Mol. Hum. Reprod. Advance Access originally published online on March 23, 2006
Molecular Human Reproduction 2006 12(3):135-144; doi:10.1093/molehr/gah247
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Transcriptome analysis of FSH and FSH variant stimulation in granulosa cells from IVM patients reveals novel regulated genes
1Maxygen, Hørsholm, 2Department of Gynecology and Obstetrics, Rigshospitalet, Blegdamsvej, Copenhagen and 3The Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark
4 To whom correspondence should be addressed at: Maxygen, Agern Allé 1, DK-2970 Hørsholm, Denmark. E-mail: TBo{at}maxygen.dk
5 Present address: Høiberg A/S, Store Kongensgade 59A, DK-1264 Copenhagen K, Denmark
6 Present address: Gastrotech Pharma A/S - Nyhavn 43B, DK-1051 Copenhagen K, Denmark
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Abstract |
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FSH is crucial for oocyte maturation and fertility and is the main component in infertility treatment in assisted reproduction. The granulosa cells expressing the FSH receptor interact with the oocyte and provide nourishing substrates controlling the oocyte maturation. Thus, transcriptome analysis of granulosa cells stimulated by FSH is of major importance in understanding the communication between oocytes and granulosa cells. In this study, gene expression profiles were assessed in human granulosa cells from normal cycling in vitro maturation (IVM) patients using oligonucleotide gene chips. Granulosa cells were stimulated for 2 h with either FSH or a previously generated glycosylated FSH variant (FSH1208) that exhibited increased in vivo activity because of prolonged half-life. The analysis identified 74 significantly FSH/FSH1208 regulated genes. Amongst these were well known FSH regulated genes as well as genes not previously described to be important in the FSH signalling pathway. These novel FSH regulated genes include transcription factors [cAMP responsive element modulator (CREM)/inducible cAMP early repressors (ICER), GATA 6, ZFN 361, Bcl11a, CITED1 and TCF 8] and other regulatory proteins and enzymes (IGF-BP3, syntaxin and PCK1) possibly important for oocyte/granulosa cell interaction and function. Array data were validated for 13 genes by northern blots or RT–PCR. Furthermore, no significant differences in gene regulation were detected between the two FSH analogs. This work uncovers novel data important for understanding the folliculogenesis. Furthermore, the results suggest that FSH1208 has a gene expression profile like FSH and thus, in the light of known prolonged in vivo activity, might be a candidate for improved infertility treatment.
Key words: FSH/gene regulation/granulosa cells/IVM patients/microarray
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Introduction |
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FSH is of major importance in regulating folliculogenesis and oocyte growth and maturation (Robker and Richards, 1998
). The hormonal control of the ovary happens in an interaction between hormones regulated by the pituitary–gonadal axis (Rao et al., 1978; Simoni et al., 1997) and other factors with gonadotropic effect (Adashi et al., 1985; Poretsky and Kalin, 1987). Furthermore, ovulation requires a complex interaction between the oocyte and the surrounding follicular cells, the granulosa cells. In the ovary, FSH receptors are exclusively located on the granulosa cells and the follicle stimulation effect of the hormone is thus mediated and regulated by granulosa cells, mainly through the cAMP signalling pathway (Habener et al., 1995).
In view of the importance of the interaction between granulosa cells and the oocyte for fertility, discovery of novel FSH regulated genes might provide new insight in understanding the fertility process. Transcriptional analyses of the reproductive process have been undertaken using northern blots, RT–PCR or differential display (Voutilainen et al., 1986; Richards, 1994; Eramaa et al., 1995; Minegishi et al., 2000; Espey and Richards, 2002) or more recently, using microarray analysis with the possibility of analysing thousand of genes simultaneously (Leo et al., 2001; Liu et al., 2001; Chin et al., 2002; McLean et al., 2002; Sasson et al., 2003). Microarray technology has so far been applied in determining gene expression profiles in the gonads, but only few studies have characterized gene expression in response to FSH stimulation using cells specifically expressing the FSH receptor, i.e. sertoli- and granulosa cells (McLean et al., 2002; Sasson et al., 2003). However, the cells used in these studies have undergone FSH-stimulated maturation in vivo before FSH stimulation in vitro. Therefore, the effect of FSH on the transcriptome of granulosa cells is yet to be fully characterized.
In this study, an approach using unstimulated human granulosa cells from in vitro maturation (IVM) (Smith, 2001) patients was established, in order to shed additional light on the fertility process at the time of assisted reproduction. The goal was to characterize the gene expression profile in cells stimulated with FSH and a hyper-glycosylated FSH variant (FSH1208) with increased in vivo activity because of prolonged half-life (Perlman et al., 2003). The use of granulosa cells collected from women undergoing IVM treatment for infertility gave a unique possibility to study the gene expression in an ex vivo system as close to the in vivo conditions as possible, as the women were not hormonally stimulated before oocyte/granulosa cell retrieval. This is the first report of gene expression profiling from IVM patients. A multitude of novel FSH-regulated genes was identified in this study. Several of these genes were selected for further analysis to confirm the gene expression profile of FSH1208 and wildtype FSH versus unstimulated cells, and to analyse whether there were differences between FSH1208 and wildtype FSH.
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Materials and methods |
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Collection of human granulosa cells
Human granulosa cells were collected at The Institute for Human Reproduction at Symbion, DK-2100 Copenhagen, a research fertility clinic collaborating with the Fertility clinic at Herlev Hospital, DK-2730 Herlev. Healthy women referred for IVF because of male factor and/or tubal disease and unexplained infertility participated in the ‘Immature oocyte retrieval with IVM project’, reviewed and accepted by The Danish Central Scientific Ethical Committee. Furthermore, the women had normal ovulatory cycles with a mean length of between 26 and 35 days. They were between 18 and 35 years of age and had a BMI between 18 and 30 kg/m2. Excluded were all patients with infertility caused by any endocrine abnormalities. All patients gave their written informed consent to participate in the study. The oocytes and the cumulus mass of cells were transvaginally aspirated from the follicles on day 7–10 (8.86 ± 1.62 days) of the natural menstrual cycles. Day 1 is defined as the beginning of the menstruation. All visible follicles (
6) ranging from 5 to 10 mm in diameter were punctured. The oocyte was retrieved for subsequent IVM, whereas the excess amounts of granulosa cells were isolated by centrifugation 216 g for 15 min. The pelleted granulosa cells were then centrifuged on a histopaque gradient (Sigma, St. Louis; MO, USA; Cat. 1077) isolating the granulosa cells in the plasma–histopaque interface after centrifugation at 863 g for 20 min. The cells were subsequently washed in media, centrifuged for 10 min at 216 g, resuspended, counted and finally seeded in 24 well plates. Cells from each patient were divided into three treatment groups: one group of cells were left unstimulated, the second group of cells was stimulated with 5 U/ml (500 ng/ml) of recombinant FSH (ß-follitropin, Organon, Oss, The Netherlands) and the third group was stimulated with 5 U/ml of a glycosylated N-terminal extended (ANITVNITV) FSH variant, FSH1208 (Perlman et al., 2003). Cells were incubated for 2 h at 37°C and subsequently lysed and homogenized according to the RNeasy protocol for total RNA isolation (Qiagen, Hilden, Germany, Cat. 74014). Cell lysates from five or eight patients were pooled and then stored at –80°C until use. In total, 50 patients were included and five replicates were performed for each treatment group. Within the majority of the pools, the included patients represented their own control versus the different FSH stimulations [unstimulated versus FSH versus FSH1208: sample 8 versus 9 versus 10, 11 versus 12 versus 13, 25 versus 26 versus 27, respectively (the sample numbers refer to the pool numbers shown on top in Figure 1)] but there were a few exceptions dependent on the amount of RNA harvested. Therefore, some of the pools were not represented in all three treatment categories [unstimulated versus FSH1208: sample 28 versus 30; FSH versus FSH1208: sample 6 versus 7, unstimulated: sample 3, FSH: sample 2, respectively (the sample numbers refer to the pool numbers shown on top in Figure 1)]. Each pool contained 1–1.4 x 106 cells, with the patients contributing more or less equally. However, equal amounts of RNA were passed on the array and compared with the other pools. The pools of patient material were generated according to cell counts and cause of infertility in order to have the same amount of RNA in each pool and to eliminate a possible bias because of different causes of infertility.
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Human RNA sample processing for microarray
First strand and second strand cDNA synthesis was performed using the MessageAmpTM aRNA Kit (Ambion, Austin, TX, USA, Cat. 1750) and 1.5–2 mg of total RNA (with a A260/A280 ratio above 1.8) as template. cDNA was purified by phenol/chloroform extraction and the resuspended pellet (about 8 µl) was added to a mixture of vacuum-concentrated biotin-UTP/CTP where 3.8 µl of each biotin NTP (Enzo Biochemicals, New York, NY, Cat. 42818, 42814) had been reduced to a volume of 1 µl. Biotin-labelled complementary RNA (cRNA) was synthesized for 12 h at 37°C using T7 polymerase and the purified cDNA as template. For each sample, 10 µg cRNA was fragmented and hybridized to Affymetrix Human Focus Chip (HG Focus) with 8795 genes. Each chip was stained with streptavidin-phycoerythrin (SAPE), restained with an IgG and SAPE to amplify the signal, washed and scanned according to the manufacturer’s instructions.
Data analysis and selection
Five Human Focus chips (HG Focus, Affymetrix) were analysed for each treatment group. The chips contain 11 oligonucleotide probe pairs per sequence. All microarray data were analysed using the GenePublisher microarray data analysis software (Knudsen et al., 2003). Gene expression on different chips was normalized by applying a non-linear cubic spline method (Workman et al., 2002). Fifteen arrays in total with five replicates in each of the three groups were tested statistically using a t-test in order to reveal differentially expressed genes between the different groups. A Benjamini–Hochberg correction for multiple testing was used to assess false positive prediction rates, correcting the P value for each gene by the total number of genes (Benjamini and Hochberg, 2003). Finally, a hierarchical gene cluster analysis was performed based on vector angle distances using the ClusterExpress software (Knudsen et al., 2003).
Semi-quantitative RT–PCR
A two-step semi-quantitative RT–PCR method was used to confirm the changes in gene regulation in human granulosa cells upon FSH treatment. RNA used for RT–PCR and northern blot confirmation of the gene regulation is identical to RNA used in the microarray. Total RNA (1 µg) was first treated at 37°C for 30 min with 1 U RQ1 DNase (1 U/µl) (Promega, Madison, WI, USA, Cat. M6101) in presence of 4 µl 5X First Strand RTase buffer (Invitrogen, Carlsbad, CA, USA, Cat. 18064–014) in a final volume of 20 µl. DNase reaction was stopped by adding 2 µl of RQ1 DNase Stop Solution (Promega, Madison, WI, USA, Cat. M6101), after which 2 µl of 500 µg/ml oligo-(dT)18 were added to the tube. DNAse-treated RNA was preheated at 65°C for 10 min to denature secondary structures and inactivate the DNAse. The mixture was chilled on ice then 4 µl 5X First Strand RTase buffer, 4 µl 100 mM DTT, 2 µl 10 mM dNTPs and 1 µl RNaseOUT (RNase inhibitor; Invitrogen, Carlsbad, CA, USA, Cat. 10777–019) were added to the tube. The mixture was incubated at 42°C for 2 min, then 1 µl of SuperScript II RT (200 U/µl) (Invitrogen, Carlsbad, CA, USA, Cat. 18064–014) was added, giving a final volume of 40 µl. RT reaction was carried out at 42°C for 50 min. Reaction was stopped by heating the tube at 70°C for 15 min, after which cDNAs were aliquoted and stored at -80°C until use.
RT–PCR reactions were performed to confirm the changes in gene regulation in granulosa cells upon FSH treatment for 11 genes and replicated 2–4 times. Primer sequences and optimal PCR conditions (number of cycles and annealing temperatures) are listed in Table III. To ensure that no false positive PCR fragments would arise from putative genomic DNA contamination, most primer sequences were designed to span intron regions, when present. Amplification yields were measured by comparison of the PCR signal to the signal generated from the co-amplification of the internal standard house-keeping gene ß-actin, whose expression levels were unaffected by the hormonal treatment. Two different sets of ß-actin primers giving rise to amplicons of different sizes were designed (Table III). This allowed alternative use of one or the other primer set to get amplicons of sizes that were separable by electrophoresis from the co-amplified target gene PCR product. PCRs were performed on a RoboCycler Gradient 96 (Stratagene, La Jolla, CA, USA) using Ready-To-Go PCR beads (Amersham Biosciences, Piscataway, NJ, USA, Cat. 407513-96), 0.5 µl cDNA and 5 pmoles of each oligonucleotide primer in a final volume of 25 µl. The PCR program initially started with a 94°C denaturation step for 1 min, followed by 20 to 35 cycles of 94°C/30 s, annealing temperature °C /30 s, 72°C/30 s (see Table III for annealing temperature). For each gene, the number of PCR cycles was adjusted in order to maintain products within the linear range of amplification. ß-actin primers were added when 21-24 cycles were remaining in the specified gene’s linear amplification range. PCR samples were size-fractionated on 2% agarose gels, stained with 10 µg/ml ethidium bromide, flushed extensively with water and finally photographed on top of a 280 nm UV light box. Amplicon sizes were estimated relative to DNA ladder standards, transcription levels were assessed visually and the sequences of all amplicons were verified by DNA sequencing.
Northern blots
Total RNA (3 µg) was size-fractionated on a 1.2% formaldehyde/formamide agarose gel in 1x MOPS buffer (N-morpholinopropanesulphonic acid) and transferred to a Hybond XL membrane (Amersham Biosciences, Piscataway, NJ, USA, Cat. RPN 2020S) using capillary blotting in 10x SSC (sodium chloride/sodium citrate buffer). Membranes were baked at 80°C for 2 h to irreversibly fix the nucleic acids to the membrane. Probes for northern blots were generated using gene-specific PCR products as template (Table III). Human genomic DNA (PCK1) or cDNA (Syntaxin 4A, ß-actin) were used as PCR template. Gel-purified PCR products were labelled with [
-32 P]dCTP using the Prime-a-Gene Labeling System (Promega, Madison, WI, USA, Cat. U1100) according to the manufacturer’s instructions. Probes were purified using Sephadex NICK Columns (Pharmacia Biotech, Piscataway, NJ, USA, Cat. 17-0855-02). Membranes were hybridized over night at 65°C in Rapid-hyb buffer (Amersham Pharmacia, England, Cat. RPN1635) in the presence of 106 cpm/ml of probe. Blots were sequentially washed as follows: 2x SSC, 0.1% sodium dodecyl sulphate (SDS) at room temperature for 20 min; 1x SSC, 0.1% SDS at 65°C for15 min; 0.1x SSC, 0.1% SDS at 65°C for 15 min Blots were exposed to a hyperfilm (Amersham Pharmacia, Piscataway, NJ, USA, Cat. RPN1676K) for 1–72 h at -80°C. Membranes were subsequently stripped in boiling 0.1% SDS before hybridizing with a new probe.
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Microarray analysis of human granulosa cells
Gene expression profiles were analysed using microarrays comparing expression patterns from untreated human granulosa cells with either FSH- or FSH1208-treated cells. Human granulosa cells were collected from women undergoing IVM fertility treatment, and thus not hormonally treated before oocyte/cumulus cell collection (Smith, 2001
). In total, cells from 50 patients with male factor and/or tubal disease and/or unexplained infertility were included in the analysis. The collected aspiration contained, apart from granulosa cells, a mixture of other cells like connective tissue cells and blood cells, which might result in interfering signals. However, previous microarray analysis have shown that it is possible to obtain conclusive information, if control samples are being treated exactly like test samples, apart from not receiving FSH, and if replicates are made (Piper et al., 2002; Sasson et al., 2003). Cells from each patient were equally divided into the three treatment groups, letting each patient serve as her own control and with a total number of 15 chips, five replicates were performed for each treatment group. Using a high number of replicates as well as using the women as internal controls diminished false positives and negatives because of biological variation and sample pooling. In a first step, we used a microarray approach to investigate whether FSH and the FSH1208 variant would exhibit the same gene regulation pattern. The comparison of microarray data for control versus FSH and control versus FSH1208 revealed that the FSH1208 variant had a highly similar gene regulation profile as human recombinant FSH. FSH-treated cells were therefore compared with FSH1208-treated cells and no significant differences in expression were found between the two FSH analogs (lowest P-value 0.002, which is not significant when correcting for multiple testing of 8795 genes). For that reason the effect of FSH on cells was assessed in a new data analysis comparing unstimulated cells against stimulated cells (FSH as well as FSH1208). The resulting list of candidate genes regulated by FSH/FSH1208 is presented in Tables I and II as well as in Figure 1.
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Validation of FSH-regulated gene expression
Semi-quantitative Northern blot and RT–PCR analyses
The differential expression of 13 genes, whose transcript levels were regulated by the FSH/FSH1208 treatments according to the microarray data, was confirmed by northern blot or RT–PCR. None of the 13 tested genes were unregulated or regulated differently than expected according to the microarray results. These genes can be classified into different groups such as transcription factors (TF), other regulatory proteins and enzymes and were selected based on their potential biological role in granulosa cells as well as the novelty of the connection with FSH. Transcript levels of the constitutively expressed ß-actin gene were also assessed in all northern blots and RT–PCR and served as internal control for loading or amplification, respectively. Transcript levels of genes from both FSH- and FSH1208-treated cells were analysed and the results strongly indicated that the two hormone variants exhibited the same gene regulation pattern (as can also be seen in Figure 1).
According to the microarray data, the levels of the PCK1 transcripts were the most affected of all transcripts by the FSH treatment (11.6-fold up-regulation). PCK1 transcript levels were assessed by northern blot as verification and the result presented in Figure 2A clearly confirms the microarray data. The syntaxin-4A transcript levels were only moderately up-regulated according to our microarray data (1.8-fold) and the northern blot (Figure 2B).
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): genes of interest. ß-actin was reprobed on the same membranes and used as constitutively expressed loading control (
). In each blot; (-): untreated pool, (FSH): FSH treated pool, (1208): FSH1208 treated pool.This study shows that many TFs and components of signalling cascades are early targets of the FSH signalling pathway. This result demonstrates the high number of transcriptional regulation events taking place in granulosa cells in response to FSH. We confirmed the differential transcript accumulation for six up-regulated TF and one down-regulated MAP kinase by RT–PCR. The moderate up-regulation of the GATA 6 gene that was observed in the microarray analysis was confirmed in RT–PCR (Figure 3A). Transcripts from the CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain (CITED1) transcriptional co-regulator, the C2H2-like zinc finger protein (ZNF361), the zinc finger protein Bcl11a and the TCF 8 TF exhibited expression fold changes of 3.2, 5, 2 and 5.3, respectively according to microarray results, and RT–PCR clearly showed an important transcript accumulation for these four genes in response to the hormonal treatment (Figure 3B–E).
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The microarray data show a moderate down-regulation (1.4-fold) of the MAPK14 gene transcript level. This observation was confirmed by RT–PCR (Figure 4A). Similarly, the accumulation of the IGF-BP3 mRNA upon FSH treatment was confirmed by RT–PCR (Figure 4B). The RNA accumulations upon FSH treatment of the HDAC5, ALTE and RASSF2 genes whose apparent up-regulation according to microarray data was 2-, 4.6- and 1.5-fold, respectively, were confirmed by RT–PCR (Figure 4C–E).
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Promoter analysis
A promoter analysis was made in order to identify GATA elements because of the up-regulation of GATA 6. The upstream region (5 kbp) for each gene was extracted using the Ensembl database (Hubbard et al., 2002). The weight matrices available in the TRANSFAC database of known transcription factor binding sites (Matys et al., 2003) were searched against the upstream regions and all positions scoring higher than 0.95 times the maximum score for a given matrix were recorded as a hit. Interestingly, the analysis revealed homologous GATA regulatory elements in more than half of the listed genes (48/74) whereas CRE-like element was found in few of the genes (7/74), not including GATA. This indicates an important role for GATA elements as target of FSH hormone in granulosa cells.
Alternative RNA processing
The cAMP responsive element modulator (CREM) is a known TF that is activated in the cAMP pathway (Habener et al., 1995; Kameda et al., 1999). The CREM gene is composed of several exons and two alternative promoters. Alternative RNA processing thus gives rise to either full-length activating CREMs or shorter internal transcripts encoding the inducible cAMP early repressors (ICER) that repress the transcription of targeted genes, because of lack of kinase-inducible or transactivation domains (Foulkes et al., 1991; Laoide et al., 1993). To date, approximately 30 different splicing variants of CREM have been reported in the databases. Only ICER isoforms have been reported to be accumulating in rat granulosa cells upon FSH treatment (Kameda et al., 1999). To investigate which isoforms of CREM mRNA accumulate in FSH-treated granulosa cells, we undertook a RT–PCR approach using isoform-specific oligonucleotides (Table III). All amplicons were gel-isolated and sequenced to confirm isoform identities. As was expected, ICER isoforms were accumulating upon FSH treatment. More specifically, the CREM variant ‘h’ and ‘g’ (ICER1
) transcript levels were clearly higher in FSH-treated granulosa cells (Figure 5A and B, respectively). A closer inspection of the electrophoresis gel revealed that a second PCR product of smaller size accumulated in response to the hormonal treatment (Figure 5B). The sequence of this amplicon corresponded to a shorter version of the ICER1 isoform, which exhibited a truncated last exon, known as CREM variant ‘e’ (ICER11). Surprisingly, the use of specific primers allowing the detection of non-ICER isoforms of CREM resulted in amplification of a product that was apparently more prominent in FSH-treated granulosa cells compared with untreated cells (Figure 5C). Sequencing showed that the amplified DNA corresponded to the activator form of CREM splicing variants (CREM variants ‘a’, ‘m’, ‘o’ or ‘u’).
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Discussion |
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The first goal of this study was to get further insight in the transcriptional regulation by FSH in human granulosa cells. A few reports have previously identified FSH-regulated genes using microarray approaches. However, our approach is novel in that the granulosa cells used were collected from women undergoing IVM treatment for infertility, giving the advantage that the patients were not receiving any hormonal treatment for stimulation of ovulation, and the oocyte/granulosa cell retrieval took place early in their normal cycle, before the LH-surge. This gave a unique opportunity to study gene regulation in an ex vivo system as close to the in vivo conditions as possible. However, the limited number of untreated cells, because of the limited number of patients participating in the ‘Immature oocyte retrieval with IVM’ project, confined the study of FSH-induced gene regulation to one time-point and restricted the validation of the gene expression to RT–PCR, mainly. A short-term treatment of 2 h was performed to identify early target genes in the FSH signalling pathway. Interestingly, besides a number of genes with recognized roles in granulosa cells/FSH signalling pathway, a large number of genes was identified whose regulation was previously not known to be dependent on FSH. More specifically, according to the microarray data, the transcript levels of 74 genes were modified significantly by 2 h of FSH stimulation, with a fold change in expression levels ranging from 1.4 to 11.6. Some of these genes have been shown to be transcriptionally regulated by FSH in previous studies, among which are components of the steroidogenic pathway such as the steroidogenic acute regulatory protein (StAR), the low density lipoprotein receptor (LDLR), the cytochrome P450 subfamily XIA (CYP11A: cholesterol side chain cleavage) and the cytochrome P450 subfamily XIX (CYP19: aromatase) (Fitzpatrick and Richards, 1991
; LaVoie et al., 1999; McLean et al., 2002; Sasson et al., 2003). This observation validates the hormonal treatment performed in our experiments. Similarly, transcripts of the two genes BTG2 and BAG1 were previously reported to accumulate upon FSH treatments in sertoli cells and follicles, respectively (Liu et al., 2001; McLean et al., 2002). The present microarray data confirm these previously reported observations and show that BTG2 is also up-regulated in human granulosa cells in response to FSH. Furthermore, these results indicate that these two genes are early targets for the FSH signalling in granulosa cells. A recent study has shown that transcripts of the metallothionin 1X and 2A genes were accumulating upon FSH treatment in granulosa cells from IVF patients (Sasson et al., 2003). Our results confirm this observation as well and extend the list of FSH-regulated metallothionin genes with three new members (Table I). For several genes, this is the first report of FSH-dependent regulation in human granulosa cells. PCK1 is involved in gluconeogenesis or pyrovate metabolism, thus this result suggests that FSH might be involved in regulation of gluconeogenesis, though additional studies on other genes in the pathway will have to be performed to characterize a possible modulating role in glucose metabolism.
Syntaxins consist of a family of proteins known to be important for regulation of membrane interaction in vesicular trafficking and membrane fusion (Bennett et al., 1992, 1993). Recently, Syntaxin 4 orthologs were shown to be expressed in sea urchin egg and in mouse oocytes, where the protein is involved in exocytosis preventing polyspermy (Conner et al., 1997; Iwahashi et al., 2003). Furthermore, it has been speculated that this protein plays a role as t-SNARE receptor in facilitative glucose uptake in the blastocyst through GLUT8-containing vesicles (Pinto et al., 2002; Wyman et al., 2003) in a similar way as previously described in insulin-stimulated glucose uptake in skeletal muscles and adipocytes (Watson and Pessin, 2001). Here the syntaxin 4A gene is reported for the first time to be up-regulated in human granulosa cells in response to FSH stimulation. Granulosa cells surround the oocyte as a part of the cumulus cell mass and nourish the oocyte with glucose metabolites (Buccione et al., 1990; Cetica et al., 1999). One role for syntaxin 4A function in the granulosa cells could be that it acts as a t-SNARE regulator of glucose uptake together with a facilitative transporter (GLUT), catalyzing the transport of glucose into the granulosa cell, analogous to the glucose uptake in the blastocyst. Furthermore, the isolated granulosa cells might include cumulus cells that produce hyaluronan for the extracellular matrix critical to ovulation (Zhuo and Kimata, 2001; Rodgers et al., 2003; Richards, 2005). It is thus possible that both PCK1 and syntaxin are involved in hyaluronan synthesis, where sugar is an important moiety. Additional experiments monitoring the trafficking patterns of the protein will be required to elucidate the exact function in granulosa cells.
GATA 6 belongs to a family of zinc finger TFs known to be important regulators of differentiation-specific gene regulation (Orkin, 1992; Molkentin, 2000). The GATA 4 and 6 genes are both expressed in the reproductive system (Heikinheimo et al., 1997; Ketola et al., 1999; Laitinen et al., 2000; Gillio-Meina et al., 2003), but whereas the GATA 4 protein is known to be associated with regulation of apoptosis-related genes (Heikinheimo et al., 1997) and steroidogenesis-related genes (Tremblay and Viger, 2001), very little is known about the possible role of GATA 6 in granulosa cells. Interestingly, GATA 6 is involved in regulation of steroidogenesis in the adrenal gland (Jimenez et al., 2003; Saner et al., 2005) and in granulosa cells it is known to be involved in transcriptional regulation of at least one of the genes appearing in the present study, the StAR gene (Gillio-Meina et al., 2003). Furthermore, GATA elements are present in the regulatory region of several steroidogenic related genes, indicating an important role of the GATA transcription factors in the regulation of steroidogenesis (Tremblay and Viger, 2003).
CITED1, a CBP/p300 binding transcriptional co-regulator, has been suggested to play a role in tissue-specific regulation of CBP/p300-dependent gene expression (Yahata et al., 2000, 2001). Bcl11a and TCF8 genes encode zinc finger TFs. Bcl11a is associated with immune system development and regulation (Liu et al., 2003), whereas TCF8 is involved in T-lymphocyte-specific interleukin 2 gene expression (Williams et al., 1992). Finally, the TF belonging to the subfamily of C2H2 zinc finger gene family has been reported to be involved in spermatogenesis (Wu et al., 2001). None of these TFs have been described in female gonads previously.
The protein encoded by the MAPK14 gene has been shown to be involved as an integration-point for multiple biochemical signals leading to various cellular processes including transcriptional regulation and development (van Biesen et al., 1996).
IGF is believed to play a significant role within the ovary in regulating steroidogenesis, augmenting estrogen in synergy with gonadotrophins and it might play a role in oocyte maturation and suppression of apoptosis (Gomez et al., 1993; Chun et al., 1994; Poretsky et al., 1999). In rodents, IGF is an obligatory mediator of FSH-induced follicular maturation and IGF-BP3 and has been shown to inhibit follicular rupture (Bicsak et al., 1991; Yoshimura et al., 1996). IGF-BP3 is an IGF binding protein, thus its gene regulation might be of regulatory importance for the IGF-mediated action. High levels of IGF-BP3 seem to be correlated to decreased infertility (Amato et al., 1999; Poretsky et al., 1999) but otherwise no consistency in the regulation of IGF-BP3 in humans has been reported (Hamori et al., 1991; Cataldo et al., 1993; Adachi et al., 1995; Barreca et al., 1996; Amato et al., 1998, 1999; Sasson et al., 2003). Clearly, the complexity of the function of IGF-BP3 in the ovary will have to be exposed to additional studies to fully understand the human physiology of the ovary. A study comparing microarray data of different causes of infertility might elucidate whether IGF-BP3 expression differs depending on the cause of infertility.
The Ac-like transposable element (ALTE) gene transcript was the most significantly regulated microarray result. Transposons are ubiquitous mobile genetic elements important for chromosomal rearrangements (Kempken and Windhofer, 2001), but the function of ALTE is unknown. Histone deacetylase (HDAC5) is known to regulate chromatin structure/folding and gene repression (Grunstein, 1997; Davie, 1998) whereas the newly identified ras association domain family 2 (RASSF2) is shown to promote apoptosis and cell cycle arrest (Vos et al., 2003).
Apparently, all these genes seemed to be of some importance early in the FSH signalling, but functional studies must be performed in order to interpret the functional relevance of these genes.
In the reproductive organs, studies on CREB and CREM have mainly been carried out in testis/sertoli cells, in which gene activation was essentially controlled by CREB. CREM, however, was shown to act as a gene transcription repressor through the alternatively spliced ICER isoform (Don and Stelzer, 2002). To our knowledge, an up-regulation of an activating isoform of the CREM gene transcript in response to FSH stimulation in human granulosa cells is thus reported here for the first time and suggests both an activating and a repressing role for CREM in regulation of the cAMP-dependent FSH signalling pathway in granulosa cells.
Surprisingly, no apoptosis-related genes were found, apart from a cystein protease (Mch6 mRNA) and RASSF2. Indeed, Sasson et al. (2003) identified apoptosis-related genes as one of the major components of the FSH transcriptome. In the study by Sasson et al., human granulosa cells from IVF patients, which have been super-stimulated with FSH and hCG, were used to identify FSH regulated genes, albeit not until the over-stimulated refractory cells had regained responsiveness to further gonadotropin stimulation during prolonged culturing. The discrepancies in the list of differentially expressed genes in the two studies may thus be a result of the differences in the duration of the hormonal treatment, or reflect that IVF cells are more luteinized than IVM cells, just as long-term culture increases the apoptotic rate of IVF cells and thus influences the interpretations of the FSH regulated genes (Breckwoldt et al., 1996). Although data sets of the different studies analysing the transcriptome of FSH stimulation display degrees of overlap, differences in study design using granulosa- or sertoli cells from humans, mice or rats or granulosa cells from IVF or IVM patients emphasize the differences in the genomics of species and the impact of assay conditions. Human granulosa cells from IVM patients will hopefully be a consistent implement in the future in understanding the transcriptional processes of FSH in folliculogenesis.
The second goal of this study was to assess whether the previously generated FSH1208 variant, containing two additional glycosylation sites in an N-terminal extension (Perlman et al., 2003) would exhibit the same pattern of gene regulation as wild-type FSH. A microarray approach that could reveal putative discrepancies in gene regulation between the two hormone variants was used. This was done in order to rule out the possibility that FSH1208 would activate or repress undesirable biological pathways, possibly by binding to receptors other than the FSH-receptor, or by stimulating the FSH receptor in a slightly different fashion than native FSH causing a different signalling cascade. Indeed, gene microarrays provide a valuable tool for the validation of drugs in early stages of development (Joyce et al., 2001; Ulrich and Friend, 2002). In the present report, it is shown that the short-term treatment of human granulosa cells with FSH and FSH1208 results in very similar transcriptomes. This observation was confirmed for all genes whose transcript levels were assessed by northern blot or RT–PCR. The minor structural changes in the FSH molecule, prolonging the in vivo activity because of increased half-life, do not change the gene expression profile apparently. The gene profile of FSH1208 combined with the pharmacological profile of the variant (Perlman et al., 2003) emphasizes the possibility of using FSH1208 as a candidate for improved infertility treatment in the future.
In conclusion, several novel differentially expressed genes involved in human folliculogenesis were found in this array study in response to FSH stimulation. Future studies with several time-points and prolonged FSH stimulation will contribute additionally to identify downstream-regulated genes, just as functional studies must elucidate the functional relevance of these genes. IVM treatment of infertility might be a promising method of assisted reproduction in the future and the use of IVM granulosa cells seems to be a valuable tool, in order to assess the gene regulation and thus the physiological processes taking place during the early fertility process.
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Professor Svend Lindenberg, The Fertility Clinic at Herlev Hospital, is thanked for useful discussions and advices. Furthermore, the authors thank Professor Bent Ottesen, Department of Gynecology and Obstetrics, Rigshospitalet, for critical reading of the manuscript. S. Perlman was supported by a PhD fellowship from the Academia of Technical Science.
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Submitted on August 25, 2005; accepted on November 11, 2005.
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