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Biology of Reproduction 63, 1205-1213 (2000)
© 2000 Society for the Study of Reproduction, Inc.

ARTICLES

Consequences of the Presence of the Booroola F Gene on the Intraovarian Insulin-Like Growth Factor System and Terminal Follicular Maturation in Mérinos d'Arles Ewes1

D. Monniaux2,,a, P. Mongeta, C. Pisseleta, J. Fontainea, and J.M. Elsenb

a Physiologie de la Reproduction et des Comportements, UMR1291 INRA-CNRS-Université F. Rabelais de Tours, INRA 37380 Nouzilly, France b INRA, Station d'Amélioration Génétique des Animaux, Chemin de Borde-Rouge, 31326 Castanet-Tolosan cedex, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In sheep, the presence of the Booroola F gene has several important consequences for ovarian function. This study investigated the consequences of the presence of the F gene for the insulin-like growth factor (IGF) system in the ewe ovary. Studies were undertaken in ovaries from F+ and ++ Mérinos d'Arles ewes to determine 1) the levels of type I IGF receptors and IGF binding proteins (IGFBPs) in follicular cells by quantitative autoradiography of [125]-IGF-I binding sites on ovarian sections; 2) the pattern of intrafollicular IGFBPs, by Western-ligand blotting on follicular fluids; and 3) the effects of IGF-I and FSH on proliferation and differentiation of granulosa cells in vitro, assessed by progesterone secretion and cytochrome P450 side-chain cleavage (P450scc) expression. The amounts of type I IGF receptors were similar in F+ and ++ follicular cells; however, at the same follicular size, F+ healthy follicles contained lower concentrations of IGFBPs smaller than 40 kDa (particularly IGFBP-2) than ++ healthy follicles. In vitro, in basal conditions as well as in IGF-I- or FSH-stimulated conditions (or both), granulosa cells from F+ follicles had a lower proliferative activity, secreted higher amounts of progesterone, and expressed higher levels of P450scc than granulosa cells from ++ follicles of the same size. When F+ and ++ preovulatory follicles were compared at the end of the follicular phase, IGFBPs <40 kDa concentrations were slightly higher, and responsiveness of granulosa cells to FSH in vitro was lower in F+ than in ++ follicles, suggesting that terminal maturation of F+ follicles, although precocious, was less complete than it was in ++ follicles. The early decrease in intrafollicular IGFBPs <40 kDa concentrations observed in F+ antral follicles, which likely leads to an early increase in IGF bioavailability, may at least partly account for the increased ovulation rate that characterizes F-carrier ewes.

follicle, granulosa cells, growth factors, IGF receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During terminal follicular growth in sheep (diameter size 1–7 mm), an inverse relationship is observed between proliferation and steroidogenesis of granulosa cells. Granulosa cells from follicles 1–2 mm in diameter actively proliferate, whereas those from follicles up to 5–7 mm in diameter lose proliferative activity and acquire a high steroidogenic activity, which leads to an increase in estradiol secretion [1]. FSH plays a central role in regulating granulosa cell differentiation because it induces and enhances expression of steroidogenic enzymes, particularly cytochrome P450 side-chain cleavage (P450scc) and cytochrome P450 aromatase (for review, see [2]). In addition, increasing evidence suggests that growth factors modulate terminal follicular development. In particular, IGF-I is a potent stimulator of granulosa cell proliferation (pig [3], sheep [4], cow [5]) and differentiation, and acts in synergy with FSH (rat [6, 7], pig [8], human [9], sheep [4, 10]). In vivo, IGF binding proteins (IGFBPs) modulate the bioavailability of IGF-I and IGF-II in follicles. In particular, intrafollicular concentrations of IGFBPs <40 kDa (mainly IGFBP-2, -4 and -5) markedly decrease during terminal follicular growth and increase in atretic follicles (reported for pig [11, 12], human [13], sheep [14], and cow [15]). From recent results, it is believed that terminal follicular development is dependent upon both circulating levels of gonadotropins and the intrafollicular concentration of bioavailable IGFs, as determined by the IGF/IGFBP ratio [16, 17].

In Mérino ewes, the presence of a hyperprolificate Booroola (or F) gene has several dramatic consequences on ovarian folliculogenesis. First, the F gene increases the number of ovulations to 3 or 4 in heterozygote F+ ewes (carriers of one mutated allele) and 5 or more in homozygote FF ewes (carriers of two mutated alleles) compared with 1 or 2 in ++ ewes (noncarriers of the mutation) [18]. Second, follicles that carry the F gene lose their proliferative activity and reach their final preovulatory stage of maturation (characterized by maximal secretion of estradiol per granulosa cell and maximal sensitivity to FSH and LH) at a smaller size than ++ follicles [19, 20]. Indeed, the diameter of preovulatory follicles is approximately 4–4.5 mm in F+ ewes and 3–4 mm in FF ewes, compared with 5–7 mm in ++ ewes [21].

The consequences of the presence of the F gene on the IGF system in the ewe ovary have not been investigated. In particular, it remains unknown if the more precocious maturation of follicles from ewes that carry the F gene is related to changes in intrafollicular levels of IGFBPs, responsiveness of granulosa cells to IGF-I, or both. The aim of this work was to investigate the effect of the presence of the F gene in Mérinos d'Arles ewes on granulosa cell maturation and responsiveness to IGF-I and FSH in vitro, and levels of IGF-I receptors and IGFBPs in ovarian follicles in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

All procedures were approved (approval A37801) by the agricultural and scientific research government committees and conducted in accordance with the National Guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching. Cyclic F+ (n = 36) and ++ (n = 36) Mérinos d'Arles ewes were treated with intravaginal fluorogestone acetate sponges (progestagen sponges, 40 mg; Intervet, Angers, France) for 13 days to synchronize estrus. Previous investigations showed that the LH preovulatory discharge occurs around 40 h after sponge removal in both genotypes following this treatment (Cognié, personal communication) and that the time of ovulation was not different between genotypes [22]. For granulosa cell cultures and experiments on follicular fluid, ovaries from both genotypes were recovered at slaughter in early and late follicular phases (at 18 and 36 h after sponge removal, respectively). For autoradiographic studies, ovaries that were recovered at slaughter in late follicular phase only were immediately embedded in cryoembedding compound (Tissue Tek, Miles Laboratories, Elkhart, IN), and stored in liquid nitrogen.

Granulosa Cell Cultures

The effects of IGF-I and FSH on granulosa cell proliferation, progesterone secretion, and P450scc expression were studied in a previously described cell culture model [4]. Briefly, in each experiment, follicles from F+ and ++ ovaries were quickly dissected and classified according to size (i.e., small follicles, 2–3 mm in diameter; medium, 3.5–4.5 mm; and large, >4.5 mm). Large follicles were found only in the ++ genotype. Large follicles from ++ ewes and medium follicles from F+ ewes were classified as fully grown follicles. Granulosa cells were isolated from each dissected follicle in modified B2 medium [23] with the addition of 10 mg/L bovine transferrin and without cholesterol, and pooled according to genotype and follicle size. The resulting five granulosa cell suspensions (small ++, medium ++, large ++, small F+ and medium F+) were seeded at 105 viable cells/cm2 and cultured in modified B2 medium with 2% ovine fetal serum (prepared from 130-day-old fetuses) at 37°C in an atmosphere of 5% CO2.

For cell proliferation studies, granulosa cells were seeded in tissue culture chambers mounted on glass microslides (Lab-Tek, Nunc, Naperville, IL) in the presence or absence of IGF-I (1 or 10 ng/ml, recombinant human IGF-I, Ciba-Geigy, Saint-Aubin, Switzerland). After 24 h of culture, cells were incubated for 2 h at 37°C in the presence of [3H]thymidine (0.5 µCi/ml, specific activity 6.7 Ci/mmol; Du Pont De Nemours, Les Ulis, France) in modified B2 medium without thymine, washed three times with modified B2 medium (with thymine), and fixed with Bohm-Sprenger fixative (15% formaldehyde, 5% acetic acid, 80% methanol) for 10 min at 20°C. Afterward, cells were stained with Feulgen (Merck, Schuchardt, Germany) and slides were dipped in Ilford K5 emulsion (Ilford, St. Priest, France), air-dried, and exposed for 6 days at 4°C. After autoradiographs were developed, the labeling index (LI; percentage of thymidine-labeled cells) was assessed by counting labeled and unlabeled cells in randomly chosen microscopic fields. For each culture well, estimation of LI was performed on about 1000 cells. For both genotypes, a total of 11 experiments was performed (5 in the early and 6 in the late follicular phases). Estimations of LI were performed on about 5000 to 6000 granulosa cells for each genotype, time of follicular phase, follicle class, and in vitro treatment.

For studies of progesterone secretion, granulosa cells were cultured in 96-well plates (Nunclon Delta, Poly Labo, Strasbourg, France) for 72 h in the presence or absence of IGF-I (1 or 10 ng/ml) and FSH (10 or 100 ng/ml, purified ovine FSH CY1767 with FSH activity = 32-fold the activity of National Institutes of Health FSH S1, INRA, Nouzilly, France). For both genotypes, a total of eight experiments was performed (four each in early and late follicular phases). In each experiment, in vitro treatments with IGF-I, FSH, or both were tested in triplicate or in duplicate. Media were changed every 24 h and stored at -20°C until assayed for progesterone. At 72 h of culture, cells were trypsinized and an aliquot of each resulting cell suspension was counted using a hemacytometer under a phase contrast microscope. Progesterone secretion by granulosa cells was studied between 48 and 72 h of culture and was expressed in pg/1000 cells per 24 h. Progesterone concentrations in the culture media were measured in a direct assay as described previously [24]. The limit of detection of the assay was 12 pg/tube. Dilution curves of samples obtained in different culture conditions were parallel to the standard curve. The intra-assay and inter-assay coefficients of variation were 10% and 11%, respectively. Progesterone concentrations in the culture media from each experiment were measured in the same assay.

For studies of P450scc expression, granulosa cells were cultured in Lab-Tek microslides (Nunc) for 72 h in the presence or absence of IGF-I (10 ng/ml) and FSH (100 ng/ml). Four experiments were performed for both genotypes (two each in early and late follicular phases). Media were changed every 24 h. At 72 h of culture, cells were fixed in PBS containing 4% paraformaldehyde and 0.1% saponin. Immunohistochemistry for P450scc was performed as previously described [25, 26]. The primary antibody was a rabbit polyclonal antibody raised against bovine P450scc (1/100 final dilution, Oxygene Dallas, Dallas, TX). Controls were performed with nonimmune rabbit immunoglobulin G (IgG). The secondary antibody was peroxidase-labeled goat IgG raised against rabbit IgG (Diagnostics Pasteur, Marnes-la-Coquette, France). 3,3'-Diaminobenzidine tetrahydrochloride (Aldrich Chimie, L'Isle d'Abeau Chesnes, France) was used as a substrate for staining.

[125I]-IGF-I Binding to Ovarian Sections

Frozen ovaries were serially sectioned with a cryostat at a thickness of 10 µm. Histological characterization of follicles and [125I]-IGF-I binding studies were systematically performed on adjacent sections. For histological examination, sections were fixed with Bohm-Sprenger fixative and stained with Feulgen. Follicles were classified according to size (<2 mm, 2–3 mm, and >3 mm), and degree of atresia was assessed by using classical histological criteria: healthy (frequent mitosis, no or rare pycnosis in granulosa cells) or atretic (numerous pycnotic bodies and no or rare mitosis in granulosa cells). These histological characteristics have been shown to be closely correlated to estradiol concentrations in ewe follicular fluid [27]. Follicles allocated to different classes of size and degree of atresia were evaluated separately in subsequent tests or evaluations.

[125I]-IGF-I binding to ovarian sections was studied by an autoradiographic method as described previously [28]. Briefly, sections were fixed for 10 min at 4°C in picric acid-formaldehyde, washed in cold PBS, and stored at -20°C overnight. Sections were then incubated in duplicate in 250 µl of PBS (0.1% BSA, pH 8) containing 100 000 cpm of [125I]-IGF-I alone (iodinated by the Iodogen method, specific activity = 200 µCi/µg), or with various concentrations of unlabeled IGF-I or insulin in order to check for the specificity of binding. At the end of the incubation period, the sections were washed twice in PBS, postfixed in 3% glutaraldehyde-PBS, washed in PBS, and air-dried. They were then dipped in Ilford K5 emulsion (Ilford), air-dried, and exposed for 2 wk at 4°C. After autoradiographs were developed, sections were stained with hematoxylin. In these conditions, nonspecific binding was obtained after incubation of [125I]-IGF-I together with 250 ng of unlabeled IGF-I. Specific binding of labeled IGF-I that had been displaced by excess insulin represented binding to a type I receptor, whereas specific binding of labeled IGF-I that had not been displaced by excess insulin represented binding to IGFBPs [28].

Quantitative autoradiographic analysis of ovarian sections was performed using a microscope-linked personal computer-based image analyzer (SAMBA TM 2005, Alcatel TITN, Meylan, France). Each section was analyzed with a 100x objective. Quantification of labeling was performed by measuring the area occupied by silver grains present in a constant area (45 µm2) of the section. In a calibration study of the system, a good correlation was obtained between the number of silver grains estimated by visual counting and their surface measured by image analysis (r = 0.94, P < 0.001). Quantification was performed on both granulosa and thecal cells from 31 ++ follicles (17 <2 mm, 8 2–3 mm, and 6 >3 mm) and 33 F+ follicles (16 <2 mm, 11 2–3 mm, and 6 >3 mm). Labeling was estimated on the basis of 40 to 50 measurements at random for each ovarian compartment (theca or granulosa) and on each follicle. Microscopic fields that were completely occupied with cells were carefully chosen for measurements. Fields containing a part of some antral cavity were not analyzed. Furthermore, the sections had a constant thickness (10 µm); thus, it can be assumed that the measurements were performed on a constant tissue quantity in both compartments of the ovary. Specific binding was obtained by subtracting the values of labeling associated with nonspecific binding from the total binding values.

Western-Ligand Blotting on Follicular Fluids

Ovarian follicles 2–7 mm in diameter were dissected from the ovaries of ++ ewes (153 follicles) and F+ ewes (145 follicles). They were individually measured and classified according to size (small, 2–3 mm; medium, 3.5–4.5 mm; and, only in the ++ genotype, large, >4.5 mm) and time of recovery (early or late follicular phase). For each follicle, follicular fluid was aspirated with a 26-gauge needle and stored at -20°C until assayed. Each follicle was then slit open in B2 medium and a suspension of granulosa cells was prepared as described previously [14]. For each cell suspension, a smear of granulosa cells was prepared on histological slides, fixed with Bohm-Sprenger fixative, and stained with Feulgen. The degree of atresia was assessed by microscopic examination using the same histological criteria described earlier. To obtain sufficient volume to perform ligand blotting experiments on each sample, follicular fluids from some small follicles were pooled between and within animals in each genotype (n = 12 and 8 pools for small healthy and small atretic follicles, respectively, in both genotypes) according to their quality. Finally, 8 to 48 different follicular fluids per class of size and atresia were submitted to Western ligand blotting analysis, except for large atretic ++ follicles, only 4 of which were available.

Western-ligand blotting was performed according to the method of Hossenlopp et al. [29] modified by Monget et al. [14]. Briefly, 1.5 µl of follicular fluid was submitted to 12% SDS-PAGE under nonreducing conditions. The separated proteins were electroblotted onto nitrocellulose filters (Schleicher and Schuell, Ecquevilly, France), which were treated with PBS (0.01 M, pH 7.4) containing 0.1% Nonidet P-40, 0.5% gelatin, and 0.1% Tween-20; then incubated overnight at 4°C with 1 x 106 cpm 125I-IGF-II (recombinant human IGF-II [Ciba-Geigy], and iodinated by the Iodogen method) in appropriate buffer. After the incubation period, membranes were washed in PBS containing 0.1% Tween-20, air-dried, and submitted to autoradiography by exposure to Amersham Hyperfilm MP with intensifying screens for one night at -70°C.

The image analysis system, SAMBA TM 2005 (Alcatel TITN), was used for densitometric analysis of autoradiographs. The amount of radiolabeled IGF-II bound to that IGFBP-2 (35 kDa) band was estimated from the ratio of the integrated optical density of this band in the sample to that of IGFBP-2 in a fetal ovine serum sample that had been run simultaneously for each ligand blot and taken as an internal standard. Without this normalization, accurate comparison of gels was difficult to perform in view of the variability between experiments. The reproducibility of this quantification was checked previously [14]. Data given in Results are the means of these determinations.

Data Analysis

All experimental data are expressed as the mean ± SEM. For in vitro cell proliferation studies, chi-square analysis was used to compare percentages of labeled cells. For in vitro studies of progesterone secretion, data were log-transformed to homogenize variances. Then, comparisons of means were made using ANOVA. Different models were used to determine the effect of genotype, follicular size, stage of follicular phase, and in vitro stimulation. Kramer post-hoc multiple-range test for groups with unequal number of replications was used to compare means [30]. For studies of type I receptors and IGFBPs in granulosa cells, theca cells, and follicular fluid, comparisons of means between different classes of follicles were also made using ANOVA to determine the effect of genotype, follicular size, and degree of atresia. A post-hoc Newman-Keuls test was used to compare means, except in the case of heterogeneity of variance, when the Kruskall-Wallis test was used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Granulosa Cell Maturation and Responsiveness to IGF-I and FSH In Vitro

Proliferation and steroidogenesis of granulosa cells were studied in response to IGF-I and FSH in vitro with the aim of comparing granulosa cell maturation between the two genotypes. The proliferation rate of granulosa cells, as assessed by thymidine LI, clearly decreased when follicular size increased in both genotypes (P < 0.001). Irrespective of the time of follicular phase and culture conditions, proliferation rate of granulosa cells was lower in F+ than in ++ follicles of the same class of size (LI in basal culture conditions: 8.9 ± 0.4% vs. 11.5 ± 0.3% small F+ vs. small ++, P < 0.001; 2.4 ± 0.1% vs. 3.9 ± 0.3% medium F+ vs. medium ++, P < 0.001). Irrespective of the time of follicular phase, IGF-I, acting at 1 ng/ml and 10 ng/ml, slightly increased (less than 1.4-fold) the proliferation rate of granulosa cells from all follicular classes (data not shown). In basal and IGF-I-stimulated conditions, granulosa cells from fully grown follicles (i.e., large ++ and medium F+) had lower proliferation rates in late phase than in early follicular phase (P < 0.01, Table 1). Such a difference was not observed in small ++, medium ++, and small F+ granulosa cells (data not shown). In addition, in IGF-I-stimulated conditions, granulosa cells from medium F+ follicles had higher proliferation rates than granulosa cells from large ++ follicles, in both early and in late follicular phases (P < 0.01, Table 1).


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  		TABLE 1. Proliferation of granulosa cells form fully grown (large ++ and medium F+) follicles in basal and IGF-I-stimulated conditions.a

Irrespective of the time of follicular phase and in basal culture conditions, the amount of progesterone secreted by granulosa cells from both genotypes increased when follicular size increased (P < 0.05, Fig. 1A). This change was accompanied by an increase in the proportion of P450scc-positive granulosa cells as well as the intensity of staining of positive cells in immunohistochemistry experiments (Fig. 2). Granulosa cells from medium F+ follicles secreted higher amounts of progesterone than granulosa cells from medium ++ follicles (P < 0.05), but not from large ++ follicles (P > 0.05, Fig. 1A). In addition, P450scc expression was higher in medium F+ than in medium ++ granulosa cells (Fig. 2). Addition of IGF-I (10 ng/ml) and FSH (100 ng/ml) to culture medium strongly enhanced progesterone secretion by granulosa cells from large ++ and medium F+ (P < 0.001), but not from small ++ and small F+ follicles (Fig. 1, A and B). In these stimulated conditions, progesterone secretion was higher in late follicular phase than it was in early phase for medium ++ (P < 0.05) and large ++ (P < 0.05), but not for medium F+ (P > 0.05) granulosa cells (Fig. 1B). In late follicular phase, responsiveness of granulosa cells to FSH in vitro was higher for large ++ than for medium F+ follicles (Fig. 3). Accordingly, P450scc expression increased during the follicular phase in FSH-stimulated granulosa cells from large ++, but not medium F+ follicles (data not shown).



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  		FIG. 1. Secretion of progesterone by ++ and F+ granulosa cells from different classes of follicles in vitro. Granulosa cells from ++ and F+ follicles of different sizes (small, 2–3 mm; medium, 3.5–4.5 mm; large, >4.5 mm in diameter) were recovered in early and late follicular phases (18 and 36 h after progestagen sponge removal, respectively), then cultured as described in Materials and Methods during 72 h without (A, basal conditions) or with (B, stimulated conditions) IGF-I (10 ng/ml) and FSH (100 ng/ml). Progesterone concentration was measured during the last 24 h of culture and expressed in pg/1000 cells per 24 h. Data represent mean ± SEM of measurements performed in four independent cultures at each time of follicular phase. Within each panel, * indicates significant differences between genotypes (F+ versus ++ for the same class of follicles and stage of follicular phase), $ indicates significant differences between follicles of different sizes (large or medium vs. small for the same genotype and stage of follicular phase), and # indicates significant differences between the two stages of follicular phase (early vs. late for the same class of follicles and genotype). Levels of significance were at least P < 0.05



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  		FIG. 2. Cytochrome P450scc expression in ++ and F+ granulosa cells from different classes of follicles in vitro. Granulosa cells from ++ and F+ follicles of different sizes (small, 2–3 mm; medium, 3.5–4.5 mm; large, >4.5 mm in diameter) were recovered in early and late follicular phases (18 and 36 h after progestagen sponge removal, respectively), then cultured during 72 h without or with IGF-I (10 ng/ml) and FSH (100 ng/ml). After culture, immunohistochemistry for P450scc expression was performed as described in Materials and Methods. This figure shows representative microscopic fields from FSH-stimulated granulosa cells from a) small ++, b) small F+, c) medium ++, d) medium F+, and e and f) large ++ follicles. Granulosa cells were recovered in early (f) or late (a through e) follicular phases. Bar = 20 µm



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  		FIG. 3. Action of IGF-I and FSH on progesterone secretion by granulosa cells from ++ and F+ preovulatory follicles in vitro. Granulosa cells were recovered from large ++ (left) and medium F+ (right) follicles 36 h after sponge removal and cultured as described in Materials and Methods during 72 h with IGF-I (0, 1, or 10 ng/ml) and FSH (0, 10, or 100 ng/ml). Progesterone concentration was measured during the last 24 h of culture and expressed in pg/1000 cells per 24 h. Data represent mean ± SEM of measurements performed in four independent cultures. Within each genotype, * indicates significant effects of IGF-I (cells cultured with IGF-I vs. without IGF-I at the same FSH concentration) and $ indicates significant effects of FSH (cells cultured with FSH vs. without FSH at the same IGF-I concentration). Levels of significance were at least P < 0.05

Overall, these results indicate that 1) the proliferative activity of granulosa cells decreased, whereas their secretion of progesterone and expression of P450scc increased during terminal follicular growth in both genotypes; 2) at the same follicular size, granulosa cells from F+ follicles had a lower proliferative activity but secreted more progesterone and expressed higher levels of P450scc than granulosa cells from ++ follicles; 3) IGF-I enhanced proliferation in granulosa cells from all follicular classes in both genotypes; 4) FSH and IGF-I stimulated progesterone secretion and P450scc expression by granulosa cells from large ++ and medium F+ fully grown follicles; and 5) responsiveness by granulosa cells to FSH from medium and large ++, but not medium F+ follicles, increased during the follicular phase of the cycle.

Type I IGF Receptors and IGFBPs in F+ and ++ Follicles In Vivo

From our in vitro results, granulosa cells responded to IGF-I with enhanced steroidogenesis, proliferation, or both according to their maturation level, which was strongly influenced by ewe genotype. The effects of the presence of the F gene on the IGF system were further investigated by studying changes in IGF binding sites (type I receptors and IGFBPs) in follicular cells and IGFBP concentrations in follicular fluid during growth and final maturation of follicles in vivo.

From binding studies performed on ovarian sections in late follicular phase, there was no difference in type I IGF receptor and IGFBP levels in granulosa cells between ++ and F+ follicles, regardless of their size and quality. In F+ ovaries, type I IGF receptor levels were higher in granulosa cells from healthy follicles with a diameter of <2 mm than from those >3 mm (P < 0.05; Fig. 4). In follicles that were <2 mm in diameter from the ++ genotype, atresia was characterized by an increase in IGFBP levels in granulosa cells (P < 0.05; Fig. 5). In the F+ genotype, IGFBP levels also tended to increase in atretic follicles <2 mm in diameter, >3 mm in diameter (both P < 0.1), and 2–3 mm in diameter (P < 0.06), compared with healthy follicles (Fig. 5). No difference in type I IGF receptor and IGFBP levels was observed in thecal cells between ++ and F+ genotypes, regardless of size and quality of the follicles; and no effect of follicular size and degree of atresia was observed in theca (data not shown).



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  		FIG. 4. Effect of genotype and follicular class on type I receptor levels in granulosa cells. Follicles were classified according to size and degree of atresia, and specific intensity of labeling of [125I]-IGF-I on type I receptor was determined by autoradiography on ovarian sections as described in Materials and Methods. Data represent mean ± SEM of measurements on 31 ++ follicles (17 <2 mm, 8 2–3 mm, and 6 >3 mm) and 33 F+ follicles (16 <2 mm, 11 2–3 mm, and 6 >3 mm). * P < 0.05, F+ healthy follicles <2 mm in diameter vs. F+ healthy follicles >3 mm in diameter



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  		FIG. 5. Effect of genotype and follicular class on IGFBP levels in granulosa cells. Follicles were classified according to size and degree of atresia, and specific intensity of labeling of [125I]-IGF-I on IGFBPs was determined by autoradiography on ovarian sections as described in Materials and Methods. Data represent mean ± SEM of measurements on 31 ++ follicles (17 <2 mm, 8 2–3 mm, and 6 >3 mm) and 33 F+ follicles (16 <2 mm, 11 2–3 mm, and 6 >3 mm). * P < 0.05, ++ atretic follicles <2 mm in diameter versus ++ healthy follicles <2 mm in diameter

IGFBP-2, -3, -4, and -5 were detected by Western ligand blotting in follicular fluid of both genotypes. There was no effect of the time of recovery (early or late follicular phase) on intrafollicular levels of IGFBP-2, -4, and-5 (all three <40 kDa, data not shown). Intrafollicular levels of IGFBPs <40 kDa were lower in ++ fully grown follicles than in small ++ healthy follicles (Fig. 6C, ++ >5 mm healthy follicles vs. Fig. 6A, ++ 2–3 mm healthy follicles; Fig. 7, P < 0.001). Intrafollicular levels of IGFBPs <40 kDa were also lower in F+ fully grown follicles than in small F+ healthy follicles (Fig. 6B, F+ 3.5–4.5 mm healthy follicles vs. Fig. 6A, F+ 2–3 mm healthy follicles; Fig. 7, P < 0.05). In both genotypes, atresia was characterized by an increase in intrafollicular levels of IGFBPs <40 kDa (Fig. 6A, healthy vs. atretic follicles; Fig. 7, P < 0.001, 0.05, and 0.05 for 2–3 mm, 3.5–4.5 mm, and >4.5 mm follicles, respectively). In addition, healthy follicles from F+ ewes 2–3 mm in diameter contained lower levels of IGFBPs <40 kDa than healthy follicles of the same size from ++ ewes (Fig. 6A; Fig. 7, P < 0.05), and in the size class of 3.5–4.5 mm in diameter, F+ healthy follicles tended to contain lower levels of IGFBPs <40 kDa than ++ healthy follicles (Figs. 6B and 7, P = 0.08). Finally, ++ fully grown follicles contained slightly lower levels of IGFBPs <40 kDa than F+ fully grown follicles (Fig. 6C, ++ >5 mm vs. F+ 3.5–4.5 mm follicles; Fig. 7, P < 0.05).



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  		FIG. 6. Representative Western ligand blotting of follicular fluid from 2–3 mm (A), 3.5–4.5 mm (B), and fully grown (C) follicles in F+ and ++ ewes. Fully grown follicles were >5 mm and 3.5–4.5 mm in diameter from ++ and F+ genotypes, respectively, and recovered in late follicular phase. Western ligand blotting experiments were performed on equal volumes (1.5 µl) of follicular fluid. S, Ovine serum; FS, fetal ovine serum; H, healthy follicle; A, atretic follicle. IGFBP-2 and IGFBP-5 were previously identified by immunoblotting (data not shown)



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  		FIG. 7. Effect of genotype and follicular class on IGFBP-2 levels in follicular fluid. Follicular fluids were submitted to Western ligand blotting and quantification of IGFBP-2 on blots was performed as described in Materials and Methods. Qualitatively, IGFBP-4 and IGFBP-5 followed a pattern similar to that observed for IGFBP-2. Data represent mean ± SEM of measurements on 8 to 48 different follicular fluids per class of size and atresia, except for large atretic follicles, only 4 of which were available. * P < 0.05 vs. healthy follicles of the same size, *** P < 0.001 vs. healthy follicles of the same size, $ P < 0.05 vs. ++ healthy follicles 2–3 mm in diameter

Overall these data indicate that in both ++ and F+ ovaries, intrafollicular levels of IGFBPs <40 kDa decreased and increased during follicular growth and atresia, respectively. The decrease in IGFBPs <40 kDa levels was more precocious but less complete in F+ than in ++ growing follicles.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have studied the consequences of the presence of the F gene in Mérinos d'Arles ewes on the IGF system in ovarian follicles in vivo and the responsiveness of granulosa cells to IGF-I and FSH in vitro. Results of this study with ++ and F+ Mérinos d'Arles ewes are in agreement with previous data in sheep showing that intrafollicular concentrations of IGFBPs <40 kDa clearly decrease and increase during terminal follicular growth and atresia, respectively [14], whereas amounts of type I IGF receptors, which mediate most actions of IGF-I and IGF-II [31] show little or no change in granulosa cells [28, 32]. In vitro, IGF-I was shown to stimulate proliferation and, in synergy with FSH, progesterone secretion as well as P450scc expression in both ++ and F+ granulosa cells, thereby confirming previous results with ovine granulosa cells [4, 10, 26]. However, some differences were observed between ++ and F+ follicles. F+ follicles were characterized by a more precocious follicular maturation, as assessed by changes in proliferation, progesterone secretion, and P450scc expression in granulosa cells, and an earlier decrease in intrafollicular concentrations of IGFBPs <40 kDa during follicular growth.

Changes in IGFBPs during follicular growth and atresia were studied by two different methods, in situ binding on ovarian sections and Western ligand blotting on follicular fluids. Results from both methods showed that follicular atresia is accompanied by an increase in intrafollicular IGFBP levels. However, in situ binding, unlike Western ligand blotting, did not show any clear decrease in follicular IGFBP levels during follicular growth and no difference between genotypes was detected by using this method. Different reasons can explain these discrepancies. First, in situ binding detects intracellular as well as extracellular matrix or cell membrane-bound IGFBPs (or both), whereas Western ligand blotting detects extracellular-soluble IGFBPs only. Second, in situ binding does not discriminate among the different IGFBPs. Third, proteolysis of soluble IGFBPs was recently shown in follicular fluid from various mammalian species, and particularly in sheep [33, 34]. Intrafollicular mechanisms of IGFBP proteolysis are not fully understood but it is clear that they can lead to important decreases in concentrations of IGFBPs <40 kDa in follicular fluid of healthy follicles and their almost complete disappearance in preovulatory ones. Differences in IGFBP proteolytic activity between F+ and ++ follicular fluids have not been investigated.

Previous results have shown that terminal follicular maturation is drastically influenced by genotype in Booroola ewes. In particular, preovulatory follicles are smaller and the number of granulosa cells per preovulatory follicle is lower in ewes that carry the F gene compared with those that do not [21]. In addition, aromatase activity [35], cAMP production in response to gonadotropins [19, 36], and inhibin production [37] were shown to increase in granulosa cells at a smaller follicular size in F carriers compared with non-F-carriers. This earlier acquisition of follicular differentiation is accompanied by an earlier loss of granulosa cell proliferation in F-carrier antral follicles [38]. Present results are in complete agreement with the existence of a more precocious differentiation of follicular cells from F-carrier ewes. First, in vitro, granulosa cells from F+ follicles had a lower proliferative activity in basal and IGF-I-stimulated conditions compared with cells from ++ follicles of the same size. Second, also in vitro, granulosa cells from F+ follicles secreted higher amounts of progesterone and expressed higher levels of P450scc than granulosa cells from ++ follicles of the same size, both in basal conditions as well as in conditions in which IGF-I-, FSH, or both were used as stimulants. Third, in vivo, F+ healthy follicles were shown to contain lower levels of IGFBPs <40 kDa than ++ healthy follicles of the same size. In a similar study, no differences between genotypes in intrafollicular IGFBP concentrations were reported [39]. Whether this discrepancy between results is explained by methodological differences in assessing follicular atresia or by breed differences is unknown. Recently, we have shown that the decrease in intrafollicular levels of IGFBPs <40 kDa accompanying terminal follicular development is due to both a decrease in expression by follicular cells (particularly for IGFBP-2) and an increase in proteolytic degradation (particularly for IGFBP-4) [33, 34, 40]. We suggest that these intrafollicular mechanisms are likely operative in smaller follicles in F-carrier than in non-F-carrier ewes.

In addition, the present results suggest that final maturation of preovulatory follicles during the follicular phase is slightly less complete (although precocious) in F+ than in ++ follicles. Indeed, at the end of the follicular phase, responsiveness of granulosa cells to FSH, as assessed by progesterone secretion and P450scc expression in vitro, was higher in granulosa cells from ++ than in F+ preovulatory follicles. Moreover, from our results, maturation of granulosa cells from ++ but not F+ fully grown follicles increased during the last stages of the follicular phase (between 18 and 36 h after sponge removal). Similarly, Henderson et al. [35] have shown that granulosa cells from ++ follicles >5 mm diameter, but not F+ or FF follicles of any size, recovered 12–24 h after injection of cloprostenol (early follicular phase) have a lower response to FSH and LH, as assessed by cAMP production, than granulosa cells recovered 36–48 h after cloprostenol (late follicular phase). In addition, Driancourt et al. [41] reported that the size of preovulatory ++ follicles continuously increases during the follicular phase, whereas F+ preovulatory follicles reach their maximal size early in the follicular phase and remain at a plateau until ovulation. Accordingly, from our results, intrafollicular IGFBP concentrations of <40 kDa did not change throughout the follicular phase in F+ fully grown follicles. However, unexpectedly, in ++ fully grown follicles, the final maturation of granulosa cells, which was observed during the follicular phase, was not accompanied by detectable changes in intrafollicular concentrations of IGFBPs <40 kDa. This result can be explained by the sensitivity of the Western ligand blotting method because IGFBPs <40 kDa are quasiundetectable in large antral follicles. Alternatively, theca cells have been previously shown to express IGFBPs <40 kDa in sheep follicles [40] and it is suggested that they may also participate in regulating intrafollicular IGFBP levels.

The role of the IGF system in the regulation of ovulation rate in sheep and other mammalian species is not clear. In this study we have shown that the presence of the F gene, which is responsible for an increase in ovulation rate in Mérinos d'Arles ewes [42], did not affect the number of type I IGF receptors on follicular cells and the responsiveness of granulosa cells to IGF-I in vitro. More generally, in different breeds of sheep, an absence of correlation was found between plasma concentrations of IGF-I and ovulation rate [43–46]. Furthermore, in Booroola Mérino ewes, no difference in intrafollicular concentrations of IGF-I were observed between FF and ++ follicles [39, 45]. However it is suggested that the concentrations of bioavailable IGFs are increased in follicles from F-carrier ewes. First, these follicles contain higher concentrations of IGF-II [39]. Second, from our results, intrafollicular IGFBP concentrations of <40 kDa were lower in F+ healthy follicles than in ++ healthy follicles of the same diameter. The resulting higher bioavailability of intrafollicular IGFs [16, 17] in F+ than in ++ follicles of the same size may be at least partly involved in the enhanced recruitment of ovulatory follicles and the lower incidence of atresia, both of which have been shown to characterize terminal follicular growth in F-carrier ewes [38]. We propose that such an earlier increase in the bioavailability of intrafollicular IGFs, in association with a slight increase in immunoreactive and bioactive FSH peripheral concentrations [47–49], may participate in the mechanisms that lead to an increase in ovulation rate in F-carrier Booroola ewes.

In conclusion, the decrease in intrafollicular concentrations of IGFBPs <40 kDa, which accompanies terminal follicular development, was shown to be more precocious in F-carrier than in noncarrier follicles, presumably leading to an increase in the concentration of bioavailable IGFs at a smaller follicular size. It is suggested that this process may at least partly account for the increased ovulation rate that characterizes F-carrier ewes. Finally, terminal maturation of preovulatory follicles, although a precocious event, may be slightly less complete in F-carrier than in non-F-carrier follicles.


   ACKNOWLEDGMENTS
 
We thank Dr. Y. Combarnous for his gift of purified ovine FSH, CY1767; and Drs. H.H. Peter and A. Hinnen from Ciba-Geigy for their gift of recombinant human IGF-I and IGF-II. We are grateful to the Boomarov team for Booroola sheep production, breeding, and characterization. We thank A. Beguey for the photographic work.


   FOOTNOTES
 
First decision: 31 March 2000.

1 Supported by AIR contract CT920232. Back

2 Correspondence. FAX: 33 47 42 77 43; monniaux{at}tours.inra.fr Back

Accepted: June 1, 2000.

Received: March 1, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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