Endocrinology Vol. 141, No. 4 1470-1476
Copyright © 2000 by The Endocrine Society
Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the Expression of Follicle-Stimulating Hormone Receptors during Cell Differentiation in Cultured Granulosa Cells1
Takashi Hirakawa,
Takashi Minegishi,
Kazuko Abe,
Hiroshi Kishi,
Kyoko Inoue,
Yoshito Ibuki and
Kaoru Miyamoto
Department of Obstetrics and Gynecology (T.H., T.M., K.A., H.K.,
K.I., Y.I.), School of Medicine, Gunma University, Maebashi, Gunma
371-8511; and Department of Biochemistry (K.M.), Fukui Medical
University, Fukui 910-1193, Japan
Address all correspondence and requests for reprints to: Takashi Minegishi, Department of Obstetrics and Gynecology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan. E-mail:
tminegis{at}sb.gunma-u.ac.jp
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Abstract
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Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is a common
environmental pollutant causing public concern. Using a cell culture
system derived from rat granulosa cells that provides unique advantages
for studying the molecular mechanisms underlying the action of
TCDD, the influences of TCDD on FSH receptor (FSH-R) induction were
examined. The treatment with FSH produced, as expected, a substantial
increase in specific FSH-R expression, whereas concurrent treatment
with the environmental amount of TCDD (10 pM) resulted in a
significant decrease in FSH-R after being cultured from 24–72 h.
Cotreatment with FSH (30 ng/ml) and increasing doses of TCDD inhibited
the levels of FSH-induced FSH-R messenger RNA (mRNA) in a
dose-dependent manner. Treatment with 8-Br-cAMP (1 mM)
produced a significant increase in FSH-R mRNA; concurrent treatment
with TCDD (10 pM) produced a significant attenuation of
8-Br-cAMP action. These findings suggest that the ability of TCDD to
interfere with FSH action, as regards the induction of FSH-Rs, is
exerted at sites distal to those involved in cAMP generation. Because a
single transcript of 5.2 kb was seen for the Ah receptor in this
granulosa cell system, the effects of TCDD may be mediated by this
specific receptor. The rates of FSH-R mRNA gene transcription, assessed
by nuclear run-on transcription assay, were decreased by the addition
of TCDD. The effect of TCDD on FSH-R mRNA stability was
determined by measuring the decay of FSH-R mRNA under conditions
known to inhibit transcription. The decay curve for the 2.4-kb FSH-R
mRNA transcript was not significantly changed after the addition of
TCDD. These findings showed that the effect of TCDD on FSH-R mRNA was,
at least in part, the result of decreased transcription.
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Introduction
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IN RECENT YEARS, several reports have
focused on certain man-made toxicants that persist in the environment
and are capable of altering the endocrine homeostasis of animals,
thereby causing serious reproductive and developmental defects (1).
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a prototype halogenated
aromatic hydrocarbon, and it is considered to be one of the most potent
toxicants studied (2). Several studies have shown that exposure to TCDD
reduced the ovulatory rate in rats. Although the factors involved in
TCDD-induced toxicity are still under investigation, several studies
have shown that the endocrine-disrupting effects of TCDD are, at least
in part, caused by a direct action on the ovary (3). TCDD exerts its
toxic effects and alters the hormonal profile of an organism, in part,
by binding to a receptor known as the aromatic hydrocarbon receptor
(AhR), and the TCDD-AhR complex acquires the ability to bind specific
sequences of DNA and subsequently accumulates in the nucleus. It has
been reported that a functional AhR is present in the rat ovary (4) and
in primate ovarian tissue, including human granulosa cells (5).
Previous studies have showed the effects of in vivo
administration of TCDD on steroid hormone production (3, 6, 7, 8, 9). These
data suggest that TCDD is capable of altering steroidogenic processes
in the rat ovary and may function in a similar fashion in humans.
The lack of a cell culture system in which toxicity can be
readily detected and analyzed in a controlled manner has hindered
efforts to understand the intracellular and molecular events induced by
TCDD. Ovarian granulosa cells undergo a complex differentiation process
during the growth and maturation of ovarian follicles (10). A widely
used model system by which to study this phenomenon has been primary
cultures of rat granulosa cells obtained from hypophysectomized or
immature female rats that had been pretreated with estradiol (11, 12).
The use of this defined system has shown that the ability of FSH to
stimulate the induction of FSH receptors (FSH-Rs) is mediated, at least
in part, by cAMP, given that exogenous cAMP or other agents that
increase intracellular levels of cAMP mimic the actions of FSH (13).
The promoters for this gene in the rat, mouse, and human FSH-R are
members of a class of promoters that lack a conventional TATA
and CCAT box and have multiple transcriptional start sites (14, 15).
Several DNA elements have been identified in the 3' proximal region of
the promoter for the FSH-R in the rat, human, and mouse FSH-R.
In the present study, we tried to assess TCDD’s effects, with the
doses that are considered to be environmentally relevant, on rat
granulosa cells to understand the actions of this toxin on normal
reproductive function; and we examined the effects of TCDD on
FSH-induced FSH-R expression.
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Materials and Methods
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Hormones and reagents
Rat FSH (I-8) was obtained from the National Hormone and
Pituitary Distribution Program (Bethesda, MD). Diethylstilbestrol
(DES), gentamicin sulfate, 8-Br-cAMP, and TCDD were purchased from
Sigma, Ltd. (St. Louis, MO). DMEM, Ham’s F-12 medium, and
fungizone were purchased from Life Technologies, Inc.
(Grand Island, NY). The RNA labeling kit and nucleic acid detection kit
were purchased from Roche Molecular Biochemicals
(Mannheim, Germany). [
-32P]UTP (3000
Ci/mmol) was purchased from Amersham Pharmacia Biotech
Japan Corporation (Tokyo, Japan).
Rat granulosa cell culture
Granulosa cells were obtained from immature female Wistar rats
that received an injection of 2 mg DES in 0.1 ml sesame oil once daily
for 4 days. The ovaries were then excised, and granulosa cells were
released by puncturing follicles with a 25-gauge needle. At all times,
the animals were treated according to NIH guidelines. Granulosa cells
were washed and collected by brief centrifugation, and cell viability
was determined by trypan blue exclusion. The granulosa cells were then
cultured in Ham’s F-12/DME (1:1 vol/vol) medium supplemented with 1.1
g/liter NaHCO3, 40 mg/liter gentamicin sulfate, 1
mg/liter fungizone, and 100 mg/liter BSA on collagen-coated plates in a
humidified atmosphere containing 5% CO2-95% air
at 37 C (16).
Receptor binding assay
Granulosa cells were cultured in Immulon-2 Removawell
(Dynatech Corp. Laboratories, Inc., Chantilly, VA).
Each well contained 1 x 105 cells in 0.1 ml
medium. After 24 h incubation, hormone was added to the medium. At
the times indicated, the cells were placed on ice and quickly washed
three times with 0.2 ml cold medium. Then the granulosa cells were
incubated in a 1:1 (vol/vol) mixture of DME; Ham’s F-12 medium
containing 0.1% BSA (pH 7.4) at 37 C with increasing amount of
125I-rat FSH. FSH was iodinated according to the
chloramine-T method. The incubation medium was removed after 2 h
of incubation, and the cells were washed twice with 0.2 ml medium. Each
well was then torn off from the Removawell strip, and the amount of
radioactivity remaining in the well (cell-bound hormone) was quantified
by ß-spectrometry. Nonspecific binding was determined by adding
excess unlabeled FSH (1.25 IU/well). Specific binding was analyzed, by
the method of Scatchard, to determine the maximum binding capacity of
the cells and the affinity of the binding sites for FSH.
RNA isolation and analysis
Granulosa cells were cultured in 60-mm dishes containing
5 x 106 viable cells in 5-ml medium, and
reagents were added to the medium after 24 h of cell culture. The
granulosa cells were further incubated, and the cultures were stopped
as indicated in the guanidinium acid-thiocyanate-phenol-chloroform
method (17). The final RNA pellet was dissolved in diethyl
pyrocabonate-treated H2O. Total RNA was
quantified by measuring the absorbance of samples at 260 nm. For
Northern blot analysis, 15 µg total RNA from each dish was separated
by electrophoresis on denaturing agarose gels and subsequently
transferred to a nylon membrane (Biodyne, ICN, Glen Cove, NY).
In accordance with the standard protocol for the nucleic acid detection
kit (Roche Molecular Biochemicals), X-Omat film
(Eastman Kodak Co., Rochester, NY) was then exposed to the
membranes. Luminescence detection was quantified with a 2202 UnitroScan
Laser Densitometer (LKB Produkter AB, Bromma, Sweden),
normalized against a corresponding relative amount of glyceraldehyde
3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) in each sample
and expressed as relative densitometric units.
PCR cloning of rat AhR complementary DNA (cDNA) from rat ovarian
cDNA library
The cDNA was amplified 30 cycles by PCR containing
Taq DNA polymerase and 1 µM each of the two
primers at 94 C for 2.5 min, 55 C 30 sec, and 72 C for 5 min in each
amplification cycle. The pair of oligonucleotides were prepared
corresponding to the published nucleotide sequence at positions
1124–1143 and 2266–2285 for rat Ah receptor (18). The isolated
PCR-synthesized cDNA fragments were subcloned into T easy and
characterized by nucleotide sequencing analysis. The sequence of the
cDNA fragment was identical to the published sequence of rat Ah
receptor cDNA.
Preparation of cRNA probe
Rat FSH-R cDNA was subcloned into the EcoRI site of
the Bluescript KS (+) vector and linearized with HindIII
(16). Digoxigenin-labeled FSH-R cRNA probes corresponding to bases
239-2368 were produced by in vitro transcription with T7 RNA
polymerase and an RNA labeling kit (Roche Molecular Biochemicals). A digoxigenin-labeled GAPDH probe was obtained by
the same method. AhR cDNA fragment was subcloned into T easy vector and
linearized BamHI. Digoxigenin-labeled AhR cRNA probes
corresponding to bases 1267–2285 were produced by in vitro
transcription with Sp6 RNA polymerase. Digoxigenin-labeled
steroidogenic acute regulatory protein (StAR) cRNA probes corresponding
to based 749-1456 were produced with Sp6 RNA polymerase.
Isolation of nuclei
Granulosa cells were cultured in 60-mm dishes containing 5
x 106 cells in 5 ml serum-free medium. After the
first 24 h, granulosa cells were further incubated without FSH,
with FSH (30 ng/ml), and with a combination of FSH (30 ng/ml) plus TCDD
(10 pM) for 48 h before isolating the nuclei. Cells
were washed three times with ice-cold Dulbecco’s PBS without calcium
and magnesium [PBS (-)], collected by scraping in PBS (-), and then
centrifuged for 5 mm at 1000 rpm at 4 C. The cell pellet was
resuspended in 500 µl nonidet P-40 lysis buffer [10 mM
Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM
MgCl2, 0.5% nonidet P-40]. Lysed cells were
incubated on ice for 10 min and centrifuged for 5 min at 3000 rpm. The
nuclear pellet was then resuspended in 500 µl nonidet P-40 lysis
buffer and centrifuged for 5 min. The final nuclear pellet was gently
resuspended in 100 µl glycerol storage buffer [50 mM
Tris-HCl (pH 8.3), 40% glycerol, 5 mM
MgCl2, 0.1 mM EDTA (pH 8.0)], frozen
in liquid nitrogen, and stored at -80 C.
Run-on transcription assay
The nuclear run-on transcription assay was performed according
to a previously described protocol (19). The relative amount of
incorporation of label into specific RNAs was determined by DNA excess
filter hybridization, using cDNAs for rat FSH-R, as described
previously (19). Ten micrograms each of FSH-R, Bluescript, AhR, and
ß-actin cDNAs were included on the DNA filter during hybridization to
correct for background and to serve as internal controls.
Autoradiographic bands were quantified by a fluoro-image analyzer (BAS
2000, Fuji Photo Film Co., Ltd., Japan).
Transcription stability analysis
Cells were preincubated with FSH alone or FSH and TCDD for
24 h before the addition of 5 µM actinomycine-D to
arrest new RNA synthesis. Cells were harvested at 0, 3, 6, and 9 h
after the addition of the inhibitor, for RNA extraction and Northern
blot analysis.
Data analysis
The relative abundance of a 2.4 kb signal for rat FSH-R
and AhR mRNA in different preparations was quantified with a
LKB 2202 UnitroScan Laser Densitometer (LKB
Produkter AB, Minami-ashigara, Japan), normalized against levels
of GAPDH mRNA in each sample, and expressed as a percentage of the
control value. The data are presented as the mean ±
SE of measurements from triplicate cultures for one
representative experiment. Comparisons between groups were performed by
one-way ANOVA. The significance of differences between the mean values
in the control group and each treated group was determined by Duncan’s
multiple-comparison test. A value of P < 0.05 was
considered statistically significant.
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Results
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To examine the possible effect of TCDD on the acquisition of
FSH-R, granulosa cells were cultured in the absence or presence of FSH
(30 ng/ml), with or without TCDD (10 pM) for 72 h
(Fig. 1
). The treatment with FSH
produced, as expected, a substantial increase in FSH-R mRNA, whereas
concurrent treatment with TCDD resulted in a significant decrease in
FSH-R mRNA after being cultured from 24–72 h. As shown in Fig. 2, cotreatment with FSH (30 ng/ml) and
increasing doses of TCDD inhibited the levels of FSH-induced FSH-R mRNA
in a dose-dependent manner. To further characterize the cellular
mechanisms underlying the interaction between FSH and TCDD, we next
investigated the effect of TCDD on the cAMP-induced FSH-R mRNA.
Treatment with 8-Br-cAMP produced a significant increase in FSH-R mRNA,
whereas concurrent treatment with TCDD (10 pM) produced
significant attenuation of 8-Br-cAMP action (Fig. 3).
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Figure 1. The effect of TCDD over time on the FSH-induced
FSH-R mRNA. A, Granulosa cells from DES-primed immature rats
were cultured alone for 24 h. These cells were then further
incubated without FSH, with FSH (30 ng/ml), and with a combination of
FSH (30 ng/ml) plus TCDD (10 pM). After various incubation
times, total RNA was extracted, and FSH-R mRNA levels were measured,
using Northern blot analysis, as described in Materials and
Methods. B, Luminesence detection of FSH-R mRNA (2.4 kb) was
quantified by densitometric scanning. The amount of FSH-R mRNA cultured
(24 h) was taken as 100%. Data were normalized for GAPDH mRNA levels
in each sample and expressed as a value relative to the control. The
absorbency values obtained from this experiment and those from two
other experiments were standardized in relation to the control and are
represented (mean ± SE; n = 3) in the bar
graphs. The data shown are means ± SE of the
three independent experiments. *, Difference from the control value at
P < 0.05; **, difference from the control value at
P < 0.01.
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Figure 2. Dose-related effect of TCDD on FSH-induced FSH-R
mRNA. A, Granulosa cells from DES-primed immature rats were cultured
for 24 h alone and were then cultured with 30 ng/ml FSH alone and
then FSH (30 ng/ml) plus increasing concentrations of TCDD for 48
h. FSH-R mRNA levels were measured using Northern blot analysis, as
described in Materials and Methods. The Northern blot
analysis is representative of the three experiments. B, Luminesence
detection of FSH-R (2.4 kb) mRNA was quantified by densitometric
scanning. The amount of FSH-R mRNA cultured alone was taken as 100%.
Data were normalized for GAPDH mRNA levels in each sample and expressed
as a value relative to the control. The absorbency values obtained from
this experiment and those from the two other experiments were
standardized in relation to the control values and are represented
(mean ± SE; n = 3) in the bar graphs.
The data shown are means ± SE of three independent
experiments. *, Difference from the control value at
P < 0.05.
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Figure 3. Effect of TCDD on 8-Br-cAMP-induced FSH-R
mRNA. A, Granulosa cells from DES-primed immature rats were cultured
for 24 h alone and were then cultured with 1 mM
8-Br-cAMP and 1 mM 8-Br-cAMP plus 10 pM TCDD
for 48 h. FSH-R mRNA levels were measured using Northern blot
analysis. The Northern blot analysis is representative of the three
experiments. B, Luminesence detection of FSH-R mRNA (2.4 kb) was
quantified by densitometric scanning. The amount of FSH-R mRNA cultured
alone was taken as 100%. Data were normalized for GAPDH mRNA levels in
each sample and expressed as a value relative to the control. The
absorbency values obtained from this experiment and those from the two
other experiments were standardized to the control and are represented
(mean ± SE; n = 3) in the bar
graphs. *, Difference from the control value at
P < 0.05.
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To determine whether the TCDD-attenuated FSH binding was caused by
decreased binding capacity, decreased binding affinity, or both,
granulosa cells were cultured in the presence of FSH, with or without
TCDD, at the 10-pM level for 3 days. Scatchard plot
analysis showed that concurrent treatment with TCDD resulted in 30%
inhibition in granulosa cell FSH binding capacity, whereas no
difference could be detected in the binding affinity of its receptors
[dissociation constant (Kd) =
4.79 ± 0.33 x 10-10 mean ±
SE (FSH) vs. 5.02 ± 0.30 x
10-10 (FSH+TCDD)]. These findings suggest that
the ability of TCDD to attenuate the FSH binding of FSH-stimulated
granulosa cells is attributable to a decrease in binding capacity but
not to affinity of FSH-R (Fig. 4).
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Figure 4. Scatchard plot is shown for rat granulosa cells
cultured for 72 h in FSH (30 ng/ml) or FSH (30 ng/ml) plus TCDD
(10 pM). B/F, Bound to free ratio.
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We next examined whether TCDD regulation of FSH-R mRNA is dependent on
gene transcription and/or receptor mRNA stability. The following set of
experiments were designed to address the potential contribution made by
changes in FSH-R gene transcription to the regulation of FSH-R mRNA. To
assess the rates of degradation of FSH-R mRNA transcripts, granulosa
cells were preincubated with FSH alone or with FSH and TCDD for 24
h. After this preincubation period, 5 µM actinomycin-D
was added to arrest new RNA synthesis. Cells were harvested at 0, 3, 6,
and 9 h after the addition of the transcription inhibitor, and
FSH-R mRNA levels were quantitated by Northern blot analysis. The
amount of FSH-R mRNA at time zero (the time of addition of
actinomycin-D) in each treatment group was assigned a value of 100%,
and all other values in each treatment group at different time-points
were expressed as a percent of this value. As shown in Fig. 5, the decay curves for the 2.4-kb FSH-R
mRNA transcript in the cells were not significantly different in the
absence and presence of TCDD.
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Figure 5. Effect of TCDD on FSH-R mRNA transcripts. A,
Granulosa cells were preincubated with FSH alone or FSH and TCDD for
48 h. After this preincubation period, 5 µM
acinomycin-D was added to arrest new RNA synthesis. Cells were
harvested at 0, 3, 6, and 9 h after the addition of the
transcription inhibitor, and FSH-R mRNA levels were quantitated by
Northern blot analysis. B, The mRNA levels at time zero were assigned a
relative value of 100%, and mRNA levels at all other times are
expressed as percentages of this value.
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To determine whether TCDD has an effect on FSH-R gene transcription in
the presence of FSH, we performed nuclear run-on assays on granulosa
cells. As shown in Fig. 6, FSH (30 ng/ml)
significantly enhanced the activity of FSH-R gene transcription, and
the presence of TCDD caused significant decrease in FSH-R
transcription.
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Figure 6. Stimulation of FSH-R gene transcription by
FSH and TCDD. A, Granulosa cells were cultured in 60-mm dishes
containing 5 x 106 cells in 5 ml serum-free medium.
After 24 h in culture, granulosa cells were further incubated in
the presence or absence of TCDD (10 pM) for 24 h, and
nuclear run-on assays were then performed as described in
Materials and Methods. B, Data acquired from the nuclear
run-on experiments shown in A were quantitated by a fluoro-image
analyzer (BAS 2000). Data were normalized for actin levels in each
sample and are expressed relative to the control value. The data shown
are means ± SE of three independent experiments. *,
Difference from the control value at P < 0.05.
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The aryl hydrocarbon receptor (AhR) is widely distributed in
vertebrates and is known to be involved in the metabolism of
xenobiotics, including man-made chemicals, most of which act as a
ligand for the receptor, although endogenous ligand has yet been
known. It has been suggested that all of the pleiotropic biological
effects of TCDD are mediated by the AhR (20, 21). In addition, Northern
blot analysis revealed the existence of Ah receptor transcript (5.2 kb)
in this cultured cell (Fig. 7). To
examine the possible effect of TCDD on the AhR, granulosa cells were
cultured in the presence of FSH (30 ng/ml), with or without TCDD (10
pM) for 48 h (Fig. 7). TCDD (10 pM) did
not affect the expression of AhR mRNA.
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Figure 7. Effect of TCDD on AhR mRNA. A, Granulosa cells
were cultured for 24 h alone and were then cultured with 30 ng/ml
FSH alone and FSH (30 ng/ml) plus TCDD (10 pM). After
various incubation times, total RNA was extracted, and AhR mRNA levels
were measured using Northern blot analysis as described in
Materials and Methods. B, Luminesence detection of AhR
mRNA (5.2 kb) mRNA was quantified by densitometric scanning. The amount
of AhR mRNA cultured (0 h) was taken as 100%. Data were normalized for
GAPDH mRNA levels in each sample and expressed as a value relative to
the control. The absorbency values obtained from this experiment and
those from the two other experiments were standardized in relation to
the control values and are represented (mean ± SE;
n = 3) in the bar graphs.
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The treatment with FSH produced, as expected, a substantial
increase in FSH-R mRNA, whereas concurrent treatment with TCDD resulted
in a significant decrease in FSH-R mRNA after being cultured from
24–72 h. Northern blot analysis of rat StAR mRNA revealed two major
transcripts of about 3.6 kb and 1.6 kb, respectively. Treatment with
8-Br-cAMP (1 mM) produced a significant increase in StAR
mRNA, whereas concurrent treatment with TCDD (10 pM) did
not affect 8-Br-cAMP action on the induction of StAR mRNA (Fig. 8).
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Figure 8. Effect of TCDD on 8-Br-cAMP-induced StAR mRNA. A,
Granulosa cells from DES-primed immature rats were cultured for 24
h alone and were then cultured with 1 mM 8-Br-cAMP and 1
mM 8-Br-cAMP plus 10 pM TCDD for 3 h. StAR
mRNA levels were measured using Northern blot analysis. The Northern
blot analysis is representative of the three experiments. B,
Luminesence detection of StAR mRNA (3.6 kb) was quantified by
densitometric scanning. The amount of StAR mRNA cultured with 1
mM 8-Br-cAMP was taken as 100%. Data were normalized for
GAPDH mRNA levels in each sample and expressed as a value relative to
the control. The absorbency values obtained from this experiment and
those from the two other experiments were standardized to the control
and are represented (mean ± SE; n = 3) in the
bar graphs.
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Discussion
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In female rats, in utero and lactational exposure
to TCDD resulted in a number of reproductive abnormalities in
adulthood, including reduced ovarian and brain weight, vaginal
deformities, delayed puberty (22), and (as reported by subsequent
experiments) decreased serum estrogen concentrations (3). Other studies
in rats, as well as in nonhuman primates (6, 7, 8, 9), have also shown
adverse effects of steroid hormone production after an in
vivo administration of TCDD. By using a system of purified human
luteinized granulosa cells (LGC), it was possible to address the direct
effects of TCDD at the single-cell level. However, there is often great
variability found when using human LGC from women undergoing ovarian
stimulation for in vitro fertilization, and this may be the
cause of the observed discrepancies between the investigators. A recent
study, using human LGC, showed that in vitro administration
of TCDD was capable of altering E2 secretion into culture
medium. These effects, however, were not consistent, with respect to
the dose of TCDD and the length of time in culture (23).
A widely used model system by which to study the effect of gonadotropin
on the ovary has been primary cultures of rat granulosa cells obtained
from hypohysectomized or immature female rats that had been pretreated
with estradiol (11, 12). The use of this defined culture system has
shown that the ability of FSH to stimulate the induction of FSH-Rs is
mediated, at least in part, by cAMP, because exogenous cAMP or other
agents that increase intracellular levels of cAMP mimic the actions of
FSH (13). The response of FSH-R mRNA to cAMP analogs was inhibited by
TCDD in granulosa cells in this experiment, suggesting that TCDD
diminished the action of FSH at sites distal to cAMP generation in the
granulosa cells. In this study, therefore, we examined the TCDD’s
effect on the FSH-induced FSH-R expression and investigated the
mechanisms of its actions.
At concentrations of TCDD that are considered to be
environmentally relevant (10 pM), TCDD significantly
inhibited FSH-induced FSH-R mRNA expression in rat granulosa cells at
24 h. Our preliminary data for TCDD supports previously reported
in vivo data and provides, for the first time, information
regarding the molecular mechanisms by which TCDD affects reproductive
functioning. The present observations indicate that the ability of TCDD
to attenuate FSH hormonal action also involves post-cAMP site(s). The
observed decrease of message levels by TCDD may be a result of
decreased FSH-R gene transcription and/or message stability. Data from
the nuclear run-on assays demonstrated that the effect of TCDD on the
FSH-induced FSH-R mRNA is brought about, at least in part, by
transcriptional mechanisms. Future studies will be aimed at determining
whether certain elements are involved in the observed TCDD regulation
of FSH-R gene transcription. It will be equally important to identify
trans-acting factors as well as any other cis-acting
elements involved.
AhR is widely distributed in vertebrates and is known to be involved in
metabolism of xenobiotics, including man-made chemicals, most of which
act as a ligand for the receptor, although no endogenous ligand has yet
been known. The induction mechanism by the AhR complex has been most
extensively studied on the CYP1A1 gene, because induction of the gene
by TCDD is so strong that it can be easily detected by the conventional
methods for gene expression. Although a list of genes other than those
for the drug-metabolizing enzymes whose expression is modulated by the
AhR complex is not yet available, it has been reported that the
expression of several genes with interesting functions was induced by
TCDD (24). These genes include those for plasminogen activator
inhibitor 2 and interleukin 1B, which are important for cell growth and
differentiation. We now show, for the first time, AhR’s presence in
the rat granulosa cells, by Northern blot analysis. Evidence of this
kind may also suggest a direct role of TCDD’s antifertility effects.
Moreover, at a concentration of TCDD that atenuated FSH action on
FSH-R, TCDD did not inhibit AhR expression in rat granulosa cells.
Therefore, it is unlikely that the decrease of mRNA for FSH-R might be
attributed to a generalized degradation of cellular RNA during the
incubation with the environmental relevant dose of TCDD (10
pM).
Other studies in rats, as well as in nonhuman primates (6, 7, 8, 9), has also
shown adverse effects on steroid hormone production after an in
vivo administration of TCDD. The rate-limiting step in steroid
hormone biosynthesis is the rate of transport of the substrate
cholesterol from the outer mitochondrial membrane, as well as cellular
pools to the inner mitochondrial membrane where P450scc locates (25, 26). Recently, StAR has been identified as a protein that is an acute
regulator of the rate-limiting transfer of cholesterol to the inner
mitochondrial membrane (27). In addition, Lin and co-workers (28) have
shown that patients suffering from congenital lipoid adrenal
hyperplasia, in which steroid synthesis from the adrenal glands and
gonads is severely impaired, have a mutation in the StAR gene.
Thus, StAR appears to be pivotal in the transfer of cholesterol to the
inner mitochondrial membrane, the proposed rate-limiting step in
steroidgenesis. The present data showed that the effect of TCDD on
steroidogensis does not involve cholesterol transport to the inner
mitochondrial membrane and is attributable to the down-regulation of
gonadotropin receptor, which is essential to response to the
gonadotropin. Although this study has provided novel information about
the effect of TCDD on the regulation of FSH-R mRNA, much more work
needs to be done to precisely define the mechanism of TCDD’s
actions on the granulosa cells.
|
Acknowledgments
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|---|
We thank the National Hormone and Pituitary Agency, National
Institute of Arthritis, Diabetes and Digestive and Kidney Diseases,
University of Maryland School of Medicine for the rat FSH.
|
Footnotes
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1 This work was supported by grants from the Ministry of Education,
Science and Culture of Japan (10044235, 10877253), Tokyo, Japan. 
Received September 10, 1999.
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Abstract
Introduction
