Expression of hepatic 3b-hydroxysteroid dehydrogenaseand sulfotransferase 2A1 in entire and castrated male pigs

Martin Krøyer Rasmussen • Carl Brunius •

Bo Ekstrand • Galia Zamaratskaia

Received: 8 November 2011 / Accepted: 16 April 2012 / Published online: 29 April 2012

� Springer Science+Business Media B.V. 2012

Abstract The present study investigated the effect of

surgical (SC) and immunological castration on the steroid

metabolizing enzymes 3b-hydroxysteroid dehydrogenase

(3b-HSD) and sulfotransferase 2A1 (SULT2A1) in male

pigs. Thirty-two male pigs were divided in four groups; in

one group the pigs were SC before the age of 7 days, two

groups were injected with Improvac� a vaccine against

gonadotropin releasing hormone (immunological castra-

tion), while the pigs in the last group remained entire males

(EMs). Immunological castration was in one group per-

formed by vaccine injection at ages 11 and 14 weeks, while

the other group received injections at ages 17 and

21 weeks. Plasma, adipose and liver tissue were collected

at the time of slaughter. Plasma was analyzed for con-

centrations of testosterone and oestradiol. The adipose

tissue was analyzed for the concentration of androstenone,

while the liver tissue was analyzed for mRNA and protein

expression of 3b-HSD and SULT2A1. Independent of

method, all castrated pigs showed greater mRNA and

protein expression of 3b-HSD and lower levels of all ste-

roids in plasma compared with EMs. Moreover, there was a

strong correlation between mRNA and protein expression

of 3b-HSD and steroid levels. The same was not valid for

expression of SULT2A1. It is concluded that steroid levels

can increase expression of the steroid metabolizing enzyme

3b-HSD and thereby influence steroid metabolism, e.g. of

androstenone.

Keywords Castration � Gene regulation � Protein

expression � Boar taint � Androstenone

Introduction

A high level of androstenone in porcine adipose tissue is one

of the major factors contributing to boar taint, an unpleasant

odour of pork meat from sexually mature entire male (EM)

pigs, both directly and indirectly, through its effect on ska-

tole metabolism [1, 2]. Androstenone is produced by the

Leydig cells of the testes of EM pigs. The production of

androstenone and other steroids is regulated by luteinising

hormone, which is released by the pituitary gland in response

to gonadotropin releasing hormone (GnRH) [3]. Andros-

tenone is metabolised in the testes and liver by similar

metabolic processes. First, androstenone is transformed to

3b-androstenol and, to a lesser extent, 3a-androstenol by 3b-

and 3a-hydroxysteroid dehydrogenase, respectively (3b-

HSD and 3a-HSD) [4, 5]. Low hepatic expression and

activity of 3b-HSD in pig liver is accompanied by reduced

clearance and increased accumulation of androstenone in pig

adipose tissue [4, 6] The relation between 3b-HSD expres-

sion in liver and androstenone accumulation in pig adipose

tissue as well as breed-dependent variations in the gene

structure for 3b-HSD have been studied [7, 8].

Phase II metabolism of 3b-androstenol is performed by

sulfotransferase 2A1 (SULT2A1) to form sulfoconjugates.

Hepatic expression of SULT2A1 (also referred to as DHEA

sulfotransferase) is related to the concentration of andros-

tenone in back fat [4, 9, 10]. Moreover, SULT2B1 has also

Martin Krøyer Rasmussen and Carl Brunius have contributed equally

to the work.

M. K. Rasmussen (&) � B. Ekstrand

Department of Food Science, Aarhus University, Blichers Alle

20, P.O. Box 50, 8830 Tjele, Denmark

e-mail: Martink.rasmussen@agrsci.dk

C. Brunius � G. Zamaratskaia

Department of Food Science, BioCenter, Swedish University of

Agricultural Sciences, Uppsala, Sweden

123

Mol Biol Rep (2012) 39:7927–7932

DOI 10.1007/s11033-012-1637-5

been shown to be involved in androstenone metabolism,

but was shown to be dependent on breed [11].

The regulation of enzymes involved in androstenone

metabolism is at present not fully elucidated. In vitro

studies using primary cultured pig hepatocytes showed that

some sex steroids might be involved in the regulation of

3b-HSD protein expression [12]. In this study, we further

investigate the role of sex steroids in the regulation of

androstenone-metabolising enzymes. To do this, we

investigated mRNA and protein expressions of hepatic 3b-

HSD and SULT2A1 and compared them with plasma

concentrations of steroids. As a model, we used surgically

castrated (SC) pigs and pigs vaccinated against GnRH with

Improvac�, which blocks the production of testicular ste-

roids. mRNA and protein expressions of hepatic 3b-HSD

and SULT2A1 in SC and immunocastrated pigs were

compared with those in EM pigs.

Materials and methods

Animals and sampling

All pigs used were male crossbreds (Swedish Yorkshire

dams 9 Swedish Landrace sires) born and raised at the

Swedish University of Agricultural Sciences Funbo Lovsta

experimental station. Eight pigs per treatment group were

randomly selected from a larger population of pigs included

in a study by Andersson et al. [13]. In the first group, piglets

were surgical castrated without anaesthesia before the age

of 7 days (SC). Pigs in the second group (early vaccination,

EV) were vaccinated with Improvac when aged 11 and

15 weeks. Pigs in the third group (standard vaccination, SV)

were vaccinated when aged 17 and 21 weeks. Pigs in the

fourth group (EMs) were kept intact throughout the study.

Pigs of the same treatment were raised in pens of 8 until

slaughter at age 25 weeks. Detailed rearing conditions for

the subpopulation are given in Brunius et al. [14] and for the

entire population in Andersson et al. [13].

Analyses of steroids

The androstenone concentration in fat was measured using

HPLC as described by Brunius et al. [14]. Testosterone

concentration in plasma was measured using a commercial

RIA kit (TKTT, Diagnostic Products Corporation, Los

Angeles, CA, USA), according to the manufacturer’s

instructions. Oestradiol concentration in plasma was mea-

sured using a commercial EIA human salivary kit (1-3702,

Salimetrics, State College, PA, USA), using a method

adopted by Brunius et al. [15]. In brief, plasma samples

were diluted with water 1:1, to make sure the samples

would be in the linear range of the standard curve, and kept

overnight at 4 �C. Two hundred microliters of the plasma

mixtures were added to pre-coated wells and incubated for

1 h at room temperature on a rotator (500 rpm) thereafter a

further 1 h without rotation. After the pre-incubation, the

following procedure was performed in accordance with the

manufacturer’s instructions.

mRNA expression

RNA isolation, reverse transcription and PCR was done in

accordance with Rasmussen et al. [16]. Briefly, total RNA

was extracted from approximately 10 mg liver tissue using

a commercial available spin column (RNeasy Mini Kit,

VWR, Herlev, Denmark). The amount of isolated RNA

was estimated from measuring the absorbance at 260 nm

and a total of 450 ng RNA was converted to cDNA using

Superscript II RNase H Reverse Transcriptase and Oli-

go(dt)12–18 primer according to the manufacturers

instruction (Invitrogen, Carlsbad, CA, USA). The mRNA

expression of the selected genes was determined by sub-

jecting equal volumes of cDNA to real-time PCR. The

samples were analysed with PCR in duplicates using a ABI

7900 HT sequence detraction system (Applied Biosystems,

Carlsbad, CA, USA). Primer and TaqMan probes are given

in [17]. Relative mRNA expression was calculated using

the Ct values obtained and normalising against the mRNA

expression of GAPDH. Expression of the group of EMs

was arbitrarily set to 1 and the value of castrated and

vaccinated pigs expressed relative to that.

The determination of mRNA expression was done by real-

time PCR using the guide lines given by Huggett et al. [18],

suggesting (1) isolation of RNA from similar tissue volumes,

(2) converting similar amounts of RNA into cDNA and (3)

normalization to a reference gene. In the initial data analysis

the obtained Ct values for the target genes was normalized

against GAPDH and b-actin, showing the same trend in data.

There was no significant effect of treatment on the Ct values

for GAPDH and b-actin (for primers and probes of b-actin

see [19]) obtained from the different treatment groups (data

not shown). The expression of GAPDH (Ct values; SC:

25.18 ± 0.85; SV: 24.89 ± 0.98; EV: 25.61 ± 0.50; EM:

25.10 ± 1.19) showed the lowest variability between the

groups and was chosen to be the housekeeping gene in the

final data analysis.

Protein expression

The expression of 3b-HSD was determined in hepatic

microsomes, while the expression of SULT2A1 was deter-

mined in the cytosolic phase resulting from the preparation

of the microsomes. Microsomes were prepared by homog-

enising frozen liver tissue in Tris–sucrose buffer (10 mM

Tris–HCl, 250 mM sucrose, pH 7.4) and ultracentrifugation

7928 Mol Biol Rep (2012) 39:7927–7932

123

(100.0009g, 60 min, 4 �C) according to Rasmussen et al.

[20]. The total amount of proteins in the cytosolic phase was

precipitated with trichloroacetic acid (TCA). TCA was

added to the cytosolic phase resulting from the microsome

preparation giving a final concentration of 12 %. After 10

min on ice, the proteins were spun down (20.0009g, 4 �C)

resulting in a small pellet. The pellet was washed twice in ice

cold acetone, and dissolved in a buffer containing 100 mM

Tris-base with 4 % SDS (pH 9.5), before being analyzed

with Western blotting. Equal amounts of protein from each

sample was subject to Western blotting conducted according

to Rasmussen et al. [20]. For detection of 3b-HSD, we used

the R1484 antibody kindly supplied by Professor J. I. Mason

(Division of Reproductive and Developmental Science,

University of Edinburgh, Scotland), and SULT2A1 was

detected with an antibody purchased from Santa Cruz (Santa

Cruz, CA, USA).

Average protein expression of the group of EMs was

arbitrarily set to 100 and the value of the castrated and

vaccinated pigs expressed relative to this.

Statistical analyses

Data were analysed using SAS 9.2 (SAS Institute, Cary,

NC, USA). Concentrations of androstenone, testosterone

and oestradiol were log-transformed prior to statistical

analyses to normalise residual distributions. Pearson cor-

relations between measured variables were calculated

using the data set from all treatment groups (n = 31). This

approach was used to investigate the overall correlation

between the measured components.

The effects of treatment (SC, EV, SV and EMs) on

androstenone, testosterone and oestradiol concentrations

and mRNA and protein expressions were evaluated with

the MIXED procedure, including treatment as a fixed

effect. Due to outlier of data for one pig in the SC group

only 7 pigs were included in that group. Significance levels

were set to p \ 0.05.

Results

Concentrations of testosterone, oestradiol

and androstenone

Plasma concentrations of testosterone, oestradiol and

androstenone are given in Table 1. There were significant

differences in plasma testosterone, oestradiol and andros-

tenone concentrations between treatments (p \ 0.0001 in

all cases). Plasma concentrations of testosterone in cas-

trated and vaccinated pigs were all below the detection

limit of 0.04 ng/ml, and lower than in EMs (p \ 0.05 in

each case). Plasma concentrations of oestradiol and

androstenone were all above the limit of detection, but

were also lower in castrated and vaccinated pigs than EMs

(p \ 0.05 in each case).

3b-HSD and SULT2A1 expression

3b-HSD and SULT2A1 expression are shown in Table 2.

There was a close to significant difference in mRNA

expression of 3b-HSD between treatments (p = 0.053),

Table 1 Effect of treatment on hormones in plasma and androstenone in adipose tissue

SC (n = 7) EV (n = 8) SV (n = 8) EM pigs (n = 8) p Value

Testosterone (ng/ml) \0.04a \0.04a \0.04a 1.77b (1.29–1.93) \0.0001

Oestradiol (pg/ml) 0.55a (0.46–1.33) 0.24a (0.13–0.68) 0.40a (ND–0.82) 8.41b (4.00–13.0) \0.0001

Androstenone (ng/ml) 0.11a (0.10–0.13) 0.15a (0.11–0.18) 0.11a (0.09–0.12) 1.40b (0.52–2.58) \0.0001

Data are presented as medians with interquartile ranges within brackets. The p value indicates the overall effect of treatments. Medians with

different superscripts within the rows differ at p \ 0.05

ND non-detectable

Table 2 Effect of treatment on mRNA and protein expressions of 3b-HSD and SULT2A1

SC (n = 7) EV (n = 8) SV (n = 8) EM pigs (n = 8) p Value

PCRHSD 2.06a (0.35) 2.28a (0.33) 1.66ab (0.33) 1.00b (0.33) 0.053

WBHSD 1.27a (0.07) 1.13ab (0.06) 1.13ab (0.06) 1.00b (0.06) 0.051

PCRSULT 1.11 (0.30) 1.37 (0.28) 0.57 (0.28) 1.00 (0.28) 0.249

WBSULT 1.19 (0.26) 1.41 (0.25) 1.34 (0.25) 1.00 (0.25) 0.660

Data are presented as LS means with SE in brackets, relative to the average expression of EM pigs. The p value indicates the overall effect of

treatments. Medians with different superscripts within the rows differ at p \ 0.05 for PCRHSD, and p \ 0.01 for WBHSD

PCRHSD relative mRNA expression of 3b-HSD, WBHSD relative protein expression of 3b-HSD, PCRSULT relative mRNA expression of

SULT2A1, WBSULT relative protein expression of SULT2A1

Mol Biol Rep (2012) 39:7927–7932 7929

123

being greater in SC and EV pigs compared with EM pigs

(post hoc test; p \ 0.05). Protein expression of 3b-HSD

was also close to being significantly different between

treatments (p = 0.051), being highest in SC pigs and

lowest in EM pigs (post hoc test; p \ 0.01). Neither mRNA

nor protein expression of SULT2A1 differed significantly

between treatments.

Pearson correlations between steroid concentrations

and enzyme expression

Correlation coefficients between mRNA and protein

expression and steroid concentrations are given in Table 3.

Expression of 3b-HSD mRNA was negatively correlated to

the plasma concentrations of testosterone and oestradiol,

and protein expression was negatively correlated to tes-

tosterone and androstenone. For SULT2A1, no correlation

was observed between mRNA or protein expression and

steroids.

Discussion

Until recently, androstenone metabolism was mainly

studied in testes [21, 22]. It was believed that the amount of

androstenone synthesized in the testes is the major factor

determining accumulation of androstenone in fat. Now it is

known that variation in hepatic metabolism is also impor-

tant in the control of androstenone accumulation. The

hepatic enzymes 3b-HSD and SULT2A1 are likely to be

the key actors in androstenone metabolism [4, 5, 7, 9].

High androstenone levels in pigs are reported to be asso-

ciated with low gene and protein expression of these

enzymes [6, 7, 9]. The expression of SULT2B1, another

enzyme reported to be active in the steroid metabolism,

was also shown to be lower in pigs with high androstenone

levels, but this association was breed-dependent and was

not observed in Landrace pigs [11]. Because crossbred

Yorkshire 9 Landrace pigs were used in this study, we

focused only on 3b-HSD and SULT2A1 and did not

investigate SULT2B1 activity.

Understanding the molecular regulation of porcine

hepatic 3b-HSD and SULT2A1 expression is important for

the development of methods to control androstenone con-

centrations in fat at slaughter; and thus improving meat

quality from EM pigs. In vitro studies using primary cul-

tured pig hepatocytes suggested that some steroids are

important in the regulation of 3b-HSD protein expression

[12]. In vitro results show low expression of 3b-HSD in

pigs with high levels of androstenone [7, 11]. Similarly, an

in vivo study using castration as a means to reduce steroid

levels, found that hepatic 3b-HSD mRNA expression was

significantly greater in SC and immunocastrated pigs

compared with a group of EM pigs with a high andros-

tenone concentration (above 0.7 ppm) in adipose tissue [6].

The same study showed no difference in hepatic 3b-HSD

mRNA expression between castrated pigs and EM pigs

with low concentrations of androstenone (below 0.7 ppm).

Our results are consistent with these findings in that both

mRNA and protein expression of 3b-HSD were lower in

the liver from EM pigs, compared to castrates. Interest-

ingly, mRNA and protein expression of 3b-HSD in EV

pigs were comparable with SC pigs, whereas no differences

were observed between SV and EM pigs. A similar pattern

was observed when studying the effect of castration on

cytochrome P450 catalytic activities [14]. The authors

explained it by the long-term suppression of testicular

steroid production which can result in similar characteris-

tics in EV and SC pigs. No previous study has related

steroid-metabolising enzymes to different time points of

vaccination and the mechanism behind this finding remains

to be elucidated. The actions of sex steroids are determined

by their local metabolism [23], and therefore up- and

downregulation of the metabolizing enzymes like 3b-HSD

is of great importance to their effect in e.g. Leydig cells

[24] or the liver [25], and steroids have been shown to be

Table 3 Pearson correlations between mRNA and protein expressions of 3b-HSD and SULT2A1, hormones in plasma and androstenone in

adipose tissue

PCRHSD PCRSULT WBHSD WBSULT Testosterone Oestradiol

PCRSULT 0.31�

WBHSD 0.46** 0.21

WBSULT -0.20 -0.16 -0.04

Testosterone -0.41* -0.05 -0.39* -0.15

Oestradiol -0.43* -0.14 -0.30 -0.13 0.84***

Androstenone -0.34� -0.01 -0.38* -0.18 0.90*** 0.84***

The correlation analysis is performed on the data set obtained by pooling data from all treatment groups (n = 31). Significance level: � p \ 0.10,

* p \ 0.05, ** p \ 0.01, *** p \ 0.001

PCRHSD relative mRNA expression of 3b-HSD, WBHSD relative protein expression of 3b-HSD, PCRSULT relative mRNA expression of

SULT2A1, WBSULT relative protein expression of SULT2A1

7930 Mol Biol Rep (2012) 39:7927–7932

123

responsible for regulation of 3b-HSD in other organs in

pigs [26].

Contrary to what could be expected, this study did not

find any difference in SULT2A1 mRNA and protein

expression among the treatment groups. Generally, this

type of sulfotransferase has very wide substrate specificity

and is involved in the conjugation of many steroids. Sin-

clair et al. [9] showed that hepatic SULT2A1 activity only

weakly correlated to the plasma concentrations of sulfo-

conjugated androstenone, although pigs with high fat

androstenone had significantly lower SULT2A1 activity.

Our present study suggested that individual capacity for

sulfoconjugation is independent of circulating hormones. A

possible explanation for this might be that the hepatic

SULT2A1 expression is not regulated by steroids of tes-

ticular origin. In humans, transcriptional regulation of

SULT2A1 is suggested to be mediated by a number of

transcription factors including: oestrogen-related receptor a[27], PXR [28], vitamin D receptor [29, 30], and thyroid

hormones [31]. Furthermore, the constitutive androstane

receptor has been suggested to be involved in rodent

SULT2A1 regulation [32]. It has also been suggested that

steroidogenic factor is important in SULT2A1 regulation in

human adrenals [33]. Al-Dujaili et al. [34] has recently

shown that bioactive compounds in plants can decrease

steroid levels by inhibiting SULT2A1 activity, but not on

the mRNA level. The exact molecular mechanisms that

control SULT2A1 mRNA and protein expression in pigs

need to be further investigated.

The statistical model in our study included the fixed

effects of treatment without taking into consideration pen

and litter effects. It is not known if these effects could have

an impact on mRNA and/or protein expression. We used

overall correlation analysis rather than studying the corre-

lations within each group. The low number of animals

included in the study versus the individual variability can

explain why the overall effect of treatment on mRNA and

protein expression of 3b-HSD only approached significant

level. It should also be noticed that with our study design

the possibility of detecting a false positive effect of treat-

ment was negligible.

However, we believe that our novel results contribute to

a more complete picture of the regulation of androstenone

metabolism in male pigs and improve our understanding of

the relationship between steroids and hepatic 3b-HSD and

SULT2A1.

In conclusion, our study, for the first time, demonstrated

that both mRNA and protein expression of 3b-HSD in pigs

are correlated with plasma concentrations of testosterone,

oestradiol and androstenone. Furthermore, the close to

significant difference observed between treatment groups,

suggests that 3b-HSD is upregulated in SC and EV pigs.

SULT2A1 mRNA and protein expression did not differ

among the groups indicating that testicular steroids are of

low if any importance in the regulation of hepatic

SULT2A1 expression in pigs.

Acknowledgments The work was financially supported by the

Swedish Board of Agriculture. Pfizer provided additional economical

support and Improvac vaccine. We thank the staff at Funbo Lovsta,

especially Ulla Schmidt and Eva Norling, for taking excellent care of

the animals. Eleonor Palmer, Johanna Zingmark and Rolf Grahm are

gratefully acknowledged for their help during sampling. We also

thank Prof. Andrzej Madej and Mattias Norrby for technical assis-

tance with hormone analysis and Michael Pearce for valuable com-

ments on the manuscript.

References

1. Bonneau M (1982) Compounds responsible for boar taint, with

special emphasis on androstenone—a review. Livest Prod Sci

9:687–705

2. Patterson RLS (1968) 5a-Androst-16-ene-3-one: compound

responsible for taint in boar fat. J Sci Food Agric 19:31–38

3. Zamaratskaia G, Squires EJ (2009) Biochemical, nutritional and

genetic effects on boar taint in entire male pigs. Animal 3:1508–

1521

4. Doran E, Whittington FM, Wood JD, McGivan JD (2004)

Characterisation of androstenone metabolism in pig liver micro-

somes. Chem Biol Interact 147:141–149

5. Sinclair PA, Hancock S, Gilmore WJ, Squires EJ (2005)

Metabolism of the 16-androstene steroids in primary cultured

porcine hepatocytes. J Steroid Biochem Mol Biol 96:79–87

6. Chen G, Bourneuf E, Marklund S, Zamaratskaia G, Madej A,

Lundstrom K (2007) Gene expression of 3b-hydroxysteroid

dehydrogenase and 17b-hydroxysteroid dehydrogenase in relation

to androstenone, testosterone, and estrone sulphate in gonadally

intact male and castrated pigs. J Anim Sci 85:2457–2463

7. Nicolau-Solano SI, McGivan JD, Whittington FM, Nieuwhof GJ,

Wood JD, Doran O (2006) Relationship between the expression

of hepatic but not testicular 3 beta-hydroxysteroid dehydrogenase

with androstenone deposition in pig adipose tissued. J Anim Sci

84:2809–2817

8. Cue RA, Nicolau-Solano SI, McGivan JD, Wood JD, Doran O

(2007) Breed-associated variations in the sequence of the pig 3b-

hydroxysteroid dehydrogenase gene. J Anim Sci 85:571–576

9. Sinclair PA, Gilmore WJ, Lin Z, Lou Y, Squires EJ (2006)

Molecular cloning and regulation of porcine SULT2A1: rela-

tionship between SULT2A1 expression and sulfoconjugation of

androstenone. J Mol Endocrinol 36:301–311

10. Sinclair PA, Squires EJ (2005) Testicular sulfoconjugation of the

16-androstene steroids by hydroxysteroid sulfotransferase: its

effect on the concentrations of 5a-androstenone in plasma and fat

of the mature domestic boar. J Anim Sci 83:358–365

11. Moe M, Grindflek E, Doran O (2007) Expression of 3b-

hydroxysteroid dehydrogenase, cytochrome P450-c17, and sul-

fotransferase 2B1 proteins in liver and testis of pigs of two breeds:

relationship with adipose tissue androstenone concentration.

J Anim Sci 85:2924–2931

12. Nicolau-Solano SI, Doran O (2008) Effect of testosterone,

estrone sulphate and androstenone on 3b-hydroxysteroid dehy-

drogenase protein expression in primary cultured hepatocytes.

Livest Sci 114:202–210

13. Andersson K, Brunius C, Zamaratskaia G, Lundstrom K (2011)

Early vaccination with Improvac�: effects on performance and

behaviour of male pigs. Animal 6:87–95

Mol Biol Rep (2012) 39:7927–7932 7931

123

14. Brunius C, Rasmussen MK, Lacoutiere H, Andersson K, Ekstrand

B, Zamaratskaia G (2012) Expression and activities of hepatic

cytochrome P450 (CYP1A, CYP2A and CYP2E1) in entire and

castrated male pigs. Animal 6:271–277

15. Brunius C, Zamaratskaia G, Andersson K, Chen G, Norrby M,

Madej A, Lundstrom K (2011) Early immunocastration of male

pigs with Improvac�—effect on boar taint, hormones and

reproductive organs. Vaccine 29:9514–9520

16. Rasmussen MK, Zamaratskaia G, Ekstrand B (2011) Gender-

related differences in cytochrome P450 in porcine liver—impli-

cation for activity, expression and inhibition by testicular ste-

roids. Reprod Domest Anim 46:616–623

17. Rasmussen MK, Brunius C, Zamaratskaia G, Ekstrand B (2012)

Feeding dried chicory root to pigs decrease androstenone accu-

mulation in fat by increasing hepatic 3b hydroxysteroid dehy-

drogenase expression. J Steroid Biochem Mol Biol 130:90–95

18. Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-

PCR normalisation; strategies and considerations. Genes Immun

6:279–284

19. Theil PK, Sørensen IL, Therkildsen M, Oksbjerg N (2006)

Changes in proteolytic enzyme mRNAs relevant for meat quality

during myogenesis of primary porcine satellite cells. Meat Sci

73:335–343

20. Rasmussen MK, Ekstrand B, Zamaratskaia G (2011) Comparison

of cytochrome P450 concentrations and metabolic activities in

porcine hepatic microsomes prepared with two different methods.

Toxicol In Vitro 25:343–346

21. Brophy PJ, Gower DB (1972) Metabolism in vitro of 16-unsat-

urated C19 keto steroids in boar testis. Biochem J 127:23–24

22. Saat YA, Gower DB, Harrison FA, Heap RB (1974) Studies on

metabolism of 5alpha-androst-16-en-3-one in boar testis in vivo.

Biochem J 144:347–352

23. Penning TM (2003) Hydroxysteroid dehydrogenases and pre-

receptor regulation of steroid hormone action. Hum Reprod

Update 9:193–205

24. Tang PZ, Tsai-Morris CH, Dufau ML (1998) Regulation of

3beta-hydroxysteroid dehydrogenase in gonadotropin-induced

steroidogenic desensitization of Leydig cells. Endocrinology

139:4496–4505

25. Mullany LK, Hanse EA, Romano A, Blomquist CH, Mason JI,

Delvoux B, Anttila C, Albrecht JH (2010) Cyclin D1 regulates

hepatic estrogen and androgen metabolism. Am J Physiol Gas-

trointest Liver Physiol 298:G884–G895

26. Sun YL, Zhang J, Ping ZG, Fan LN, Wang CQ, Li WH, Lu C,

Zheng LW, Zhou X (2011) Expression of 3 beta-hydroxysteroid

dehydrogenase (3 beta-HSD) in normal and cystic follicles in

sows. Afr J Biotechnol 10:6184–6189

27. Seely J, Amigh KS, Suzuki T, Mayhew B, Sasano H, Giguere V,

Laganiere J, Carr BR, Rainey WE (2005) Transcriptional regu-

lation of dehydroepiandrosterone sulfotransferase (SULT2A1) by

estrogen-related receptor a. Endocrinology 146:3605–3613

28. Duanmu ZB, Locke D, Smigelski J, Wu W, Dahn MS, Falany

CN, Kocarek TA, Runge-Morris M (2002) Effects of dexa-

methasone on aryl (SULT1A1)- and hydroxysteroid (SULT2A1)-

sulfotransferase gene expression in primary cultured human

hepatocytes. Drug Metab Dispos 30:997–1004

29. Echchgadda I, Song CS, Roy AK, Chatterjee B (2004) Dehy-

droepiandrosterone sulfotransferase is a target for transcriptional

induction by the vitamin D receptor. Mol Pharmacol 65:720–729

30. Song CS, Echchgadda I, Seo YK, Oh T, Kim S, Kim SA, Cho

SW, Shi LH, Chatterjee B (2006) An essential role of the CAAT/

enhancer binding protein-alpha in the vitamin D-induced

expression of the human steroid/bile acid-sulfotransferase

(SULT2A1). Mol Endocrinol 20:795–808

31. Huang YH, Lee CY, Tai PJ, Yen CC, Liao CY, Chen WJ, Liao

CJ, Cheng WL, Chen RN, Wu SM, Wang CS, Lin KH (2006)

Indirect regulation of human dehydroepiandrosterone sulfo-

transferase family 1A member 2 by thyroid hormones. Endocri-

nology 147:2481–2489

32. Saini SPS, Sonoda J, Xu L, Toma D, Uppal H, Mu Y, Ren S,

Moore DD, Evans RM, Xie W (2004) A novel constitutive

androstane receptor-mediated and CYP3A-independent pathway

of bile acid detoxification. Mol Pharmacol 65:292–300

33. Saner KJ, Suzuki T, Sasano H, Pizzey J, Ho C, Strauss JF, Carr

BR, Rainey WE (2005) Steroid sulfotransferase 2A1 gene tran-

scription is regulated by steroidogenic factor 1 and GATA-6 in

the human adrenal. Mol Endocrinol 19:184–197

34. Al-Dujaili EAS, Kenyon CJ, Nicol MR, Mason JI (2011)

Liquorice and glycyrrhetinic acid increase DHEA and deoxy-

corticosterone levels in vivo and in vitro by inhibiting adrenal

SULT2A1 activity. Mol Cell Endocrinol 336:102–109

7932 Mol Biol Rep (2012) 39:7927–7932

123