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Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853
Address all correspondence and requests for reprints to: Dr. J. E. Fortune, Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853. E-mail: jf11{at}cornell.edu.
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Abstract |
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7 mm in diameter), at about the expected time of size divergence (group 2; largest follicle, 8 mm) or after a dominant follicle was clearly present (group 3; largest follicle, ≥9 mm). The four largest follicles (F1–F4) were dissected and concentrations of steroids, IGFBPs and free IGF-I and levels of proteolytic activity for IGFBP-4 and -5 in the follicular fluid were determined. Follicles in group 1 did not differ significantly in size or estradiol concentrations, but levels of proteolytic activity against IGFBP-4 and -5 were higher in F1–F2 than in F3–F4. However, in group 2 the largest follicle (F1) had higher estradiol, free IGF-I, and IGFBP-4 and -5 proteolytic activity than F2–F4, whereas only slight (dissected) or no (ultrasound) differences in diameters could be detected. Differences between F1 and F2–F4 in diameter, estradiol, free IGF-I, and IGFBP-4 and -5 proteolytic activity were even greater in group 3. In addition, the hormonal regulation of IGFBP-4 and -5 proteolysis was evaluated in vivo by injecting heifers with small doses of recombinant bovine FSH to induce codominant follicles (group 4). The induced codominant follicles were larger and had higher IGFBP-4 and -5 proteolytic activity than subordinate follicles. The results suggest that follicular selection is a progression of changes starting with acquisition of an FSH-inducible IGFBP-4/-5 proteolytic activity, leading to an increase in intrafollicular concentration of free IGF-I that, in turn, synergizes with FSH to promote greater estradiol production by the follicle destined for dominance. |
Introduction |
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It was hypothesized that granulosa cells of the dominant follicle acquire LH receptors that allow them to increase estradiol synthesis in response to both LH and FSH and thereby increase their production of estradiol. Although the dominant follicle has higher levels of mRNA for gonadotropin receptors than subordinate follicles (13), this change appears to be a result, rather than a cause, of selection for dominance (7). Another possibility is that the dominant follicle amplifies the FSH signal via an intrafollicular mechanism. Because follicles of the Igf1 null mouse arrest at the late preantral/early antral stage (14) and IGF synergizes with FSH to promote estradiol production by granulosa cells (15), the intrafollicular IGF system became a target for studies of follicular dominance. Although concentrations of total (free plus bound) IGF-I and -II were reported to be the same in dominant and subordinate follicles of cattle (16, 17), more recent observations and experiments in vivo have suggested a positive association between free IGF and selection for dominance (18, 19).
Consistent with the idea that an increase in free IGF is important in follicular selection, low molecular weight IGFBPs (IGFBP-2, -4, -5), which are believed to inhibit IGF actions on follicular cells by sequestering and thereby decreasing the bioavailability of IGFs, are barely detectable in follicular fluid of dominant and preovulatory follicles but are readily detectable in subordinate follicles of humans (20) and several domestic species (21, 22, 23). Closer to the time of follicular selection in cattle, shortly after one follicle can be distinguished as larger than others of the cohort, the largest follicle has lower levels of low molecular weight IGFBPs than the other follicles of the cohort (10). In addition, a recent study by Mihm et al. (24), in which follicular fluid was sampled in vivo from cohort follicles before morphological differentiation (i.e. size difference) of the dominant follicle in cattle, showed that the follicle with the lowest concentration of IGFBP-4 always became dominant. The negligible levels of low molecular weight IGFBPs in dominant follicles could potentially be achieved by a decrease in gene expression or by an increase in proteolytic degradation (20, 21). We showed that, shortly after morphological selection, the dominant follicle of cattle has an FSH-inducible proteolytic activity against IGFBP-4 (10, 25). More recently, we found that pregnancy-associated plasma protein-A (PAPP-A) in bovine follicular fluid accounts for proteolysis of both IGFBP-4 and -5 and that IGFBP-5 degradation is clearly associated with follicular dominance in cattle (26).
Although, taken together, the results cited above suggest the hypothesis that FSH-induced proteolytic activity against IGFBP-4 and -5 in one of the recruited follicles is a key component of the mechanism of selection of a dominant follicle, direct experimental evidence is lacking. Herein, we report the results of experiments aimed at examining this hypothesis by evaluating temporal relationships between changes in proteolytic activity against IGFBP-4 and -5 and morphological selection of the dominant follicle, using cattle as the experimental model. Cattle provide an excellent model for studies of follicular selection for dominance because the development of large antral follicles occurs in waves at regular intervals of 7–10 d throughout the 21-d estrous cycle. The development and regression of follicles in these waves of follicular development can be easily tracked in vivo by repeated ultrasonography and the follicles are large enough to provide abundant follicular fluid and cells for studies in vitro. Bovine follicles were isolated before, around the time of, or just after selection of the dominant follicle and follicular fluid analyzed for steroids, IGFBPs, free IGF-I and IGFBP-4/-5 proteolytic activity. In addition, the potential role of FSH in the regulation of intrafollicular degradation of IGFBP-4 and IGFBP-5 was investigated.
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Materials and Methods |
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(25 mg im; Lutalyse, Pharmacia \|[amp ]\| Upjohn Co., Kalamazoo, MI) during the mid-luteal phase to induce luteolysis, a follicular phase and ovulation. To characterize intrafollicular changes in components of the IGF system, follicles were isolated after ovariectomy performed at different times during the first follicular wave of the next estrous cycle. Starting the day before prostaglandin F2 injection, the ovaries of each heifer were examined daily for the first 48 h and every 12 h thereafter by transrectal ultrasonography to monitor follicular dynamics, as described previously (7). Carefully planned ultrasonographic studies of bovine ovaries have shown previously that when the largest follicle in the cohort of growing follicles reaches approximately 8.3 mm in diameter the future dominant follicle begins to grow at a faster rate than the others (27); this rapid divergence in size is referred to as "follicular deviation" or "morphological differentiation." Accordingly, ovariectomies were performed before the expected time of follicular deviation as detected by ultrasonography in group 1 (largest follicle, 7 mm in diameter; n = 5), at about the expected time of follicular deviation in group 2 (largest follicle, 8 mm; n = 4), or after differentiation of a dominant follicle in group 3 (largest follicle, ≥9 mm; n = 4). In the current study, sampling times were designed to occur at predetermined follicular sizes rather than at predetermined intervals after the emergence of the follicular wave as previously reported by our laboratory (7, 10). This is a new approach used in our laboratory that was specifically designed to unveil previously uncharacterized temporal associations between biochemical and morphological changes during follicular development.
The regulation in vivo of components of the intrafollicular IGF system by FSH was also investigated (group 4; n = 4). To this end, codominant follicles were induced during the first follicular wave by injecting small doses of FSH (25) to prevent the spontaneous decline in serum FSH concentrations that occurs during selection and the establishment of follicular dominance (28). When the largest follicle of the first follicular wave reached 6 mm in diameter, recombinant bovine (rb) FSH (lot no. IG-07-11088; Granada Biosciences, Marquez, TX) was injected im every 12 h (three injections of 2 mg each, total dose = 6 mg) and ovaries were removed 12 h after the last injection of rbFSH. Thus, the FSH treatment was slightly modified (smaller total dose and shorter treatment) in the present experiments compared with the FSH regime used in our previous study (25). A diagram of the experimental design is shown in Fig. 1
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Analysis of IGFBP-4 and IGFBP-5 proteolytic activity
The ability of intrafollicular proteases to degrade IGFBP-4 and -5 (Austral Biologicals, San Ramon, CA) was assessed by incubating 5 µl of follicular fluid plus substrate for 18 h at 37 C in a solution of 20 mM Tris (pH 7.5) containing 137 mM NaCl and 0.1% BSA (final volume, 20 µl). When recombinant human (rh) IGFBP-4 (50 ng) was used as substrate, SDS-PAGE was followed by Western ligand blotting/phosphorimaging (Fuji BAS 1000, Tokyo, Japan) to quantify the percent of substrate loss as described (10). Proteolysis of IGFBP-5 was assessed by incubating follicular fluid with approximately 50,000 cpm of [125I]- labeled rhIGFBP-5 for 18 h at 37 C. This assay was chosen to assess proteolytic activity against IGFBP-5 because the presence of abundant endogenous IGFBP-5 in subordinate follicles could interfere with determinations by Western ligand blotting if non-radioactive rhIGFBP-5 were used as substrate. Iodination of rhIGFBP-5 was performed by the chloramine-T method as previously described (29). IGFBP-5 protease assay samples were resolved by 12% SDS-PAGE and the gels subjected to autoradiography and phosphorimaging.
Western ligand blot analysis
Western ligand blot analysis was performed as previously described (10). Briefly, samples were subjected to electrophoresis in 12% sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and transferred onto nitrocellulose membranes. The membranes were incubated with 1.2 x 106 cpm of [125I]IGF-II (SA: 340–430 µCi/µg) overnight at 4 C. The nitrocellulose membranes were washed, air-dried, and subjected to autoradiography and phosphorimaging as previously described (10). Relative abundance (%) of endogenous IGFBPs was expressed as the ratio 100 x IIGFBPx/ IIGFBP, where IIGFBPx = intensity (arbitrary units) of the particular IGFBP (either IGFBP-2, -3, -4, or -5) band, and IIGFBP = sum of intensity (arbitrary units) for all IGFBPs (-2, -3, -4, and -5) for samples from each individual follicle incubated for 0 h. For each follicular fluid sample, proteolytic activity was expressed as percent of substrate loss after 18 h of incubation relative to non-incubation. Molecular weights of intact IGFBP species were estimated by running the samples in parallel with protein molecular weight standards (2,850–43,000 molecular weight range; Life Technologies, Inc., Grand Island, NY) on the same gel.
Determination of steroids, free IGF-I, and protein concentrations in follicular fluid
Duplicate aliquots (0.1–5 µl) of follicular fluid from each follicle were assayed without extraction for estradiol and progesterone as described previously (7). Free IGF-I concentrations in follicular fluid were determined by a two-site immunoradiometric assay kit (Diagnostic Systems Laboratories, Webster, TX). Serial dilutions of a pool of bovine follicular fluid resulted in a dose-response curve similar to the standard curve. The sensitivity of the assay was 0.03 ng/ml and the intraassay coefficient of variation for quality control samples was 12%. All the samples were run in a single assay. Total protein concentrations in samples of follicular fluid were measured in duplicate according to the Bradford method (30) using reagents purchased from Bio-Rad Laboratories, Inc. (Melville, NY).
Statistical analysis
The four largest follicles per heifer were ranked by size (diameter) in descending order (F1–F4). When follicles had similar sizes (one animal in group 1), the follicle with higher estradiol in the follicular fluid was assigned the higher rank. Data were analyzed by ANOVA with a nested design to evaluate the effects of groups 1–4, animal (nested within group), follicle type (F1–F4) and the interaction of group by follicle type using the General Linear Models procedure of the Statistical Analysis System (SAS Institute, Cary, NC). Bartlett’s test was used to test for heterogeneity of variance. When appropriate, logarithmic or square root transformations were used to yield variance homogeneity. After ANOVA, means within-follicle type and across-groups or within-groups and across-follicle type were compared by Tukey’s multiple comparison test. Comparisons with P ≤ 0.05 were considered statistically significant. Values are presented as means ± SEM of untransformed variables. Simple correlation coefficients (Pearson) were calculated to assess relationships among IGFBP-4 or -5 proteolysis and follicular diameter (after dissection), estradiol, IGFBP-4, IGFBP-5, and free IGF-I concentrations in follicular fluid.
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Results |
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. Comparisons among follicles within groups showed no differences in either ultrasound or dissected diameters in group 1, the earliest time of follicle isolation. In group 2, the largest follicle (F1) was significantly larger than the rest of follicles examined when dissected diameters were compared, but not when ultrasound diameters were compared. In contrast, in group 3 the largest follicle had greater ultrasound and dissected diameters than the rest of the follicles examined. Comparison of follicles in the same category (F1, F2, F3, or F4) across experimental groups showed that the fairly rapid development of a size difference between the future dominant (F1) and the future subordinate follicles (F2–F4) from group 1 to group 3 was due to an increase in the diameter of F1, but not F2–F4.
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, G4) that were similar in diameter. Mean diameters of codominant follicles (F1/F2) of group 4 did not differ from that of the single dominant follicle (F1) of group 3. Codominance was also indicated by the significantly greater diameter of F2 in group 4 compared with groups 1–3 (Fig. 2).
Intrafollicular concentrations of steroids and protein in follicles obtained at different times during the first follicular wave and after FSH stimulation
Follicles in group 1 had similar concentrations of estradiol in the follicular fluid (Fig. 3). However, in groups 2 and 3, estradiol concentrations were higher in follicular fluid of the largest follicle than in the rest of follicles examined. Comparisons of follicles in the same category (F1, F2, F3, or F4) across experimental groups showed that estradiol concentrations in F1 increased progressively from group 1 to group 3. Estradiol levels in F2 did not differ between groups 1 and 2 but were slightly higher in group 3. Thus, as follicular selection proceeded (group 1 to group 3), intrafollicular estradiol concentrations increased dramatically in F1, modestly in F2, and not at all in F3–F4, so that by the time follicles of group 3 were isolated, functional differentiation of the dominant follicle was obvious. In contrast, concentrations of progesterone in follicular fluid did not differ in F1–F4, within or among groups (Fig. 3).
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). As in groups 1–3, the follicles of group 4 did not differ in progesterone concentrations.
No significant differences were observed in protein concentrations in the follicular fluid, either among follicle categories within groups or among groups within a given follicle category (Fig. 3). Therefore, the analyses of IGFBPs and their proteases reported below were performed on samples of follicular fluid that did not differ in protein concentrations.
Relative abundance of IGFBPs in follicular fluid of follicles obtained at different times during the first follicular wave and after FSH stimulation
Follicular fluid samples from each of the four largest follicles (F1–F4) obtained per animal were subjected to SDS-PAGE followed by ligand blotting. The identities of the different IGFBPs were determined by their molecular mass. Immunoprecipitation studies (16) have previously shown that bovine follicular fluid contains four IGFBPs (Mr 10-3): IGFBP-3 (49 and 43), IGFBP-2 (35), IGFBP-5 (30), and IGFBP-4 (28 and 22). Representative ligand blots of follicular fluid samples from one animal from each of the four experimental groups are shown in Fig. 4 (endogenous IGFBPs, lanes 1–4) and Fig. 5 summarizes the quantitative data derived from all the replicates. Statistical analysis showed no differences in levels of IGFBP-3 among follicle categories within groups or during the course of follicular selection (groups 1–3; quantitative data not shown). In contrast, differences in levels of the low molecular weight IGFBPs (IGFBP-2, -5, and -4) are readily apparent. Relative abundance of IGFBP-2 did not vary among follicle categories in groups 1 and 2 (Fig. 5). However, IGFBP-2 levels were lower in the dominant (F1) than in subordinate follicles of group 3 and lower in the F1 of group 3 than in the F1 of groups 1 and 2. The difference in IGFBP-2 levels between F1 and the other follicles in group 3, but not in groups 1 and 2, suggests that down-regulation of levels of IGFBP-2 occurred after morphological differentiation of the dominant follicle. On the other hand, levels of endogenous IGFBP-5 and -4 seemed to increase gradually with decreasing follicle size (F1–F4) in groups 1–3. Relative abundance of IGFBP-5 did not vary among follicle categories in group 1. However, relative levels of IGFBP-5 were lower in the largest follicle than in the other follicles in both groups 2 and 3. Thus, a difference in IGFBP-5 levels in the largest vs. future subordinate follicles occurred at about the time the dominant follicle could first be detected as the largest. In group 1, relative abundance of IGFBP-4 did not differ between F1 and F2, but was lower in F1 vs. F3–F4. In groups 2 and 3, the largest follicle had less IGFBP-4 than the other follicles. Therefore, lower levels of IGFBP-4 preceded morphological differentiation of follicles presumably destined for dominance.
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and 5). Levels of IGFBP-2, -5, and -4 did not differ in the codominant follicles (F1–F2), but they were higher in F4 and intermediate in F3 (Fig. 5). In addition, the levels of low molecular weight IGFBPs in the codominant follicles of FSH-treated animals (group 4) did not differ from those of the single dominant follicle of group 3. Codominance was also indicated by the significantly lower levels of low molecular weight IGFBPs in F2 of group 4, compared with F2 of groups 1–3 (Fig. 5).
Proteolytic activity for IGFBP-4 and IGFBP-5 and concentrations of free IGF-I in follicular fluid of follicles obtained at different times during the first wave and after FSH stimulation
Proteolytic activity for IGFBP-4.
Proteolytic activity for IGFBP-4 was determined by incubating 5 µl of follicular fluid with 50 ng of rhIGFBP-4 followed by ligand blot analysis/phosphorimaging. Representative ligand blots of IGFBP-4 protease assays of follicular fluid from one animal from each group are shown in Fig. 4 (lanes 5–9) and quantitative data derived from all animals in Fig. 6. In group 1, proteolytic activity for IGFBP-4 was significantly higher in F1 + F2 than in F3 + F4 and higher in F1 vs. F3-F4, but not vs. F2. In contrast, in groups 2 and 3 IGFBP-4 proteolytic activity was higher in the largest vs. all other follicles examined. Across-group comparisons of follicles in the same category showed decreased IGFBP-4 proteolytic activity in F2 of groups 2–3 vs. group 1. In contrast, IGFBP-4 proteolysis did not differ for F1 among groups, suggesting that increased IGFBP-4 proteolysis is a very early change during differentiation of the future dominant follicle.
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and 6). Codominance was also indicated by the significantly higher levels of IGFBP-4 proteolytic activity in F2 of group 4, compared with groups 2 and 3 (Fig. 6).
Proteolytic activity for IGFBP-5.
Proteolytic degradation of IGFBP-5 was assessed by incubating 5 µl of follicular fluid with [125I]IGFBP-5 as a substrate followed by quantification of substrate loss by phosphorimaging (see Fig. 7 for representative ligand blots and Fig. 6 for quantitative data). This assay was chosen to assess proteolytic activity against IGFBP-5 because the presence of abundant endogenous IGFBP-5 in subordinate follicles could interfere with determinations by Western ligand blotting if nonradioactive rhIGFBP-5 were used as substrate. Substrate loss was accompanied by the expected appearance of radiolabeled fragments of approximately 19 and 17 kDa after 18 h of incubation (Fig. 7). In group 1, IGFBP-5 proteolytic activity in F1 + F2 was significantly higher than in F3 + F4 (Fig. 6). However, in groups 2 and 3, IGFBP-5 proteolytic activity was higher in the largest follicle than in all other follicles examined. Comparisons of follicles in the same category (F1, F2, F3, or F4) across experimental groups showed increased IGFBP-5 proteolytic activity in the largest follicle of group 3 vs. groups 1 and 2. Conversely, IGFBP-5 proteolytic activity in F2 and F3 was lower in groups 2 and 3 vs. group 1. Thus, as follicular selection proceeded, proteolytic activity against IGFBP-5 increased in the dominant follicle but decreased in subordinate follicles.
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and 7). Levels of IGFBP-5 proteolytic activity in the codominant follicles of FSH-treated animals (group 4) did not differ from those of the single dominant follicle of group 3. Codominance was also indicated by the significantly higher levels of IGFBP-5 proteolytic activity in F2 of group 4, compared with groups 2 and 3 (Fig. 6).
Concentrations of free IGF-I.
Concentrations of free IGF-I in follicular fluid exhibited a pattern similar to that for IGFBP-4 proteolytic activity (Fig. 6). In group 1, free IGF-I was higher in F1 vs. F3–F4, but not vs. F2. In contrast, free IGF-I concentrations were higher in the largest follicle than in the rest of the follicles examined in groups 2 and 3. Comparisons within follicle category and across-groups showed higher concentrations of free IGF-I in the largest follicle of group 3 vs. group 2 and intermediate levels in group 1. In contrast, concentrations of free IGF-I in the F2, F3, and F4 did not differ among groups 1–3. In FSH-treated animals (group 4), no differences in free IGF-I were detected among F1–F3, but free IGF-I concentrations were higher in F1 vs. F4.
Correlations between follicular parameters
To examine further which characteristics, of those examined, are predictive of follicular dominance, overall correlation coefficients (Pearson) between IGFBP-4 or -5 protease activity in follicular fluid and follicular diameters (after dissection), estradiol, IGFBP-4, IGFBP-5, and free IGF-I concentrations were determined (Table 1). IGFBP-4 and -5 protease activities were positively correlated with follicular diameters, estradiol and free IGF-I concentrations in follicular fluid. In contrast, proteolytic activities for IGFBP-4 and -5 were negatively correlated with intrafollicular concentrations of IGFBP-4 and IGFBP-5.
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shows partial correlation coefficients between several characteristics of follicles obtained at different times during the first follicular wave and after FSH stimulation. In group 1, no significant association between estradiol and follicular size (dissected diameter) or IGFBP-4 or IGFBP-5 protease activity was observed. Of the correlations tested for group 1, follicular diameter was positively associated only with IGFBP-4 or -5 protease activities. In contrast, in groups 2–4, the pairs of variables were all positively correlated. Thus, the lack of association between estradiol and diameter or IGFBP-4 or -5 proteolytic activity in group 1 (in contrast to the positive association between these variables in groups 2–4) and the positive correlations between diameters and IGFBP-4 and -5 proteolytic activity in all the experimental groups (groups 1–4) suggest that increased proteolysis of IGFBP-4/-5 precedes the increase in estradiol in the follicular fluid and that it is one of the earliest changes leading to morphological differentiation of the dominant follicle.
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Discussion |
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As reviewed in the Introduction, previous studies established higher intrafollicular concentrations of estradiol, lower concentrations of IGFBP-4/-5, and higher proteolytic activity against IGFBP-4 and -5 (PAPP-A) as characteristics of dominant follicles isolated shortly after morphological selection. These findings suggested the hypothesis that before morphological selection FSH induces PAPP-A, which lowers levels of IGFBP-4/-5, increasing the amount of free IGF available to synergize with FSH to increase estradiol secretion. To test this hypothesis requires determining the relationship among these factors in the future dominant vs. subordinate follicles. Therefore, our approach in the current studies involved isolating follicular cohorts at specific and carefully selected times: 1) before, 2) during, and 3) after the development of a size differential among the cohort. The results showed that these three experimental groups were developmentally distinct, based on follicular diameter and endocrinological/biochemical characteristics. Most important is group 1, the follicles isolated before any significant differences in follicular diameter, but groups 2 and 3 are also essential to show the developmental progression of follicular characteristics, as follicles differentiate as either dominant or subordinate. Taken together, the results show clearly that an increase in proteolytic activity for IGFBP-4/-5 (PAPP-A) precedes the increase in estradiol in the dominant follicle, suggesting that the increased PAPP-A degrades IGFBP-4 and -5 in the future dominant follicle, freeing more IGF to synergize with FSH and increase estradiol production.
Interestingly, although no significant differences in follicular diameter were detected in group 1 and only a slight difference in group 2, size does appear to matter at those stages. There was a gradation in follicular characteristics based on diameter, even when the differences were not significant, especially in group 1. Although the earliest detectable significant differences among the follicles of a cohort were in levels of IGFBP-4, IGFBP-4/-5 proteolytic activity and free IGF-I, the two largest follicles of group 1 were not significantly different from each other in these characteristics. In agreement with this, Austin et al. (31) reported that the two largest of cohorts of five bovine follicles isolated on d 2.5 of the cycle had lower IGFBP-4 and -5 concentrations than the two smallest, with intermediate levels in the F3. However by the time the follicles of group 2 were isolated in the current study, just a few hours later than group 1, the F2 was clearly out of the running, and the F1 was clearly different from all other follicles, having higher PAPP-A, free IGF, and estradiol and lower levels of low molecular weight IGFBPs. This suggest not only that increased intrafollicular PAPP-A is the earliest change during selection of the dominant follicle, but also that initially there is a back-up follicle, the F2, very similar to the future dominant follicle. Regardless, the correlational analyses of the relationships between the various follicular characteristics examined in the present study clearly point to the increase in proteolytic activity for IGFBP-4/-5 as the earliest developmental change, among those examined, that predicts future dominance.
The results for group 3 show a further developmental progression in which the differences between the dominant and subordinate follicles in diameter, estradiol, free IGF-I, IGFBP-4 and -5, and proteolytic activity for IGFBP-4 and -5 were even more pronounced than in group 2, and an additional difference, lower levels of IGFBP-2 in the follicular fluid of the dominant vs. subordinate follicles, first emerged. Thus, the decrease in IGFBP-2 seems to follow, rather than to precede, follicular selection for dominance. Evidence from previous studies suggests that the decrease in IGFBP-2 is due to a decrease in mRNA for IGFBP-2 (32, 33), rather than induction of proteolytic activity for IGFBP-2 (26, 29).
The results for group 4 show that the characteristics critical for dominance can be induced by FSH in an experimental situation. These results are consistent with studies in vitro showing that FSH increased IGFBP-4 protease activity in cultures of human granulosa cells (34) and stimulated the production by rat granulosa cells of a protease that degrades both IGFBP-4 and -5 (35). More recently, an FSH-inducible IGFBP-5 metalloprotease produced by rat granulosa cells was characterized (36).
Interestingly, intrafollicular concentrations of free IGF-1 in the largest follicle (F1) seemed to fluctuate among G1-G3 (with an apparent, although nonsignificant, decrease in G2 vs. G1 and a significant increase in G3), whereas intrafollicular estradiol concentrations increased steadily in the largest follicle from G1-G3. A recent, independent study has shown a very similar pattern of fluctuation for the concentration of free IGF-I, but not for estradiol, in the follicular fluid of the largest follicle of a cohort of bovine follicles sampled at similar stages of development (37). The physiological significance, if any, of fluctuations in free IGF levels in the follicular fluid of the largest follicle is as yet unknown. Efficient proteolysis of IGFBP-4 requires the presence of IGF, whereas the cleavage of IGFBP-5 by PAPP-A is slightly inhibited by IGF as shown in vitro with purified recombinant proteins (38). In addition, recent studies also suggest that PAPP-A is capable of cleaving synthetic peptides (with no IGF binding capacity) derived from 22–26 residues spanning the cleavage site of IGFBP-4 (39) and of autocleavage (40). Taken together, those studies clearly demonstrate that IGF is not a direct activator of PAPP-A and that IGFBP-4 becomes a better substrate of PAPP-A when complexed with IGF. It is tempting to speculate that earlier expression of PAPP-A activity in one of the follicles of the cohort may initially trigger IGFBP-5 degradation (no IGF required) and increase the pool of IGF available for complexing with IGFBP-4, which then would became a better substrate for subsequent degradation by PAPP-A. Clearly, testing of this and related hypotheses will require additional work.
In summary, these results provide unique insight into the temporal sequence of changes that comprises the mechanism of follicular selection for dominance (Fig. 8). The data suggest a sequence in which the follicle targeted for selection first acquires an FSH-induced protease (PAPP-A), which, by degradation of IGFBP-4 and -5, increases the intrafollicular concentration of free IGF. The increased availability of IGF for interaction with its receptor then leads to increased FSH-responsiveness and greater estradiol production by the future dominant follicle. It is not clear why only one follicle responds to FSH with differentiative changes that lead to dominance, but the correlation between follicular size and IGFBP-4/-5 proteolytic activity suggests that the future dominant follicle is, by chance, slightly more advanced than others in the recruited cohort and that the induction of proteolytic activity for IGFBP-4/-5 quickly amplifies that small initial advantage into a much greater capacity to produce estradiol. The increase in circulating estradiol decreases circulating FSH and thereby limits the growth of future subordinate follicles and destines them for atresia. Although the temporal sequence of biochemical and developmental changes reported in this study suggests a critical role for PAPP-A in follicular selection, direct evidence for the above will require the experimental control of levels of expression and/or activity of the intrafollicular PAPP-A proteolytic system.
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Acknowledgments |
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Footnotes |
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1 Current address: Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030-3301. 
Abbreviations: IGFBP, IGF binding protein; PAPP-A, pregnancy- associated plasma protein-A; rb, recombinant bovine; rh, recombinant human.
Received November 25, 2002.
Accepted for publication February 24, 2003.
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 M. Matsui, B. Sonntag, S. S. Hwang, T. Byerly, A. Hourvitz, E. Y. Adashi, S. Shimasaki, and G. F. Erickson Pregnancy-Associated Plasma Protein-A Production in Rat Granulosa Cells: Stimulation by Follicle-Stimulating Hormone and Inhibition by the Oocyte-Derived Bone Morphogenetic Protein-15 Endocrinology, August 1, 2004; 145(8): 3686 - 3695. [Abstract] [Full Text] [PDF]
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L. J. Spicer Proteolytic Degradation of Insulin-Like Growth Factor Binding Proteins by Ovarian Follicles: A Control Mechanism for Selection of Dominant Follicles Biol Reprod, May 1, 2004; 70(5): 1223 - 1230. [Abstract] [Full Text] [PDF]
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O.J. Ginther, E.L. Gastal, M.O. Gastal, and M.A. Beg Critical Role of Insulin-Like Growth Factor System in Follicle Selection and Dominance in Mares Biol Reprod, May 1, 2004; 70(5): 1374 - 1379. [Abstract] [Full Text] [PDF]
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A.C.O. Evans, J.L.H. Ireland, M.E. Winn, P. Lonergan, G.W. Smith, P.M. Coussens, and J.J. Ireland Identification of Genes Involved in Apoptosis and Dominant Follicle Development During Follicular Waves in Cattle Biol Reprod, May 1, 2004; 70(5): 1475 - 1484. [Abstract] [Full Text] [PDF]
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 S. Jonard and D. Dewailly The follicular excess in polycystic ovaries, due to intra-ovarian hyperandrogenism, may be the main culprit for the follicular arrest Hum. Reprod. Update, March 1, 2004; 10(2): 107 - 117. [Abstract] [Full Text] [PDF]
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O.J. Ginther, D.R. Bergfelt, M.A. Beg, C. Meira, and K. Kot In Vivo Effects of an Intrafollicular Injection of Insulin-Like Growth Factor 1 on the Mechanism of Follicle Deviation in Heifers and Mares Biol Reprod, January 1, 2004; 70(1): 99 - 105. [Abstract] [Full Text] [PDF]
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S. M. Quirk, R. G. Cowan, R. M. Harman, C.-L. Hu, and D. A. Porter Ovarian follicular growth and atresia: The relationship between cell proliferation and survival J Anim Sci, January 1, 2004; 82(13_suppl): E40 - 52. [Abstract] [Full Text] [PDF]
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R. Webb, P. C. Garnsworthy, J.-G. Gong, and D. G. Armstrong Control of follicular growth: Local interactions and nutritional influences J Anim Sci, January 1, 2004; 82(13_suppl): E63 - 74. [Abstract] [Full Text] [PDF]
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