Molecular Human Reproduction, Vol. 5, No. 8, 703-707,
August 1999
© 1999 European Society of Human Reproduction and Embryology
Evidence for the presence of hepatocyte growth factor expression in human ovarian follicles
1 Department of Obstetrics and Gynecology, University of Tokyo, Tokyo 113-8655, and 2 CREST, Japan Science and Technology, Kawaguchi 332-0012, Japan
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Abstract |
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The presence of hepatocyte growth factor (HGF) in follicular fluid (FF) relative to concentrations of sex steroid hormones and human chorionic gonadotrophin (HCG) was investigated. A total of 69 FF samples were obtained during oocyte retrieval for in-vitro fertilization (IVF) from 11 patients with no apparent endocrine disorders. The concentrations of HGF, oestradiol, progesterone, HCG and testosterone in FF samples were measured by enzyme-linked immunosorbent assay. Transcription of HGF and its receptor, c-met, was detected by reverse transcription–polymerase chain reaction (RT–PCR). Human FF samples contained ~90-fold higher amounts of HGF (24.2 ± 1.2 ng/ml), compared with those of serum (0.28 ± 0.04 ng/ml). Concentrations of HGF in FF were positively correlated with those of progesterone (r = 0.649, P < 0.0001) and HCG (r = 0.264, P = 0.026) concentrations in FF. However, HGF concentrations were not significantly correlated with oestradiol and testosterone. HGF in FF was detected by Western blotting, as a single 90 kDa band, corresponding to a single chain form. Additionally, mRNA for both HGF and its receptor were detected in a crude granulosa cell preparation from the pre-ovulatory follicles. These findings suggest that HGF is produced locally in human ovarian follicles and may have a physiological role as an autocrine/paracrine factor.
follicular fluid/hepatocyte growth factor/IVF embryo transfer/ovary
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Hepatocyte growth factor (HGF), which is identical to scatter factor (Weidner et al., 1991
) and tumour cytotoxic factor (Shima et al., 1991), is a heparin-binding glycoprotein that consists of a 60 kDa 
-chain and a 30 kDa ß-chain linked by disulphide bonds (Gherardi et al., 1989). HGF was initially discovered as a hepatotrophic factor present in plasma from patients with fulminant hepatic failure (Gohda et al., 1986). Later on, several lines of evidence have implicated HGF in mitogenic, motogenic and morphogenic functions on various epithelial cells derived from rodents and humans (Nakamura et al., 1986; Tajima et al., 1992).
Recently, HGF mRNA has been detected in murine ovaries (Liu et al., 1994) and bovine thecal cells (Parrott et al., 1994); in the adult mouse, the HGF receptor, c-met, was found in growing follicles (Yang and Park, 1995). Furthermore, HGF protein has been detected in conditioned medium from bovine thecal cell cultures (Parrott et al., 1994). The expression of HGF has been shown to be stimulated by human chorionic gonadotrophin (HCG), a luteinizing hormone (LH) agonist, in bovine thecal cells (Parrott and Skinner, 1998).
The biological effects of HGF within the ovary are complex. For instance, HGF induces proliferation of bovine granulosa cells but not of bovine (Parrott et al., 1994) or rat (Zachow et al., 1997) thecal cells. HGF reversibly suppresses LH-dependent androgen synthesis while enhancing LH-dependent progesterone synthesis in rat thecal cells (Zachow et al., 1997). A recent study has provided a framework for a positive feedback loop between thecal cells and granulosa cells by demonstrating that HGF stimulates the expression of Kit ligand (KL) in bovine cultured granulosa cells and KL reciprocally stimulates the expression of HGF in thecal cells (Parrott and Skinner, 1998).
Given these observations, it is intriguing to speculate that HGF may play a role during ovarian follicular development and ovulation by mediating gonadotrophin action. However, there is no information regarding HGF in human ovaries. In order to determine the physiological role of HGF in human ovaries, we asked whether HGF is present in follicular fluid (FF). Here we demonstrate the presence of a considerable amount of HGF in FF suggesting a possible autocrine/paracrine role for HGF in the human ovary.
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Materials and methods |
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Subjects
After obtaining informed consent, 11 infertile women undergoing in-vitro fertilization (IVF) and embryo transfer for tubal factor or male factor infertility were recruited in this study. Their ages were 26–37 years (mean ± SEM, 32.0 ± 1.0 years). The women were pretreated with a gonadotrophin-releasing hormone (GnRH) analogue (buserelin acetate, Suprecur®; Hoechst, Tokyo, Japan), starting in the mid-luteal phase of the preceding cycle. After verifying ovarian suppression by observing the ovaries and endometrium by ultrasonography, daily i.m. injections of 150–300 IU human menopausal gonadotrophin (HMG; Nikken, Tokyo, Japan) were given, depending on individual responses. HCG at a dose of 10 000 IU (HCG; Mochida, Tokyo, Japan) was administered when the diameter of the leading follicle was
17 mm. Transvaginal ultrasound-guided oocyte retrieval was performed 35 h later. During oocyte collection, each follicle was aspirated separately and the FF was collected into a sterile plastic tube by electricity-driven suction. The oocyte was isolated from the aspirate and its degree of maturity was assessed according to the classification of Hoshi et al. (Hoshi et al., 1983). After isolation of the oocyte, aspirated FF was immediately centrifuged (1500 g), and the supernatant was divided into aliquots and stored at –80°C until use. Only follicles in which an oocyte was clearly identified were analysed, so that 69 FFs were assessed. Serum was also sampled from each woman just before oocyte retrieval.
Hormone measurement
Concentrations of oestradiol, progesterone, testosterone, and HCG in FFs were measured in duplicate, using a Serono SR1 analyser (Serono, Allentown, PA, USA). The standard preparation of HCG was the World Health Organization (WHO) first International Reference preparation (IRP) (75/537).
HGF concentrations in FFs and sera were measured in duplicate, using a specific enzyme-linked immunosorbent assay (ELISA) (Institute of Immunology, Tokyo, Japan). The limit of sensitivity of this ELISA was 0.1 ng/ml. The intra-assay coefficients of variation were <10%. All assays were conducted by investigators who had been blinded to the clinical data.
Western blotting
The HGF in FF were partially purified by heparin–Sepharose affinity chromatography. The slurry of 0.5 g Sepharose CL-6B (Pharmacia Inc, Piscataway, NJ, USA) was incubated with ~100 ml FF with gentle stirring for 3 h at 4°C. The mixture was poured into an open column. The packed column was washed with phosphate-buffered saline (PBS) and eluted with 15 ml of 1 mol/l NaCl. The elutant was further concentrated to ~400 µl by Bio-Max 30 (Millipore, Bedford, MA, USA). The partially-purified HGF in 1:80 dilution was resolved by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions in the parallel lane with recombinant human HGF (Institute of Immunology, Tokyo, Japan). The proteins were transferred to nitrocellulose membranes and incubated with anti-human HGF heavy chain mouse monoclonal antibody (Institute of Immunology, Tokyo, Japan) using the ECL Western blotting system (Amersham, Buckinghamshire, UK).
Reverse transcription–polymerase chain reaction (RT–PCR)
In order to detect HGF mRNA, cellular components were obtained from FF by centrifugation at 150g for 5 min; cells were resuspended in PBS. To minimize the contamination of blood cells, clear FFs obtained on the first puncture were collected for analysis. The suspension was layered onto Ficoll–Paque (Pharmacia, Uppsala, Sweden) and centrifuged at 300 g for 15 min. The crude granulosa cell subpopulation was recovered from the interface, washed with PBS, pelleted and snap-frozen in liquid nitrogen. Total RNA was extracted and 1 µg was reverse-transcribed in a 20 µl volume using TaKaRa RNA PCR kit (Takara Shuzo, Tokyo, Japan), according to the manufacturer's instructions. A 5 µl aliquot of the reaction was used for amplification with the following primers : 5'-GCC TGA AAG ATA TCC CGA CA-3', sense primer, corresponding to nucleotides 839–858 of the published sequence (Miyazawa et al., 1989), and 5'-TTC CAT GTT CTT GTC CCA CA-3', antisense primer (nucleotides 1342–1361). In the genomic DNA, the paired primers span four introns. A total of 35 cycles were carried out, each consisting of 94°C for 1 min; 60°C for 1 min; and 72°C for 1 min. An aliquot of the reaction was then analysed by agarose gel electrophoresis. The size of the predicted product was 523 bp. To detect c-met mRNA, a high affinity receptor for HGF, reverse-transcribed was amplified with the following primers: 5'-TCT TGG GAC ATC AGA GGG TC-3', sense primer, corresponding to nucleotides 1362–1381 of the published sequence (Park et al., 1987), and 5'-TGA CTG CAG GAC TGG AAA TG-3', antisense primer (nucleotides 1564–1583). In genomic DNA, the paired primers span two introns. A total of 35 cycles were performed, each consisting of 94°C for 30 s; 66°C for 30 s; and 72°C for 1 min. The size of the predicted product is 222 bp. For both PCR procedures, negative controls, i.e. without cDNA or primers, were negative for amplification.
Statistical analysis
Data are expressed as means ± SEM. Correlations were calculated by linear regression analysis. P < 0.05 was considered to be statistically significant.
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Results |
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In all the FF samples and sera examined, HGF concentrations were above the limit of the sensitivity of the ELISA. Concentrations of HGF in FF were 24.2 ± 1.2 ng/ml, which were ~90-fold higher compared to those of serum (0.28 ± 0.04 ng/ml). The concentrations of oestradiol, progesterone, testosterone and HCG in FFs are shown in Table I
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As shown in Figure 1
, simple regression analysis showed that the concentrations of HGF in FFs were positively correlated with those of progesterone (r = 0.649, P < 0.0001) and HCG (r = 0.264, P = 0.0258). In contrast, there was no significant correlation between the concentrations of HGF and those of oestradiol or testosterone.
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The HGF concentrations in the follicles containing grade I and II oocytes (2.58 ± 0.15 ng/ml) tended to be higher than those in the follicles containing grade III and IV oocytes (2.16 ± 0.19 ng/ml), between-group differences were not significant. No correlation was detected between HGF concentration and follicular size.
Western blotting of HGF is shown in Figure 2. A single band of ~90 kDa was detected in the lane of partially purified HGF from FF. In the lane of recombinant human HGF, a single band of ~60 kDa was detected. The 90 and 60 kDa bands correspond to a single chain form and a heavy chain of HGF respectively.
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As depicted in Figure 3,
a PCR product of total RNA extracted from granulosa cell-rich population demonstrated a clear band for HGF mRNA at ~523 bp, consistent with its predicted size. Likewise, a PCR product for c-met mRNA was detected as a band at ~222 bp, consistent with its predicted size.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The present study is the first to demonstrate that FF samples aspirated at the time of oocyte retrieval in women undergoing an IVF/embryo transfer programme contain considerable amounts of HGF, which are almost 100 times higher than those found in serum obtained at the same time. HGF in FF was shown to be a single chain form. In addition, our RT–PCR study demonstrated that mRNA of both HGF and its receptor, c-met, were expressed in crude granulosa cell subpopulations of human preovulatory follicles.
To date, the regulatory mechanism of HGF production in human ovaries has not been investigated. A number of growth factors or cytokines (e.g. epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin-like growth factor-I, tumour necrosis factor-, interleukin-1), have been shown to stimulate HGF expression in non-reproductive cells and tissues (Ekberg et al., 1992; Tamura et al., 1993; Gohda et al., 1994). As these growth factors are also detected in the human ovary, they might regulate the production of HGF in ovarian follicles in human. Furthermore, in mouse ovary, the hgf gene is transcriptionally regulated by oestrogen, via a direct interaction of the oestrogen receptor complex with cis-acting oestrogen responsive elements identified in the mouse hgf gene (Liu et al., 1994; Jiang et al., 1997). However, oestrogen concentrations in FF were not correlated with those of HGF. In this regard, it is known that oestradiol concentrations in FFs decline close to ovulation (Chaffin et al., 1999). In this study, FFs were collected at the time of oocyte retrieval when oestradiol concentrations in FFs were supposed to drastically decline. This may explain the failure to detect a correlation between HGF and oestradiol concentrations in FFs.
Recently, HCG and Kit ligand (KL) have been shown to stimulate HGF transcription in cultured bovine thecal cells. Interestingly, HCG has been shown to stimulate KL transcription in cultured bovine granulosa cells (Parrott and Skinner, 1998). Taken together with our observations that the HGF concentration is positively correlated with the HCG concentration in human FF, it is intriguing to speculate that HCG might have a direct stimulatory effect and/or an indirect stimulatory effect via an enhanced KL production from granulosa cells on HGF production in preovulatory human follicles.
The current data also demonstrate that the concentrations of HGF in FF are highly correlated with those of progesterone. Previously, it has been shown that HGF stimulates basal and LH-dependent progesterone production in cultured rat thecal cells (Zachow et al., 1997). Therefore, the positive correlation observed here gives rise to assumption that the stimulatory effect of HGF on progesterone production may also be at work in human follicles.
Mature HGF is a heterodimeric protein consisting of a heavy chain and a light chain held together by a disulphide bond (Gohda et al., 1988). The two chains are produced from a single chain precursor by proteolytic processing (Shimomura et al., 1995). This proteolytic processing is reported to be necessary for HGF to exert its mitogenic activity on hepatocytes in primary culture as well as other cultured cells (Hartmann et al., 1992; Lokker et al., 1992; Naka et al., 1992). In this study, the HGF detected in FFs was shown to be a single form. Physiological roles of HGF in human follicles remain to be delineated. As yet, we cannot be certain whether the HGF present in FFs is bioactive.
Apoptosis of the surface epithelial cell layer that overlies the ovulatory follicle has been demonstrated prior to ovulation and is considered to be essential for ovulation (Murdoch, 1995). In cultured rat ovarian surface epithelial cells, HGF reduces cell contact and induces apoptosis (Gulati and Peluso, 1997). Based on these findings, it is postulated that ovarian surface epithelial cells, if exposed to HGF, become apoptotic. Relatively higher concentrations of HGF in pre-ovulatory FFs as shown in this study may be in line with the hypothesis.
Regarding the localization of HGF production, it has been shown that HGF mRNA was expressed in thecal cells and not in bovine granulosa cells (Parrott et al., 1994), whereas other studies have shown that HGF mRNA was expressed both in thecal cells and granulosa cells of rat ovaries (Zachow et al., 1997). The present results, showing that the mRNA of both HGF and its receptor are expressed in crude granulosa cell subpopulations should be tempered because of a possible contamination of a few thecal cells into granulosa cell subpopulation given that granulosa cells were obtained through follicle aspiration. Further study is needed to specify the localization of HGF in human follicles. At any rate, it seems logical to assume that HGF present in FF is locally produced in human follicles.
In summary, our study demonstrated that human FFs contain considerable amounts of HGF, which were significantly correlated with progesterone and HCG concentrations in FFs. Given the presence of HGF in FF along with the detection of mRNAs for both HGF and its receptor, c-met, in a crude granulosa cell subpopulation, it is reasonable to speculate that HGF might have a possible physiological role in folliculogenesis and ovulation in an autocrine/paracrine fashion.
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Acknowledgments |
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The authors thank Yuko Kai for her technical assistance.
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Notes |
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3 To whom correspondence should be addressed
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References |
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Submitted on October 6, 1998; accepted on May 11, 1999.
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