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Biology of Reproduction 66, 1640-1648 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Effect of Follicle Size on In Vitro Production of Steroids and Insulin-Like Growth Factor (IGF)-I, IGF-II, and the IGF-Binding Proteins by Equine Ovarian Granulosa Cells1

Tonya R. Davidsona, Connie S. Chamberlaina, Tamara S. Bridgesa, and Leon J. Spicer2,a

a Department of Animal Science, Oklahoma State University, Stillwater, Oklahoma 74078


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Little is known regarding the hormonal regulation of granulosa cell steroidogenesis and the ovarian insulin-like growth factor (IGF) system in the mare. The objectives of this study were to determine, first, if estradiol, insulin, and/or FSH affect steroid production by equine granulosa cells (experiment 1) and, second, if the components of the IGF system are produced by equine granulosa cells in culture as well as whether estradiol, insulin, and/or FSH affects IGF and/or IGF-binding protein (IGFBP) production by equine granulosa cells (experiment 2). Granulosa cells from small (6–15 mm), medium (16–25 mm), and large (25–48 mm) follicles were collected from cyclic mares (n = 14), 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 or without added hormones. In experiment 1, large-follicle granulosa cells produced less progesterone and more estradiol than did medium- and/or small-follicle granulosa cells (P < 0.05). Progesterone production was inhibited (P < 0.05) by FSH and insulin in small- and medium- but not in large-follicle granulosa cells; estradiol was without effect. Insulin increased (P < 0.05) estradiol production in small- and medium-follicle granulosa cells but had no effect in large-follicle granulosa cells. In experiment 2, IGF-I production was inhibited (P < 0.05) by insulin across all follicle sizes but was not affected by estradiol or FSH. Granulosa cells of medium and large follicles produced more IGF-II than did granulosa cells of small follicles (P < 0.05). Insulin and FSH inhibited (P < 0.05) IGF-II production by granulosa cells of large and medium but not of small follicles; estradiol was without effect. Only IGFBP-2 and -5 were produced by equine granulosa cells. Production of IGFBP-2 was less (P < 0.10) in granulosa cells of large versus those of small and medium follicles, whereas medium-follicle granulosa cells produced more (P < 0.05) IGFBP-5 than did small- or large-follicle granulosa cells. Averaged across follicle sizes, estradiol increased (P < 0.05) IGFBP-2 production, FSH increased (P < 0.10) IGFBP-2 and -5 production, and insulin was without effect. These results indicate that IGF-I, IGF-II, IGFBP-2, and IGFBP-5 are produced by equine granulosa cells and that insulin, FSH, and estradiol play a role in the regulation of steroidogenesis and the IGF system of equine granulosa cells.

estradiol, follicle, follicular development, granulosa cells, insulin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The insulin-like growth factor (IGF) system, which is composed of IGF-I, IGF-II, IGF receptors, and IGF-binding proteins (IGFBPs), plays an essential role in ovarian function in several species (for reviews, see [1–4]). In most mammalian species evaluated, IGF-I and insulin stimulate granulosa and thecal cell proliferation and mitogenesis and synergize with gonadotropins to stimulate granulosa and thecal cell steroidogenesis (for reviews, see [1–4]). However, to our knowledge, the effect of IGF-I or insulin on steroidogenesis of equine granulosa cells is unknown.

The IGFs are found in the systemic circulation bound to high-affinity, soluble, carrier proteins (for reviews, see [2, 5, 6]). These binding proteins have a greater affinity for the IGFs compared to the IGF receptors and, therefore, are thought to act as regulators of IGF availability for target cells [5, 6]. The presence of IGFBP mRNA in ovarian tissue was first reported in 1989 [7–9], and since that time, additional IGFBPs (i.e., IGFBP-1 through -6) have been identified in the ovary of several species (for reviews, see [2, 5, 6]). Within the follicle IGFBP-3 levels do not change much during development, yet it is the most predominant IGFBP found in the ovarian follicular fluid of horses [10, 11], pigs [12–15], cattle [16–19], and sheep [20, 21]. In contrast, follicular fluid IGFBP-2, -4, and -5 activity is less in growing (i.e., estrogen active) dominant versus subordinate (i.e., estrogen inactive, atretic) follicles in cattle [16, 18], pigs [15, 22], sheep [20, 21], and horses [10, 11]. These studies suggest that follicular fluid levels of IGFBP-2, -4, and -5 are closely related to the physiological status of follicles. Some of these IGFBPs are produced within the follicle by granulosa cells of some species (for review, see [2, 4]). However, again to our knowledge, which IGFBPs are produced by equine granulosa cells is unknown.

In the mare, one follicle is selected from a cohort of follicles to become dominant. After selection, the dominant follicle continues to grow until ovulation, whereas the remaining cohort, or subordinate, follicles become atretic and regress (for review, see [23]). Limited information is available regarding the physiological mechanism of follicle selection and maturation in the mare. During preovulatory development in the mare, follicular fluid levels of IGF-I increase [10, 24], whereas levels of IGFBP decrease [10, 11]. In cattle and other species, it has been hypothesized that estradiol and the gonadotropins induce changes in the amount of IGF-I and IGFBPs produced by granulosa and thecal cells during follicular development [1–4]. We hypothesize that, in the mare, follicular development may involve steroid- or gonadotropin-induced changes in the intrafollicular IGF system. Specifically, we hypothesize that FSH and insulin will stimulate, whereas estradiol will inhibit, steroid, IGF-I, IGF-II, and IGFBP production by equine granulosa cells and that these effects will be more pronounced in cells from small versus medium or large follicles. Therefore, the specific objectives of these experiments were, first, to compare the effect of estradiol, insulin, and/or FSH on steroid production by equine granulosa cells obtained from small, medium, and large follicles and, second, to determine what components of the IGF system are produced by equine granulosa cells in culture as well as to compare the effect of estradiol, insulin, and/or FSH on IGF and IGFBP production by equine granulosa cells obtained from small, medium, and large follicles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Hormones

The reagents used were as follows: Dulbecco modified Eagle medium (DMEM), Ham F-12, sodium bicarbonate, gentamicin, insulin (bovine; 28.5 U/mg), trypan blue, fetal calf serum (FCS), estradiol, and acrylamide, all of which were obtained from Sigma Chemical Company (St. Louis, MO.); ovine FSH (F1913; FSH activity, 15x NIH-FSH-S1 U/mg), obtained from Scripps Laboratories (San Diego, CA); nitrocellulose transfer membrane (pore size, 0.45 µm), obtained from Midwest Scientific (St. Louis, MO); protease-free BSA, obtained from Integrin (Purchase, NY); 20% (w/v) SDS, obtained from Amresco (Solon, OH); and recombinant bovine IGF-II, obtained from Monsanto Co. (St. Louis, MO).

Cell Culture

In late May 1998, 28 ovaries were obtained at a commercial abattoir from 14 mares of various breeds and ages and were processed as described previously [10]. Briefly, follicular fluid was aspirated from individual follicles, and without removing the needle from the follicle, follicular fluid was injected back into the follicle and then aspirated out again while the follicle was hand-massaged to loosen granulosa cells. This was repeated 2 times per follicle. Follicles were separated into 3 groups based on surface diameter: small (6–15 mm), medium (16–25 mm), and large (25–48 mm) [10, 25]. Granulosa cells from individual follicles were separated from follicular fluid by centrifugation (200 x g for 5–7 min), combined to form 3 pools for each of the 3 size categories, washed twice in serum-free medium by centrifugation (200 x g for 5–7 min), and resuspended in medium containing 1.25 mg/ml of collagenase and 0.05 mg/ml of DNase to disperse and to prevent clumping of the cells. Each of the three pools of granulosa cells within each size category was derived from pooling granulosa cells of individual follicles from 3 to 5 mares. The percentage of viable granulosa cells was determined using a hemocytometer and the trypan blue-exclusion method and averaged 74.9% ± 8.9%, 79.2% ± 5.5%, and 79.9% ± 11.0% from small, medium, and large follicles, respectively.

Medium consisted of a 1:1 (v/v) mixture of DMEM and Ham F-12 containing 0.12 mM gentamicin, 2.0 mM glutamine, and 38.5 mM sodium bicarbonate [26, 27]. For each pool of cells, 4 x 105 viable cells in 100 µl of medium were added to each well of Falcon 24-well plates (no. 3047; Becton Dickinson and Co., Lincoln Park, NJ) containing 1 ml of medium with 10% FCS. Cultures were kept at 38.5°C in a 95% air:5% CO2 atmosphere [26, 27]. A total of 18 wells for each of the nine pools of cells were plated. To obtain optimal attachment, cells were maintained in 10% FCS without added hormones for the first 2 days of culture. After 48 h, cells were washed twice with 0.5 ml of serum-free medium to remove FCS and nonadherent cells, and incubations continued in serum-free medium (0.5 ml) containing 500 ng/ml of testosterone (as an estradiol precursor) and 0.25% BSA (to minimize loss of the IGFBPs) [28] with or without added hormones for an additional 48 h. Throughout the 4-day culture, medium was changed every 24 h.

Experiment 1 was designed to determine whether estradiol, insulin, and/or FSH affected steroid production by equine granulosa cells. Granulosa cells from small (6–15 mm), medium (16–25 mm), and large (25–48 mm) follicles were cultured in medium containing 10% FCS for 48 h as described earlier. After 48 h, the media was replaced with serum-free medium containing testosterone and either no hormone (control), estradiol (500 ng/ml), insulin (100 ng/ml), FSH (50 ng/ml), insulin (100 ng/ml) plus estradiol (500 ng/ml), or insulin (100 ng/ml) plus FSH (50 ng/ml) were added and then incubated for an additional 48 h. Each treatment was applied to 3 replicate wells for each of the 9 pools of cells. At the end of the first 24-h incubation with treatments (i.e., during Days 2–3 of culture), medium was collected for estradiol and progesterone measurement, and fresh medium was added containing the same treatments used to evaluate their effects on the IGF system in experiment 2.

Experiment 2 was designed to determine which components of the IGF system are produced by equine granulosa cells in culture as well as to determine whether estradiol, insulin, and/or FSH affected IGF and/or IGFBP production by granulosa cells. Granulosa cells from small, medium, and large follicles were cultured as described in experiment 1. Treatments were no additions (control), estradiol (500 ng/ml), insulin (100 ng/ml), FSH (50 ng/ml), insulin (100 ng/ml) plus estradiol (500 ng/ml), or insulin (100 ng/ml) plus FSH (50 ng/ml). At the end of the second 24-h incubation with treatments (i.e., during Days 3–4 of culture), medium was collected for IGF and IGFBP assessment, and cell numbers were determined. For both experiments, medium from each well was collected individually and stored at -20°C until analysis.

Determination of Cell Numbers

Numbers of granulosa cells were determined at the termination of experiments (i.e., Day 4 of culture) using a Coulter counter (Model Zm; Coulter Electronics, Hialeah, FL) as previously described [27, 29]. Briefly, cells were exposed to 0.5 ml of trypsin (0.25% [w/v] in 0.15 M NaCl) for 20 min at 25°C, then scraped from each well, diluted in 0.15 M NaCl, and enumerated.

Concentration of Spent Medium

Media samples collected for IGF and IGFBP assessment were ultrafiltrated using Centricon concentrators with a molecular weight limit of 3000 (Amicon, Inc., Beverly, MA). The spent medium was concentrated 5- to 18-fold. Briefly, 400 µl of the spent media were placed inside the sample reservoir of the concentrator and then centrifuged at 5322 x g for approximately 80 min. After centrifugation, the filtrate vial was discarded, and the sample reservoir was inverted and recentrifuged for 5 min at 591 x g to transfer the retentate into the retentate vial. Final volumes ranging from 22 to 77 µl were measured to record the concentration factor of the sample; this value was used to correct IGF and IGFBP data (see Statistical Analysis). As shown in Figure 1, addition of 0.25% BSA to culture medium during a 24-h incubation was required for quantitative recovery of IGFBP from culture medium incubated in culture wells and concentrated using the Centricon concentrators.



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  		FIG. 1. Effect of BSA on recovery of IGFBP-3 after 24 h of culture in serum-free medium without cells. Additions of 0, 25, and 100 ng/ml of IGFBP-3 to medium with (---) or without (—) 0.25% BSA were compared. Medium was collected and concentrated using Centricon ultrafiltration devices (see Materials and Methods).

Radioimmunoassays

Progesterone RIA Concentrations of progesterone in culture medium collected 24 h after hormone treatments were determined with a double-antibody RIA as previously described [27, 29]. Assay sensitivity (i.e., 95% of total binding) was 0.016 ± 0.007 ng/tube, and intra- and interassay coefficients of variation were 12.9% and 17.5%, respectively.

Estradiol RIA Concentrations of estradiol-17ß in culture medium collected 24 h after hormone treatments were determined with a double-antibody RIA as previously described [26, 30]. Assay sensitivity (i.e., 90% of total binding) was 0.29 ± 0.05 pg/tube, and the intra- and interassay coefficients of variation were 12.5% and 10.4%, respectively.

IGF-I RIA Concentrations of IGF-I in concentrated culture medium collected 48 h after hormone treatments were determined with a double-antibody RIA after acid-ethanol extraction (16 h at 4°C) as previously described [24, 31]. Assay sensitivity (i.e., 95% of total binding) was 4.89 ± 0.72 pg/tube, and the intra- and interassay coefficients of variation were 14.5% and 18.8%, respectively.

IGF-II RIA Concentrations of IGF-II in concentrated culture medium collected 48 h after hormone treatments were determined with a double-antibody RIA as previously described for bovine and ovine follicular fluid [18, 21] and validated for equine samples. Increasing volumes of extracted equine follicular fluid as well as concentrated spent culture medium from equine granulosa cell cultures created displacement curves parallel to the standard curve. Assay sensitivity (i.e., 95% of total binding) was 22.98 ± 9.40 pg/tube, and intraassay coefficient of variation was 9.17%.

Gel Electrophoresis of IGFBPs

The IGFBPs in concentrated culture medium collected 48 h after hormone treatment were analyzed by one-dimensional, nonreducing SDS-PAGE based on molecular weight as previously described [10, 16, 32]. Briefly, 12.5 µl of concentrated culture medium were mixed with 12.5 µl of Laemmli sample buffer (Bio-Rad, Hercules, CA), heat-denatured (3 min at 100°C), and then electrophoresed on a 12% polyacrylamide gel at a constant current (25–35 A) and varying voltage. After separation, proteins in gels were electrophoretically transferred to nitrocellulose paper (Midwest Scientific, St. Louis, MO) and subsequently ligand-blotted (16 h at 4°C) using recombinant bovine [125I]IGF-II. The next day, the nitrocellulose blots were washed, dried, and exposed to x-ray film at -80°C for 4 days. Individual band intensity on autoradiographs was densitometrically analyzed using a Molecular Analyst (Bio-Rad).

Statistical Analysis

Data were analyzed using PROC MIXED of the Statistical Analysis System [33]. For experiments 1 and 2, interaction of size by treatment was assessed. In the absence of interaction, orthogonal contrasts to assess the main effects of estradiol, FSH, and insulin were utilized. In the presence of interaction, contrasts were formed to evaluate the simple effects of estradiol, FSH, and insulin on each follicle size. Multiple mean comparisons (Fisher protected least significant difference mean test) [34] were performed only if a main effect was significant. For experiment 2, correction factors were utilized to standardize the volume of concentrated media. For both experiments 1 and 2, cell numbers assessed on Day 4 of culture were used to correct data on a per 105-cell basis. Data are presented as means ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Effect of Insulin, FSH, and Estradiol on Granulosa Cell Steroid Production

Basal levels of progesterone production were lower (P < 0.05) in large- versus small- and medium-follicle granulosa cells (Fig. 2). Progesterone production was inhibited (P < 0.05) by insulin, FSH, and a combination of insulin plus FSH in cultures of small- and medium- but not of large-follicle granulosa cells (treatment x size interaction, P < 0.001). Estradiol alone had no effect (P > 0.10) on progesterone production in medium- and large-follicle granulosa cells but inhibited (P < 0.05) progesterone production in small-follicle granulosa cells. Progesterone production in the presence of insulin plus FSH did not differ (P > 0.10) from that in the presence of either insulin alone or FSH alone (Fig. 2). When estradiol was combined with insulin, progesterone production was inhibited (P < 0.05) in cultures of small- and medium- but not of large-follicle granulosa cells; this inhibition was similar to that seen with insulin alone (Fig. 2).



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  		FIG. 2. Effects of insulin (100 ng/ml), estradiol (500 ng/ml), and/or FSH (50 ng/ml) on progesterone production by granulosa cells collected from small (6–15 mm), medium (16–25 mm), and large (25–48 mm) follicles of mares. a,b,cWithin a follicle size, means (n = 9) without a common superscript differ (P < 0.05).

Basal levels of estradiol production were greater (P < 0.05) in cultures of medium- and large- than of small-follicle granulosa cells (Fig. 3). Insulin stimulated (P < 0.05) estradiol production by small- and medium-follicle granulosa cells but was without effect (P > 0.10) in large-follicle granulosa cells (treatment x size interaction, P < 0.01). Treatment with FSH alone had no effect (P > 0.10) on estradiol production by small- and large-follicle granulosa cells but inhibited (P < 0.05) estradiol production by 90% in medium-follicle granulosa cells. FSH also blocked (P < 0.05) the insulin-induced increase in estradiol production by medium follicles. In small follicles, the combined treatment of insulin and FSH-stimulated (P < 0.05) estradiol production fivefold compared to controls and eightfold compared to FSH-treated cultures; estradiol production by granulosa cells of large follicles was not affected (P > 0.10) by the insulin and FSH combination (Fig. 3).



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  		FIG. 3. Effects of insulin (100 ng/ml) and/or FSH (50 ng/ml) on estradiol production by granulosa cells collected from small (6–15 mm), medium (16–25 mm), and large (25–48 mm) follicles of mares. a,b,cWithin a follicle size, means (n = 9) without a common superscript differ (P < 0.05).

Experiment 2: Effect of Insulin, FSH, and Estradiol on Granulosa Cell IGF and IGFBP Production

Equine granulosa cells produced 10- to 50-fold more IGF-II (Fig. 4) than IGF-I (Table 1) in culture. When averaged across follicle size, insulin inhibited (P < 0.05) IGF-I production from 24% to 36%, but IGF-I production was not affected (P > 0.10) by estradiol or FSH (Table 1). Medium-follicle granulosa cells produced more (P < 0.01) IGF-I than did small- or large-follicle granulosa cells (0.36 ± 0.02 vs. 0.28 or 0.29 ± 0.03 ng/105 cells/24 h, respectively). In comparison, singular treatments of insulin or FSH inhibited (P < 0.05) IGF-II production by medium- and large- but not by small-follicle granulosa cells (treatment x size interaction, P < 0.01). Combined treatments of insulin plus FSH also inhibited (P < 0.05) IGF-II production by medium- and large- but not by small-follicle granulosa cells; this inhibition by combined treatments did not differ (P > 0.10) from that of either singular treatment (Fig. 4). Estradiol alone inhibited (P < 0.05) IGF-II production by large-follicle granulosa cells but had no effect (P > 0.05) on small- or medium-follicle granulosa cells. Medium- and large-follicle granulosa cells produced more (P < 0.05) basal IGF-II than did small-follicle granulosa cells (Fig. 4).



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  		FIG. 4. Effects of insulin (100 ng/ml), estradiol (500 ng/ml), and/or FSH (50 ng/ml) on IGF-II production by granulosa cells collected from small (6–15 mm), medium (16–25 mm), and large (25–48 mm) follicles of mares. a,bWithin a follicle size, means (n = 9) without a common superscript differ (P < 0.05).


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  		TABLE 1. Effect of estradiol, insulin, and FSH on equine granulosa cell production of IGF-I, IGFBP-2, and IGFBP-5

Equine granulosa cells produced two forms of IGFBP: a 32- to 36-kDa IGFBP (IGFBP-2), and a 29- to 31-kDa IGFBP (IGFBP-5) (Figs. 5A, 6A, and 7A). Size of follicle influenced (P < 0.05) production of IGFBP-5 such that medium-follicle (0.46 ± 0.03 arbitrary densitometric units [ADU]/105 cells/24 h) granulosa cells produced 2.1- to 2.4-fold more IGFBP-5 than did small-follicle (0.22 ± 0.02 ADU/105 cells/24 h) or large-follicle (0.19 ± 0.04 ADU/105 cells/24 h) granulosa cells. Production of IGFBP-2 by granulosa cells of large follicles (1.68 ± 0.65 ADU/105 cells/24 h) tended (P < 0.10) to be from 35% to 59% less than that of small (2.60 ± 09.53 ADU/105 cells/24 h) and medium (4.10 ± 0.53 ADU/105 cells/24 h) follicles. Treatment affected both IGFBP-2 (P < 0.01) and IGFBP-5 (P < 0.10) production (Table 1). When averaged across follicle size, estradiol increased (P < 0.05) only IGFBP-2 production, whereas FSH increased (P < 0.10) both IGFBP-2 and IGFBP-5 production (Table 1). Insulin alone was without effect (P > 0.10) on IGFBPs-2 and -5 production but blocked (P < 0.10) the stimulatory effect of estradiol on production of IGFBP-2 and the stimulatory effect of FSH on production of IGFBP-2 and -5 (Table 1).



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  		FIG. 5. A) Representative ligand blot of IGFBP production by equine granulosa cells from small follicles. Lanes 1 and 2: control; lanes 3 and 4: estradiol; lanes 5 and 6: insulin; lane 7: estradiol plus insulin; lane 8: FSH; lane 9: FSH plus insulin. B) Representative example of control cultures of equine granulosa cells from small follicles under phase-contrast microscopy. C) Representative example of insulin-treated cultures of equine granulosa cells from small follicles under phase-contrast microscopy. Magnification x200 (B and C).

Representative examples of cultured equine granulosa cells from small, medium, and large follicles are depicted in Figures 5, B and C, 6, B and C, and 7, B and C. For each follicle size, granulosa cells maintained a fibroblastic appearance during culture and were nearly confluent at the end of culture. Insulin stimulated (P < 0.05) granulosa cell numbers in cultures from small, medium, and large follicles (Fig. 8). Estradiol and FSH treatments alone increased (P < 0.05) granulosa cell numbers in cultures from small and medium follicles. Estradiol increased (P < 0.05), whereas FSH had no effect on, cell numbers in cultures from large follicles (Fig. 8). When estradiol or FSH was combined with insulin, granulosa cell numbers increased (P < 0.05), but these increases were similar to those seen with insulin alone (Fig. 8).



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  		FIG. 8. Effects of insulin (100 ng/ml), estradiol (500 ng/ml), and/or FSH (50 ng/ml) on granulosa cell numbers from small (6–15 mm), medium (16–25 mm), and large (25–48 mm) follicles of mares. a,b,cWithin a follicle size, means (n = 9) without a common superscript differ (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results of the present study revealed that 1) of the IGFBPs found in follicular fluid, only IGFBP-2 and -5 were produced by equine granulosa cells; 2) FSH and/or estradiol increased IGFBP-2 and -5 production, had no effect on IGF-I production, and decreased IGF-II production by equine granulosa cells; and 3) estradiol and progesterone production by equine granulosa cells were differentially regulated by insulin and FSH, and these effects were dependent on follicle size.

For the first time, to our knowledge, IGFBP production by equine granulosa cells has been evaluated. Of the IGFBPs detected in equine follicular fluid (i.e., IGFBP-2, -3, -4, and -5) [10, 11], only IGFBP-2 and -5 were produced by equine granulosa cells, and both were responsive to hormone treatment. Previous research suggests that granulosa cell production of IGFBPs is species specific. For example, granulosa cells from mice [35] and cattle [28] produce IGFBP-2, -4, and -5, whereas porcine granulosa cells produce IGFBP-2, -4, and -5 as well as IGFBP-3 [22]. In the present study, IGFBP-5 production was twofold greater in medium- than in large- or small-follicle granulosa cells, whereas IGFBP-2 production tended to be from 35% to 59% less in large- versus small- and medium-follicle granulosa cells. In this same study, IGFBP-2, -4, and -5 levels were less in large versus small and medium follicles [10]. The present study also revealed that FSH treatment increased both IGFBP-2 and -5 production, whereas estradiol treatment increased only IGFBP-2 production, by equine granulosa cells. In ovine granulosa cells, FSH and IGF-I are needed for maximal IGFBP-2 production, but size of follicle did not affect IGFBP-2 production [36]. These results are in contrast with those of studies in pigs [37, 38] that found FSH to be one of the most potent inhibitors of IGFBP-2. Similarly, FSH has been observed to inhibit granulosa cell IGFBP-2 production in rats [39] and IGFBP-2 mRNA in cattle [40, 41], but it was not observed to affect IGFBP-2 production in bovine granulosa cells [28], human luteinizing granulosa cells [42], or ovine granulosa cells [43]. In comparison, estradiol inhibits granulosa cell IGFBP-2 production in rats [44] and pigs [37] but increases plasma IGFBP-5 levels in cattle [32]. In the mare, granulosa cell production of IGFBP-2 and -5 may play an inhibitory role in intrafollicular IGF-I action, as reported in other species [45–47]. High expression of these binding proteins within a follicle may limit free IGF-I, thus slowing follicular growth and allowing emergence of a dominant follicle, as observed in cattle [18, 48]. Therefore, inhibition of intrafollicular production of IGFBP-2 and -5 may be necessary, at least in part, for selection of the dominant follicle in the mare.

Follicular fluid levels of IGFBP-2, -4, and -5 decrease with increases in estradiol concentrations in mares [10], cattle [16–18, 48], pigs [13], and sheep [21]. Therefore, follicular fluid levels of IGFBP-2, -4, and -5 likely are initially high at follicular emergence due to increased granulosa cell production in response to high levels of FSH in circulation. We further speculate that, when follicles reach approximately 20 mm in diameter, just before selection of the dominant follicle in the mare (for review, see [23]), follicular fluid levels of IGFBP-4 and -5 begin to decrease, most probably due, in part, to actions of specific IGFBP proteases [10] concomitant with the increase in intrafollicular estradiol and the decline in FSH. In response to declining FSH levels, granulosa cell production of IGFBP-2 and -5 slows. Once the dominant follicle has been selected, estradiol levels increase, further stimulating granulosa cell IGFBP-2 and -5 production in atretic, cohort follicles.

For the first time, to our knowledge, hormonal regulation of equine granulosa cell-derived IGFs was observed. The present study revealed that FSH and estradiol treatment had no effect on IGF-I production but decreased IGF-II production by equine granulosa cells obtained from large follicles. Also, insulin inhibited IGF-II production by medium- and large- but not by small-follicle granulosa cells, suggesting that IGF-II production by small-follicle granulosa cells is unresponsive to trophic factors in the mare. In comparison to our findings, FSH treatment has no effect on IGF-I production by bovine granulosa cells [49, 50] but stimulates IGF-I production by porcine granulosa cells [51–53]. In humans, FSH stimulates IGF-II, but not IGF-I, production and mRNA by granulosa cells [54]. In bovine granulosa cells, estradiol enhances the insulin-induced decreases in IGF-I and IGF-II production but has no effect on basal IGF-I and IGF-II production [50, 55]. However, in cultured ovine [56] and porcine [51, 52] granulosa cells, estradiol increases IGF-I production, and in vivo treatment of immature hypophysectomized rats with diethylstilbestrol increases IGF-I mRNA but decreases IGF-II mRNA within the ovary [57]. Thus, species differences may exist in regard to hormonal control of ovarian IGF production.

The present study also revealed that estradiol and progesterone production by equine granulosa cells were differentially regulated by insulin and FSH. We found that insulin decreased progesterone production but increased estradiol production in small- and medium-follicle granulosa cells but had no effect on steroidogenesis of large-follicle granulosa cells. Similarly, insulin stimulates estradiol production by small-follicle bovine granulosa cells [49, 58, 59] but either has no effect on or decreases estradiol production in pigs [60, 61] and humans [62]. However, insulin and FSH stimulate progesterone production by granulosa cells of cattle [49, 63], swine [64], and rats [65]. We also observed that FSH alone decreased progesterone production by small- and medium-follicle granulosa cells but had no effect on large-follicle granulosa cells. Why insulin and FSH are inhibitory to progesterone production in mares but stimulatory in other species remains to be elucidated, but it may involve some species-specific mechanism unique to the mare. FSH alone did not affect estradiol production by small- and large-follicle granulosa cells, but it dramatically decreased estradiol production by medium-follicle granulosa cells. However, in the presence of insulin, FSH enhanced the stimulatory effect of insulin on estradiol production by small-follicle granulosa cells and inhibited the stimulatory effect of insulin on estradiol production by medium-follicle granulosa cells. Why FSH exhibited both stimulatory and inhibitory effects is unclear, but it may be dependent on the differentiated state of the granulosa cells as well as on the dose of FSH and the hormonal milieu. In support of this suggestion, Saumande [66], using bovine granulosa cells, demonstrated that low doses (i.e., <5 ng/ml) of FSH increased estradiol production but that higher doses of FSH blocked the stimulatory effect of insulin on estradiol production. Also, FSH decreases aromatase activity of bovine granulosa cells in the presence of high doses (i.e., >=100 ng/ml) of insulin [67].

Whether variation in the amount of estradiol produced by the different treatment groups (i.e., 0.2–7.0 ng/ml) influenced the results of the present study is uncertain. Sirois et al. [68] reported that, in the presence of insulin, FSH was unable to stimulate estradiol production in large equine follicles during the early estrous phase but was able to significantly stimulate progesterone production by granulosa cells from large (early and late-estrous stage) equine follicles. Also, equine granulosa cell responsiveness to FSH in terms of estradiol and progesterone production decreased as follicle diameter increased in early to late-estrous follicles (39–47 mm) [69], which is consistent with the results of the present study. Therefore, high circulating levels of FSH, as seen during early follicular growth (peak FSH occurs when growing follicles are <15 mm in diameter) [23], may inhibit premature granulosa cell differentiation and/or luteinization in small and medium follicles, as indicated by the decreased levels of progesterone and estradiol in the present study. In addition, because estradiol inhibited small-follicle progesterone production, high levels of estradiol within the preovulatory follicle may be an additional paracrine mechanism to prevent premature differentiation of small-follicle granulosa cells. We hypothesize that, once follicles reach a large diameter (>25 mm), FSH-induced steroidogenesis may become down-regulated, making steroid production by these follicles unresponsive to FSH. Further research is needed to verify these suggestions and to elucidate further the hormonal regulation of granulosa cell steroidogenesis in the mare.

Insulin increased the numbers of equine granulosa cells in the present study and in previous studies using bovine [49, 70], porcine [29, 64], murine [71, 72], and ovine [73] granulosa cells in vitro. The stimulatory effect of insulin on granulosa cell numbers was observed for small, medium, and large equine follicles, but the stimulatory effect of insulin was less in cells from large versus small or medium follicles, as observed in cattle [27] and rats [71]. Interestingly, estradiol treatment increased the numbers of granulosa cells from all three size categories, but the stimulation by estradiol was less than that of insulin in small- and medium-follicle granulosa cells. In contrast, estradiol has no effect on the numbers of bovine granulosa cells [27] but inhibits insulin-dependent mitosis of small-follicle granulosa cells isolated from immature but not eCG-primed rats [72]. Also, FSH was able to increase numbers of small- and medium- but not of large-follicle granulosa cells of mares in the present study. Previously, FSH has been shown either to inhibit [70] or to have no effect [27] on bovine granulosa cell proliferation. Similarly, FSH has been shown to inhibit porcine granulosa cell proliferation [74] but to have no effect on ovine granulosa cell proliferation [75] in vitro. Reasons for these inconsistent mitogenic responses to estradiol and FSH are unclear, but they likely depend on the physiologic state of the animal or follicle at the time of collection, culture conditions, and/or species differences. Because estradiol and FSH did not stimulate IGF-I or IGF-II production and either had no effect or increased IGFBP production, the stimulatory effect of estradiol and FSH on numbers of equine granulosa cells cannot be explained by changes in the IGF system.

In summary, the results of the present study indicate that IGF-I, IGF-II, IGFBP-2, and IGFBP-5 are produced by equine granulosa cells and that insulin, FSH, and estradiol play a role in the regulation of steroidogenesis and the IGF system of equine granulosa cells. Decreased levels of low molecular weight IGFBPs in large, estrogen-active follicles may allow an increase in free IGF-I and, thus, increase gonadotropin responsiveness and growth of the future dominant follicle in the mare. In contrast, increased levels of the low molecular weight IGFBPs in small cohort follicles may act to sequester IGF-I, causing a low responsiveness to IGF-I and FSH and, thus, slowing the growth rate of future subordinate follicles.



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  		FIG. 6. A) Representative ligand blot of IGFBP production by equine granulosa cells from medium follicles. Lane 1: control; lane 2: estradiol; lane 3: insulin; lanes 4 and 5: estradiol plus insulin; lanes 6 and 7: FSH; lanes 8 and 9: FSH plus insulin; lane 10: equine follicular fluid; lane 11: no sample; lane 12: bovine follicular fluid. B) Representative example of control cultures of equine granulosa cells from medium follicles under phase-contrast microscopy. C) Representative example of insulin-treated cultures of equine granulosa cells from medium follicles under phase-contrast microscopy. Magnification x200 (B and C).



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  		FIG. 7. A) Representative ligand blot of IGFBP production by equine granulosa cells from large follicles. Lane 1: control; lane 2: estradiol; lane 3: insulin; lanes 4 and 5: estradiol plus insulin; lanes 6 and 7: FSH; lanes 8 and 9: FSH plus insulin. B) Representative example of control cultures of equine granulosa cells from large follicles under phase-contrast microscopy. C Representative example of insulin-treated cultures of equine granulosa cells from large follicles under phase-contrast microscopy. Magnification x200 (B and C).


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Bel-Tex (Ft. Worth, TX) for their generous donation of equine ovaries; N.R. Mason (Lilly Research Laboratories, Indianapolis, IN) for the generous donation of estradiol antiserum; the National Hormone and Pituitary Program (Rockville, MD) for supplying the IGF-I antibody; Monsanto Co. for supplying the recombinant bovine IGF-II; the Oklahoma State University Recombinant DNA/Protein Resource Facility for use of its molecular imager and scanning densitometer; Dr. Mark Payton for providing assistance with statistical analysis; and Paula Cinnamon for secretarial assistance.


   FOOTNOTES
 
First decision: 21 September 2001.

1 Supported in part under project H-2329 (to L.J.S.). Approved for publication by the Director, Oklahoma Agric. Exp. Sta. Back

2 Correspondence. FAX: 405 744 7390; igf1leo{at}okstate.edu Back

Accepted: December 19, 2001.

Received: August 20, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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