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RESEARCH |
1 Department of Physiology and Biophysics, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA and 2 Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08901, USA
Correspondence should be addressed to R Zachow; Email: zachowrj{at}umdnj.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The precise signals, which determine follicular fate (ovulation vs atresia) are not known; however, several ligand-directed cellular mechanisms that promote either growth (cell survival) or atresia (apoptosis) have been identified. It appears that FSH, and the modulation of FSH bioactivity by select intraovarian growth factors, can protect GC from apoptosis (Hussein 2005).
Hepatocyte growth factor (HGF) exerts autocrine and paracrine regulation of GC and theca cell growth and steroidogenesis (Liu et al. 1994, Parrott et al. 1994, Zachow et al. 1997, 2000, Lail-Trecker et al. 1998, Parrott & Skinner 1998a,b, Osuga et al. 1999, Zachow & Woolery 2002, Shimizu et al. 2003). Despite its well-documented anti-apoptotic bioactivity in other cells (Lail-Trecker et al. 1998), the ability of HGF to control the onset of apoptosis in GC has not been previously reported.
Although HGF and its receptor (c-met) are expressed within the ovaries of numerous mammalian species (e.g., human, rat, pig, cow, sheep, and mouse) (Liu et al. 1994, Parrott et al. 1994, Zachow et al. 1997, Osuga et al. 1999, Ito et al. 2001, Shimizu et al. 2003), some controversy exists regarding the cellular origin of intraovarian HGF and c-met. It has been shown that HGF is secreted by theca cells (Parrott et al. 1994); whereas c-met is expressed within GC (Parrott et al. 1994, Zachow et al. 2000). This model fits the general pattern of mesenchymal HGF targeting epithelial c-met that has been documented in other tissues (Gheradi & Stoker 1991). In this context, the effects of theca cell-derived HGF would then be predicted to be exclusive within GC. However, HGF can regulate rat theca cell steroidogenesis in vitro (Zachow et al. 1997), and this would suggest that theca cells contain c-met. In fact, theca cells and GC express HGF mRNA (Zachow et al. 1997); therefore, we propose a broader function for intraovarian HGF. Importantly, this more elaborate pattern for the expression of follicular HGF and c-met has been corroborated in other species (Parrott et al. 1994, Zachow et al. 1997, 2000, Osuga et al. 1999, Ito et al. 2001, Shimizu et al. 2003). Although some question does exist regarding which follicular cells produce HGF and c-met proteins, there is precedent for the expression of HGF and c-met by both theca cells and GC.
A third component of the HGF system, HGF activator (HGFA) protein, is a serine protease that cleaves the immature HGF precursor molecule to yield mature bioactive HGF (Miyazawa et al. 1993). The presence of ovarian HGFA has not been previously reported; however, HGFA represents a potential control mechanism for regulating local bioactive HGF concentrations.
In order to better define the role of HGF during folliculogenesis, this study investigated the effect of HGF on apoptosis in cultures of primary rat GC and isolated follicles. To complement this, the cellular distribution of the ovarian HGF system (HGF, c-met, and HGFA) during rat folliculogenesis was also evaluated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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GCs were harvested in M199 using follicle puncture. Collected GC were centrifuged (5 min, 200g, 4 °C), and resuspended in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL) containing antibiotics and 5% FBS. Cell viability and number were determined via the Trypan Blue exclusion test, using a hemacytometer. Intact preantral follicles were isolated from rat ovaries by microdissection.
Upon isolation, 5x105 viable GC/well (6-well plates, Falcon Primaria) or follicles (5–6/well, 24-well plates, Falcon Primaria) were incubated at 37 °C, in a humidified atmosphere containing 5% CO2 in air. Cultures were incubated for the first 24 h in DMEM containing 5% FBS; then media was changed to serum-free, and hormones were added as described later.
Detection of apoptotic GC
Following serum withdrawal, follicles, and GC were incubated in the presence and absence of HGF (rhHGF, R&D Systems, Minneapolis, MN, USA; 30, 60, and 90 ng/ml). Following the addition of HGF, follicle, and GC cultures were incubated for 24 h, and at 24 h, YO-PRO-1 (final concentration=0.1 µM; Molecular Probes, Inc., Eugene, OR, USA) was added to the wells based upon the method described by Peluso et al.(2001). In brief, a loss in plasma membrane integrity enables YO-PRO-1 to enter apoptotic cells, while healthy cells remain impermeable to this nucleic acid stain. After 10 min in the presence of YO-PRO-1 (37 °C), cultures were examined via fluorescence microscopy (200x) using a fluorescein isothiocyanate (FITC) filter set. For isolated GC, separate fields were evaluated in each treatment group so that 300 GCs were examined for quantitative analysis. For follicle cultures, a minimum of three size-matched follicles was compared for each treatment.
Immunohistochemistry
Twenty-three-day-old Sprague–Dawley rats were injected with either equine chorionic gonadotropin (eCG) (15 IU, Sigma) to induce the growth of large antral follicles, or vehicle (normal saline). Five non-primed and five eCG-primed rats (ten ovaries for each) were used in three experiments. Forty-eight hours after the injections, rats were euthanized as described previously. Ovaries were removed and placed in ice-cold M199 that was supplemented with 10% FBS before being fixed in Bouin’s fluid for 4 h (room temperature). Following the incubation in Bouin’s fluid, ovaries were washed approximately ten times in 70% ethanol, and then stored in 70% ethanol (4 °C) pending immunohistochemistry (Uzumcu et al. 2000).
Fixed ovarian tissue was embedded in paraffin and then sectioned at 4–5 µm. The sections were deparaffinized, rehydrated, and heated (using a microwave oven) to boiling 0.01 M citrate buffer (pH 6.0). Upon boiling, the sections were subsequently heated (low heat setting) for an additional 15 min. The sections were then blocked in 1.5% normal serum for 10 min at room temperature. The following antisera (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used: HGF (sc-7949), c-met (sc-162), and HGFA (sc-1371).
The ovarian sections were incubated with antisera overnight at room temperature using the following dilutions for anti-HGF, anti-c-met, and anti-HGFA antisera respectively: 1:50, 1:200, 1:200. Immunoreactivity (as identified by brown color) to the respective protein was then visualized using the ABC Staining System (Santa Cruz Biotechnology) following the manufacturer’s protocol. The negative control sections for HGF, c-met, or HGFA were incubated with normal rabbit or goat IgG instead of primary antibody. Additional negative control sections for c-met and HGFA were incubated with c-met and HGFA primary antibodies that were preadsorbed with ten times c-met and HGFA-blocking peptides (Santa Cruz Biotechnology) respectively prior to conducting the detection protocol.
Sections were counterstained with Mayer’s hematoxylin. The sections were then photographed using a Nikon imaging station equipped with a Nikon Eclipse E800 light microscope and Nikon DXM1200F digital camera.
Scoring of the relative immunohistochemical staining intensity
Differences in staining intensity were evaluated using a subjective scoring system from 0 to +4, with a half-point increments (0, no staining and +4, the strongest staining). Immunostaining intensities from two entire serial sections from all ovaries were evaluated for each antibody. The total score was divided by the number of sections, in which a respective structure (e.g., mid/large antral follicles, blood vessels) was observed.
Western-blot analysis
To determine the specificity of the antibodies used in immunohistochemistry, western-blot analysis was performed as described previously (Uzumcu et al. 2002). Briefly, harvested immature rat ovaries were lysed with 1x cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA). The protein concentrations within the supernatants were measured using the DC Protein Assay Kit II (BioRad). Approximately, 50 µg protein in sample-loading buffer was boiled for 5 min and then loaded into 10% (HGF and c-met) or 15% (HGFA) SDS gels. Upon transfer onto an Immobilon P membranes (Millipore, Bedford, MA, USA), proteins were probed with the specific antibodies that were used for HGF, c-met, or HGFA IHC as described previously (Santa Cruz Biotechnology). Following incubation with a secondary antibody, the specific antigen–antibody complex was visualized using ECL Advance Western Blotting Detection Kit (GE Biosciences, Piscataway, NJ, USA) and viewed by autoradiography. Molecular weights were estimated by comparing visible bands against Kaleidoscope Prestained Standards (cat. #161-0324; BioRad).
Statistical analyses
All experiments were repeated three times. For GC counts, mean values from independent experiments were statistically analyzed using one-way ANOVA followed by Tukey’s test. Values were determined to be significant when P<0.05.
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Results |
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, arrows). This elevated level of fluorescence is indicative of a higher incidence of apoptosis in these cultures (Peluso et al. 2001). Counting of brightly fluorescent GC showed that when compared to control GC, HGF induced a statistically significant (P<0.05) reduction in the number of apoptotic GC (Fig. 1, bar chart).
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). This effect was most apparent with the higher doses of HGF (60 and 90 ng/ml) that were given.
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, Table 1). Select GC within antral follicles also contained immunoreactive HGF protein. In contrast, a relatively low HGF signal was apparent within some theca cells in non-primed ovaries.
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). Populations of GC within large antral follicles showed a muted signal for HGF; however, a general shift in staining from the GC to the theca–interstitial cell layers occurred following exposure to eCG (Fig. 3b, d and f, Table 1). The negative control sections showed no immunoreactivity for HGF (Fig. 3e).
c-met
In non-primed ovaries, staining was detected in GC within, and theca cells surrounding, preantral follicles (Fig. 4a and c, Table 1). Some intense levels of immunoreactive c-met were present in GC within certain small preantral follicles, as well as distinct GC within antral follicles (Fig. 4c). Within the c-met-positive preantral follicles, the level of immunoreactive c-met that was observed in the GC was arguably greater than that detected in theca cells. Structures resembling blood vessels within interstitium also showed immunoreactive c-met (Fig. 4a).
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) when compared to that detected in size-matched follicles within non-primed ovaries (Fig. 4a). Moreover, there were no noticeably exclusive populations of intensely stained GC within large antral follicles, in contrast to what was observed in non-primed ovaries. In large antral follicles, immunoreactive c-met was seen in the theca cells and regions of the interstitium, concomitant with a virtual absence of staining within the GC (Fig. 4d, Table 1). The negative control sections showed no or minimal immunoreactivity (Fig. 4e).
HGFA
In non-primed ovaries, a somewhat uniform HGFA signal was observed in GC within, and theca cells surrounding, preantral follicles (Fig. 5a and c, Table 1). The same general pattern of immunoreactive HGFA was apparent within the antral follicles that were present within these ovaries (Fig. 5a). Relatively low signal for HGFA was detected within the interstitium.
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, Table 1). This pattern for HGFA was especially apparent in the theca cells associated with large antral follicles (Fig. 5d and f). The GC within large antral follicles showed no immunoreactive HGFA (Fig. 5f). The theca externa and the interstitium were devoid of HGFA immunoreactivity (Fig. 5f, Table 1). Immunoreactive HGFA was not detected in the negative control sections (Fig. 5e).
Western analysis was used to verify the specificity of the antisera used in this study. As shown in Fig. 6, a major bands corresponding HGF, c-met, and HGFA were detected (A–C respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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These new data extend those from previous studies, which have shown that HGF modulates theca cell and GC steroidogenesis in vitro (Zachow et al. 1997, Parrott & Skinner 1998a). As reported using cultured immature rat theca cells, HGF dampens luteinizing hormone (LH)-stimulated androgen production (Zachow et al. 1997); and in immature rat GC, HGF suppresses FSH-directed estrogen synthesis in vitro (Parrott & Skinner, 1998a, Zachow et al. 2000). The downregulatory effects of HGF on ovarian steroidogenesis have implications with regard to guiding preantral folliculogenesis and selection of the dominant follicle. It can be suggested that by impairing androgen and estrogen secretion in younger follicles, HGF would block an abnormal accumulation of intrafollicular steroids that could lead to follicle atresia (Hutz et al. 1990, Billig et al. 1993, Hillier & Tetsuka 1997). We would propose this steroid-modulatory action is one mechanism by which HGF could impede the induction of apoptosis in GC in vivo. On the other hand, the culture conditions that were used in the present study to investigate the effect of HGF on apoptosis did not include the addition of exogenous gonadotropins and/or steroid hormones. Therefore, it can be argued that an accumulation of androgens and/or estrogens within the culture microenvironment did not instigate the onset of apoptosis in GC. Rather, in the present study, it is likely that HGF exerted a direct anti-apoptotic effect within GC. Future studies will be required in order to investigate the intracellular mechanisms of action that are affected by HGF in this context. Certainly, the effects of HGF within the so-called survival signaling cascades (e.g., those mediated by the Bcl family of proteins) would be useful in more thoroughly modeling the molecular mechanisms by which HGF suppresses apoptosis in GC.
Prior reports have shown that HGF and c-met are expressed within the ovary. For example, HGF has been measured in human follicular fluid; whereas the mRNAs for both HGF and c-met were detected in isolated human GC (Osuga et al. 1999). In the cow, HGF and c-met were measured in isolated theca cells and GC respectively (Parrott et al. 1994). In the pig, Shimizu et al. showed that theca cells expressed c-met, with HGF transcript expressed in GC Shimizu et al.(2003). Finally, in the rat, HGF mRNA was measured in both theca cells and GC (Zachow et al. 1997), with hormone-dependent changes in the relative level of c-met mRNA reported in GC (Zachow et al. 2000). To our knowledge, prior reports have not, however, described changes in the relative abundance of HGF and c-met proteins during in vivo folliculogenesis. Using freshly isolated bovine follicles, Parrott et al. have showed that the largest follicles contained the greatest abundance of c-met and HGF mRNAs (Parrott & Skinner, 1998a); although, comparison of the specific cellular origin (i.e., theca cells vs GC) for HGF and c-met transcripts was not reported. In another study, hCG was shown to stimulate HGF mRNA expression in bovine theca cells in vitro (Parrott & Skinner, 1998a). Interestingly, the present data showed that eCG priming induced a shift in the location of immunoreactive HGF from predominantly GC in non-primed rats, to theca cells following the administration of eCG. Therefore, it can be proposed that the production of HGF within theca cells is stimulated by gonadotropins; however, the physiologic implications for this change in HGF content are uncertain.
It has previously been shown that both rat GC and theca cells respond to HGF stimulation in vitro (Zachow et al. 1997, 2000); and although Zachow et al. described the presence of theca cell c-met mRNA Zachow et al.(1997), this finding has been challenged to suggest that the effects of HGF in theca cells were actually a phenomenon mediated by undefined factors produced within the theca cell cultures (Lail-Trecker et al. 1998). The present data do, however, show that theca cells contain c-met protein and this extends the previous data showing that these cells express c-met mRNA (Zachow et al. 1997). Interestingly, in porcine theca cells, the relative abundance of c-met mRNA did not change following in vivo treatment with eCG (Shimizu et al. 2003), and LH did not induce changes in the amount of c-met mRNA in rat theca cells in vitro (Zachow et al. 1997). These data contrast with the observations in the present study in which eCG induced an increase in the presence of immunoreactive c-met in the rat theca cells in vivo. On the other hand, in primary rat GC cultures, c-met expression was reduced by FSH (Zachow et al. 2000), and as shown in this report, eCG caused a noticeable decrease in immunoreactive c-met in rat GC in vivo. Thus, data from independent investigations, using both in vivo and in vitro approaches, show that c-met content within GC can be reduced by gonadotropin stimulation. These findings support a model in which the effects of HGF within preovulatory GC can be controlled in response to hormonally mediated levels of c-met. Specifically, a gonadotropin-induced reduction in c-met within large antral follicles would likely impair follicular HGF bioactivity. Moreover, the present study is the first to show that immunoreactive HGFA is located within the ovary. Whether or not ovarian HGF bioactivity depends upon, or is modulated by, local HGFA is unknown at this time. It is noteworthy, however, that eCG did cause a shift in the cellular distribution of ovarian HGFA. Therefore, like HGF and c-met, it appears that stage-specific changes in available HGFA occur during follicle development.
Based upon the data presented in this report, and those previously published by this Lab. and others, it is very likely that HGF exerts multiple levels of control over GC and theca cell function which collectively enable the progression of folliculogenesis. We suggest that a complete HGF system within preantral follicles (as identified in this study) down-modulates gonadotropin-stimulated steroidogenesis, and concomitantly supports GC survival via an anti-apoptotic effect. The aforementioned would be complemented by the paracrine mitogenic effects of the previously described HGF-kit ligand system (Parrott & Skinner 1998b). As antral follicle growth proceeds, gonadotropin-orchestrated changes in the distribution of HGF, c-met, and/or HGFA are likely to regulate HGF bioactivity, perhaps leading to the alleviation of the HGF-directed suppression of gonadotropin-stimulated androgen and estrogen production within the dominant follicle. It is thus enticing to predict that manipulation of the intraovarian HGF system may provide a novel approach to controlling the balance between follicular selection and atresia.
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Acknowledgements |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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