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Department of Molecular Cell Biology, The Weizmann Institute of Science (K.H., A.D., C. S.-L., A.A.), Rehovot 76100, Israel; Department of Obstetrics and Gynecology, Kaplan Hospital (A.B.), Rehovot 76100, Israel; Department of Obstetrics and Gynecology, Fukui Medical University (K.H., Y.Y., F.K.), Fukui 910-1193, Japan; and Department of Oncology, Hadassah-Hebrew University Hospital (I.V.), Jerusalem 91120, Israel
Address all correspondence and requests for reprints to: Abraham Amsterdam, Department of Molecular Cell Biology, Weigmann Institute of Scince, Rehovot 761000, Israel. E-mail: lhamster{at}weizmann
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxy-4-pregnen-3-one (20-OH-progesterone) comparable with
amounts produced by highly differentiated primary human
granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment
the cAMP-stimulated progesterone production, whereas testosterone and
PRL enhanced cAMP-induced progesterone synthesis only moderately.
Estradiol, insulin-like growth factor I, and insulin showed no
significant effect on cAMP-induced steroidogenesis. The phorbol ester
TPA, and basic fibroblast growth factor, dramatically suppress
cAMP-induced production of progesterone, whereas bovine corneal
endothelial cell ECM (BCE/ECM) enhanced cAMP-induced progesterone and
antagonized basic fibroblast growth factor suppression of cAMP-induced
steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in
control cells, and its expression was augmented by FK, whereas the
steroidogenic acute regulatory protein showed low expression in the
nonstimulated cells but was clearly elevated upon cAMP stimulation and
was slightly decreased by TPA in cAMP-stimulated cells. Expression of
the electron carrier adrenodoxin (ADX), which is a part of the
cytochrome P450scc enzyme system, was very low in nonstimulated cells
but was dramatically elevated in FK- and 8-Br-cAMP-stimulated cells,
whereas no reduction of ADX was evident in cells costimulated with FK
and TPA. Immunocytochemical studies revealed a weak staining of ADX in
mitochrondria of nonstimulated cells and intensive staining in highly
clustered mitochondria of FK- or 8-Br-cAMP-stimulated cells. Only
moderate reduction in ADX staining was evident in cells costimulated
with FK and TPA. These unique cell lines can provide a useful model for
the investigation of induced steroidogenesis in human granulosa cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Basal progesterone production declined when granulosa-luteal cells obtained from IVF units were cultured in the absence of hCG (12, 13). Culturing such cells for several days on a native basement membrane produced by bovine corneal endothelial (BCE) cells increased their basal progesterone output as well as their steroidogenic response to hCG, compared with cells on uncoated culture dishes (14). Moreover, increasing formation of gap junctions, which serves as the anatomical basis for intercellular communication, was observed when cells were cultured on the ECM and stimulated by hCG (14). It was accepted that the rate-limiting step in steroid biosynthesis in mammalian steroidogenic tissue, including human granulosa cells, is the conversion of cholesterol to pregnenolone, catalyzed by the mitochondrial cytochrome P450scc enzyme system (15, 16). Nevertheless, it was discovered recently that the steroidogenic acute regulatory protein (StAR) is an essential and limiting factor in steroidogenesis, responsible for the transport of cholesterol into mitochondria (17). Pregnenolone, in turn, serves as the substrate for the formation of progesterone and aromatization of androgens to estrogen is then catalyzed by the P450 aromatase (15, 18). Steroidogenic transcription factor Ad4BP/SF-1 (19, 20) was found to be expressed in steroidogenic tissue including human granulosa cells obtained from IVF patients (11). However, no regulation of its expression in long-term cultures of human granulosa cells was demonstrated.
In recent reports, human granulosa cells obtained from IVF patients were immortalized with the E6 and E7 regions of the human papilloma viruses (21) or by SV40 large T antigen (22). These cells responded to 8-Br-cAMP, dibutyryl cAMP, or forskolin (FK) (21, 22), and aromatase messenger RNA levels were 4- to 5-fold after FK treatment (21). However, the control of expression of Ad4BP/SF-1 as well as StAR and the cytochrome P450scc enzyme system, believed to be limiting steps in steroidogenesis, were not characterized in human immortalized granulosa cells.
In the present paper, we characterize the steroidogenic regulation of newly established human granulosa cell lines in terms of steroid hormone production and expression of the cytochrome P450scc enzyme system, Ad4BP/SF-1 and StAR. Moreover, we demonstrate the effect of glucocorticoid hormones, growth factors, and ECM on cAMP-induced steroidogenesis. In the accompanying paper, we demonstrate the characteristic of the tumor suppressor p53- and cAMP- generated signals on apoptosis in these cells and cross-talk between the apoptotic and survival signals exerted by growth factors and ECM (23).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Antibodies
Antibodies against pregnenolone, progesterone, and
20-OH-progesterone were generous gifts of Dr. F. Kohen, Department
of Biological Regulation, Weizmann Institute of Science, Rehovot,
Israel. Goat antirabbit IgG coupled to horseradish peroxidase (HRP) was
obtained from Biomakor (Rehovot, Israel). Monoclonal antibody PAb419,
directed against SV40 large T antigen, was kindly provided by Dr. M.
Oren. p53-specific monoclonal antibody, PAb421 was kindly provided by
Dr. D. P. Lane (University of Dundee, Dundee, UK). Antibovine
Ad4BP/SF-1 antibodies were kindly provided by Dr. K. Morohashi (Okazaki
National Research Institutes, Okazaki, Japan). Antihuman adrenodoxin
antibodies were kindly provided by Dr. W. L. Miller
(University of California, San Francisco, CA). Antihuman StAR
antibodies were kindly provided by Dr. J. F. Strauss III
(University of Pennsylvania Medical Center, Philadelphia, PA).
Reagents
FK (a potent activator of adenylate cyclase), 8-Br-cAMP and 4',
6-diamido-2-phenylindole hydrochloride (DAPI, for DNA staining) were
purchased from Sigma Chemical Co. (St. Louis, MO). Highly
purified basic fibroblast growth factor (bFGF) was generously provided
by Dr. A. Yayon (Department of Molecular Cell Biology, Weizmann
Institute of Science, Rehovot, Israel).
Establishment of human granulosa cell lines
Transfection of granulosa cells. Granulosa cells were
obtained from women undergoing in vitro fertilization (IVF)
at Kaplan Hospital (Rehovot, Israel). Patients received a GnRH analog
(GnRH-a) in combination with FSH or human menopausal gonadotropin
(hMG), followed by administration of human CG (hCG). Granulosa cells
were isolated from aspirated follicular fluid after ovum retrieval. The
follicular fluid was centrifuged at 300 x g for 5 min
to separate granulosa cells from red blood cells. The resulting pellet
was resuspended and cultured in Nunc tissue culture dishes (100 mm)
with DMEM/Ham’s F12 (DMEM/F12) (1:1) containing 5% FCS, penicillin
(100 IU/ml), and streptomycin (100 µg/ml), for 48 h (11).
Primary cultures were washed 3 times in PBS to remove the remaining red
blood cells and transfected simultaneously with 2 µg of pSVBam, 5
µg of pEJ6.6, and 5 µg of p53val135, by the calcium phosphate
precipitation procedure (27).
Isolation of colonies.Densely growing foci of transformed cells were visualized and selected after 2 weeks and transferred to 24-well plastic culture dishes. After 4 days, stably growing cells were transferred to 60-mm plastic culture dishes and finally cultured in 100-mm culture dishes. The cells were collected, placed in freezing vials, and kept in liquid nitrogen (27).
Preparation of dishes coated with ECM
Cultures of bovine corneal endothelial (BCE) cells established
as described (28, 29, 30) were dissociated from stock cultures (passage
2–5), and plated (0.5 x 105 cells/dish) into 35-mm
tissue culture dishes in medium containing 5% dextran T-40 and no
bFGF. Six to eight days after the cells reached confluency, the
subendothelial ECM was exposed by dissolving the cell layer (3 min at
22 C) with a solution containing 0.5% Triton X-100 and 20
mM NH4OH in PBS, followed by four washes in PBS
(28, 29, 30). The BCE/ECM remained intact, free of cellular debris, and
firmly attached to the entire area of the tissue culture dish.
Biochemical assays
Pregnenolone, 20--OH-progesterone and progesterone
measurement. Pregnenolone, progesterone, and 20-OH-progesterone
accumulated in the culture medium were determined by RIA (27, 31) at
the end of cell stimulation.
Protein assay.Protein was quantified by the Bradford method (32).
Western blot analysis
Cells were washed with cold PBS and harvested with rubber
policemen using lysis buffer containing 50 mM HEPES (pH
7.2), 150 mM NaCl, 1.5 mM MgCl2, 1
mM EGTA, 1 mM PMSF, 1% Triton X-100, 10
µg/ml leupeptin, 10% glycerol, 30 mM NaF, 30
mM Na-pyrophosphate, 1 mM orthovanadate, and 5
µg/ml aprotinin. Lysates were boiled in sample buffer for 10 min.
Samples containing equal amounts of protein (25–40 µg) were
separated by 12% (to detect Ad4BP/SF-1 and StAR) or 15% (to detect
ADX) SDS-PAGE and transferred onto nitrocellulose membranes. As
positive control for Ad4BP, StAR, and ADX we used whole cell extract
from rat adrenal cortex (33), mitochondrial fraction of cAMP-stimulated
MA-10 mouse Leydig tumor cells (17) or purified bovine ADX (16),
respectively. The blots were then blocked using 5% milk powder in PBS
plus 0.05% Tween 20 and reacted (overnight, 4 C) with the
corresponding first antibody followed by 1 h incubation at room
temperature with goat antirabbit IgG conjugated to HRP. The detection
was carried out using the enhanced chemiluminescence (ECL) kit
(Amersham Ltd., Buckinghamshire, UK).
Phase contrast and immunofluorescent microscopy
Phase contrast and fluorescent microscopy of cells labeled with
antiadrenodoxin antibodies and DAPI was carried out as described
earlier (16, 34). Cultures were fixed with 3% paraformaldehyde in PBS
(pH 7.4) at 24 C and permeabilized for 4 min with 1% Triton X-100 in
PBS at 24 C. Cells were incubated for 60 min with rabbit
antiadrenodoxin antibodies followed by 30 min incubation with
FITC-labeled goat antirabbit antibodies solution containing 0.5 µg/ml
DAPI. Cells were washed intensively with PBS and mounted in Mowiol
(34). Microscopic examination of the specimens was carried out using a
Zeiss Axioskop microscope (Carl Zeiss, Oberkochen,
Germany) in both phase and fluorescent modes.
Statistical analysis
Analysis of pregnenolone, progesterone, 20-OH-progesterone,
and densitometer tracing was performed using the t test for
comparison of means (35). Differences between treatment groups were
considered statistically significant at P 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Expression of SV40 large T antigen
was low in HO-9, -12, -13, -23, and GTS-5 lines compared with high
amounts of p53, most of which is probably due to the expression of the
ts mutant protein because the expression of p53 in cells not
transfected with the ts mutant is considerably lower (Fig. 1B). The
expression of SV40 large T antigen in cells not transfected with the ts
mutant was considerably higher, suggesting that ts mutant expression
may lead to reduced amounts of SV40 large T antigen.
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).
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-OH-progesterone. Stimulation
with 50 µM FK for 24 h at 37 C resulted in a sharp
increase of at least 33-fold of pregnenolone, progesterone, and
20-OH-progesterone (P < 0.001) compared with their
levels in nonstimulated cells. The amount of progesterone production in
stimulated cells was 6.3-fold higher than pregnenolone and 8.2-fold
higher than 20-OH-progesterone (P < 0.005),
suggesting that progesterone was the main steroid hormone produced in
these cells (Fig. 3).
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). Dexamethasone (100 nM) or
hydrocortisone (100 nM) enhanced progesterone production in
FK-stimulated cells by 6.2-fold and 2.9-fold, respectively
(P < 0.001) (Fig. 4) but had no effect on basal
production of progesterone (not shown). Testosterone (100
nM) enhanced FK-stimulated progesterone by 1.4-fold
(P < 0.001), whereas estradiol (100 nM)
did not show any significant effect (Fig. 4).
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).
It should be noted that the lack of response to IGF and to insulin was
not due to a masking effect of 50 µM FK because 1 µg/ml
insulin did not exert any significant effect on FK induced production
of progesterone even in the presence of lower doses of FK (10 and 25
µM, Fig. 5).
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).
Expression of steroidogenic factors in HO-23 cells
The expression of Ad4BP/SF-1, StAR, and the electron carrier ADX
was examined by Western blots in nonstimulated cells and in cells
stimulated for 24 h with 50 µM FK in the absence or
presence of 100 nM of TPA.
The 53-kDa Ad4BP/SF1 was clearly expressed in nonstimulated cells and
was moderately augmented (1.9 times P < 0.005) in
FK-treated cells. No significant reduction in Ad4BP SF1 expression was
observed in cells costimulated with FK and TPA (Fig. 4). In contrast,
the expression of the 30-kDa StAR, which was low in control cells, was
enhanced by 5.4-fold (P < 0.001) in FK-treated cells,
and this effect was reduced (by 4.3-fold) (P < 0.005)
in cells costimulated with FK and TPA (Fig. 6). Nonstimulated cells show low
expression of the 11 kDa ADX. The expression of ADX was dramatically
elevated following FK stimulation (14 times) (P <
0.001) and remained constant in cells costimulated with FK and TPA
(Fig. 6).
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). Nonstimulated cells (a, a') showed
cytoplasmic processes. Dark granules, which probably represent lipid
droplets characteristic of granulosa cells, were located in the
perinuclear region. Mitochondria, showing a weak staining, were
abundant both in the perinuclear region and cell processes.
FK-stimulated cells (b, b') showed some shorter cell processes, and
mitochondria were intensely labeled both in the perinuclear region and
in cell processes. Cells treated with 8-Br-cAMP (c, c') completely
rounded up and the mitochondria were highly clustered in the
perinuclear region. Cells costimulated with FK and TPA (d, d') also
rounded up and showed no decrease in the fluorescence intensity of the
mitochondria.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the substrate of progesterone is pregnenolone and it can be
metabolized to 20-OH-progesterone (15), we compared the accumulation
of these three steroids following FK stimulation. Although the level of
20-OH-progesterone was much lower than progesterone in stimulated
cells as compared with immortalized rat granulosa cells transfected
with SV40 DNA and Ha-ras oncogene (35), it was far higher
than in control cells. The possibility that these cells can also
synthesize estradiol is being investigated.
The immortalized HO cell lines retain some of the properties of primary granulosa cells. Although they did not respond to gonadotropic hormones, they were highly responsive to FK and to 8-Br-cAMP. The lack of gonadotropin response is probably due to down-regulation of the expression of the receptors at the time of transfection, 24–48 h following extensive stimulation with hCG in vivo (11). Another possibility is that the cells lost the receptors due to transformation (35). At present we are conducting experiments transfecting these cells with either human LH/CG or human FSH receptors (41, 42).
HO-23 cells preserved their response to glucocorticoid hormones. This result is in line with the preservation of response to glucocorticoid hormones in SV40-ras transfected rat granulosa cells (4, 42, 43). Glucocorticoid hormones also enhance gonadotropin-/cAMP-stimulated progesterone production in primary granulosa cells (1, 2, 44). The response to testosterone in HO-23 cells suggests that the newly established immortalized human granulosa cells also preserve responses to androgen, similar to primary granulosa cells (1, 2). Because both mural and antral granulosa cells respond to testosterone (45), we cannot assign the HO-23 cells to either mural or antral preovulatory granulosa cells.
In spite of the rapid proliferation of nonstimulated HO-23 cells, they express a significant amount of the steroidogenic factor (Ad4BP/SF-1), which is characteristic of steroidogenic tissue (19, 20) and immortalized rat steroidogenic granulosa cells (33). The fact that cAMP only moderately elevates the expression of this factor in HO-23 cells is in line with the observation that Ad4BP/SF-1 appears at an early stage of gonadal development before the acquisition of steroidogenic activity (19, 20). In contrast, cAMP dramatically elevates both StAR and the electron carrier ADX, which is an intrinsic part of the P450scc enzyme system (15, 16, 43). This observation suggests that these cells preserve cAMP-induced steroidogenesis similar to primary granulosa cells. The phorbol ester TPA, which suppresses steroidogenesis, did not affect significantly Ad4BP/SF-1 and ADX expression. These data accord with the cytochemical analysis of ADX appearance in cells costimulated with cAMP and TPA. We have previously demonstrated that TPA did not suppress cAMP accumulation following FK stimulation of SV40-ras transformed granulosa cells but dramatically suppressed cAMP-induced progesterone production (35). Moreover, TPA was able to inhibit FSH-induced progesterone production both in primary (46, 47) and immortalized GFSHR-17 cells (48). The moderate suppression of StAR expression by TPA suggests that some of the cross-talk between the protein kinase C signaling pathway and cAMP-induced steroidogenesis is exerted via modulation of StAR (49). TPA was also found to inhibit progesterone production within less than 2 h following its addition to cultures prestimulated with FK (our unpublished results). This may suggest that protein kinase C activation could lead to rapid inhibition of progesterone production by posttranslational modification of Ad4BP/SF-1 or StAR, which may suppress their activities in steroidogenesis (50).
bFGF and extracellular matrix produced by bovine corneal endothelial cells were found to enhance basal and gonadotropin-/cAMP-induced progesterone production both in preovulatory rat and human granulosa cells (14, 51, 52). Here, in contrast to primary cells, growth of HO-23 cells in the presence of bFGF or on BCE/ECM had no effect on basal progesterone production. This is probably due to the fact that nonstimulated HO-23 cells show a very low expression of StAR and ADX. However, in cAMP-stimulated cells, whereas bFGF inhibited cAMP-stimulated steroidogenesis, most batches of BCE/ECM exerted a stimulatory effect. Because BCE/ECM contains a significant amount of sequestered bFGF (30), it may well be that yet unknown component of the ECM exert its stimulatory effect on granulosa cell steroidogenesis.
The establishment of HO human granulosa cell lines provides a novel system for inducible steroidogenesis. Moreover, the moderate response to PRL in enhancing cAMP-induced steroidogenesis suggests that these cells can provide a good model of luteinized cells particularly if the cells could be transfected with an expression vector for PRL receptors.
In the following paper (23), we demonstrate that HO-23 cells can undergo rapid and massive apoptosis after shifting the temperature of growth of cAMP-stimulated cells from 37 C to 32 C, which allows manifestation of the wild-type p53 activity. Therefore, HO cells can serve also as an interesting model for investigation of the mechanism of apoptosis in highly luteinized human granulosa cells.
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Acknowledgments |
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Footnotes |
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2 Incumbent of the Joyce and Ben B. Eisenberg Chair of Molecular
Endocrinology and Cancer Research.
Received February 26, 1998.
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