Biological roles of angiotensin II via its type 2 receptor
during rat follicle atresia
Eri
Kotani1,
Masataka
Sugimoto2,
Hachiroh
Kamata2,
Nobuharu
Fujii2,
Masahiro
Saitoh1,
Satoshi
Usuki1,
Takeshi
Kubo1,
Keifu
Song3,
Mizuo
Miyazaki3,
Kazuo
Murakami2, and
Hitoshi
Miyazaki2
2 Gene Experiment Center,
Institute of Applied Biochemistry, and
1 Department of Obstetrics and
Gynecology, Institute of Clinical Medicine, University of Tsukuba,
Ibaraki 305-8572; and
3 Department of Pharmacology,
Osaka Medical College, Osaka 569, Japan
 |
ABSTRACT |
Type 1 angiotensin II (ANG II) receptors play
crucial roles in the regulation of blood pressure and fluid osmolarity,
whereas the physiological roles of type 2 ANG II receptors
(AT2) remain unclear. Because
AT2 is expressed in atretic
follicles where granulosa cells undergo apoptosis, we examined the
space and time relationship between
AT2 expression and follicle
atresia in vivo and the effect of
AT2 on follicle-stimulating
hormone (FSH) actions in vitro. Binding studies, autoradiography, and
RT-PCR of AT2 revealed that the
AT2 content in granulosa cells was
time dependently increased at both protein and mRNA levels in equine
chorionic gonadotropin-treated immature female rats. This increase
paralleled the progression of atresia. ANG II suppressed FSH-caused
prevention of DNA fragmentation, increases in luteinizing hormone
receptor content, and estrogen production through
AT2 in cultured granulosa cells.
Moreover, FSH-induced stimulation of extracellular signal-regulated
kinase activity, critical for cell survival, was inhibited by
AT2 stimulation. These results
suggest that AT2 mediates the
progression of follicle atresia through granulosa cell apoptosis by
inhibiting FSH actions.
ovarian renin-angiotensin system; follicle-stimulating hormone; luteinizing hormone receptor; estrogen; polycystic ovary syndrome
 |
INTRODUCTION |
ANGIOTENSIN (ANG) II is a vasoactive peptide that
exerts profound hypertensive effects through actions on various tissues and organs such as the vasculature and the adrenal glands (1). Recent
pharmacological and molecular biological studies have identified two
distinct subpopulations of ANG II receptors. ANG II receptor type 1 (AT1) is widely distributed
throughout adult tissues, whereas ANG II receptor type 2 (AT2) is abundantly expressed
throughout fetal tissues but decreases dramatically and rapidly after
birth (1, 8, 14, 21, 22, 28). Most of the known physiological functions
of ANG II, such as regulation of blood pressure and fluid osmolarity,
are mediated by AT1. In contrast,
the functional roles of AT2 are
not yet apparent.
The granulosa layer and theca interna in ovarian follicles contain ANG
II receptors that are predominantly
AT2 (12, 18, 27, 29). In addition,
human follicular fluid contains angiotensinogen, prorenin, ANG I, and
ANG II, the concentration of which is more than 11 times greater than
that found in plasma (3, 7). The concentration of rat ovarian ANG II is
also 8- to 75-fold higher than that found in plasma and is not reduced
in bilaterally nephrectomized rats (12). These data indicate that ANG
II is locally produced in the ovary, but the physiological roles of ANG
II via AT2 in the ovary have not
yet been defined. Several reports suggest that ANG II is involved in
the regulation of follicle atresia where granulosa cells undergo
apoptosis. Daud et al. (5) first showed that
AT2 is exclusively expressed in
atretic follicles of adult rats during all stages of the estrous cycle,
whereas AT1 is associated with
other ovarian structures including blood vessels. Pucell et al. (26)
demonstrated that AT2 expression is inhibited in rat cultured granulosa cells by follicle-stimulating hormone (FSH), a crucial factor for survival and differentiation of
granulosa cells, whereas the expression of most receptors for hormones
and growth factors related to follicle maturation, such as epidermal
growth factor, luteinizing hormone (LH), and prolactin, is induced by
FSH (9). We recently demonstrated that the
AT2 content in cultured granulosa
cells increases under conditions in which apoptosis of the cells is
observed (30). Kitzman and Hutz (15) indicated that ANG II treatment of
hamster follicles cultured in vitro reduces the estradiol-to-androgen
ratio and induces morphological changes in the theca interna that are
observed in atresia. Mukhopadhyay et al. (20) showed that levels of the renin precursor prorenin in follicular fluid are associated with follicle atresia in the bovine ovary. Regardless of these findings, no
concrete evidence has shown that ANG II regulates the onset and/or progression of follicle atresia via
AT2. This is because the
localization of AT2 and the change
in its expression level with the onset and progression of follicle
atresia are not clear, and the effect of ANG II via
AT2 on the roles of FSH also
remains obscure.
Recent reports indicated that FSH activates extracellular
signal-regulated kinases (ERK1 and ERK2) in rat and porcine cultured granulosa cells (2, 4). ERKs belong to the mitogen-activated protein
kinases (MAPKs), which are activated in response to a variety of
stimuli involved in cell proliferation, survival, and differentiation
(17). For example, withdrawal of nerve growth factor (NGF), a survival
and differentiation factor for neural cells, from rat PC12
pheochromocytoma cells led to inhibition of ERKs, resulting in
apoptosis of these cells (31). Also, experiments with PC12 cells
suggested that the difference in the duration of ERK activation may be
critical for decisions about proliferation vs. differentiation (17).
Therefore, the effect of ANG II via AT2 on FSH-induced ERK activation
must be very important for determining the fate of granulosa cells.
Moderate doses of equine chorionic gonadotropin (eCG) stimulate the
growth and development of ovarian follicles for 2-3 days, after
which the follicles undergo atresia because of waning levels of trophic
support caused by metabolism of the gonadotropin (24). This eCG-induced
model shows follicle atresia morphologically identical to that
occurring in untreated adult animals (23). In the present study, we
examined AT2 expression at both
the mRNA and protein levels during the onset and progression of atresia in vivo with this rat model. Furthermore, we studied the effect of ANG
II on the abilities of FSH to prevent apoptosis and to differentiate
ovarian granulosa cells and to activate ERKs in vitro prepared from
immature female rats treated with diethylstilbestrol (DES) or eCG.
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MATERIALS AND METHODS |
Reagents. Ovine FSH was
kindly provided by the National Institutes of Health (Bethesda, MD).
This compound (NIH-FSH) is also known as NIDDK-oFSH-15 or AFP-5529C.
The potency of NIH-FSH is 20 times greater than that of
NIH-FSH-S1. By weight, the LH potency of NIH-FSH is 0.04 times that of NIH-LH-SI, and that of prolactin is less than 0.001 times
that of NIH-PRL. eCG, DES, Hanks' balanced salt solution (HBSS), and
myelin basic protein were purchased from Sigma (St. Louis, MO).
Modified McCoy's 5A medium (pH 7.4) was prepared with McCoy's 5A
(GIBCO Life Technologies) supplemented with 25 mM HEPES, 26 mM
NaHCO3, 2 mM
L-glutamine (Sigma), 100 µg/ml
streptomycin, and 100 IU/ml penicillin (GIBCO Life Technologies). ANG
II, leupeptin, and antipain were purchased from the Peptide Institute
(Osaka, Japan). The nonpeptide antagonists DuP-753 and PD-123319 were
provided by Dr. R. D. Smith (DuPont Merck Pharmaceutical, Wilmington
DE) and Dr. J. A. Keiser (Warner-Lambert, Ann Arbor, MI),
respectively. The selective
AT2 ligand CGP-42112B was provided by Dr. Marc de Gasparo (Ciba-Geigy, Basel, Switzerland). The
radioligands [
-32P]dCTP,
[
-32P]ATP, and
125I-[Sar1,Ile8]ANG
II were obtained from Amersham (Bucks, UK) and
125I-human chorionic gonadotropin
(125I-hCG) was obtained from New
England Nuclear (Boston, MA).
Hormone administration. Immature
female Wistar-Imamichi rats obtained from the Imamichi Institute for
Animal Reproduction (Ibaraki, Japan) were housed in air-conditioned
quarters with a 12:12-h light-dark cycle. For in vivo experiments,
23-day-old rats were subcutaneously injected with 20 IU of eCG for
3-6 days. The rats were killed by decapitation under mild ether
anesthesia on each of days
3-6
after injection, and the ovaries were removed. For
autoradiographic studies, ovaries were snap-frozen in a dry ice-isopentane bath and stored at
80°C. For in situ
3'-end labeling analysis, ovaries were immersion-fixed in 10%
buffered formalin for 7 days at room temperature. Granulosa cells were
prepared as described by Knecht et al. (16) to extract total RNA and for binding studies. For in vitro experiments, one set of rats was
implanted subcutaneously with Silastic 10-mm capsules containing DES at
21 days of age to stimulate granulosa cell proliferation. The
other set of animals (23 days old) was subcutaneously injected with 20 IU of eCG to induce preovulatory follicles. Five days after DES
implantation and 48 h after eCG injection, the animals were
anesthetized with ether and decapitated, and then the ovaries were removed.
Preparation of granulosa cells.
Granulosa cells were prepared according to the method of Knecht et al.
(16). Briefly, after the intercellular gap junctions were disrupted
with 6.8 mM EGTA and 0.5 M sucrose, granulosa cells were released by
puncturing the ovaries with a 27-gauge needle and pressing the
remaining cells through a 40-mesh stainless steel grid. Thereafter,
cells were washed and pelleted by gentle centrifugation (200 g, 5 min, room temperature) and then
snap-frozen in liquid nitrogen at
80°C until DNA and total
RNA extraction. The remaining cells were resuspended in McCoy's 5A
medium in 12 × 75-mm polystyrene tubes and were cultured in an
incubator at 37°C with a humidified gas mixture of 5%
CO2-95% room air for 48 h with or
without various additives.
In subsequent experiments of MAPK activity, the granulosa cell cultures
were plated for 48 h in 10% fetal calf serum and cultured for an
additional day in serum-free media.
In situ 3'-end labeling. Ovaries
were paraffin-embedded, sectioned at 8-µm thickness from the center,
and mounted on microscope slides. The sections were deparaffinized by
heating for 30 min at 60°C followed by two 5-min washes in xylene
and were rehydrated through a graded ethanol series and
double-distilled water. Protein was removed from tissues by an
incubation with 20 µg/ml proteinase K (Sigma) for 15 min at room
temperature; then endogenous peroxidase was inactivated by immersion in
3%
H2O2
in ethanol for 5 min. DNA was labeled at 3'-ends with
biotinylated 2'-deoxyuridine 5'-triphosphate biotin-16-dUTP; Boehringer Mannheim, Mannheim, Germany) by
an incubation with reaction buffer consisting of 300 mM
Tris · HCl (pH 7.4), 140 mM
(CH3)2AsO2Na,
and 1 mM CoCl2 containing terminal deoxynucleotidyl transferase for 30 min at 37°C. The sections were
incubated for 1 h at 37°C in Vectastain avidin-biotin
complex-peroxidase standard solution (Vector Laboratories, Burlingame,
CA). Diaminobenzidine (Dojin, Kumamoto, Japan), a
substrate for peroxidase, was applied to the slides for the color
reaction, and then sections were stained with hematoxylin and eosin.
Negative controls were sections incubated with reaction mixtures in the
absence of terminal deoxynucleotidyl transferase.
ANG II receptor autoradiography.
Ovarian sections (20 µm) were cut in a cryostat, attached to
gelatinized slides (Wako Pure Chemical, Osaka, Japan), air-dried at
4°C, and stored at
80°C. Sections were incubated for 15 min at room temperature in incubation buffer containing 0.2% BSA and
0.5 mg/ml bacitracin (Nakarai Tesque, Kyoto, Japan) and were placed for
1 h at room temperature in incubation buffer containing 0.5 nM
125I-[Sar1,Ile8]ANG
II, 0.2% BSA, and 0.5 mg/ml bacitracin with or without several agonists and antagonists. The sections were then sequentially rinsed in
ice-cold wash buffer (10 mM
Na2HPO4/150
mM NaCl/5 mM EDTA/0.02% NaN3)
four times and dried under a stream of cold air for 1 h. Slides were
exposed to X-ray film (Eastman Kodak, Rochester, NY) for 5 to 6 days
and then stained with hematoxylin and eosin.
DNA isolation and analysis.
Low-molecular-weight DNA was extracted from granulosa cells isolated
from the ovaries of rats killed on each of
days
3-6
after eCG injection or from cultured granulosa cells. Cells were gently
homogenized in a 300 mM Tris · HCl (pH 7.4)
containing 100 mM NaCl, 10 mM EDTA, and 200 mM sucrose, and then
incubated in 0.5% SDS at 65°C for 30 min to facilitate
membrane and protein disruption. The samples were cooled on ice for 60 min in 1 M potassium acetate to precipitate protein and were clarified
by centrifugation at 5,000 g for 10 min at 4°C. The supernatant was then extracted with
phenol-chloroform, precipitated in ethanol, and resuspended in TE
buffer (10 mM Tris · HCl-1 mM EDTA, pH 8.0). After
RNase digestion (100 µg/ml) at 37°C for 60 min, samples were
again extracted with phenol-chloroform, precipitated in ethanol, and
resuspended in water. Thereafter, DNA samples (4 µg) were resolved
through 2% agarose gels, stained with ethidium bromide, and visualized
under ultraviolet light.
Quantitative RT-PCR analysis of
AT2
mRNA. Total RNA (1.2 µg) isolated from granulosa
cells was reverse transcribed with random primers. The resultant cDNA
mixtures were amplified by PCR to selectively detect
AT2 mRNA in the presence of a
known amount of deletion-mutated
AT2 cDNA with a trace amount of
[
-32P]dCTP with the
following primers (6): 5'-TATGCTCAGTGGTCTGCT-3' (sense
primer, nucleotides 610-627 of a cDNA for rat
AT2) and 5'-CCACTAACAGATTTAAGACAC-3' (antisense primer, nucleotides
1,084-1,104) (14, 21). Denaturation, annealing, and the polymerase
reaction proceeded at 94°C for 1 min, at 55°C for 1 min, and at
72°C for 1.5 min, respectively. After 20 cycles of amplification,
the incubation was continued at 72°C for another 8.5 min to
complete polymerization. Native
AT2 cDNA produces a 495-bp
fragment, whereas deletion-mutated AT2 cDNA generates a fragment of
425 bp. The PCR products were size-fractionated on 5% acrylamide gels.
The gels were dried and examined with a BAS-2000 imaging analyzer (Fuji
Film, Tokyo, Japan). The radioactivity of the PCR product obtained from
the target cDNA was divided by that from the deletion-mutated cDNA. The
resultant values were normalized to the amount of
-actin mRNA, which
was also determined in the same method as
AT2 mRNA at the same time, for
correcting variations in RNA loading and RT-PCR efficiency.
Binding of
125I-[Sar1,Ile8]
ANG II. Cells (1 × 106 cells/tube) were immersed in
acid buffer (50 mM glycine-150 mM NaCl, pH 3.0) on ice for 10 min,
washed twice with HBSS, incubated with 4 mM DTT at 22°C for 15 min,
and then reacted with
125I-[Sar1,Ile8]ANG
II at a final concentration of 50 pM in 200 200 µl of binding buffer
consisting of HBSS containing 0.2% crystallized BSA, 1 mM
phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, and 25 µg/ml antipain at 4°C for 2 h. Nonspecific binding was determined by incubating with 1 µM unlabeled
[Sar1,Ile8]ANG
II. Free and bound radioligands were separated by centrifugation, and
then radioactivity was assayed by
-scintillation counting.
Binding of
125I-hCG. After
culture, cells (1 × 106
cells/tube) were immersed in acid buffer, washed twice with HBSS, and
then reacted with 125I-hCG at a
final concentration of 10 ng/ml in 200 µl of binding buffer at
22°C for 17 h. Nonspecific binding was determined by incubating the
cells with 25 µg/ml unlabeled hCG. Free and bound radioligands were
separated by centrifugation; then radioactivity was assayed by
-scintillation counting.
Measurement of estradiol-17
.
Estradiol-17
was measured with an enzyme immunoassay (EIA) kit
(Cayman Chemical, MI). The cross-reactivity of the antiserum used in
the estradiol-17
assay was 100% for estradiol-17
, 7.5% for
estrone, 0.3% for estriol, 0.1% for testosterone and
dihydrotestosterone-5
, and <0.01% for
C21 and other
C19 and C18 steroids. The minimum
detectable amount of the steroid assay was 9.2 pg/ml. Intra- and
interassay coefficients of variation were <10%.
MAPK activity. Control and various
additive-treated granulosa cell cultures were rinsed with three changes
of ice-cold phosphate-buffered saline (pH 7.4) and immediately scraped
on ice in lysis buffer [20 mM HEPES, pH 7.2, 25 mM NaCl, 2 mM
EGTA, 1 mM EDTA, 0.2 mM DTT, 0.1% Triton X-100, 50 mM NaF, 1 mM
Na3VO4,
25 mM
-glycerophosphate, and protease inhibitor mixture tablet
(Boehringer Mannheim)]. Cell lysates were centrifuged at 12,000 g for 10 min at 4°C. Supernatants were removed, and protein concentrations were assayed with the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). MAPK
activity within the supernatant was analyzed with the in-gel kinase
method. Cell extracts (25 µg total protein/lane) were separated by
10% SDS-polyacrylamide gel electrophoresis with 0.5 mg/ml of myelin
basic protein copolymerized in the running gel. After electrophoresis, the gel was washed twice for 30 min with 50 mM Tris (pH 8.0) and 20%
isopropyl alcohol followed by two changes of a buffer containing 50 mM
Tris (pH 8.0) and 5 mM
-mercaptoethanol
(buffer
1). The gel was then treated with 6 M guanidine HCl in buffer
1 for 1 h to denature the proteins.
The kinases in the gel were renatured with
buffer
1 containing 0.04% Tween 40 for 16 h
at 4°C, and then incubated with 40 mM HEPES buffer (pH 7.5)
containing 2 mM DTT, 0.1 mM EGTA, and 20 mM
MgCl2 for 1 h. The kinase assay
was performed by layering 10 ml of the same buffer containing 25 µCi
of [
-32P]ATP onto
the renatured gel followed by incubation at 25°C for 1 h.
Thereafter, the gel was washed with 5% trichloroacetic acid and 1%
sodium pyrophosphate to remove unreacted
[
-32P]ATP.
Thereafter, the gel was dried and examined with a BAS-2000 imaging analyzer.
Data analysis. Data are presented as
means ± SE of several independent experiments. The effects of
various treatments on different culture groups were compared by one-way
ANOVA, followed by the post hoc Student-Newman-Keuls test. A difference
of P < 0.05 was considered significant.
 |
RESULTS |
Effects of eCG on ovarian morphology and DNA
fragmentation. The apoptic death of granulosa cells is
the molecular mechanism underlying follicle atresia (10). We initially
examined the time course of the effect of eCG injection on ovarian DNA
degradation, a characteristic of apoptosis, by treating immature female
rats with 20 IU eCG at 23 days of age. The electrophoretic profile of
low-molecular-weight DNA fragments obtained from granulosa cell lysates
on each of days
3-6
after eCG injection is shown in Fig. 1.
Granulosa cells obtained 3 days postinjection lacked signals indicating
low-molecular-weight DNA. However, light fragments were discernible on
day
4. On
days
5 and
6, oligonucleosomal length DNA
fragments formed an obvious ladder.

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Fig. 1.
Electrophoretic analysis of DNA fragments in granulosa cells obtained
on days
3-6
after injection with 20 IU equine chorionic gonadotropin (eCG).
Low-molecular-weight DNA (4 µg) extracted from granulosa cells was
loaded onto a 2% agarose gel, resolved by electrophoresis at 100 V,
and visualized by ultraviolet transillumination. Data represent 3 separate experiments. Similar results were obtained in 2 other
experiments. Lanes
1, 2,
3, and
4 represent postinjection
days
3, 4,
5, and
6, respectively.
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To study the specific ovarian cell types exhibiting apoptotic DNA
degradation after eCG injection, DNA on histological sections of
ovaries was 3'-end labeled with biotin-16-dUTP as shown in Fig.
2. Incorporation of biotin-16-dUTP after
the terminal transferase reaction was detected with avidin conjugated
to peroxidase. In ovaries obtained 4 but not 3 days after eCG
injection, low levels of DNA were 3'-end labeled in granulosa
cells in a small population of atretic follicles. In contrast, in
ovaries obtained on days 5 and
6, biotin-16-dUTP was incorporated
into the granulosa cell layer of many atretic follicles. Although
oocytes were also labeled in some follicles, theca cells were not
labeled in the same follicles where granulosa cells were heavily
labeled. In follicles with antral cavities, antral granulosa cells were
frequently labeled more heavily than mural granulosa cells. In some of
these follicles, the antral cavity was also positive, probably because
of the presence of cell-free DNA fragments.

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Fig. 2.
In situ 3'-end labeling of DNA on ovarian sections from rats
obtained on days
3-6
after injection with 20 IU eCG. Ovaries were fixed in 10% buffered
formalin, and DNA on sections (8 µm) was in situ 3'-end labeled
with biotinylated 2'-deoxyuridine 5'-triphosphate
(biotin-16-dUTP). Incorporated biotinylated biotin-16-dUTP was detected
by avidin conjugated to peroxidase, as described in
MATERIALS AND METHODS. Sections were
examined by light microscopy at 100×
(top) and 400×
(bottom) magnification. gc,
Granulosa cells; tc, theca cells; ic, interstitial cells; solid arrows,
healthy multilaminar granulosa cell layer; open arrows, degenerating
cumulus oophorus complex; arrowheads, detached granulosa cell layer;
* antral cavity.
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Effects of eCG on
AT2 content at
the protein and mRNA levels. To investigate the levels
of AT2 during the onset and
progression of atresia, granulosa cells were obtained from rats on each
of days
3-6
after eCG injection. To specifically detect
AT2, cells were exposed to DTT,
which considerably decreases the ligand binding activity of
AT1 and enhances that of
AT2 without affecting antagonist selectivity (18). Figure 3 shows that the
binding of
125I-[Sar1,Ile8]ANG
II, a subtype-nonselective antagonist, was noticeably and time
dependently increased 3-6 days after eCG injection.

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Fig. 3.
125I-[Sar1,Ile8]ANG
II binding to AT2 in granulosa
cells obtained on days
3-6
after injection with 20 IU eCG. Binding of
125I-[Sar1,Ile8]ANG
II was assayed in granulosa cells isolated from ovaries without culture
as described in MATERIALS AND METHODS.
Data are means ± SE (vertical bars) of 3 independent experiments,
each performed in duplicate.
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Because follicles containing AT2
in the adult rat ovary are mainly atretic throughout the estrous cycle
(5), we studied the cellular localization of
AT2 during atresia by
autoradiography of
125I-[Sar2,Ile8]ANG
II binding. To investigate the possibility of heterogeneous ANG II
receptor types localizing in the ovaries of rats injected with eCG, we
used the selective nonpeptide ANG II receptor antagonists DuP-753 for
AT1 and PD-123319 for
AT2. Figure
4, top,
shows that ANG II receptors in granulosa cells were
AT2, whereas those on other
ovarian structures were AT1. As
indicated in Fig. 4, bottom, the
radioligand binding localizing in the granulosa cell layer was markedly
and time dependently increased from
days
3 to
6.

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Fig. 4.
Binding sites for
125I-[Sar1,Ile8]ANG
II in ovaries obtained on days
3-6
after injection with 20 IU eCG. Ovarian sections (20 µm) were
prepared as described in MATERIALS AND
METHODS.
A-D:
autoradiograms of 4 adjacent ovarian sections 6 days after injection of
eCG. A, total
125I-[Sar1,Ile8]ANG
II binding; B,
125I-[Sar1,Ile8]ANG
II binding in presence of PD-123319 (5 µM) to selectively block
AT2;
C,
125I-[Sar1,Ile8]ANG
II binding in presence of DuP-753 (5 µM) to selectively block
AT1;
D, nonspecific
125I-[Sar1,Ile8]ANG
II binding in presence of ANG II. Nonspecific radioligand binding was
negligible.
E-H:
autoradiograms of ovaries obtained 3-6 days after injection with
20 IU eCG. E,
F, G,
and H represent postinjection
days
3, 4,
5, and
6, respectively.
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To determine the levels of AT2
mRNA during follicle atresia, total RNA was extracted from ovaries on
each of days
3-6
after eCG injection and analyzed by reverse transcription (RT)-PCR. Figure 5 shows that the
AT2 mRNA level increased from
days
3 to 6 in a time-dependent fashion.
Therefore, the time course of the increased
AT2 content at both protein and
mRNA levels agrees well with that of the progression of follicle
atresia, suggesting a close relationship between
AT2 and atresia.

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Fig. 5.
Content of AT2 mRNA in granulosa
cells obtained on days
3-6
after injection with 20 IU eCG.
AT2 mRNA in uncultured granulosa
cells isolated from ovaries was examined by RT-PCR as described in
MATERIALS AND METHODS.
A: representative autoradiogram
showing native and deletion-mutated cDNA encoding
AT2
(top) and -actin
(bottom).
B: quantification of
AT2 mRNA. One arbitrary unit
indicates value of AT2 mRNA level
3 days after eCG injection. Data are means ± SE (vertical bars) of
3 independent experiments, each determined in triplicate.
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Effects of ANG II on FSH actions in cultured granulosa
cells. To determine the biological roles of ANG II via
AT2 in follicle atresia, we
studied the effects of ANG II on the actions of FSH in
cultured granulosa cells, because FSH is a crucial differentiation and
survival factor for these cells. We focused on the abilities of FSH to
prevent apoptosis (10), to induce the LH receptor expression (9), and
to enhance steroidogenesis (9).
As shown in Fig. 6, DNA prepared from rats
2 days after eCG injection was cleaved in serum-free media during a
48-h culture. ANG II did not affect DNA fragmentation. Although FSH
decreased apoptotic DNA fragmentation compared with untreated controls, this decrease was prevented by ANG II.

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Fig. 6.
Effect of ANG II on DNA fragmentation in cultured granulosa cells from
ovaries of immature rats 2 days after eCG injection. Granulosa cells
were cultured in presence or absence of follicle-stimulating hormone
(FSH) (200 ng/ml) with or without ANG II (1 µM) for 48 h.
Low-molecular-weight DNA extracted from cells was loaded on to a 2%
agarose gel, resolved by electrophoresis and visualized by ultraviolet.
Data represent 3 separate experiments. Similar results were obtained in
2 other experiments. Lane
1, control;
lane
2, ANG II;
lane
3, FSH;
lane
4, FSH plus ANG II.
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Figure 7 shows the effect of ANG II on
125I-hCG binding to the LH
receptor of granulosa cells prepared from DES-treated rats. After a
48-h incubation, FSH caused an 8.5-fold increase in
125I-hCG binding compared with
untreated controls (P < 0.05). In contrast, ANG II inhibited the FSH-stimulated binding of
125I-hCG by 48%
(P < 0.05). ANG II-induced
suppression of this FSH effect was reversed by the
AT2-selective antagonist PD-123319 (P < 0.05) but not by the
AT1-selective antagonist DuP-753.

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Fig. 7.
Effect of ANG II on luteinizing hormone receptor content in cultured
granulosa cells isolated from ovaries of immature rats treated with
diethylstilbestrol (DES). Granulosa cells were cultured in presence (+)
or absence ( ) of FSH (50 ng/ml) or ANG II (100 nM) with or
without DuP-753 (10 µM) and PD-123319 (10 µM). Binding of
125I-labeled human chronionic
gonadotropin (125I-hCG) was
assayed as described in MATERIALS AND
METHODS. Data are means ± SE (vertical bars) of 3 independent experiments, each performed in triplicate.
* P < 0.05.
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Similarly, Fig. 8 indicates the effect of
ANG II on estrogen production in cultured granulosa cells. After a 48-h
incubation, FSH induced a 2.4-fold increase in estrogen production
compared with untreated controls (P < 0.05). ANG II suppressed FSH-stimulated estrogen production by 36%
(P < 0.05). This ANG II effect was reversed by PD-123319 (P < 0.05) but
not by DuP-753.

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Fig. 8.
Effect of ANG II on estradiol-17 production in cultured granulosa
cells from ovaries of immature rats treated with DES. Experimental
conditions were identical to those in Fig. 7. Enzyme immunoassay was
performed as described in MATERIALS AND
METHODS. Data are means ± SE (vertical bars) of 3 independent experiments, each performed in triplicate.
* P < 0.05.
|
|
ERKs (ERK1 and ERK2) are key enzymes in the regulation of a variety of
cellular events, including cell differentiation, survival, and
apoptosis. To know the mechanism of the inhibitory effects of ANG II on
FSH actions described above, we measured FSH-induced ERK activation in
the presence or absence of ANG II by in-gel kinase assay. The
activities of both ERK1 and ERK2 were rapidly increased after FSH
treatment, with a peak at 2 min, and declined to basal levels within 30 min (data not shown). ANG II inhibited FSH-stimulated ERK activities
(data not shown). To selectively investigate the role of
AT2, we examined the effect of the
AT2-specific agonist CGP-42112B on
FSH-stimulated ERK activities. CGP-42112B is known to act as an agonist
for AT2 but as an antagonist for AT1 at a concentration in this
study (1 µM). As shown in Fig. 9, after a
2-min incubation, FSH induced a 1.8-fold and a 1.6-fold increase in
ERK1 and ERK2 activities, respectively
(P < 0.05), whereas CGP-42112B
inhibited these FSH-caused increases
(P < 0.05). This CGP-42112B effect
was reversed by PD-123319 (P < 0.05).

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Fig. 9.
Effect of CGP-42112B on FSH-stimulated extracellular signal-regulated
kinase (ERK) 1 and ERK2 activities in cultured granulosa cells from
ovaries of immature rats treated with DES. Serum-starved granulosa
cells were incubated at 37°C in presence (+) or absence ( )
of FSH (100 ng/ml) or CGP-42112B (1 µM) with (+) or without ( )
PD-123319 (100 µM) for 2 min. Cell lysates were subjected to in-gel
analysis of ERK1 and ERK2 activities as detailed in
MATERIALS AND METHODS.
A: representative autoradiogram
showing ERK1 and ERK2 activities in each treatment situation. Nos. at
right indicate molecular mass in kDa.
B: quantification of ERK1 and ERK2
activities. Data are means ± SE (vertical bars) of 5 independent experiments.
* P < 0.05.
|
|
 |
DISCUSSION |
In addition to gonadotropins, several growth factors, cytokines, and
vasoactive peptides are implicated in the regulation of follicle
development and atresia. In contrast to the regulatory mechanisms
underlying follicle development, little is known about the regulation
of atresia, which is the ultimate fate of most follicles. The
vasoactive peptide ANG II is one of such ovarian factors. Ovarian ANG
II receptors are predominantly
AT2, but their physiological roles
remain to be resolved. Therefore, the ovary is a useful
model with which to investigate novel roles of the renin-angiotensin
system (RAS) through AT2,
functions that are unrelated to the regulation of blood pressure and
fluid osmolarity. However, although
AT2 is predominantly expressed in
atretic follicles, AT2 has not
been conclusively implicated in the regulation of atresia. In this
study, we examined immature model rats injected with eCG, in which
ovarian follicles undergo growth and development with subsequent
atresia, in addition to investigating granulosa cells in primary
culture. Our data suggest that AT2
stimulation causes the progression of follicle atresia through
granulosa cell apoptosis by inhibiting FSH actions, mainly because of
the following findings. First, the
AT2 content in vivo was increased
at both protein and mRNA levels in parallel with the progression of
follicle atresia. This increase was not observed before the onset of
atresia. If AT2 is involved in the
fate of follicles, considerable levels of
AT2 should be detected in some
healthy follicles. Thus the implication of
AT2 in the progression of atresia
is more likely than in the onset of atresia. Second,
AT2 stimulation inhibited the in
vitro actions of FSH, which plays crucial roles for survival and
differentiation of granulosa cells. That is, FSH-induced DNA fragmentation and FSH-caused increases in the LH receptor content and
estrogen production were all inhibited. Also,
AT2 stimulation inhibited
FSH-induced ERK activation, enzymes that are critical for cell survival
and differentiation. Enhancement of estrogen biosynthesis and the
increase in the LH receptor content are the parameters for
FSH-stimulated differentiation of cultured granulosa cells (9).
In contrast to our findings, several reports (25, 33, 34) indicate that
ANG II may be involved in the induction of follicle development,
steroidogenesis, oocytes maturation, and ovulation, rather than with
atresia. For example, ANG II stimulated estrogen production, oocytes
maturation, and ovulation in in vitro-perfused rabbit ovaries, and
these ANG II effects were mediated by
AT2. No convincing rationale can
explain this discrepancy, but a species difference might have some
relevance. An autoradiographic study has demonstrated a high degree of
localization of AT2 in the
granulosa cell layers and the stroma of rabbit preovulatory follicles
(33, 34), whereas our results and those of another (5) revealed intense
signals for AT2 on atretic
follicles in the rat ovary. Also, the rabbit estrous cycle differs from
cycles of rats and humans. That is, copulatory stimulation induces
ovulation in the rabbit. Pucell and co-workers (25, 27) have also
demonstrated that ANG II stimulated estrogen secretion in quartered
ovaries from eCG-treated immature rats and that ANG II did not
affect aromatase activity in cultured granulosa cells prepared from
DES-treated rats. Our data are not in agreement with these findings.
This can be explained by experimental conditions. Pucell et al. (27) showed that ANG II (1-100 nM) does not affect FSH (100 ng/ml)-induced estrogen production. We examined the effect of ANG II on
FSH-stimulated estrogen production in the presence of 50, 100, and 200 ng/ml FSH with or without 1, 10, or 100 nM ANG II (data not shown). FSH
significantly stimulated estrogen production in a
concentration-dependent manner. ANG II did not affect the estrogen
production induced by 100 or 200 ng/ml FSH, whereas 100 nM ANG II
suppressed the increase in estrogen production induced by 50 ng/ml FSH.
Nevertheless, our findings that the increase in
AT2 expression exhibits close space and time correlation with follicle atresia in vivo and that AT2 mediates
inhibition of FSH actions in vitro are consistent with the fact that
AT2 is predominantly
expressed in atretic follicles of adult rats at all stages of the
estrous cycle. It has been proposed that ANG II receptors would become
undetectable in healthy follicles because of downregulation or by
blockade of the binding of exogenous radiolabeled ANG II in receptor
binding studies, because preovulatory follicles contain high levels of
endogenous ANG II. However, we found that
AT2 mRNA levels increased in
parallel with the progression of atresia, suggesting that this notion
is unlikely.
The AT2-mediated induction of
apoptosis in granulosa cells is consistent with the results of several
investigations. For example, Kakuchi et al. (13) found by in situ
hybridization that sites of AT2
expression overlap closely with those of a specific group of cells
undergoing apoptosis during nephrogenesis in fetal mice. Yamada et al.
(32) reported that ANG II induces the apoptosis of PC12W cells cultured
in the presence of low concentrations of NGF via
AT2. Xia et al. (31) also found
that withdrawal of NGF from PC12 cells led to inhibition of ERK
activity, resulting in apoptosis of these cells.
Androgen is a potential atretogenic factor (10). Estrogen is thought to
prevent the apoptosis of granulosa cells, in part by suppressing the
atretogenic effect of androgen (11). We demonstrated that ANG II
inhibits FSH-induced estrogen production via
AT2 in granulosa cells. This
finding further supports the thought that AT2 is involved in follicle
atresia involving granulosa cell apoptosis. On the other hand, FSH
induces follicle maturation with an increase in the LH receptor
content. Preovulatory follicles expressing increased levels of LH
receptors ovulate in response to an LH surge. The present study showed
that this FSH-induced increase in the LH receptor content was
considerably inhibited by AT2. Therefore, AT2 stimulation should
decrease the response of follicles to LH, which would lead to a
decrease in the rate of ovulation and progression of atresia.
Some reports suggest that the ovarian RAS is associated in humans with
polycystic ovary syndrome (PCOS), which can be identified by various
symptoms such as chronic anovulation, inappropriate gonadotropin
secretion, and hyperandrogenism, depending on the patient (33). Basal
plasma levels of prorenin in PCOS patients are higher than those in
follicular-phase controls and significantly correlate with peripheral
androgen concentrations. Serum concentrations of total renin are also
particularly enhanced in women with PCOS. Moreover, both theca and
granulosa cells in large cystic follicles of PCOS intensely immunostain
for renin and ANG II, as do granulosa cells of atretic follicles of
normal ovaries. These findings raised the possibility that increased
levels of ANG II in the ovary may contribute to the cause of PCOS
through AT2. The
estrogen-to-androgen ratios are similarly low in follicles from PCOS
and in atretic follicles from normal ovaries. The number of LH
receptors in cell samples from cystic follicles obtained by wedge
resection from patients with PCOS is significantly lower than that in
normal preovulatory follicles. These findings suggested that
dysregulation of the RAS is involved in the development of PCOS,
because AT2 may be able to induce
follicle atresia with a low ratio of estrogen to androgen and low
levels of LH receptors as described above. The ovarian RAS may be one
target to consider when designing a strategy to treat such functional
ovarian disorders.
Recent studies indicated that AT2
activates protein tyrosine phosphatase, serine-threonine phosphatase,
and the delayed rectifier K+
current and inhibits T-type Ca2+
channels (1, 19). However, the signaling pathways of
AT2 are still far from being
completely understood. Our data revealed that stimulation of
AT2 inhibits FSH-induced ERK
activation in granulosa cells. Further studies are necessary to gain
insight as to how AT2 mediates
apoptosis and how AT2 mediates
inhibition of ERK activities and to understand the relation between
apoptosis and ERK activities in these cells. Ovarian granulosa cells
provide a useful tool for studying not only unknown signaling pathways of AT2 but also novel function of
the RAS.
 |
ACKNOWLEDGEMENTS |
We thank the Hormone Distribution Office of National Institute of
Diabetes and Digestive and Kidney Diseases for providing ovine FSH
(NIH-FSH-15) and Norma Foster for help in preparing the manuscript.
 |
FOOTNOTES |
This work was supported by a grant-in-aid from the Ministry of
Education, Science, and Culture of Japan and by a grant from Tsukuba
Advanced Research Alliance of the University of Tsukuba.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: H. Miyazaki, Gene Experiment Center,
Univ. of Tsukuba, Ibaraki 305-8572, Japan.
Received 18 June 1998; accepted in final form 4 September 1998.
 |
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