Mol. Hum. Reprod. Advance Access originally published online on March 16, 2004
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Molecular Human Reproduction, Vol. 10, No. 5, pp. 299-311, 2004
© European Society of Human Reproduction and Embryology 2004
Gonadotrophin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases
1Department of Molecular Cell Biology, The Weizmann Institute of Science Rehovot, 76100 and 2Department of Obstetrics and Gynecology, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 64239 Israel *These two authors contributed equally to this work.
3 To whom correspondence should be addressed. e-mail: abraham.amsterdam{at}weizmann.ac.il
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ABSTRACT |
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Gonadotrophins exert a major effect on ovarian development and on the control of fertilization. By stimulating cells with forskolin (FK), it is possible to study which genes are activated by gonadotrophins via the cAMP cascade, and which by alternative pathways. Using RNA isolated from stimulated cells, we found that 59% of the total genes modulated by LH were also modulated by FK, while 69% of the genes modulated exclusively by FSH were also modulated by FK. Gene transcripts involved in steroidogenesis/progesterone production were highly elevated, while 17ß-hydroxysteroid dehydrogenase was down-regulated. This suggests that a decrease in the conversion of androstenedione to testosterone and estrone to estradiol occurs during luteinization. Down-regulation of genes coding for actin cytoskeleton proteins and cytokeratin 18 was observed in response to gonadotrophin and cAMP stimulation. Several of the genes coding for the microtubule network were also modulated, implying that rearrangement of the cytoskeletal proteins permits better coupling between organelles involved in steroidogenesis. A dramatic change in gene transcripts coding for signalling enzymes was observed following LH stimulation. This includes the down-regulation of adenylyl cyclase 7 and 9, elevation of cAMP-dependent phosphodiesterase, and the up-regulation of a negative regulator of G-protein signalling (RGS16) that may negate gonadotrophin signalling via guanine nucleotide binding proteins. Thus luteinized cells, despite increased gene transcripts to LH/chorionic gonadotrophin (CG) receptors, respond inefficiently to gonadotrophin stimulation, due to attenuation of signal transduction in the cAMP cascade at multiple steps. Novel genes involved in the regulation of apoptosis were found for the first time to be up-regulated by gonadotrophin stimulation, including: BAX inhibitor-1, granulysin and apoptosis repressor with caspase recruitment domain (ARC). These proteins may be involved in a unique alternative pathway of ovarian cell death. Such a pathway could temporarily preserve the mitochondria and progesterone production during the initial stages of granulosa cell apoptosis.
Key words: Key words: adenylyl cyclase/apoptosis repressor with caspase recruitment domain/DNA array/human ovary/regulators of G-protein signalling
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Introduction |
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TopABSTRACTIntroductionMaterials and methodsResults and discussionConclusionREFERENCES |
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FSH and LH/chorionic gonadotrophin (CG) are the primary triggers of folliculogenesis and ovulation/luteinization, which act via specific ovarian membrane receptors. These receptors are G-coupled to the activation of adenylyl cyclase, but may also exert their effects via alternative pathways (Amsterdam and Rotmensch, 1987; Amsterdam et al., 1989; Amsterdam and Aharoni, 1994; Amsterdam and Selvaraj, 1997). Although intensive and extensive research has been conducted during the last few decades, the entire spectrum of genes modulated by LH/CG and FSH has not yet been described. Also, it is not yet clear which of the gonadotrophin-sensitive genes are modulated by the cAMP/protein kinase A (PKA) cascade and which are regulated by alternative mechanisms. Several attempts have been made to reveal the changes in gene expression in the intact rat ovary (Leo et al., 2001) and in the micro-dissected isolated ovarian follicle (Liu et al., 2001). However, it should be kept in mind that the intact ovary is composed of different types of cells and tissues (e.g. oocytes, granulosa cells, theca interna and externa cells, interstitial tissue, and blood vessels), which may not necessarily contain the same repertoire of actively expressed genes. Moreover, the granulosa cells may differ at different follicular layers with respect to gonadotrophin receptors (Amsterdam et al., 1975) and steroidogenic enzyme expression (Amsterdam and Rotmensch, 1987; Amsterdam et al., 1989). This may affect changes in gene activity modulated by gonadotrophins during specific stages of follicular cell differentiation (Amsterdam, 2003).
In previous studies we have succeeded in immortalizing human and rat granulosa cell lines, thereby preserving their steroidogenic response to gonadotrophin/cAMP stimulation in a homogeneous manner (Amsterdam and Selvaraj, 1997; Amsterdam et al., 1999). Moreover, we have achieved prolonged culturing of primary granulosa–lutein cells obtained from IVF patients (1 week in hormone-free medium), causing re-sensitization of the cells to gonadotrophic hormones following their intensive exposure to saturating doses of gonadotrophin in vivo (Breckwoldt et al., 1996). These cells were recently shown to respond homogeneously to gonadotrophin stimulation (Sasson and Amsterdam, 2002).
In the present study we report on a wide spectrum of genes, shown for the first time to be modulated by gonadotrophins and forskolin (FK). These results were obtained by using mRNA isolated from stimulated and non-stimulated human granulosa cell cultures hybridized to Affymetrix DNA microarrays. These data may shed light on the indispensable pleiotrophic role of granulosa cells in the cyclic differentiation of the follicles, oocytes and corpora lutea of the human ovary.
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Materials and methods |
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Antibodies
Rabbit polyclonal antibodies against apoptosis repressor with caspase recruitment domain (ARC) were purchased from Santa Cruz Biotechnology, Inc. (USA). These antibodies (H-150) were raised against recombinant protein corresponding to amino acids 1–150 mapping the amino acid terminus of human ARC. Monoclonal anti-ß-tubulin antibodies, raised against rat tubulin and cross-reacted with human ß-tubulin, were purchased from Sigma Chemical Co. (T4026); goat anti-rabbit and anti-mouse IgG coupled to horseradish peroxidase (HRP) were obtained from BioMakor (Israel). Polyclonal anti-progesterone antibody, raised in rabbits, was provided by Dr F.Kohen (Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel). Anti-human steroidogenic acute regulatory protein (StAR) polyclonal antibodies raised in rabbits were provided by Dr J.F.Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA, USA). Anti-vinculin (monoclonal) antibodies were provided by Dr B.Geiger (Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel). Anti-26S proteasomes antibodies were generated in rabbits (polyclonal) in collaboration with Prof. F.Baumeister (Department of Structural Biology, Max-Planck-Institute for Biochemie, Germany) (Amsterdam et al., 1993).
Reagents
Forskolin was purchased from Sigma Chemical Co. Recombinant hFSH Lot AFP8468A and hLH Lot AFP4395A were kindly provided by the NIH and Dr A.Parlow of the National Hormone and Pituitary Program.
Primary human granulosa cell cultures.
Primary granulosa cells were obtained from women 26–32 years old undergoing IVF at Sheba Medical Center, Tel-Hashomer, Israel, due to mechanical or male factor infertility. Patients received a GnRH analogue in combination with FSH or hMG, followed by administration of hCG.
Detailed IVF protocol.
The GnRH analogue protocol used: D-Ser (TBU) 6-ethylamide-GnRH (burserelin; Aventis Pharma, Germany) administered intranasally at a daily dosage of 600 µg, starting on day 2 of the cycle or D-TRP-GnRH [3.2 mg CR triptorelin (Ferring, Germany)/hMG] i.m. on day 2 of the cycle. This was followed by hMG from day 15 of the same cycle, after hormonal and ultrasonic demonstration of ovarian quiescence.
The hMG treatment protocol included daily 225 IU FSH and 225 IU LH (Pergonal; Israel) from day 3 of the cycle. hMG administration and ovarian response was evaluated repeatedly in all patients by serum estradiol concentrations and vaginal ultrasonographic scans to define ovarian follicular development. The dosage of hMG was adjusted on an individual basis according to clinical judgment. When follicles reached a diameter >18 mm and serum estradiol concentrations were >400 pg/ml, hCG (10 000 IU, Chorigon; Teva) was administered. Ultrasonographic-guided follicular aspiration for oocyte retrieval was performed 34–35 h after hCG administration.
Purification of granulosa cells.
Granulosa cells were isolated from aspirated follicular fluid after ovum retrieval. The follicular fluid was centrifuged at 300 g for 5 min. The resulting pellets were resuspended in 10 mmol/l Tris, 0.84% NH4Cl, pH 7.4 to lyse red blood cells (15 min shaking at 37°C). Several washings in phosphate-buffered saline (PBS) eliminated excess cell debris (Breckwoldt et al., 1996). Cells were plated in Dulbecco’s modified Eagle’s medium [DMEM/Ham’s F-12 (1:1)], supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml) and 5% fetal calf serum (FCS). Attachment of the granulosa cells to the bottom of the dishes (35 or 60 mm plastic dishes; Nunc) was achieved after 24 h of incubation 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 non-adherent white blood cells (lymphocytes), as well as cell debris. Co-staining of the cells on the culture dish with 4,6-diamidino-2-phenylindole for general nuclear DNA and with anti-StAR antibodies revealed that 95.28 ± 1.8% of the total population (random counting in six cultures, n = 6) was positive for staining of mitochondria StAR (typical in steroidogenic cells) and showed in phase microscope typical morphology of granulosa cells (e.g. lipid droplets). The remaining cells showed fibroblast- or monocyte-like morphology with negative staining for StAR (Sasson and Amsterdam, 2002). Cells were cultured for an additional 6 days in 5% FCS and washed every 24 h with PBS (twice) to remove any minor contamination of non-adherent red and white blood cells. Cells were cultured for a total of 7 days in hormone-free medium to release them from possible gonadotrophin desensitization, since these cells were over-exposed to LH and FSH in vivo for induction of ovulation (Breckwoldt et al., 1996; Sasson and Amsterdam, 2002). Cells washed on day 7 of culture with PBS were incubated for 24 h with FSH (3 IU/ml), LH (3 IU/ml) or FK (50 µmol/l) in DMEM/F-12 culturing medium containing 5% FCS.
Determination of progesterone and protein levels
Progesterone released from the cells to the culture medium was assayed by radioimmunoassay (Keren-Tal et al., 1996). Protein was assayed according to Bradford (1976).
Western blot analysis
Western blot analysis was carried out as described previously (Sasson and Amsterdam, 2002; Tajima et al., 2003). In brief, after stimulation primary cultures at the end of incubation were rinsed with ice-cold PBS and harvested in lysis buffer containing 50 mM HEPES (pH 7.2), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 30 mM NaF, 30 mM sodium pyrophosphate, 1 mM orthovanadate, 1 mM phenylmethylsulphonylfluoride, 10 µg/ml leupeptin and 5 µg/ml aprotinin. Lysates were boiled in sample buffer for 10 min and subjected to western blot analysis to detect the different proteins. Samples containing equal amounts of protein (40 µg) were separated by 12% (to detect ARC, actin, ß-tubulin and StAR), or 7.5% (to detect vinculin) acrylamide sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The relevant proteins were detected on blots using their specific antibodies. Western blots were repeated three times with three groups of women undergoing IVF treatment. Each group contained six women. Representative western blots are shown in Figure 1.
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Expression profiling
Total RNA was collected from six individual women using Tri reagent (Molecular Research Center, Inc.). The average amount of pooled total RNA was
120 µg/six women. Biotin-labelled cRNA was created from pooled total RNA, following protocols provided by Affymetrix (USA). Briefly, total RNA was used in a reverse transcription reaction (Life Technologies, Inc.) with an oligo-dT primer containing a T7 polymerase promoter. Double-stranded cDNA was generated and used in an in vitro transcription reaction containing biotinylated UTP and CTP (Enzo Diagnostics, USA). Equal amounts of biotinylated cRNA from each sample were used to probe Affymetrix U133 and U95 expression array GeneChips. These arrays contained 33 000 or 11 000 genes and EST respectively. Once all samples of all treatments (LH-, FSH- or FK-stimulated cells) were hybridized and scanned, the difference between expression of gene transcripts in stimulated compared to non-stimulated cells in each treatment was calculated according to Affymetrix programming and was normalized between the U95 chip and the U133 chip as follows. Quantitative changes in gene expression in the U133 chip were reported as changes in signal log ratio (log scale base 2), whereas changes in the U95 chip were calculated using a linear normal scale. Since log base 2 scale was used for calculating signal log ratio in the U133 chip, the values obtained were converted to anti-log base 2 to normalize the data. As a result, the U133 and U95 microarrays were both expressed as fold changes. This manipulation was performed according to the Statistical Algorithms Reference Guide of Affymetrix 2001, using Tukey’s Biweight method for calculation of the significance in changes in gene expression. The data from the two types of arrays were calculated and given as a mean of four determinations. Gene transcripts were identified as ‘absent’ (A) or ‘present’ (P) by Affymetrix software. Gene transcripts identified as absent throughout all of the samples were removed from the analysis. In addition, only gene transcripts identified as ‘increased’ (I) or ‘decreased’ (D) between informative samples by Affymetrix software were recorded as significant changes following LH, FSH or FK stimulation. Changes of 
2–3-fold in gene activity were considered significant because changes in this range or higher were previously found to be highly replicable and reliable between triplicate samples or more (Cho et al., 1998; Owens et al., 2002; Burns et al., 2003). Moreover, it was recently demonstrated that the minimum detectable fold change for differential expression is 1.4 (Yue et al., 2001). In addition, signals and change in signals between samples were detected in the new generation of Affymetrix DNA chips, in the present work, by plotting on a logarithmic, rather than a linear, scale. This increases the sensitivity and the accuracy of the calculated fold change even further to values with statistical significance of 1.3. Therefore, only gene transcript activities (Owens et al., 2002), which changed by more than 2–3-fold, and those that were identified as ‘present’ (P), ‘increased’ (I), or ‘decreased’(D) as a result of a particular treatment, were accepted and reported in each cluster, aside from a few genes whose differential expression was <2 but highly significant according to Affymetrix software (P < 0.05). It should be noted that the quantitative analysis for each gene transcript and the difference between the different treatments was based on 16 different oligonucleotides present on the U95 DNA array and 12 oligonucleotides present on the U133 array. The oligonucleotides derived from the particular gene were examined on the chip for their binding to specific mRNA. As a control for non-specific binding of mRNA, obtained from the biological samples, 16 or 12 mismatched (MM) oligonucleotides were present. The results for specific binding were calculated for all the nucleotides present by Affymetrix programming and were reported only if statistically significant [confidence level >95%, P < 0.05 calculated according to Statistical Algorithms Reference Guide by Affymetrix, Affymetrix Technical Note: ‘New Statistical Algorithms for Monitoring Gene Expression on GeneChip® Probe Arrays.’ (2001)]. Also the increase (I) or decrease (D) in gene activity were proven to be statistically significant by the Affymetrix software. In contrast, no change (NC) was concluded when tentative changes were found insignificant (P > 0.05, see above). In this study, we report only on genes modulated by LH and FSH and compare their effects to FK-stimulated cells. We do not report on any gene with constitutive activity or genes modulated by FK but not by LH or FSH, thus avoiding any gene activity not stemming from granulosa cells, assuming that modulation of genes by LH or FSH must arise from granulosa cells and not from minor non-granulosa contaminant cells in the primary cultures.
Electron microscopy
Primary cultures were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde and cryopreserved. Ultrathin cryo-sections were prepared and incubated with either antibodies to StAR or ARC and further incubated with gold protein A particles (diameter of 10 nmol/l) and visualized by the Philips CM12 Transmission Electron Microscope. Random fields at 42 000 magnification were taken and the number of gold particles, representing the location of StAR or ARC in each cell compartment, was scored on 12 random fields for each treatment.
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Results and discussion |
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In order to verify whether the cells from IVF patients incubated for 7 days in hormone-free medium expressed functional LH/CG and FSH receptors, we measured progesterone release following 24 h of stimulation with LH/CG, FSH and FK at 37°C from the same cultures as those from which RNA was isolated. Progesterone release from LH/CG-, FSH- and FK-treated cells was 2.9-, 2.7- and 3.7-fold higher respectively, compared to non-stimulated cells (Figure 1). These data are in line with our recent observations on the recovery of IVF human granulosa cells from desensitization to cAMP/gonadotrophin stimulation (Breckwoldt et al., 1996; Tajima et al., 2003).
In the present study we discovered a marked change in gene activity in numerous genes coding for steroidogenic enzymes (Table I), cytoskeletal proteins (Table II) and signalling molecules coding for pro- and anti-apoptotic processes (Table III) as well as genes coding for signalling molecules including GTP, cAMP and various kinases and phosphatases (Table IV). However, it has yet to be seen whether modulation of gene activity is followed by similar changes in the expression of their conjugate proteins. In three of the gene products—StAR, vinculin and ARC—the validity of the data was substantiated by examination of protein levels by western blot analysis (Figure 1). ARC protein, which can protect against mitochondrial destruction (Neuss et al., 2001) was clearly expressed in granulosa cells (Figure 1). Moreover, the intracellular level of the protein is elevated by LH, FSH and FK (Figure 1). In contrast, as expected the intracellular levels of vinculin were diminished by gonadotrophin and FK treatment (Ben-Ze’ev and Amsterdam, 1989), and the levels of StAR were up-regulated by the same treatments (Tajima et al., 2003) (Figure 1). However, a perfect match between gene activity and gene products is not always linear; as observed in FK-stimulated cells, much higher accumulation of intracellular ARC was apparent at the protein level, despite only minor changes in gene activity (compare Figure 1 to Table III). This suggests that possible attenuation in protein turnover, including blocking of its degradation, could also lead to elevation of ARC protein levels. Using electron microscopy for localization of ARC on ultrathin cryo-sections showed that most of the ARC labelling was found in the cytoplasm, occasionally in close proximity to the mitochondria in contrast to accumulation of StAR which was found mainly on the mitochondrial cristae (inner mitochondrial membrane) (Figure 2). The localization of ARC in the cytoplasm may suggest spatial interaction between caspases necessary for their down-regulation (Koseki et al., 1998).
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Steroidogenesis
The human granulosa cells were cultured in hormone-free medium for 7 days so as to free them from desensitization to LH and FSH, as was clearly demonstrated in earlier studies (Breckwoldt et al., 1996; Sasson and Amsterdam, 2002). In this model, cellular response to these gonadotrophins was demonstrated mainly at the level of progesterone and estradiol secretion (when the tissue culture medium was supplemented with androgens), as well as at the protein and post-translational levels (Sasson and Amsterdam, 2002; Tajima et al., 2003), but not at the level of gene expression. In the present study, a comprehensive screening of the total human genome was performed. It was found that after 24 h of stimulation with either LH, FSH or FK there was a clear elevation in steroidogenic acute regulatory protein (StAR) gene activity (reviewed in Amsterdam and Selvaraj, 1997; Christenson and Strauss, 2000) and in cytochrome P450, subfamily XIA (cholesterol side chain cleavage) (CYP11A) gene transcripts (Gizard et al., 2002). Elevation of gene activity of the adrenodoxin gene and adrenodoxin reductase gene following gonadotrophins was more modest (Table I) (see also Sasson et al., 2003). Elevation of aromatase: cytochrome P450 subfamily XIX (CYP19 estrogen synthetase) gene (Harada, 1988; Meinhardt and Mullis, 2002a,b), which catalyses the aromatization of androgens to estrogen, was clearly observed (Table I). Also gene transcripts for hydroxysteroid dehydrogenases 11-ß (11HSD and HSD11B1) (Thomas et al., 1998; Yong et al., 2002) were elevated markedly. The enzyme encoded by this gene catalyses conversion of 11-hydroxy- and 11-deoxycorticosterone (DOC) to corticosterone, and dexamethasone to 11-dehydrodexamethasone. These reactions are reversible (Thomas et al., 1998). Elevation in the activity of this enzyme was reported in earlier studies during ovulation in the rat ovary (Tetsuka et al., 1999). However, no correlation was found between enzymatic activity in granulosa–lutein cells obtained from IVF patients and IVF outcome (Thomas et al., 1998).
3ß-Hydroxysteroid dehydrogenases (HSD3ß1 and HSD3ß2) gene activities were elevated by gonadotrophin stimulation. HSD3ß1 was significantly up-regulated by LH but not by FSH or FK. In contrast, HSD3ß2 was elevated by LH, FSH and FK to the same extent. HSD3ß1 is expressed in classic steroidogenic tissues: the adrenals and the gonads. However, HSD3ß2 expression was found in the rat liver and kidney (Payne et al., 1997). Interestingly, the expression of these genes in the human ovary has not been fully characterized. Gene transcripts for hydroxysteroid 17ß-dehydrogenase 1 (HSD17B1) (Gunnarsson et al., 2003) were down-regulated which may result, most likely, from the cell’s ability to produce more progesterone and less estradiol during luteinization. It was also found that the Neiman–Pick disease, type 2 gene (Watari et al., 1999) was down-regulated by FSH and LH (Table I). This may cause a feedback down-regulation in cholesterol availability upon excessive progesterone production. Progesterone receptor membrane component 1 (PGRMC1) (reviewed in Levine et al., 2001) was up-regulated by gonadotrophins (Table I). The activity of gene transcripts of the cytosolic progesterone receptor did not show changes in activity following LH/FSH or FK treatments (data not shown). In contrast, a 54 kDa progesterone receptor-associated FKBP54 (Sinars et al., 2003) was moderately elevated by LH but not by FSH or FK, whereas HSP70 and HSP90 were markedly elevated both by LH and FSH but not by FK. Since these proteins are known to be a part of the cytosolic progesterone receptor complex, possible changes in the protein production of these genes may modulate progesterone receptor activity (Renoir et al., 1993; Sinars et al., 2003). This may suggest a shift in progesterone response upon elevation of progesterone production in these cells. Gene transcript activity of sterol-C4-methyl oxidase (SC4MOL/ERG25) (Bard et al., 1996) and dehydroxycholesterol reductase (DHCR7) (Lee et al., 2002) were elevated, suggesting enhanced biosynthesis of sterols, which serve as a precursor to steroid hormone production. Glucocorticoid-binding protein (GRLF-1) (LeClerc et al., 1991) was elevated, indicative of modulation in glucocorticoid response to the luteinizing human granulosa cells. It should be noted that elevation of the progesterone membrane receptor was evident only in LH- and FSH- but not in FK-stimulated cells, suggesting that the modulation did not occur via cAMP/PKA, but rather by an alternative pathway.
Cytoskeleton
Analysis of genes coding for cytoskeletal and associated proteins revealed a marked down-regulation in gene transcripts coding for the actin cytoskeleton mRNA (Zamir and Geiger, 2001; Strelkov et al., 2003) upon LH, FSH and FK stimulation (Table II). These proteins include: actin binding LIM protein (ABLIM), actin alpha 1 and 2 (ACTA1 and ACTA2), actin gamma 2, smooth muscle enteric (ACTG2), actin-related protein 2/3 complex, subunit 4 (ARPC4), alpha-actinin 2-associated LIM protein (ALP), actinin alpha 1 (ACTN1), filamin C, gamma (FLNC) and tropomyosin 1 alpha and beta (TMP1 and TPM2) (Zamir and Geiger, 2001). Down-regulation of the actin cytoskeleton both in human (Ben-Ze’ev and Amsterdam, 1989) and in rat granulosa cells (Ben-Ze’ev and Amsterdam, 1987; Ben-Ze’ev et al., 1987; Amsterdam and Rotmensch, 1987; Amsterdam et al., 1989, 1992; Amsterdam and Aharoni, 1994) was shown to coincide with reorganization of the actin cytoskeleton, which enhances steroidogenesis (Ben-Ze’ev and Amsterdam, 1987; Ben-Ze’ev et al., 1987). Increased steroidogenesis is probably due in part to clustering of the steroidogenic organelles (e.g. lipid droplets, lysosomes, mitochondria and the smooth endoplasmic reticulum) (Amsterdam and Aharoni, 1994; Strauss et al., 1998; Amsterdam et al., 1999). Although some changes in cytoskeletal elements possibly reflect changes imposed by cell culture, it has already been shown that these changes are tightly coupled to progesterone production stimulated by gonadotrophin and cAMP stimulation, which is typical of luteinized cells (Amsterdam et al., 1989; Strauss et al., 1998).
Down-regulation of gene transcripts of talin 1 (TLN1), vinculin (VCL) (Table II) and villin 2 (VC2) genes may indicate that adherence junctions, which increase cell contact, are down-regulated. The same down-regulation of catenin delta-1 (CTNNF1), integrin-linked kinase (ILK), integrin alpha-3 (ITGA3), integrin beta-5 (ITGB5) and SW/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) gene products was observed (Zamir and Geiger, 2001), thus indicating a potential loss of contact between granulosa cells and the matrix. Moreover, down-regulation of these genes may explain the partial dissociation of the granulosa cell layers during ovulation prior to remodelling of the tissue to form the corpus luteum (Amsterdam and Rotmensch, 1987; Ben-Ze’ev and Amsterdam, 1987; Ben-Ze’ev et al., 1987; Amsterdam et al., 1989, 1992; Amsterdam and Aharoni, 1994). Reduced expression of thrombospondin 1 (THBS1) gene reported in the present work, modulated by LH/FSH and FK, was also observed in pre-ovulatory rat granulosa cells (Dreyfus et al., 1992). Increase in actin beta (ACTB) gene expression, actin-related protein 2, yeast homologue (ARP2) and actin-related protein 2/3 complex, subunit 1A (ARPC1A), as shown in Table II, suggests that there is not only down-regulation of the actin cytoskeletal proteins, but more probably rearrangement of these proteins in the cortical area of the cells (Ben-Ze’ev and Amsterdam, 1987; Ben-Ze’ev et al., 1987; Amsterdam et al., 1992). There is also evidence for up-regulation of gene transcripts of proline-4 hydroxylase (P4HA2) collagen types XV, alpha 1 (COL15A1) and collagen type XVIII, alpha 1 (COL18A1) genes (Table II). These gene activities are probably important for collagen replacement during breakdown associated with follicular rupture (Ny et al., 1993; Rodgers et al., 1999; Tsafriri and Reich, 1999).
Intermediate filaments comprise the complementary cytoskeleton network (Strelkov et al., 2003). Down-regulation of Keratin 18 (KRT 18) gene transcripts was observed subsequent to LH, FSH and FK stimulation of the human granulosa cells obtained from IVF patients (Table II). Down-regulation of the protein was found to be associated with the maturation and luteinization of human granulosa cells (Ben-Ze’ev and Amsterdam, 1989).
Increase in gene transcripts coding for microtubule-associated protein 1A/1B chain 3 (MAP1A/1BLC3) was found to be stimulated by LH, FSH and FK. Microtubule-associated protein RP/EB family member 2 (MAPRE2) gene (Howard and Hyman, 2003) was found to be down-regulated by the same stimulant. Possible modulation in microtubule organization during granulosa cell maturation and luteinization is not well characterized, and therefore it is not clear how their possible rearrangement may affect steroidogenesis. No elevation in steroidogenesis was found in primary rat granulosa cells upon treatment with colchicine, known to interfere with microtubule organization (Ben-Ze’ev and Amsterdam, 1987; Ben-Ze’ev et al., 1987). However, there was an increase in progesterone production in cytochalasin B-treated cells (Ben-Ze’ev et al., 1987), emphasizing the role of the actin filament network in modulation of steroidogenesis.
It seems that the vast majority of genes coding for cytoskeletal and matrix proteins modulated by LH and FSH are mediated by cAMP. This conclusion stems from the fact that there was no marked difference between LH- or FSH-stimulated cells. However, most changes were amplified by FK, which is known to elevate cAMP and progesterone production to a greater extent than the gonadotrophic hormones (Keren-Tal et al., 1997), with some exceptions including: (i) actin, beta and actin-related protein 2 (ARP2) genes, which are down-regulated by FK in contrast to up-regulation by LH or FSH; (ii) several genes discovered to be down-regulated by LH or FSH in the present study did not show down-regulation in expression upon FK stimulation. These include ARPC4, capping protein muscle Z-line alpha 1 (CAPZA1), SMARCA4, tubulin beta polypeptide (TUBB), and villin 2 (VIL2). Therefore, we cannot exclude the possibility that an alternative pathway to the cAMP/PKA cascade could modulate the change in expression of these genes.
Apoptosis
Modulation of genes that may be involved in apoptosis were evident following stimulation of the cells with LH, FSH or FS (Table III). Testis-enhanced gene transcript (TEGT), known also as BAX inhibitor 1 (Jean et al., 1999) was up-regulated by LH, FSH but not by FK. The same was true for Bcl-2-antagonist of cell death (BAD). In contrast, Bcl-2-associated athanogene (BAG1) (Doong et al., 2002; Kudoh et al., 2002) was up-regulated both by gonadotrophins and cAMP. Bcl-2 was shown to function as a survival factor in primary human granulosa cells (Sasson and Amsterdam, 2002), whereas BAX knockout in female mice reduced ovarian apoptosis (Perez et al., 1997). The elevation of transcripts in the above-mentioned three genes supports the general view that LH and FSH are major survival factors in the mammalian ovary. However, since FK did not up-regulate TEGT and BAD genes, it is possible that some of the apoptotic factors are up-regulated by cAMP-independent signals (Amsterdam, 2003). Interestingly, Bcl-2/Adenovirus E1B 19 kDa interacting protein 3-like (BNIP3L) is considered to be an apoptotic factor (Imazu et al., 1999), and its gene activity is elevated by gonadotrophins (Table III), which supports the view that cross-talk among different survival and death factors rather than a single factor determines cell fate (Amsterdam et al., 1999, 2003).
It was found that Dead-box protein abstract (DBPA) gene transcript was elevated by LH, FSH and FK (Table III). The members of DBPA are essential for cell viability (Irion and Leptin, 1999), and therefore may be considered anti-apoptotic. DAP kinase 1 (DAPK) gene activity was evident and shown to be down-regulated by the gonadotrophins and cAMP. DAPK is considered to up-regulate p53 (Wang et al., 2002), and is therefore pro-apoptotic. Gene transcripts associated with the tumour suppressor protein p53, such as tumour protein p53 (TP53) and p53-induced protein (PIG11), were down-regulated by LH and FSH and to a greater extent by FK. Interestingly, the p53-inducible protein (P53IP) was also down-regulated, consistent with p53 down-regulation. The same was true for the candidate mediator of p53-dependent G2-arrest (REPRIMO) (Ohki et al., 2000). These observations suggest that gonadotrophin survival activity involves reduction in p53-related proteins, in contrast to the elevation of intracellular p53 levels in apoptotic follicular cells (Tilly et al., 1995) and its involvement in granulosa cell apoptosis (Keren-Tal et al., 1995; Amsterdam and Selvaraj, 1997; Hosokawa et al., 1998). Beclin 1 (BECN1) gene transcript was down-regulated by gonadotrophins, but not by cAMP, and is considered to be a tumour suppressor (Weinmann et al., 2001). The granulysin (GNLY) gene was up-regulated by gonadotrophins. This gene product is known to reside in T lymphocytes and is known to induce apoptosis in target cells when it is released from specific granules upon specific stimulation (Pardo et al., 2001). This is in line with our recent findings of granzyme B expression in granulosa cells; which was previously reported to reside specifically in T lymphocytes and natural killer cells of the immune system (Amsterdam, 2003; Sasson et al., 2003). This may indicate that accumulation of granulysin in granulosa cells may prepare them to undergo apoptosis in the presence of an appropriate stimulus. Also, it may suggest that apoptotic granulosa cells may induce apoptosis in neighbouring granulosa cells, thereby expediting the demise of apoptotic follicles.
Apoptotic repressor (ARC) protein, which was previously thought to reside exclusively in heart muscle tissue (Neuss et al., 2001), was found for the first time to be expressed in granulosa cells (Figures 1 and 2). The gene product (30 kDa) may significantly contribute to the protection of the mitochondria against its destruction following apoptotic signals, thus ensuring uninterrupted steroidogenesis in the cellular organelles harbouring StAR and the P450scc enzyme system (Amsterdam et al., 1999) during the initial stages of apoptosis. ARC contains caspase recruitment domains, which interact and inactivate caspase activities, thus preserving mitochondrial integrity and function (Neuss et al., 2001; Shelke and Leeuwenburgh, 2003). Elevation of ARC intracellular levels by LH, FSH and FK was demonstrated by western blot analysis (Figure 1). Intracellular localization of ARC protein by electron microscopy was evident in the cytoplasm often in close proximity to the outer mitochondrial membrane. No labelling was found in the nucleus (Figure 2). Such localization can permit interaction with caspases, which are also normally found in the cytoplasm. Gene activity of ARC was clearly seen in non-stimulated cells (not shown) and was elevated by both LH and FSH but not by FK (Table III).
Proteasomes are multicatalytic proteinases that are essential for degradation of many cellular proteins. Proteasomes change their distribution during the cell cycle in immortalized mammalian granulosa cells (Amsterdam et al., 1993). As shown in Table III, genes coding for proteasome 26S subunit, ATPase 1 (PSMP1) and proteasome 26S subunit, ATPase 3 (PSMP3) were up-regulated following 24 h stimulation with LH or FSH. Increased activity of proteasomes could lead to activation of apoptosis, since proteasomes are involved in the degradation of pro-apoptotic Bcl-2 family members such as BAX and tBid (Yang and Yu, 2003). Since BAX is a significant pro-apoptotic factor in ovarian cell death (Perez et al., 1997), enhanced proteasome formation can account in part for the survival activity of gonadotrophins. As shown in Table III, gene transcripts coding for caspase 9 apoptosis-related cysteine protease and caspase 9 beta are elevated by LH and FK with no significant changes in FSH-stimulated cells. In contrast, caspase 8 and FADD-like apoptosis regulator (CFLAR), caspase 4 and caspase recruit domain protein 10 were moderately down-regulated by LH and FK stimulation. Western blot analysis (not shown) revealed intact caspase 8, 57 kDa, in control and stimulated cells with no specific cleavage product, 18 kDa, of activated caspase (Earnshaw et al., 1999). Gonadotrophins function as a prominent survival factor that may change the intracellular levels of these caspases, probably without activation of these cysteine proteases, as was demonstrated for caspase 8 (Chen and Wang, 2002). It should be noted that no changes were found for caspases 1, 2, 3, 6 and 7 under LH, FSH and FK stimulation (data not shown).
Signal transduction
Stimulation of the granulosa cells by LH/FSH or FK showed variation in a wide spectrum of related genes, of which the vast majority were not reported to be mediated by gonadotrophins in human granulosa cells. This modulation by gonadotrophins is probably a reflection of an extensive shift in signalling by gonadotrophins upon granulosa cell luteinization. We have divided these genes into four categories: G-related genes, kinases, phosphotases and others (Table IV).
One of the most interesting findings was the down-regulation of genes coding for adenylate cyclase 7 and 9 (ADCY7 and 9), adenylyl cyclase-associated protein 2 (CAP2), and adenylyl cyclase-associated protein (CAP) (Conti, 2002; Kuznetsova, 2002). This may suggest lower response to LH/hCG despite the elevation of gene transcripts to these receptors (Figure 3). As was observed in our studies, attenuation of transmitting gonadotrophin signals downstream to gonadotrophin signalling may serve as a limiting factor in steroidogenic signal transduction (Zeleznik and Somers, 1999; Seger et al., 2001). Interestingly, FK did not down-regulate ADCY7 and 9. Adenylate cyclase 3 was not modulated either by LH, FSH or FK (not shown), suggesting that gonadotrophins may affect only specific subtypes of AC.
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Increase in Rho guanine nucleotide exchange factor 5 (ARHGEFS) gene transcript and decrease in Rho GTPase-activating protein 1 (ARHGAP1) gene transcript was evident both after LH and FSH cAMP stimulation (Table IV). The change in the activity of these gene transcripts is probably closely related to the marked change in genes coding for cytoskeletal proteins, since they are known to be involved in cytoskeleton remodelling (Lee and Gotlieb, 2002; Masuda-Robens et al., 2003). Moreover, cytoskeletal alterations caused by FK were even more extensive than those resulting from gonadotrophin activity. This observation is in-line with our data (Table II), which show that genes coding for cytoskeletal proteins are more markedly changed in FK-treated cells compared to gonadotrophin-stimulated cells (Amsterdam and Aharoni, 1994). We, for the first time, have proven the existence of a comprehensive change elicited in LH and FSH by gene transcripts coding for RAB GTPase. RAB1, 2, 4, 5A, 14, 27A, 31 expression increased, whereas RaB5C and 33B decreased. The RAB family is part of the Ras superfamily of small GTPases (Stenmark and Olkkonen, 2001). The different RAB GTPases are localized at the cytosolic face of specific intracellular membranes, where they function as regulators in distinct steps of membrane trafficking. In the GTP-bound form, the Rab GTPases recruit specific sets of effector proteins onto the membrane. Through their effectors, Rab GTPases regulate vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion (Stenmark and Olkkonen, 2001). A possible change in these proteins caused by gonadotrophins may be involved in mobilization of cholesterol esters in lysosomes to form free cholesterol substrates for steroid hormone production (Choi and Freeman, 1999). The RAB proteins may also be involved in endocytosis associated with the plasma membrane and exocytosis, which may take place following the secretion of growth factors such as epiregulin, amphyregulin, inhibin
, follistatin and vascular endothelial growth factor. The elevation of their synthesis may be expected due to the up-regulation of gene transcripts coding for these peptide hormones. RAB proteins may also be important in the traffic of membrane residues associated with cytoskeletal proteins, which are regulated during luteinization. However, RAB proteins are probably not involved in steroid hormone secretion since there is no up-regulation of RAB1, 14, 2, 5A in FK-stimulated cells, despite enhanced steroidogenesis by FK. Mitogen-activated protein kinase 1, 5 and 7 (MAP2K1, MAP3K5 and MAP3K7) genes were up-regulated by gonadotrophins, whereas only MAP3K5 was also up-regulated by FK. By contrast, MAP2K3, MAPK4K4, MAPK6 and MAPK14 gene activities decreased. The last two gene transcripts were not affected by FK. We have recently demonstrated that MAPK activation is stimulated by gonadotrophins and FK (phosphorylation of ERK1 and ERK2) in rat and human granulosa cells (Seger et al., 2001; Tajima et al., 2003). It is possible that activation of these kinases may lead to changes in the expression of kinases involved in this cascade, although we do not know which of them are phosphorylated in response to gonadotrophin or cAMP stimulation. An increase in the protein kinase gene and the cAMP-dependent regulatory type 1 alpha (PRKAR1A) gene both by gonadotrophins and cAMP is expected, since gonadotrophin and FK significantly elevated intracellular cAMP in the rat ovary (Richards and Hedin, 1988). However, it has not yet been reported whether PRKAR1A gene activity is elevated in the human ovary. Up-regulation of serum/glucocorticoid-regulated kinase (SGK) gene activity was shown to increase by LH and FSH (Table IV). Elevation of SGK by gonadotrophins in the rat ovary was demonstrated by Gonzalez-Robayna et al. (2000). Interestingly, we found no elevation in gene transcripts coding for SGK by FK, suggesting that this gene is not modulated by cAMP.
Gonadotrophin stimulation leads to up-regulation of the following gene transcripts for protein phosphatases: PPFIA4, PPP2R1A, PPP2R5B, PTP4A1, PTPRF, PTPRN and PTPN21. By contrast, there was a reduction in the expression of other protein phosphatase genes including: MKP-L,PPP2R5B, PPP3R1 and PTPN1. Since protein phosphatases can abrogate the activity of protein kinases (Seger et al., 2001), the net outcome of protein phosphorylation is hard to predict especially in view of phosphorylation/dephosphorylation of post-translational events.
Gene transcripts to phosphodiesterase 4D, cAMP-specific (PDNHD) was found to be considerably up-regulated by LH (Table IV), which may contribute to the attenuation of LH-dependent cAMP during granulosa cell luteinization. By contrast, FSH did not attenuate the expression of this gene, thus a different mechanism may be involved in the regulation of the cAMP cascade by FSH compared to LH. Regulators of G-protein signalling (RGS) proteins are known to negate positive G-coupled-mediated signalling (Chen et al., 1997; Tesmer et al., 1997; Mao et al., 1998). Gene transcript to RGS 16 (Wieland and Mittmann, 2003) was up-regulated by gonadotrophins, but not by FK. Since RGS 16 is controlled specifically by gonadotrophins it seems more likely that gonadotrophin signalling and thus RGS 16 up-regulation may contribute to down-regulation of gonadotrophins during granulosa cell luteinization (Figure 3). In contrast, gene transcripts of RGS 4 and 5 were down-regulated by gonadotrophins and much more extensively by FK. This modulation may also contribute to the regulation of gonadotrophin response to luteinized granulosa cells. However, which RGS protein interacts with gonadotrophin signalling has yet to be determined.
Modulation of gene expression by FSH compared to LH
Most gene stimulation induced by LH was similar to that of FSH, despite higher numbers of LH/CG receptors compared to FSH receptors in the IVF cells (not shown). In contrast, elevation in gene transcripts coding for HSD11B1, StAR, Sterol-C4-methyl oxidase-like (ERG25) and aromatase were >3-fold higher in LH-stimulated cells compared to FSH-stimulated cells, suggesting that LH compared to FSH is a better stimulant for luteinization and progesterone production. Indeed, women treated for IVF with FSH alone needed support of endogenous progesterone during pregnancy (Rimon,E. et al., 2004). Gene transcripts coding for collagen type XV alpha 1 (COL15A1) were highly elevated in LH- versus FSH-stimulated cells (by 3-fold), suggesting a higher synthesis of collagen XV alpha 1 during luteinization, while actin alpha 1 skeletal muscle (ACTA1) gene activity was considerably down-regulated by LH compared to FSH (7.2 fold), suggesting a sharp down-regulation of actin during luteinization and tissue remodelling during granulosa cell differentiation. In contrast, alpha-actinin-2-associated LIM protein (ALP) gene activity was down-regulated to a greater extent by FSH compared to LH stimulation (4.4-fold). Tumour necrosis factor alpha-induced protein 3 (TNFA3) gene transcript was up-regulated to a higher extent in FSH-stimulated cells than in LH-stimulated cells (3.3-fold), suggesting higher cytokine activity during luteinization than during granulosa cell differentiation (Matsubara et al., 2000). Up-regulation of hexokinase (HK2) and protein tyrosine phosphatase type IVA, member 1 (PTP4A1) gene transcripts and down-regulation of phospholipase C epsilon (PLCE) gene transcripts were much more marked in LH-treated cells than in FSH-treated cells. By contrast, glycerol kinase (GK) gene was significantly more activated after FSH stimulation compared to LH. These findings suggest that, despite the same degree of cell differentiation, LH and FSH may alter the extent to which the same genes are expressed.
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Conclusion |
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TopABSTRACTIntroductionMaterials and methodsResults and discussionConclusionREFERENCES |
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The changes in gene expression presented in this study were determined by RNA obtained from six individual women undergoing stimulated cycles as part of IVF treatment. Changes were reproducible in duplicate microarrays and also using two types of arrays (U95, U133). Deviation from the mean values was
35%. We included only changes >2–3-fold, which strengthens the validity of our data. We have not reported on constitutive expression of genes, in order to eliminate data which may represent the minor contamination of other cell types in the cultures. Our data suggest that the regulation of StAR by gonadotrophins is significant. However, its potential to be enhanced by other hormones that can elevate cAMP levels in the cell, such as the PGE family, should be considered, especially in view of the considerable elevation of gene transcripts to StAR by FK. Although aromatase transcriptional activity is a prime target for FSH stimulation, our data suggest that LH has an even higher potential to stimulate this enzyme. LH/CG receptors are believed to be down-regulated by LH or hCG. However, our data suggest that isolated granulosa cells luteinized in response to LH stimulation have a higher receptor density, similar to the in vivo intact corpus luteum. Moreover, despite higher receptor density in the corpus luteum, there is less response to hormone stimulation. This can now, for the first time, be explained in view of the pronounced down-regulation of adenylyl cyclase and up-regulation of phosphodiesterase in response to LH stimulation. Rearrangement of the cytoskeleton was demonstrated to be an integral part of luteinization in granulosa cells. The cytoskeleton rearrangement permits coupling between organelles involved in steroidogenesis. The data from the DNA chips strongly suggest that changes in transcriptional activity of genes mainly coding for actin and actin-binding proteins are involved in cytoskeletal rearrangement and enhanced steroidogenesis. LH and FSH are known to stimulate steroidogenesis, but at the same time, they could exert a mitogenic effect. In the present study we found an appreciable elevation of gene transcripts coding for several kinases, especially MAPKK1, MAPKKK5 and MAPKKK7, which are in the centre of the activation of the MAPK cascade. This gonadotrophin activity could contribute significantly to the stimulatory effects on cell proliferation. The induction of follicular rupture by LH may elevate the risk of follicular cell damage during ovulation and formation of the corpus luteum. Nevertheless, the sharp increase in BAX inhibitor-1 and ARC are novel observations, which may explain the anti-apoptotic activity of LH during corpus luteum formation and maintenance. Therefore, the modulation in the expression of these genes may contribute to the structural and functional integrity of the corpus luteum in ensuring successful implantation of the embryo and the progression of pregnancy.
Further examination concerning the physiological relevance of the genes reported in this study is necessary. The role of gonadotrophins in primary human granulosa cells can be followed by in situ localization of these gene products in the intact ovary and complemented by knockout or over-expression of genes that were modulated in vitro by gonadotrophins. It should be noted that the level of most of the gene products was not measured in this study. Further studies may involve western blot analysis or proteomics and immunocytochemistry in cultured granulosa cells and/or the intact ovary. This study offers new insights from which we can improve our understanding of gonadotrophin stimulation in the human ovary and the dynamic process of luteinization at the transcriptional level.
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Acknowledgements |
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We thank Dr J.F.Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA, USA) and Dr B.Geiger (Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel) for the generous gifts of anti-StAR and anti-vinculin antibodies, respectively. We thank Drs S.Saban and R.Ophir of the Bioinformatics and Biological Computing Unit at the Weizmann Institute of Science for excellent technical assistance. The work was supported by the Yad Avraham Center for Cancer Research and by the Center for Scientific Excellence 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.
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Submitted on November 27, 2003; resubmitted on December 15, 2003; accepted on January 15, 2004.
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