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Biology of Reproduction 66, 120-126 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Follicle Selection in Cattle: Dynamics of Follicular Fluid Factors During Development of Follicle Dominance1

M.A. Bega, D.R. Bergfelta, K. Kota,b, and O.J. Ginther2,a,b

a Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706 b The Eutherian Foundation, Cross Plains, Wisconsin 53528


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Follicle diameter deviation during follicular waves in cattle begins with a reduction in growth rates of developing subordinate follicles, in contrast to the maintenance of a constant growth rate by a developing dominant follicle. In experiment 1, the temporal changes encompassing deviation in concentrations of follicular fluid factors relative to one another in the three largest follicles (F1, F2, and F3) were studied. Follicular fluid samples were collected when F1 reached diameter ranges of 7.0–7.9, 8.0–8.9, 9.0–9.9, and 10.0–10.9 mm (n = 12 per range). The first increase (P < 0.05) in the difference between F1 and F2 for estradiol occurred at the 8.0- to 8.9-mm range, which was one range earlier than for diameter (P < 0.05). Free insulin-like growth factor (IGF)-1 concentrations in F1 were similar among diameter ranges, but concentrations in F1 were higher (P < 0.05) than in F2 for each range except 7.0–7.9 mm. Concentrations of free IGF-1 in F2 decreased (P < 0.05). No significant differences were detected in concentrations of progesterone, androstenedione, total inhibin, and inhibin-A. Averaged over follicles, inhibin-B decreased (P < 0.05) between the 8.0- to 8.9- and 10.0- to 10.9-mm ranges, and activin-A increased (P < 0.05) between the 7.0- to 7.9- and 9.0- to 9.9-mm ranges. However, no differences were found among follicles. In experiment 2, changes associated with the development of dominance by F2 were studied using ablation of F1 at the beginning of expected deviation (F1, 8.5 mm; Hour 0) as the reference point. Follicular fluid factors were compared at Hour 12 between F2 of a control group (F1 intact; n = 10) and an ablated group (F1 ablated; n = 10). Diameter (P < 0.02), estradiol (P < 0.001), free IGF-1 (P < 0.002), and progesterone (P < 0.003) were greater and IGF-binding protein-2 was lower (P < 0.01) in F2 of the ablated group at Hour 12. No differences were detected in concentrations of androstenedione, total inhibin, and inhibin-A. The results of the two experiments indicated, on a temporal basis, that intrafollicular changes in estradiol and the IGF system, but not in the inhibin/activin system, could account for a reported greater FSH responsiveness by the future dominant follicle than by the future subordinate follicles by the beginning of diameter deviation in cattle.

follicle, growth factors, inhibin, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Development of a follicular wave in monovular species is a complex process during which a single follicle continues to grow and develop while the rest of the follicles cease growing and regress. The beginning of the difference in growth rates between the two largest follicles is termed follicle deviation [1] and, in cattle, occurs when the largest follicle reaches a mean diameter of 8.5 mm [2]. The process of follicle selection is under systemic regulation by FSH and LH and local regulation by factors that modulate the actions of gonadotropins [3]. Although not convincingly demonstrated, other factors may act independently of gonadotropins in follicle selection [4].

It has been proposed [5] that a close, two-way functional coupling between FSH and the follicles is an integral component of the deviation mechanism. During the common growth phase that precedes deviation, FSH concentrations decline, and the FSH/follicle two-way coupling involves multiple follicles [5, 6]. However, the coupling involves only the future dominant follicle by the beginning of deviation [5]. The change from multiple- to single-follicle coupling is attributed to the development of greater responsiveness by the future dominant follicle than by the future subordinate follicles to the low FSH concentrations at the beginning of deviation. To our knowledge, the developmental changes that increase the FSH responsiveness of the future dominant follicle have not been clarified.

Candidates for a role in increasing the FSH responsiveness by the developing dominant follicle in cattle include estradiol, insulin-like growth factors (IGFs), and inhibin/activin peptides [3]. Estradiol enhances aromatase activity, promotes expression of LH receptors, increases sensitivity of granulosa cells to FSH and LH [7], and increases the synthesis of IGF-1 from granulosa cells [8]. At the beginning of deviation, follicular fluid concentrations of estradiol in heifers begin to increase in the future dominant follicle, but not in the future largest subordinate follicle [9, 10]. The concentrations of other follicular fluid steroids at deviation and their potential role in deviation are not clear. The IGFs are potent mitogens [11], and they function as modulators of gonadotropin action on granulosa and theca cells [3]. The IGF-binding proteins (IGFBPs) exert a pivotal role in regulation of IGF bioavailability by selectively binding to IGFs and making them unavailable to their receptors [4]. Changes in total IGF-1 in follicular fluid have been reported, although not in reference to deviation [12–14]. In a recent study [10], no significant changes in the concentration of free IGF-1 were detected in the largest follicle encompassing deviation, but the concentrations of IGFBP-2 decreased beginning at deviation. Follicular fluid concentration of free IGF-1 decreased and of IGFBP-2 increased in the second-largest follicle when the diameter of the largest follicle ranged between 7.5 and 11.2 mm, a period encompassing deviation. Activins and inhibins also have autocrine/paracrine actions on granulosa and theca cells [15]. No significant changes have been detected in total inhibin and inhibin-A concentrations in the two largest follicles encompassing deviation [10]. The changes in follicular fluid concentration of inhibin-B in the three largest follicles during deviation in cattle have, apparently, not been studied. In women, follicular fluid concentrations of inhibin-A, but not of inhibin-B, increase in the largest follicle as both diameter and maturity of the follicle increased [16]. However, the temporal relationship between the inhibin-A increase and the beginning of presumptive deviation has not been determined in women.

After ablation of the largest follicle at the expected beginning of deviation in cattle, the second-largest follicle usually becomes the dominant follicle [5, 17]. The increase in diameter of the second-largest follicle for 12 h after ablation of the largest follicle at the beginning of deviation is comparable to the growth of the largest follicle 12 h before the beginning of deviation [17]. Thus, a future subordinate follicle that was destined to regress in the presence of a dominant follicle changed its course after removal of the future dominant follicle. This phenomenon provides a model in which the time of ablation of the largest follicle serves as a reference point to establish the sequence of events leading to the development of dominance by the second-largest follicle.

In the present study, experiment 1 was done to characterize the temporal changes and relationships encompassing follicle deviation in follicular fluid concentrations of estradiol, estrone, progesterone, androstenedione, free IGF-1, total inhibin, inhibin-A, inhibin-B, and activin-A in the three largest follicles. The results of experiment 1 served as the rationale for the hypotheses that follicular fluid concentrations of estradiol, estrone, and free IGF-1 increase; that concentrations of IGFBP-2 decrease; and that concentrations of androstenedione, progesterone, total inhibin, and inhibin-A do not change within 12 h during establishment of dominance. These hypotheses were tested in experiment 2 by studying the response of the second-largest follicle to ablation of the largest follicle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Ultrasound Scanning

Two experiments were conducted during the wave that emerges in the periovulatory period (wave 1). The animals were Holstein heifers between 24 and 36 mo of age and weighing from 490 to 680 kg. The feeding program and the prostaglandin F2{alpha} (Lutalyse; Pharmacia and Upjohn Co, Kalamazoo, MI) protocol for inducing luteolysis to schedule ovulation as well as the equipment and techniques for transrectal ultrasound scanning of ovaries and measuring follicles have been described elsewhere [5]. Scanning was done every 24 h for measuring and tracking the follicles, beginning on the day of induced luteolysis at mid-diestrus and continuing until the largest follicle of the new wave reached 6.0 mm. Follicular fluid samples were collected by ultrasound-guided, transvaginal aspiration with a 20-gauge needle using a 5.0-MHz convex-array transducer equipped with a 50-cm extension for intravaginal access as described elsewhere [9]. The follicular fluid samples were centrifuged at 500 x g for 10 min, decanted, and stored at -20°C until hormone analyses. The cell pellet was discarded.

Experiment 1

Forty-eight heifers were randomized into four groups (n = 12 per group) and scanned daily to monitor the follicular growth until aspiration of follicular fluid. Follicular fluid was aspirated from the three largest follicles when the largest follicle reached 7.0–7.9, 8.0–8.9, 9.0–9.9, or 10.0–10.9 mm. These diameter ranges were chosen to encompass the expected time of deviation or when the future dominant follicle reached 8.5 mm based on the results of previous studies [3]. At the time of sampling, the three follicles were designated as F1, F2, and F3, based on descending diameter. Follicular fluid samples were assayed for estradiol, free IGF-1, progesterone, androstenedione, estrone, total inhibin, inhibin-A, inhibin-B, and activin-A.

Experiment 2

Twenty heifers were randomized into a control group and an ablated group (n = 10 per group). The ultrasound scanning was done every 12 h, beginning when the largest follicle was 6.0 mm and continuing until sampling of follicular fluid. Ablation (Hour 0) was done when the largest follicle (F1) was >=8.2 mm, and the next-largest follicles at Hour 0 were designated as F2 and F3. Ablation of F1 when it reached >=8.2 mm was done so that the actual diameter would approximate the expected diameter at the beginning of deviation (8.5 mm [3]). Follicle ablations were done by an ultrasound-guided, transvaginal procedure as described elsewhere [17]. This method of follicle ablation is functionally effective, as discussed previously [17]. Follicular fluid was collected at Hour 12 from F1, F2, and F3 in the control group and from F2 and F3 in the F1-ablated group. The follicular fluid samples were assayed for estradiol, free IGF-1, IGFBP-2, androstenedione, estrone, total inhibin, and inhibin-A.

Hormone Assays

Follicular fluid concentrations of estradiol [18], progesterone, estrone, androstenedione, free IGF-I, IGFBP-2, total inhibin, and dimeric inhibin-A [10] were determined by procedures that have been described and validated for bovine follicular fluid in our laboratory. The intra- and interassay coefficient of variations (CVs) for quality-control samples and the mean assay sensitivity, respectively, were as follows: estradiol, 7.0%, 6.8%, and 2.0 pg/ml; progesterone, 3.8%, <1.0%, and 0.02 ng/ml; estrone, 4.9%, 2.0%, and 0.4 pg/ml; androstenedione, 1.5%, 4.5%, and 0.07 ng/ml; free IGF-1, 4.2%, 3.4%, and 0.005 ng/ml; total inhibin, 4.7%, 1.8%, and 6.8 ng/ml; and inhibin-A, 4.2%, 3.9%, and 4.2 pg/ml. The intraassay CV and sensitivity for IGFBP-2 were 9.8% and 0.7 ng/ml, respectively.

Follicular fluid concentrations of inhibin-B were determined using a sandwich-type ELISA kit (MCA1312KZZ; Serotec, Inc., Raleigh, NC) that was developed for use with human serum [19]. Monoclonal antibodies were directed against ß-B subunit for capture and {alpha}-C subunit for detection. Color intensity of the enzyme substrate was directly proportional to the concentration. The kit has been documented for use with human follicular fluid [20] and adapted and validated for use with bovine follicular fluid in our laboratory. The kit-supplied standards (15.6–1000 pg/ml) were reconstituted with fetal calf serum, which also served as the zero standard. Serial dilutions (0.25–5 µl) of a pool of bovine follicular fluid in total volume of 100 µl resulted in a dose-response curve that was similar to the standard curve. A working dilution of 1:100 (v/v) in fetal calf serum was used for follicular fluid samples, because 1 µl of pooled follicular fluid resulted in an optical density that was central to the range of the standard curve. Limited cross-reactivity (<0.5%) has been reported with inhibin-A, activin-A, activin-B, follistatin, and pro-{alpha}-C subunits [19]. The intra- and interassay CVs for quality-control samples were 12.2% and 5.6%, respectively. Determinations were made from a pool of bovine follicular fluid. The sensitivity was 1.9 pg/ml (n = 2 assays).

Follicular fluid concentrations of activin-A were determined using a sandwich-type ELISA kit (MCA1426KZZ; Serotec). A monoclonal antibody was directed against ß-A subunit for capture and detection. Color intensity of the enzyme substrate was directly proportional to the concentration. The kit has been documented for use with bovine follicular fluid [21] and adapted and validated for use in our laboratory. The kit-supplied standards (0.078–5 ng/ml) were reconstituted with phosphate-buffered saline containing 5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO), which also served as the zero standard. Serial dilutions (0.025–0.2 µl) of a pool of bovine follicular fluid in total volume of 100 µl resulted in a dose-response curve that was similar to the standard curve. A working dilution of 1:2000 in 5% (v/v) bovine serum albumin was used for follicular fluid samples, because 0.05 µl of pooled follicular fluid resulted in an optical density that was central to the range of the standard curve. The assay has limited cross-reactivity (<0.5%) with a range of related molecules, including inhibin-A, inhibin-B, activin-B, follistatin, and pro-{alpha}-C subunits [21]. The intra- and interassay CVs for quality-control samples were 3.0% and 11.1%, respectively. Determinations were made from a pool of bovine follicular fluid. The sensitivity was 0.02 ng/ml (n = 2 assays).

Statistical Analyses

In experiment 1, the follicular and hormonal data were tested for normality using the Kolmogorov-Smirnov test [22], and suspected outliers were challenged using the Dixon outlier test [23]. The hormonal data for estradiol, progesterone, free IGF-1, estrone, androstenedione, and inhibin-B lacked normality and were transformed logarithmically before the statistical analyses. Follicle diameter and follicular fluid concentrations of estradiol, progesterone, androstenedione, estrone, free IGF-1, total inhibin, inhibin-A, inhibin-B, and activin-A were analyzed with a general linear model using the Statistical Analysis System [24] to determine the effects of follicle (F1, F2, and F3), diameter range, and their interactions. A significant (P < 0.05) effect of follicle, diameter range, or interaction was further analyzed by the Duncan multiple-range test to locate the differences among follicles or diameter ranges. Probabilities between P > 0.06 and P < 0.1 were considered as approaching significance. In experiment 2, differences between control and ablated groups for diameter and various follicular fluid factors were tested by unpaired t-tests.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

Of the initial 48 heifers, follicular fluid could not be collected from 5; therefore, from 9 to 12 heifers were available per group for follicular fluid hormone analyses. In addition, a reduced quantity of follicular fluid was obtained from some follicles of the 9–12 heifers, resulting in 40 follicular fluid samples for analysis of F1 and 38 samples for analysis of F2 and F3. Outlying observations were detected for estradiol, progesterone, estrone, androstenedione, free IGF-1, and total inhibin. For these end points, one to six observations (0.9–5%) were determined as being extreme values and were excluded from further statistical analysis. For inhibin-A, inhibin-B, and activin-A, no extreme values were detected.

Mean changes in diameter and follicular fluid concentrations of estradiol and free IGF-1 for the three largest follicles are shown with the results of statistical analyses in Figure 1. Diameter of F1 increased (P < 0.05) progressively over the four diameter ranges, whereas that of F2 increased (P < 0.05) only between the 7.0- to 7.9- and 8.0- to 8.9-mm ranges. No increase (P > 0.05) was observed in the diameter of F3 over the ranges. The first increase (P < 0.05) in the diameter difference between F1 and F2 occurred at the 9.0- to 9.9-mm range and between F1 and F3 at the 8.0- to 8.9-mm range. Follicular fluid concentrations of estradiol increased (P < 0.05) in F1, but not in F2 or F3, after the 7.0- to 7.9-mm diameter range. The first increase (P < 0.05) in the difference of estradiol concentrations between F1 and F2 and between F1 and F3 occurred at the 8.0- to 8.9-mm range. No significant changes in follicular fluid concentrations of free IGF-1 in F1 were detected over the diameter ranges, but concentrations in F1 were higher (P < 0.05) than in F2 for each diameter range except the first (7.0–7.9 mm) and were higher in F1 than in F3 for each range. Concentrations decreased (P < 0.05) in F2 between the 7.0- to 7.9- and 8.0- to 8.9-mm ranges.



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  		FIG. 1. Mean (±SEM) follicle diameter and follicular fluid estradiol and free IGF-1 concentrations of the largest (F1), second-largest (F2), and third-largest (F3) follicles (n = 9–12 heifers/diameter range) in experiment 1. Main effects and interaction that were significant are shown for each end point (F, main effect of follicle; R, main effect of diameter range; RF = interaction of range by follicle). An asterisk indicates the first increase (P < 0.03) in the difference between F1 and F2 for diameter and estradiol. Within a follicle, means with different letters (ab) among ranges are significantly different (P < 0.05)

Mean changes in follicular fluid concentrations of progesterone, androstenedione, and estrone for the three largest follicles are shown with the results of statistical analyses in Figure 2. No significant changes in follicular fluid concentrations of progesterone or androstenedione were detected. For androstenedione, however, the differences among ranges approached significance (P = 0.12), apparently due, in part, to a higher (P < 0.05) concentration in F1 than in F2 and F3 at the 10.0- to 10.9-mm diameter range. No significant changes in follicular fluid concentrations of estrone in F1 and F2 were detected over the diameter ranges, but the concentrations were higher (P < 0.05) in F1 than in F2 and F3 for each range. Concentrations in F3 decreased (P < 0.05) between the 8.0- to 8.9- and 10.0- to 10.9-mm ranges.



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  		FIG. 2. Mean (±SEM) follicular fluid hormone concentrations of the largest (F1), second-largest (F2), and third-largest (F3) follicles in experiment 1 (n = 9–12 heifers/diameter range). Main effects and interaction that were significant or approached significance are shown for each end point (F, main effect of follicle; R, main effect of diameter range; RF, interaction of range by follicle). Within a follicle, means with different letters (ab) among ranges are significantly different (P < 0.05)

Mean changes in follicular fluid concentrations of total inhibin, inhibin-B, and activin-A for the three largest follicles and of inhibin-A for the two largest follicles are shown with the results of statistical analyses in Figure 3. Adequate follicular fluid was not available for assay of inhibin-A in F3. No significant changes in follicular fluid concentrations of the inhibins and activin-A were detected among the three largest follicles within the diameter ranges. However, averaged over all follicles, changes in concentrations over the diameter ranges approached significance (P < 0.09) for activin-A and were significant (P < 0.02) for inhibin-B. The concentrations of activin-A increased (P < 0.05) between the 7.0- to 7.9- and 9.0- to 9.9-mm ranges, whereas inhibin-B decreased (P < 0.05) between the 8.0- to 8.9- and 10.0- to 10.9-mm ranges.



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  		FIG. 3. Mean (±SEM) follicular fluid hormone concentrations of the largest (F1), second-largest (F2), and third-largest (F3) follicles in experiment 1 (n = 9–12 heifers/diameter range). Main effects and interaction that were significant or approached significance are shown for each end point (R, main effect of diameter range). Means with different letters (ab) among ranges averaged over the three follicles are significantly different (P < 0.05)

Experiment 2

Mean diameters of F1, F2, and F3 at Hours 0 and 12 in the control and ablated groups are shown in Table 1, and comparisons of the concentrations of follicular fluid factors at Hour 12 between the control and ablated groups are shown in Figure 4. Diameter (P < 0.02) was greater; concentrations of follicular fluid estradiol (P < 0.001), free IGF-1 (P < 0.002), and progesterone (P < 0.003) were higher; but IGFBP-2 was lower (P < 0.01) in F2 for the ablated group compared to the control group. An increase in follicular fluid concentrations of estrone in the ablated group approached significance (P < 0.07); however, no differences were detected between the ablated and control groups for androstenedione, total inhibin, and inhibin-A. For F3 in the ablated group compared to the control group, diameter was less (P < 0.04), progesterone was higher (P < 0.02), and estradiol concentrations tended to be higher (P < 0.06), but free IGF-1, IGFBP-2, estrone, androstenedione, total inhibin, and inhibin-A concentrations did not differ significantly (data not shown). In the control group, comparisons were also available between F1 and F2 at Hour 12 for concentrations (mean ± SEM) of estradiol (277.8 ± 47.0 vs. 69.5 ± 25.2 ng/ml, P < 0.001), free IGF-1 (14.3 ± 2.0 vs. 5.4 ± 2.0 ng/ml, P < 0.008), IGFBP-2 (527.9 ± 95.0 vs. 660.2 ± 81.3 ng/ml, P < 0.03), estrone (31.4 ± 9.7 vs. 11.3 ± 5.3 ng/ml, P < 0.008), progesterone (117.4 ± 13.5 vs. 64.5 ± 8.6 ng/ml, P < 0.04), androstenedione (28.6 ± 7.1 vs. 12.6 ± 3.4 ng/ml, P < 0.04), total inhibin (375.0 ± 21.0 vs. 311.1 ± 22.8 µg/ml, P < 0.03), and inhibin-A (432.7 ± 37.9 vs. 285.5 ± 49.6 ng/ml, P < 0.01).


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  		TABLE 1. Effect of ablating F1 at the expected beginning of deviation on follicle diameters.a



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  		FIG. 4. Mean (±SEM) follicular fluid hormone concentrations of F2 in the control (F1 intact) and ablated (F1 ablated at >=8.2 mm) groups in experiment 2 (n = 10 heifers/group) 12 h after F1 was ablated at >=8.2 mm (Hour 0). An asterisk indicates a difference (P < 0.02) between groups, and a pound symbol marks a tendency (P < 0.07)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In experiment 1, the beginning of deviation was assigned to the diameter range preceding the range at which the diameter difference between the two largest follicles significantly increased [10]. That is, at the defined beginning of diameter deviation, growth rates of the future subordinate follicles had not yet decreased. The first increase in the diameter difference between F1 and F2 occurred at the 9.0- to 9.9-mm range; therefore, the beginning of deviation occurred at the 8.0- to 8.9-mm range. The mean diameter of F1 at the beginning of deviation was 8.5 mm in experiment 1 and 8.4 and 8.6 mm for the expected deviation for the two groups in experiment 2. These values are close to those reported for the observed beginning of deviation in previous studies [1, 25]. In experiment 2, the diameter of F2 at Hour 12 was greater in the F1-ablated group than in the controls and was comparable to the diameter of F1 in both groups at Hour 0. Thus, the experimental model successfully provided a means for F2 to assume morphologic dominance as assessed from a defined reference point (ablation of F1) for study of the physiologic changes associated with the initiation of deviation. A larger mean diameter of F3 in the control group compared to the ablated group at Hour 12 was unexpected. In this regard, no differences in follicular fluid factors were found between groups for F3 at Hour 12.

The first increase in the difference in follicular fluid estradiol concentrations between F1 and F2 occurred at the 8.0- to 8.9-mm range, or one diameter range earlier than the first increase in the diameter difference between F1 and F2. A similar differential increase in estradiol concentrations in F1 preceded diameter deviation in ponies [26]. In earlier studies with cattle, however, the first increase in the difference between F1 and F2 for diameter and follicular fluid estradiol occurred at the same time [9, 10]. In experiment 2, the hypothesis that estradiol concentrations in F2 would increase within 12 h of ablation of F1 was supported by the higher concentrations in the ablated group than in the control group. These results are compatible with the interpretation that estradiol has a role in the initiation of diameter deviation or the putative increase in FSH sensitivity of the future dominant follicle. Follicular fluid concentrations of estrone were higher in F1 than in F2 and F3 during each of the diameter ranges in experiment 1, and an increase in estrone concentrations in F2 within 12 h after ablation of F1 approached significance in experiment 2. In a study using excised bovine ovaries, follicular fluid estrone concentrations were greater in the two largest follicles than in a pool of small follicles and changed over days of the estrous cycle in both follicle populations [27]. In the present study, estrone was already higher in F1 than in F2 and F3 at the first diameter range studied (7.0–7.9 mm) and remained higher throughout the study. These results indicated that the activity of the aromatase system in F1 increased before the beginning of diameter deviation, but the increase in follicular fluid estradiol after an increase in estrone needs clarification, especially in association with deviation.

Results of experiment 1 indicated that differential changes among follicles in progesterone concentrations were not associated with differential changes in diameter or estradiol concentrations. Similarly, earlier studies did not detect progesterone changes apparently encompassing deviation [28, 29] or following deviation [14]. In contrast to the hypothesis developed from the results of experiment 1 and of earlier reports, concentrations of progesterone in F2 increased within 12 h after ablation of F1. This finding agrees with recent reports that concentrations were higher in F1 than in F2 at the expected time of deviation [30, 31] and that progesterone appeared to differentially increase in F1 in association with an increase in estradiol and the beginning of diameter deviation [10]. The increase in progesterone may reflect higher steroidogenic activity [32] of the developing dominant follicle, but a direct role in the deviation phenomenon cannot be excluded.

Differences in follicular fluid androstenedione concentrations averaged over the three follicles approached significance and seemed to reflect, in part, higher concentrations in F1 than in F2 and F3 at the 10.0- to 10.9-mm diameter range. These results are consistent with those of previous studies [12, 14, 33]. In experiment 2, concentrations did not change in F2 within 12 h of ablation of F1. Thus, the reported and present studies indicated that androstenedione concentrations increased in the largest or dominant follicle after, but not before, the beginning of deviation. Therefore, androstenedione apparently is not directly involved in the initiation of deviation.

Follicular fluid concentrations of free IGF-1 decreased in F2 after the 7.0- to 7.9-mm diameter range and were significantly lower than in F1 at the beginning of deviation (i.e., 8.0- to 8.9-mm diameter range). Similar results have been reported [10]. In experiment 2, free IGF-1 concentrations increased and IGFBP-2 decreased in F2 within 12 h of ablation of F1 and, therefore, temporally supported the hypothesis that the IGF system, like estradiol, is involved in the initiation of deviation. This interpretation is consistent with reports that an intraovarian injection of IGF-1 resulted in an increased diameter of the largest follicle [34], and that IGF-1 stimulated the mitosis of cultured theca and granulosa cells [35, 36] and increased the synthesis of androgen and estradiol [36, 37]. In experiment 2, it could not be determined whether IGF-1 and estradiol began to increase synchronously or asynchronously in F2 after ablation of F1 because of the length of the interval (12 h) between F1 ablation and F2 sampling. In vitro studies have shown that IGF-1 increased the production of estradiol from bovine granulosa cells [38]. Conversely, estradiol has been shown to stimulate IGF-1 production from porcine granulosa cells [8]. The timing of the changes in follicular fluid estradiol versus IGF factors in association with follicle deviation will require more study.

Changes in follicular fluid concentrations of total inhibin and inhibin-A were not different among follicles over the diameter ranges in experiment 1. A previous study in cattle found no difference in follicular fluid concentrations of inhibin-A and various molecular weight forms of inhibin in the three largest follicles of wave 1 when the largest follicle was a mean of 7.6 mm [39]. Follicular fluid concentrations of total inhibin and dimeric inhibin-A were not different among estrogen-active follicles 1 and 3 days after estrus [40]. A decrease in inhibin-A, apparently after deviation, has been shown by lower concentrations in the dominant follicle a mean of 4.8 days [28] and 6 days [40] after estrus. In a recent study, inhibin-A concentrations in plasma were low on the day of ovulation, increased and reached maximum levels approximately 3 days after ovulation, and declined thereafter [41]. The changes in concentration were not studied in relation to follicle diameter, but the decline of inhibin-A in the systemic circulation in the recent study appears to be consistent with the decline in concentration in follicular fluid after the beginning of deviation in the present study.

No differences were detected among follicles for inhibin-B and activin-A concentrations. However, when concentrations were averaged over the three largest follicles, inhibin-B decreased between the 8.0- to 8.9- and 10.0- to 10.9-mm ranges, and activin-A increased between the 7.0- to 7.9- and 9.0- to 9.9-mm ranges. Changes in concentrations of follicular fluid inhibin-B have not been previously reported, and circulating concentrations apparently have not been studied in cattle. In other studies, follicular fluid concentrations of activin-A in cattle were similar among the three largest follicles of wave 1 when the largest follicle was a mean of 7.6 mm [39]. In addition, concentrations increased in the largest follicle after the periovulatory FSH peak [42] and appear to be consistent with the present findings. In contrast, it has been reported that concentrations of activin-A were similar among small follicles 3 days and among dominant and subordinate follicles 4.8 days after estrus [28]. To our knowledge, circulating concentrations of activin-A have not been reported in cattle.

In contrast to the present and reported results in cattle, follicular fluid inhibin-A concentrations in women increased linearly as diameter increased (6.5–23.5 mm [16]) and tended to be higher in mature follicles during the late follicular phase [19]. Inhibin-B and activin-A did not change consistently with follicle size [16]. Changes in concentrations of inhibin-A and inhibin-B in the systemic circulation in women were similar to the changes in follicular fluid [19]. Whether the reported changes in inhibin/activin in women are associated with a presumed deviation phenomenon is not known. In cattle, the present study and previous reports have not detected an association with the beginning of deviation; therefore, inhibin/activin apparently does not differentially increase the FSH sensitivity of the future dominant follicle.

Although circulating concentrations of FSH and LH were not examined in the present studies, FSH has been shown to increase within 12 h after ablation of F1 at the expected beginning of deviation [25], and an LH elevation encompasses deviation [3] in cattle. The increased growth of F2 after ablating F1 likely was a function of an increase in systemic FSH, and the accompanying production of estradiol and the effects on the IGF system likely were functions of both gonadotropins. In regard to the effects of LH, experimental reduction of circulating LH concentration resulted in decreased intrafollicular concentrations of estradiol, free IGF-1, progesterone, estrone, and androstenedione and in increased IGFBP-2 at the expected beginning of deviation [31]. In addition, FSH stimulated the production of IGF-1 [43] and inhibited the mRNA expression of IGFBP-2 [44] from cultured granulosa cells; LH stimulated the production of IGFBP-4 from theca cells [44]. Both FSH and LH also stimulated the production of IGFBP proteases from cultured granulosa and theca cells [44–46]. Thus, the literature supports a role for systemic regulation involving both FSH and LH in the development of follicle dominance, and the present results temporally implicate intrafollicular factors in the modulation of gonadotropic actions.

In conclusion, an increase in estradiol concentrations in F1 but not in F2 were detected just before the beginning of diameter deviation. Concentrations of free IGF-1 in F1 encompassing deviation remained constant (no significant differences) but decreased in F2. In contrast, no differential changes between F1 and F2 in concentrations of inhibin-A, inhibin-B, or activin-A were found in association with the beginning of deviation; the changes that were found involved all three follicles (F1–F3). Within 12 h after ablation of F1 at the expected time of deviation, F2 was larger and had higher follicular fluid concentrations of estradiol and free IGF-1 and lower concentrations of IGFBP-2 compared to control values. Total inhibin and inhibin-A did not change. The results of the two experiments indicated that, on a temporal basis, intrafollicular changes in estradiol and the IGF system, but not in the inhibin/activin system, could account for the reported greater FSH responsiveness of the future dominant follicle than for the future subordinate follicles by the beginning of diameter deviation in cattle.


   ACKNOWLEDGMENTS
 
The authors thank the Pharmacia and Upjohn Company for the gift of Lutalyse and Susan C. Jensen for technical assistance.


   FOOTNOTES
 
First decision: 26 July 2001.

1 Supported by the University of Wisconsin, Madison, and by Equiservices Publishing and The Eutherian Foundation, Cross Plains, Wisconsin. Back

2 Correspondence: O.J. Ginther, Department of Animal Health and Biomedical Sciences, 1656 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706. FAX: 608 262 7420; ojg{at}ahabs.wisc.edu Back

Accepted: August 22, 2001.

Received: July 2, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ginther OJ, Kot K, Kulick LJ, Wiltbank MC. Emergence and deviation of follicles during the development of follicular waves in cattle. Theriogenology 1997; 48:75-87
  2. Ginther OJ, Wiltbank MC, Fricke PM, Gibbons JR, Kot K. Selection of the dominant follicle in cattle. Biol Reprod 1996; 55:1187-1194[CrossRef][Medline]
  3. Ginther OJ, Beg MA, Bergfelt DR, Donadeu FX, Kot K. Follicle selection in monovular species. Biol Reprod 2001; 65:638–647
  4. Armstrong DG, Webb R. Ovarian follicular dominance: the role of intraovarian growth factors and novel proteins. Rev Reprod 1997; 2::139-146[Abstract]
  5. Ginther OJ, Bergfelt DR, Kulick LJ, Kot K. Selection of dominant follicle in cattle: role of two-way functional coupling between follicle-stimulating hormone and the follicles. Biol Reprod 2000; 62:920-927[Abstract/Free Full Text]
  6. Gibbons JR, Wiltbank MC, Ginther OJ. Functional interrelationships between follicles greater than 4 mm and the follicle stimulating surge in heifers. Biol Reprod 1997; 57:1066-1073[Abstract]
  7. Hillier SG. Paracrine control of follicular estrogen synthesis. Semin Reprod Endocrinol 1991; 9:332-340
  8. Hsu CJ, Hammond JM. Gonadotropins and estradiol stimulate immunoreactive insulin-like growth factor-I production by porcine granulosa cells in vitro. Endocrinology 1987; 120:198-207[Abstract]
  9. Ginther OJ, Kot K, Kulick LJ, Wiltbank MC. Sampling follicular fluid without altering follicular status in cattle: estradiol concentrations early in a follicular wave. J Reprod Fertil 1997; 109:181-186[Abstract/Free Full Text]
  10. Beg MA, Bergfelt DR, Kot K, Wiltbank MC, Ginther OJ. Follicular fluid factors and granulosa-cell gene expression associated with follicle deviation in cattle. Biol Reprod 2001; 64:432-441[Abstract/Free Full Text]
  11. Wetterau LA, Moore MG, Lee KW, Shim ML, Cohen P. Novel aspects of insulin-like growth factor binding proteins. Mol Genet Metab 1999;; 68:161-181[CrossRef][Medline]
  12. Echternkamp SE, Howard HJ, Roberts AJ, Grizzle J, Wise T. Relationships among concentrations of steroids, insulin-like growth factor-I, and insulin-like growth factor binding proteins in ovarian follicular fluid of beef cattle. Biol Reprod 1994; 51:971-981[Abstract]
  13. De la Sota RL, Simmen FA, Diaz T, Thatcher WW. Insulin-like growth factor system in bovine first wave-dominant and subordinate follicles. Biol Reprod 1996; 55:803-812[Abstract]
  14. Stewart RE, Spicer LJ, Hamilton TD, Keefer BE, Dawson LJ, Morgan GL, Echternkamp SE. Levels of insulin-like growth factor (IGF) binding proteins, Luteinizing hormone and IGF-I receptors, and steroids in dominant follicles during the first follicular wave in cattle exhibiting regular estrous cycles. Endocrinology 1996; 137:2842-2850[Abstract]
  15. Knight PG, Glister C. Potential local regulatory functions of inhibins, activins and follistatin in the ovary. Reproduction 2001; 121:503-512[Abstract]
  16. Schneyer AL, Fujiwara T, Fox J, Welt CK, Adams J, Messerlian GM, Taylor AE. Dynamic changes in the intrafollicular inhibin/activin/follistatin axis during human follicular development: relationship to circulating hormone concentrations. J Clin Endocrinol Metab 2000; 85::3319-3330[Abstract/Free Full Text]
  17. Ginther OJ, Bergfelt DR, Kulick LJ, Kot K. Selection of the dominant follicle in cattle: establishment of follicle deviation in less than 8 hours through depression of FSH concentrations. Theriogenology 1999; 52::1079-1093[CrossRef][Medline]
  18. Bergfelt DR, Kulick LJ, Kot K, Ginther OJ. Follicular and hormonal response to experimental suppression of FSH during follicular deviation in cattle. Theriogenology 2000; 54:1191-1206[CrossRef][Medline]
  19. Groome NP, Illingworth PJ, O'Brien M, Pai R, Rodger FE, Mather JP, McNeilly AS. Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab 1996; 81:1401-1405[Abstract]
  20. Robertson DM, Cahir N, Findlay JK, Burger HG, Groome N. The biological and immunological characterization of inhibin A and B forms in human follicular fluid and plasma. J Clin Endocrinol Metab 1997; 82:889-896[Abstract/Free Full Text]
  21. Knight PG, Muttukrishna S, Groome NP. Development and application of a two-site enzyme immunoassay for determination of total activin-A concentrations in serum and follicular fluid. J Endocrinol 1996; 148:267-279[Abstract/Free Full Text]
  22. Zar JH. Biostatistical Analysis. 2nd ed. Englewood Cliffs, NJ: Prentice-Hall; 1984: 88–96
  23. Kanji GK. 100 Statistical Tests. London: SAGE Publications; 1993: 45, 171
  24. SAS Institute, Inc. SAS/STATTM User's Guide, version 6, vol 2, 4th ed. Cary, NC: Statistical Analysis System Institute, Inc.; 1989
  25. Ginther OJ, Bergfelt DR, Beg MA, Kot K. Follicle selection in cattle: relationships among growth rate, diameter ranking, and capacity for dominance. Biol Reprod 2001; 65:345-350[Abstract/Free Full Text]
  26. Gastal EL, Gastal MO, Wiltbank MC, Ginther OJ. Follicle deviation and intrafollicular and systemic estradiol concentrations in mares. Biol Reprod 1999; 61:31-39[Abstract/Free Full Text]
  27. Wise T. Biochemical analysis of bovine follicular fluid: albumin, total protein, lysosomal enzymes, ions, steroids and ascorbic acid content in relation to follicle size, rank, atresia classification and day of estrous cycle. J Anim Sci 1987; 64:1153-1169
  28. Mihm M, Good TEM, Ireland JLH, Ireland JJ, Knight PG, Roche JF. Decline in serum follicle-stimulating hormone concentrations alters key intrafollicular growth factors involved in selection of the dominant follicle in heifers. Biol Reprod 1997; 57:1328-1337[Abstract]
  29. Evans ACO, Fortune JE. Selection of the dominant follicle occurs in the absence of the differences in the expression of messenger ribonucleic acid for gonadotropin receptors. Endocrinology 1997; 138:2963-2971[Abstract/Free Full Text]
  30. Ginther OJ, Bergfelt DR, Beg MA, Kot K. Follicle selection in cattle: role of luteinizing hormone. Biol Reprod 2001; 64:197-205[Abstract/Free Full Text]
  31. Ginther OJ, Bergfelt DR, Beg MA, Kot K. Effect of LH on circulating estradiol and follicular fluid factors during follicle deviation in cattle. Reproduction 2001; 122:103-110[Abstract]
  32. Fortune JE. Bovine theca and granulosa cells interact to promote androgen production. Biol Reprod 1986; 35:292-299[Abstract]
  33. Singh J, Pierson RA, Adams GP. Ultrasound image attributes of bovine ovarian follicles and endocrine and functional correlates. J Reprod Fertil 1998; 112:19-29[Abstract/Free Full Text]
  34. Spicer LJ, Alvarez P, Prado TM, Morgan GL, Hamilton TD. Effects of intraovarian infusion of insulin-like growth factor-I on ovarian follicular function in cattle. Domest Anim Endocrinol 2000; 18:265-278[CrossRef][Medline]
  35. Khamsi F, Armstrong DT. Interaction between follicle-stimulating hormone and growth factors in regulation of deoxyribonucleic acid synthesis in bovine granulosa cells. Biol Reprod 1997; 57:684-688[Abstract]
  36. Spicer LJ, Stewart RE. Interactions among basic fibroblast growth factor, epidermal growth factor, insulin and insulin-like growth factor-I (IGF-I) on cell numbers and steroidogenesis of bovine thecal cells: role of IGF-I receptors. Biol Reprod 1996; 54:255-263[Abstract]
  37. Spicer LJ, Alpizar E, Echternkamp SE. Effects of insulin, insulin-like growth factor I and gonadotropins on bovine granulosa cell proliferation, progesterone production, estradiol production, and(or) insulin-like growth factor I production in vitro. J Anim Sci 1993; 71:1232-1241[Abstract]
  38. Yang MY, Rajamahendran R. Effects of gonadotropins and insulin-like growth factors-I and -II on in vitro steroid production by bovine granulosa cells. Can J Anim Sci 1998; 78:587-597
  39. Mihm M, Austin EJ, Good TEM, Ireland JLH, Knight PG, Roche JF, Ireland JJ. Identification of potential intrafollicular factors involved in selection of dominant follicles in heifers. Biol Reprod 2000; 63:811-819[Abstract/Free Full Text]
  40. Sunderland SJ, Knight PG, Boland MP, Roche JR, Ireland JJ. Alterations in intrafollicular levels of different molecular mass forms of inhibin during development of follicular- and luteal-phase dominant follicles in heifers. Biol Reprod 1996; 54:453-462[Abstract]
  41. Bleach ECL, Glencross RG, Feist SA, Groome NP, Knight PG. Plasma inhibin A in heifers: relationship with follicle dynamics, gonadotropins, and steroids during the estrous cycle and after treatment with bovine follicular fluid. Biol Reprod 2001; 64:743-752[Abstract/Free Full Text]
  42. Austin EJ, Mihm M, Evans ACO, Knight PG, Ireland JLH, Ireland JJ, Roche JF. Alterations in intrafollicular regulatory factors and apoptosis during selection of follicles on the first follicular wave of the bovine estrous cycle. Biol Reprod 2001; 64:839-848[Abstract/Free Full Text]
  43. Khalid M, Haresign W, Luck MR. Secretion of IGF-1 by ovine granulosa cells: effects of growth hormone and follicle stimulating hormone. Anim Reprod Sci 2000; 58:261-272[CrossRef][Medline]
  44. Armstrong DG, Baxter G, Gutierrez CG, Hogg CO, Glazyrin AL, Campbell BK, Bramley TA, Webb R. Insulin-like growth factor binding protein-2 and -4 messenger ribonucleic acid expression in bovine ovarian follicles: effect of gonadotropins and developmental status. Endocrinology 1998; 139:2146-2154[Abstract/Free Full Text]
  45. Iwashita M, Kudo Y, Takeda Y. Effect of follicle stimulating hormone and insulin-like growth factors on proteolysis of insulin-like growth factor binding protein-4 in human granulosa cells. Mol Hum Reprod 1998; 4:401-405[Abstract/Free Full Text]
  46. Nicholas BL, Webb R, Armstrong DG. Characterization of insulin-like growth factor binding protein-2 (IGFBP-2) protease activity in bovine theca cell conditioned media. J Reprod Fertil Abstr Ser 2000; 25: (abstract 52)



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