Ontogeny of expression and localization of steroidogenic enzymes in the neonatal and prepubertal pig testes
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Ontogeny of Expression and Localization of SteroidogenicEnzymes in the Neonatal and Prepubertal Pig Testes
INHO CHOI,* JI-YOUNG KIM,{ EUN JU LEE,* YOO YONG KIM,{ CHUNG SOO CHUNG,§
JONGSOO CHANG,5 NAG-JIN CHOI," HAK-JAE CHUNG," AND KI-HO LEE#
From the *School of Biotechnology, Yeungnam University, Gyeongsan, Republic of Korea; the �Department of
Biochemistry and Molecular Biology, School of Medicine, Kyunghee University, Seoul, Republic of Korea; the `School
of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea; the §Department of Animal
Science, Chungbuk National University, Cheongju, Republic of Korea; the 5Department of Agricultural Sciences, Korea
National Open University, Seoul, Republic of Korea; the "Hanwoo Experimental Station, National Institute of Animal
Sciences, Pyeongchang, Republic of Korea; and the #Department of Biochemistry and Molecular Biology and Center of
Anti-aging, School of Medicine, Eulji University, Daejeon, Republic of Korea.
ABSTRACT: The early neonatal development of boars is charac-
terized by significant testicular production of androgens and estro-
gens, including an anabolic steroid hormone, 19-nortestosterone. The
present study was conducted to determine the expression and
presence of steroidogenic and steroid hormone metabolism–related
enzymes in the testes of neonatal and 4-month-old prepubertal pigs.
Quantitative analyses with real-time polymerase chain reaction and
Western blotting were utilized to reveal mRNA and protein expression,
respectively. The localization of the molecules in the testes was
determined by immunohistochemistry. mRNA expressions of the
molecules tested were mostly significantly increased between 1 and 3
weeks of age and decreased at 4 months of age, compared with those
at 0 weeks of age. The protein levels of cytochrome P450 aromatase
and carbonyl reductase 1 were significantly increased between 1 and
3 weeks of age and decreased at 4 months of age. However, protein
expression patterns of other molecules differed from those of mRNA
expression, which implied the existence of posttranscriptional gene
regulation. Immunohistochemical analysis revealed that all of the
molecules were present in Leydig cells of the pig testis, regardless of
age, except cytochrome P450 side chain cleavage in germ cells and
17b-hydroxysteroid dehydrogenase 4 on the blood-testis barrier at
4 months of age. Aldose reductase and 3b-hydroxysteroid dehydro-
genase were localized in both Leydig and Sertoli cells. We postulate
that marked rises in the expression of steroidogenic enzymes in the
pig testis during early neonatal development could be associated with
peak production of 19-nortestosterone, thus eventually leading to the
early growth of male pigs.
Key words: Leydig cells, male reproductive tract, steroid hor-
mones, estradiol, nandrolone.
J Androl 2009;30:57–74
A remarkable feature of the domestic boar (Sus
scrofa) is high circulating estrogen concentrations
(Claus and Hoffman, 1980; Setchell et al, 1983).
Estrogen concentrations peak during neonatal develop-
ment, between 1 and 3 weeks of age, and transiently
decrease and remain at low level until pubertal
development (Ford, 1983). Changes in the serum levels
of free androgens and conjugated steroids also show
similar patterns to those of estrogens during postnatal
development of male pigs (Colenbrander et al, 1978;
Schwarzenberger et al, 1993). Along with androgens and
estrogens, 19-nortestosterone (17b-hydroxy-19-nor-4-
androsten-3-one, also known as nandrolone) is normally
found at high levels in pig serum during early neonatal
development (Schwarzenberger et al, 1993). Particular
attention is paid to nandrolone because of its high
anabolic activity (Kuhn, 2002). Because the production
of steroid hormone requires actions of a number of
steroidogenic enzymes, it is suggested that there is a
strong association between elevated steroid production
and enhanced expression of steroidogenic enzymes
during the early neonatal period.
Dynamic morphologic and histochemical changes in
the pig testis appear during the early neonatal period.
Increases of Sertoli cell proliferation and Leydig cell
volume occur during the first month after birth (Franca
et al, 2000). In addition, the majority of testicular
volume is made up of Leydig cells in the early neonatal
pig, predominantly between 2 and 3 weeks of age (van
Straaten and Wensing, 1978). In mammal testes,
syntheses of androgens and estrogens occur mostly in
Supported by a grant from the Biogreen 21 program (20050401-034-
712), Rural Development Administration, Republic of Korea.
Correspondence to: Dr Ki-Ho Lee, Department of Biochemistry and
Molecular Biology, College of Medicine, Eulji University, 143-5
Yongdoo-dong, Joong-goo, Daejeon, Republic of Korea (301-110)
(e-mail: kiholee@eulji.ac.kr).
Received for publication January 29, 2008; accepted for publication
August 27, 2008.
DOI: 10.2164/jandrol.107.004796
Journal of Andrology, Vol. 30, No. 1, January/February 2009Copyright E American Society of Andrology
57
Leydig cells, and require a number of steroidogenic
enzymes. In fact, the expression and presence of
steroidogenic enzymes in the domestic pig testis are welldocumented (Sasano et al, 1989; Hall, 1991; Clark et al,
1996; Conley et al, 1996; Conley and Bird, 1997; Moran
et al, 2002). A number of investigations have demon-
strated that the expressions and activities of steroido-
genic enzymes in pig testis are dependent on a variety of
extragonadal and intragonadal factors (Chuzel et al,
1996; Clark et al, 1996; Lejeune et al, 1998; Moran et al,
2002). Estrogens are synthesized from the aromatizationof androgens through the action of cytochrome P450
aromatase (CYP19). Differential expression of CYP19
has been found during different stages of pig develop-
ment. In fetal pig testis, CYP19 is present in Leydig cells
and/or gonocytes (Conley et al, 1996; Parma et al, 1999;
Haeussler et al, 2007), whereas the expression of CYP19
is exclusively limited to Leydig cells of immature and
mature pigs (Fraczek et al, 2001; Mutembei et al, 2005).During early neonatal development, an increase of
CYP19 activity has been detected between 1 and 7 days
after birth (Moran et al, 2002). However, the ontogeny
of CYP19 expression in the pig testis during early
neonatal development has not yet been determined, in
spite of the peak production of estrogen during the first
month after birth (Schwarzenberger et al, 1993).
Differential expressions of other steroidogenic enzymesin the pig testis during fetal and postnatal development
have also been reported (Conley et al, 1994; Moran et al,
2002; Haeussler et al, 2007). However, a detailed
examination of the expression of these steroidogenic
enzymes during early neonatal development is needed,
because of the significant production of steroid hor-
mones in pigs during the neonatal period (Schwarzen-
berger et al, 1993).
As noted above, nandrolone is a potent anabolicsteroid that is found at high levels in male pig serum,
particularly during early neonatal development and after
puberty (Schwarzenberger et al, 1993; Choi et al, 2007).
Endogenous production of nandrolone is also detected in
mares (Sterk et al, 1998) and some ruminants, including
goat, cow, and sheep (Mayer et al, 1992; De Brabander et
al, 1994; Sterk et al, 1998). The mechanism of nandrolone
synthesis in the pig testis has not been revealed in detail.Kao et al (2000) showed that the porcine CYP19 is
capable of converting testosterone into nandrolone via
demethylation. In addition, Corbin et al (1999) demon-
strated the catalytic activity of the porcine CYP19 on the
formation of nandrolone using testosterone as a sub-
strate. These findings imply that the presence of a high
serum level of nandrolone in the male pig during early
neonatal development would be associated with theexpression of CYP19, as well as other steroidogenic
enzymes, in the pig testis. Table
1.
Prim
er
sequences
and
expecte
dpro
duct
siz
es
of
ste
roid
ogenic
enzym
es
teste
dfo
rre
al-tim
eP
CR
Mole
cule
Forw
ard
Prim
er
Sequence,
59-
39a
Revers
eP
rim
er
Sequence,
59-
39a
Tem
pera
ture
,
uCE
xpecte
dP
roduct
Siz
e,
bp
GenB
ank
Access
ion
Num
ber
CY
P19
GT
CC
TG
GC
TA
TT
TT
CT
GG
GA
AT
TG
G(2
16–240)
TG
GA
AT
CG
GC
AC
AG
AC
GG
TC
AC
CA
T(5
48–572)
50
356
U37312
CY
P11A
1T
TT
AC
AG
GG
AG
AA
GC
TC
GG
CA
AC
(297–319)
TT
AC
CT
CC
GT
GT
TC
AG
GA
CC
AA
C(4
87–509)
53
213
X13768
HS
D17B
4T
GC
AG
AT
CG
TG
AT
GT
GT
TG
A(1
716–1735)
TT
CT
TC
AC
CA
TT
TC
TT
GC
CC
(1987–2006)
53
291
X78201
CB
R1
AC
CA
GC
TG
GA
CA
TC
AT
AG
AC
(286–305)
AG
AT
CC
TG
GA
CA
AC
AC
AG
AG
(710–729)
53
444
M80709
CY
P17A
CA
CT
GT
TG
CG
GA
CA
TC
TT
TG
(979–998)
CT
GA
TA
GA
TG
GG
GC
AC
GA
TT
(1112–1131)
50
152
M63507
ALR
2G
GC
AA
AA
GC
AA
CG
AA
GA
GA
C(8
75–894)
CT
GC
CA
TA
GT
CC
AG
TG
GG
TT
(1148–1167)
53
293
AF
202775
HS
D3B
TC
CA
CA
CC
AG
CA
GC
AT
AG
AG
(534–553)
AT
AC
AT
GG
GC
CT
CA
GA
GC
AC
(720–739)
53
206
NM
_001004049
PP
IAA
GC
AC
TG
GG
GA
GA
AA
GG
AT
T(1
22–141)
GC
CA
TC
CA
AC
CA
CT
CA
GT
CT
(357–375)
61
255
AY
266299
Abbre
via
tions:
ALR
2,
ald
ose
reducta
se;
CB
R1,
carb
onyl
reducta
se
1;
CY
P11A
1,
cyto
chro
me
P450
sid
e-c
hain
cle
avage;
CY
P17A
,17
a-h
ydro
xyla
se;
CY
P19,
cyto
chro
me
P450
aro
mata
se;
HS
D17B
4,
17b-h
ydro
xyste
roid
dehyd
rogenase
4;
HS
D3B
,3b-h
ydro
xyste
roid
dehyd
rogenase;
PP
IA,
cyclo
phili
n.
aN
um
bers
inpare
nth
eses
indic
ate
the
positio
ns
of
bases
inG
enB
ank.
58 Journal of Andrology N January �February 2009
Figure 1. Expression and immunolocalization of cytochrome P450 aromatase in pig testes. CYP19 mRNA (A) and protein (B) were detected inneonatal and prepubertal pig testes. Different letters indicate significant differences among groups (P , .05). M indicates 100-bp size marker;CYP19, cytochrome P450 aromatase; PPIA, cyclophilin, an internal control for real-time PCR analysis. (C) Immunohistochemical localization ofCYP19 in neonatal and prepubertal pig testes. At all ages, Leydig cells (L) were immunopositive for CYP19 protein, whereas the sex cords (SC)in the neonatal pig testes and seminiferous tubules (ST) at 4 months of age were immunonegative. Bars 5 100 mm. 0w indicates 0 weeks ofage; 1w, 1 week of age; 2w, 2 weeks of age; 3w, 3 weeks of age; and 4M, 4 months of age.
Choi et al N Steroidogenic Enzymes in Pig Testes 59
Figure 2. Expression and immunolocalization of cytochrome P450 side chain cleavage transcript and protein in pig testes. CYP11A1 mRNA (A)and protein (B) were detected in neonatal and prepubertal pig testes. Different letters indicate significant differences among groups (P , .05).M indicates 100-bp size marker; CYP11A1, cytochrome P450 side chain cleavage; PPIA, cyclophilin, an internal control for real-time PCR
60 Journal of Andrology N January �February 2009
Comprehensive evaluation of differential gene expres-
sion in the pig testis during postnatal development has not
been studied. Our recent, unpublished cDNA microarray
data have shown dramatic expressional changes of a
variety of molecules in pig testis between 2 weeks of age
and prepuberty. Based on preliminary data and other
investigations, we hypothesized that peak production of
steroid hormones in the male pig during early neonatal
development would relate to increases of gene expression
of steroidogenic enzymes in the pig testis. To test this
hypothesis, based on our cDNA microarray results, we
selected a total of 7 genes that are involved in the synthesis
and metabolism of steroid hormones in the pig testis.
These molecules were CYP19, cytochrome P450 side
chain cleavage (CYP11A1), 17b-hydroxysteroid dehydro-
genase 4 (HSD17B4), 3b-hydroxysteroid dehydrogenase
(HSD3B), carbonyl reductase 1 (CBR1), aldose reductase
(ALR2), and 17a-hydroxylase (CYP17A). In the present
study, we first attempted to evaluate the differential
expression of mRNA and protein by real-time polymerase
chain reaction (PCR) and Western blot analyses, respec-
tively. In addition, immunohistochemical analysis was
performed to localize the molecules in the pig testis at
different neonatal ages (0, 1, 2, and 3 weeks of age). We
also included the testis at 4 months of age for comparison
in the present study, because steroid hormones were
present at the basal level in circulating blood at this age
(Schwarzenberger et al, 1993).
Materials and Methods
Animals and Tissue Collection
Male reproductive tracts were obtained from boars (Sus scrofa
domestica; Yorkshire 6 Landrace 6 Duroc) during routine
castrations at the local animal farm and the National Institute
of Animal Science, Republic of Korea. Five experimental
groups were used at the following ages: 1) 0 weeks (within 3
days of age after birth: n 5 4), 2) 1 week (n 5 5), 3) 2 weeks (n
5 5), 4) 3 weeks (n 5 4), and 5) 4 months (n 5 3). One side of
the male reproductive tract from each pig was fixed in Bouin
fixative for the detection of steroidogenic enzymes in the testes
by immunohistochemistry. The other side of the male
reproductive tract was rapidly dissected in ice-cold phos-
phate-buffered saline (PBS), and the testes were separated
from the rest of the reproductive tract. The testes were quickly
frozen in liquid nitrogen and stored at 280uC until isolation of
total RNA or protein for real-time PCR or Western blotting,
respectively.
Total RNA and Protein Isolation
Total RNA was isolated according to the instructions provided
with TRIzol RNA extraction solution (Invitrogen, Carlsbad,
California). In brief, 50–100 mg of fresh testis tissue was
homogenized in 1 mL of extraction buffer using a Polytron
homogenizer (Fisher Scientific, Pittsburgh, Pennsylvania),
followed by chloroform and isopropanol total RNA precipi-
tation. The isolated RNA pellets were dissolved in RNA
storage buffer (Ambion, Austin, Texas) and stored at 280uCuntil used for the reverse transcription (RT) reaction. The
Table 2. Summary of immunohistochemical analyses of molecules tested
Molecule
Age
0 wk 1 wk 2 wk 3 wk 4 mo
L SC G L SC G L SC G L SC G L S G
CYP19 + 2 2 + 2 2 + 2 2 + 2 2 +/2 2 2
CYP11A1 + 2 2 + 2 2 + 2 2 + 2 2 + 2 +/2
HSD17B4a + 2 2 + 2 2 + 2 2 + 2 2 2 2 2
CBR1 + 2 2 + 2 2 + 2 2 + 2 2 + 2 2
CYP17A + 2 2 + 2 2 + 2 2 + 2 2 + 2 2
ALR2 + + 2 + + 2 + + 2 + + 2 +/2 + 2
HSD3Ba +/2 + 2 + + 2 + + 2 +/2 + 2 +/2 +/2b 2
Abbreviations: +, positive; +/2, weakly positive; 2, negative. ALR2, aldose reductase; CBR1, carbonyl reductase 1; CYP11A1, cytochrome
P450 side-chain cleavage; CYP17A, 17a-hydroxylase; CYP19, cytochrome P450 aromatase; G, germ cell; HSD17B4, 17b-hydroxysteroid
dehydrogenase 4; HSD3B, 3b-hydroxysteroid dehydrogenase; L, Leydig cell; S, Sertoli cell; SC, sex cord.a Positive immunoreaction on blood-testis barrier at 4 months of age.b Not all cells immunopositive.
r
analysis. (C) Immunohistochemical localization of CYP11A1 in neonatal and prepubertal pig testes. Leydig cells (L) in the neonatal pig testeswere immunopositive for CYP11A1 protein, whereas the sex cords (SC) were immunonegative. At 4 months of age, spermatids (Sp) inseminiferous tubules (ST) became weakly immunopositive, whereas Leydig cells were still strongly immunopositive. Bars 5 100 mm. Bar in 4M(E) 5 20 mm. 0w indicates 0 weeks of age; 1w, 1 week of age; 2w, 2 weeks of age; 3w, 3 weeks of age; 4M, 4 months of age; 4M (E), enlargedpicture of the testis at 4 months of age.
Choi et al N Steroidogenic Enzymes in Pig Testes 61
Figure 3. Expression and immunolocalization of carbonyl reductase 1 in pig testes. CBR1 mRNA (A) and protein (B) were detected in neonataland prepubertal pig testes. Different letters indicate significant differences among groups (P , .05). M indicates 100-bp size marker; PPIA,cyclophilin, an internal control for real-time PCR analysis. (C) Immunohistochemical localization of CBR1 in neonatal and prepubertal pig testes.
62 Journal of Andrology N January �February 2009
purity and yield of the total RNA were determined by an
ultraviolet (UV) spectrophotometer (Eppendorf, New York,
New York), and the qualities of the total RNAs were checked
by gel electrophoresis prior to the RT reaction.
The protein from the testes was prepared in ProPrep protein
extraction solution (iNtRON Biotech, Sungnam, Republic of
Korea). The 10–20 mg of testicular tissue was homogenized in
600 mL of lysis buffer using a Polytron homogenizer (Fisher
Scientific), followed by incubation at 220uC for 20–30 minutes
and centrifugation at 16 6096 g (4uC) for 10 minutes. The total
protein concentration of the supernatant was determined by the
Bradford method (BioRad, Hercules, California), with bovine
serum albumin (BSA) as standard. Isolated protein was kept at
280uC until used for Western blot analysis.
RT and Real-time PCR
RT was carried out according to the instructions in the ImProm-
II RT system (Promega, Madison, Wisconsin). Briefly, 1 mg of
total RNA was reverse-transcribed in a total volume of 20 mL
using oligo-dT primer. The RT reaction was performed at 25uCfor 5 min, 42uC for 1 hour, and 70uC for 15 minutes. One
microliter of cDNA was used as a template for real-time PCR in
a 25 mL reaction mixture, including 0.75 U of GoTaq DNA
polymerase (Promega), 5 mL of 56 buffer, 0.2 mM of deoxyri-
bonucleotide triphosphate (Promega), 2.5 mL of 30006 SYBR
Green I (BMA, Rockland, Maine), and 10 pmol of each primer.
Oligonucleotide primers for real-time PCR were prepared either
by using Primer 3 software (Whitehead Institute/MIT Center
for Genomes Research, Cambridge, Massachusetts; http://
www.bioneer.co.kr/cgi-bin/primer/primer3.cgi) or utilizing pub-
lished information. Information and sequences of primers of
steroidogenic enzymes tested in the present study are summa-
rized in Table 1. The PCR program employed an initial step of
95uC for 5 minutes for predenaturation, followed by denatur-
ation at 94uC, annealing, and extension at 72uC using the PTC-
200 Chromo 4 real-time system (Bio-Rad Laboratories). The
final extension was carried out for 10 minutes at 72uC. No
RNA, no cDNA template, and no primer controls were
included for PCR control purposes. The PCR products were
visualized on 1.2% agarose gel and photo-captured under UV
light using an image documentation system (Vilber Loumat,
Marne-la-Vallee, France). Cyclophilin (PPIA) was included as
an internal PCR control. For quantification of real-time PCR
results, the relative standard curve method was used to obtain
quantitative values. Each sample was replicated 3 or 4 times,
and the normalized mean value to PPIA was used for final
comparison.
Immunohistochemistry
The male reproductive tract was fixed in Bouin fixative for 18–
24 hours. The testes were separated from other parts of the
reproductive tract. The testes were dehydrated in a serial of
ethanol, cleared in xylene, and infiltrated with paraffin.
Paraffin-embedded testes were sectioned at thicknesses of 4–
5 mm. For immunohistochemistry, tissue sections were depar-
affinized in xylene and rehydrated in a series of ethanol. After
microwaving in 0.01 M citrate buffer, pH 6.0, for 10 minutes
for antigen retrieval, tissue sections were placed in 0.3% H2O2/
methanol for 15 minutes to inactivate endogenous peroxidase.
After washing in PBS, tissue sections were incubated in 10%
normal goat (Chemicon International, Temecula, California) or
rabbit serum (Jackson ImmunoResearch Laboratories Inc,
West Grove, Pennsylvania), for 30 minutes at room tempera-
ture to block nonspecific binding. Diluted primary antibodies
were applied on the tissue sections and incubated in a
humidified chamber at 4uC overnight. The dilutions of the
primary antibodies were selected after a series of multiple
preliminary trials for each antibody. We used dilutions of 1:1000
for CYP19 (polyclonal rabbit anti-CYP19; a generous gift from
Dr Nobuhiro Harada, Fujita Health University, Japan), 1:400
for CYP11A1 (AB1244; Chemicon), 1:2000 for HSD17B4
(monoclonal mouse anti-HSD17B4; a kind gift from Dr
Gabriele Moller, GSF-Research Center for Environment and
Health, Neuherberg, Germany), 1:500 for CBR1 (ab4148;
Abcam Ltd, Cambridge, United Kingdom), 1:500 for CYP17A
(polyclonal rabbit anti-CYP17A; a generous gift from Dr Anita
Payne, Stanford University, Stanford, California), 1:200 for
ALR2 (polyclonal rabbit anti-ALR2; a gracious gift from Dr
Motoko Takahashi, Saga University, Saga, Japan), and 1:500
for HSD3B (polyclonal rabbit anti-HSD3B; a benevolent gift
from Dr Ian Mason, University of Edinburgh, Edinburgh,
United Kingdom). Excess primary antibodies were washed off
the tissue sections using PBS. Tissue sections were then
incubated with biotinylated goat anti-rabbit IgG (DAKO
Corporation, Carpinteria, California) for CYP19, CYP11A1,
CYP17A, ALR2, and HSD3B, biotinylated goat anti-mouse
IgG (DAKO) for HSD17B4, or biotinylated rabbit anti-goat
IgG secondary antibody (DAKO) for CRB1 in a humidified
chamber at room temperature for 1 hour. Unbound secondary
antibodies were washed off with PBS, and elite avidin-biotin
peroxidase (Vector Laboratories, Burlingame, California) was
placed on slides in a humidified chamber at room temperature
for 30 minutes. After three 5-minute washes in PBS, the tissue
sections were treated with a mixture of 3,39-diaminobenzidine
(Sigma, St Louis, Missouri), 0.05 M Tris-HCl buffer, and 5%
hydrogen peroxide to detect the peroxidase. The tissue sections
were then counterstained with hematoxylin, followed by
dehydration in ethanol and mounting. For negative controls,
tissue sections were treated with normal rabbit, mouse
(Chemicon), or goat serum at the same dilutions in the place
of primary antibodies. Immunostaining was evaluated with
digitalized images captured with an Olympus-CoolSNAP cf
color/OL camera (Olympus America, Melville, New York)
using RSImage version 1.1 software (Roper Scientific, Duluth,
r
At all ages, Leydig cells (L) were strongly immunopositive for CBR1 protein, whereas the sex cords (SC) in the neonatal pig testes andseminiferous tubules (ST) at 4 months of age were devoid of immunoreactivity for CBR1. Bars 5100 mm. 0w indicates 0 weeks of age; 1w,1 week of age; 2w, 2 weeks of age; 3w, 3 weeks of age; and 4M, 4 months of age.
Choi et al N Steroidogenic Enzymes in Pig Testes 63
Figure 4. Expression and immunolocalization of 17b-hydroxysteroid dehydrogenase 4 in pig testes. HSD17B4 mRNA (A) and protein (B) weredetected in neonatal and prepubertal pig testes. Different letters indicate significant differences among groups (P , .05). M indicates 100-bpsize marker; PPIA, cyclophilin, an internal control for real-time PCR analysis. (C) Immunohistochemical localization of HSD17B4 in neonatal
64 Journal of Andrology N January �February 2009
Georgia). The photographic images were processed in Photo-
Shop software (Adobe Systems, San Jose, California).
Western Blotting Analysis
Forty micrograms of protein were fractionated on 12% SDS-
PAGE polyacrylamide gel (Invitrogen) and electrotransferred
to a nitrocellulose membrane. After rinsing in Tris-buffered
saline with Tween (TBST; 0.2M Tris, 1.37M NaCl, 0.05%
Tween-20), nonspecific binding was blocked by incubation of
the membrane in TBST with 1% BSA (Sigma) for 1 hour at
room temperature. Blotting membranes were incubated with
primary antibodies diluted in TBST at 4uC overnight. The
same antibodies used for immunohistochemistry were em-
ployed, but at different dilutions: 1:2500 for CYP19, 1:5000 for
CYP11A1, 1:10 000 for HSD17B4, 1:10 000 for CBR1, 1:5000
for CYP17A, 1:5000 for ALR2, and 1:5000 for HSD3B. After
washing in TBST, blotting membranes were incubated with a
goat anti-rabbit or anti-mouse HRP-conjugated IgG or rabbit
anti-goat HRP-conjugated IgG antibody (Santa Cruz Bio-
technology, Inc, Santa Cruz, California) diluted at 1:2000 in
TBST at room temperature for 1 hour. The membranes were
then washed 5 times with TBST, and blotting results were
detected with the enhanced chemiluminescence detection
system (Amersham Biosciences, Pittsburgh, Pennsylvania). b-
actin (SC-47778; Santa Cruz Biotechnology) served as an
internal control for Western blot analysis. Blotting results were
analyzed using image analysis software, Image J, released from
the National Institutes of Health (Bethesda, Maryland; http://
rsb.info.nih.gov/ij/download.html). Each sample was analyzed
3 times, and a mean value that was normalized to b-actin was
used in the final comparison.
Data Presentation and Statistical Analysis
Data for mRNA and protein abundance were expressed
relative to 0 weeks of age as arbitrary units. In the figures,
data are presented as mean 6 SD. A lack of bars indicates an
insignificant SD. Comparison of mean differences among
neonatal and prepubertal ages were made using 1-way analysis
of variance, followed by Tukey’s test, using SPSS software
(SPSS Inc, Chicago, Illinois). In all cases, results were
considered significant if P , .05.
Results
Expression and Immunohistochemical Localization ofCYP19 Transcript and Protein
The presence and expression of CYP19 mRNA andprotein were detected in neonatal and prepubertal pig
testes (Figure 1). The level of CYP19 mRNA expression
was not significantly different between 0 and 1 weeks of
age (Figure 1A). However, a significant increase in the
CYP19 mRNA level was observed at 2 weeks of age
(Figure 1A). The abundance of CYP19 transcript at
3 weeks of age was similar to that seen at 0 and 1 weeks
of age (Figure 1A). A significant decrease in the CYP19
mRNA level was detected at 4 months of age, when the
level was approximately 20-fold lower than the abun-
dance of CYP19 mRNA at 0 weeks of age (Figure 1A).A similar expression pattern was found for the protein
level (Figure 1B). A significant increase of the CYP19
protein level was observed at 2 weeks of age (Fig-
ure 1B). Compared with the level at 0 weeks of age, the
levels of CYP19 (<50 kDa) at 1 week and 3 weeks of
age were not significantly changed (Figure 1B). How-
ever, the abundance of CYP19 was significantly lower at
4 months of age than at 0 weeks of age (Figure 1B).
Immunohistochemical analysis showed an exclusivelocalization of CYP19 in Leydig cells of the testis,
regardless of the postnatal ages (Figure 1C; Table 2).
Sex cords (SCs) in the neonatal testis and seminiferous
tubules (STs) in the prepubertal testis were devoid of
CYP19 staining (Figure 1C). Strong immunopositive
staining of CYP19 in Leydig cells was found at all
neonatal ages (Figure 1C). However, the immunoreac-
tivity of CYP19 was visibly reduced in Leydig cells at
4 months of age (Figure 1C; Table 2).
Differential Expression and Immunolocalization ofCYP11A1 mRNA and Protein
The expression level of CYP11A1 mRNA was signifi-
cantly increased at 1 week of age, compared with the
expression level at 0 weeks of age (Figure 2A). The
abundance of CYP11A1 mRNA remained significantly
high at 2 and 3 weeks of age (Figure 2A), but theexpression of CYP11A1 mRNA was significantly re-
duced at 4 months of age (Figure 2A). In contrast to the
mRNA expression pattern, the highest level of CYP11A1
protein (<52 kDa) was found at 0 weeks of age, followed
by significantly decreased levels of CYP11A1 at 1, 2, and
3 weeks of age (Figure 2B). At 4 months of age, the testes
possessed the lowest level of CYP11A1 (Figure 2B).
Restricted immunoreactivity of CYP11A1 was found in
Leydig cells (Figure 2C; Table 2). No positive immuno-staining of CYP11A1 was observed in SCs during the
neonatal period (Figure 2C). However, at 4 months of
age, CYP11A1 was immunolocalized in some germ cells,
r
and prepubertal pig testes. Leydig cells (L) in the neonatal pig testes were immunopositive for HSD17B4 protein, whereas the sex cords (SC)were immunonegative. At 4 months of age, strong immunoreactivity of HSD17B4 was found on the blood-testis barrier (BTB, blue arrows),whereas Leydig cells and cells in seminiferous tubules (ST) were immunonegative for HSD17B4 protein. Bars 5 100 mm. Bar in 4M (E) 520 mM. 0w indicates 0 weeks of age; 1w, 1 week of age; 2w, 2 weeks of age; 3w, 3 weeks of age; 4M, 4 months of age; and 4M (E), enlargedpicture of the testis at 4 months of age.
Choi et al N Steroidogenic Enzymes in Pig Testes 65
Figure 5. Expression and immunolocalization of 17a-hydroxylase in pig testes. CYP17A mRNA (A) and protein (B) were detected in neonataland prepubertal pig testes. Different letters indicate significant differences among groups (P , .05). M indicates 100-bp size marker. CYP17A,17a-hydroxylase. PPIA, cyclophilin, an internal control for real-time PCR analysis. (C) Immunohistochemical localization of CYP17A in neonatal
66 Journal of Andrology N January �February 2009
including secondary spermatocytes and round sperma-
tids, as well as Leydig cells (Figure 2C; Table 2).
Immunohistochemical Localization and Expression ofCBR1 mRNA and Protein
The abundance of CBR1 mRNA increased with age
during the neonatal period (Figure 3A). The highest
expression of CBR1 mRNA was detected at 3 weeks of
age, and the testes expressed the lowest level of CBR1
mRNA at 4 months of age (Figure 3A). Western blot
analysis also showed significant increases of CBR1 protein(<30 kDa) at 2 and 3 weeks of age (Figure 3B). As in
CBR1 mRNA, the expression of CBR1 protein in the testis
was significantly reduced at 4 months of age (Figure 3B).
Regardless of age, the strong immunoreactivity of CBR1
was exclusively localized in Leydig cells, but not in SCs or
Sertoli or germ cells in STs (Figure 3C; Table 2).
Expression and Immunohistochemical Localization ofHSD17B4 Transcript and Protein
The expression and immunohistochemical localization of
HSD17B4 mRNA and protein are shown in Figure 4. The
level of HSD17B4 mRNA was increased with neonatal age,
followed by a significant decrease at 4 months of age
(Figure 4A). The highest mRNA expression of HSD17B4
was found at 3 weeks of age (Figure 4A). Western blot
analysis showed a single band of HSD17B4 protein(<32 kDa) in the testis (Figure 4B). Interestingly, the
highest level of HSD17B4 protein was found at 0 weeks of
age, followed by a significant decrease at 1 week of age
(Figure 4B). However, the amounts of HSD17B4 protein
at 2 and 3 weeks of age were not significantly different from
the level at 0 weeks of age (Figure 4B). As seen in mRNA
expression, the lowest expression of HSD17B4 protein was
found at 4 months of age (Figure 4B). During the neonatalperiod, HSD17B4 expression was localized in Leydig cells,
as determined by immunohistochemistry (Figure 4C;
Table 2). However, at 4 months of age, the Leydig cells
were devoid of HSD17B4 (Figure 4C), and the blood-testis
barrier (BTB) along the Sertoli cells was strongly immu-
nostained for HSD17B4 (Figure 4C; Table 2). Neither
Sertoli cells nor germ cells were immunopositive for
HSD17B4 at 4 months of age (Figure 4C; Table 2).
Expression and Localization of CYP17A Transcriptand Protein
The expression of CYP17A mRNA was significantly
increased at 1, 2, and 3 weeks of age, compared with
that at 0 weeks of age (Figure 5A). A significant
decrease in the CYP17A mRNA level was detected in
the testis at 4 months of age (Figure 5A). The expres-
sion pattern of CYP17A protein (<50 kDa) during the
neonatal period differed from that of mRNA expression
(Figure 5B). A significant reduction of the CYP17A
protein level was found at 2 weeks of age, whereas the
levels of CYP17A protein at 1 and 3 weeks of age were
not significantly different from the CYP17A protein
level at 0 weeks of age (Figure 5B). A significant
decrease in the CYP17A protein level was also detected
at 4 months of age (Figure 5B). Immunohistochemical
analysis showed strong immunoreactivity of CYP17A in
Leydig cells of the testis at all ages (Figure 5C; Table 2).
The Sertoli cells and germ cells were immunonegative
for CYP17A (Figure 5C; Table 2).
Changes of Expression and Localization of HSD3BmRNA and Protein
The expression of HSD3B mRNA was significantly
increased at 1 week of age (Figure 6A). The abundance
of HSD3B mRNA at 2 and 3 weeks of age was
significantly decreased compared with that at 1 week of
age (Figure 6A). A significant decrease of HSD3B
mRNA expression was seen at 4 months of age
(Figure 6A). Western blot analysis showed that the level
of HSD3B protein (<45 kDa) was the highest at 0 weeks
of age (Figure 6B). A significant decrease of HSD3B level
was found at 1 week of age, followed by further
significant decrease at 3 weeks of age (Figure 6B). The
lowest level of HSD3B protein in the boar testis was
detected at 4 months of age (Figure 6B). Immunohisto-
chemistry revealed the localization of HSD3B in Leydig
cells and SCs at neonatal ages (Figure 6C; Table 2).
Strong immunoreactivity of HSD3B was detected in SCs
at all neonatal ages, whereas the intensity of the positive
reaction of HSD3B in Leydig cells varied to some extent
with age (Figure 6C; Table 2). At 4 months of age, the
immunoreactivity of HSD3B became visibly weaker and
was found in Leydig cells and Sertoli cells, as well as the
BTB (Figure 6C; Table 2).
Expression and Localization of ALR2 mRNA and Proteinin the Pig Testis
The expressions of ALR2 mRNA and protein are shown
in Figure 7A and 7B, respectively. Compared with 0
weeks of age, significant increases of ALR2 mRNA
expression were noticed at 1 and 2 weeks of age, whereas
r
and prepubertal pig testes. At all ages, Leydig cells (L) were strongly immunopositive for CYP17A protein, whereas the sex cords (SC) in theneonatal pig testes and seminiferous tubules (ST) at 4 months of age were devoid of immunoreactivity for CYP17A. Bars 5 100 mm. 0windicates 0 weeks of age; 1w, 1 week of age; 2w, 2 weeks of age; 3w, 3 weeks of age; and 4M, 4 months of age.
Choi et al N Steroidogenic Enzymes in Pig Testes 67
Figure 6. Expression and immunolocalization of 3b-hydroxysteroid dehydrogenase in pig testes. HSD3B mRNA (A) and protein (B) weredetected in neonatal and prepubertal pig testes. Different letters indicate significant differences among groups (P , .05). M indicates 100-bpsize marker; PPIA, cyclophilin, an internal control for real-time PCR analysis. (C) Immunohistochemical localization of HSD3B in neonatal and
68 Journal of Andrology N January �February 2009
no change of the ALR2 mRNA level was found at
3 weeks of age (Figure 7A). A significant decrease of
ALR2 mRNA abundance was detected at 4 months of
age (Figure 7A). Western blot analysis revealed that
there was no significant change of the ALR2 protein
(<36 kDa) level until 2 weeks of age (Figure 7B). A
significant decrease of the protein level was found at
3 weeks of age, and the lowest level of ALR2 protein
was observed at 4 months of age (Figure 7B). Immu-
nohistochemical analysis showed a strong positive
reaction of ALR2 in Leydig cells and SCs at all neonatal
ages (Figure 7C; Table 2). At 4 months of age, the
immunoreactivity of ALR2 in Leydig cells was visibly
reduced, whereas a strong immunostaining of ALR2
was found in Sertoli cells (Figure 7C; Table 2).
Discussion
This study examined the expression and localization of
the enzymes involved in steroidogenesis and metabolism
of steroid hormone in the early neonatal and prepuber-
tal pig testes. Quantitative real-time PCR and Western
blotting analyses were used to determine the expressions
of the mRNA and proteins of enzymes, respectively. In
addition, the localization of these molecules in the pig
testes was evaluated by immunohistochemistry. Criteria
used to select the molecules tested in the present study
were based on our unpublished DNA microarray
analysis, which showed differential expression of pig
testicular genes between 2 weeks of age and prepuberty.
To our knowledge, this is the first time that the
expression and localization patterns of a number of
enzymes related to the synthesis and metabolism of
steroid hormones in the pig testis have been investigated
during early neonatal development. Significant increases
of the mRNA levels of all of the molecules examined
were clearly observed between 1 and 3 weeks of age.Interestingly, except in the cases of CYP19 and CBR1,
changes of protein abundance during early neonatal
development were not consistent with the patterns of
mRNA expression. However, the expression pattern of
mRNA in the prepubertal pig testis at 4 months of age
was similar to the protein expression pattern. Thus,
these results suggest the existence of posttranscriptional
regulatory mechanisms on the expression of steroido-
genic enzymes in the pig testis during early neonatal
development. Results of immunohistochemical analysis
are summarized in Table 2. Immunohistochemistry
revealed the presence of steroidogenic enzymes in
Leydig cells, and Sertoli cells in the cases of ALR2
and HSD3B, during neonatal development. However, as
seen in CYP11A1 and HSD17B4, differential localiza-
tion of molecular expression was found in the prepu-
bertal pig testis, indicating a change and/or addition to
the functional roles of these molecules in the pig testis
during postnatal development.
The synthesis and metabolism of steroid hormones
require a variety of steroidogenic enzymes. The boars
have extraordinarily high plasma and testicular levels of
estrogens, compared with the males of other species and
females of the same species (Claus and Hoffman, 1980;
Setchell et al, 1983; Schwarzenberger et al, 1993).
During postnatal development, the first peak of
estrogen concentration occurs during the first month
after birth, followed by transient decreases until the
second peak, which occurs after puberty (Christenson et
al, 1984; Schwarzenberger et al, 1993). The production
of estrogens is catalyzed by the action of CYP19, which
results in irreversible conversion of androgens to
estrogens (Carreau and Levallet, 1997). Thus, a surge
of estrogen production in the pig testis during early
neonatal development would result in an increase of
CYP19 expression. Indeed, the present study showed
marked increases of CYP19 mRNA and protein levels in
the pig testis at 2 weeks of age, in parallel with our
previous finding (Choi et al, 2007). A slight but
significant increase of the CYP19 protein level was
observed at 1 and 3 weeks of age. In addition, the
present study demonstrated the exclusive localization of
CYP19 in Leydig cells, in agreement with the findings of
other investigators (Conley et al, 1996; Mutembei et al,
2005; Haeussler et al, 2007). In spite of dramatic
increases of plasma estrogen concentrations in the first
few weeks (Schwarzenberger et al, 1993), increases of
CYP19 mRNA and protein levels were unexpectedly
low, 1.7 times or less. A similar finding for CYP19
activity was found in the neonatal pig testes (Moran et
al, 2002). However, because the interstitial volume,
density, and cytoplasmic volume of Leydig cell in pig
testis increase greatly between birth and 1 month of age
(van Straaten and Wensing, 1978; Franca et al, 2000), it
is reasonable to consider that overall CYP19 level and
activity in the pig testes during early neonatal develop-
ment would be greater than observed in the findings
from the present study, as well as previous studies
r
prepubertal pig testes. Leydig cells (L) and sex cords (SC) in the neonatal pig testes were immunopositive for HSD3B protein. At 4 months ofage, positive immunoreactivity of HSD3B was found on the blood-testis barrier (BTB, black arrows) and Sertoli cells (S, blue arrows) inseminiferous tubules (ST), as well as Leydig cells. Bars 5 100 mm. Bar in 4M (E) 5 20 mM. 0w indicates 0 weeks of age; 1w, 1 week of age;2w, 2 weeks of age; 3w, 3 weeks of age; 4M, 4 months of age; and 4M (E), enlarged picture of the testis at 4 months of age.
Choi et al N Steroidogenic Enzymes in Pig Testes 69
Figure 7. Expression and immunolocalization of aldose reductase in pig testes. ALR2 mRNA (A) and protein (B) were detected in neonatal andprepubertal pig testes. Different letters indicate significant differences among groups (P , .05). M indicates 100-bp size marker; ALR2, aldosereductase; PPIA, cyclophilin, an internal control for real-time PCR analysis. (C) Immunohistochemical localization of aldose reductase in
70 Journal of Andrology N January �February 2009
(Moran et al, 2002). Thus, it is speculated that such
increase of CYP19 activity during the first 2 weeks after
the birth would strongly correlate with a significant
secretion of nandrolone from the neonatal pig testis.
Interconversion of 17-ketosteroids with the corre-
sponding 17b-hydroxysteroids requires the action of
HSD17B. Of a number of HSD17B isoforms, HSD17B4
is responsible for the inactivation of estradiol and
androstene-3b, 17b-diol into estrone and dehydroepian-
drosterone, respectively (Labrie et al, 1997). In the pig
testis, HSD17B4 is localized in Leydig cells and
predominantly directs the oxidation of estradiol to
estrone (De Launoit and Adamski, 1999). High plasma
estrone concentrations have been measured in boars
during neonatal development (Claus and Hoffmann,
1980; Ford, 1983). In the present study, the lowest level
of HSD17B4 mRNA in the pig testis during the early
neonatal period was detected at 0 weeks of age, whereas
the highest level of HSD17B4 protein was found at the
same age. These data indicate the existence of posttran-
scriptional regulation of HSD17B4 expression. Even
though the present study showed lower levels of
HSD17B4 protein between 1 and 3 weeks of age
compared with that at 0 weeks of age, it is speculated
that overall HSD17B4 protein levels would remarkably
increase because of apparent increases of Leydig cell
volume and density during the neonatal period (van
Straaten and Wensing, 1978; Franca et al, 2000). An
unexpected finding was the alternation in HSD17B4
localization in the pig testis at 4 months of age. A strong
immunoreactivity of HSD17B4 was localized on the
BTB at 4 months of age, whereas only Leydig cells were
immunopositive for HSD17B4 during early neonatal
development. The role of HSD17B4 on the BTB is not
currently known. Booth (1983) showed the stimulatory
effect of estrone on the development of male character-
istics in the boar. Entry of steroid hormones such as
testosterone and dehydroepiandrosterone into rete testis
fluid through the BTB has been demonstrated in rats
(Cooper and Waites, 1975). Rete testis fluid in the boar
testis contains a significant concentration of estrogens
(Setchell et al, 1983). Thus, it is presumed that
HSD17B4 on the BTB would play a role in the
accumulation of estrone in rete testis fluid through the
active conversion of estradiol synthesized from Leydig
cells. Another possible role of HSD17B4 on the BTB
would be a stimulatory effect on spermatogenesis and/or
Sertoli cell proliferation by estrone. Additional investi-
gation should be conducted to resolve the role of
HSD17B4 on BTB in the pig testis.
The synthesis of androgens from a cholesterol
precursor requires a number of steroidogenic enzymes,
including CYP11A1, CYP17A, and HSD3B. The
CYP11A1 is the rate-limiting enzyme for steroidogen-
esis and converts cholesterol into pregnenolone, which
is then metabolized into progesterone by the action of
HSD3B. The CYP17A is a pivotal enzyme that
converts pregnenolone or progesterone to 17-hydro-
xypregnenolone or 17-hydroxyprogesterone, respective-
ly. These 2 intermediates serve as precursors for
androstenedione that is further converted into testos-
terone by the action of HSD17B. The expression and
localization of these 3 enzymes in the pig testis have
been demonstrated from the findings of other studies
(Suzuki et al, 1992; Clark et al, 1996; Moran et al, 2002;
Weng et al, 2005). Androstenone, dehydroepiandros-
terone, and testosterone are types of androgens that are
found in pig plasma at relatively high levels (Sinclair et
al, 2001). The initial peaks in plasma androgen
concentrations are seen within the first month after
birth during postnatal development (Sinclair et al,
2001), which implies a requirement for marked
increases of gene expression for CYP11A1, CYP17A,
and HSD3B. In fact, the present study showed
significant increases of mRNA levels of these enzymes
between 1 and 3 weeks of age. However, protein levels
during early neonatal development were lower or
equivalent to those at 0 weeks of age. The discordance
between the mRNA and protein expressions of these
enzymes implies the existence of posttranscriptional
modulation on gene expression during early neonatal
development. In addition, we could not rule out the
possibility of posttranslational regulation, leading to
the enhancement of enzyme activities during neonatal
development. Immunohistochemical analysis revealed
the primary localization of CYP11A1, CYP17A, and
HSD3B in Leydig cells, regardless of age. Interestingly,
we also found a positive immunoreaction of CYP11A1
in germ cells of the prepubertal pig testis. Moreover,
positive immunoreactivity of HSD3B was found not
only in Leydig cells, but also in Sertoli cells in the
neonatal testis and Sertoli cells and BTB in the
prepubertal testis. Similar observations were made for
CYP11A1 in the bear testis (Tsubota et al, 1993) and
r
neonatal and prepubertal pig testes. Leydig cells (L) and sex cords (SC) in the neonatal pig testes were immunopositive for ALR2 protein. At4 months of age, positive immunoreactivity of ALR2 was found in Sertoli cells (S, blue arrows) in seminiferous tubules (ST), as well as Leydigcells. Bars 5 100 mm. Bar in 4M (E) 5 20 mM. 0w indicates 0 weeks of age; 1w, 1 week of age; 2w, 2 weeks of age; 3w, 3 weeks of age; 4M,4 months of age; and 4M (E), enlarged picture of the testis at 4 months of age.
Choi et al N Steroidogenic Enzymes in Pig Testes 71
for HSD3B in the monkey testis (Liang et al, 1999).
Such differential testicular expression would indicate
distinguishable roles of steroidogenic enzymes in thepig testes during postnatal development. To our
knowledge, the present study is the first report to
demonstrate the differential localization of CYP11A1
and HSD3B in the domestic pig testis. Further
examinations are needed to determine the functional
roles of steroidogenic enzymes in different cell types of
the pig testis.
In the present study, we examined the expression
and presence of 2 metabolic enzymes, ALR2 andCBR1. ALR2 is a member of the aldo-keto reductase
superfamily, whereas CBR1 is a member of the short-
chain dehydrogenase/reductase superfamily (Hoff-
mann and Maser, 2007). Both of these enzymes share
a common characteristic: NADPH-dependent reduc-
tion. Porcine testicular CBR catalyzes the reduction of
ketones on androgens and progesterone (Tanaka et al,
1992). The CBR1 is expressed and localized only inLeydig cells of the neonatal pig testis (Kobayashi et al,
2002), and this is in agreement with our present
finding. The expression of CBR1 mRNA and protein
during early neonatal development increases according
to age, and shows a transient decrease at 4 months of
age. Similar findings on CBR1 mRNA expression and
activity in the neonatal pig testes have been demon-
strated in previous studies (Ohno et al, 1992; Tanaka etal, 1992). It is believed that CBR1 is responsible for the
conversion of 17a-hydroxyprogesterone to 17a,20b-
dihydroxy-4-pregnen-3-one, which is present in the
neonatal pig testis (Ghosh et al, 2001). The CBR has 2
distinct activities, 20b-HSD (Tanaka et al, 1992) and
3a- and 3b-HSDs (Ohno et al, 1992), thus implying a
diverse role in the metabolism of steroid hormones.
Thus, it is speculated that multifunctional actions ofCBR1 in the pig testis would play an important role in
metabolic reactions of steroid hormones synthesized in
Leydig cells, eventually leading to adequate testicular
function during early neonatal development. The
expression and localization of ALR2 in the domestic
boar testis have not yet been determined. It has been
demonstrated that progesterone is a substrate for the
reducing activity of ALR2 with 20a-HSD activity(Warren et al, 1993). The present study demonstrates
immunolocalization of ALR2 in Leydig and Sertoli
cells of the pig testes. In the rat testis, ALR2 is
exclusively present in Sertoli and spermatogenic cells
(Kobayashi et al, 2002), suggesting species-specific
cellular expression of ALR2 in the testis. The
functional role of ALR2 in the pig testis is not
understood at this point. However, significant increas-es of mRNA and protein levels during early neonatal
development indicate that ALR2 would be involved in
the metabolism of steroid hormones in pig testes
following exposure to high concentrations of steroid
hormones. In fact, Kobayashi et al (2002) suggested a
potential role of ALR2 on the reduction of steroid
hormones in the rat testis. Detailed information for arole of ALR2 in the pig testis should be addressed in
future studies.
A number of investigations have shown a correlation
between the remarkable increase of the Leydig cell
number and size and steroidogenic activity in the pig
testis during the first month after birth (Franca et al,
2000; Herrera et al, 1983; Schwarzenberger et al, 1993;
van Straaten and Wensing, 1978). In addition, van
Straaten and Wensing (1977) reported that a marked
increase of the volume percentage of the Leydig cells inthe pig testis reaches the highest value at 3 weeks of
age after the birth. These findings imply that a
proportional increase of the Leydig cells relative to
the testicular interstitium and STs would contribute to
enhanced expression of steroidogenic enzymes in the
pig testis during the early neonatal period. In pigs, the
total body weight shows an almost 10-fold increase,
with maximal growth in the skeletal muscle, during thefirst month of birth (Sarkar et al, 1977). As stated
earlier, nandrolone, an androgen having 10 times
higher anabolic activity than testosterone, is found at
high concentrations in pigs during early neonatal
development (Schwarzenberger et al, 1993). Thus, it
is believed that anabolic steroid hormones synthesized
from the pig testis may play a role in early postnatal
development of pigs. In conclusion, the present studydemonstrates that differential gene and protein expres-
sions of various steroidogenic and steroid metabolism–
related enzymes in the neonatal pig testes would
contribute to the significant increases of plasma and
testicular steroid hormone concentrations during early
neonatal development, eventually leading to overall
growth of the pig.
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