Gene expression profiles in testis of pigs with extreme high and low levels of androstenone

The production of uncastrated male pigs is profitable to the swine production, because it leads to improved feed efficiency and greater lean yield of the carcass [1]. However, production of entire males is a challenge due to boar taint, an unpleasant odour and flavour often present in the meat of un-castrated male pigs [2]. Castration is undesirable both for ethical as well as for economical reasons and thus alternative methods are needed to prevent tainted meat. Boar taint is primarily caused by high levels of the two components androstenone and/or skatole in the pig carcasses [3, 4]. Moderate to high heritabilities have been shown for both androstenone and skatole levels in fat [5–7]. For example, a recent study of Duroc and Norwegian Landrace showed heritabilities ranging from 0.5–0.6 for androstenone and 0.23–0.56 for skatole [8].

Androstenone (5α-androst-16-en-3-one) is a 16-androstene steroid produced from pregnenolone in the steroid hormone pathway in boar testis near sexual maturity. It is released into the blood and transported to salivary glands [9], where it regulates reproductive functions in female pigs [10]. Due to its lipophilic properties, it is also easily stored in the adipose tissue [11], causing boar taint when the fat is heated. Another steroid produced from pregnenolone in the same pathway is testosterone. Testosterone stimulates growth and fertility and the challenge is to reduce the level of androstenone without affecting the level of testosterone. Genes specifically affecting the production of androstenone in the testis are therefore of interest, as well as genes involved in degradation and elimination of this steroid.

Several candidate genes have been suggested to affect levels of androstenone. The biosynthesis of 16-androstenes from pregnenolone is catalysed by the andien-β synthase enzyme system [12], which consists of cytochrome P450-C17 (CYP17) and cytochrome b5 (CYB5) [13]. Although CYP17 has been proposed as a very potent candidate gene affecting levels of androstenone, studies so far have not found significant effects of SNPs within the CYP17 gene and androstenone levels [14, 15]. Similarly, it has not been possible to find a significant correlation between levels of CYP17 and levels of 16-androstene steroids in fat [14]. CYB5 has been found positively correlated to the production of androstenone [14]. In addition, a SNP in the 5' un-translated region of porcine CYB5 is found to be associated with decreased fat androstenone levels [16].

Another potent class of candidate genes is the sulphotransferase (SULT) enzymes whose main function is to transfer a sulfo group to a range of molecules including for example steroid hormones [17]. The SULTs have been proposed to regulate the amount of unconjugated 5α-androstenone available for accumulation in fat [18], and high proportions of sulphoconjugated to unconjugated 16-androstene steroids are observed in porcine plasma and Leydig cells [18, 19].

The levels of androstenone vary between breeds, with e.g. higher androstenone levels in Duroc and Hampshire compared to Landrace and Yorkshire [20]. It is therefore of interest to investigate gene expression patterns in individuals with high or low levels of androstenone in different breeds. In this study boars with extreme levels of androstenone were selected from the two commercial breeding populations in Norway, Duroc and Norwegian Landrace, for gene expression analysis in testis using microarray technology. The objective of the study was to identify differentially expressed genes that could point towards pathways associated with extreme levels of androstenone in pigs. To our knowledge, this is the first microarray experiment performed in this context.

The present study describes the transcriptional profiles of testicle from animals with extreme high and low levels of androstenone in D and NL boars. Some of these genes have already been shown to affect levels of androstenone, although most of them have not previously been described in this context. Both CYB5 and CYP17 were up-regulated in both D high (DH) and NL high (NLH) animals and this was confirmed by real-time PCR. Over-expression of CYB5 cDNA clones in high androstenone animals agrees with results previously seen in protein expression studies [13, 14]. Earlier studies on CYP17 have shown that polymorphisms in the coding region of the gene are not associated with androstenone levels [15], and that levels of the CYP17 protein are not correlated to levels of 16-androstene steroids in fat [14]. Notably, up-regulation of the gene expression of CYP17 in this study may be explained by its direct interaction with CYB5 in the andien-β synthase enzyme system. Differential expression of both genes was confirmed by real-time PCR in the D boars of our study.

StAR is involved in the transport of cholesterol from the outer to the inner mitochondrial membrane, where CYP11A1 converts cholesterol to pregnenolone. This has been described as the rate-limiting step in steroid hormone synthesis, after which pregnenolone may exit the mitochondria and can be metabolised to steroid hormones [21]. The cholesterol side chain cleavage enzyme system contains ferredoxin (FDX1) which functions as an electron transport system, transferring electrons from NADPH-dependent ferredoxin reductase to CYP11A1 [22, 23]. StAR was over-expressed in NLH, while FDX1 and CYP11A1 were over-expressed in both the DH and NLH groups. Our rcPCR results show that both StAR and CYP11A1 are highly up-regulated in the DH and NLH groups, even though the microarray study was not able to detect differences in the gene expression of StAR in DH. Based on results from the rcPCR study, StAR seems to particularly affect the androstenone level since this gene is up-regulated as much as five times in DH pigs and twelve times in NLH pigs. Emopamil-binding protein (EBP), sterol-C4-methyl oxidase-like isoform 1 (SC4MOL) and the cytochrome P450 enzyme lanosterol 14-alpha-demetylase (CYP51) are enzymes involved in the cholesterol biosynthesis pathway [24–26]. EBP was up-regulated in NLH, while SC4MOL and CYP51 were up-regulated in the DH. Isopentenyl-diphosphate delta isomerase (IDI1) catalyses the inter-conversion of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP), which is the substrate for the reaction that ultimately results in cholesterol, and this gene was up-regulated in both DH and NLH. The over-expression of all these genes may indicate that this early stage of steroid hormone biosynthesis is important for the production levels of androstenone. However, since cholesterol ultimately metabolises into both androstenone and testosterone, it might be impossible to use these genes as markers for low androstenone without simultaneously affecting levels of testosterone. However, further studies are needed to address this hypothesis.

In addition to FDX1, two additional ferritin cDNA clones were significantly up-regulated in the NLH and DH groups: FTL and the ferritin heavy-chain (FTH1). The differential expression of FTL was also confirmed by real-time PCR. Ferritin is an iron storage protein and plays a central role in numerous essential cellular functions [27]. Ferritin may also affect levels of androstenone through the CYB5/CYP450 electron transfer as the haem-containing cytochrome P450s need to receive electrons, e.g. from interaction with CYB5 [28], to be activated. 5-aminolevulinate synthase (ALAS1) is the first and rate-limiting enzyme in the haem biosynthesis pathway, providing haem for e.g. CYP450s [29]. This gene is up-regulated in DH. The gene ontology results also suggest an important role for ferric iron/iron ion binding for the levels of androstenone in adipose tissue.

Cytochrome P450 c19 (CYP19) encodes the enzyme aromatase, which catalyses the synthesis of estrogens from androgens. The pig secretes unusually high levels of estrogens from the testes [30] and is the only mammal known to express functionally distinct isoforms of this gene [31]. Our results show an up-regulation of CYP19A2 isoform in both NLH and DH boars, whereas the CYP19A1 isoform was up-regulated in DH. Estrogens are shown to be positively correlated with androstenone levels in fat [32] and estrone sulfate levels in plasma have been found positively correlated with both plasma and fat levels of androstenone [33]. SMPD1 is involved in the conversion of sphingomyelin to ceramide, which has been shown to inhibit CYP19 activity through induction of transcription factors [34]. SMPD1 was down-regulated in DH animals in the microarray study, but we were not able to confirm this in the rcPCR analyses.

HSD17B4 was found up-regulated for both DH and NLH, in both microarray and rcPCR analyses. The 17β-HSDs catalyse the last step in the formation of androgens and estrogens, and the HSD17B4 inactivates hormones very efficiently [35]. This has been reported for the substrates 17β-estradiol and 5-androstene-3β,17β-diol [36] but the enzyme also accepts other substrates [37]. The enzymes have previously been assigned to porcine Leydig and Sertoli cells [38] and porcine tissues express HSD17B4 as a predominant dehydrogenase [39]. Progesterone, a metabolite of pregnenolone, has been shown to increase porcine HSD17B4 activity [39] and the progesterone receptor membrane component 1 (PGRMC1) was over-expressed in DH. Another 17β-HSD, isoform 11, also called dehydrogenase/reductase family member 8 (DHRS8) was down-regulated in DH boars.

AKR1C4 belongs to the cytosolic aldo-keto reductases that act as 3α-/3β-/17β-/20α-hydroxysteroid dehydrogenases (HSDs) in human [40, 41] and was highly up-regulated in DH and NLH pigs. This was also confirmed by rcPCR. Penning et al. [36] showed that all the isoforms AKR1C1-AKR1C4 could interconvert active androgens and oestrogens with their associated inactive metabolites, which prevents an excess of circulating steroid hormones and makes the steroids into substrates for conjugation reactions [40].

Nuclear receptor subfamily 5, group A, member 1 (NR5A1), also called steroidogenic factor 1 (SF-1), was over-expressed in DH boars. This transcription factor is involved in the regulation of numerous genes, including StAR [42], CYP11A [43], CYP17 [44] and CYP19 [45]. Also, nuclear receptor co-activator 4 (NCOA4), an androgen receptor (AR) activator often referred to as ARA70, was up-regulated in the DH group. The nuclear receptor co-activator 2 (NCOA2), another AR activator, was significantly over-expressed in NLH. Other genes regulating transcription and translation that were differentially expressed in this study include class III POU transcription factor (POU3F2), microtubule-associated protein light chain 3 isoform A (MAP1LC3A) and EEF1A1 which were up-regulated in the DH and NLH groups, eukaryotic translation elongation factor 1 beta (EEF1B2) which was up-regulated in DH boars, and nuclear receptor subfamily 3, group C, member 2 (NR3C2) which was down-regulated in DH pigs.

Conjugation reactions contribute to the levels and pattern of steroids present in the plasma circulation of the boar. The enzymes involved in conjugation reactions were first thought to strictly inactivate and eliminate the compounds by rendering them more water-soluble. However, the high levels of conjugated steroids that are present in the plasma of the boar may suggest that the biological role of these reactions is more complex [46, 47]. A common family of conjugation enzymes is the sulfotransferases. SULT2A1, also called hydroxysteroid sulfotransferase (HST), was up-regulated in both DH and NLH boars in the microarray experiment and subsequently confirmed by rcPCR. SULT2A1 is a key sulfotransferase enzyme in terms of the 16-androstene steroids, and its activity in the testis is negatively correlated to androstenone concentrations in fat [19, 48]. Sulfotransferase family 2 isoform B1 (SULT2B1) is also a HST enzyme, showing selectivity for the conjugation of 3β-hydroxysteroids [49]. The SULT2B1 was significantly up-regulated in NLH pigs. The over-expression of the sulfotransferase genes in this study does not correspond to the negative correlation to levels of androstenone previously reported for this enzyme family [48].

Another conjugation reaction is catalysed by the glutathione S-transferases (GSTs). They have been shown to bind hormones [50] and influence their transport, metabolism and action [51]. One isoform of these enzymes (GSTalpha) is shown to be active in the pig testis Leydig and Sertoli cells and positively regulated by both estradiol and testosterone [52]. Glutathione S-transferase omega (GSTO1) and glutathione S-transferase (MGST1) were over-expressed in DH. These enzymes have not previously been studied in connection with androstenone; however, no correlation was found between fat skatole levels and glutathione S-transferase activity [53].

D and NL show many similar molecular functions and biological processes in this study, although they also differ with respect to which genes are differentially expressed. [54]. This miscellany might be due to the generally higher androstenone levels observed in D compared to NL [8]. The variability in the potential for androstenone production or elimination may to some extent be explained by breed differences in age at sexual maturity [55]. Differences between D and NL have also been shown by differential expression of 3β-hydroxysteroid dehydrogenase and SULT2B1 proteins, both enzymes critical for steroid production [54]. The higher number of significant genes in D compared to NL may be explained by the more extreme androstenone values in this breed (See Additional file 9: Androstenone values), giving higher contrasts.

In addition to the genes listed, we also identified several highly differentially expressed cDNA clones with no homology to known human or mouse sequences. Further characterisation of these genes may uncover new and unexpected roles in association with androstenone. The over-expression of for example AKR1C4, SULT2A1 and SULT2B1 in high androstenone animals does not explain a role for these genes as inactivation enzymes and the role of these genes needs to be further studied in relation to androstenone levels. Additional studies of all the genes are necessary to see if their proteins show the same differential expression. Furthermore, identification of single nucleotide polymorphisms (SNPs) in the genes or in association with the genes can be used for breeding purposes. The exact function of genes from interesting gene ontology pathways, like ferric iron binding, iron ion binding, electron transport activities and oxidoreductase activities, also needs to be clarified.

Our study detected differentially expressed genes that are previously found to affect androstenone levels in boars, as well as genes from pathways not formerly described in this aspect. We confirm the involvement of CYP17 and CYB5 and detect a number of other genes involved in the steroid hormone pathway that seem to be essential for androstenone levels. Besides SULT2A1 we identified other conjugation enzyme genes that might be important, including SULT2B1, AKR1C4, GSTO1, MGST1 and HSD17B4. The genetic profiles identified should be further examined to clarify their potential as molecular markers for reduced boar taint.