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Messenger Ribonucleic Acid Content in Rat Ovarian Granulosa Cells by Hepatocyte Growth Factor and Follicle-Stimulating Hormone1
a Southern Illinois University (SIU) School of Dental Medicine, Department of Applied Dental Medicine, Alton, Illinois 62002-4700
ABSTRACT
Hepatocyte growth factor (HGF) suppresses FSH-dependent estradiol-17ß (E2) production in ovarian granulosa cells (GC). The mechanisms of action for HGF in GC are unknown; however, activation of the HGF receptor, c-Met, can induce c-Akt/protein kinase B (PKB)-mediated signal transduction in nonovarian cells. Using immature rat GC, the present study investigated the effects of HGF within the estrogen biosynthetic pathway, concomitant with changes in c-Met and PKB
mRNA expression. Granulosa cells were incubated with androstenedione and FSH, HGF, and/or dibutyryl-cAMP (Bu2-cAMP). Follicle-stimulating hormone and Bu2-cAMP each stimulated estrone (E1) and E2 synthesis at 48 h. Hepatocyte growth factor suppressed FSH-dependent E2, but not E1, synthesis. Semiquantitative reverse transcription–polymerase chain reaction showed that HGF impaired FSH-supported 17ß-hydroxysteroid dehydrogenase type-1 (17ß-HSD) and cytochrome P450 aromatase (P450arom) mRNA levels. Hepatocyte growth factor did not reduce E2 synthesis or 17ß-HSD and P450arom mRNA expression in the presence of Bu2-cAMP at 48 h. The FSH and HGF each down-modulated c-Met mRNA accumulation, whereas Bu2-cAMP increased c-Met mRNA content. Between 0 and 48 h a biphasic change in PKB mRNA content occurred with either FSH or HGF; however, PKB mRNA accumulation was augmented by HGF. Collectively, results suggest that HGF can suppress E2 production in GC by disrupting cAMP-dependent 17ß-HSD and P450arom. Changes in c-Met and PKB mRNA content provide a potential link between HGF signaling and the FSH-dependent mechanisms that control the steroidogenic differentiation of GC.
Follicle-stimulating hormone directs granulosa cell (GC) differentiation into estrogenic cells. This process enables GC to convert theca cell androgens into estradiol-17ß (E2). Bioactivity of FSH is itself mediated by cAMP-dependent signal transduction [1]. The FSH–cAMP-dependent signaling supports the expression and function of the estrogenic enzymes in GC:cytochrome P450 aromatase (P450arom) and 17ß-hydroxysteroid dehydrogenase type-1 (17ß-HSD) [2]. The P450arom and 17ß-HSD orchestrate FSH-dependent conversion of androgens into estrone (E1) and E2, respectively.
Bioactivity of FSH in GC is subject to modulation by intraovarian cytokines [3]. Hepatocyte growth factor (HGF) shows promise as one such regulatory intraovarian cytokine. Hepatocyte growth factor was initially characterized due to its mitogenic and motogenic actions in hepatocyte cultures [4]. Hepatocyte growth factor protein has been measured in human follicular fluid [5], and HGF mRNA has been detected in GC and theca cells obtained from immature rat ovaries [6]. Hepatocyte growth factor is part of an autocrine/paracrine system that exerts multiple levels of control within GC and theca cells [7]. For example, HGF blocks LH-dependent androgen production by theca cells [6]. Moreover, HGF stimulates bovine GC mitosis [8] but impairs the FSH-dependent conversion of androstenedione into E2 in vitro [9, 10].
Although HGF can mediate GC growth and steroidogenesis, virtually nothing is known regarding how HGF functions in GC. One study has shown that HGF suppresses P450arom activity in bovine and diethylstilbestrol (DES)-primed rat GC in vitro [9]; implying that the conversion of androstenedione into E1 is selectively blocked by HGF. The comparative effects of HGF on E1 and E2 production, as well as any effects of HGF upon P450arom and 17ß-HSD expression, would enable more complete understanding about how HGF alters the estrogen biosynthetic pathway in GC.
The HGF receptor, c-Met, is expressed in GC [11]. In isolated bovine follicles, c-Met mRNA accumulation increased relative to follicle size [9]. Therefore, it appears as if c-Met expression is hormonally regulated during folliculogenesis; moreover, it is likely that changes in c-Met expression dictate GC responsiveness to HGF. To date, the temporal and hormonal regulation of c-Met expression in GC has not been directly tested.
In nonovarian cells, HGF binding and activation of the c-Met tyrosine kinase induces a complex array of phospholipid-dependent signal transduction [7]. This signaling motif incorporates the stimulation of one or more c-Akt/protein kinase B isozymes (PKB, ß, and 
) [12]. Because this HGF–PKB-dependent signaling pathway has been observed, it is possible that similar mechanisms may function in HGF-stimulated GC.
By determining changes in the expression of c-Met and HGF signaling molecules (i.e., PKB) in GC, we can begin to elaborate upon how HGF blocks FSH-dependent E2 production. In this study, HGF- and FSH-stimulated alterations in the estrogen biosynthetic pathway are compared with changes in the relative abundance of both c-Met and PKB mRNA expression in GC.
MATERIALS AND METHODS
Reagents and Supplies
Recombinant human HGF (lyophilized with BSA as a carrier, >97% purity) was purchased from R&D Systems (Minneapolis, MN). Human recombinant FSH was generously donated by the National Hormone and Pituitary Program of the NIDDK and Dr. Parlow (Harbor-UCLA, Torrance, CA). McCoy's 5a medium (M5a, serum-free) and Medium 199 were purchased from GIBCO-BRL (Grand Island, NY). Oligonucleotide polymerase chain reaction (PCR) primers were synthesized by GIBCO-BRL. Culture plates were purchased from Falcon (Lincoln Park, NJ). [32P]dCTP (3000 Ci/mmol) was obtained from Dupont NEN (Boston, MA). The E2 and E1 RIA kits were obtained from Diagnostic Products Corporation (Los Angeles, CA) and Diagnostic Systems Laboratories (Webster, TX), respectively. Microcarrier was generously donated by Molecular Research Products, Inc. (Cincinnati, OH). Unless otherwise specified, all assay reagents were purchased from Sigma (St. Louis, MO), and reverse transcription (RT)-PCR reagents were purchased from Perkin Elmer Cetus (Foster City, CA).
Granulosa Cell Culture
All animal procedures were approved by the SIU-Edwardsville Institutional Animal Care and Use Committee. Immature (25–27-day-old) Sprague-Dawley rats (SASCO, Wilmington, MA) were euthanized via CO2 inhalation followed by cervical dislocation. Ovaries were removed and placed in ice-cold Medium 199 supplemented with 0.1% BSA (containing PSG: penicillin, 100 U/ml; streptomycin sulfate, 100 µg/ml; and L-glutamine, 2 mM). Ovaries were cleaned of bursa and other extraneous tissues, and GC were collected from the surrounding media following follicle puncture. Granulosa cells were centrifuged (250 x g), then resuspended in a known volume of M5a that was supplemented with PSG. Granulosa cell number and viability were determined by trypan blue exclusion using a hemacytometer.
Aliquots of GC (1 x 105 viable cells/well) were placed in 24-well culture plates. All GC were incubated in a final volume of 500 µl M5a/well containing 0.1 µM androstenedione, and GC identified as controls in this report did not receive treatments in addition to androstenedione. Designated GC were given FSH (0.3, 3.0, or 30 ng/ml) with or without HGF (30 ng/ml). The HGF concentration was chosen in order to approximate the reported Kd (0.3 nM) for HGF binding [4], as well as HGF concentrations measured in human follicular fluid [5]. The effect of HGF on dibutyryl-cAMP (Bu2-cAMP) steroidogenic differentiation was investigated by challenging GC with Bu2-cAMP (50, 100, and 250 µM), in the presence and absence of HGF (30 ng/ml). Cultures were conducted at 37°C in a humidified atmosphere containing 5% CO2 in air.
Incubations to measure E1 and E2 production were terminated at 48 h. The GC-conditioned media were collected and frozen at -20°C pending RIA to measure E1 and E2 content. Radioimmunoassays were conducted according to the kit manufacturers' protocols.
Reverse Transcription–Polymerase Chain Reaction
To measure the relative level of P450arom, 17ß-HSD, and c-Met mRNAs, GC were incubated for either 48 h (17ß-HSD and P450arom), or 12, 24, 36, and 48 h (c-Met), in the presence and absence of FSH (0.3, 3.0, or 30 ng/ml), HGF (30 ng/ml), or Bu2-cAMP (100, 250, or 500 µM). For analysis of PKB mRNA content, GC were cultured with FSH or HGF for 12, 24, 36, and 48 h. An aliquot of GC was frozen immediately upon harvest from the follicles (t = 0 GC) in order to establish basal levels of c-Met and PKB mRNAs.
The RT-PCR procedures were based upon the initial methods described by Orly et al. [13]. In brief, upon collection of the GC-conditioned media for measurement of estrogen content, the RNA extraction was performed using Trizol reagent containing 5 µl microcarrier per well. The RNA was resuspended in diethyl pyrocarbonate-treated MilliQ H2O. The RNA samples from duplicate treatment groups were pooled and RNA concentration was measured at A260. The RNA was stored at -80°C pending RT-PCR.
Aliquots of RNA (1 µg RNA/reaction) were used as templates for the synthesis of cDNA in reactions containing 1x PCR buffer II, 0.5 mM dNTPs, 100 ng oligo (dT)12–18, 1 U/µl recombinant placental RNase inhibitor, 200 U M-MLV reverse transcriptase, and 4 mM MgCl2. After incubation at 37°C for 30 min, the reaction was heat inactivated at 95°C for 5 min and then cooled to 22°C. Specific cDNA primers (10–50 pmol) for P450arom [14], c-Met [15], PKB [16], and 17ß-HSD type-1 [17] were used during PCR reactions as previously described. The PCR primer sequences are listed in Table 1. Each reaction also contained 1–5 µCi of [-32P]dCTP and 0.5 U AmpliTaq Gold polymerase in 1x PCR buffer II. Gene-specific cycles of PCR (GeneAmp 2400 thermocycler, Perkin Elmer Cetus) were run as follows: (1) 17ß-HSD and P450arom: 25 cycles: 94°C, 1 min; 55°C, 1 min; 72°C 2 min; (2) c-Met and PKB: 28 cycles: 94°C, 45 sec; 60°C, 45 sec; 72°C, 1 min. Both thermocycler protocols were terminated with a 10-min extension phase at 72°C. All RT-PCR reactions included a negative control in which no RNA was added. The amplification of all target mRNAs was determined to be linear with respect to increasing concentrations of RNA (1, 2, 5, 10, and 20 µg/reaction), using the above indicated cycle numbers.
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Primers designed to amplify a specific 194-bp region of the cDNA for the rat ribosomal protein, L19, were included in each PCR reaction [13]. This provided an internal control for differences in total RNA concentration among samples and variability among individual PCR amplifications. This method reflects a direct correlation between RNA concentration and L19 levels using the aforementioned PCR cycles, as has been demonstrated in our preliminary studies (not shown) and published reports by others using immature rat GC [13].
To validate the effectiveness of each set of oligonucleotide primers in amplifying the desired target rat cDNA sequence, a preliminary RT-PCR reaction was performed using GC RNA. In this experiment, the P450arom, 17ß-HSD, c-Met, and PKB PCR products were digested with restriction endonucleases. Specific restriction endonucleases were chosen that selectively cleaved each mRNA product into two smaller fragments of known base pair sizes.
All PCR products were visualized via electrophoresis using 2% agarose gels stained with ethidium bromide. For semiquantitative studies, the individual bands were cut from the gels and counted in a ß-spectrometer. The cpm present in the bands representing 17ß-HSD, P450arom, PKB, and c-Met mRNAs were normalized to the cpm in the L19 mRNA bands for each treatment. Relative mRNA abundance is presented in this report as the ratio of target gene/L19 in arbitrary units.
Statistical Analyses
Treatments were administered in duplicate in all studies. All experiments were repeated a minimum of three times. Mean values from independent experiments were statistically analyzed by unpaired t-test and multiple comparisons were performed using one-way ANOVA followed by Tukey's test. Values were determined to be significant when P < 0.05.
RESULTS
The Effects of HGF on Estrogen Synthesis in GC
The effect of HGF on FSH-dependent E1 and E2 production Results showed that FSH (0.3 and 3.0 ng/ml) increased E2 over control levels. The stimulatory actions of FSH were suppressed by HGF (Fig. 1a); but importantly, FSH-stimulated E1 production was not diminished by HGF (Fig. 1b). This novel finding indicates that FSH-supported 17ß-HSD expression and/or activity is blocked by HGF, thereby disrupting the enzymatic conversion of E1 into E2. In order to better define these steroid data, 17ß-HSD and P450arom mRNA levels were evaluated by semiquantitative RT-PCR.
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The effect of HGF on 17ß-HSD and P450arom mRNA expression As shown in Fig. 2, FSH (0.3, 3.0, and 30 ng/ml) stimulated the basal level of 17ß-HSD (Fig. 2a) and P450arom (Fig. 2b) mRNAs. Hepatocyte growth factor did not significantly alter the basal amount of P450arom and 17ß-HSD mRNAs. However, FSH-supported 17ß-HSD mRNA content was significantly reduced by HGF (Fig. 2a), and this effect correlated with the suppressive effect of HGF on FSH-dependent E2, but not E1, accumulation. Surprisingly, FSH-stimulated P450arom mRNA content was also impaired by HGF (Fig. 2b), and this observation somewhat contradicts the data showing that FSH-dependent E1 accumulation was not attenuated by HGF. Such results raise several questions as to the effect of HGF on 17ß-HSD and P450arom protein in GC as FSH-dependent E1 production was not blocked.
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The effect of HGF on Bu2-cAMP-stimulated E2 synthesis and expression of 17ß-HSD and P450arom mRNAs Results showed that Bu2-cAMP (50, 100, and 250 µM) stimulated the synthesis of E2 when compared to control GC (Fig. 3). However, HGF did not suppress Bu2-cAMP-supported E2 production (Fig. 3). Because Bu2-cAMP-dependent E2 production was not attenuated by HGF, E1 concentrations were not measured in these cultures.
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Because the addition of Bu2-cAMP overcame the suppressive actions of HGF on E2 synthesis in GC, the effect of HGF on Bu2-cAMP-stimulated P450arom and 17ß-HSD mRNA expression was also examined. As shown in Fig. 4, HGF did not significantly change Bu2-cAMP-supported 17ß-HSD and P450arom mRNA levels in GC. Collectively, the preceding data indicate that HGF affects one or more sites of FSH-stimulated cAMP production, but nothing is presently known about the molecular regulatory mechanisms used by HGF in GC. The available evidence shows that c-Met is the first step in HGF signal transduction; therefore, the regulation of c-Met mRNA expression was examined in GC.
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Modulation of c-Met mRNA Expression in GC by FSH, HGF, and Bu2-cAMP
Initial studies compared the effects of FSH, HGF, and Bu2-cAMP on c-Met mRNA expression at 48 h (Fig. 5a,b). As shown in Fig. 5a, c-Met mRNA levels were reduced in the presence of FSH (0.3, 3.0, and 30 ng/ml) or HGF (30 ng/ml) when compared to control GC. No significant difference in c-Met mRNA content was measured when comparing the suppressive effect of FSH versus that of HGF (Fig. 5a).
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Cyclic AMP is a predominant second messenger that is stimulated by FSH. Because FSH caused a reduction in c-Met mRNA, the ability of a cAMP analogue (Bu2-cAMP) to alter c-Met expression was investigated. Data revealed that Bu2-cAMP (100, 250, and 500 µM) stimulated a significant increase in c-Met mRNA content when compared to control (Fig. 5b, 0 µM Bu2-cAMP) or FSH-treated GC (Fig. 5a) at 48 h in vitro.
Time-course In order to investigate changes in c-Met expression during the early course of GC differentiation in vitro, a detailed time-course study was conducted (Fig. 6). The relative abundance of c-Met mRNA in control GC did not change between t = 0 and 12 h. However, when compared to freshly isolated GC (t = 0) and the 12-h cultures, c-Met mRNA levels were increased at 24 and 48 h.
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In GC that were challenged with HGF, c-Met mRNA content was not different from controls at 12 h (Fig. 6). When compared to control GC, HGF caused a precipitous decline in c-Met mRNA content at 24 h, and this suppressive action was also detected at 48 h.
Dibutyryl-cAMP significantly augmented c-Met mRNA accumulation over that measured in control GC at 12 h in vitro (Fig. 6, 100 µM concentration shown). However, at 12 h, c-Met mRNA content was not different in Bu2-cAMP- or HGF-treated GC. When compared to the 12-h cultures, no additional increase in c-Met mRNA was induced by Bu2-cAMP at 24 and 48 h. However, in contrast to either the control GC or GC challenged with HGF, c-Met mRNA content was elevated at 24 and 48 h in the presence of Bu2-cAMP.
The above studies have shown that c-Met mRNA content is distinctly modulated by FSH, HGF, and Bu2-cAMP. The HGF-dependent signaling molecules have not been reported in GC. However, others have demonstrated that HGF stimulation is coupled with c-Met and PKB signal transduction. As an initial step in linking HGF with PKB in GC, regulation of PKB mRNA expression was investigated.
Regulation of PKB mRNA by HGF
Using RT-PCR, our initial studies detected the mRNA for the three known PKB isoforms (, ß, and ) in preparations of whole ovarian cell dispersates from immature rats (Zachow, unpublished observations). Subsequent screening by RT-PCR revealed that PKB mRNA was observed in GC cultures (Fig. 7: 1096 bp, lane 1, control; lane 2, FSH). Although PKBß and PKB were not detected (not shown), the presence of PKBß and PKB in GC cannot be discounted. If HGF uses PKB as a signaling molecule in GC, it follows that PKB mRNA expression can be modulated by HGF in GC.
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At 12, 24, 36, and 48 h in vitro, the relative abundance of PKB mRNA was not different between control GC and those cells challenged with FSH (Fig. 8). In control and FSH-stimulated GC, PKB mRNA content increased at 12 h, relative to mRNA levels detected in t = 0 GC. In the presence and absence of FSH, PKB mRNA declined significantly at 24 h and 36 h, but mRNA levels rose sharply at 48 h (Fig. 8).
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In HGF-stimulated GC, a similar biphasic temporal pattern of PKB mRNA accumulation was observed. However, when compared to control and FSH-stimulated GC, at 12, 36, and 48 h, the HGF challenge resulted in a significantly greater relative level of PKB mRNA (Fig. 8).
DISCUSSION
Hepatocyte growth factor can down-modulate ovarian steroidogenesis at two critical levels: de novo androgen synthesis in theca cells [6] and the conversion of androgen (i.e., androstenedione) into E2 in GC. This study has extended this information by showing that HGF suppresses FSH-dependent E2 synthesis while suppressing the expression of P450arom and 17ß-HSD mRNAs in rat GC. Based upon our steroid hormone and mRNA data, HGF exerts multiple levels of control within the estrogen biosynthetic pathway. Specifically, the FSH-stimulated aromatization of androstenedione into E1 was not attenuated; whereas, the metabolism of E1 into E2 appears to have been blocked by HGF. Although not directly tested, this would indicate that 17ß-HSD activity is impaired. Because the levels of 17ß-HSD and P450arom mRNA were each reduced by HGF at 48 h in vitro, we suggest that sufficient translation and activity of P450arom protein occurred prior to 48 h. By this selective action, the P450arom-mediated conversion of androstenedione into E1 would not be disrupted by HGF. A previous report has demonstrated that HGF reduces FSH-stimulated P450arom activity in DES-primed rat GC in vitro [9], thus indicating that the aromatization of androstenedione into E1 can be blocked by HGF. Importantly, the effect of DES (e.g., substantially elevated estrogen concentrations) upon the molecular regulation of HGF activity in GC is unpredictable. That is, the selective targeting of P450arom versus 17ß-HSD by HGF may be affected by the presence or absence of estrogens.
Stimulation by FSH is arguably central to regulating GC function within selected and recruited follicles. Follicle-stimulating hormone induces the sequential activation of (1) stimulatory G proteins (Gs proteins), (2) adenylyl cyclase-directed generation of cAMP, and (3) cAMP-dependent protein kinases. Although HGF suppressed FSH-dependent E2 production, HGF did not impair E2 synthesis in the presence of the cAMP analogue, Bu2-cAMP. Similarly, Bu2-cAMP-stimulated expression of P450arom, and 17ß-HSD mRNAs were not attenuated by HGF. One explanation of these data is that HGF interferes with the FSH-induced cAMP pathway prior to the generation of cAMP. In fact, cytokine regulation at Gs proteins and adenylyl cyclase has been indicated in GC [18, 19]. Alternatively, HGF could mobilize Gi proteins, as has been demonstrated in cultured rat hepatocytes [20]. In so doing, the FSH-directed activation of adenylyl cyclase, cAMP production, and ultimately estrogen production would be collectively prevented. The involvement of Gi proteins as an inhibitory mechanism would also be overcome by the addition of Bu2-cAMP. Because nothing is currently known about HGF signal transduction in GC, further studies are required in order to determine any interactions between HGF and the FSH-cAMP pathway in GC.
Based upon the observed effects of HGF in GC, as reported in this study and others [9], HGF functions as a negative modulator of GC steroidogenic differentiation. If operational in young growing follicles in vivo, this HGF-directed action would be beneficial in preventing an untimely rise in E2 secretion by GC. However, suppression of E2 production by HGF would have to be reversible so that E2 secretion would rise during the preovulatory phase of the cycle. In theory, HGF bioactivity could be neutralized by one or more of the following mechanisms: (1) down-regulation in the synthesis of intraovarian HGF, (2) a reduction in c-Met expression and/or autophosphorylation, or (3) interruption of post-c-Met signal transduction. First, consider regulation of c-Met as a potential mechanism. In bovine follicles, c-Met mRNA concentrations increased in direct relation to follicle size [9]. This observation would suggest that down-regulation of c-Met is not involved in reversing HGF bioactivity in GC. The present report has shown that the relative level of c-Met mRNA in GC cultures is in fact regulated by FSH, HGF, and Bu2-cAMP. As observed at 12 h in vitro, c-Met mRNA levels were increased by either Bu2-cAMP or HGF but not FSH (not shown). Whereas at 48 h, the stimulatory effect of Bu2-cAMP was preserved; however, FSH and HGF each caused a precipitous decline in the amount of c-Met mRNA. These experimental observations suggest that several mechanisms can alter c-Met expression during GC differentiation, all of which warrant further investigation. For example, the HGF-dependent increase in c-Met mRNA early on (e.g., 12 h in vitro) could be a primary event in establishing a key link between HGF and intracellular coupling that dampens FSH-directed E2 synthesis. Whereas, once the HGF signaling machinery is operational, an HGF-directed negative feedback loop could down-modulate c-Met expression (e.g., 48 h in vitro). By this proposed mechanism, a sustained block in FSH-induced E2 synthesis would be prevented in GC should HGF concentrations remain unchallenged in mature (preovulatory) follicles.
An interesting observation was that FSH (a potent stimulator of cAMP signal transduction) reduced the level of c-Met mRNA in GC, but Bu2-cAMP induced a rapid and sustained elevation of c-Met mRNA content in vitro. This paradox is perhaps best rationalized when considering that FSH can signal via cascades in addition to that mediated by cAMP. For example, FSH can activate mitogen-activated protein kinase and tyrosine kinase pathways in GC [13, 21, 22]. Therefore, the opposite actions of FSH and Bu2-cAMP on c-Met expression implies that multiple FSH-dependent signaling molecules can selectively up- and down-modulate c-Met expression in GC. Because the cAMP analogue increased c-Met expression (present study), and c-Met mRNA was increased relative to follicle size in bovine GC [9], it appears that the cAMP-rich environment in preovulatory follicles fosters c-Met gene expression. Any effects of FSH and HGF on the pattern of c-Met protein and tyrosine kinase activity in GC remain to be determined.
Interactions between c-Met activity and signaling molecules in GC have not been investigated. However, in nonovarian cells, HGF stimulates the activation of c-Met and phospholipid-dependent cascades [7] and PKB [12]. In that HGF stimulated PKB mRNA accumulation, a preliminary link between HGF and PKB has been identified in GC. Interestingly, at 12 h in vitro, HGF up-regulated an increase in both PKB mRNA and c-Met mRNA. One compelling interpretation of this observation is that during GC steroidogenic differentiation, HGF establishes two components of its signaling apparatus: c-Met and PKB.
In forming a physiologic model for HGF during GC growth and differentiation, the collective constituents of the follicular microenvironment must be considered. For example, insulin-like growth factor-I (IGF-I) and transforming growth factor-ß (TGFß) are present in young follicles [23, 24]. In vitro, IGF-I and TGFß each augment FSH-dependent E2 production in GC [25, 26] and therefore can be considered antagonists of HGF. With this evidence in hand, we hypothesize that HGF functions as part of a negative modulatory cytokine system. This system could postpone the steroidogenic differentiation of GC that would otherwise be induced by combinations of FSH, IGF-I, and TGFß. Therein, a precocious and reproductively detrimental increase in E2 secretion would be blocked, and experimental evidence supports a role for HGF in this fundamental model.
FOOTNOTES
First decision: 30 November 1999.
1 This work was funded by an SIU-Edwardsville Graduate School Research Grant (R.J.Z.) and an SIU-SDM Dean's Student Fellowship DSRF99-3 (B.E.R.). 
2 Correspondence: Rob Zachow, SIU School of Dental Medicine, 2800 College Ave., Alton, IL 62002-4700. FAX: 618 474 7150; rzachow{at}siue.edu
Accepted: January 28, 2000.
Received: October 26, 1999.
REFERENCES
inhibits gonadotropin hormonal action in nontransformed ovarian granulosa cells. J Biol Chem 1989; 264:11591–11597. inhibits follicle-stimulating hormone-induced differentiation in cultured rat granulosa cells. Biochem Biophys Res Commun 1989; 163:1038–1046.[CrossRef][Medline] and TGF-ß in human ovarian tissues. J Clin Endocrinol Metab 1992; 74:973–980.[Abstract]This article has been cited by other articles:
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  R. Zachow and M. Uzumcu The hepatocyte growth factor system as a regulator of female and male gonadal function J. Endocrinol., December 1, 2007; 195(3): 359 - 371. [Abstract] [Full Text] [PDF]
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 M. Uzumcu, Z. Pan, Y. Chu, P. E Kuhn, and R. Zachow Immunolocalization of the hepatocyte growth factor (HGF) system in the rat ovary and the anti-apoptotic effect of HGF in rat ovarian granulosa cells in vitro. Reproduction, August 1, 2006; 132(2): 291 - 299. [Abstract] [Full Text] [PDF]
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 R. J. Zachow and J. K. Woolery Effects of Hepatocyte Growth Factor on Cyclic Nucleotide-Dependent Signaling and Steroidogenesis in Rat Ovarian Granulosa Cells In Vitro Biol Reprod, August 1, 2002; 67(2): 454 - 459. [Abstract] [Full Text] [PDF]
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