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(The FASEB Journal. 2003;17:1256-1266.)
© 2003 FASEB

Novel genes modulated by FSH in normal and immortalized FSH-responsive cells: new insights into the mechanism of FSH action

RAVID SASSON, ADA DANTES, KIMIHISA TAJIMA and ABRAHAM AMSTERDAM1

Department of Molecular Cell Biology, Weizmann Institute of Science Rehovot, Israel

1Correspondence: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 76100. E-mail: abraham.amsterdam{at}weizmann.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Follicle-stimulating hormone (FSH) controls the development of follicle-enclosed oocytes in the mammalian ovary by interacting with specific receptors located exclusively on granulosa cells. Its biological activity involves stimulation of intercellular communication, intracellular signaling, and up-regulation of steroidogenesis; the entire spectrum of genes regulated by FSH is not yet fully characterized. We have established monoclonal rat FSH-responsive granulosa cell lines that express FSH receptors at 20-fold higher rates than with primary cells, and thus increased the probability of yielding a distinct spectrum of genes modulated by FSH. Using Affymetrix DNA microarrays, we discovered 11 genes not reported earlier to be up-regulated by FSH and 9 genes not reported earlier to be down-regulated by FSH. Modulation of signal transduction associated with G-protein signaling, phosphorylation of proteins, and intracellular-extracellular ion balance was suggested by up-regulation of decay accelerating factor GPI-form precursor (DAF), membrane interacting protein RGS16, protein tyrosine phosphatase (PTPase), oxidative stress-inducible protein tyrosine phosphatase (OSIPTPase), and down-regulation of rat prostatic acid phosphatase (rPAP), Na+, K+-ATPase, and protein phosphatase 1ß. Elevation in granzyme-like proteins 1 and 3, and natural killer (NK) cell protease 1 (NKP-1) along with reduction in carboxypeptidase E indicates possible FSH-mediated preparation of the cells for apoptosis. Up-regulation of vascular endothelial growth factors indicates the ability of FSH to produce angiogenic factors upon their maturation; whereas, reduction in insulin-like growth factor binding protein (IGFBP3) indicates its increased potential to promote p53-induced apoptosis. Striking similarities in FSH modulation of gene expression were found in primary cultures of human granulosa cells obtained from IVF patients although these cells expressed only 1% of FSH receptor compared with immortalized rat cells, as indicated by microarray technique, which probably is in the normal range of expression of this receptor in nontransformed cells. These findings should increase our understanding of the mechanism of FSH action in stimulating development of the ovarian follicular cells, of intracellular and intercellular communication, and of increasing the potential of ovarian follicular cells to undergo apoptosis during the process of selection of the dominant follicle.—Sasson, R., Dantes, A., Tajima, K., Amsterdam, A. Novel genes modulated by FSH in normal and immortalized FSH-responsive cells: new insights into the mechanism of FSH action.


Key Words: ovary • apoptosis • DNA microarray • granzyme • steroidogenesis • human granulosa cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
GRANULOSA CELLS constitute the vast majority of follicular cells in the mammalian ovary. On the one hand they nurse the oocyte via gap junction communication; on the other hand, they are the main source of the female sex hormones estradiol and progesterone, which control the estrus cycle and reproduction (reviewed in refs 1 2 3 4 ). These cells are organized as multilayers around the oocyte (cumulus oophorus) and as a major part of the follicular wall (membrana granulosa). They are surrounded by a basement membrane separating the granulosa and the theca-interna cells (1 2 3) . Before ovulation, these cells and the oocyte do not have a direct connection with the nervous and immune systems, since the follicular antrum is filled with the follicular fluid, which is mainly devoid of the cellular components of the blood (5) . Therefore, it is reasonable to expect that granulosa cells adapted themselves in a unique way to communicate within their population and with the oocyte via gap junctions (6 , 7) . However, it is still unclear how they communicate with the immune and the nervous systems.

The development of granulosa cells is uniquely controlled by follicle-stimulating hormone (FSH) during folliculogenesis, causing them to proliferate and subsequently differentiate and contribute to the formation of the follicular antrum, secreting fluids, ions, and proteins characteristic of the follicular fluid, and finally to become steroidogenic via de novo synthesis of steroidogenic factors, steroidogenic enzymes, and transcription factors that control this process (reviewed in refs 1 2 3 4 ; see refs 8 9 10 11 12 ). This requires a dramatic plasticity in gene function, modulated predominantly by a single glycoprotein hormone via activation of a G-coupled 7 transmembrane domain receptor located uniquely on the membrane of granulosa cells (reviewed in ref 13 ). Therefore, it is important to analyze the genes and proteins modulated by FSH in a comprehensive manner. Such an analysis may yield a complete set of genes modulated by FSH that can explain the physiological role of granulosa cells in intra- and extrafollicular communication. Since there is a centrifugal gradient of differentiation among the granulosa cells (1 , 14) , this may obscure a possible shift in gene expression upon FSH stimulation when cellular mRNA obtained from these cells is analyzed by DNA microarray. Therefore, we decided to use immortalized granulosa cells that were uniquely engineered in our laboratory and demonstrate a clear response to gonadotropin/cAMP in steroidogenesis (15 16 17 18 19 20 21 22 23 24) , intercellular communication (25) , formation of follistatin (26) , synaptosome-associated protein-25 (SNAP-25) (27) , and synthesis of non-neuronal acetylcholine (28) as the source of mRNA for analyzing modulation of gene expression evoked by FSH. Since these cells are monoclonal, we expect them to respond in a homogeneous fashion to FSH stimulation.

Human granulosa cells (HGC) obtained from in vitro fertilization (IVF) patients after stimulation with gonadotropic hormones and retrieved from the follicular antrum during aspiration of the ovulated egg also represent a considerably homogeneous population of granulosa-lutein cells (29) . It was demonstrated that after 7 days of culturing these cells as monolayers in gonadotropin free medium, they recover from hormone desensitization and respond well in considerably uniform fashion to luteinizing hormone (LH)/chorionic gonadotropin (CG) and FSH stimulation (29 , 30) . We found these primary cultures adequate to be compared with immortalized granulosa cells in modulation of gene expression evoked by FSH stimulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Materials
Granzyme B antibodies were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Forskolin and 4', 6-diamido-2-phenylindole hydrochloride (DAPI, for DNA staining) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Recombinant hFSH was kindly provided by the National Institutes of Health and by Dr. A. Parlow of the National Hormone and Pituitary Program.

Cell culture
Rat cells
rFSH-17 cells (18) were cultured in DMEM/F12 medium containing 5% fetal calf serum (FCS) in 35 mm or 100 mm plastic culture dishes to 25% of confluence. Cultures were stimulated with either 0.2 IU/mL or 1.0 IU/mL FSH for 24 h at 37ºC. In some experiments, they were stimulated for an additional 24 h with 50 µM forskolin. For analysis of RNA by DNA microarray, cells at 50% confluence were incubated without or with 3 IU/mL of FSH for 8 h. B3Z T cells were kindly provided by Dr. L. Eisenbach at the Weizmann Institute of Science (31) .

Primary human granulosa cells
Primary granulosa cells were obtained from women undergoing IVF at Sheba Medical Center, Tel-Hashomer, Israel. Patients received a GnRH analog (GnRH-a) in combination with FSH or human menopausal gonadotropin (hMG), followed by administration of human chorionic gonadotropin CG (hCG) (29 , 30) . 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 in 10 mM Tris, 0.84% NH4Cl pH 7.4 to lyse red blood cells (15 min shaking at 37ºC). Several washings in phosphate-buffered saline (PBS) achieved elimination of the debris. Cells were plated in Dulbecco's modified Eagles' medium [DMEM/Ham's F12 (1:1)] supplemented with penicillin (100 IU/mL), streptomycin (100 µg/mL), and 5% FCS. Attachment of the granulosa cells to the bottom of the dishes (35 mm or 60 mm plastic dishes of NUNC Brand Products) was already achieved after 24 h at 37°C. Medium was removed and cells were washed five times with PBS (1 mL for 35 mm plastic dish and 4 mL for 60 mm plastic dish) to remove remaining red and nonadherent white blood cells (lymphocyte), as well as cell debris. Co-staining of the cells on the culture dish with DAPI for general nuclear DNA and with anti-StAR antibodies revealed that 95.28 ± 1.8% of the total population (random counting in 6 cultures, n=6) was positive in staining of mitochondria StAR (typical for steroidogenic cells) and showed in phase microscope typical morphology of granulosa cells (e.g., lipid droplets). The rest of the cells demonstrated fibroblast or monocyte-like morphology with negative staining for StAR. Cells were cultured an additional 48 h in 5% FCS and washed every 24 h with PBS (twice) to remove any minor contamination of nonadherent red and white blood cells. The 7 days of cell culture before stimulation with FSH were essential to release the cells from refractoriness to FSH stimulation (29 , 30) . Cells were washed on day 7 of culture with PBS and incubated with FSH (3 IU/mL for 24 h). The increment in progesterone production in stimulated cells compared with nonstimulated was 3.46 ± 0.2 (n=3, P<0.001, 4.5 ng/mL/10 5 cells/24 h progesterone in FSH stimulated cells compared with 1.3 ng/mL in nonstimulated cultures). The extended 24 h of FSH stimulation was selected because of expected lower rate of metabolism and lower levels of FSH receptor compared with the immortalized rat cells.

Analysis of RNA on DNA microarray
Total RNA was extracted using Tri Reagent (Molecular Research Center) and double-stranded complementary (c) DNA synthesized by reverse transcription. Biotin-labeled cRNA was produced from the cDNA and used to probe Affymetrics Rat DNA microarray (rat genome U34 array) and human genome (U133 array). Hybridization and washes were performed using Affymetrix gene chip system according to the manufacturer’s instructions. Average difference and expression level of genes were calculated according to absolute and comparison analysis algorithms specified in appendix 5 of the manufacturer’s instructions. Experiments with rat cells were repeated once with the same RNA and once with a new batch of nonstimulated and FSH-stimulated cells (3IU/mL for 8 h) using fresh sets of DNA microarrays. Experiments with FSH stimulation were repeated three times with different batches of culture cells obtained from six women undergoing the same IVF treatment

Assays
Progesterone released to the culture medium was assayed by RIA. Protein was assayed according to Bradford (32) .

Preparation of rat cells for fluorescent and confocal microscopy
Cells were grown on glass slides in plastic tissue culture dishes. At the end of stimulation, cells were fixed with 3% paraformaldehyde and stained with DAPI (32) . In some experiments, cells were incubated with mouse anti-rat monoclonal antibodies to granzyme B, followed by fluorescein-labeled rabbit anti-mouse antibodies using the indirect immunofluorescent technique (22) . For general morphology, cells were visualized by phase contrast microscopy, for DAPI staining by fluorescent microscopy (Zeiss, Thornwood, NY, USA), and for visualization of granzyme B by laser confocal microscopy (Bio-Rad, Hercules, CA, USA).

For analysis of apoptosis, six random fields of each treatment stained with DAPI were photographed by fluorescent microscopy at magnification x400. Nuclei with highly condensed and fragmented DNA were identified as apoptotic nuclei.

Blots
Western blots for granzyme B were carried out on human and rat cell lysates. Proteins were chromatographed on 10% acrylamide gels (32) . Northern blots of mRNA coding for vascular endothelial growth factor 165 (VEGF-165), estrogen receptor {alpha} (ER{alpha}), ERß, glucocorticoid receptor (GR), 20 {alpha} hydroxysteroid dehydrogenase (20 {alpha} HSD), 3ß HSD, and cytochrome p450 side cleavage chain enzyme (p450scc) were performed as described elsewhere (26) .


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Steroidogenesis
Isolation of RNA of rFSH-17 cells after 8 h of stimulation with FSH revealed up-regulation of mRNA coding for steroidogenic acute regulatory protein (StAR) (Table 1A ) obligatory for rapid up-regulation of steroidogenesis and adrenodoxin (ADX), an intrinsic part of the cytochrome P450scc enzyme system (reviewed in refs 2 , 16 , 33 ). Similar amplification of StAR expression by FSH was evident in HGC. Other genes coding for components of the steroidogenic machinery such as P450scc, adrenodoxin reductase, steroid carrier protein 2 (SCP2) (34) , AD4 binding protein steroidogenic factor 1 (AD4BP/SF1) (29 , 24) , and 20{alpha} HSD (35) showed constitutive expression, suggesting that in these cells 8 h is insufficient to elevate their message levels (Table 1) . However, it demonstrates that the pathways converting cholesterol to progesterone and then to 20{alpha} dihydroprogesterone, characteristic of granulosa cells, are well preserved in these immortalized cells. In contrast, in HGC a clear elevation in transcriptional activity of P450scc, adrenodoxin reductase, StAR, and SCP2 was evident after 24 h stimulation with hFSH for 24 h (Table 2 ). The expression of 20{alpha} HSD, 3ß HSD, and P450scc as well as GR and ER{alpha} in FSH-17 cells was confirmed by Northern blot analysis (Fig. 1 A). Moreover, ovarian mRNA coding for 20{alpha} HSD was moderately elevated in ovaries of female rats 48 h after pregnant mare serum gonadotropin injection (9) . There was no change in mRNA expression of p450scc after 8 h of stimulation. Stimulation of its expression was found only after 16 h of incubation with gonadotropins/cAMP in immortalized granulosa cells (16) . Indeed, a clear elevation of P450scc was evident in FSH stimulated HGC for 24 h (Table 2) . The FSH receptor is highly expressed in these cells (18) ; that no change in its mRNA expression after 8 h of stimulation was observed suggests that down-regulation of the response to FSH due to continuous stimulation with the hormone does not involve rapid attenuation of mRNA coding for the receptor. The amount of mRNA coding for FSH receptors in HGC was only 1% of rFSH-17 cells, which were expressing much higher levels of the receptor. However, the signal-to-noise ratio of FSH receptor gene activity as evident from the U133 DNA array was 12.1± 1.5 (P<0.05) (Table 2) .


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  		Table 1. A. Genes up-regulated by FSH in immortalized granulosa cells (rFSH-17)


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  		Table 2. A. Genes up-regulated by FSH in primary human granulosa cells



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  		Figure 1. Modulation of gene expression by FSH in rFSH-17 cells. A) Northern blot analysis of specific genes constitutively expressed in rFSH-17 granulosa cells. Numbers indicate markers of DNA in kb. GR, glucocorticoid receptor, ER, estrogen receptor. B) Northern blot of VEGF 165 after FSH stimulation (1 IU/mL) of rFSHR-17 cells. Aldolase serves as a constitutive marker. C) Chip images of FSH regulated genes. Cont, nonstimulated cells, FSH, 8 h after stimulation with 1 IU/mL FSH. Bright (fluorescent) square boxes indicate intensity of interaction of cmRNA derived from rFSH-17 cells with specific sequences of cDNA located on chip coding for the specific gene (see Materials and Methods). Each pair of boxes (Cont and FSH) contains a different sequence of oligonucleotides of the specific gene. The upper cluster of 8 squares indicates down-regulation of expression of Na+/K+ ATPase ß subunit. The lower cluster of 8 squares shows up-regulation of PTPase expression.

Proteolysis: alternative apoptotic pathway
Analysis of the mRNA expression pattern revealed the existence and elevation by FSH of granzyme-like proteins I, III (36) and the natural killer cell protease PNKP1 in rFSH-17 cells (Table 1A) (37 , 38) . All these mRNA sequences show high homology to granzyme B (39) . In HGC, elevation of RNA coding for granzyme B evidenced a 1.49-fold change. Using specific antibodies to granzyme B revealed, in a Western blot, a 31 kDa protein band whose intensity was elevated upon FSH stimulation in FSH-17 cells and HGC (Fig. 2 A). The same pattern of granzyme B intracellular levels was observed in primary rat granulosa cells (data are not shown). Granzyme B is uniquely located in intracellular granules of T cytotoxic lymphocytes, and serves as an apoptotic protease upon releasing its granule content into the target cell after fusion of the granule membrane with the cell membrane of the target cells (reviewed in ref 40 ). The release and activation of granzyme B can activate directly specific caspases (such as caspase-8), leading to the death of the target cells (36 , 38) . We first verified that the granulosa cells 31 kDa protein interacts with granzyme B antibodies that co-migrate with a 31 kDa protein of a line of T cytotoxic lymphocytes (B3Z) (Fig. 2A ). We localized the protein in small cytoplasmic granules, using specific antibodies to granzyme B for immunostaining of the cells and confocal laser microscopy (Fig. 2D ). Moreover, the incidence of the granules was elevated significantly after 24 h of stimulation with FSH.



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  		Figure 2. Expression of granzyme B like protein and its involvement in cAMP-induced apoptosis in rFSH-17 cells. A) Western blot analysis of granzyme B without stimulation (con) after 24 h of stimulation by hFSH (FSH) and with 50 µM of forskolin (Fc) for an additional 24 h. P.C., positive control of 23Z cells. B) Percentage of apoptosis as calculated from cultures stained with DAPI after stimulation and aldehyde fixation. C) Progesterone release to the medium (measured by RIA) after the same stimulation shown in panel B. D) Immunostaining of FSH-17 cells in the absence (upper) and the presence (lower) of hFSH for 24 h at 37°C. Fluorescent granules containing granzyme B (arrows) are evident in the cytoplasm. Laser confocal microscopy x1000. E) DAPI staining of cells stimulated by FSH (1 IU/mL) for 24 h, followed by hormone-free incubation for 24 h (a), and FSH-stimulated cells, followed by 50 µM of forskolin for 24 h at 37°C (b). Apoptotic cells in b demonstrate nuclei with condensed and fragmented DNA x400. Arrows point to highly condensed and fragmented nuclei.

To verify whether granzyme B release and activation are part of inducible apoptosis in rFSHR-17 cells, we stimulated the cells for 24 h with FSH, followed by 24 h stimulation with 50 µM forskolin in serum-free medium. This treatment increased progesterone release from the cells by threefold compared with stimulation with FSH alone (Fig. 2C ). On the other hand, it increased the incidence of apoptosis to 40% compared with 5–6% found in non- or FSH-stimulated cells (Fig. 2B ). Levels of 31 kDa granzyme B were markedly elevated in forskolin-treated cells with the appearance of a 28 kDa protein band, probably indicating cleavage and activation of granzyme B, which suggests its participation in granulosa cell apoptosis (41) . Since cytolysin could not be detected on the DNA microarray and perforin is absent in the DNA microarray (36 37 38) , we suggest this is the first demonstration of granzyme B participation in apoptosis in the same cells where it was synthesized. In HGC we could identify by Western blot the 31 kDa granzyme B protein, also evident in nonstimulated cells (Fig. 3 ) with gradual elevation in intracellular levels of FSH- and forskolin-stimulated cells. A weak but clear band of the 26–28 kDa cleavage product was evident in FK-treated cells, which induced apoptosis to a lesser extent compared with rFSH-17 cells. This phenomenon can explain the consistent observations that steroidogenesis is not attenuated and the mitochondria remain intact during initial steps of apoptosis in primary and immortalized rat and in human granulosa cells (3 , 42) .



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  		Figure 3. Expression of granzyme B like protein in primary human granulosa cells. A) Western blot analysis of granzyme B without stimulation (CONT) after 24 h of stimulation by hFSH (FSH) or with 50 µM of forskolin (FK). A.D.U., arbitrary densitometric units. B) Percentage of apoptosis observed in cultures stained with DAPI after stimulation and aldehyde fixation (see Materials and Methods).

We have shown that initial stages of apoptosis in granulosa cells do not involve release of cytochrome c, as revealed by immunoelectron microscopy and Western blot of lysed mitochondria using specific antibodies to cytochrome c (data not shown). Bypass of mitochondria destruction during initial steps of apoptosis suggests alternative pathways for apoptosis in steroidogenic cells that do not require release of mitochondrial cytochrome c. Such an alternative pathway has the advantage of preserving steroidogenesis until total cell collapse. Granzyme B inhibitor-9, an intracellular serpin proteinase and the only mammalian protein able to inhibit the activity of granzyme B (43) , was recently localized in granulosa cells surrounding the primordial and primary follicle (44) . This suggests protection of immature granulosa cells and the enclosed oocyte against granzyme B activity at early stages of follicular development. The DNA microarray analysis revealed expression of caspase 6 mRNA (Table 1C and Table 2 ). Caspase 11 was found to be moderately elevated during in vitro maturation of mouse preantral follicles (45) , suggesting that these caspases may be activated by Granzyme B upon its release from the specific granules in response to apoptotic stimuli.

Carboxypeptidase E mRNA was found to be expressed and attenuated by FSH stimulation (Table 1B and Table 2 ). In HGC, however, it was elevated by FSH, suggesting that modulation of some genes are species specific and may be related to the degree of granulosa cell differentiation (2 , 12) . This protease is considered to process prohormones in a variety of endocrine systems (46 , 47) . However, its possible role in the early stages of folliculogenesis and granulosa cell differentiation is still unknown.

Growth factors and cytokines
rFSHR-17 cells and HGC contained mRNA coding for VEGFs (48) . Activity of genes coding for these growth factors was clearly elevated by FSH (Table 1A) , and the elevation of VEGF 165 mRNA was evident already after 6 h of FSH stimulation (Fig. 1B ). VEGFs have the ability to stimulate vascular endothelial cell mitosis; on the other hand, they are able to induce vascular leakage (49 50 51) . It is assumed that during ovulation and formation of the corpus luteum, VEGFs may play an important role in the intensive angiogenesis characteristic of luteinization, stimulated by luteinizing hormone (52) . Our observations suggest a potential role for gonadotropins in the activation of these genes, which may lead to accumulation of growth factors that may be released during follicular rupture and the formation of the corpus luteum in the enhancement of vascular permeability and angiogenesis. The discovery that genes coding for VEGF are targets for gonadotropin stimulation raises the possibility that the syndrome of gonadotropin hyperstimulation or resistance to gonadotropins may involve modulation in signal transduction pathways that lead to accumulation/release of these potent effectors of angiogenesis and vascular permeability (51 , 53) .

Early observations of human and rat granulosa cells revealed a clear response to leptin in enhancing steroidogenesis induced by co-stimulation with gonadotropin/cAMP and insulin or glucocorticoid (54) . Leptin could accelerate puberty in immature female rats (55) . Formation of leptin by the mammalian ovary was recently reported, but whether ovarian leptin biosynthesis is due to resident ovarian cells or adipocytes adhering to the ovarian tissue is under debate. Our present data indicate formation of mRNA coding for leptin both in primary and immortalized granulosa cells, which highlights the importance of this cytokine in ovarian homeostasis.

It was recently revealed that tumor necrosis factor {alpha} (TNF-{alpha}) can modulate granulosa cell proliferation, steroidogenesis and apoptosis depending on the cell system and the species (56 , 57) . The existence of TNF-{alpha}-converting enzyme and TNF-{alpha} receptor 1 mRNAs in HGC and FSHR-17 cells revealed by DNA microarray (Table 1C , Table 2C ) supports the notion that TNF receptor signaling is essential for ovarian homeostasis. Insulin growth factor binding protein 3 (IGF-BP3) mRNA was found to be expressed and down-regulated by FSH in immortalized granulosa cells (Table 1B) . Unfortunately, we could not measure mRNA activity coding for IGF-BP3 in HGC due to the lack of oligonucleotides coding for this gene in UA133 DNA chip. IGF-BP3 was demonstrated to act directly on ovarian granulosa cells in addition to its ability to neutralize insulin growth factor 1 activity (58 , 59) . Since IGFBP3 may decrease cell sensitivity to p53-induced apoptosis, as FSH stimulates differentiation of granulosa cells it may increase their sensitivity to other possible signals that can induce granulosa cell apoptosis. Therefore, maturation of granulosa cells may include preparation of these cells to undergo apoptosis at later stages of folliculogenesis or during the luteolytic process.

Intercellular communication and connection to the nervous system
We recently found that expression of synaptosome-associated protein 25 (SNAP-25) (27) , expression of muscarinic receptor (28) , and synthesis of acetylcholine were characteristic of the mammalian ovary and are synthesized in rFSHR-17 granulosa cells in response to FSH/cAMP stimulation (27 , 28) . In the present study, we found that human and rat granulosa cells both express mRNA coding for neuropeptide Y-1 (NPY-1) receptor (60 , 61) , which is elevated under FSH stimulation (Table 1A and Table 2A ). However, it is not yet known whether granulosa cells produce NPY-1 peptide since it is absent on the DNA chips. Rat cells also express the nicotinic acetylcholine receptor {alpha}7 subunit, the muscarinic receptor M2, and SNAP-23 (62) (Table 1C) . In HGC we found a dramatic elevation in SNAP-23, which may suggest a higher degree of differentiation than in the immortalized rFSH-17 cells, and a constitutive expression of nicotinic acetylcholine receptor {alpha}4 subunit (nAChR) and the cholinergic receptor muscarinic 2 (CHRM2), which is in line with the observation found in FSH-stimulated rFSH-17 cells. These observations suggest that the granulosa cells preserved or developed special means of intercommunications and/or communication with the oocyte and the external component of the follicle, since innervations probably do not penetrate the multilayer granulosa cells, which are surrounded by the basement membrane (63) .

rFSH-17 cells and primary HGC are reported here for the first time to express chemokine CX3C mRNA (64 , 65) , which is attenuated by FSH (Tables 1 and 2) . It is possible that the formation of chemokines may resemble a means of communication and attraction among granulosa cells or between granulosa cells and the oocyte at early stages of folliculogenesis required for tissue remodeling. Attenuation of expression of Na+/K+ ATPase ß subunit mRNA (66) by FSH, reported here for the first time (Fig. 1C , Table 1B ), may indicate loss of polarity of granulosa cells in the transition from a monolayer of granulosa cells surrounding the oocyte in the primordial follicle to a multilayer nonpolar organization in the preantral and antral follicle, where only the corona radiata cells show some polarity toward the oocyte. This phenomenon is consistent with the presence of desmosomes in primordial and primary follicles and their disappearance at more advanced stages of folliculogenesis (6 , 7) .

Protein phosphorylation and signal transduction
The DNA microarray analysis in rFSH-17 cells and HGC revealed up-regulation in genes coding for protein tyrosine phosphatase (PTPase) (67) , oxidative stress inducible protein phosphatase (OSIPTPase) (68) (Table 1A) , protein tyrosine phosphatase type IVA member 1, and acid phosphatase prostate, respectively (Table 2A) . Activation of tyrosine phosphatase is essential for mediating inhibitory growth signals (69) . OSIPTPase is highly inducible by oxidative stress and heat shock in skin cells and may be important for cellular response to environmental stress (68) , including oxidants and hyperthermia. Expression of two phosphatases was down-regulated by FSH: rat prostatic acid phosphatase (rPAP) and protein phosphatase 1ß (PTP1B) (Table 1B) . PTP1B acts at a point of counter-regulation of insulin action by agents that elevate cAMP and activate PTP1B (70) . rPAP expression suggests the origin of granulosa cells from pluripotent endoderm (71) . The function of the latter phosphatase in the ovary is unknown. Human PAP activity is exerted by c-ERB-2/neu dephosphorylation (72) .

We found a constitutive expression of serum- and glucocorticoid-dependent kinase (sgk) (Table 1C , Table 2C ). This kinase and protein kinase B (PKB/Akt) were recently found to be activated by FSH in granulosa cells. The PKB and sgk pathway appeared to be enhanced in terminally differentiated granulosa cells, suggesting that they act in concert as survival factors (73) .

We found that expression of the regulator of G-protein signaling (RGS16) mRNA is elevated by FSH stimulation in HGC and rFSH-17 cells (Table 1A and 2A) . RGS-16 enhances the GTPase activity of heterotrimeric G-protein {alpha} subunits to turn off signaling between cell surface receptors and intracellular effectors. RGS-16 was first cloned from mouse pituitary (74) and it was proposed that palmitoylation may be important in regulating its activity in mammalian cells (75) . Modulation of its expression by FSH may suggest a turning point in the transmission of G-coupled signals subsequent to FSH stimulation of granulosa cells.

A dramatic attenuation of phosphatidylserine-specific phospholipase A1 (Ps-PLA1) mRNA is observed after FSH stimulation in rFSH-17 cells (Table 1B) . A modest but significant reduction in its expression was also found in HGC (Table 2) . The serine-phosphatidyl-specific phosphatase has been cloned from the rat and murine genome (76 , 77) . Phosphatidyl serine migration from the inner to the outer face of the cell membrane is implicated in apoptosis, but it is not yet clear how PS-PLA1 activity could play a role in this process.

Decay accelerating factor GPI-form precursor (DAF) mRNA increased dramatically after 8 h exposure of rFSH-17 cells to FSH (Table 1A) . Moreover, a gene coding for a similar decay accelerating factor complement was enhanced in FSH-stimulated HGC (Table 2A) . The activity of DAF-2 insulin receptor is required for C. elegans reproduction, growth, and normal adult life span (78) . Adult rat testis showed an increased expression of GPI-DAF, suggesting that DAF may be involved in enabling sperm to survive a complementary attack in the mucus of the female genital organ and/or interact with the ovum (79) . The significance of the increase of GPI-DAF during FSH-induced granulosa cell maturation is obscure, although GPI-DAF cross-linking may be involved in modulation of apoptosis (79) .

Antioxidants, antitoxicants
Expression of metallothionein-1 and metallothionein-2 (MTs) genes was clearly elevated in response to FSH in normal and immortalized granulosa cells (Table 1 , Table 2 ). MTs, a family of proteins of which four isoforms have been described (80) , are small molecular mass proteins (6–7 kDa) that contain ~60 amino acids, 20 of which are cysteine and none of which is aromatic. MTs have been associated with zinc homeostasis and metal detoxification by binding coordinately to several (7 8 9 10 11 12) heavy metals, such as arsenic. MT-1 and MT-2, the major isoforms, are ubiquitous and inducible in many species and organs. However, there is no information about their expression in the ovary. MT-1 and MT-2 induced by FSH may protect follicular cells and the oocyte from toxic metals and, together with OSIPTPase, protect follicles and the enclosed oocytes from oxidative stress. It was found that oxidative stress-induced protein mRNA expression was elevated during in vitro LH- and FSH-stimulated maturation of mice preantral follicles to graafian follicles (45) .


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
This report demonstrates the effectiveness of immortalized FSH responsive granulosa cells to reveal genes that have not yet been reported to be modulated by FSH. The striking similarities of FSH gene regulation found in primary HGC stress the importance of these findings in the present work on normal granulosa cells. Modulation or constitutive expression of genes typical of steroidogenic cells, especially ovarian granulosa cells, as steroidogenic enzymes and receptors to steroidogenic hormones emphasize that these cells, although immortalized by oncogenes, can serve as authentic models for the study of gene regulation by FSH. A novel aspect of this work is the discovery of granzyme like proteins, which opens the possibility of an alternative pathway for apoptosis. Inducible VEGFs point to the high potential of these cells to stimulate angiogenesis and vascular permeability during a critical phase of the estrous cycle such as ovulation. Modulation of kinases and phosphatases, DAF and RGS16, may indicate a shift in signal transduction during differentiation. The elevation of proteins involved in neutralizing stress induced by oxygen, heat, and toxic metals indicates the protective effect of granulosa cells on the normal development of the oocyte.

The discovery of modulation of Na+/K+ ATPase and chemokines indicates the plasticity in the development and organization of granulosa cells during folliculogenesis. The existence of typical components of neuronal signal transmission suggests that the granulosa cells in the follicular phase, lacking a direct innervation, can still communicate with the extracellular milieu and nervous system by expressing typical components that fulfill this mission.

Whether the unique gene activity of granulosa cells indicates the ontogeny of their development or late adaptation needs further investigation. In spite of immortalization of primary granulosa cells to make them a monoclonal line, we believe that the discovery of novel genes with a potential to be modulated by FSH will shed light on the pluralism of granulosa cell function in guarding the oocyte and in keeping ovarian homeostasis.


   ACKNOWLEDGMENTS
 
We thank Drs. A. M. Kaye, D. Michael, and D. Einsberg for helpful discussions, Dr. L. Eisenbach for providing us with B3Z T cells, Drs. S. Saban and R. Ophir of the Functional Genomic Unit at the Weizmann Institute of Science for excellent technical assistance, and Dr. G. Gibori at the University of Illinois at Chicago for excellent assistance in performing the Northern blots. The work was supported by the Levine Center of Applied Research at the Weizmann Institute of Science. A.A. is the incumbent of the Joyce and Ben B. Eisenberg Professorial Chair of Molecular Endocrinology and Cancer Research.

Received for publication August 28, 2002. Accepted for publication March 10, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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