Ontogeny of expression and localization of steroidogenic enzymes in the neonatal and prepubertal pig testes

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: [email protected]).

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


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.


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.


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.


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


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.


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


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.


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


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.


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


(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.


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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Ontogeny of expression and localization of steroidogenic enzymes in the neonatal and prepubertal pig testes

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