Molecular and Cellular Endocrinology, 90 (1992) 39-46 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/92/$05.00

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MOLCEL 02873

Characterization of insulin-like growth factor binding proteins secreted by cultured bovine theta and granulosa cells Edna Sakal a, Arieh Gertler a, Liliane Aflalo b and Rina Meidan b Departments of ’ Biochemistry and Human Nutrition and ‘Animal Science, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehocot 76100, Israel

(Received 27 May 1992; accepted 21 August 1992)

Key words: Insulin-like growth factor binding protein; Theta cell; Granulosa cell; Follicle; (Bovine)

Summary

Insulin-like growth factor binding proteins (IGFBPs) secreted by bovine granulosa and theta interna cells cultured in the presence of different luteinizing factors - insulin (2 pg/ml), forskolin (10 PM), or a combination of the two were examined and characterized. Direct binding of [‘25111GF to the conditioned media was compared to progesterone production under these different treatments. In theta cells, maximal secretion of IGFBPs was achieved using forskolin alone, whereas maximal progesterone production was induced by the insulin + forskolin treatment. In contrast, maximal secretion of both IGFBPs and progesterone in granulosa cells was achieved using forskolin alone. IGFBP species secreted by the two cell types under the different treatments were detected by ligand blotting. Conditioned media from theta cells in serum-free medium collected on the seventh day of culture exhibited three bands of 34, 40 and 44 kDa when treated with insulin or forskolin. The intensity of the 40-44 kDa complex was enhanced and a 21 kDa band appeared when cells were treated with a combination of insulin plus forskolin. Conditioned media of granulosa cells stimulated with insulin or forskolin exhibited 21, 27, 29, 34 and 40-44 kDa bands. Treatment with insulin + forskolin greatly increased the intensity of a 40-44 kDa complex. A similar shift towards high molecular weight binding proteins was observed when these media were analyzed by high-performance liquid chromatography gel filtration. These findings substantiate the secretion of IGFBPs by bovine theta and granulosa cells and show it to be dependent on culture treatment. The positive correlation between the secretion of a 40-44 kDa complex (possibly IGFBP-3) and cell luteinization indicates the former’s importance in ovarian function.

Introduction

Although pituitary gonadotropins are the major regulators of ovarian granulosa and theta cell differentiation, an important modulatory role has been attributed to peptide growth factors, including insulin-like growth factors (IGFs) (Hammond et al., 1991b). Ovarian cells have specific receptors for IGF-I and IGF-II and express IGF-I mRNA (Hammond et al., 1991a). IGF-I alone stimulates steroid production in bovine and porcine granulosa cells, as well as in gonadotropinstimulated rat granulosa cells (Adashi et al., 1991).

Correspondence to: Rina Meidan, Department of Animal Science, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel. Tel. 972-8-481394; Fax 972-8-465763.

IGF-I and IGF-II present in the body fluids and in conditioned media of various cell types are associated with specific binding proteins (IGFBPs). These IGFBPS constitute a family of heterogeneous proteins originating from discrete genes, which bind both IGF-I and IGF-II with high affinity. Insulin does not bind to these proteins despite its structural similarity to IGFs. Six distinct IGFBPs designated IGFBP l-6 have been characterized and cloned (Clemmons, 1990; Shimasaki et al., 1991). Human (Suikkari et al., 1989; Giudice et al., 19911, rat (Adashi et al., 19901 and porcine (Mondschein et al., 1990) cultured granulosa cells have been found to produce and secrete IGFBPs. However, the existence/secretion of IGFBPs in bovine cultured granulosa cells or in cultured theta cells from any species has not been documented. We previously reported (Meidan et al., 1990) that luteinization of bovine

theca and granulosa cells is achieved by incubating those cells with forskolin and growth factors, such as insulin or IGF-I. Different responses to IGF-I and to insulin were observed for both theta and granulosa cells, despite the fact that at the insulin dose used (2 fig/ml), both insulin and IGF-I are expected to act through the IGF-I receptor. Such findings are consistent with the existence of IGFBPs in the media. In the present study we examined the regulation of IGFBP secretion from bovine theta and granulosa cells, using direct binding, ligand blotting and high-performance liquid chromatography (HPLC) gel filtration.

For ligand blotting and HPLC gel filtration of IGFIGFBP complexes, ceils were attached and grown in serum-containing media (as above) for the first 3 days. Subsequently, cells were cultured for 4 days in serumfree media supplemented with insulin (2 ,ug/ml), forskolin (10 PM) or a combination of the two. Progesterone determination

Progesterone content in the media was measured by radioimmunoassay as described previously (Meidan et al., 1990). Determination of IGF binding activity

Materials and methods &agents

Dulbecco’s modified Eagle’s medium/ nutrient mixture F12 Ham (1: 1) was obtained from Grand Island Co. (Grand Island, NY, USA). Antibiotics and fetal calf serum were from Biological Industries (Kibbutz Beit HaEmek, Israel). [1,2,6,7-3H]Progesterone was purchased from NEN (Boston, MA, USA). Tissue culture plates were Nunc brand (Kompstrup, Denmark). A kit for determining estradiol was purchased from DPC (Los Angeles, CA, USA). Antiserum against progesterone was generously provided by Dr. F. Kohen (the Weizmann Institute of Science, Rehovot, Israel). Electrophoretic materials were purchased from BioRad (Richmond, CA, USA) and nitr~ellulose membranes from Schleicher & Schuell (Dassel, Germany). Crystalline bovine insulin and forskolin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). IGF-I and IGF-II were generously provided by Dr. R.J. Collier, Monsanto (St. Louis, MO, USA). Follicular cell cultures

Granulosa and theta cells were isolated from healthy preovulatory follicles, and cultured as previously described (Meidan et al., 1990). Briefly, granulosa cells were removed in culture medium containing 0.1% hyaluronidase, 0.1% collagenase and 5 pg/ml DNase I. The theta interna layer was incubated for 15 min in sterile phosphate-buffered saline containing 0.25% trypsin and 0.02% EDTA at 37°C. The theta interna was then incubated for 45 min at 37°C in the presence of 3 mg/ml collagenase and 10 kg/ml DNase I. Cells from individual follicles were cultured for 9 days in 24-well plates under basal conditions or in the presence of forskolin (10 PM), insulin (2 pg/ml) or a combination of the two agents. To avoid competition of IGF-I in the direct binding analysis we used a pharmacological ~ncentration of insulin (2 pgg/mlI throughout. Medium was collected every second day, cells from one well were trypsinized and counted in a Coulter counter (Coulter Electronics, Luton, UK) and the rest were retreated.

IGF-I and IGF-II were iodinated with chloramine-T as described by Gertler et al. (1984). IGF-binding capacity of conditioned media was determined by the polyethylene glycol precipitation method (Busby et al., 1988). Aliquots (50 ~1) of conditioned media were incubated overnight with 40,000 cpm [‘251]IGF in 250 ~1 of buffer (100 mM Hepes, 44 mM NaHCO,, 0.02% NaN,, 0.01% Triton X-100, 0.01% bovine serum albumin (BSA), pH 6.0) in the presence or absence of excess unlabeled IGF (2 pg per tube). For IC,, determination, 50-~1 aliquots of media from forskolintreated cells on the seventh day of culture were incubated with [‘241]IGF-I (48,000 cpm) and decreasing concentrations of IGF-I, IGF-II and insulin. The nonspecific binding for theta cells and granulosa cell-conditioned media was 5425 cpm (ll%), while the specific binding was 6600 cpm (14%) and 18,110 cpm (37%) respectively. Ligand blot analysis

Ligand blotting was performed as described by Hossenlopp et al. (1986). Serum-free conditioned media (80 ~1) were run on 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. The proteins were then electroblotted onto nitrocellulose. Blots were blocked with 3% Nonidet NP-40 (NP~), 1% BSA and 0.1% Tween 20 and then incubated overnight at 4°C in 0.01 M Tris, 0.15 M NaCI, 1% BSA and 0.1% Tween 20, pH 7.4 with [‘251]IGF (60,000 cpm/ml). After washing, bound radioactivity was detected by autoradiography. Initial exposure was 5 days and was than adjusted to optimize resolution. Molecular sizes of the major IGFBP species were calculated based on the positions of prestained Sigma molecular weight standards. Nonspecific binding was demonstrated by incubating part of the IGFBPcontaining nitrocellulose with excess unlabeled IGF. Dete~inat~on of IGF-~GFB~ complexes aping size Edelusion high-performance gel chromatography

IGF binding activity in conditioned media was also analyzed under neutral conditions by gel filtration chromatography on a Superdex 75 HR lo/30 column

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(Pharmacia, Upsala, Sweden). Medium samples (200 ~1) were incubated overnight with 2 x lo5 cpm of [‘251]IGF-I or [‘zI]IGF-II in 200 ~1 of buffer (100 mM Hepes, 44 mM NaHC03, 0.02% NaN,, 0.01% Triton X-100,0.01% BSA, pH 6.0) in the absence or presence of 2 pg of unlabeled IGF-I or IGF-II. After incubation, 200 ~1 of media were applied to a column preequilibrated with 44 mM NaHCO, buffer, pH 7.4, then eluted in the same buffer. Fractions of 0.5 ml were collected at a flow rate of 0.5 ml/min and radioactivity was counted in each fraction. Values for nonspecific

binding (complexes made in the presence of unlabeled IGF-I or IGF-II) were subtracted from those of total binding (complexes made in the absence of unlabeled IGF). Results Time- and treatment-dependent secretion of IGFBPs and progesterone by cultured theca and granulosa cells

The secretion of IGFBPs and progesterone by theta cells cultured under the different treatments is shown

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DAYS OF CULTURE Fig. 1. A representative experiment demonstrating progesterone content (A, D), and IGF-I (B, E) and IGF-II (C, F) binding activity in theta (A-C) and granulosa (D-F) cell culture media. Cells were cultured with basal medium CO), or in the presence of forskolin (A 1, insulin (0) or insulin+forskolin (A). Each point represents the value measured in media pooled from six wells. The results are expressed as percentage of specific binding of [lzI]IGF-I, normalized for cell number.

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in Fig. 1A, B and C. Maximal IGFBP secretion was observed in cultures treated with forskolin alone throughout the experimental period. Maximal progesterone production was obtained with a combination of insulin plus forskolin. In granulosa cells (Fig. lD, E and F) maximal IGFBP secretion as well as progesterone production were induced in cells cultured in the presence of forskolin, except on day 9, when progesterone production with this treatment was attenuated. In granulosa cells, insulin alone had the least stimulatory effect on IGFBP and progesterone secretion. It is worth noting that maximal IGFBP secretion by granulosa cells was approximately 3-fold higher than that of theta cells. The affinity of IGF-I, IGF-II and insulin to IGFBPs in theta and granulosa cell-conditioned media collected on the seventh day of culture was determined by competitive binding assays in media collected from cells incubated with forskolin (Fig. 2). The IC,, values from theta cell media treated with IGF-I, IGF-II or insulin (Fig. 2A) were 3.5, 2 and 350 ng/tube, respectively. The affinity of IGF-II to theta cell-conditioned medium was found to be 1.75 times higher than that of IGF-I, while that of insulin was 100-fold lower. The IC,, values for IGF-I, IGF-II and insulin in media from granulosa cells (Fig. 2B) were 0.6, 0.35 and 14 ng/tube, respectively. IGF-II’s affinity to granulosa cell-conditioned medium was twice that of IGF-I, while that of insulin was 24 times greater (Fig. 2). Ligand blotting Ligand blot analysis of IGFBPs secreted by theta and granulosa cells was carried out in serum-free media. IGFBPs secreted by theta cells incubated with insulin or forskolin consisted of three bands of 44, 40 and 34 kDa (Fig. 3A). Incubation with insulin plus forskolin resulted in an increased amount of the 40-44 kDa complex and in the appearance of a weak 21 kDa band (Fig. 3A). These bands could only be detected with [ 1251]IGF-II, whereas no bands were revealed even after 20 days of exposure using [ ‘251]IGF-I as the tracer (data not shown). Analysis of IGFBPs secreted by granulosa cells incubated with insulin or forskolin (Fig. 4A and C) revealed a 21 kDa band, three poorly resolved bands corresponding to 27, 29 and 34 kDa, and two faint bands of 40-44 kDa. Incubation with insulin plus forskolin resulted in a remarkable intensification of the 40-44 kDa bands while the 27 and 29 kDa bands became undetectable (Fig. 4). The same bands were detected with either [‘2sI]IGF-I or [1251]IGF-II (Fig. 4A and C) but the intensity of the bands using IGF-II was higher. No bands were detected in media collected from either cell type grown under control conditions (Figs. 3A, 4A and 40 These media also came up negative in competitive binding assays (data not shown).

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Fig. 2. Competition of [ ‘251]IGF-I binding with unlabeled IGF-I ( A ), IGF-II (A) or insulin (0) in theta (A), and granulosa cell-conditioned media (B). Competition assays were carried out in media

from cells cultured with forskolin, collected on day 7 of culture. The results are expressed as percentage of specific binding of [ 1251]IGF-I, normalized for cell number.

Incubation of blots with excess unlabeled IGF-I or IGF-II revealed the specificity of the binding (Figs. 3B, 4B and 40). Gel filtration of conditioned media Fig. 5 presents specific elution profiles of [ 125I]IGF-II complexes with IGFBPs in theta (Fig. 5A, B and C) and granulosa (Fig. 5D, E and F) cell-conditioned media. No specific radioactive binding could be detected for control cells grown in serum-free medium without the addition of insulin or forskolin (not shown). Elution of complexes from theta cell-conditioned medium showed a marked difference between cells treated with insulin (Fig. 5A) or forskolin (Fig. 5B) only, and cells treated with insulin plus forskolin (Fig. 50. The first two treatments yielded a main complex

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Discussion

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Fig. 3. Ligand blot analysis of IGFBPs in conditioned medium from unstimulated theta cells (1) or cells stimulated with insulin (2), forskolin (3) or insulin + forskolin (4). Cells were cultured for 4 days in medium containing 1% fetal calf serum (FCS) then transferred to serum-free media. On the seventh day of culture, samples of conditioned media were run on 10% SDS-polyactylamide gels under nonreducing conditions. After transfer to nitrocellulose the blots were incubated with [‘Z51]IGF-II with (B) or without (A) an excess amount of unlabeled IGF-II.

in tubes 38-44, with a peak at tube 40, which corresponded to molecular weights of 28-45 and 40 kDa respectively. Incubation with insulin plus forskolin shifted the complex to higher molecular weights with peaks at 64 and 92 kDa. A similar upward shift was observed for IGFBPs secreted by granulosa cells and cultured under the same treatments. Insulin (Fig. 5D) or forskolin (Fig. 5E) treatments yielded a main peak at 42 kDa and a small peak at 95 kDa. Incubation with insulin plus forskolin shifted the complex to higher molecular weight with a peak at 95 kDa (Fig. 5F). The same complexes were observed in granulosa cell-conditioned medium incubated with [ ‘25111GF-I(not shown), whereas no radioactivity could be detected in theta cell-conditioned medium incubated with [‘25111GF-I. It should be noted that almost identical elution profiles were observed with excess of unlabeled IGF-I and IGF-II.

This study demonstrates that theta interna and granulosa cells derived from bovine preovulatory follicles secrete IGFBPs into their culture media. It further indicates this secretion to be affected by time and treatment of the cells in culture. Ligand blot studies showed that theta cells secrete IGFBPs of 34, and 40 and 44 kDa, while granulosa cells secrete five IGFBPs with apparent molecular weights of 21, 27, 29, 34, and 40-44 kDa. Gel filtration analysis validated these results by indicating the presence of low and high molecular weight IGF-IGFBP complexes in conditioned media from these cells. In both cell types, incubation with forskolin plus insulin caused a shift to high molecular weight IGFBP species as compared to cells treated with each of these agents alone. The high molecular weight (40-44 kDa) IGFBP appearing on the ligand blot, and the 95 kDa intact complex detected by gel filtration (Figs. 3, 4 and 5) are consistent with the characteristics of IGFBP3 (Clemmons, 1990). These findings therefore may suggest that this BP is present in both cell-conditioned media. It is possible that the lower molecular weight IGFBPs, i.e. those of 34, and 27-29 kDa, are IGFBP-2 and 1 respectively. All the IGFBPs exhibited higher affinity for IGF-II than IGF-I, consistent with the properties of the well-described IGFBP-1, 2 and 3 (Clemmons, 1990). However, positive identification awaits validation by immunoprecipitation with antisera specific to these IGFBPs. The presence of IGFBPs in the ovarian tissue of several species has been documented in recent years (Suikkari et al., 1989; Adashi et al., 1990; Mondschein et al., 1990). IGFBPs 1, 2 and 3 were detected in human follicular fluid and in granulosa cells of women undergoing in vitro fertilization (Giudice et al., 1990; Martikainen et al., 1991). Rat granulosa cells secrete a low molecular weight (23-29 kDa) IGFBP, possibly IGFBP-1 (Adashi et al., 1990). IGF binding activity has

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Fig. 4. Ligand blot analysis of IGFBPs in conditioned medium from unstimulated granulosa cells (1) or cells stimulated with insulin (2) forskolin (3) or insulin +forskolin (4). Cells were cultured for 4 days in medium containing 1% FCS then transferred to serum-free media. On the seventh day of culture, samples of conditioned media were run on 10% SDS-polyacrylamide gels under nonreducing conditions. After transfer to nitrocellulose the blots were incubated with [lasI]IGF-I (A) or [1251]IGF-II (C) only, or in the presence of excess amounts of IGF-I (B) or IGF-II (D).

also been demonstrated in ewe ovarian follicles (Monget et al., 1989) but the molecular species have not yet been identified. The presence of IGFBPs in follicular fluid and their secretion from granulosa cells has been well characterized in pig (Hammond et al., 19911, and IGFBPs 2 and 3, with apparent molecular weights of 34 and 40-45 kDa respectively, have been identified as the major binding species in pig ovaries (Mondschein et al., 1990).

findings match those found here of IGFBPs secreted by bovine granulosa cells. In contrast to granulosa cells, the production of IGFBPs in theta cells has not been previously described. This study provides evidence that this other ovarian steroidogenic cell also secretes IGFBPs and is probably a site for their production. It is interesting to note that theta cells do not produce IGFs as do some of the other IGFBP-secreting cells (Hammond et al., These

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Fig. 5. Gel filtration of IGF-II-IGFBP complexes derived from theta (A, B and C) and granulosa (D, E and F) cell-conditioned media cm a Superdex 75 HR lo/30 column. The media were taken from cells treated with insulin (A and DI, forskolin (B and E) or insulin+forskolin (C and F). Rliquots (200 ~1) of media that has been incubated overnight with 1125111GF-11 were injected into the column. Fractions of 0.5 ml were collected at a flow rate of 0.5 mI/min and radioactivity was counted. Nonspecific binding was measured by the addition of 2 fig of IGF-I (of or IGF-II (*I to the incubation medium., Results are expressed as total binding minus nonspecific binding.

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1991).IGFBP secretion by. theta cells may modulate granulosa cell function, thereby adding a new dimension to the known cooperation between these two cell types. While the existence of IGFBPs is well established, their role and regulation in terms of ovarian function is still far from being understood. Here we demonstrate that the production of IGFBPs by bovine granulosa and theta cells can be altered by different culture treatments. Our two main findings were: (1) cells which are grown under basal conditions, i.e. in serum-free medium, do not secrete IGFBPs, and (2) a shift to a high molecular weight IGFBP (possibly IGFBP-3) is observed when cells are incubated with forskolin plus insulin. This last point may be of particular significance, in view of our previous observation (Meidan et al., 1990) that treatment of granulosa and theta cells with forskolin plus insulin for 7-9 days induces the differentiation of these cells into large and small luteal like cells, as judged by morphological and functional parameters. Regulation of IGFBP secretion was examined by Mondschein et al. (1990), who claimed that cultured granulosa cells from small porcine follicles secreted IGFBPs without stimulation; however, their ‘basal’ media contained insulin and hydrocortisone. Accordingly, our results show that the addition of insulin alone to the culture media stimulates IGFBP secretion, to the same extent as that induced by forskolin. Mondschein et al. (1990) also found that treatment of cells recovered from small follicles with FSH plus estradiol stimulated progesterone production but downregulated IGFBP-3 secretion. We have previously shown that follicular cells incubated with forskolin plus insulin undergo differentiation to luteal-like cells which are derived from the corpus luteum (Meidan et al., 1990). The results obtained in this study therefore demonstrate a positive relationship between induction of a 40-44 kDa IGFBP and the cell’s differentiation, which is not necessarily identical to its ability to produce progesterone. Our understanding of the function of IGFBP3 as well as that of other IGFBPs is only now beginning to take shape. IGFBP-3 is known to be secreted by the liver and to be the principal carrier of IGFs in serum (Clemmons, 1990). However, this IGFBP is also produced by other cell types, suggesting a role outside the vascular system. Both inhibitory and stimulatory effects have been ascribed to IGFBPs, specifically to IGFBP-3 (Ooi, 1990). For instance, IGFBP-3 inhibited lipogenesis and glucose oxidation in adipose tissue (Walton et al., 19891, as well as FSH-stimulated estradiol secretion by rat granulosa cells (Shimasaki et al., 1990). However, De Mellow and Baxter (1988) observed that, depending on whether it is pre or co-incubated with IGFs IGFBP-3 can stimulate or inhibit IGF-induced

cell function in human skin fibroblasts. Using bovine fibroblasts, Conover et al. (1990) demonstrated a similar stimulatory effect of IGFBP-3 on IGF-I-induced biological activity. IGF-I mRNA, as well as IGF-I itself, has been reported to increase in concentration as the cow’s corpus luteum develops (Einspanier et al., 1990). In addition, IGF-I action is essential to the induction of steroidogenesis (Hammond et al., 1991). Therefore, parallel induction of IGF-I and IGFBP-3 (as suggested by this study) would seem paradoxical if the binding proteins’ action were exclusively inhibitory to IGF-I-induced effects. Our findings may therefore be in line with studies demonstrating IGFBPs’ stimulatory role. One cannot, however, exclude the possibility of IGFBPS’ dual (stimulatory and inhibitory) activity. Whatever the case, the findings of this study contribute to the growing body of information which strongly suggests the importance of IGFBPs in ovarian physiology. Acknowledgement

This study was supported IS-1839-90.

by the BARD (to R.M.),

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46 Ooi, G.T. (1990) Mol. Cell. Endocrinol. 71, C3Y-C43. Shimasaki, S., Shimonaka, M., Ui, M., Inouye, S., Shibata, F. and Ling, N. (1990) J. Biol. Chem. 265, 2198-2202. Shimasaki, S., Gao, L., Shimonaka, M. and Ling, N. (1991) Mol. Endocrinol. 4. 1451-1458.

Suikkari, A.M., Jalkanen, J., Koistinen, R., Butzow, R., Ritvos, 0.. Ranta, T. and Seppala, M. (1989) Endocrinology 124. 1088- 1090. Walton, P.E., Gopinath, R. and Etherton, T.D. (1989) Proc. Sot. Exp. Biol. Med. 190, 315-319.

Characterization of insulin-like growth factor binding proteins secreted by cultured bovine theca and granulosa cells.

Insulin-like growth factor binding proteins (IGFBPs) secreted by bovine granulosa and theca interna cells cultured in the presence of different lutein...
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