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Department of Endocrine Pharmacology, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan 192-0392
ABSTRACT
Insulin-like growth factor (IGF)-binding protein (IGFBP) 7 is a secreted protein that regulates cellular proliferation, adhesion, and angiogenesis, and has low affinity for IGF compared with that of IGFBP1-IGFBP6. We sought to determine whether IGFBP7 is present in follicular fluid and to elucidate whether IGFBP7 participates in the steroidogenesis of rat mature follicles. Follicular fluid and granulosa cells (GCs) were collected from immature rats 2 days after their treatment with equine chorionic gonadotropin (eCG). IGFBP7 protein was detected in the follicular fluid and the conditioned medium of cultured ovarian GCs by immunoblot analysis. When subconfluent GCs were cultured and treated with FSH and activin, coincubation with FSH and activin markedly increased GC expression of Cyp19a1 (aromatase) mRNA and 17beta-estradiol (E2) secretion. The addition of recombinant murine IGFBP7 to these cultures decreased in the activin-enhanced, FSH-stimulated Cyp19a1 mRNA levels in the cells and suppressed the 17beta-E2 levels in the culture medium. Treatment of GCs with Igfbp7-specific small interfering RNA (siRNA), which knocked down Igfbp7 expression, increased the FSH-stimulated levels of Cyp19a1 but not Cyp11a1 expression. Basal and FSH-stimulated 17beta-E2 secretion into the culture medium was also enhanced by Igfbp7 siRNA. These results suggest that IGFBP7 suppresses estrogen production in GCs. These observations support the notion that this protein, which is secreted into the follicular fluid, may serve as an intraovarian factor that negatively regulates GC differentiation.
estradiol, follicle, granulosa cells, IGFBP7, insulin, ovary, ovarian, steroidogenesis
Follicular development and ovulation are dependent on proliferative and differentiation changes in granulosa cells (GCs) and thecal cells, which undergo steroidogenesis upon stimulation with gonadotropins and intraovarian cytokines [1, 2]. The preovulatory follicles produce high levels of estradiol (E2), which is synthesized by the key steroidogenic enzymes, CYP11A1 (cytochrome P450scc, or cholesterol side-chain cleavage enzyme) in the theca and by CYP19A1 (cytochrome P450aromatase, or aromatase enzyme) in GCs; these enzymes are the rate-limiting enzymes for the conversion of cholesterol to pregnenolone and the synthesis of E2 from androgen precursors, respectively [1, 3, 4]. The enzymatic changes that take place upon stimulation with gonadotropins and intraovarian cytokines are also associated with the abundant expression of luteinizing hormone (LH) receptors on GCs, which is characteristic of preovulatory follicles [5, 6].
Insulin-like growth factor (IGF)-binding proteins (IGFBPs) classically comprise 6 isoforms (IGFBP1-IGFBP6) [7]. IGFBPs bind to IGF and thereby modify its metabolism, distribution, and ability to bind to the IGF receptor. IGFBPs 1, 3, 4, and 5 are expressed by the blastocyst and the endometrium in rats [8–10], while IGFBPs 4 and 5 may be involved in the selection of dominant follicles in ruminants [10]. Recently, IGFBPs that exhibit a low affinity for IGF were reclassified as IGFBP-related proteins (IGFBPrP) [7]. One of these IGFBPrPs is Igfbp7, which was originally identified as a gene that shows reduced mRNA expression in meningioma cell lines as compared to normal cells [11]. IGFBP7 shares high homology with IGFBPs and binds to IGF1/2 and insulin, but its binding affinity for IGFs is considerably lower than those of IGFBPs 1–6 [12]. It is possible that the IGFBPrP family may have unique physiological activities that do not involve regulating insulin and IGF1 activity [8].
IGFBP7 is expressed by the GCs of the large antral follicles in the porcine ovary [13] and by the bovine corpus luteum [14]. It is characterized by the presence of a highly conserved follistatin sequence [15]. Follistatin is produced by small antral and preovulatory follicles [16] and binds activin, which neutralizes the activity of activin. Activin induces immature GCs to proliferate and promotes the expression of the FSH receptor and CYP19A1 [17]. Mature GCs also differentiate in response to activin [16]. Thus, follistatin and activin act as autocrine and paracrine factors in the development of follicles toward ovulation. Given the presence of the follistatin sequence in IGFBP7, we speculated that IGFBP7 may also bind activin and regulate its activity, thereby modulating follicle development and ovulation. To test this notion, we searched for IGFBP7 expression in the GCs of developing follicles and the presence of this protein in the follicular fluid from rat ovaries. We also harvested the GCs from mature rat follicles and examined the effects of recombinant IGFBP7 protein treatment and knocking down Igfbp7 expression on FSH-stimulated GC steroidogenesis in vitro.
Ovine FSH (NIDK oFSH-19-SIAFP: AFP-4117A) was obtained from Dr. A.F. Parlow of the National Hormone and Pituitary Program (Harbor/UCLA Medical Center, Torrance, CA). Porcine collagen type I (Cellmatrix) was purchased from Nitta Gelatin (Osaka, Japan). Transferrin and fibronectin were acquired from Wako Pure Chemical Co. (Tokyo, Japan). Androstenedione and hydrocortisone were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Human extracellular matrix (ECM), comprised of laminin, collagen IV, and heparin sulfate proteoglycan (HSPG), was obtained from BD Biosciences (Franklin Lakes, NJ). Recombinant rat activin A and murine IGFBP7 were purchased from R&D Systems, Inc. (Minneapolis, MN). Rabbit anti-murine IGFBP7 antibody (H-102) and normal rabbit immunoglobulin (Ig) G were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Vector Lab, Inc. (Burlingame, CA), respectively. Another antibody against a partial IGFBP7 polypeptide was raised by Peptide Institute, Inc. (Minoh-shi, Osaka, Japan). Igfbp7-specific small interfering RNA (siRNA) (siGENOME SMARTpool reagent) was synthesized by Darmacon, Inc. (Chicago, IL). As a control treatment, the irrelevant control siRNA (sc-37007) was obtained from Santa Cruz Biotechnology.
Ovaries were collected 2 days after treating immature rats with equine chorionic gonadotropin (eCG). The follicular fluid was collected and concentrated by using a Centricon filter device (10 kDa cut-off, YM-10; Millipore, Minato-ku, Tokyo) while the GCs were harvested, cultured initially as described below, and then cultured in subconfluent conditions for 24 h in serum-free Dulbecco modified Eagle medium (DMEM). The uteri of rats in their first proestrus were also flushed out and the flushing medium was retained and concentrated. Cell lysates obtained from the cultured GCs and the conditioned medium samples were subjected to immunoprecipitation using anti-IGFBP7 antibody, as described below. The immunoprecipitates and the concentrated follicular and uterine fluids were then subjected to Western blotting for detection of the IGFBP7 protein levels. In a separate experiment, the GCs continued to be cultured in serum-free DMEM in the presence of FSH, activin, and/or IGFBP7. Furthermore, to determine the effect of knocking down IGFBP7 in GCs on their steroidogenesis, the cells were treated for 24 h with the Igfbp7-specific or control siRNA before FSH stimulation, as described below.
Immature 21-day-old female Wistar-strain rats were supplied by the Imamichi Institute for Animal Reproduction (Ibaraki-ken, Japan). All procedures using animals were performed in accordance with institutional guidelines for experimental animal care at the Tokyo University of Pharmacy and Life Sciences. Animals (24-days old) were injected s.c. with 20 IU eCG (Teikoku Hormone Mfg. Co., Ltd., Tokyo, Japan) dissolved in saline. After 48 h, the 26-day-old ovaries were removed and the GCs were harvested as previously described [18]. Briefly, GCs were collected by repeated puncture of antral follicles with a 26-gauge needle, and the cells that were released were placed into PBS containing 1% BSA. The collected cells were a mixture of the GCs derived from antral follicles of various sizes. After centrifugation at 200 x g for 10 min, the cells were washed three times with PBS and then initially cultured in collagen type I-coated dishes at a density of 2 x 105 cells/well and maintained for 2 days in 0.5 ml DMEM (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) plus 100 µg/ml gentamicin. The medium was then changed to a serum-free culture medium supplemented with 5 µg/ml transferrin, 40 ng/ml hydrocortisone, 1 µg/ml fibronectin, 30 ng/ml androstenedione, and 2 µg/ml ECM, and the subconfluent cells were incubated for another 24 h to promote cell differentiation. The cells were then harvested for Western blot analysis or cultured for another 24 h in DMEM supplemented with androstendione with or without FSH, activin, and/or IGFBP7. The latter cells were then analyzed for steroidogenic activity and mRNA expression, as described below.
Immunoblotting of IGFBP7 in Follicular Fluid, Cultured GCs, and Conditioned Medium
The follicular fluid samples (10 or 25 µg protein) were resolved on SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking with 5% nonfat dry milk, the blots were probed with anti-IGFBP7 antibody (H-102) and stained by using a chemiluminescence kit (PerkinElmer Inc., Wellesley, MA) after incubating the blots with horse radish peroxidase-conjugated anti-rabbit IgG, as described previously [18]. The cell lysates and conditioned medium samples were immunoprecipitated by incubation with anti-IGFBP7 antibody, followed by incubation with immobilized protein G (Pierce, Rockford, IL). After washing, the protein G-bound complexes were subjected to SDS-PAGE and Western blotting with the polyclonal anti-IGFBP7 antibody raised against a synthetic IGFBP7 polypeptide by the Peptide Institute, Inc. Detection was performed as described above.
Ovaries collected from immature rats 2 days after 20 IU eCG injection were fixed for 8 h in 0.15 M phosphate buffer (pH 7.45, room temperature) containing 4% paraformaldehyde. To examine the expression of IGFBP7 in the corpus luteum, some rats were injected intraperitoneally with human CG (10 IU) 57 h after eCG treatment, and their luteinized ovaries were removed 3 days later. Paraffin-embedded sections (4 µm) were prepared for in situ hybridization, as described previously [18]. Bound probes were visualized as a purple color using an alkaline phosphatase-conjugated anti-digoxigenin antibody and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Promega Corp., Madison, WI) as substrates.
Measurement of E2 and Progesterone Secretion into the Culture Medium
17ß-Estradiol content was assayed by using an E2 ELISA kit (Japan EnviroChemicals, Ltd., Minato-ku, Tokyo, Japan). The concentration of progesterone in the medium was determined by radioimmunoassay, as described previously [19]. The intra- and interassay coefficients of variation were, respectively, 5.8% and 18.1% for E2 and 3.4% and 10.5% for progesterone.
RT-PCR Analysis of Igfbp7, Cyp19a1, and Cyp11a1 Expression
Poly(A)+ RNA from cultured GCs was extracted by using the QuickPrep micro mRNA purification kit (GE Healthcare Bio-Science Corp., Piscataway, NJ), and PCR amplification of Cyp19a1 and Cyp11a1 mRNAs was performed as described previously [20]. The Igfbp7 mRNA levels were also examined by using a one-step RNA PCR kit (TaKaRa, Otsu, Japan) and specific primers (sense, 5'-TGCTGCAGAGGCAGGG AGCCC-3'; antisense, 5'-AGGGATCCTCTTCCCATT CCA-3'). The PCR reaction involved 32 cycles of 94°C for 45 sec, 55°C for 45 sec, and 72°C for 1 min [21]. After agarose gel electrophoresis and visualization with ethidium bromide, the PCR products were photographed under ultraviolet transillumination and subjected to densitometric analysis. The mRNA levels were normalized to the G3pdh mRNA levels.
Small Interfering RNA Transfection
Transfection with Igfbp7-specific siRNA (10 pmole) and the irrelevant control siRNA was performed by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. GCs at 40%–50% confluency after the initial 48 h of culture in DMEM supplemented with 10% FBS plus gentamicin were treated with either Ibfbp7-specific siRNA or control siRNA for 24 h in DMEM lacking serum and antibiotics. The inhibition of Igfbp7 mRNA expression was observed within 24 h, and this knockdown was maintained for at least 72 h after the medium containing the siRNA was removed. Consequently, the cells were stimulated with FSH 24 h after the end of siRNA treatment and cultured for 24 h.
The EIA and densitometric analysis results are presented as means ± SEM and examined by one-way ANOVA with Fisher protected least significant difference test. Differences were considered to be significant when P values were less than 0.05.
Detection of IGFBP7 Expression in Follicular Fluid and Cultured GCs
To obtain follicular fluid and GCs from preovulatory rat follicles, immature rats were injected with eCG and their ovaries were harvested 48 h later. At this stage, the ovaries had many mature follicles. The ovaries were punctured with a needle and the follicular fluid and GCs were separated by centrifugation at 200 x g. The follicular fluid was diluted with PBS during the process of puncturing of the follicles and, thus, had to be concentrated. The GCs were cultured in subconfluent conditions for 48 h with serum, and then further cultured for 24 h in serum-free medium. The GC lysates and conditioned medium samples were then immunoprecipitated with a specific anti-IGFBP7 antibody, and the immunoprecipitates and follicular fluid were subjected to Western blot analysis with another anti-IGFBP7 antibody. This analysis revealed the presence of the 31-kDa IGFBP7 protein in the follicular fluid, the GC lysates, and the conditioned medium (Fig. 1, A and B). An intense band was also detected in the medium used to flush out the uteri of rats in their first proestrus (Fig. 1A, left lane). Based upon these experiments, we estimated that the follicles expressed about 50 ng of IGFBP7 per rat, while the conditioned medium had approximately 2 ng/ml of IGFBP7. The levels in the follicular fluid were lower than the level of IGFBP7 in the uterine fluid. We examined the distribution of Igfbp7 mRNA by in situ hybridization with a cRNA probe. Ifgbp7 mRNA was localized in the GCs and theca cells of healthy growing follicles (Fig. 1C, left panel) and at the edge of the corpus luteum (Fig. 1C, right panel) in gonadotropin-primed immature rat ovary. No specific signals were detected when ovarian sections were hybridized with the sense probe (data not shown).
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Effect of IGFBP7 Treatment on FSH-Stimulated Steroidogenesis in GCs
To examine the effect of IGFBP7 on steroidogenesis in cultured GCs, the GCs were incubated for 24 h in serum-free medium containing 25 ng/ml FSH and a range of IGFBP7 concentrations (0–30 ng/ml). As expected, FSH increased the 17ß-E2 and progesterone production of the GCs (Fig. 2 and data not shown). However, coincubation with 30 ng/ml IGFBP7 significantly inhibited FSH-stimulated E2 production (Fig. 2), although it had no effect on FSH-stimulated progesterone production (data not shown). We then examined the effect of FSH and IGFBP7 cotreatment on GC expression of Cyp19a1 mRNA (Fig. 3). Semiquantitative RT-PCR revealed FSH strongly promoted Cyp19a1 expression but cotreatment with 30 ng/ml IGFBP7 significantly attenuated this effect. In contrast, IGFBP7 had no effect on FSH-upregulated expression of Cyp11a1, steroidogenic acute regulatory protein (Star), or LH receptor (Lhcgr) mRNA (data not shown).
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Effect of IGFBP7 Cotreatment on GC Steroidogenesis Stimulated by Treatment with Both FSH and Activin
Activin has been shown to cooperate with FSH to stimulate GC proliferation and oocyte maturation, to upregulate the FSH receptor, and to promote CYP19A1 activity, thereby accelerating follicular development [1, 16, 17]. Consequently, we examined whether IGFBP7 had an effect on the enhanced steroidogenesis induced by simultaneous treatment with FSH and activin A (FSH/activin). As expected, we found that coincubation of GCs with FSH/activin significantly increased the E2 levels in the culture media (Fig. 4A) and the expression of Cyp19a1 mRNA (Fig. 4B) as compared with cells treated with FSH or activin alone. However, these effects were both downregulated when the FSH/activin-treated GCs were cotreated with IGFBP7. Activin did not improve the ability of FSH to upregulate GC progesterone secretion (Fig. 4A) and Cyp11a1 mRNA expression (data not shown). Moreover, while IGFBP7 cotreatment tended to diminish the FSH-upregulated expression of progesterone and Cyp11a1 mRNA levels, this effect was not statistically significant (Fig. 4, A and B). IGFBP7 alone had no effect on the E2 or progesterone levels secreted by the GCs (data not shown).
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Effect of Igfbp7 siRNA Pretreatment on FSH-Stimulated Steroidogenesis and Steroid mRNA Expression in GCs
We next examined the effect of knocking down IGFBP7 expression on steroidogenesis in GCs (Fig. 5). The levels of Igfbp7 were suppressed 24 h after the siRNA treatment (data not shown), at which point the cells were treated with or without FSH for another 24 h. Representative data in Figure 5A shows the low Igfbp7 mRNA levels were maintained at even this timepoint. As shown in this figure, FSH stimulation decreased Igfbp7 mRNA levels in control cells. With regard to the effect of IGFBP7 knockdown on the expression of the Cyp19a1 and Cyp11a1 steroidogenic enzymes, Igfbp7 siRNA treatment enhanced FSH-stimulated mRNA expression of Cyp19a1 but not Cyp11a1 (Fig. 5, A and B). The decreased levels of Igfbp7 mRNA tended to increase the basal levels of Cyp19a1 mRNA (Fig. 5B, bottom panel) and elevated the basal and FSH-stimulated levels of E2 in the medium (Fig. 5C).
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Previous reports have revealed that IGFBP7 is expressed in porcine GCs of preovulatory follicles [13] and in human granulosa lutein cells [22]. Wandji et al. [13] have indicated, in the pig, that IGFBP7 is particularly abundant in the mural GCs close to the theca interna that express aromatase (the product of the Cyp19a1 gene) at high levels. However, to our knowledge, the biological functions of IGFBP7 in ovarian cells have not been reported to date. Our data in this study demonstrate that the follicular fluid in preovulatory rat ovaries contains immunoreactive IGFBP7 protein and that GCs from these ovaries express Igfbp7 mRNA and secrete this protein. It should be noted, however, that the follicular fluid had considerably lower concentrations of IGFBP7 than the uterine fluid. This disparity may relate to the following observations: (1) IGFBP7 has a high affinity for heparin sulfates on the cell surface [23] and (2) GCs express HSPG at high levels [24]. Thus, the IGFBP7 levels in the follicular fluid may be low because most of the IGFBP7 molecules have been adsorbed to the GC surfaces. We also show that when GCs were cultured with exogenous IGFBP7, their mRNA expression of Cyp19a1 and secretion of E2 were inhibited. Conversely, the knockdown of endogenous Igfbp7 expression in GCs enhanced their FSH-stimulated steroidogenesis and Cyp19a1 expression. Thus, it appears that IGFBP7 attenuates FSH-stimulated E2 production in cultured GCs, and that this may be at least partly due to its inhibitory effect on Cyp19a1 mRNA expression. These observations suggest that IGFBP7 secreted into follicular fluid inhibits E2 production. Thus, IGFBP7 may negatively control the steroidogenesis of GCs, perhaps in an autocrine or paracrine fashion, thereby, maintaining appropriate steroid levels.
IGFBP7 has also been implicated in cell senescence and tumor suppression [25, 26], although it has also been shown to enhance the growth of fibroblastic cells [27]. We have shown previously that IGFBP7 inhibits the proliferation of uterine stromal cells, resulting in cell cycle arrest in G1 phase [21]. In eCG-primed immature rats, approximately 10% of the GCs are in S phase, while most of the rest are in G1 phase [28]. However, the mechanism that prevents preovulatory GCs from exiting G1 phase and entering into S phase remains unknown. Interestingly, an ovulatory stimulus elicits a general decline in the proliferation of mural GCs, suggesting that mural GCs exit the cell cycle under these conditions. In contrast, cumulus GCs continue to proliferate after an ovulatory stimulus [28]. IGFBP7 might be a candidate for cell cycle regulator of GC proliferation in rat ovarian follicles. Thus, the proliferation of GCs in preovulatory follicles might be restricted by the levels of IGFBP7 secreted into the follicular fluid. However, as subconfluent cells growing under serum-free conditions were used in the present study, we could not evaluate the effect of IGFBP7 on GC proliferation.
IGFBP activity is generally controlled in part by IGFBP-degrading proteases, including matrix metalloproteinases [7–9]; however, IGFBP7 activity may also be controlled by other mechanisms. In the preovulatory follicles, IGFBP7 may bind to HSPG, which is abundant on GCs, as described above. This may serve to concentrate the IGFBP7 produced by the GCs in the follicles. Similarly, because IGFBP7 also binds to type-IV collagen [29] and syndecan-1 [30], the IGFBP7 that is produced by mature follicles may become concentrated in the ECM of the mural GCs. The bound forms of IGFBP7 may then become cleaved off and activated by the various proteases that are activated by the preovulatory gonadotropin surge. The release of free IGFBP7 either activates it or makes it accessible to posttranslational modifications that influence its activity. Supporting this notion is the study by Ahmed et al. [31], who reported that a membrane-bound serine proteinase, matriptase, cleaves IGFBP7 to produce a 25-kDa short form that has a different character when compared to one of 31 kDa. Such mechanisms would regulate the ability of IGFBP7 to inhibit E2 production, resulting in the exertion of this activity only after the gonadotropin surge, when E2 production declines. This ensures that IGFBP7 function remains strongly associated with the mechanism of ovulation and/or luteinization of the follicles rather than with follicular function.
The transforming growth factor ß superfamily includes activin, growth differentiation factor 9, and several types of bone morphogenic proteins, and these have been shown to regulate the growth of primary follicles [32]. We found, as expected, that treatment of cultured GCs with both FSH and activin markedly enhanced their secretion of E2 and Cyp19a1 mRNA expression as compared with the effect of FSH treatment alone. This activin-induced enhancement of FSH-stimulated E2 secretion and Cyp19a1 expression was also significantly inhibited by IGFBP7 treatment. IGFBP7 treatment lowered progesterone secretion and Cyp11a1 expression slightly, although this effect did not attain statistical significance. These observations may relate to the observation that IGFBP7 bears a highly conserved sequence found in the follistatin protein that suggests that it too may bind to activin [15]. If IGFBP7 does bind to activin, it is possible that it blocks activin activity and thereby inhibits the activin-mediated augmentation of FSH activity and attenuates E2 production and Cyp19a1 expression. Another possibility is that IGFBP7 suppresses the signaling pathway of GC steroidogenesis by binding to an as-yet unidentified IGFBPrPs receptor on GCs [8]. However, the presence of unique receptors for IGFBPrPs, like IGFBP7, actually remains unclear.
IGF1 clearly plays an important autocrine role in murine ovarian follicular development [9], although we observed that IGF1 and IGF2 did not promote steroid production in the present culture system (data not shown). With regard to the classical IGFBPs, IGF1 and IGF2, although they do play a role in follicular function, it is unlikely that IGFBP7 acts through modulation of IGF1 or IGF2 action, since it has very low affinity for them [12]. A recent study indicated that Igfbp7 mRNA expression is upregulated in the bovine corpus luteum and that its levels appear to increase during the late stage of the luteal phase [14]. The authors speculated that IGFBP7 either directly inhibits the proliferation of luteal cells or acts indirectly by inhibiting IGF1 and IGF2 activity on luteal cell growth. We also observed in the present study that an intense signal of Igfbp7 mRNA is observed in the early stage of the corpus luteum. Moreover, morphological abnormalities have been observed in the corpus luteum of IGFBP7-knockout mice [33]. Further experiments will need to be undertaken to determine the roles that this molecule plays in ovarian follicles and corpus luteum physiology.
Correspondence: 1Kazuhiro Tamura, Department of Endocrine Pharmacology, Tokyo University of Pharmacy and Life Sciences, 1432-1, Hachioji-shi, 192-0392, Japan. FAX: 81 42 676 4536; e-mail: hiro{at}ps.toyaku.ac.jp
Received: 9 November 2006.
First decision: 23 December 2006.
Accepted: 18 May 2007.
REFERENCES
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