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Developmentally Regulated Responses of Human Granulosa Cells to Insulin-Like Growth Factors (IGFs):IGF-I and IGF-II Action Mediated Via the Type-I IGF Receptor¹

Imperial College School of Medicine at St. Mary’s, Norfolk Place, London, W2 1PG, United Kingdom

Address all correspondence and requests for reprints to: Debbie S. Willis, Department of Obstetrics and Gynaecology, Imperial College School of Medicine at St. Mary’s, Norfolk Place, London, W2 1PG, United Kingdom.

Abstract

In experimental animal models, insulin-like growth factors (IGFs) have been found to be more potent stimulators of ovarian function than insulin. In human theca cells, however, insulin, IGF-I, and IGF-II have similar effects on androgen production. The relative effects of insulin and IGFs on human granulosa cell steroidogenesis is unknown. Furthermore, it is unclear whether effects of IGF-II on steroidogenesis are mediated by the type-I or type-II IGF receptor.

The effects of insulin, IGF-I, and IGF-II on human granulosa cell steroidogenesis were compared in vitro. As expected, insulin, IGF-I, and IGF-II enhanced steroidogenesis. Previously, IGF-II has been shown to enhance granulosa cell steroid production after insulin preincubation. In this study, an effect of IGF-II, independent of insulin priming, also was observed. In granulosa cell cultures from small antral follicles (13 mm), insulin and IGF-I stimulated steroid production to a similar degree, whereas IGF-II was less effective. In contrast, IGFs were more effective than insulin (IGF-I > IGF-II > insulin) in granulosa cells isolated from preovulatory follicles. IGF-I and IGF-II actions were mediated via the type-1 IGF receptor.

The increased responsiveness of mature granulosa cells to IGFs may be an important mechanism by which granulosa cells increase their steroidogenic output in the preovulatory follicle.

INSULIN, insulin-like growth factor (IGF)-I, and IGF-II have all been shown to stimulate ovarian steroidogenesis in primate and nonprimate species (1, 2, 3, 4, 5, 6, 7). IGFs are generally more effective stimulators of ovarian function than insulin (1, 8). In human theca cells, however, insulin, IGF-I, and IGF-II have been shown to have similar activities (5); but this may not be the case in human granulosa cells. In a recent publication, IGF-I and IGF-II were found to be more effective than insulin in suppressing IGF binding protein (IGFBP)-I production in luteinized granulosa cells obtained from women undergoing in vitro fertilization treatment (9). The relative activities of insulin, IGF-I, and IGF-II in nonluteinized granulosa cells, isolated from women having natural cycles, remain unknown.

We have previously shown that IGF-I action in human granulosa cells is mediated via the type-I IGF receptor (10). This has subsequently been shown to be the case in granulosa-lutein cells (11). The receptor that mediates IGF-II action in human granulosa cells is unknown. It has been suggested that it may be mediated via the type-II IGF receptor (6, 9). This seems unlikely, however, because IGF-I and IGF-II exert their effects via the type-I IGF receptor in other systems, including rat granulosa cells (12).

The aims of this study, therefore, were: 1) to compare insulin, IGF-I, and IGF-II effects on steroidogenesis in human granulosa cells from follicles at various stages of development, isolated from women having natural cycles; and 2) to determine the receptor that mediates IGF-II action in human granulosa cells.

Materials and Methods

Patients

Ovarian tissue was obtained from 12 ovulatory women undergoing bilateral oophorectomy for benign, nonovarian, gynecological disease. No patient had been on treatment for suppression or stimulation of the ovaries for at least 3 months before surgery. Approval for the use of ovarian tissue in this study was obtained from Kensington, Chelsea and Westminister Health Authority Ethical Committee, and informed consent was obtained from each patient.

Granulosa cell cultures

Granulosa cells, obtained from women having natural cycles were isolated and cultured, as previously described (7). Briefly, granulosa cells were plated at a density of approximately 5 x 10⁴ viable cells/well in 200 µL of serum-free Medium 199 (Life Technologies, Paisley, Scotland).

Comparison of insulin and IGF effects

Granulosa cell cultures were incubated with human recombinant insulin (Boehringer Mannheim Biochemica, Lewes, UK), IGF-I, or IGF-II (ICN Pharmaceuticals, Oxford, UK) (0.1–500 ng/mL) for 48 h, in the presence of testosterone (5 x 10^-7 mol/L) as substrate for aromatase. When possible, a full range of insulin doses was used; but in some experiments, insufficient cells were obtained to perform full-dose responses. In these studies, the effects of insulin, IGF-I, and IGF-II were compared at a dose of 10 ng/mL. This dose has previously been shown to produce a submaximal steroidogenic response (7).

Blocking of the type-I IGF receptor

Granulosa cells were incubated with IGF-I and IGF-II (1–100 ng/mL) with or without the addition of 0.1–10 µg/mL IR3 [an anti-type-I IGF receptor antibody that blocks the type-I IGF receptor (13); Oncogene Science, Cambridge, UK] in the presence of testosterone (5 x 10^-7 mol/L) for 48 h.

At the end of the incubation period, the overlying medium was removed and stored at -20 C until measurement of estradiol (E₂) and progesterone (P) levels by RIA, as previously described (7). The cells were either solubilized for measurement of total DNA, as previously described (7), or the number of viable cells was assessed using a commercially available mitochondrial tetrazolium salt assay (Celltiter 96 nonradioactive cell proliferation assay kit from Promega, Southampton, UK). Statistical analysis was performed by Student’s t test and ANOVA. Significance was assumed when P < 0.05.

Results

As shown previously, insulin and IGF-I augmented steroid accumulation in the granulosa cell cultures. We have previously reported that IGF-II stimulates human granulosa cell steroidogenesis after insulin preincubation (6). In the series of experiments presented here, an effect of IGF-II on granulosa cell steroidogenesis was observed in the absence of preincubation of the cells with insulin. Nevertheless, in these studies, insulin preincubation of granulosa cells was found to enhance the subsequent effect of IGF-II (Fig. 1).

View larger version (23K): [in this window] [in a new window]   Figure 1. P response in granulosa cells from 3- to 7.5-mm follicles isolated from one patient challenged with IGF-II (10 ng/mL), either immediately on culturing (open bars) or after 48-h control (medium alone) preincubation (solid bars) or insulin (10 ng/mL) preincubation (hatched bars). Data are the mean and SE of triplicate wells. Where not visible, the error bars are contained within the symbol. *, P < 0.05, compared with relevant control.

Comparison of insulin, IGF-I, and IGF-II

Follicles 13 mm. The effects of insulin and IGFs on steroidogenesis were compared in granulosa cell cultures from follicles 13 mm, from eight ovulatory women (anovulatory women were not studied). Insulin and IGF-I stimulated steroidogenesis to a similar degree, whereas IGF-II was less effective in stimulating E₂ and P accumulation (Figs. 2 and 3). The effects of insulin, IGF-I, and IGF-II (10 ng/mL) in granulosa cell cultures from a range of follicle sizes (from three experiments) are shown in Fig. 2. Figure 3A illustrates superimposable P dose response curves to insulin and IGF-I (ED₅₀: 8.3 ng/mL) across a wide range of concentrations, in granulosa cells isolated from a dominant 13-mm follicle.

View larger version (37K): [in this window] [in a new window]   Figure 2. Effect of insulin, IGF-I, and IGF-II (10 ng/mL) on E2 (A) and P (B) accumulation in granulosa cell cultures from three different ovulatory patients from 2- to 5-mm follicles (i), a 6-mm follicle (ii), and 3- to 7-mm follicles (iii). C, Control. Data are the mean and SE of triplicate wells. Where not visible, the error bars are contained within the symbol. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control.

View larger version (25K): [in this window] [in a new window]   Figure 3. The effect of insulin and IGF-I (A) and insulin, IGF-I, and IGF-II (B) on P accumulation in granulosa cells from a dominant 13-mm follicle (A) and pooled results from three different preovulatory follicles (B). In A (data are the mean and SE of triplicate wells; and, where not visible, the error bars are contained within the symbol), insulin and IGF-I significantly stimulated P accumulation (P < 0.0001). There was no significant difference between the responses to insulin and IGF-I. Insufficient cells were obtained to compare the effect of IGF-II. a and b, Minimal effective dose of insulin and IGF-I, respectively. In B (data are the mean and SE of three experiments performed in triplicate wells; and, where not visible, the error bars are contained within the symbol), insulin, IGF-I, and IGF-II significantly stimulated P accumulation (P < 0.0001). The response to IGF-I and IGF-II were significantly higher than the response to insulin (P < 0.04). c, Minimal effective dose of IGF-I; d, minimal effective dose of insulin and IGF-II.

Effect of anti-type-I IGF receptor antibodies on IGF action

As shown previously for granulosa cells from small antral follicles < 10 mm, IGF-I action was found to be mediated via the type-I IGF receptor in mature granulosa cells from preovulatory follicles (Fig. 4). In four experiments in which the effect of the anti-type-I IGF receptor (IR3) on IGF-II was assessed, IGF-II action was found to be mediated via the type-I IGF receptor in granulosa cells from small antral follicles (data not shown) and preovulatory follicles (Fig. 4). Both IGF-I and IGF-II (10 and 100 ng/mL) significantly enhanced P accumulation (P < 0.001) in the granulosa cell cultures. Incubation of the granulosa cell cultures with IGF-I or IGF-II, in the presence of IR3, significantly inhibited IGF-I- and IGF-II-stimulated steroidogenesis (Fig. 4).

View larger version (33K): [in this window] [in a new window]   Figure 4. Effect of IR3 on IGF-stimulated P accumulation in granulosa cell cultures from a preovulatory follicle. Antibody (IR3) concentrations are shown in the insert. a, Significantly different from control (P < 0.001); *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with IGF alone.

Comparison of insulin and IGF effects on granulosa cell steroidogenesis have highlighted differences between the human and other species. IGFs were more potent stimulators of steroidogenesis than insulin in rat (1, 8) and porcine granulosa cells from small, medium, and large follicles (14). In the bovine ovary, insulin and IGF-I had similar activities in granulosa cells from all follicle sizes (15). In the human, however, there seems to be a developmentally regulated response to IGFs in granulosa cells of the maturing follicle. In the study presented here, insulin and IGF-I had similar activities in granulosa cells from small antral follicles, whereas in mature granulosa cells from preovulatory follicles, IGFs were more effective than insulin. The exact point in development at which IGFs become more effective than insulin is not evident from this study. However, the results presented here indicate that this occurs somewhere between the 13-mm and preovulatory stages of follicle development. Actions of IGF-I and IGF-II in human granulosa cells were mediated via the type-I IGF receptor. This is consistent with the findings in other systems, including rat granulosa cells (12).

The developmentally regulated responses to IGFs that are apparent in ovulatory women (anovulatory women were not investigated) suggest that IGFs may be more important in the latter stages of follicle development, when P production is greatly increased. IGF-II is presumably the physiological ligand, because it is the predominant IGF in human granulosa cells (16, 17). In the preovulatory follicle, there is increased granulosa cell IGF-II production and increased type-I IGF receptor expression, compared with granulosa cells from small antral follicles (16, 17, 18). There is also the presence of IGFBPs in vivo that modulate IGF action. In the preovulatory follicle, there are decreased levels of IGFBPs (16, 17, 18). Hence, the intraovarian IGF system seems to be regulated so that there is increased bioavailable IGF-II to interact with increased type-I IGF receptors in mature granulosa cells. Coupled to the ability of IGFs to amplify the effects of gonadotropins, this may be one mechanism by which human granulosa cells increase their steroidogenic activity in preovulatory follicles.

Footnotes

¹ This work was supported by funding from the Medical Research Council, UK (D.S.W. and H.W.).

Received July 24, 1997.

Revised November 13, 1997.

Accepted December 10, 1997.

References

1. Adashi EY, Resnik CE, D’Ercole AJ, Svoboda ME, van Wyk JJ. 1985 Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr Rev. 6:400–420.[Abstract]
2. Giudice LC. 1992 Insulin-like growth factors and ovarian follicular development. Endocr Rev. 13:641–669.[CrossRef][Medline]
3. Garzo VG, Dorrington JH. 1984 Aromatase activity in human granulosa cells during follicular development and the modulation by follicle stimulating hormone and insulin. Am J Obstet Gynecol. 148:657–662.[Medline]
4. Erickson GF, Garzo VG, Magoffin DA. 1989 Insulin-like growth factor-I regulates aromatase activity in human granulosa and granulosa luteal cells. J Clin Endocrinol Metab. 69:716–724.[Abstract]
5. Nahum R, Thong KJ, Hillier SG. 1995 Metabolic regulation of androgen production by human thecal cells in vitro. Hum Reprod. 10:75–81.[Abstract/Free Full Text]
6. Mason HD, Willis DS, Holly JMP, Franks S. 1994 Insulin preincubation enhances insulin-like growth factor-II (IGF-II) action on steroidogenesis in human granulosa cells. J Clin Endocrinol Metab. 78:1265–1267.[Abstract]
7. Willis DS, Mason HD, Gilling-Smith C, Franks S. 1996 Modulation by insulin of follicle-stimulating hormone and luteinizing hormone actions in human granulosa cells of normal and polycystic ovaries. J Clin Endocrinol Metab. 81:302–309.[Abstract]
8. Davoren JB, Kasson BG, Li CH, Hsueh AJW. 1986 Specific insulin-like growth factor (IGF)-I and -II binding sites on rat granulosa cells: relation to IGF action. Endocrinology. 119:2155–2162.[Abstract]
9. Poretsky L, Bai C, Liu HC, Rosenwaks Z. 1996 Insulin-like growth factor II (IGF-II) inhibits insulin-like growth factor binding protein 1 (IGFBP-1) production in luteinized human granulosa cells with a potency similar to insulin-like growth factor I (IGF-I) and higher than insulin. J Clin Endocrinol Metab. 81:3412–3414.[Abstract]
10. Willis DS, Franks S. 1995 Insulin action in human granulosa cells from normal and polycystic ovaries is mediated by the insulin receptor and not the insulin-like growth factor receptor. J Clin Endocrinol Metab. 80:3788–3790.[Abstract]
11. Poretsky L, Chandrasekher YA, Bai C, Liu HC, Rosenwaks Z, Giudice L. 1996 Insulin receptor mediates inhibitory effect of insulin, but not insulin-like growth factor (IGF)-I, on IGF binding protein (IGFBP-1) production in human granulosa cells. J Clin Endocrinol Metab. 81:493–496.[Abstract]
12. Adashi EY, Resnik CE, Rosenfeld RG. 1990 Insulin-like growth factor-I (IGF-I) and IGF-II hormonal action in cultured rat granulosa cells: mediation via type I but not type II IGF receptors. Endocrinology. 126:216–222.[Abstract]
13. Kull FC, Jacobs S, Su YF, Svoboda ME, van Wyk JJ, Cuatrecasas P. 1983 Monoclonal antibodies to receptors for insulin and somatomedin C. J Biol Chem. 258:6561–6566.[Abstract/Free Full Text]
14. Maruo T, Hayashi M, Matsuo H, Ueda Y, Morikawa H, Mochizuki M. 1988 Comparison of the facilitative roles of insulin and insulin-like growth factor I in the functional differentiation of granulosa cells: in vitro studies with the porcine model. Acta Endocrinol (Copenh) 117:230–240.
15. Gong JG, McBride D, Bramley TA, Webb R. 1994 Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on bovine granulosa cell steroidogenesis in vitro. J Endocrinol. 143:157–164.[Abstract]
16. El-Roeiy A, Chen X, Roberts VJ, LeRoith D, Roberts CT, Yen SSC. 1993 Expression of insulin-like growth factor-I (IGF-I) and IGF-II and the IGF-I, IGF-II and insulin receptor genes and localisation of the gene products in the human ovary. J Clin Endocrinol Metab. 77:1411–1418.[Abstract]
17. Voutilainen R, Franks S, Mason HD, Martikainen H. 1996 Expression of insulin-like growth factor (IGF), IGF-binding protein and IGF receptor messenger ribonucleic acids in normal and polycystic ovaries. J Clin Endocrinol Metab. 81:1003–1008.[Abstract]
18. Giudice LC. 1995 The insulin-like growth factor system in normal and abnormal human ovarian follicle development. Am J Med. [Suppl 1A] 98:48S–54S.

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