-fold greater in pigs with low backfat androstenone compared with animals with a high backfat androstenone level (P < 0.001) and compared with expression of the testicular 3ß-HSD (P < 0.001). In contrast, expression of 3ß-HSD protein in testis did not differ significantly between animals with high and low androstenone levels (P = 0.26).
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). No relationship between these 2 parameters was found for testis (r2 = 0.004; P = 0.84; Figure 6B).
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show that in pigs with high backfat androstenone, the hepatic 3ß-HSD mRNA content was lower (P < 0.01) compared with pigs with low androstenone levels.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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; Simard et al., 2005). The current study investigated the relationship between expression of the pig hepatic and testicular 3ß-HSD protein and deposition of androstenone in adipose tissue and established that: 1) 3ß-HSD is expressed at higher levels in pig liver compared with pig testis and 2) expression of the hepatic but not testicular 3ß-HSD protein is repressed in pigs with high back-fat androstenone levels.
The fact that in the current study the low expression of the hepatic 3ß-HSD protein in high androstenone pigs was accompanied by a low expression of 3ß-HSD mRNA may indicate inhibition of the 3ß-HSD expression at the level of transcription. The mechanism of regulation of hepatic 3ß-HSD gene expression in pig is unknown. One of the key roles in this process might be played by sex hormones. Positive correlations between the levels of sex hormones and androstenone level were reported by a number of authors (Andresen, 1976; Lundstrom et al., 1978; Zamaratskaia et al., 2004).
The current study established that 3ß-HSD protein expression was repressed only in the liver but not in the testis of high androstenone pigs. The tissue-specific expression of 3ß-HSD has been reported for other species and linked to the tissue-specific distribution of 3ß-HSD isoforms. Thus, six 3ß-HSD isoforms were reported for mice (Payne et al., 1995). Murine 3ß-HSD isoform I is expressed in the gonads and the adrenal glands; isoforms II, III, and V are present in the liver (Bain et al., 1991; Park et al., 1996); isoforms II, III, and IV are found in kidney (Bain et al., 1991; Clarke et al., 1993), and isoform VI is predominantly expressed in placenta and skin (Abbaszade et al., 1997). Four 3ß-HSD isoforms were identified in rats (Zhao et al., 1991; Simard et al., 1993), 3 in the hamster (Rogerson et al., 1998), and 2 in the human (Luu-The et al., 1990; Rheaume et al., 1991). The distribution of these isoforms is also tissue-specific. So far only one 3ß-HSD isoform has been found in the pig (Von Teichman et al., 2001). Cloning and sequencing of the coding regions of the pig hepatic and testicular 3ß-HSD, performed in the current study, did not reveal any differences in cDNA sequences from these 2 tissues. The pig hepatic and testicular 3ß-HSD cDNA sequences were similar to pig adipose tissue 3ß-HSD cDNA, reported by Von Teichman et al. (2001) and shared 98% similarity with the human 3ß-HSD isoform I. Furthermore our Western blot experiments detected only 1 band at approximately 50 kDa in the microsomal preparations from pig liver and testis. In other species, when more than one 3ß-HSD isoform was reported, more than 1 band was detected with Western blotting (Keeney et al., 1993). In addition, the hepatic and testicular 3ß-HSD could be inhibited in a similar manner by trilostane, which is, according to Thomas et al. (2004), specific toward the human 3ß-HSD isoform I. Trilostane is a well-known inhibitor of gonadal and adrenal 3ß-HSD (Kawai et al., 1991; Cooke, 1996). However, its inhibitory action was also demonstrated in nonsteroidogenic tissues, including rat liver (Naville et al., 1991; Coirini et al., 2003). The fact that in our experiments androstenone metabolism in testis and liver can be completely inhibited by 25 µM trilostane is in agreement with data of Coirini et al. (2003). They observed 90% inhibition of steroid metabolism (conversion of pregnenolone to progesterone) by 25 µM trilostane in rat sciatic nerve homogenates.
Taken together, the results of the present paper indicate that a single 3ß-HSD gene in the pig is regulated differently in different tissues. Factors that are involved in regulation of 3ß-HSD expression in pig liver but not in testis remain to be determined. The liver-specific regulation of 3ß-HSD expression in pigs is likely to contribute to the low rate of hepatic androstenone metabolism and consequent androstenone accumulation in adipose tissue.
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Footnotes |
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2 Corresponding author: e.udovikova{at}bristol.ac.uk
Received for publication October 17, 2005. Accepted for publication May 17, 2006.
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-androstenone and plasma testosterone in boars selected for rate of body weight gain and thickness of back fat during growth, sexual maturation and after mating. J. Reprod. Fertil. 48:51–59.
5-4 isomerase and differential expression in gonads, adrenal glands, liver and kidneys of both sexes. Proc. Natl. Acad. Sci. USA 88:8870–8874.5-4 isomerase. Mol. Endocrinol. 7:1569–1578.-androst-16-ene-3-one content and the sex odor intensity in boar fat. Swed. J. Agric. Res. 1:233–237. 5-4 isomerase in mouse tissues: Male-specific isoforms are expressed in the gonads and liver. Endocrinology 133:39–45.-androstenone and testosterone in boars. Swed. J. Agric. Res. 8:171–180.5-4 isomerase from human placenta. Ann. N. Y. Acad. Sci. 595:386–388.-androst-16-ene-3-one in boar peripheral plasma and back fat. Acta Agric. Scand. 25:92–96.-Androst-16-ene-3-one, compound responsible for taint in boar fat. J. Sci. Food Agric. 19:31–38.[CrossRef]5-4 isomerase in human adrenals and gonads. Mol. Endocrinol. 5:1147–1157.5-4 isomerase. J. Mol. Endocrinol. 20:99–110.[Abstract]5-4 isomerase in the hamster. J. Steroid Biochem. Mol. Biol. 55:481–487.[CrossRef][Medline]5-4 isomerase in the zebrafish central nervous system. J. Compar. Neurol. 439:291–305.[CrossRef][Medline]5-4 isomerase (3ß-HSD) family. The exclusive 3ß-HSD Gene expression in the skin. J. Biol. Chem. 268:19659–19668.5-4 isomerase gene family. Endocr. Rev. 26:525–582.5-4 isomerase in different regions of the avian brain. Brain Res. 818:536–542.[CrossRef][Medline]5-4 isomerase. Anim. Genet. 32:298–302.[CrossRef][Medline]5-4 isomerase cDNAs and differential tissue-specific expression of the corresponding mRNAs in steroidogenic and peripheral tissues. J. Biol. Chem. 266:583–593.This article has been cited by other articles:
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  G. Chen, R.-A. Cue, K. Lundstrom, J. D. Wood, and O. Doran Regulation of CYP2A6 Protein Expression by Skatole, Indole, and Testicular Steroids in Primary Cultured Pig Hepatocytes Drug Metab. Dispos., January 1, 2008; 36(1): 56 - 60. [Abstract] [Full Text] [PDF]
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 M. Moe, E. Grindflek, and O. Doran Expression of 3{beta}-hydroxysteroid dehydrogenase, cytochrome P450-c17, and sulfotransferase 2B1 proteins in liver and testis of pigs of two breeds: Relationship with adipose tissue androstenone concentration J Anim Sci, November 1, 2007; 85(11): 2924 - 2931. [Abstract] [Full Text] [PDF]
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G. Chen, E. Bourneuf, S. Marklund, G. Zamaratskaia, A. Madej, and K. Lundstrom Gene expression of 3{beta}-hydroxysteroid dehydrogenase and 17{beta}-hydroxysteroid dehydrogenase in relation to androstenone, testosterone, and estrone sulphate in gonadally intact male and castrated pigs J Anim Sci, October 1, 2007; 85(10): 2457 - 2463. [Abstract] [Full Text] [PDF]
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R.-A. Cue, S. I. Nicolau-Solano, J. D. McGivan, J. D. Wood, and O. Doran Breed-associated variations in the sequence of the pig 3{beta}-hydroxysteroid dehydrogenase gene J Anim Sci, March 1, 2007; 85(3): 571 - 576. [Abstract] [Full Text] [PDF]
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) testicular microsomes. Microsomes were incubated in the presence of 0.5 mM androstenone and 1 mM NADH. Values are plotted as mean ± SE. Each point represents an average for 3 pigs, for which each incubation was run in duplicate.
