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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Department of Food Science, Swedish University of Agricultural Sciences, PO Box 7051, SE-750 07 Uppsala, Sweden;
and
Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, PO Box 597, SE-751 24 Uppsala, Sweden; and
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, PO Box 7011, SE-750 07 Uppsala, Sweden
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: androstenone • estrone sulphate • 3ß-hydroxysteroid dehydrogenase • 17ß-hydroxysteroid dehydrogenase • pig • testosterone
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-androst-16-en-3-one) is known to be one of the main compounds responsible for boar taint (Patterson, 1968
). The QTL for fat androstenone has been detected on pig chromosome 4 (SSC4; Quintanilla et al., 2003; Lee et al., 2005). The gene encoding 3ß-hydroxysteroid dehydrogenase (3ßHSD) has been mapped within this QTL to the position SSC4q16-q21 (Von Teichman et al., 2001).
The enzyme 3ßHSD in humans can catalyze the oxidative conversion of 
5-3ß-hydroxysteroids to the 4-3-keto configuration, and also has 3-ketosteroid reductase function (see Simard et al., 2005 for review). The 17ß-hydroxysteroid dehydrogenase (17ßHSD) enzyme in humans catalyzes the final conversion of inactive 17-ketosteroid into its active 17ß-hydroxy form. Both 3ßHSD and 17ßHSD belong to the same phylogenetic protein family, namely the short-chain alcohol dehydrogenase reductase superfamily (see Payne and Hales, 2004, for review). The 17ßHSD7 gene, encoding one of the newly found 17ßHSD isotypes, is located on human chromosome 1q23 (Törn et al., 2003), a region that has been comparatively mapped to SSC4q1.5 (Moller et al., 2004). Both 3ßHSD and 17ßHSD are important in testicular steroid biosynthesis, the same pathway as for androstenone production (see Simard et al., 2005, for review). Therefore, it is of interest to investigate their gene expression and association with other testicular steroids, particularly androstenone, which is implicated in boar taint.
This study was designed to investigate the expression of 3ßHSD and 17ßHSD mRNA in relation to accumulation of fat androstenone and other testicular steroids and to determine how the variation of 3ßHSD and 17ßHSD gene expression is affected by immunocastration and surgical castration, respectively.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The care of the pigs and the experimental design of this study were approved by the local Animal Ethics Committee in Tierp, Sweden.
Pigs from a Landrace sire x Yorkshire dam cross were used. Two separate experiments were conducted. In Exp. 1, fat and testis samples from 22 gonadally intact male pigs were included. Androstenone concentrations in fat were measured as described below, and the pigs were classified in 2 groups based on androstenone concentration. The median androstenone concentration (0.74 µg/g) in the samples was arbitrarily chosen as a level below which pigs were included in the low androstenone group (LA), and above which they were included in the high androstenone group (HA). The mean fat androstenone level in the LA group was 0.368 µg/g (SD 0.275, n = 12) and in the HA group 3.445 µg/g (SD 3.882, n = 10).
In Exp. 2, a total of 20 male pigs were raised. The experimental design included 4 groups. One group consisted of 5 male piglets surgically castrated without anesthesia before 1 wk of age. A second group consisted of 5 male pigs treated with Improvac (Pfizer Ltd., formerly CSL Ltd., Parkville, Victoria, Australia) containing a modified form of GnRH in an aqueous adjuvant system (200 µg of GnRH-protein conjugate/mL). Vaccination was performed twice at 8 and 4 wk before slaughter [i.e., at 16 wk of age (60 kg of BW) for the first injection and at 20 wk of age (90 kg of BW) for the second injection]. The remaining 10 male pigs were kept intact throughout the study. The gonadally intact pigs were classified in 2 groups, one with 5 pigs with the 5 lowest fat androstenone concentrations and the other with 5 pigs with the greatest fat androstenone concentrations.
All pigs in both experiments were slaughtered at 24 wk of age (120 kg of BW) in a commercial slaughterhouse. In connection with slaughter, pigs were withheld from feed overnight but had free access to water. They were transported 5 km to the slaughterhouse and kept in lairage for 2 h. Pigs were immobilized by CO2 and killed by exsanguination. Fat samples from the neck region as well as testis and liver samples were taken, immediately frozen in liquid nitrogen, transported to the laboratory within 1 h, and then stored at –80°C until analyses. A blood sample (10 mL) was taken 1 d before slaughter and was collected via jugular venipuncture into a heparinized Vacutainer tube. Plasma was separated by centrifugation at 2,000 x g for 15 min at 4°C and kept at –80°C until steroid analysis.
Steroid Analyses
Fat androstenone was analyzed by an HPLC method (Chen et al., 2007). In brief, 150 µL of tissue-free liquid fat was extracted by using 750 µL of methanol containing 0.33 µg/mL of androstanone as the internal standard. After centrifugation, 140 µL of the supernatant was derivatized by dansylhydrazine (Sigma-Aldrich, Steinheim, Germany) and analyzed by HPLC. Chromatography was conducted using a Merck-Hitachi D-7000 HPLC system (Tokyo, Japan), and signals were detected by a fluorescence detector with the wavelength at 346 nm for excitation and 521 nm for emission. An external standard curve from fat spiked with androstenone was used for quantification.
Testosterone and estrone sulphate (E1S) in plasma were measured using commercial RIA kits from Coat-A-Count (Diagnostic Products Corporation, Los Angeles, CA) and DSL (DSL-5400, Cherwell Innovation Center, Upper Heyford, UK), respectively. According to the manufacturer’s instructions, the limit of detection for testosterone was 0.04 ng/mL, and the intra- and interassay CV were 4.0 to 18.0% and 5.9 to 12.0%, respectively. For E1S, the limit of detection was 0.01 ng/mL, and the intra- and interassay CV were 4.6 to 9.2% and 5.1 to 8.8%, respectively.
Total RNA Isolation and Reverse Transcription
Total RNA was extracted from testis and liver by using the TRIzol reagent (Invitrogen/Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. The concentration of total RNA was quantified at 260 nm using the NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE). The quality of total RNA was assessed using Agilent’s 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) with RNA chip kits (for nanogram assay, Caliper Technologies, Mountain View, CA), and samples showing a good RNA quality (i.e., low degradation and presence of 2 ribosomal peaks) were selected for further reverse transcription. Reverse transcription was performed using the First-Strand cDNA Synthesis Kit (Amersham Biosciences UK Limited, Little Chalfont, Buckinghamshire, UK) according to the manufacturer’s protocol. A total of 3 µg of total RNA in 20 µL was used for reverse transcription, and pd(N)6 was used for cDNA synthesis. The cDNA were further purified using QIAquick PCR purification columns (Qiagen Inc., Valencia, CA).
Real-Time PCR
The primers and probes information for the two 3ßHSD and 17ßHSD target genes as well as for the 2 housekeeping internal control genes [transferrin receptor (TFR) and hypoxanthine phosphoribosyltransferase (HPRT)] are listed in Table 1. All primers and probes were designed using the Primer Express software v2.0 (Applied Biosystems, Foster, CA). Probes were dual-labeled with 6-FAM (6-Carboxyfluorescein) at the 5' end and with TAMRA (tetramethylrhodamin) at the 3' end (Bustin, 2000). To avoid genomic amplification, at least one of the primers of each gene was designed to span 2 exons.
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Ct value was obtained by subtracting the housekeeping gene Ct value from the target gene Ct value; e.g., Ctt1 = Ctt1 – Cth1, where t1 was target mRNA 1 and h1 was housekeeping gene 1. The Ct value was obtained by subtracting a calibrator Ct from the target sample Ct value (e.g., Ctt1 = Ctt1 – Ctc1, where c1 was calibrator 1). The calibrator sample selected was the sample showing the greatest Ct value. Statistical Analysis
The GLM procedure (SAS Inst. Inc., Cary, NC) was performed to evaluate the difference between groups for concentrations of fat androstenone, plasma E1S, testosterone, 3ßHSD, and 17ßHSD mRNA using the PDIFF statement. Pearson correlation coefficients were calculated for the relationship between androstenone, E1S, testosterone, and the relative quantity of 3ßHSD and 17ßHSD mRNA. Correlations were considered statistically significant at P < 0.05. Differences in the 3ßHSD and 17ßHSD relative mRNA quantities within the same tissue were compared by Student’s paired t-test, and the results were considered to be significant at P < 0.05.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The amplification efficiency for all amplicons was above 90% and considered to be adequate for real-time PCR quantification taking into account the observed differences between amplicons regarding amplification efficiency. The BestKeeper software (Pfaffl et al., 2004) was used to validate TFR and HPRT as internal reference genes. The TFR gene showed the smallest variation among testis samples and was less sensitive to treatments, but proved to be inadequate for liver samples. The HPRT gene appeared to be suitable as a liver sample endogenous control.
Analysis of Gene Expression in Testis from the LA and HA Groups
Real-time PCR analysis showed that in the 22 gonadally intact male pigs from Exp. 1 with different fat androstenone concentrations, the 3ßHSD gene expression in testis in the LA group was greater than in the HA group (4.88 ± 1.36 vs. 3.28 ± 1.14, mean ± SD; P < 0.01), whereas the 17ßHSD gene expression was not different between groups (2.13 ± 1.08 vs. 2.13 ± 0.68; P = 0.996). Comparison of the 17ßHSD and 3ßHSD gene expression in the same tissue showed that 3ßHSD mRNA levels were much greater than 17ßHSD mRNA levels (P < 0.001; Figure 1).
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Steroids, as well as relative gene expression levels, were determined in pigs treated with surgical castration (SC), immunocastration (IC), and gonadally intact male pigs with low (NC1) or high (NC2) fat androstenone (Exp. 2). Androstenone concentrations in NC1 and NC2 from Exp. 2 were separated to a greater extent than in the groups from Exp. 1. None of the steroids could be detected in either of the SC or IC groups. In the NC2 group with greater fat androstenone, testosterone and E1S concentrations in plasma were also greater than in the NC1 group (P < 0.001; Table 2).
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). Furthermore, the 17ßHSD in liver in the NC2 group was also less compared with the other groups. The expression of 3ßHSD gene in liver did not differ between the SC, IC, and NC1 groups, but 17ßHSD mRNA levels in liver in the IC group were greater than in the SC group (P = 0.022) and did not differ between IC and NC1 (P = 0.121) and between SC and NC1 (P = 0.387).
For testis samples, 3ßHSD mRNA levels in the NC2 group were less than in the IC and NC1 groups (P < 0.001 and P = 0.021, respectively). Moreover, the expression of 3ßHSD in testis in the IC group was greater than in the NC1 group (P < 0.01; Figure 2). The expression of 17ßHSD in testis in the IC group was greater than in the 2 gonadally intact male groups (NC1 and NC2, P = 0.043 and P = 0.036, respectively), but did not differ between the gonadally intact male groups (P = 0.745; Figure 2).
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The correlation between steroid hormones and gene expression in liver was only investigated in gonadally intact male pigs because steroids in castrated pigs were not detectable. Fat androstenone was negatively correlated with 3ßHSD gene expression (r = –0.69; P < 0.05; Figure 3a) and 17ßHSD gene expression (r = –0.81; P < 0.01), but positively correlated with plasma E1S (Figure 3b) and testosterone (r = 0.88 and 0.84, respectively; P < 0.01). Level of E1S correlated negatively with 3ßHSD gene expression (r = –0.75; P < 0.05) and 17ßHSD gene expression (r = –0.95; P < 0.001; Figure 3c). Testosterone was negatively correlated with 17ßHSD gene expression (r = –0.81; P < 0.01), but not with 3ßHSD gene expression (r = –0.55; P = 0.10). The positive correlation between E1S and testosterone was also high (r = 0.94; P < 0.001). In addition, the gene expressions of 3ßHSD and 17ßHSD were positively correlated (r = 0.86; P < 0.01; Figure 3d).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Bonneau et al. (1982) reported that androstenone can be mobilized from adipose tissue into the blood circulation when castration of boars occurs at 175 d. A recent study showed that the enzyme 3ßHSD can catalyze the transformation of androstenone to ß-androstenol (Doran et al., 2004), which is accumulated in low concentration in fat due to its 3-OH hydrophilic structure and is therefore less important in causing boar taint (Garcia-Regueiro and Diaz, 1989). The low 3ßHSD enzyme activity is associated with high fat androstenone accumulation (Nicolau-Solano et al., 2006), and a negative correlation between fat androstenone and 3ßHSD mRNA levels in liver has also been reported (Doran et al., 2004). In our study, we used real-time PCR analysis to show that in gonadally intact male pigs from the same cross breed, high 3ßHSD gene expression was associated with low fat androstenone accumulation in liver and testis. It should be noted that the gonadally intact male pigs were separated based on their fat androstenone concentrations, with no gap in androstenone concentrations between the groups. If the pigs had been selected more stringently, we would expect a greater negative association between 3ßHSD gene expression and androstenone. In addition, we also found a strong negative correlation between fat androstenone and liver 3ßHSD mRNA levels in the gonadally intact male pigs; this further confirmed 3ßHSD enzyme as a likely candidate in catalyzing androstenone reduction in gonadally intact male pigs. The full pig 3ßHSD cDNA sequence has been published (Von Teichman et al., 2001; GenBank accession No. AF232699), and no polymorphism was found on cDNA in 2 pig breeds (Meishan and Large White) exhibiting different backfat androstenone concentrations (Doran et al., 2004). More recently, the 5'-flanking region (from –741 to +378 bp) of the 3ßHSD gene has been sequenced, and 3 SNP and 1 microsatellite (TTAT repeats) have been found to be associated with the pig breed, but not with fat androstenone concentrations (Cue et al., 2007). The fact that no association between 3ßHSD polymorphisms and fat androstenone has been reported may indicate that this regulation is likely due to specific transcription factors rather than a regulatory mutation carried by 3ßHSD. Future studies are necessary to better understand the differential expression of this gene.
In our study, surgical castration and immunocastration caused a significant increase of the liver 3ßHSD gene expression compared with the gonadally intact male pigs with high fat androstenone concentrations. To the best of our knowledge, this has not been reported before, and the mechanism is not obvious. However, it is well known that steroid hormones are synthesized via LH stimulating testicular Leydig cells, and that castration will sufficiently block this pathway. Thus, the induction of 3ßHSD gene expression might be caused by the absence of steroids after castration. Indeed, there is evidence showing that androgens can downregulate human type II 3ßHSD transcription in the cortical cells and in testicular Leydig cells (see Simard et al., 2005, for review). Surgical castration is routinely used to stop androstenone production in testis. Immunocastration, as an alternative to surgical castration, is the active immunization against GnRH. Its inhibitive function to androstenone as well as to other testicular steroids has also been illustrated (Dunshea et al., 2001; McCauley et al., 2003). Our study showed that immunocastrated pigs and gonadally intact male pigs with low fat androstenone concentrations differed significantly in 3ßHSD gene expression in testis, but not in liver. We assume that immunocastration induces more 3ßHSD gene expression in testis than in liver because this procedure directly changes the testicular physiological properties. In addition, a significant negative correlation between 3ßHSD gene expression and fat androstenone was obtained in liver, but not in testis of gonadally intact male pigs. Liver 3ßHSD mRNA was also negatively correlated with plasma E1S, probably because of the high positive correlations between the analyzed steroids. Bonneau et al. (1982) explained that the fat androstenone disappearance in boars castrated at 175 d was dependent on the intensity of plasma androstenone catabolism and elimination. Here we assume this catabolism might be achieved by the 3ßHSD metabolic function. Androstenone exists in abundance in boar salivary glands and acts as a pheromone to stimulate standing response in oestrous sows (Booth, 1987). The catabolism of androstenone to ß-androstenol in the salivary gland might also be achieved by 3ßHSD enzyme due to its presence in this site (Booth, 1977). Like 3ßHSD, 17ßHSD gene expression in liver was also significantly increased after castration, and the increase in immunocastrated pigs was more prominent than in surgically castrated pigs. However, 17ßHSD mRNA levels in liver did not differ between immunocastrated pigs and low androstenone gonadally intact male pigs or between surgically castrated pigs and low androstenone gonadally intact male pigs. This induction of 3ßHSD and 17ßHSD gene expression in the castrated pigs suggests that the absence of testicular steroids not only affects 3ßHSD transcription regulation, but also the 17ßHSD transcription.
The involvement of 17ßHSD in transformation of estrone to active estradiol has been confirmed in humans (see Payne and Hales, 2004, for review), but its function in pigs has not been determined. Our study of the gonadally intact male pigs revealed a strong negative correlation between liver 17ßHSD gene expression and plasma E1S, suggesting an important role of the 17ßHSD enzyme in the metabolism of pig estrogens. A previous study showed that estrogens present in peripheral blood in mature boars were greater than in postpubertal female pigs, possibly because the boar requires estrogens as well as androgens for normal activity (Joshi and Raeside, 1973). We found that in gonadally intact male pigs of about 120 kg of BW, the E1S concentration in plasma was even greater than the main anabolic steroid hormone, testosterone. This increased amount of E1S in the blood circulation in gonadally intact male pigs has been reported elsewhere (Schwarzenberger et al., 1993). It has also been reported that estrogens in pigs are mainly present as sulphated form and that the unconjugated estrone and estradiol 17ß only represent <10% and <5% of the total estrogens, respectively (Schwarzenberger et al., 1993). Estrone sulphate is the main sulphated form of estrogens, and it has been suggested to be an indicator for evaluating steroid hormonal status in gonadally intact male pigs (Babol et al., 1999). Plasma E1S is also highly correlated with fat androstenone accumulation (Zamaratskaia et al., 2005), which was further confirmed in our study. We report here that between gonadally intact male pigs with low and high fat androstenone concentrations, respectively, the 17ßHSD gene expression varied significantly in liver, but not in testis. One possible explanation for this is that 17ßHSD is more stable expressed in testis because it is an important enzyme catalyzing 17-ketosteroids transformation in testis as mentioned above. However, less is known about its function in liver. The different pattern of 17ßHSD gene expression suggests that the 17ßHSD enzyme catalytic activity might vary between tissues. In addition, the expression of liver 3ßHSD and 17ßHSD mRNA levels was strongly positively correlated in the gonadally intact male pigs. This might further suggest that both enzymes are involved in a similar steroid biosynthetic pathway and are regulated by concurrent transcription.
Taken together, by performing the gene expression analysis in samples from gonadally intact male and castrated pigs, we confirmed the important role of 3ßHSD in androstenone metabolism. In addition, we also found a strong negative correlation between 17ßHSD and E1S. Moreover, the noteworthy induction of 3ßHSD and 17ßHSD gene expression after castration was exhibited. We also showed that liver 3ßHSD and 17ßHSD gene expression in the gonadally intact male pigs were positively correlated. Thus, if the regulation of 3ßHSD expression affects androstenone accumulation, its effects on other genes that are involved in anabolic steroid metabolism need to be considered.
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Footnotes |
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2 Corresponding author: Gang.Chen{at}lmv.slu.se
Received for publication February 9, 2007. Accepted for publication June 12, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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5-4 isomerase gene family. Endocr. Rev. 26:525–582.5– 4 isomerase. Anim. Genet. 32:298–302.[CrossRef][Medline]
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