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Endocrinology 145(3):1227–1237
Copyright © 2004 by The Endocrine Society
doi: 10.1210/en.2003-1475
Di(n-Butyl) Phthalate Impairs Cholesterol Transport and
Steroidogenesis in the Fetal Rat Testis through a Rapid
and Reversible Mechanism
CHRISTOPHER J. THOMPSON, SUSAN M. ROSS,
AND
KEVIN W. GAIDO
CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709-2137
In utero exposure to di(n-butyl) phthalate (DBP) leads to a
variety of male reproductive abnormalities similar to those
caused by androgen receptor antagonists. DBP demonstrates
no affinity for the androgen receptor, but rather leads to diminished testosterone production by the fetal testis. The purpose of this study was to determine the onset and reversibility
of DBP effects on the fetal testis and to determine at a functional level the points in the cholesterol transport and steroidogenesis pathways affected by DBP. Starting at gestational
day (gd) 12, pregnant rats were gavaged daily with 500 mg/kg
DBP or corn oil control. Significant decreases in testosterone
production and mRNA expression of scavenger receptor B1,
P450SCC, steroidogenic acute regulatory protein, and cytochrome p450c17 were observed as early as gd 17. Testosterone,
mRNA, and protein levels remained low 24 h after withdrawal
of DBP treatment but increased 48 h after cessation of DBP
exposure. In another experiment, pregnant dams were
treated with DBP until gd 19, with the start of DBP treatment
A
N ARRAY OF INCREASINGLY common male reproductive abnormalities such as testicular germ cell cancers, poor semen quality, cryptorchidism, and hypospadias
may be manifestations of one condition termed “testicular
dysgenesis syndrome.” Evidence suggests that this syndrome arises from disruption of embryonic gonadal development due either to genetic defects or to environmental
factors (1, 2). Administration of the phthalate ester di(nbutyl) phthalate (DBP) to female rats at a dose of 500 mg/
kg䡠d during pregnancy leads to a variety of male reproductive malformations, including underdeveloped or absent
reproductive organs, malformation of the external genitalia,
cryptorchidism, decreased anogenital distance, diminished
sperm counts (3, 4), and Leydig cell adenomas (5). These
abnormalities correspond closely with the human conditions
that comprise testicular dysgenesis syndrome. Male fetuses
exposed to DBP in utero also exhibit abnormal gonocyte
development (6), which is the putative etiologic event of
human testicular carcinoma (7).
Phthalate esters are commonly used as plasticizers (8, 9)
Abbreviations: Bt2-cAMP, Dibutyryl cAMP (Bt2-cAMP)DBP, di(nbutyl) phthalate; CYP17, cytochrome p450c17; gd, gestational day; HSD,
17␤-hydroxysteroid dehydrogenase; MBP, monobutyl phthalate;
MEHP, mono (2-ethylhetyl)phthalate; P450scc, side-chain cleavage enzyme; RNase, ribonuclease; SF-1, steroidogenic factor-1; SR-B1, scavenger receptor B1; StAR, steroidogenic acute regulatory protein.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
moved 1 d later into gestation for each treatment group, with
the final group dosed only on gd 19. Significant decreases in
testosterone, mRNA expression, and protein expression were
evident as early as 3 h after treatment with DBP, with full
repression apparent 24 h after treatment. Using a testis explant system, we determined that DBP treatment led to diminished transport of cholesterol across the mitochondrial
membrane as well as diminished function at each point in
the testosterone biosynthesis pathway except 17␤-hydroxysteroid dehydrogenase. The transcriptional repression
caused by DBP does not appear to be mediated via interference with steroidogenic factor-1 as determined by reporter
assays. We conclude that high-dose DBP exposure leads to
rapid and reversible diminution of the expression of several
proteins required for cholesterol transport and steroidogenesis in the fetal testis, resulting in decreased testosterone
synthesis and consequent male reproductive maldevelopment. (Endocrinology 145: 1227–1237, 2004)
and can be found in such disparate products as cosmetics (10)
and infant formula (11). As such, the potential for human
exposure to phthalates is quite high, particularly among
those using polyvinyl chloride-based medical devices (12,
13). Phthalate esters are metabolized in the gut to the corresponding monoester and alcohol (14 –16), with toxicity ascribed to the monoester metabolite (17, 18). A study of urinary levels of phthalate ester metabolites in the general
population showed that women of childbearing age (20 – 40
yr) displayed significantly higher levels of monobutyl phthalate (MBP) than all other groups examined (19). The high
levels of MBP in women of reproductive age are a matter of
concern given the clear adverse effects of the diester precursor DBP on male reproductive development in animal models (3, 11, 20).
Endocrine-disrupting chemicals are defined as exogenous
agents that interfere with normal endocrine signaling (21).
Typically, endocrine-disrupting chemicals are thought to
work at the level of the estrogen or androgen receptor. However, phthalate esters do not interact with the androgen receptor (4, 22). Rather, these chemicals disrupt testosterone
synthesis by the fetal testis (6, 20, 22), probably through
diminished expression of several genes in the cholesterol
transport and testosterone biosynthesis pathways (20, 23).
Cholesterol, synthesized de novo in the testis or acquired from
serum lipoproteins via scavenger receptor B1 (SR-B1), is
transported from the outer to the inner mitochondrial membrane by steroidogenic acute regulatory protein (StAR).
Transport across the mitochondrial membrane is the rate-
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Endocrinology, March 2004, 145(3):1227–1237
limiting step of testosterone biosynthesis (24). A six-carbon
moiety is cleaved from cholesterol by the side-chain cleavage
enzyme (P450scc) to generate the steroid pregnenolone. The
remaining enzymatic reactions required for production of
testosterone take place in the smooth endoplasmic reticulum
(Fig. 1) (25). Expression of SR-B1, StAR, P450SCC, 3␤-hydroxysteroid dehydrogenase (HSD), and cytochrome p450c17
(CYP17) have all been shown to be diminished in the fetal
testis after treatment with DBP (20, 23).
The aim of these studies was to determine the onset and
reversibility of DBP effects on the fetal testis and to determine
at a functional level the points in the cholesterol transport
and steroidogenesis pathways affected by DBP. We show
that the effects of treatment with phthalate esters are rapid
and reversible and that diminution of steroidogenesis is due
to diminished protein function at several points in the testosterone synthesis pathway. The transcriptional repression
induced by DBP does not appear to be mediated through
interference with the activity of the steroidogenic factor-1
(SF-1) transcription factor.
Materials and Methods
Animals
This study was approved by the Institutional Animal Care and Use
Committee of the CIIT Centers for Health Research and followed Federal
guidelines for the care and use of laboratory animals (26). Pregnant
timed-mated Sprague Dawley outbred rats were purchased from
Charles Rivers Laboratories, Inc. (Raleigh, NC) on gestation d 0 (gd 0 ⫽
the day sperm is detected in the vaginal smear). Animals were housed
in the AAALAC-accredited animal facility at CIIT in a humidity- and
temperature-controlled, HEPA-filtered, mass air-displacement room.
The room is maintained on a 12-h light, 12-h dark cycle at approximately
18 –25 C with a relative humidity of approximately 30 –70%. Rodent diet
NIH-07 (Zeigler Brothers, Gardners, PA) and reverse-osmosis water
were provided ad libitum. Animals were acclimatized for the time period
Thompson et al. • Steps in Steroidogenesis Altered by DBP
before dosing. Allocation of animals to treatment groups (four to five
dams per group) was done by body weight randomization. Dams were
dosed by oral gavage with 1 ml/kg corn oil (Sigma Chemical, Co., St.
Louis, MO) or 500 mg/kg DBP (Aldrich Chemical Co., Milwaukee, WI).
DBP was prepared at a concentration of 500 mg/ml corn oil. This dose
was chosen because previous studies have shown near 100% incidence
of testicular pathology (23) and altered gene expression (20) at this dose
without evidence of maternal toxicity (27). Dosing schedules for different study groups are detailed in Table 1. Animals were euthanized by
CO2 anesthesia and exsanguination by abdominal aorta transection.
Fetuses were removed by Cesarean section and euthanized by decapitation. The sex of fetuses was determined by internal inspection of the
gonads. Female fetuses were discarded. Testes for explant culture were
removed and placed in ice-cold PBS supplemented with penicillin and
streptomycin. All other testes were snap-frozen in liquid nitrogen and
stored at ⫺70 C.
Testis explant cultures
The culture method used was based on that previously described (28).
Fetal testes were placed in Petri dishes containing PBS supplemented
with penicillin (100 U/ml) and streptomycin (0.1 mg/ml) on ice. Testes
were cut in half and both halves of one testis placed on a 0.45-␮m filter
(Millipore Corp., Billerica, MA) in a six-well plate. One milliliter of
culture media (Ham’s F12/DMEM with 100 mm glutamine, 50 ␮g/ml
gentamicin, and 15 mm HEPES, pH 7.4) was placed on the filter, and
testes were cultured for 3 h (overnight for 3H-cholesterol uptake) at 37
C, 5% CO2/ 95% air. All media components were purchased from Life
Technologies (Gaithersburg, MD). The cultures included supplements
chosen to bypass points in the cholesterol transport and steroidogenesis
pathways (Table 2). The supplement concentrations used in this study
have been shown to be maximally stimulating in Leydig cell cultures
(29). 3H-cholesterol was purchased from PerkinElmer Life Sciences, Inc.
(Boston, MA). SR-B1 has been shown to efficiently transport nonlipoprotein cholesterol (30). All other supplements were purchased from Sigma.
RIA
Fetal testis testosterone levels were determined as previously described (20). Briefly, the testis was homogenized in 100 ␮l PBS-gel buffer
followed by extraction with a mixture of ethylacetate and chloroform
(4:1). Extraction was performed three times using a total of 1 ml ethylacetate:chloroform. Extracts were dried under nitrogen and resuspended in 100 ␮l RIA zero standard. Culture medium was assayed to
determine testosterone production by testis explants. Testosterone concentration was determined using the ImmuChem Coated Tube Testosterone 125I RIA Kit (ICN Biomedicals, Inc., Costa Mesa, CA) according
to manufacturer’s instructions. Samples were counted in a Cobra D5005
␥ counter (Packard Instrument Co., Downers Grove, IL).
RNA isolation and cDNA synthesis
FIG. 1. Testosterone biosynthesis in the rat testis. Cholesterol is produced de novo in the testis or acquired from plasma lipoproteins. StAR
protein transports cholesterol from the outer to the inner mitochondrial membrane, where it is converted to pregnenolone by P450SCC.
Pregnenolone is converted to testosterone by a series of enzymatic
reactions in the smooth endoplasmic reticulum. Genes previously
shown to have diminished expression in the fetal testis after DBP
treatment are underlined. Events or reactions affected by DBP treatment are shown with broken arrows.
Total RNA was isolated from frozen tissues using STAT-60 reagent
(Tel-Test, Inc., Friendswood, TX) according to manufacturer’s instructions. Each total RNA sample was checked for integrity and DNA contamination by measurement of OD and size-fractionation of 18S and 28S
rRNA on an agarose gel. All reagents for reverse transcription were
purchased from Applied Biosystems (Foster City, CA) unless otherwise
noted. After isolation, total RNA was incubated for 1 h at 37 C, in a
reaction mixture containing ribonuclease (RNase) inhibitor, dithiothreitol, 5⫻ transcription buffer, and RQ1 RNase-free deoxyribonuclease
(Promega Corp., Madison, WI). Deoxyribonuclease was inactivated by
incubating for 5 min at 75 C. One microgram of total RNA was reversetranscribed for 65 min at 42 C in a 20-␮l reaction containing 5 mm MgCl2,
1⫻ GeneAmp PCR buffer II [50 mm KCl, 10 mm Tris-HCl (pH 8.3)], 1
mm each deoxynucleotide transferase, random hexamers, 20 U RNase
inhibitor, and 50 U murine leukemia virus reverse transcriptase. The
reverse transcriptase reaction was terminated by heating to 95 C for 5
min; 0.4 ␮l cDNA was used for subsequent PCRs.
Real-time quantitative RT-PCR
Real-time quantitative RT-PCR was performed on an ABI Prism 7900
HT Sequence Detection System (Applied Biosystems). cDNA prepared
Thompson et al. • Steps in Steroidogenesis Altered by DBP
Endocrinology, March 2004, 145(3):1227–1237 1229
TABLE 1. Dosing schedule for DBP toxicity onset and recovery studies
Treatment
Corn oil
500 mg/kg䡠d
Corn oil
500 mg/kg䡠d
Corn oil
500 mg/kg䡠d
500 mg/kg䡠d
500 mg/kg䡠d
500 mg/kg䡠d
500 mg/kg䡠d
500 mg/kg䡠d
500 mg/kg䡠d
500 mg/kg䡠d
500 mg/kg䡠d
500 mg/kg䡠d
DBP
DBP
DBP
DBP
DBP
DBP
DBP
DBP
DBP
DBP
DBP
DBP
Dosing period
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
Study conclusion
12–17
12–17
12–18
12–18
12–19
12–17
12–18
12–19
13–19
14 –19
15–19
16 –19
17–19
18 –19
19
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
gd
17
17
18
18
19
19
19
19
19
19
19
19
19
19
19
Purpose
Onset of DBP effects
Onset of DBP effects
Onset of DBP effects
Onset of DBP effects
Recovery from and timing of DBP effects
Recovery from DBP
Recovery from DBP
Timing of DBP effects
Timing of DBP effects
Timing of DBP effects
Timing of DBP effects
Timing of DBP effects
Timing of DBP effects
Timing of DBP effects
Timing of DBP effects
TABLE 2. Fetal testis explant culture supplements
Pathway activity
Media supplement
Assay
LH receptor
cAMP response
SR-B1
StAR
P450SCC
3␤-HSD
CYP17: 17␣-hydroxylase
CYP17: 17, 20 lyase
17␤-HSD
100 ng/ml LH
100 ␮M Bt2-cAMP
1 ␮Ci 3H-cholesterol
1 ␮Ci 3H-cholesterol
50 ␮M 22(R)-hydroxycholesterol
20 ␮M Pregnenolone
20 ␮M Progesterone
20 ␮M 17-hydroxyprogesterone
20 ␮M Androstenedione
Testosterone RIA
Testosterone RIA
Whole-cell radioactivity
Mitochondrial radioactivity
Testosterone RIA
Testosterone RIA
Testosterone RIA
Testosterone RIA
Testosterone RIA
as described above was amplified in a 25 ␮l reaction mix containing 1⫻
SYBR Green PCR Master Mix (Applied Biosystems) and 64 nm each
primer. After a 10-min Taq activation step at 95 C, reactions were subjected to 50 cycles of 15 sec denaturation at 94 C, and 1 min annealing/
extension at 60 C. Primers were purchased from Operon, Inc. (Alameda,
CA). After PCR, reaction products were melted for 3 min at 95 C, and
then the temperature was lowered to 50 C in 0.5 C increments, 10 sec per
increment. Optical data were collected over the duration of the temperature drop, with a dramatic increase in fluorescence occurring when
the strands reannealed. This was done to ensure that only one PCR
product was amplified per reaction. Relative expression of the RT-PCR
products was determined using the method described by Pfaffl (31). One
of the control samples was chosen as the calibrator sample and used in
each PCR. Each sample was run in triplicate and the mean Ct used for
determination of relative expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization as described previously (20). Primer pairs used are as follows: GAPDH sense 5⬘-GAAGGTGAAGGTTCGGAGTC-3⬘ and antisense 5⬘-GAAGATGGTGATGGGATTTC-3⬘; P450SCC sense 5⬘-TTCCCATGCTCAACATGCCTC-3⬘
and antisense 5⬘-ACTGAAAATCACATCCCAGGCAG-3⬘; CYP17 sense
5⬘-TGGCTTTCCTGGTGCACAATC-3⬘ and antisense 5⬘-TGAAAGTTGGTGTTCGGCTGAAG-3⬘; StAR sense 5⬘-ACCACATCTACCTGCACGCCAT-3⬘ and antisense 5⬘-CCTCTCGTTGTCCTTGGCTGAA-3⬘;
SR-B1 sense 5⬘-CCATTCATGACACCCGAATCCT-3⬘ and antisense 5⬘TCGAACACCCTTGATTCCTGGT-3⬘. The following primers were used
to amplify the full-length rat SF-1 cDNA: sense 5⬘-CGAATTCACCATGGACTATTCGTACGACG-3⬘, and antisense 5⬘-GCGGCCGCAGTCTGCTTGGCCTGCAGCATC-3⬘. The sense primer was designed to include an EcoR1 restriction site and a Kozak initiation sequence. The
antisense primer contained a Not1 restriction site downstream from the
coding region. Thermal cycling parameters were 2 min at 95 C; 1 cycle
of 30 sec denaturation at 94 C, 60 sec annealing at 42 C, and 90 sec
extension at 72 C; 40 cycles of 30 sec denaturation at 94 C, 30 sec annealing
at 60 C, and 90 sec extension at 72 C; and 1 cycle of 10 min at 72 C.
After overnight culture, explants were washed twice in ice-cold PBS.
Samples for whole-cell cholesterol uptake assessment were homogenized in lysis buffer [0.1 m Tris-HCl (pH 8.0), 0.05 m EDTA, 0.1 m NaCl,
1% wt/vol sodium dodecyl sulfate, 1% wt/vol sarcosyl] and incubated
for 30 min at 65 C. Samples for mitochondrial uptake assessment were
prepared as described (33). Testes were incubated for 10 min on ice in
a hypotonic buffer [10 mm Tris-HCl (pH 7.5), 10 mm NaCl, 1.5 mm
MgCl2], followed by homogenization. The homogenate was then spiked
with 0.4 vol 2.5⫻ buffer [525 mm mannitol, 175 mm sucrose, 12.5 mm
Tris-HCl (pH 7.5), 12.5 mm MgCl2]. This was centrifuged twice at 1000 ⫻
g for 10 min at 4 C. The supernate was then centrifuged at 10,000 ⫻ g for
20 min at 4 C. The mitochondrial pellet was resuspended in lysis buffer and
incubated at 65 C for 30 min. Total DPM were counted on a Tri-Carb
1900CA Liquid Scintillation Analyzer (Packard Instrument Co.).
Immunoblotting
Cloning
For immunoblot analysis, total protein was extracted from paired
testis by homogenizing in 50 ␮l lysis buffer [0.1 m Tris-HCl (pH 8.0), 0.05
For expression of the full-length rat SF-1 cDNA, purified PCR products were cloned into pGEM-T Easy Vector (Promega Corp.) for se-
m EDTA, 0.1 m NaCl, 1% wt/vol sodium dodecyl sulfate, 1% wt/vol
sarcosyl] supplemented with Complete Protease Inhibitor Cocktail
(Roche Molecular Biochemicals, Mannheim, Germany). Total protein
was quantitated using the bicinchoninic acid protein assay reagent
(Sigma). Thirty micrograms of total protein were run on SDS-PAGE and
transferred to polyvinylidene difluoride membranes. The following antibodies were used to probe the membrane: rabbit antimouse SR-B1
(Novus Biologicals, Inc., Littleton, CO), rabbit antirat cytochrome
P450SCC (U.S. Biological, Swampscott, MA), rabbit antirat StAR (Affinity
Bioreagents, Inc., Golden, CO), and rabbit antiporcine CYP17 (a kind gift
of Dr. D. B. Hales, University of Illinois at Chicago, Chicago, IL) (32).
Immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibodies to rabbit IgG (Amersham Biosciences, Piscataway, NJ) and ECL Plus Western Blotting Detection Reagents (Amersham) and visualized with a MultiImage Light Cabinet (Alpha
Innotech Corp., San Leandro, CA). Quantitation of expressed protein
levels was done using FluorChem 8000 software (Alpha Innotech).
Cholesterol transport
1230
Endocrinology, March 2004, 145(3):1227–1237
quence verification. Clones that matched the predicted sequence for SF-1
were digested with EcoR1 and Not1 (both purchased from New England
Biolabs, Beverly, MA) and ligated into expression vector pEF1/V5 HisA
(Invitrogen, Corp., Carlsbad, CA). This vector adds the V5 epitope tag
onto the expressed protein. Expression of SF-1 was confirmed by
immunoblotting.
Cell culture
HepG2 cells were purchased from American Type Culture Collection
(Manassas, VA) and cultured in phenol red-free MEM (Mediatech, Inc.,
Herndon, VA) with 4 mm l-glutamine, 1 mm sodium pyruvate (all Life
Technologies), and 10% charcoal-stripped fetal bovine serum (HyClone,
Logan, UT). MA-10 cells were provided by Dr. Mario Ascoli, University
of Iowa (Iowa City, IA). Cells were maintained in Waymouth MB 752/1
media supplemented with 4 mm l-glutamine, 62.5 ␮g/ml gentamicin,
20 mm HEPES, pH 7.4 (all Life Technologies), and 15% horse serum
(Atlanta Biologicals, Inc., Norcross, GA). Both cell lines were maintained
at 37 C in an atmosphere of 5% CO2/95% air.
Transfection and luciferase assay
HepG2 or MA-10 cells were grown to 80% confluence in 24-well
plates. Plasmid DNA was mixed with TransIT-LT1 Transfection reagent
(Mirus Corp., Madison, WI) in 0.65 ml phenol red-free MEM (Mediatech,
Inc.) and added to cells. Our laboratory has obtained the following
luciferase reporter constructs: StAR (from Dr. Doug Stocco, Department
of Cell Biology and Biochemistry, Texas Tech University Health Science
Center); SR-B1 (from Dr. Kaoru Miyamoto, Department of Biochemistry,
Fukui Medical University, Fukui, Japan); and CYP17 (from Dr. Synthia
Mellon, Department of Obstetrics, Gynecology, and Reproductive Sciences and the Metabolic Research Unit, University of California). A
␤-galactosidase plasmid was used as a transfection control. Cells were
transfected with reporter constructs with or without the SF-1 expression
vector in the presence of 10⫺5 m MBP (Aldrich) or dimethylsulfoxide
control. After 48 h, cells were lysed in 65 ␮l lysis buffer [25 mm Tris/
EDTA (pH 7.8), 10% glycerol, 0.5% Triton X-100, 3 mm dithiothreitol].
One hundred microliters of Luciferase Assay Reagent (Promega Corp.)
were added to 20 ␮l of lysate and luminescence read on an LMax microplate luminometer (Molecular Devices Corp., Sunnyvale, CA). Thirty
microliters of lysate were used for ␤-galactosidase activity determination. Eighty micrograms of chlorophenol red-␤-d-galactopyranoside
(CPRG, Roche Molecular Biochemicals, Indianapolis, IN) in 170 ␮l of
CPRG buffer [60 mm Na2HPO4, 40 mm NaH2PO4, 10 mm KCl, 1 mm
MgSO4, and 50 mm ␤-mercaptoethanol (pH 7.8)] were added to lysate
and absorbance read at 575 nm at 1-min intervals for 30 min in a
SpectraMax340 microplate reader (Molecular Devices Corp.) to obtain the
Vmax for the reaction. Luciferase activity was normalized to ␤-galactosidase activity.
Thompson et al. • Steps in Steroidogenesis Altered by DBP
Statistical analyses
The Student’s t test or one-way ANOVA with Tukey post hoc analysis
were performed using JMP statistical software (SAS Institute, Inc., Cary,
NC), version 5.0.1. P ⬍ 0.05 was considered to be statistically significant.
Results
Onset and duration of adverse effects of DBP on fetal
testicular steroidogenesis
A significant decrease in fetal testicular testosterone concentration was observed as early as gd 17 after in utero exposure of fetuses to DBP (Fig. 2A). The mean testosterone
concentration per DBP-treated testes was actually slightly
lower on gd 18 than on gd 17, although this difference was
not statistically significant. The percentage difference in testicular testosterone between control and treated testes was
much higher on gd 18 than on gd 17. Testosterone concentration in the gd 17 testis was 46.6% of that seen in the
untreated control, whereas the level at gd 18 was 17.8% of
that seen in the control samples. The previously observed
differences in expression of mRNA for SR-B1, StAR, P450SCC,
and CYP17 after DBP exposure (20, 23) were detected at gd
17, although the diminished expression of P450SCC mRNA
was not significant at this gestational day (Fig. 2B). As was
the case with testosterone, the percentage difference between
DBP-treated and control gene expression was considerably
less at gd 17 than at gd 18. Each of the genes demonstrated
a similar relative expression level at both gd 17 and 18, but
the mean percentage of expression of the four genes relative
to age-matched control was 46.4% in the gd 17 DBP-exposed
fetuses vs. 15.4% in the gd 18 fetuses exposed to DBP.
Because the mechanism of DBP interference with steroidogenesis appears to be the same at different developmental
time points, we next sought to determine the persistence of
this effect. Pregnant dams were dosed with DBP via oral
gavage from gd 12–17 or gd 12–18. All animals were killed
at gd 19 and compared with gd 19 vehicle control animals.
Testosterone concentration was significantly diminished in
both the 24-h recovery and 48-h recovery groups (Fig. 3A).
However, testosterone concentration was 3.6-fold higher 48 h
FIG. 2. Testosterone content and gene expression of fetal testes after exposure to DBP. A, Total testicular testosterone from fetuses exposed
in utero to DBP or corn oil control was measured by RIA at gd 17 and 18. Results are expressed as mean testosterone per milliliter testis lysate ⫾
SE n ⫽ 4 fetuses per group, with each fetus taken from a separate litter. *, P ⬍ 0.05; **, P ⬍ 0.01 compared with age-matched control by Student’s
t test. B, Total RNA was extracted from fetal testes at gd 17 or gd 18 after in utero exposure to DBP or corn oil. Real-time RT-PCR was performed
to assess expression of genes in the cholesterol transport and steroidogenesis pathways previously shown to be affected by phthalate esters (20).
Results are expressed as mean expression relative to one gd 17 control sample used as a calibrator. Error bars show SE n ⫽ 4 fetuses per group,
with each fetus taken from a separate litter. *, P ⬍ 0.05; **, P ⬍ 0.01 compared with age-matched control by Student’s t test.
Thompson et al. • Steps in Steroidogenesis Altered by DBP
Endocrinology, March 2004, 145(3):1227–1237 1231
FIG. 3. Increasing fetal testicular function after withdrawal of DBP treatment. Fetuses were exposed to DBP in
utero from gd 12–17 or 12–18 before animals were killed on gd 19. A, Total testicular testosterone was measured by
RIA. Results are expressed as mean testosterone per ml testis lysate ⫾ SE n ⫽
4 fetuses per group, with each fetus
taken from a separate litter. *, P ⬍ 0.01
when compared with corn oil control; †,
P ⬍ 0.01 when compared 24-h recovery
samples by one-way ANOVA with
Tukey post hoc analysis. B, Real-time
RT-PCR was performed as described in
Fig. 2 with a gd 19 corn oil control sample used as the calibrator. Error bars
show SE n ⫽ 4 fetuses per group, with
each fetus taken from a separate litter.
*, P ⬍ 0.05 compared with control; †,
P ⬍ 0.05 compared with 48-h recovery
group by one-way ANOVA with Tukey
post hoc analysis. C and D, Total protein
lysates were prepared and 30 ␮g of each
sample electorphoresed on 10% SDSPAGE gels and probed with antibodies
to StAR, SR-B1, P450SCC, and CYP17.
C, Immunoblots showing diminished
expression of proteins 24 h after DBP
exposure, with expression increasing
48 h after withdrawal of DBP. Photo is
representative of three separate blots.
D, Densitometric quantitation of protein expression for StAR, SR-B1,
P450SCC, and CYP17. Error bars show
SE. n ⫽ 5 fetuses per group, with each
fetus taken from a separate litter. *, P ⬍
0.01 compared with untreated control;
†, P ⬍ 0.01 compared with 24 h recovery
animals by one-way ANOVA with
Tukey post hoc analysis.
after cessation of DBP exposure relative to the 24-h recovery
group, indicating that the steroidogenic capability of the
testis was not irreversably affected by DBP. The suppression
of transcription by DBP was also a transient effect because
mRNA levels for SR-B1, StAR, P450SCC, and CYP17 all returned to control levels 48 h after DBP withdrawal (Fig. 3B).
To determine whether the observed changes in mRNA
expression were mirrored by changes in protein expression,
we performed immunoblotting for SR-B1, StAR, P450scc, and
CYP17. Each of these proteins showed an expression pattern
similar to that of testosterone production, with strong expression in the controls, minimal expression in the 24-h recovery animals, and rising expression in the 48-h recovery
animals (Fig. 3C). CYP17 and StAR were not detected in the
24-h recovery group. SR-B1 protein showed the most rapid
recovery from phthalate exposure, with protein expression
67% that of the control animals 48 h after withdrawal of DBP
(Fig. 3D). StAR, P450SCC, and CYP17 protein were all expressed at a similar level (approximately 40 – 45% of control
expression) at this time point. The level of protein expression
for StAR and CYP17, both 40% of the control level, correlated
well with observed testosterone production by testes in the
48-h recovery group (36% of control steroidogenesis).
To determine whether DBP exposure leads to diminished
testicular testosterone through interference with a critical
event in male reproductive development, pregnant dams
were dosed with corn oil from gd 12–19 or with 500 mg/kg
DBP. The start of DBP treatment was shifted from gd 12 1 d
later in gestation for each dose group, so that the final group
was dosed only on gd 19 approximately 3 h before the
animals were killed. Testosterone was repressed in all DBP
dose groups (Fig. 4A). The mean testosterone concentration
in the fetuses dosed only on gd 19 was 44% of that seen in
the control group. The mean testosterone concentration of all
1232
Endocrinology, March 2004, 145(3):1227–1237
Thompson et al. • Steps in Steroidogenesis Altered by DBP
FIG. 4. Fetal testicular toxicity after exposure to DBP for different time periods. Pregnant dams were dosed with 500 mg/kg DBP via oral gavage
until gd 19. The commencement of dosing was shifted 1 d later into gestation for each dose group such that groups were dosed gd 12–19, gd
13–19, gd 14 –19, etc. Panel A, Total testicular testosterone from fetuses exposed in utero to DBP or corn oil control was measured by RIA on
gd 19. Results are expressed as mean testosterone per ml testis lysate ⫾ SE. n ⫽ 4 fetuses per group, with each fetus taken from a separate
litter. *, P ⬍ 0.01 when compared with corn oil control; †, P ⬍ 0.01 compared with gd 19 dose group by one-way ANOVA with Tukey post hoc
analysis. B, Total RNA was extracted from fetal testes on gd 19 after in utero exposure to DBP for varying lengths of time. Real-time RT-PCR
was performed as described in Fig. 2 with a corn oil control sample used as the calibrator. Error bars show SE. n ⫽ 4 fetuses per group, with
each fetus taken from a separate litter. Panel C, Immunoblot showing expression of proteins after DBP treatment of varying duration. C, Corn
oil control; 12, treatment gd 12–19; 13, treatment gd 13–19, etc. Photo representative of three separate blots. D, Protein expression in animals
dosed solely on gd 19. Results represent specific band densitometry values expressed relative to the mean control value. Error bars show SE
n ⫽ 4 fetuses per group, with each fetus taken from a separate litter. *, P ⬍ 0.05 compared with corn oil control by Student’s t test.
the other dose groups was 13% of control levels. mRNA
levels were similarly repressed at all time points (Fig. 4B).
The only genes that did not show significantly diminished
expression relative to control were CYP17 in the gd 19 dose
group and P450SCC in the gd 12–19 dose group, although the
lack of significance in the latter group is attributable to one
sample displaying 3-fold higher expression than all of the
other samples in this group. Expression of these four genes
was significantly higher in the group dosed only on gd 19
when compared with all the other groups exposed to DBP,
except P450SCC in the gd 12–19 and gd 17–19 dose groups.
Each of these groups had one sample that showed elevated
expression. The pattern of protein expression followed that
of mRNA expression, with minimal protein expressed in the
animals dosed gd 12–19 through gd 18 –19 and moderately
decreased expression in the animals dosed exclusively on gd
19 (Fig. 4, C and D).
Alterations in cholesterol transport after in utero exposure
to DBP
In our study, DBP exposure led to rapid and reversible
changes in testosterone biosynthesis and expression of key
genes in the cholesterol and steroidogenesis pathways at
mRNA and protein levels. A testis explant system was em-
ployed to determine whether the observed changes in mRNA
and protein correlated with altered levels of protein function.
Pregnant dams were gavaged with 500 mg/kg DBP from gd
12–19. Animals were killed on gd 19, fetuses delivered by
Cesarean section, and testes removed and cultured ex vivo for
assessment of steroidogenic function. To determine DBP effects on cholesterol transport, explanted testis were cultured
overnight with 3H-cholesterol. Whole tissue and mitochondrial lysates were prepared, and cholesterol uptake was measured by liquid scintillation counting. At the level of transport across the cell membrane, there was a slight but
statistically insignificant difference (P ⫽ 0.1) in cholesterol
uptake between the control and DBP-exposed explants (Fig.
5A). This suggests that there was little functional diminishment of SR-B1 activity after DBP exposure. There is a significant decrease in the amount of cholesterol transported
across the mitochondrial membrane in the testes from DBPtreated fetuses, indicating that the observed decrease in StAR
mRNA correlated with diminished protein function.
Stimulation of testosterone production in
DBP-exposed explants
We also sought to determine the capacity of DBP-exposed
testes to produce testosterone in response to external signals.
Thompson et al. • Steps in Steroidogenesis Altered by DBP
Endocrinology, March 2004, 145(3):1227–1237 1233
FIG. 5. Functional assessment of fetal
testis explants after exposure to DBP.
Dams were gavaged with 500 mg/kg䡠d
DBP from gd 12–19. Testes were removed and put into culture on gd 19. A,
Explants were cultured overnight with
1 ␮Ci 3H-cholesterol. Whole-cell or mitochondrial lysates were prepared and
assayed for radioactivity. n ⫽ 12 with
results combined from two separate experiments. *, P ⬍ 0.01 compared with
corn oil control by Student’s t test. Error
bars show SE (B and C). Control or DBPexposed testis explants were supplemented with steroidogenesis stimulators (B) or testosterone precursors (C)
and assayed for testosterone production
by RIA after 3-h culture. Testosterone
concentration was normalized to total
protein content of the explants. These
values were log transformed to normalize the distribution of the data. n ⫽ 12
with results combined from two separate experiments. Error bars show SE. †,
P ⬍ 0.01 compared with unsupplemented explants from fetuses exposed
similarly in utero; *, indicates P ⬍ 0.01
compared with similarly supplemented
explants from control fetuses by ANOVA
with Tukey post hoc analysis.
Explants were treated with LH or dibutyryl cAMP (Bt2cAMP) for 3 h, followed by measurement of testosterone in
the culture media by RIA. Both control and treated explants
demonstrated increased testosterone production after exposure to stimulants (Fig. 5B). However, the magnitude of the
response was significantly less in the treated explants, with
testosterone production approximately 40% of the control
value for each molecule (nontransformed values). Bt2-cAMP
was a more efficient stimulant than was LH, with the fold
increase over control double that seen with LH for both
control and the DBP-exposed explants. The magnitude of the
increase in steroidogenesis was actually greater in the DBP
group, with the fold increase over unstimulated explants
2.5⫻ that of the corn oil controls in response to both LH and
Bt2-cAMP.
Determination of enzymatic steps in steroidogenesis affected
by DBP
Explants were supplemented with steroidal precursors to
testosterone to pinpoint the steps in the testosterone synthesis pathway altered by DBP. Both control and DBP-exposed
explants showed a significant increase in testosterone production after exposure to all supplements. However, the
level of testosterone production corresponding to each supplement in the DBP-exposed explants was significantly lower
than the accompanying control, except in the case of androstenedione (Fig. 5C). These data suggest that 17␤-HSD is the
only enzyme in the testosterone biosynthesis pathway unaffected by exposure to DBP.
Effects of MBP on SF-1-mediated transcription
The genes SR-B1, StAR, P450SCC, and CYP17 have all been
shown previously to be regulated by the transcription factor
SF-1 (34). However, our laboratory has shown SF-1 gene
expression not to be affected after exposure to DBP. To ascertain whether the mechanism of DBP toxicity might be
through interference with SF-1-mediated transcription, we
cloned the rat SF-1 cDNA into a mammalian expression
vector and coexpressed this construct in HepG2 cells along
with reporter plasmids for StAR, SR-B1, or CYP17. In each
case, SF-1 increased transcription of the reporter construct.
However, monobutyl phthalate, the metabolite of DBP to
which reproductive toxicity has been ascribed (17, 18), had
no effect on SF-1-mediated transcription (Fig. 6). This was
also the case when reporter assays were performed in the
mouse Leydig tumor cell line MA-10 (not shown).
Discussion
The ubiquity of phthalate esters in the environment makes
the reported adverse effects of these chemicals on male reproductive development a matter of concern. Several previous studies have shown that in utero exposure to
di(2-ethylhexyl) phthalate (22, 35) or DBP (5, 20) leads to
1234
Endocrinology, March 2004, 145(3):1227–1237
FIG. 6. SF-1-driven luciferase activity in cells treated with MBP.
Results represent mean luciferase activity normalized to ␤-galactosidase activity. n ⫽ 3. Error bars show SE. A, HepG2 cells were
transfected with a StAR luciferase reporter construct and 400 ng
empty expression vector or rat SF-1 expression vector with or without
10 ␮M MBP. B, SR-B1 reporter construct cotransfected with 400 ng
empty vector or SF-1 expression vector. C, CYP17 reporter construct
cotransfected with 400 ng empty vector or SF-1 expression vector.
diminished testosterone production by the fetal rat testis. We
have proposed that phthalate esters inhibit steroidogenesis
by repressing expression of several genes required for testosterone production (20, 23). In this study, we determined
the onset and persistence of DBP toxicity and used an ex vivo
Thompson et al. • Steps in Steroidogenesis Altered by DBP
culture system to demonstrate the steps in testicular steroidogenesis impaired by in utero exposure to DBP.
Our data indicated that the effects of DBP on testicular
mRNA expression and steroidogenesis were evident as early
as gd 17. The lack of a statistically significant down-regulation of testosterone production by DBP at gd 16 reported by
Shultz et al. (20) is probably due to the low level of steroidogenesis in control animals at this developmental time
point. The Shultz study demonstrated significant decreases
in testicular progesterone concentration, StAR mRNA expression, and SR-B1 mRNA expression. Steroidogenesis
commences with the differentiation of fetal Leydig cells at gd
15 (36) and increases gradually until gd 18, at which time
there is a rapid increase to the peak of testosterone production at gd 19 (37). Our findings correlate well with previous
reports of maldevelopment of the reproductive tract in male
fetuses exposed in utero on gd 15–17 to DBP or its monoester
metabolite MBP (38, 39) and also with data indicating that
DEHP, another phthatlate ester that causes testicular toxicity,
interferes with testosterone production at gd 17 (22).
The rapid functional recovery of the testis after phthalate
exposure could provide insight into the mechanism of phthalate transcriptional regulation. The elimination half-life of
phthalate monoesters is approximately 12 h (40). Assuming
an equivalent elimination half-life for animals exposed in
utero, the level of toxic phthalate to which the fetus is exposed
should be below the previously reported NOAEL for DBP of
50 mg/kg䡠d (27) 48 h after cessation of treatment. Our results
demonstrate that transcription returns to normal as toxic
phthalate is cleared from the system. This indicates that the
mechanism of transcriptional repression by DBP involves
direct interaction of DBP or its metabolites with the factors
responsible for regulation of the genes responsible for testosterone synthesis. It is not clear at this time whether DBP
acts directly on Leydig cells or on the production of molecules by other testicular cell populations that stimulate steroidogenesis in the Leydig cells. Future studies will aim to
determine this point of interaction.
Although mRNA expression had returned to control levels
48 h post DBP treatment, testosterone levels as well as levels
of SR-B1, StAR, P450SCC, and CYP17 protein were still significantly lower than those seen in control animals. The observed differences between mRNA and protein expression
may simply have been a result of translation lagging transcription. Previous studies have shown that protein expression of CYP17, P450SCC (41), and StAR (42) corresponds to
mRNA expression in stimulated Leydig cells. However, peak
expression of StAR protein in MA-10 Leydig tumor cells
occurs approximately 2 h after peak expression of the mRNA
(42), and expression of StAR protein has been shown to
increase up to 20 h after mRNA expression has reached its
peak in granulosa cells (43) and 16 h after mRNA peaks in
Y-1 adrenal cells (44). A more detailed time course study of
DBP recovery will determine whether the exposed testes are
capable of complete recovery of steroidogenesis.
The effects of DBP treatment become evident rapidly, with
full repression of steroidogenesis apparent 24 h after first
exposure and some repression apparent as early as 3 h after
exposure. These data agree with previously reported in vitro
Thompson et al. • Steps in Steroidogenesis Altered by DBP
and in vivo studies on testicular toxicity of phthalate esters.
MA-10 Leydig tumor cells are less responsive to human
chorionic gonadotropin-stimulated progesterone production
after 24-h exposure to mono(2-ethylhetyl)phthalate (MEHP)
(45). Primary cultures of Leydig cells display diminished
testosterone production 2 h after dosing with MEHP (46), and
expression of mRNA for TRPM2, a gene negatively regulated
by androgens, is elevated within 3 h of MEHP gavage in
28-d-old rats (47). It has been suggested that the toxic effects
of phthalates on Sertoli cells (48) and Leydig cells (22) are a
result of arrested development of these cells. Our results
indicate that phthalate esters exert toxic effects on fetal Leydig cells via transcriptional repression well after development of full testosterone biosynthetic capability by these
cells.
Despite diminished mRNA and protein expression for
SR-B1, there was not a significant reduction in whole-cell
cholesterol transport observed in testis explants after in utero
exposure to DBP. This finding is in contrast to the findings
of Barlow et al. (23), who showed diminished Leydig cell lipid
content after in utero exposure to DBP. A possible explanation
for this discrepancy is that SR-B1 activity is not required for
basal steroidogenesis in the fetal testis. Most of the cholesterol used in steroidogenesis is produced de novo in the adult
testis, with selective uptake of cholesteryl esters from highdensity lipoprotein particles increasing only after prolonged
stimulation with gonadotropins, a treatment regimen that
also results in increased expression of SR-B1 (49). Although
the source of cholesterol for the fetal rat Leydig cell has not
been clearly defined, studies indicate that there is high de
novo synthesis of cholesterol in several tissues of the rat
embryo (50 –52) and that the human fetal testis uses cholesterol produced internally as a testosterone precursor (53). In
this study, cholesterol uptake by the explants was assessed
under basal culture conditions. Supplementing the cultures
with LH or Bt2-cAMP led to dramatic increases in testosterone production. Culture of the explants under stimulating
conditions should lead to greater cholesterol uptake on the
whole-cell level and, consequently, a greater disparity in
selective uptake between control and DBP-exposed testis.
Alternatively, the lack of difference in whole-cell cholesterol
transport between the control and DBP-exposed explants
might indicate that the amount of SR-B1 protein in the DBPexposed explants, although decreased, was still sufficient for
movement of cholesterol across the cell membrane.
There was a significant difference in mitochondrial transport of 3H-cholesterol between control and DBP-exposed
testes, indicating that the observed repression of StAR
mRNA and protein by DBP was of functional importance.
Transport of cholesterol from the outer to the inner mitochondrial membrane by the StAR protein is the rate-limiting
step of acute steroidogenesis (24) and as such represents a
key point in the regulation of testosterone synthesis. StAR
protein is rapidly expressed in response to stimulating signals such as gonadotropins (54). However, in regard to basal
steroidogenesis in the fetal testis cultures, the observed 28%
decrease in mitochondrial transport alone is unlikely to account for the 83% reduction in testosterone production after
DBP exposure.
Both control and DBP-exposed cultures demonstrated sig-
Endocrinology, March 2004, 145(3):1227–1237 1235
nificant increases in testosterone production in response to
testosterone precursors. The response of the DBP-exposed
explants was lower than that of the controls for all supplements, with the exception of explants supplemented with
androstenedione. This finding correlates well with the reported lack of change in expression of 17␤-HSD mRNA after
in utero exposure to DBP (23). These results suggest that the
combined effects of diminished expression of StAR, P450SCC,
3␤-HSD, and CYP17 led to the observed alterations in steroidogenesis after DBP exposure.
DBP-exposed explants responded to stimulation with both
LH and Bt2-cAMP. However, as was the case with steroid
precursors, the response of explants was dampened in DBPexposed testes. These data suggest that the reduction in steroidogenesis was not due to altered responsiveness to LH or
cAMP signaling but rather due to reduced expression of the
downstream factors necessary for steroidogenesis. The factors involved in regulating steroidogenesis in the fetal testis
are unknown. Testosterone production by the fetal testis
begins on gd 15 (36) and peaks on gd 19 (37). However, LH
levels in the fetus do not reach appreciable levels until gd 19.5
(37), indicating that fetal testicular steroidogenesis, unlike
that of mature Leydig cells, is initiated by factors other than
LH. Pituitary adenylate cyclase-activating polypeptide and
vasoactive intestinal peptide have both been shown to stimulate testosterone production by fetal Leydig cells (37). However, mRNA expression of these genes and their receptors are
not altered in the fetal testis after DBP exposure (our unpublished observations). Future studies will employ this explant culture system to identify potential regulators of fetal
steroidogenesis.
All the genes shown to have diminished expression after
DBP exposure are regulated by the orphan nuclear receptor
SF-1 (34). SF-1 mRNA expression is unchanged by treatment
with phthalate esters (20). Numerous studies have shown
interactions between SF-1 and other nuclear proteins leading
to potentiation or attenuation of SF-1-mediated transcription
(55–59). However, MBP, the metabolite of DBP to which
reproductive toxicity has been ascribed (17, 18), did not affect
the SF-1-regulated transcription of SR-B1, StAR, or CYP reporter constructs in either HepG2 or MA-10 cells. Because
MA-10 cells are steroidogenic (45), any cofactors required for
expression of these genes are most likely present in this cell
line. This finding indicates that MBP does not interfere with
transcriptional regulation by preventing SF-1 binding to response elements or recruitment of cofactors. Because there
are differences in the regulation of steroidogenesis between
fetal and adult Leydig cell populations (36, 37) and in the
susceptibility of these cell types to phthalate ester toxicity (5,
11, 60), exposure to DBP might lead to altered gene expression through interaction with a fetal Leydig cell-specific transcriptional regulator. Alternatively, DBP treatment could
lead to expression of a novel transcriptional repressor in the
fetal testis. The future aim of our laboratory is to elucidate the
mechanism of transcriptional repression by DBP.
In summary, these studies demonstrate that suppression
of testosterone production in the fetal testis by DBP is coincident with diminished transcription of several genes in the
cholesterol transport and steroidogenesis pathways as early
as gd 17. The effect of DBP on gene expression is rapid and
1236
Endocrinology, March 2004, 145(3):1227–1237
independent of the stage of development of the fetal Leydig
cell. Also, diminished expression of the genes and proteins
necessary for testicular steroidogenesis is reversed as DBP
and its metabolite are cleared. The rapid and reversible effect
of DBP on steroidogenesis indicates that DBP directly interferes with the signaling processes necessary for maintenance
of steroidogenesis or with the transcriptional regulators required to maintain coordinate expression of the genes involved in cholesterol transport and testosterone biosynthesis. Future work will focus on determining the extent of
testicular recovery after cessation of DBP treatment and on
determining the minimal timing of exposure to DBP required
for altered expression of the relevant genes.
Acknowledgments
The authors thank Dr. Katie Turner, Dr. Christopher Bowman, and
Ms. Kimberly Lehman for their assistance in this study, as well as the
CIIT animal care and necropsy staff. We also thank Dr. D. B. Hales for
providing us with CYP17 antisera, Dr. Doug Stocco for the StAR reporter
construct, Dr. Kaoru Miyamoto for the SR-B1 reporter construct, and Dr.
Synthia Mellon for the CYP17 reporter construct.
Thompson et al. • Steps in Steroidogenesis Altered by DBP
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
Received October 31, 2003. Accepted November 5, 2003.
Address all correspondence and requests for reprints to: Dr. Kevin
Gaido, CIIT Centers for Health Research, 6 Davis Drive, P.O. Box 12137,
Research Triangle Park, North Carolina 27709-2137. E-mail: [email protected].
This work was supported by NIH Grant R01ES011803.
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