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Endocrinology Vol. 139, No. 11 4679-4687
Copyright © 1998 by The Endocrine Society

ARTICLES

Induction of Ad4BP/SF-1, Steroidogenic Acute Regulatory Protein, and Cytochrome P450scc Enzyme System Expression in Newly Established Human Granulosa Cell Lines1

Kumiko Hosokawa, Ada Dantes, Carolina Schere-Levy, AMIHAI Barash, Yoshio Yoshida, Fumikazu Kotsuji, Israel Vlodavsky and Abraham Amsterdam2

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 µM of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20{alpha}-hydroxy-4-pregnen-3-one (20{alpha}-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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HUMAN GRANULOSA cells are subjects of extensive research due to their crucial role in successful reproduction. Surrounding and nursing the oocyte, they support its maturation. The high level of steroid hormone secretion by these cells ensures a receptive environment for the implantation and development of the early embryo (for reviews see Refs. 1, 2, 3, 4). Numerous reports on granulosa cells obtained from women participating in in vitro fertilization (IVF) programs confirm that these cells become highly steroidogenic due to their previous overstimulation with gonadotropic hormones (5, 6, 7). Freshly prepared cells fail to show consistent response to human CGs (hCG)/LH; no response to FSH was observed in short-term cultures (8, 9, 10), probably due to their refractory state as a consequence of intensive in vivo gonadotropin stimulation. However, prolonged culture of the cells in gonadotropin-free medium reestablished responsiveness to both FSH and LH/CG (11), as was evident by cAMP accumulation and production of estradiol and progesterone.

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).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
pSVBam contains the entire SV40 genome (24). pEJ.6.6 encodes activated human Ha-ras oncogene (25). pLTRp53cGval135 contains a chimera of mouse p53 complementary DNA and genomic DNA, including introns 2–9, under the transcriptional control of a Harvey sarcoma virus long terminal repeat. It encodes a mutant protein with a substitution from alanine to valine at position 135, which is temperature sensitive and possesses wild-type activity at 32 C, but not at 37 C (26).

Antibodies
Antibodies against pregnenolone, progesterone, and 20{alpha}-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-{alpha}-OH-progesterone and progesterone measurement. Pregnenolone, progesterone, and 20{alpha}-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{alpha}-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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Progesterone production and expression of SV40 large T antigen and p53 in the newly established human granulosa cell lines
Primary cultures of human granulosa cells were triply transfected with SV40 DNA, Ha-ras oncogene, and the ts mutant of p53 (p53val135), 48 h after plating the cultures; 41 clones were isolated, expanded, and analyzed for their steroidogenic response following stimulation with 50 µM FK for 24 h at 37 C. While levels of progesterone production in nonstimulated cultures were <0.20 ng/106 cells, the amount of progesterone in the different cell lines varied from 0.5 ng/106 cells (e.g. HO-9, HO-12) to approximately 100 ng/106 cells in HO-13 and HO-23 cells. The latter amounts were 10 times higher than in the rat granulosa cell line, GTS-5 transfected with similar plasmids (P < 0.001) and 40 times higher than the rat granulosa cell line POGRS-1 transfected with SV40 DNA and Ha-ras oncogene alone (P < 0.001) (Fig. 1AGo). 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. 1BGo). 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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  		Figure 1. Progesterone production and expression of SV40 large T antigen and p53 in human and rat immortalized granulosa cells. A, Cells were incubated for 24 h with 50 µM FK at 37 C in serum-free medium (DMEM/F12 1:1) and progesterone release to the medium was measured by RIA. Values are means ± SD for three culture plates. Values with asterisks in the appropriate cell lines were significantly different from HO-23 cells (*, P < 0.001; ***, P < 0.05). The amount of progesterone in nonstimulated cells of all lines was <0.1 ng/106 cells/24 h. B, Western blot analysis of large T antigen and tumor suppressor gene product p53. Cell extract proteins were resolved on 15% SDS/polyacrylamide gel and electrotransferred to a nitrocellulose membrane. The nitrocellulose membrane was reacted with monoclonal antibody PAb419, directed against SV40 large T antigen, or monoclonal antibody PAb421 directed against p53. Protein interaction with the specific antibodies was visualized by the ECL reaction. Molecular weight markers in kilodaltons are indicated. Arrowheads show the expected position of SV40 large T antigen at 90 kDa and p53 at 53 kDa. HO-9, HO-12, HO-13, and HO-23 are human granulosa cell lines established by triple transfection with SV40 DNA, Ha-ras oncogene and the ts mutant of p53 (p53val135). GTS-5 is a rat granulosa cell lines transfected with the same DNAs (27 ). POGRS-1 is a rat granulosa cell line transfected with SV40 DNA and Ha-ras oncogene (35 ).

 
Steroidogenic response in HO-23 cell line
We examined the dose response to FK in a typical highly steroidogenic cell line (HO-23). A characteristic dose-response curve was obtained, reaching saturation at 100 µM of FK. The calculated ED50 was 17 ± 3 µM and at 50 µM, a concentration used in most of the experiments, 84% saturation was achieved (Fig. 2Go).



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  		Figure 2. Dose response in HO-23 cells to stimulation with increasing concentrations of FK. HO-23 cells were incubated for 24 h at 37 C in the absence and presence of increasing concentrations of FK. Progesterone released into the medium was assayed by RIA. Data are means ± SD.

 
We further examined in HO-23 cells whether induction of progesterone production is accompanied by elevation of its precursor pregnenolone and its first steroid metabolite 20{alpha}-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{alpha}-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{alpha}-OH-progesterone (P < 0.005), suggesting that progesterone was the main steroid hormone produced in these cells (Fig. 3Go).



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  		Figure 3. Steroidogenesis in HO-23 cells. Cells were incubated for 24 h at 37 C in the absence (-) or presence (+) of 50 µM FK. Pregnenolone, progesterone, and 20{alpha}-OH-progesterone released into the medium were assayed by RIA. Data are means ± SD (n = 3). *, P < 0.001 compared with nonstimulated cultures.

 
To examine the steroidogenic response of HO-23 cells to stimulants that are known to affect primary granulosa cells, we incubated HO-23 cells for 24 h with different combination of stimulants. The cells did not respond to hCG even at high concentrations of 800 ng/ml. However, they clearly responded to 50 µM FK and to 1 mM 8-Br-cAMP, which stimulated 3.6-fold higher progesterone production than FK (P < 0.001) (Fig. 4Go). 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. 4Go) 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. 4Go).



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  		Figure 4. Modulation of progesterone production by cAMP, steroid hormones, growth factors, and ECM. HO-23 cells were incubated for 24 h at 37 C in serum-free medium without any stimulant (CONT) or in the presence of 50 µM FK, 1 mM 8-Br-cAMP, FK + 100 nM dexamethosone; FK + 100 nM hydrocortisone; FK + 100 nM testosterone; FK + 100 nM estradiol; FK + 10 ng/ml IGF-I; FK + 10 ng/ml insulin; FK + 10 ng/ml PRL; FK + 10 ng/ml bFGF; FK + 100 nM TPA; FK + extracellular matrix (ECM) deposited by bovine corneal endothelial cells; or FK + bFGF + ECM. Data are means ± SD for duplicate assays in triplicate plates. *, **, ***, Values are different from cultures stimulated by FK alone P < 0.001, P < 0.005, P < 0.05, respectively. Values of progesterone in cultures stimulated with steroid hormones, growth factors, TPA, or ECM alone were below 0.2 ng/105 cells/24 h at 37 C.

 
Insulin-like growth factor I (IGF-I) (10 ng/ml) and insulin (10 ng/ml and 1 µg/ml) did not enhance FK-stimulated progesterone production, whereas PRL (10 ng/ml) increased progesterone production by 1.2-fold (P < 0.05). On the other hand, bFGF (10 ng/ml) reduced progesterone production by 73%, whereas 12-O-tetradecanoylphorbol 13-acetate (TPA) reduced it by 83% (P < 0.001) (Fig. 4Go).

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. 5Go).



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  		Figure 5. Effect of insulin on FK-induced progesterone production in HO-23 cells. Cells were incubated for 24 h at 37 C in the absence or presence 1 µg/ml insulin (INS), with and without different doses of FK. Progesterone release into the medium was determined by RIA. The slight increase in progesterone production in the presence of insulin plus 20 µM FK (compared with FK alone) and the slight decrease in the presence of insulin plus 50 µM FK was found statistically insignificant [P > 0.1 (n = 3)].

 
Growing the cells on a native basement membrane deposited by bovine corneal endothelial cells (BCE/ECM) did not affect progesterone production (not shown) but enhanced cAMP-induced progesterone production by 2.3-fold (P < 0.001). Growing the cells on BCE/ECM partially blocked the inhibitory effect of bFGF on cAMP-induced progesterone production [only 35% reduction in progesterone production compared with FK-stimulated cells (P < 0.05)] (Fig. 4Go).

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. 4Go). 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. 6Go). 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. 6Go).



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  		Figure 6. Expression of proteins involved in steroid synthesis in HO-23 cells. Cells were incubated in serum-free medium in the absence of stimulants (CONT), in the presence of 50 µM FK, or in the presence of FK + 100 nM TPA for 24 h, at 37 C. Left panel, Western blotting of Ad4BP/SF-1, StAR and adrenodoxin. Molecular weight markers are indicated. Arrowheads indicate the expected position of Ad4BP/SF-1 at 52 kDa, StAR at 30 kDa, and ADX at 11 kDa. Right panel, Densitometric tracing of the same blots. Values of the densitometer tracing are means ± SD for three independent measurements. Values are different from control cultures (CONT). *, P < 0.001; **, P < 0.005. ADU, arbitrary densitometric units.

 
Localization of ADX in HO-23 cells
To reveal the morphology of HO-23 cells and the distribution of the steroidogenic mitochondria, we doubly stained the cells for nuclear DNA with DAPI, and with antiadrenodoxin for steroidogenic mitochondria, and visualized the cells under phase and fluorescent microscopy (Fig. 7Go). 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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  		Figure 7. Localization of adrenodoxin in HO-23 cells. Cells were cultured without stimulants (a, a') or with 50 µM FK (b, b'), 1 mM 8-Br-cAMP (c, c'), or 50 µM FK + 100 nM TPA (d, d') for 24 h at 37 C. At the end of the incubation period, cells were fixed with 3% paraformaldehyde and doubly stained with DAPI (a–d) and antiadrenodoxin (a'–d'). Dark lipid droplets, characteristic of granulosa cells, are evident in the perinuclear cellular region with all treatments (arrowheads). Nuclei are stained blue. Weak appearance of ADX is evident in nonstimulated cells (a'), and bright staining is evident following FK (b'), 8-Br-cAMP (c') and FK + TPA treatments (arrows). Rounding of the cells and clustering of mitochondria are seen following 8-Br-cAMP and FK + TPA treatments. a–d, Combined phase and fluorescence microscopy. a'–d', Fluorescence microscopy. Exposure time for a' photograph was 2-fold longer than b'–d' to visualize the mitochondria in nonstimulated cells. Original magnification x1000.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have succeeded in immortalization of highly differentiated human granulosa cells without losing their high steroidogenic capacity. Our earlier attempts using SV40 DNA and Ha-ras for transfection and immortalization of highly differentiated human granulosa cells obtained from an IVF program failed, probably because these cells, following excessive gonadotropin stimulation, are in their final differentiation stage (our unpublished results). However, transfection of the cells with mutated tumor suppressor gene p53val35 together with SV40 DNA and Ha-ras oncogene resulted in a successful immortalization of 41 lines of which some produced high levels of progesterone as primary cells from which they were derived following FK or 8-Br-cAMP stimulation (11). One possibility for the necessity of expression of the mutant p53 in immortalization may be by exhibiting a dominant negative effect (36) that may lead to suppression of the endogenous p53, which could arrest the cell cycle. Indeed, gel electrophoresis of metabolically labeled rat cells transfected with the ts p53 mutant show no expression of the endogenous p53 in the highly steroidogenic clones (27). The high capacity of steroidogenesis in the lines expressing the mutated p53 may be due, in part, to suppression of the T antigen, which is a transforming product of the SV40 genome (24). Indeed, cells transfected with SV40 alone (POGS-5) showed high expression of T antigen and low steroidogenic capacity (35, 37, 38). Also, we cannot exclude the possibility that cells expressing high levels of either normal or mutated p53 gain the property of inducible steroidogenesis in a mechanism not yet understood (39). As for the importance of expressing the ras protein in the immortalized cells, we demonstrated earlier that the expression of this protein was important for inducible steroidogenesis in immortalized rat granulosa cells (4, 33, 37). Moreover, we have previously demonstrated that corpus luteal cells contain higher amounts of ras protein than immature granulosa cells which are in their proliferative stage (40).

Because the substrate of progesterone is pregnenolone and it can be metabolized to 20{alpha}-OH-progesterone (15), we compared the accumulation of these three steroids following FK stimulation. Although the level of 20{alpha}-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.


   Acknowledgments
 
We thank Dr. A. M. Kaye for helpful discussion, Dr. W. L. Miller for generous provision of anti-ADX antibodies, Dr. J. F. Strauss III for anti-StAR antibodies, Dr. F. Kohen for antiprogesterone antibodies, Dr. K. Morohashi for anti Ad4BP/SF1 antibodies, Dr. D. P. Lane for anti-p53 antibodies, Dr. A. Yayon for generous provision of bFGF, and Mrs.V. Laufer for excellent secretarial assistance.


   Footnotes
 
1 This work was supported by grants from the Israel Academy of Sciences (to I.V. and A.A.), by the Israeli Ministry of Science (to A.A.), by the Leo and Julia Forchheimer Center of Molecular Genetics at the Weizmann Institute of Science (to A.A.), and by the Grants-in-Aid 0704424 from the Ministry of Education, Science and Culture of Japan (to F.K., K.H., and A.A.). Back

2 Incumbent of the Joyce and Ben B. Eisenberg Chair of Molecular Endocrinology and Cancer Research. Back

Received February 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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