Mol. Hum. Reprod. Advance Access originally published online on August 3, 2006
Molecular Human Reproduction 2006 12(10):593-599; doi:10.1093/molehr/gal068
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PGE2 up-regulates EGF-like growth factor biosynthesis in human granulosa cells: new insights into the coordination between PGE2 and LH in ovulation
1Department of Molecular Cell Biology, 2Department of Biological Regulation, the Weizmann Institute of Science, Rehovot and 3IVF and Fertility Unit, Assaf Harofeh Medical Centre, Tel Aviv University, Tel Aviv, Israel
4 To whom correspondence should be addressed at: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 76100 Israel. E-mail: idorit{at}netvision.net.il
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Abstract |
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LH and prostaglandin E2 (PGE2) share many similar effects on the pre-ovulatory follicle. They can induce independently cumulus expansion, the resumption of meiosis and progesterone production. However, cyclooxygenase-2 (COX-2) inhibitors were found to hinder most of the LH-induced effects. Recently, EGF-like growth factors amphiregulin (Ar) and epiregulin (Ep) were found to be produced in response to LH stimulation and to induce cumulus expansion and oocyte maturation. We aimed at evaluating whether PGE2 induces Ar and Ep syntheses in human granulosa cells and whether the inhibition of PGE2 production by selective COX-2 inhibitor, nimesulide, affects LH-induced Ar and Ep biosynthesis. Ar and Ep mRNA levels increased following PGE2 stimulation, in a dose- and time-dependent manner, which resembled those of LH. The blockade of protein kinase A (PKA) (by H89) and mitogen-activated protein kinase (MAPK) (by UO126) reduced the expression of PGE2-induced Ar and Ep biosynthesis. Although the stimulation of the cells with LH in the presence of nimesulide did not change the progesterone levels, it resulted in a significant reduction of Ar and Ep biosynthesis. In conclusion, PGE2 may mimic LH action, at least in part, by the induction of Ar and Ep biosynthesis, which involves cAMP/PKA and MAPK pathways. The negative effect of nimesulide on the ovulatory process may be due to the reduction of Ar and Ep biosynthesis, which implies a possible collaborative role between PGE2 and LH on their induction.
Key words: amphiregulin/epiregulin/LH/PGE2/oocyte maturation
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Introduction |
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The ovulatory process, which ultimately leads to follicle rupture, oocyte release and luteinization of the follicle wall, involves a complex series of biochemical and biophysical events. Among the various mediators of this process, prostaglandins (PGs), which play a central role in inflammation, have been recognized as key mediators for more than three decades (reviewed in Armstrong, 1981
).
LH surge selectively induces the biosynthesis of cyclooxygenase-2 (COX-2) in granulosa cells of different mammalian species, which in turn produces a rapid rise in follicular fluid prostaglandin E2 (PGE2) (Wong and Richards, 1991; Sirois, 1994; Sirois and Dore, 1997). In cultured follicles, PGE2 can replace LH in inducing the resumption of meiosis and in changing the pattern of steroidogenesis to progesterone dominant (reviewed in Lindner et al., 1980). In vivo studies revealed its critical role in cumulus expansion and restructuring of the pre-ovulatory follicle, as documented by the effects of COX-2 and PG receptor knockouts in mice (Davis et al., 1999; Hizaki et al., 1999). However, the mechanisms by which PGs induce these LH-like effects are still not clear (Conti et al., 2006).
Non-selective COX inhibitors, such as indomethacin, effectively block ovulation while not impairing progesterone production, as demonstrated in rats and rabbits (reviewed in Sirois et al., 2004). This luteinized unruptured-follicle syndrome was subsequently demonstrated in numerous species including pigs, sheep, cows and humans (reviewed in Norman, 2004). Interestingly, indomethacin was still effective in blocking ovulation when given after the LH surge has passed its peak, and the block was not restored by exogenous LH. By contrast, the administration of PGE2 was proved to induce ovulation in indomethacin-treated rats (Lindner et al., 1980). Similar results were demonstrated while using selective COX-2 inhibitors (reviewed in Sirois et al., 2004).
Recently, LH stimulation of human granulosa cells was found to induce amphiregulin (Ar) and epiregulin (Ep) biosynthesis (Freimann et al., 2004). These EGF-like growth factors were found to interact with the cumulus–oocyte complex (COC) inducing the mucification of the cumulus cells and the resumption of meiosis of the oocyte in rodents (Park et al., 2004).
Because both LH and PGE2 share similar effects on the pre-ovulatory follicle and LH exerts most of these effects by the induction of Ar and Ep biosynthesis, we hypothesized that PGE2 may exert its effects in a similar manner, namely by the induction of Ar and Ep. Therefore, we tested whether PGE2 could induce the formation of Ar and Ep as well as the effect of selective COX-2 inhibitor on their biosynthesis in human granulosa cells.
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Materials and methods |
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Cultures of primary human granulosa cells
Primary granulosa cells were obtained from women, aged 22–38 years, undergoing IVF treatment in Assaf Harofeh Medical Centre, because of male factor infertility. Patients were treated according to the long protocol guidelines, i.e. received a GnRH agonist at the mid-luteal phase, followed by FSH or human menopausal gonadotrophin and eventually by the administration of hCG. Granulosa cells were isolated from aspirated follicular fluid after oocyte retrieval. The follicular fluid was centrifuged at 300 g for 5 min at room temperature. The resulting pellets were resuspended in 10 mM Tris, 0.84% NH4Cl, pH 7.4, to cause the lysis of blood cells (15 min shaking at 37°C). Several washings in phosphate-buffered saline (PBS) eliminated debris. Cells were plated in Dulbecco’s modified Eagle’s medium (DMEM/Ham F12 1:1), supplemented with penicillin (100 IU/ml), streptomycin (100 mg/ml) and 10% fetal calf serum (FCS). Cells were cultured for an additional 6 days in medium containing 10% FCS and washed every 24 h with PBS in a hormone-free medium as described previously (Breckwoldt et al., 1996
; Sasson and Amsterdam, 2002).
Cultures of primary human cumulus cells
Immediately following oocyte retrieval, cumulus cells were stripped from the oocyte with a micropipette. Cells were plated immediately and treated like granulosa cells.
Antibodies
Goat polyclonal antibodies against human amphiregulin were obtained from Santa Cruz (Santa Cruz, CA). Mouse monoclonal anti-diphospho-extracellular signal-regulated kinase (ERK; anti-active ERK/MAPK) (anti-DP-ERK) antibodies and anti-general ERK (anti-G-ERK) antibody were obtained from Sigma (Rehovot, Israel). Monoclonal antibodies against human ß-tubulin (Sigma, St Louis, MI), rabbit anti-goat (R&D Systems, Minneapolis, MN), goat anti-rabbit and goat anti-mouse IgG (Sigma, St Louis, MI), coupled to horse-radish peroxidase (HRP) were applied as second antibodies, respectively. Anti-progesterone antibodies were provided generously by Dr F.Kohen (Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel).
Reagents
H89, a specific protein kinase A (PKA) inhibitor, and UO126, a specific MEK inhibitor, were purchased from Sigma (St Louis, MI). Recombinant hLH was kindly provided by the NIH and Dr A.Parlow of the National Hormone and Pituitary Program (Bethesda, MD). PGE2 was purchased from Pharmacia (Puurs, Belgium). Nimesulide, a specific COX-2 inhibitor, was obtained from A.G. Scientific (San Diego, CA).
LH and PGE2 stimulations
LH (1 IU/ml) or PGE2 (10–6–10–9M) was administered after 16 h of starvation (FCS-free medium). Thereafter, cells were harvested up to 24 h of stimulation and collected for further RNA and protein analyses.
Determination of progesterone and protein levels
Progesterone accumulated in the culture medium was determined by radioimmunoassay at the end of cell stimulation using [H3]-labelled progesterone (Amersham Biosciences Piscataway, NJ) (Kohen et al., 1975). Protein was assayed according to Bradford (1976).
Western blot analysis
The analysis of specific protein levels was carried out as previously described (Tajima et al., 2003). Briefly, following hormone stimulation, primary cultures were rinsed with ice-cold PBS, harvested in lysis buffer containing 50 mM Hepes (pH 7.2), 150 mM NaF, 30 mM sodiumpyrophosphate, 1 mM orthovanadate, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml of leupeptin and 5 µg/ml of aprotinin and were subjected to western blot analysis to detect different proteins. Samples containing equal amounts of protein (50 µg) were separated on 12% acrylamide SDS–PAGE. Equal protein loading was detected by Ponceau staining. The relevant proteins were detected on blots using their specific antibodies. Western blots were repeated three times with three groups of women (each group consisting of three women undergoing IVF treatment).
Determination of ERK activity by phosphorylation
Serum-starved human granulosa cells were stimulated with PGE2 (10–6 M), and the phosphorylation of the activation TEY motif of ERK was then assessed using western blot analysis with DP-ERK antibodies, as previously described (Yung et al., 1997).
RNA isolation, reverse transcription and RT–PCR
Total RNA was isolated using a commercial kit (Gentra, Minneapolis, MN). First-strand cDNA was created by RT (Promega Reverse Transcription System, Madison, WI) from total RNA. The reaction mix contained 1 µg of total RNA, 0.5 µg of oligo(dT)15 primer, 1 mM each dNTP and 50 U of M-MLV reverse transcriptase (Promega). One microlitre of the RT reaction was taken for quantitative PCR of Ar and Ep gene expressions using TaqMan® Master Mix (Applied Biosystems) probes for Ar (assay no. Hs00155832_m1), Ep (assay no. Hs00154995_m1) and GAPDH (assay no. Hs99999905_m1) using sequence detection system PRISM 7000 (Applied Biosystems).
Animals
Twenty-one-day-old C57 black/6J Ola Hsd female mice were treated by 10 IU of pregnant mare’s serum gonadotrophin (PMSG) i.p. After 24 h, mice were randomly divided to receive either nimesulide (n = 3) or vehicle (n = 3) treatment. Following additional 24 h, all mice were injected with 5 IU of hCG i.p. Mice were killed, and ovaries were removed 24 h after hCG administration and fixed in 4% paraformaldehyde for subsequent Haematoxylin and eosin staining as previously described (Almog et al., 2001).
Statistics
Real-time quantitative RT–PCR data are presented as mean ± SE. Calculations were performed using SPSS software (student’s t-test, Version 11, Chicago, IL, USA). P-values of <0.05 were considered statistically significant.
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Results |
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Quantitative RT–PCR revealed a time-dependent pattern of both Ar and Ep expression levels following treatment with either PGE2 (10–6 M) or LH (1 IU/ml) up to 24 h. Ar mRNA was up-regulated 2–8 h following PGE2 treatment, reaching maximal expression level at 4 h, and decreased thereafter (Figure 1A). Ep was found to be elevated 2–8 h following PGE2 stimulation, reaching peak level at 8 h, and decreased thereafter (Figure 1B). Interestingly, equivalent time courses of Ar and Ep mRNA expression levels were observed following LH stimulation.
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In order to examine whether Ar and Ep gene expression acts in a dose-dependent manner following PGE2 stimulation, human granulosa cells were treated with either increasing doses of PGE2 or LH (1 IU/ml) for 4 h (Figure 2). Although PGE2 stimulation significantly increased Ar (P < 0.05) and Ep (P < 0.01) gene expression at 10–7M (Figure 2a, 2b respectively), reaching a similar level as with 1IU/ml of LH at 10–6M of PGE2. The release of progesterone to the culture media was coordinated with the expression of Ar and Ep and likewise reached peak level at 10–6 M of PGE2, which was similar to 1 IU/ml of LH (Figure 2C).
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Western blot analysis demonstrated a clear elevation of Ar intracellular protein level in response to either PGE2 (10–6 M) or LH (1 IU/ml) stimulation for 24 h as compared with control (Figure 3A). This up-regulation followed the changes in Ar mRNA expression, suggesting a regulation of Ar synthesis in the transcription level.
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Because PGE2 receptors have been detected in mouse cumulus cells (Hizaki et al., 1999
), whereas LH receptor expression is restricted in cumulus cells, we examined whether Ar is regulated in response to PGE2 stimulation in isolated human cumulus cells. Western blot analysis of cell lysates treated by PGE2 (10–6 M) for 24 h revealed a clear elevation in the protein level of Ar following PGE2 stimulation, in a similar way to granulosa cells (Figure 3B).
Progesterone secretion gradually increased following PGE2 (10–6 M) stimulation, starting from baseline level of 307 ± 6 ng/ml up to 753 ± 1.6 ng/ml (P = 0.008) at 24 h. A similar time course was observed following LH (1 IU/ml) stimulation as reported by us earlier (Freimann et al., 2004), reinforcing the similarity between LH and PGE2.
Signaling cascades of PGE2-induced Ar and Ep biosynthesis
PGE2 is known to activate G-protein-coupled receptors (GPCR, reviewed in Jabbour and Sales, 2004). To test whether the elevation of Ar and Ep following PGE2 stimulation was due to the activation of the cAMP-PKA pathway, we stimulated cells with (10–6 M) PGE2 with or without PKA inhibitor, H89. The expression level of Ar (Figure 4A) and Ep (Figure 4B) mRNA was significantly lower in the presence of H89 compared with control cells (P < 0.01 and P < 0.001, respectively), indicating that PGE2-dependent Ar and Ep biosynthesis is regulated by PKA activity.
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Because the ERK cascade was implicated in the signalling of GPCR (Naor et al., 2000), we examined whether the ERK cascade is also activated in human granulosa cells in response to PGE2 stimulation. As shown in Figure 5A, pERK1 and pERK2 were not detected in granulosa cells without PGE2, whereas incubation with PGE2 (10–6 M) resulted in the phosphorylation of ERK. This response was observed 5 min after exposure to PGE2, reaching maximal level after 10 min, followed by a decline at 20–60 min. To further examine the involvement of the ERK cascade in PGE2-induced biosynthesis of Ar and Ep in human granulosa cells, its action was impeded by using the MEK inhibitor UO126. The expression level of PGE2-induced Ar (Figure 5B) and Ep (Figure 5C) mRNA was significantly lower in the presence of UO126 compared with control cells (P < 0.01 and P < 0.001, respectively), indicating that Ar and Ep biosynthesis by PGE2 is mediated, at least in part, by MAPK activity.
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Nimesulide effect on LH-induced Ar and Ep biosynthesis
Because COX-2 inhibitors were shown to block most of the LH-induced effects, we further examined the effect of COX-2 inhibition on LH-induced Ar, Ep and progesterone expressions. Cells were pre-incubated with 50 µM nimesulide for 60 min, followed by LH (1 IU/ml) stimulation. Interestingly, although co-stimulation of the cells with nimesulide and LH did not change the progesterone levels (Figure 6A), compared with LH-treated cells, it resulted in a significant reduction of Ar (Figure 6B) and Ep (Figure 6C) mRNA biosynthesis (P < 0.05). This reduction was evident 2 h following LH stimulation and lasted up to 16 h.
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At the protein level, although a clear elevation of Ar was observed following LH treatment, as compared with control, no change in Ar was observed when co-stimulation with LH and nimesulide was carried out (Figure 6D).
To examine the in vivo effect of nimesulide on cumulus expansion following LH treatment, mice were treated with PMSG followed by LH either in the presence or in the absence of nimesulide. Although pre-ovulatory follicles of control animals exhibited a clear expansion of the cumulus cells around the oocyte, follicles of nimesulide-treated mice demonstrated firmly clustered and closely packed cumulus cells (Figure 7).
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Discussion |
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The LH surge triggers a cascade of events in the ovarian follicle, including the resumption of meiosis of the oocyte, cumulus expansion and oocyte maturation (reviewed in Amsterdam and Rotmensch, 1987
; Amsterdam et al., 1989). However, the restricted expression of LH receptors on cumulus cells and the oocyte itself raised the possibility that many LH effects were indirect (Amsterdam et al., 1975; Peng et al., 1991). Recently, it was demonstrated that the incubation of germinal vesicle-stage follicles of mouse with Ar, Ep and betacellulin recapitulates the morphological and biochemical events triggered by LH, including cumulus expansion and oocyte maturation (Park et al., 2004). It was hypothesized that Ar and Ep are synthesized in granulosa cells upon LH surge and act in a paracrine manner on the COC, inducing cumulus expansion and oocyte maturation.
In a similar way as LH, PGE2 was found to induce the resumption of the meiotic division of the oocyte in cultured follicles (reviewed in Sirois et al., 2004). However, the mechanism by which PGE2 induces these LH-like effects is yet unknown. In this study, we provide a novel observation that PGE2 induces Ar and Ep gene expressions in human granulosa cells. Similarly, Ar protein levels were elevated following PGE2 stimulation. Moreover, because cumulus cells express low or undetectable levels of LH receptors, we assumed that PGE2 might induce EGF-like growth factor expression in these cells. In this study, we demonstrate for the first time that PGE2 stimulation of isolated human cumulus cells induces Ar protein synthesis. These preliminary results indicate that cumulus cells can produce EGF-like growth factors, thus providing a possible autocrine mechanism on top of the paracrine effect exerted by granulosa cells. Collectively, it is suggested that LH-like effects of PGE2 may be accounted for by the induction of Ar and Ep both in granulosa and in cumulus cells.
LH initiates ovulation through the activation of GPCR, which activates adenylate cyclase, PKA and cAMP-responsive elements (Furman et al., 1986; Aharoni et al., 1993; Selvaraj and Amsterdam, 1997). Recently, it was demonstrated by us that LH-induced biosynthesis of Ar and Ep in primary human granulosa cells is mediated at least in part by the cAMP/PKA cascade (Freimann et al., 2004; Freimann et al., 2005). Likewise, PGE2 receptors are members of the GPCR family, which include four subtypes (EP1–EP4). The stimulation of EP2 and EP4 receptors activates adenylate cyclase, thus increasing intracellular cAMP concentrations (reviewed in Breyer et al., 2001; Funk, 2001). Because a significant attenuation of Ar and Ep biosynthesis occurred by H89 in PGE2-treated cells, it seems likely that the effect of PGE2 on Ar and Ep formation is mediated to a large extent by the cAMP/PKA cascade in a similar way to LH.
LH is also known to activate other signalling pathways than cAMP/PKA cascade. ERK1 and ERK2, members of MAPK family, were phosphorylated by MEK1 in mouse and porcine COCs during oocyte maturation and are known to be essential for cumulus expansion (Su et al., 2002). Herein, we show that when human granulosa cells were cultured with PGE2, ERK1/2 phosphorylation was evident. Furthermore, the significant attenuation of Ar and Ep occurred by UO126 in PGE2-treated cells implies that PGE2-induced Ar and Ep biosynthesis is also mediated via the activation of the MAPK cascade.
Notably, both the kinetic patterns of Ar and Ep mRNA and their expression magnitude following LH stimulation at 1 IU/ml resemble those achieved by PGE2 at 10–6 M. This further suggests a similarity in signal cascades between LH and PGE2 with regard to Ar and Ep biosynthesis.
However, the time course of PGE2-induced progesterone biosynthesis does not fit the same pattern as either Ar or Ep, suggesting that their induction by PGE2 involves different signal transduction pathways. This finding resembles the differential signal cascades that were found to be activated in granulosa cells by LH in steroidogenesis pathway versus proliferation (Seger et al., 2001).
Much of the knowledge about the role of PGE2 in ovulation originated from the use of COX-2 inhibitors. Several selective COX-2 inhibitors were tested for their effect on the ovulatory process (reviewed in Sirois et al., 2004). Testing nimesulide in female mice revealed similar results as in other selective COX-2 inhibitors, namely the impairment of cumulus expansion. Because Ar and Ep were shown to induce cumulus expansion in mice, we assumed that COX-2 inhibition might impair the LH-induced Ar and Ep biosynthesis. Herein, we demonstrate that whereas nimesulide significantly impaired LH-induced Ar and Ep mRNA biosynthesis, as well as Ar protein level, its administration did not change the LH-induced progesterone levels in human granulosa cells. A recently published study further supports this finding. Ar and Ep mRNA and protein levels were reduced significantly in COCs and ovaries collected from COX-2-null mice and PG receptor-null mice at 4 and 8 h after hCG stimulation, respectively (Shimada et al., 2006). Taken together, these findings suggest that PGE2 is essential for Ar and Ep induction by LH in human granulosa cells. Indeed, PGs are known to modulate the action of various hormones and transmitter substances, reinforcing or antagonizing their effects (Lindner et al., 1980). Furthermore, the failure of LH in restoring the ovulation of COX-2 inhibitor-treated animals could be explained by the nimesulide blockage of LH-induced Ar and Ep biosynthesis. Finally, luteinized unruptured-follicle syndrome could also be related, at least in part, to the suppression of Ar and Ep biosynthesis by nimesulide while not impairing progesterone production.
In conclusion, we demonstrated that PGE2 may mimic LH action at least in part by the activation of Ar and Ep biosynthesis in human granulosa cells. Furthermore, we found that PGE2 induced Ar expression in isolated human cumulus cells, suggesting an autocrine loop of EGF-like factors on these cells, in addition to the paracrine effect induced by granulosa cells, which should be further investigated. Finally, we showed that COX-2 inhibitors effectively blocked LH-induced Ar and Ep biosynthesis, signifying a novel role of PGE2 in LH-induced signal cascade of these EGF-like growth factors. Further research is needed to elucidate this collaborative role between PGE2 and LH on the induction of Ar and Ep biosynthesis.
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Acknowledgements |
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This study was supported by Yad-Avraham Center for Cancer Research, by a research grant from Mr Edgar L.Cadden and La Fundation Raphael et Regina Levy and by the Women Health Research Center at the Weizmann Institute of Science, Rehovot, Israel. A.A. is the incumbent of the Joyce and Ben B.Eisenberg Professorial Chair in Molecular Endocrinology and Cancer Research at the Weizmann Institute of Science.
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References |
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Submitted on May 19, 2006; resubmitted on June 2, 2006; accepted on July 5, 2006.
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