BIOLOGY OF REPRODUCTION 56, 1458-1465 (1997) Insulin-Like Growth Factor-Binding Protein-2 and -3: Their Biological Effects in Bovine Thecal Cells' L.J. Spicer,2 R.E. Stewart, P. Alvarez, C.C. Francisco, and B.E. Keefer Department of Animal Science, Oklahoma State University, Stillwater, Oklahoma 74078 ABSTRACT INTRODUCTION The insulin-like growth factor (IGF) system is composed of IGF-I and -II, IGF receptors, and IGF-binding proteins (IGFBPs) (for reviews see [1-3]). Both IGF-I and IGF-II have been shown to be relevant promoters of ovarian follicular function of several species including cattle (for reviews see [3-5]). In most species, IGF-I stimulates mitogenesis of granulosa and thecal cells in the absence of gonadotropins, whereas in the presence of gonadotropins, IGF-I stimulates steroidogenesis of granulosa and thecal cells (for reviews see [3, 51). It was more than 10 yr after the determination of the amino acid sequence of IGF-I and IGF-II in 1978 [6, 7] that the structure of an IGFBP and its mRNA in ovarian Accepted January 22, 1997. Received March 11, 1996. 'Approved for publication by the Director, Oklahoma Agricultural Experiment Station. This research was supported under project H-2088, project number HR4-032 from the Oklahoma Center for the Advancement of Science and Technology, and by the Cooperative State Research Service, U.S. Department of Agriculture, under Agreement No. 93-372039023. 2Correspondence. FAX: (405) 744-7390; e-mail: igfleo@okway.okstate.edu MATERIALS AND METHODS Reagents and Hormones Reagents used were Dulbecco's Modified Eagle medium (DMEM), Ham's F-12, insulin (bovine; 25.7 U/mg), pronase E, collagenase, hyaluronidase, DNase, and fetal calf serum (FCS), all obtained from Sigma Chemical Company (St. Louis, MO); bovine LH (USDA-bLH-B5, LH activity 2.1 NIH-LH-S1 U/mg; FSH activity < 1.0% by weight) obtained from the National Hormone and Pituitary Program (Baltimore, MD); recombinant human IGFBP-2 (31 000 molecular weight) obtained from Austral Biologicals (San Ramon, CA); and recombinant human IGFBP-3 (47 000 molecular weight) obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Cell Culture Ovaries were obtained at a nearby commercial abattoir from beef and dairy cattle after slaughter. After transport to the laboratory on ice (< 120 min), the ovaries were processed and thecal cells were obtained as previously described [17, 18]. Briefly, large ( 8 mm) follicles were dissected from the ovary, follicular fluid was aspirated, follicles were bisected, and each follicle wall was scraped and 1458 Downloaded from https://academic.oup.com/biolreprod/article-abstract/56/6/1458/2760761 by guest on 01 June 2020 This study was aimed at testing the hypothesis that the insulin-like growth factor-binding proteins (IGFBP)-2 and -3 can modulate the hormone-dependent differentiation of thecal cells in vitro. Thecal cells from large ( 8 mm) follicles were collected from cattle, cultured for 2 days in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 days in serum-free medium with bovine LH (100 ng/ml), recombinant human insulin-like growth factor (IGF)-I (0 or 30 ng/ml), recombinant human IGFBP-2 (0, 200, or 400 ng/ml; i.e., 0, 6.5, or 12.9 nM), or recombinant human IGFBP-3 (0, 200, or 400 ng/ml; i.e., 0, 4.3, or 8.5 nM). IGFBP-2 (200 and 400 ng/ml) inhibited (p < 0.05) IGF-l-induced androstenedione production by 18-30% but did not influence (p > 0.10) progesterone production or thecal cell proliferation inthe presence of LH and/or IGF-I. In contrast, IGFBP-3 (200 ng/ml) inhibited the IGF-l-induced increase in thecal cell numbers by 76%, and thecal cell progesterone and androstenedione production by 52% and 89%, respectively. A higher dose of IGF-I (i.e., 100 ng/ml) overcame the inhibitory effects of IGFBP-3 on IGF-l-induced cell proliferation and on progesterone and androstenedione production by thecal cells. As with IGFBP-2, IGFBP-3 had no effect (p > 0.10) on LH-induced progesterone or androstenedione production by thecal cells in the absence of IGF-I. Both IGFBP-2 and IGFBP-3 directly inhibited [12511GF-I and -II binding to thecal cells; IGFBP-2 was a weaker inhibitor of thecal [1251]IGF-I and -II binding than IGFBP-3. These results indicate that IGFBP-3 has a more pronounced inhibitory effect than IGFBP-2 on IGF-I action in cultured bovine thecal cells. Thus, IGFBP-3 may play a more significant role than IGFBP-2 in regulating thecal cell proliferation and steroidogenesis during follicular development in cattle. tissue were first reported [8-10]. Since that time, the presence of several additional IGFBPs (i.e., IGFBP-1 through -6) in the ovary of several species has been described (for reviews see [3, 4, 11]). In particular, it appears that follicular fluid levels of IGFBP-2 decrease whereas IGFBP-3 remains unchanged as follicles enlarge and become steroidogenically active in swine, cattle, and sheep (for reviews see [3, 5]). IGFBP-3 and IGFBP-2 are the first and second most prevalent IGFBPs, respectively, in ovarian follicular fluid of pigs, cattle, and sheep [3, 12-14]. In particular, IGFBP-2 activity in follicular fluid is lower in the growing dominant vs. subordinate follicles [13], whereas androstenedione and estradiol concentrations in follicular fluid are greater in the growing dominant follicle vs. subordinate follicles early in the estrous cycle of cattle [13]. Whether the lower steroid production by subordinate follicles is a direct result of the greater IGFBP-2 levels is unknown. Although previous studies have shown that IGFBPs can directly inhibit FSH-induced steroid production by rat granulosa cells [10, 15, 16], previous investigations have not evaluated the effect of IGFBPs on steroidogenesis of thecal cells in any species or on mitogenesis of ovarian cells in any species. Potential in vivo effects of IGFBP-2 and -3 could be classified as endocrine or paracrine as well as autocrine (for review see [3]). Moreover, direct effects of IGFBPs on ovarian follicular cell mitogenesis and steroidogenesis in domestic animals have not been reported. Therefore, we set out to test the hypothesis that IGFBP-2 and IGFBP-3 can modulate proliferation and steroidogenesis of bovine thecal cells in vitro. THECAL CELLS AND INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS IGFBP-3 on LH (dose dependent)-stimulated thecal cell steroidogenesis. Theca cells were cultured for 2 days in 10% FCS and then cultured in serum-free medium for an additional 2 days with bovine LH (0, 1, 3, 10, 30, or 100 ng/ml), IGFBP-3 (0 or 200 ng/ml), and insulin (100 ng/ml). The doses of LH, IGFBP-3, and insulin were selected based on previous studies [18] and as described for experiment 2. Experiment 6 was conducted to determine whether IGFBP-2 and IGFBP-3 inhibit the binding of [ 25 I]IGF-I or [125 I]IGF-II to their receptors. Thecal cells were obtained from large follicles, cultured for 3 days in 10% FCS, washed twice with serum-free medium, and then used for IGF-I binding assays. Binding of [' 2 5I]IGF-I and [125 I]IGF-II to thecal cells was determined as previously described [21, 26]. To further evaluate the relative potency of IGFBP-2 and IGFBP-3 on competition for IGF-I binding, experiment 7 was conducted to determine whether IGFBP-2 or IGFBP-3 could inhibit binding of [ 25I]IGF-I to the IGF-I antiserum in an IGF-I RIA. Various concentrations of IGFBP-2 and IGFBP-3 were added to total binding tubes in the IGF-I assay. The IGF-I assay was conducted as previously described [27]. Determination of Thecal Cell Numbers Numbers of thecal cells were determined at the termination of experiments using a Coulter counter (Model Zm; Coulter Electronics, Hialeah, FL) as previously described [17]. Briefly, cells were exposed to 0.5 ml of trypsin (0.25% [w:v] in 0.15 M NaCI) for 20 min at 25 0C and then scraped from each well, diluted in 0.15 M NaCl, and enumerated. Androstenedione RIA Concentrations of androstenedione in culture medium collected on Day 4 of culture were determined using solidphase RIA kits (ICN Biomedicals, Costa Mesa, CA) as previously described [18]. Intra- and interassay coefficients of variation were 14% and 10%, respectively. Progesterone RIA Concentrations of progesterone in culture medium collected on Day 4 of culture were determined with an RIA as previously described [17]. Intra- and interassay coefficients of variation were 16% and 20%, respectively. IGF-II RIA Concentrations of IGF-II in culture medium collected on Day 4 of culture were determined in one RIA as previously described [14]. Culture medium was concentrated 3- to 10-fold using Centricon-3 microconcentrators (Amicon, Danvers, MA) prior to extraction. Assay sensitivity (i.e., 90% of total binding) was 60 pg/tube, and intraassay coefficient of variation was 4.9%. StatisticalAnalyses Experimental data are presented as the least squares means SE of measurements for triplicate culture wells from three or more experiments. Each experiment was performed with different pools of thecal cells collected from approximately 30 ovaries for each pool. Main effects and interactions on dependent variables (i.e., cell numbers and steroid production) were assessed using General Linear Downloaded from https://academic.oup.com/biolreprod/article-abstract/56/6/1458/2760761 by guest on 01 June 2020 flushed with Ham's F-12 to remove any remaining granulosa cells. The theca interna layer was microdissected from the follicle wall and torn into small pieces. These pieces were digested for 1 h at 37°C in Ham's F-12 containing 1 mg/ml of collagenase, 1 mg/ml of hyaluronidase, 1 mg/ml of pronase E, and 0.01 mg/ml of DNase on a rocking platform shaker. After incubation, undigested tissue was removed from the cell suspension by filtration, and the dispersed cells were washed and resuspended in serum-free medium [18]. The number of viable cells was determined using the trypan blue exclusion method, and averaged 90 ± 3% of total thecal cells. Contamination of thecal cells by granulosa cells is minimal using this procedure (i.e., < 10% [19, 20]). Medium was a 1:1 (v:v) mixture of DMEM and Ham's F-12 containing 0.12 mM gentamicin and 38.5 mM sodium bicarbonate. Approximately 2 x 105 viable cells in 45-110 Rl of medium were added to Falcon multiwell plates (#3047; Becton Dickinson and Co., Lincoln Park, NJ) containing 1 ml of medium. Cultures were kept at 38.5°C in a 5% CO 2 atmosphere. To obtain optimal attachment, cells were maintained in the presence of 10% FCS for the first 2 days of culture, with a medium change occurring on Day 1. After this time, cells were washed twice with 0.5 ml of serum-free medium, and incubations were continued in serum-free medium with or without added hormones for an additional 48 h (i.e., from Day 2 to 4 of culture) without a change of media. Experiments 1 and 2 were conducted to evaluate the dose-response effect of IGFBP-2 and IGFBP-3, respectively, on the action of LH and IGF-I on thecal cell proliferation and steroidogenesis. Thecal cells were cultured for 2 days in 10% FCS and then cultured in serum-free medium for an additional 2 days with 100 ng/ml of LH in the absence or presence of 30 ng/ml of IGF-I and either IGFBP-2 (0, 200, and 400 ng/ml; i.e., 0, 6.5, and 12.9 nM) or IGFBP-3 (0, 200, and 400 ng/ml; i.e., 0, 4.3, and 8.5 nM). The doses of IGF-I and LH were selected based on previous studies [18, 21]. The 30 ng/ml dose of IGF-I was selected as a dose of IGF-I that elicits a steroidogenic response so that the IGFBP concentrations used would be 2-3 times greater than IGF-I concentrations; this ratio has been reported to be effective in inhibiting IGF-I action [22, 23]. Concentrations of immunoreactive IGFBP-2 in blood of cattle and women have been reported to be between 100 and 500 ng/ml [24, 25]. Concentrations of immunoreactive IGFBP-3 have not been reported for cattle but average between 2 and 4 g/ml in humans [25]. Experiment 3 was conducted to evaluate the effect of IGFBP-3 on IGF-I (dose dependent)-stimulated thecal cell proliferation and steroidogenesis. Thecal cells were cultured for 2 days in 10% FCS and then cultured in serumfree medium for an additional 2 days with 100 ng/ml of LH in the absence or presence of IGF-I (0, 30, or 100 ng/ml) and IGFBP-3 (0 or 200 ng/ml). The doses of IGF-I, LH, and IGFBP-3 were selected based on previous studies [18] and as described for experiments 1 and 2. Experiment 4 was conducted to evaluate the effect of IGFBP-2 on LH (dose dependent)-stimulated thecal cell steroidogenesis. Thecal cells were cultured for 2 days in 10% FCS and then cultured in serum-free medium for an additional 2 days with bovine LH (0, 3, or 10 ng/ml), IGFBP-2 (0 or 200 ng/ml), and insulin (100 ng/ml). The doses of LH, IGFBP-2, and insulin were selected based on previous studies [18] and as described for experiment 1. Experiment 5 was conducted to evaluate the effect of 1459 SPICER ET AL. 1460 .1IL M .1 N MI *UL Nl-I l la A 'o _ ' I- X 5 0.5 I +F-I h 1.5 1.0 _ m A 2.0r 1.0- . 0. - _ 0.0 o - A 2 60oB 60 0I '- 10 o b .. b b r e 2 0 0 -C {L a- "o - ME 10 _ a Le 6O 5 C - - 0 400 IGFBP-2, nlmL 600oo C - I w Cia * 400 300 sAA. uu d C * * 200 B W 100 C S. I0 < 200 a v 400 IQFBP-2, ng/mL 200 400 IQFBP-3, ng/mL o co 0 0 b 15 O01 -00 200 c 20 06 e 0 B Sa[ b 40 10 400 IGFBP-3, ng/mL T W -- --200 0 20 . 0.0- b 0 0 200 400 IGFBP-S, n/mL FIG. 1. Effects of IGFBP-2 on IGF-I-stimulated thecal cell proliferation (A), progesterone production (B), and androstenedione production (C) by thecal cells in the presence of LH (experiment 1). Thecal cells were cultured for 2 days in the presence of 10% FCS and then treated in serumfree media with 100 ng/ml of LH, IGF-I (0 or 30 ng/ml), and IGFBP-2 (0, 200, or 400 ng/ml) for an additional 2 days. Values are means from three separate replicate experiments. Within a panel, means without a common superscript differ (p < 0.05). FIG. 2. Effects of IGFBP-3 on IGF-I-stimulated thecal cell proliferation (A), progesterone production (B), and androstenedione production (C) by thecal cells in the presence of LH (experiment 2). Thecal cells were cultured for 2 days in the presence of 10% FCS and then treated in serumfree media with 100 ng/ml of LH, IGF-I (0 or 30 ng/ml), and IGFBP-3 (0, 200, or 400 ng/ml) for an additional 2 days. Values are means from three separate replicate experiments. Within a panel, means without a common superscript differ (p < 0.05). Models procedures of the Statistical Analysis System [28]. Each well was a replicate, and each experiment contained three replicates per treatment. When steroid production was expressed as nanograms or picograms per 105 cells per 48 h, cell numbers at the termination of the experiment were used for this calculation. Specific differences in steroid production and cell numbers between treatments were determined using the Fisher's protected least significant difference procedure [29]. Experiment 2 RESULTS Experiment 1 The dose-response effects of IGFBP-2 on IGF-I- and LH-induced thecal cell steroidogenesis and cell numbers are shown in Figure 1. In the absence of IGFBP-2, 30 ng/ml of IGF-I increased (p < 0.001) thecal cell numbers by 1.4fold, and LH induced progesterone and androstenedione production by 1.7- and 3.9-fold, respectively (Fig. 1). In the absence of IGF-I, 200 and 400 ng/ml of IGFBP-2 had no effect (p > 0.10) on cell numbers or on LH-induced progesterone and androstenedione production. However, 200 and 400 ng/ml of IGFBP-2 decreased (p < 0.05) the IGF-I-induced increase in androstenedione production by 30% and 18%, respectively. In contrast, 200 and 400 ng/ml of IGFBP-2 had no effect on IGF-I-induced thecal cell proliferation or progesterone production (Fig. 1). The dose-response effects of IGFBP-3 on IGF-I- and LH-induced thecal cell steroidogenesis and cell numbers are shown in Figure 2. In the absence of IGFBP-3, 30 ng/ml of IGF-I increased (p < 0.01) thecal cell numbers by 1.3fold and LH-induced progesterone and androstenedione production by 1.7- and 3.2-fold, respectively (Fig. 2). In the absence of IGF-I, IGFBP-3 had no effect (p > 0.10) on cell numbers or on LH-induced progesterone or androstenedione production. In contrast, 200 and 400 ng/ml of IGFBP-3 inhibited (p < 0.05) the IGF-I-induced increase in thecal cell proliferation, progesterone production, and androstenedione production by 71% and 41%, 63% and 66%, and 81% and 51%, respectively (Fig. 2). Experiment 3 To determine whether higher doses of IGF-I could overcome the inhibitory effects of IGFBP-3, we conducted studies summarized in Figure 3. In the absence of IGFBP-3, 30 and 100 ng/ml of IGF-I increased (p < 0.001) thecal cell numbers and LH-induced progesterone and androstenedione production in a dose-dependent manner (Fig. 3). In the absence of IGF-I, 200 ng/ml of IGFBP-3 had no effect (p > 0.10) on cell numbers or on LH-induced progesterone and androstenedione production. However, 200 ng/ml of IGFBP-3 reduced (p < 0.05) cell numbers (by 56%) and Downloaded from https://academic.oup.com/biolreprod/article-abstract/56/6/1458/2760761 by guest on 01 June 2020 O oe 200 400 IQFBP-2, ng/mL THECAL CELLS AND INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS I ,o .11 L M IH RB- I [A - 2.5A Y c co v .0r, _ 1.5 i 1.0 m 0.5 150 90 60 0° o 0 - 80 o 30 0 100 .r 0 IQF-I, ngl/ml 2 10 o 800 C40 600 O u>O 0La 400 O >- 200 - a 4) 4 20 b b b i LO 0_ 0 c C Z:~t . c 4.O 4)" - ' aP o 0 1000 oo 100 30 IQF-I, ng/mL 60.. L 800 c 600 400 - 200 0 0 30 100 OF-I, nglmL FIG. 3. Effects of IGFBP-3 (BP-3) on IGF-I-stimulated thecal cell proliferation (A), progesterone (B), and androstenedione (C) production by thecal cells in the presence of LH (experiment 3). Thecal cells were cultured for 2 days in the presence of 10% FCS and then treated in serum-free media with 100 ng/ml of LH, IGF-I (0 or 30 ng/ml), and IGFBP-3 (0 or 200) for an additional 2 days. Values are means from four separate replicate experiments. Within a panel, means without a common superscript differ (p < 0.05). androstenedione production (by 55%) stimulated by 30 ng/ml of IGF-I and 100 ng/ml of LH. In the presence of 100 ng/ml of IGF-I and LH, however, IGFBP-3 had no effect (p > 0.10) on cell numbers or androstenedione production (Fig. 3). Basal and IGF-I-induced progesterone production was not significantly affected by IGFBP-3 (Fig. 3B), although IGF-I-induced progesterone was reduced by 31% by IGFBP-3; IGFBP-3 significantly inhibited IGF-Iinduced progesterone in two of the four experiments summarized in Figure 3B (data not shown). Experiment 5 To determine whether IGFBP-3 could influence the stimulatory effects of low doses of LH on progesterone and androstenedione production, we conducted studies summarized in Figure 5. LH caused a dose-dependent increase 10 FIG. 4. Effects of IGFBP-2 on IGF--stimulated progesterone (A) and androstenedione (B) production by thecal cells (experiment 4). Thecal cells were cultured for 2 days in the presence of 10% FCS and then treated in serum-free media with 100 ng/ml of insulin, IGFBP-2 (0 or 200 ng/ml), and LH (0, 3, or 10 ng/ml) for an additional 2 days. Values are means from three separate replicate experiments. Within a panel, means without a common superscript differ (p < 0.05). (p < 0.05) in both progesterone and androstenedione production by thecal cells (Figure 5). However, IGFBP-3 had no effect (p > 0.10) on basal or LH-induced progesterone or androstenedione production (Figure 5). Experiment 6 To determine whether IGFBP-2 or IGFBP-3 inhibits binding of [125I]IGF-I or [ 25 I]IGF-II to their receptors in I e co -IGFBP-3 +IGCFBP-3 150 120 90 cL. 00 O° L - c Experiment 4 To determine whether IGFBP-2 could influence the stimulatory effects of low doses of LH on progesterone and androstenedione production, we conducted studies summarized in Figure 4. Both 3 and 10 ng/ml of LH increased (p < 0.01) progesterone and androstenedione production by thecal cells (Fig. 4). However, IGFBP-2 had no effect (p > 0.10) on progesterone or androstenedione production induced by LH (Fig. 4). 3 LH, ng/mL o d rC a 60 30 0 0 1 0 1 3 10 30 LH, ng/mL 100 1000 800 4o C = 600 0 C0 O 400 200 0 3 10 30 100 LH, ng/mL FIG. 5. Effects of IGFBP-3 on LH-stimulated progesterone (A) and androstenedione (B) production by thecal cells (experiment 5). Thecal cells were cultured for 2 days in the presence of 10% FCS and then treated in serum-free media with 100 ng/ml of insulin, IGFBP-3 (0 or 200 ng/ml), and LH (0, 1, 3, 10, 30, or 100 ng/ml) for an additional 2 days. Values are means from three separate replicate experiments. Within a panel, means with a common superscript differ (p < 0.05). Downloaded from https://academic.oup.com/biolreprod/article-abstract/56/6/1458/2760761 by guest on 01 June 2020 a w. B 1000 c } h 40 0o 3 LH, ng/mL c 0 6 orB 0o [ a *IGFBP-2 -IGFBP-2 120 0, o.o C cc 1461 1462 SPICER ET AL. '251-11F- I M c 120 100 80 60 40 20 0 C m ~0 o I-_ M on subsequent specific binding of [ 25I]IGF-I thecal cells; specific binding of [12 5I]IGF-I to thecal cells averaged 5330 109 cpm/105 cells for control and ± 103 and 5598 IGFBP-3-treated cultures, respectively (n = 5 separate experiments). "' I-IGF-11 b Experiment 7 b Av C c: 25 5 FIG. 6. Effects of IGFBP-2 and -3 on [' 111GF-I and ['2111GF-II binding to thecal cells (experiment 6). Thecal cells were cultured for 3 days in the presence of 10% FCS, washed with serum-free media, and then treated with 0 or 200 ng/ml of either IGFBP-2, IGFBP-3, IGF-I, or IGF-II during the binding assay. Values are means of three separate replicate experiments and expressed as a percentage of buffer control (0 ng/ml of IGFBP-2 or -3) incubations. a,b,,,d For each respective tracer, means without a comn\ in li(ffr -nn clnrc.rnt -UIIIJCI"I ...II, I -- UJ. es summarized in Figure 6. thecal cells, we conducted studied As depicted in Figure 6, 20C ng/ml of IGFBP-2 and IGFBP-3 inhibited (p < 0.05) [ 1251]IGF-I and [ 25 I]IGF-II binding to thecal cells; IGFBP- 3 inhibited [ 25 1]IGF-I and -II binding to a greater degree than did IGFBP-2. In cornparison, 200 ng/ml of IGF-I inlhibited (p < 0.05) specific [1 25I]IGF-I binding to thecal ce lls, whereas 200 ng/ml of IGF-I had a weak inhibitory efeffect on thecal [12 51]IGF-II binding. An additional set of experimients was conducted to determine whether IGFBP-3 treatnnent, rather than competing for IGF-I binding, could alter t]he number of thecal IGF-I receptors and thereby reduce the effectiveness of IGF-I. Thecal cells were cultured for 2 days in 10% FCS and then cultured for an additional 2 daays in serum-free medium containing 10 ng/ml of insulin in the presence or absence of 50 ng/ml of IGFBP-3. Cells were washed and the IGF-I binding assay was conducted. Exposure of thecal cells to 50 ng/ml of IGFBP-3 for 2 day s had no effect (p > 0.05) - rh-IGF-I Pg °-- IcGFBP-2 .. ,' IGFBP-3 ng ng 100 80 aI 60 I- Us 40 20 0 0.1 , , -,I , 1 , 10 100 1000 pg or njg per tube FIG. 7. Effects of IGFBP-2 and -3 on [['25111GF-I binding to the IGF-I antiserum in an IGF-I RIA. IGFBP-2 or IC FBP-3 was added to the assay at 0.5, 1.25, 5.0, and 10.0 ng/tube; recomibinant human IGF-I was added at pg/tube. Values are means of ' 7.8, 15.6, 31.25, 62.5, 125, 250, and 500 duplicate determinations from a representative assay and expressed as a percentage of total binding (0 pg/tube (of IGF-I). DISCUSSION Results of the present study revealed that 1) IGFBP-2 weakly inhibited IGF-I-induced androstenedione production by thecal cells but had no effect on IGF-I-induced thecal cell proliferation or progesterone production; 2) IGFBP-3 (200 ng/ml) inhibited thecal cell numbers and androstenedione production induced by 30 ng/ml of IGF-I, while addition of 100 ng/ml of IGF-I reversed this inhibitory effect; 3) IGFBP-2 and IGFBP-3 had no effect on LH-induced progesterone and androstenedione production by thecal cells in the absence of IGF-I; and 4) IGFBP-3 was a more potent inhibitor of [ 125I]IGF-I and-II binding to thecal cells and to IGF-I antiserum than was IGFBP-2, but IGFBP-3 had no effect on numbers of IGF-I receptors. For the first time, the actions of IGFBP-2 and -3 on thecal cell steroidogenesis have been evaluated. Previous studies using cultured rat Leydig cells showed that recombinant human IGFBP-2 inhibited IGF-I-induced testosterone production but not hCG-induced testosterone production [23]. We observed that IGFBP-2 weakly inhibited IGFI-induced androstenedione production by bovine thecal cells, whereas IGFBP-2 had no effect on LH-induced androstenedione production in the absence of IGF-I. In addition, we observed that IGFBP-2 had no effect on LHand IGF-I-induced progesterone production. Why IGFBP-2 had a significant effect on IGF-I-induced androstenedione production but not progesterone production is unclear, but a likely reason is that thecal cell androstenedione production is more sensitive to low doses of IGF-I than is progesterone production [18]. As with IGFBP-2, we found that IGFBP-3 had no effect on LH-induced androstenedione or progesterone production by bovine thecal cells. In contrast to findings for IGFBP-2, we observed that IGFBP-3 reduced the stimulatory effect of IGF-I on progesterone and androstenedione production and that these inhibitory effects of IGFBP-3 were overcome with higher doses of IGF-I. Previous studies have shown that purified porcine IGFBP-2 and IGFBP-3 and purified rat IGFBP-4 and IGFBP-5 can inhibit, whereas recombinant human IGFBP-6 has no effect on, FSH-induced estradiol and progesterone production by cultured rat granulosa cells [10, 15, 16, 30]. In cultured human granulosa cells, purified human IGFBP-1 and recombinant human IGFBP-3 inhibited FSH-induced estradiol and progesterone production and IGF-I-induced amino acid uptake but had no effect on FSH-induced progesterone production [31-33]. In contrast, purified porcine IGFBP-3 in- Downloaded from https://academic.oup.com/biolreprod/article-abstract/56/6/1458/2760761 by guest on 01 June 2020 BP-2 BP-3 IGF-I IGF-11 Treatment 0 To determine whether the potency of IGFBP-2 or IGFBP-3 differed in terms of their ability to inhibit binding of [ 25 1]IGF-I to the IGF-I antiserum in an IGF-I RIA, studies were conducted as summarized in Figure 7. IGFBP-3 at 5 and 10 ng/tube nearly completely blocked all binding of [125 I]IGF-I to the IGF-I antiserum (Fig. 7). In contrast, IGFBP-2 only weakly competed for [ 125 I]IGF-I binding; at 10 ng/tube, IGFBP-2 reduced binding to 60% of total binding. As estimated from linearized log-logit plots, IGFBP-3 and IGFBP-2 cross-reacted in the IGF-I assay at 2.9% and 0.2%, respectively. THECAL CELLS AND INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS ulosa and thecal cells in cattle both contain IGF-I mRNA [3, 49], and IGF-II mRNA exists in thecal cells of sheep [14] and rats [50], there has been no previous evidence for bovine thecal or granulosa cell IGF-II production. We find that bovine thecal cells produce - 1.5 ng of IGF-II/10 5 cells per 24 h and - 6 ng of IGF-I/10 5 cells per 24 h ([49]; unpublished results). Thus, IGFBP-2, with a greater affinity to bind IGF-II than IGF-I [3, 11], may be inactivated by IGF-II produced within the theca interna, allowing for the effects of locally produced or systemic IGF-I to be manifested. Alternatively, IGFBP-2 may be selectively degraded by bovine thecal cells, since porcine granulosa cells can degrade IGFBP-3 [51] and rat granulosa cells can degrade IGFBP-5 [52, 53]. Further research will be required to verify this suggestion. Moreover, the hormonal milieu within the follicle may alter the biological effects of the IGFBPs, since IGF-I stimulates the production of both IGFBP-2 and IGFBP-3 [54] and attenuates IGFBP-3 degradation [51] in porcine granulosa cells. A recent study has shown that IGF-I but not LH increases IGFBP-2 and -4 production by rat thecal cells in vitro [55]. In cultured bovine dermal fibroblasts, IGFBP-3 can prevent IGF-I-induced receptor down-regulation, a process that renders cells refractory to further stimulation by IGF-I [56]. Thus, numerous factors may be involved in determining the cellular response to IGFBPs and IGF-I. Whether IGFBP-2 and IGFBP-3 are acting as endocrine, paracrine, or autocrine regulators of ovarian follicular function in cattle is uncertain. IGFBP-2 and IGFBP-3 are the predominant IGFBPs found in bovine [12, 13] and ovine [14, 35] follicular fluid. Moreover, IGFBP-2 levels are greater in small follicles than in peripheral blood, and they decrease as follicles enlarge and become estrogen active in sheep [14], cattle [12, 13], and humans [38, 39, 57]. Evidence suggests that IGFBP-2 is produced by porcine granulosa and thecal cells [36, 54, 58, 59] and human granulosa cells [60] but not rat granulosa cells [15, 61]. Also, IGFBP-3 appears to be produced by rat thecal cells but not rat granulosa cells [15, 61-63] or granulosa or thecal cells of preovulatory porcine follicles [59]. On the basis of ligand blotting of concentrated spent culture medium, we find that there is no detectable IGFBP-3 or IGFBP-2 produced by culture bovine thecal cells (unpublished results). Furthermore, using a charcoal exchange assay [27], IGF binding protein activity is not detectable in spent thecal cell culture medium (unpublished results). Collectively, these results indicate that IGFBP-2 and -3 may be produced by ovarian follicular cells of some species and thus may act as autocrine or paracrine regulators of ovarian follicular steroidogenesis during follicular growth and atresia. However, based on results of the present study, it appears that IGFBP-2 and -3 may be acting as primarily endocrine regulators of ovarian follicular function in cattle. Regardless of the source of IGFBPs, imbalances between IGFs and IGFBPs, such as those that occur during severe feed restriction [64], may be involved in the pathology of reproductive inefficiency and therefore warrant further study. ACKNOWLEDGMENTS The authors thank the National Hormone and Pituitary Program (University of Maryland School of Medicine, Baltimore) for supplying bovine LH and IGF-I antiserum (UB3-189), Wellington Quality Meats (Wellington, KS) for their generous donations of bovine ovaries, and Paula Cinnamon for her excellent secretarial assistance. Downloaded from https://academic.oup.com/biolreprod/article-abstract/56/6/1458/2760761 by guest on 01 June 2020 creased hCG-induced progesterone accumulation by intact rat follicles cultured for 24 h [34]; IGFBP-3 significantly decreased hCG-induced estrogen production and attenuated the inhibitory effect of hCG and IGF-I on follicular cell apoptosis in this same study. Collectively, it seems that regardless of the species from which they are derived, IGFBP-1 to -5 consistently inhibit FSH-induced estradiol production by granulosa cells of rats and humans, whereas the effects of IGFBPs on LH/hCG-induced granulosa-cell steroidogenesis are inconsistent. Our studies further indicate that IGFBPs may have no effect on gonadotropin-induced steroidogenesis by thecal cells. In vivo, follicular fluid levels of IGFBP-2 and IGFBP-3 are negatively correlated with estradiol and androstenedione in cattle [12, 13]. Also, growing dominant follicles have lower IGFBP-2 than nongrowing static dominant follicles [13]. Moreover, levels of IGFBP-2 and other lower molecular weight IGFBPs in follicular fluid increase as follicles become atretic and lose their ability of produce estradiol, whereas IGFBP-3 levels do not change in bovine [12], ovine [14, 35], porcine [36], and human [37, 38] follicles. In contrast, others have reported that IGFBP-3 activity decreases during human follicle development [39]. Recently, IGFBP-3 has been shown to inhibit the ability of FSH to suppress apoptosis in cultured rat follicles [40]. Thus, in vitro and in vivo data suggest that the presence of IGFBP-3 and increased IGFBP-2 in follicles undergoing atresia may hasten the atretic process by inhibiting androstenedione and estradiol production, and that IGF-I at physiological concentrations can attenuate this inhibitory effect. In addition to effects on thecal cell steroidogenesis, IGFBP-3 but not IGFBP-2 inhibited thecal cell proliferation. No previous reports have evaluated the effect of IGFBPs on ovarian cell mitosis or proliferation. Because our steroid production data were corrected for the decrease in cell numbers, it is unlikely the decrease in steroid production was due indirectly to a decrease in cell numbers. Previously, IGFBP-2 and/or IGFBP-3 have been shown to inhibit IGF-I-induced mitosis of cultured fibroblasts [41, 42], rat osteoblasts [43], and estradiol-stimulated breast cancer cells [44]. However, other studies have reported that IGFBP-2 and/or IGFBP-3 can enhance IGF-I-stimulated mitosis of some reproductive and endocrine cells such as fetal rat islets of Langerhans [45], human breast cancer cells [46], and prostate carcinoma cells transfected with androgen receptor cDNA [47]; nontransfected prostate cells were not affected by IGFBP-3 [47]. Collectively, these studies along with those reviewed by Jones and Clemmons [2] suggest that numerous factors may interplay to determine the response of a particular cell type to IGFBP-2 or IGFBP-3. The mechanism by which IGFBP-2 and IGFBP-3 antagonize the stimulatory action of IGF-I on follicular mitogenesis and steroidogenesis in vivo is unclear; however, on the basis of the present and previous [48] in vitro studies, it is reasonable to propose that the mechanism involves the binding of systemic and endogenously produced IGF-I. Results from our studies clearly indicate that IGFBP-3 blocks IGF-I binding to its receptors and competes for [125I]IGF-I binding to IGF-I antiserum in an IGF-I RIA, but 2-day treatment with IGFBP-3 in vitro has no effect on the number of IGF-I receptors in thecal cells. Also, IGFBP-2 blocked IGF-I binding to its receptors to a lesser degree than IGFBP-3 and weakly competed for [12 5I]IGF-I binding to IGF-I antiserum in the IGF-I RIA, suggesting that the affinity of thecal IGF-I receptors for IGF-I is greater than the affinity of IGFBP-2 for binding IGF-I. Although gran- 1463 1464 SPICER ET AL. REFERENCES 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. Downloaded from https://academic.oup.com/biolreprod/article-abstract/56/6/1458/2760761 by guest on 01 June 2020 1. Sara VR, Hall K. Insulin-like growth factors and their binding proteins. Physiol Rev 1990; 70:591-614. 2. Jones JI, Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 1995; 16:3-34. 3. Spicer LJ, Encternkamp SE. The ovarian insulin and insulin-like growth factor system with an emphasis on domestic animals. Domest Anim Endocrinol 1995; 12:223-245. 4. Hammond JM, Mondschein JS, Samaras SE, Canning SE The ovarian insulin-like growth factors, a local amplification mechanism for steroidogenesis and hormone action. J Steroid Biochem Mol Biol 1991; 40:411-418. 5. Giudice LC. Insulin-like growth factors and ovarian follicular development. Endocr Rev 1992; 13:641-669. 6. Rinderknecht E, Humble RE. The amino acid sequence of human insulin-like growth factor-I and its structural homology with proinsulin. J Biol Chem 1978; 253:2769-2776. 7. Rinderknecht E, Humble RE. The primary structure of human insulinlike growth factor-II. FEBS Lett 1978; 89:283-286. 8. Margot JB, Binkert C, Mary JL, Landwehr J, Heinrich G, Schwander J. A low molecular weight insulin-like growth factor binding protein from the rat: cDNA cloning and tissue distribution of its messenger RNA. Mol Endocrinol 1989; 3:1053-1060. 9. Shimasaki S, Koba A, Mercado M, Shimonaka M, Ling N. Complementary DNA structure of a high molecular weight rat insulin-like growth factor binding protein (IGF-BP-3) and tissue distribution of its mRNA. Biochem Biophys Res Commun 1989; 165:907-912. 10. Ui M, Shimonaka M, Shimasaki S, Ling N. An insulin-like growth factor-binding protein in ovarian follicular fluid blocks follicle-stimulating hormone-stimulated steroid production by ovarian granulosa cells. Endocrinology 1989; 125:912-916. 11. Rechler MM. Insulin-like growth factor binding proteins. Vitam Horm 1993; 47:1-114. 12. Echternkamp SE, Howard HJ, Roberts AJ, Grizzle J, Wise T. Relationship among concentrations of steroids, insulin-like growth factor-I, and insulin-like growth factor binding proteins in ovarian follicular fluid of beef cattle. Biol Reprod 1994; 51:971-981. 13. Stewart RE, Spicer LJ, Hamilton TD, Keefer BE, Dawson LJ, Morgan GL, Echternkamp SE. Levels of insulin-like growth factor (IGF) binding proteins, luteinizing hormone and IGF-I receptors, and steroids in dominant follicles during the first follicular wave in cattle exhibiting regular estrous cycles. Endocrinology 1996; 137:2842-2850. 14. Spicer LJ, Echternkamp SE, Wong EA, Hamilton TD, Vernon RK. Serum hormones, follicular fluid steroids, insulin-like growth factors and their binding proteins, and ovarian IGF mRNA in sheep with different ovulation rates. J Anim Sci 1995; 73:1152-1163. 15. Liu X, Malkowski M, Guo Y, Erickson GE Shimasaki S, Ling N. Development of specific antibodies to rat insulin-like growth factorbinding proteins (IGFBP-2 to -6): analysis of IGFBP production by rat granulosa cells. Endocrinology 1993; 132:1176-1183. 16. Bicsak TA, Shimonaka M, Malkowski M, Ling N. Insulin-like growth factor-binding protein (IGF-BP) inhibition of granulosa cell function: effect on cyclic adenosine 3',5'-monophosphate, deoxyribonucleic acid synthesis, and comparison with the effect of an IGF-I antibody. Endocrinology 1990; 126:2184-2189. 17. Langhout DJ, SpicerLJ, Geisert RD. Development of a culture system for bovine granulosa cells: effects of growth hormone, estradiol, and gonadotropins on cell proliferation, steroidogenesis, and protein synthesis. J Anim Sci 1991; 69:3321-3334. 18. Stewart RE, Spicer LJ, Hamilton TD, Keefer BE. Effects of insulinlike growth factor-I and insulin on proliferation, and basal and LH-induced steroidogenesis of bovine thecal cells: involvement of glucose and receptors for IGF-I and LH. J Anim Sci 1995; 73:3719-3731. 19. Roberts AJ, Skinner MK. Hormonal regulation of thecal cell function during antral follicle development in bovine ovaries. Endocrinology 1990; 127:2907-2917. 20. Spicer LJ, Stewart RE. Interaction among bovine somatotropin, insulin, and gonadotropins on steroid production by bovine granulosa and thecal cells. J Dairy Sci 1996; 79:813-821. 21. Spicer LJ, Stewart RE. Interactions among basic fibroblast growth factor, epidermal growth factor, insulin, and insulin-like growth factor-I (IGF-I) on cell numbers and steroidogenesis of bovine thecal cells: role of IGF-I receptors. Biol Reprod 1996; 54:255-263. 22. Raisz LG, Fall PM, Gabbitas BY, McCarthy TL, Kream BE, Canalis E. Effects of prostaglandin E2 on bone formation in cultured fetal rat calvariae: role of insulin-like growth factor-I. Endocrinology 1993; 133:1504-1510. Wang D, Nagpal ML, Lin T, Shimasaki S, Ling N. Insulin-like growth factor-binding protein-2: the effect of human chorionic gonadotropin on its gene regulation and protein secretion and its biological effects in rat Leydig cells. Mol Endocrinol 1994; 8:69-76. Vicini JL, Buonomo FC, Veenhuizen JJ, Miller MA, Clemmons DR, Collier RJ. Nutrient balance and stage of lactation affect responses of insulin, insulin-like growth factors I and II, and insulin-like growth factor-binding protein 2 to somatotropin administration in dairy cows. J Nutr 1991; 121:1656-1664. Baxter RC. Insulin-like growth factor binding proteins in the human circulation: a review. Horm Res 1994; 42:141-144. Spicer LJ, Aplizar A, Vernon RK. Insulin-like growth factor-I receptors in ovarian granulosa cells: effect of follicle size and hormones. Mol Cell Endocrinol 1994; 102:69-76. Echternkamp SE, Spicer LJ, Gregory KE, Canning SE Hammond JM. Concentrations of insulin-like growth factor-I in blood and ovarian follicular fluid of cattle selected for twins. Biol Reprod 1990; 43:814. SAS. SAS/STAT User's Guide. Cary, NC: Statistical Analysis Systems Institute, Inc.; 1988. Ott L. An Introduction of Statistical Methods and Data Analysis. North Scituate, MA: Duxbury Press; 1977: 384. Rohan RM, Ricciarelli E, Kiefer MC, Resnick CE, Adashi EY. Rat ovarian insulin-like growth factor-binding protein-6: a hormonally regulated theca-interstitial-selective species with limited antigonadotropin activity. Endocrinology 1993; 132:2507-2512. Mason HD, Willis D, Holy JMP, Cwyfan-Hughes SC, Seppala M, Frank S. Inhibitory effects of insulin-like growth factor-binding proteins on steroidogenesis by human granulosa cells in culture. Mol Cell Endocrinol 1992; 89:RI-R4. Hillensjo T, Bergh C, Selleksog U. Insulin-like growth factor-I stimulates amino acid accumulation in culture human granulosa cells. Hum Reprod 1992; 7:1094-1097. Iwashita M, Adachi T, Katayama E, Kido Y, Takeda Y. Regulation and physiological role of insulin-like-growth-factor-binding protein-I in human granulosa cells. Horm Res 1994; 41(suppl 1):22-28. Chun S-Y, Billig H, Tilly JL, Furuta I, Tsafriri A, Hsueh AJW. Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor-I. Endocrinology 1994; 135:1845-1953. Monget P, Monniaux D, Pisselet C, Durand P Changes in insulin-like growth factor-I (IGF-I), IGF-II and their binding proteins during growth and atresia of ovine ovarian follicles. Endocrinology 1993; 132:1438-1446. Grimes RW, Guthrie HD, Hammond JM. Insulin-like growth factorbinding protein-2 and -3 are correlated with atresia and preovulatory maturation in the porcine ovary. Endocrinology 1994; 135:19962000. Huang ZH, Matson P Lieberman BA, Morris ID. Insulin-like growth factor binding proteins in serum and follicular fluid from women undergoing ovarian stimulation with and without growth hormone. Human Reprod 1994; 9:1421-1426. Cataldo NA, Giudice LC. Insulin-like growth factor-binding proteins profiles in human ovarian follicular fluid correlate with follicular functional status. J Clin Endocrinol & Metab 1992; 74:821-829. San Roman GA, Magoffin DA. Insulin-like growth factor-binding proteins in healthy and atretic follicles during natural menstrual cycles. J Clin Endocrinol & Metab 1993; 76:625-632. Flaws JA, DeSanti A, Tilly KI, Javid RO, Kugu K, Johnson AL, Hirshfield AN, Tilly JL. Vasoactive intestinal peptide-mediated suppression of apoptosis in the ovary: potential mechanisms of action and evidence of a conserved antiatretogenic role through evolution. Endocrinology 1995; 136:4351-4359. Schwander J, Mary JL, Landwehr J, Boni M, Margot JB, Brinkert C. The regulation of mRNA of an insulin-like growth factor binding protein (IGF-2) in the rat. In: Drop SLS, Hintz RL (eds.), InsulinLike Growth Factor Binding Proteins. Excerpta Medica International Congress Series 881. Amsterdam: Elsevier Science Publishers; 1989: 125-131. DeMellow JSM, Baxter RC. Growth hormone dependent insulin-like growth factor (IGF) binding protein both inhibits and potentiates IGF-I stimulated DNA synthesis in skin fibroblasts. Biochem Biophys Res Commun 1988; 156:199-204. Schmid C, Rutishauser J, Schlapfer I, Froesch ER, Zapf J. Intact but not truncated insulin-like growth factor binding protein-3 (IGFBP-3) THECAL CELLS AND INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS 44. 45. 46. 48. 49. 50. 51. 52. 53. 54. Grimes RW, Hammond JM. Insulin and insulin-like growth factors (IGFs) stimulate production of IGF-binding proteins by ovarian granulosa cells. Endocrinology 1992; 131:553-558. 55. Erickson GF, Li D, Shimasaki S, Ling N, Weitsman SR, Magoffin DA. Insulin-like growth factor-I (IGF-I) stimulates the IGF binding protein system in rat theca interstitial cells. Endocrine 1995; 3:525531. 56. Conover CA, Powell DR. Insulin-like growth factor (IGF)-binding protein-3 blocks IGF-I-induced receptor down-regulation and cell desensitization in cultured bovine fibroblasts. Endocrinology 1991; 129: 710-716. 57. Schuller AG, Lindenbergh-Kortleve DJ, Pache TD, Zwarthoff EC, Fauser BC, Drop SL. Insulin-like growth binding protein-2, 28 kDa and 24 kDa insulin-like growth factor binding protein levels are decreased in fluid of dominant follicles obtained from normal and polycystic ovaries. Regul Pept 1993; 48:157-163. 58. Samaras SE, Guthrie HD, Barber JA, Hammond JM. Expression of the mRNAs for the insulin-like growth factors and their binding proteins during development of porcine ovarian follicles. Endocrinology 1993; 133:2395-2398. 59. Gadsby JE, Lovdal JA, Sarmaras S, Barber JS, Hammond JM. Expression of the messenger ribonucleic acids for insulin-like growth factor-I and insulin-like growth factor binding proteins in porcine corpora lutea. Biol Reprod 1996; 54:339-346. 60. El-Roiey A, Chen X, Roberts VJ, Shimasaki S, Ling N, LeRoith D. Expression of the genes encoding the insulin-like growth factors (IGF-I and II), the IGF and insulin receptors, and IGF-binding proteins-1-6 and the localization of their gene products in normal and polycystic ovary syndrome ovaries. J Clin Endocrinol &Metab 1994; 78:1488-1496. 61. Nakatani A, Shimasaki S, Erickson GE Ling N. Tissue-specific expression of four insulin-like growth factor-binding proteins (1, 2, 3, and 4) in the rat ovary. Endocrinology 1991; 131:553-558. 62. Ricciarelli E, Hernandez ER, Hurwitz A, Kokia E, Rosenfeld RJ, Schwander J. The ovarian expression of the antigonadotropic insulinlike growth factor binding protein-2 is theca-interstitial cell selective: evidence for hormonal regulation. Endocrinology 1991; 129:22662268. 63. Ricciarelli E, Hernandez ER, Tedeschi C, Botero LF, Kokia E, Rohan RM. Rat ovarian insulin-like growth factor binding protein-3: a growth hormone-dependent theca-interstitial cell-derived antigonadotropin. Endocrinology 1992; 130:3092-3094. 64. Clemmons DR, Underwood LE. Nutritional regulation of IGF-I and IGF binding proteins. Annu Rev Nutr 1991; 11:393-412. Downloaded from https://academic.oup.com/biolreprod/article-abstract/56/6/1458/2760761 by guest on 01 June 2020 47. blocks IGF-I-induced simulation of osteoblasts: control of IGF signaling to bone cells by IGFBP-3-specific proteolysis? Biochem Biophys Res Commun 1991; 179:579-585. Huynh H, Yang X, Pollak M. Estradiol and antiestrogens regulate a growth inhibitory insulin-like growth factor binding protein 3 autocrine loop in human breast cancer cells. J Biol Chem 1996; 271:10161021. Hogg J, Han VKM, Clemmons DR, Hill DJ. Interactions of nutrients, insulin-like growth factors (IGFs) and IGF-binding proteins in the regulation of DNA synthesis by isolated fetal rat islets of Langerhans. J Endocrinol 1993; 138:401-412. Chen JC, Shao ZM, Sheikh MS, Hussian A, LeRoith D, Roberts CT Jr. Insulin-like growth factor-binding protein enhancement of insulinlike growth factor-I (IGF-I)-mediated DNA synthesis and IGF-I binding in human breast carcinoma cell line. J Cell Physiol 1994; 158: 69-78. Marcelli M, Haidacher SJ, Plymate SR, Birnbaum RS. Altered growth and insulin-like growth factor-binding protein-3 production in PC3 prostate carcinoma cells stably transfected with a constitutively active androgen receptor complementary deoxyribonucleic acid. Endocrinology 1995; 136:1040-1048. Conover CA, Ronk M, Lombana F, Powell DR. Structural and biological characterization of bovine insulin-like growth factor binding protein-3. Endocrinology 1990; 127:2795-2803. Spicer L, Alpizar E, Echternkamp SE. Effects of insulin, insulin-like growth factor I, and gonadotropins on bovine granulosa cell proliferation, progesterone production, estradiol production, and(or) insulinlike growth factor I production in vitro. J Anim Sci 1993; 71:12321241. Hernandez ER, Roberts CT Jr, LeRoith D, Adashi EY. Rat ovarian insulin-like growth factor-I (IGF-I) gene expression is granulosa cellselective: 5'-untranslated mRNA variant representation and hormonal regulation. Endocrinology 1989; 125:572-574. Grimes RW, Hammond JM. Proteolytic degradation of insulin-like growth factor (IGF)-binding protein-3 by porcine ovarian granulosa cells in culture: regulation by IGF-I. Endocrinology 1994; 134:337343. He H, Herington AC, Roupas P Effects of phorbol ester and staurosporine on the actions of insulin-like growth factor-I on rat ovarian granulosa cells. Endocrine 1995; 3:159-167. Fielder PJ, Pham H, Adashi EY, Rosenfeld RG. Insulin-like growth factors (IGFs) block FSH-induced proteolysis of IGF-binding protein-5 (BP-5) in cultured rat granulosa cells. Endocrinology 1993; 133: 415-418. 1465