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Regular Article |
a Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706 Eutherian Foundation, Cross Plains, Wisconsin 53528
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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activin, estradiol, follicle-stimulating hormone, follicular development, growth factors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A close two-way functional coupling between FSH and the follicles has been reported to be an integral component of the deviation mechanism [2]. During the common-growth phase, multiple follicles contribute to a sustained decline in circulating FSH concentrations [3], yet the follicles continue to require the FSH [2]. At the end of the common-growth phase or beginning of deviation, the largest or future dominant follicle, alone, is responsible for the continuing FSH decline [4, 5] and is the only follicle that continues to use the declining FSH [2]. It has been suggested that the low FSH concentrations increase the synthesizing capacity of estradiol [6] in the most developed or largest follicle [1]. In this regard, insulin-like growth factor I (IGF-I) enhances the ability of FSH to stimulate estradiol secretion in vitro but only at low doses of FSH [7]. Furthermore, only low concentrations of FSH stimulate in vitro estradiol production by granulosa cells from medium (5–8 mm) and large (>8 mm) but not small (<5 mm) bovine follicles [8]. The increased responsiveness of the large follicle to low FSH apparently becomes established rapidly as reflected by the difference in diameter between the two largest follicles at the beginning of deviation (equivalent to <8 h of follicle growth in cattle [1]). Thus, the largest follicle differentially develops enhanced responsiveness to low FSH and a continued capacity to suppress circulating FSH concentrations before the second largest follicle can reach a similar developmental stage. Differential responsiveness among follicles to LH may also be involved in deviation [1]. In cattle, concentrations of LH are transiently higher during deviation [1, 9–11], and an increase in the differences between the 2 largest follicles in granulosa LH-receptor mRNA [12] occur 8 h before an increase in the differences between the two follicles in diameter [13].
Candidates for an intrafollicular role in increasing the gonadotropin responsiveness of the developing dominant follicle within 8 h in cattle include estradiol, IGFs, and inhibin/activin peptides [1]. Based on in vitro studies, estradiol enhances aromatase activity, increases the sensitivity of granulosa cells to FSH and LH [14, 15], potentiates granulosa cell expression of gonadotropin receptors [15], and increases the synthesis of IGF-I from granulosa cells [16]. Follicular-fluid concentrations of estradiol in heifers begin to increase differentially in the future dominant follicle at [12, 17] or just before [18] the beginning of deviation. Similarly, an increase in circulating estradiol begins when diameter deviation begins and is attributable to the largest follicle [4].
The IGFs are potent mitogens [19] and stimulate the mitosis of cultured theca and granulosa cells [20, 21], increase the synthesis of androgens and estradiol [21, 22], and modulate gonadotropin action on granulosa and theca cells [1, 7]. Estradiol and IGF-I also influence one another; in vitro studies indicated that FSH and estradiol have a positive effect on IGF-I production in porcine granulosa cells [16] and that IGF-I has a positive effect on estradiol production in bovine granulosa cells [8]. 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 [23]. In recent studies that encompassed deviation, concentrations of free IGF-I did not change in the largest follicle, whereas those of free IGF-I decreased and those of IGFBP-2 increased in the second largest follicle [10, 13, 18]. These studies indicate that the higher ratio of free IGF-I to IGFBP-2 in the largest follicle favors continued growth, whereas the higher ratio of IGFBP-2 to IGF-I in the smaller follicles favors regression. Activins and inhibins also have autocrine/paracrine actions on granulosa and theca cells, thereby modulating follicle growth, gonadotropin responsiveness, and steroidogenesis [24].
When the largest follicle (F1) is ablated at the expected beginning of diameter deviation in cattle, the second largest follicle (F2) usually (86% of the time) becomes the dominant follicle [5, 25]. The diameter of F2 by 12 h after ablation of F1 is comparable to the diameter of F1 at the beginning of deviation [5, 18]. Thus, a follicle that is destined to regress changes its course within 12 h after removal of the future dominant follicle. This phenomenon provides a model in which the time of ablation of F1 serves as a reference point to establish the sequence of events leading to the experimental development of dominance by F2. In an initial study [18], the F2 concentrations of follicular-fluid factors 12 h after the ablation of F1 were compared with concentrations in F2 in control heifers; estradiol, free IGF-I, and progesterone were higher; IGFBP-2 was lower; and androstenedione, total inhibin, and inhibin A were unchanged. However, because of the 12-h interval, the order of changes in concentration for these factors was not determinable. The present experiments concerned the effects of ablating F1 at the expected beginning of deviation on the sequential changes in diameter and concentrations of follicular-fluid factors during the conversion of F2 to dominant status.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two experiments were done during the wave that emerges during the periovulatory period (wave 1). The animals were Holstein heifers between 24 and 36 mo of age and weighed 490–680 kg. The feeding program and the prostaglandin F2
(Lutalyse; Pharmacia Co., Kalamazoo, MI) protocol for inducing luteolysis to schedule ovulation and the equipment and techniques for transrectal ultrasound scanning of ovaries and measuring follicles have been described [2]. 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 6.5 mm. Thereafter, scanning was done at 12-h intervals until the largest follicle (F1) reached an average of 8.5 mm (Hour 0) and again just before the scheduled sampling of follicular fluid. Because of the 12-h interval between scanning sessions, ablation was done when F1 first reached >8.1 mm to obtain a range of actual diameters with an average of approximately 8.5 mm. Heifers in which the second largest follicle at Hour 0 was <7.2 mm were not used because of the report [25] that such follicles do not have high capacity for assuming dominance after ablation of F1. The second largest follicle at the time of follicular-fluid collection in the follicle-retained control groups and the largest remaining follicle in F1-ablated groups were designated F2. Follicle ablation was done by ultrasound-guided transvaginal aspiration of follicle contents [5]. Similarly, a follicular-fluid sample was collected by aspirating until the antrum collapsed [18]. The follicular-fluid collections were centrifuged at 500 x g for 10 min, decanted, and stored at -20°C as assay samples.
Hormone Assays
Plasma concentrations of FSH were determined as described for this laboratory [25]. The intraassay coefficient of variation (CV) and assay sensitivity were 4.5% and 0.04 ng/ml, respectively. Follicular-fluid concentrations of estradiol [9], estrone, progesterone, androstenedione, free IGF-I, IGFBP-2, and dimeric inhibin A [12], inhibin B, and total activin A [18] were determined by procedures that have been described and validated for bovine follicular fluid in this laboratory. For experiment 1, the intraassay and interassay CV for quality control samples and the mean assay sensitivity, respectively, were as follows: estradiol: 6.6%, 10.9%, and 0.4 pg/ml; progesterone: 6.2%, 18.2%, and 0.2 ng/ml; free IGF-I: 10.2%, 1.6%, and 0.01 ng/ml; inhibin A: 6.6%, 0.4%, and 0.13 pg/ml; inhibin B: 2.2%, 9.9%, and 1.9 pg/ml; and activin A: 6.5%, 5.7%, and 0.1 ng/ml. For androstenedione, the intraassay CV and sensitivity were 8.9% and 0.03 ng/ml, respectively. For experiment 2, the corresponding values were as follows: estradiol: 6.4% and 0.5 pg/ml; free IGF-I: 3.6% and 50 pg/ml; IGFBP-2: 20.4% and 0.23 ng/ml; activin A: 14.4% and 0.05 ng/ml; and progesterone: 4.1% and 0.02 ng/ml.
Experiment 1
At Hour 0 (expected beginning of deviation), the heifers were randomly assigned to 4 control groups and 6 F1-ablated groups (n = 7 heifers per group). In the control groups, follicular fluid of F1 and F2 was collected at Hours 0, 4, 8, or 12. In the 6 F1-ablated groups, follicular fluid from the largest remaining follicle (F2) was collected at Hours 4, 8, 12, 16, 20, or 24. Follicular-fluid samples were assayed for estradiol, free IGF-I, progesterone, androstenedione, activin A, inhibin A, and inhibin B. For the follicle end points, the follicles were designated F1C (F1 in control groups), F2C (F2 in control groups), and F2A (F2 in F1-ablated groups).
Experiment 2
At Hour 0, heifers were randomly assigned to 5 F1-ablated groups for follicular-fluid collection from the largest remaining follicle (F2A) at Hours 4, 6, 8, 10, or 12 (n = 9 heifers per hour). Control (F1-retained) heifers were not used in this study because of the large number of heifers that would be required and because considerable information is available for controls on follicular-fluid factors and circulating FSH [1]. The follicular-fluid samples were assayed for estradiol, free IGF-I, IGFBP-2, activin A, and progesterone. Jugular venous samples were collected every 12 h from the time the largest follicle was 
6.5 mm until Hour 0 and then every 2 h until the hour of follicular-fluid collection. Plasma samples were assayed for FSH until Hour 10 for the groups with follicular-fluid collection at Hours 10 or 12. This was done so that FSH values would come from sequential samples taken from the same animals for most of the experiment.
Statistical Analyses
Data for follicular end points were challenged for extreme values with the Dixon outlier test [26]. Data were tested for normality using the Kolmogorov-Smirnov test [27]. When the normality test was significant (P < 0.05), data were transformed by either natural logarithm or square root. In experiment 1, data for F1C and F2C were analyzed by a 2 (follicles) by 4 (Hours 0, 4, 8, 12) factorial ANOVA. Data for F2C and F2A were compared by a 2 by 3 (Hours 4, 8, 12) factorial ANOVA. In addition, a one-way ANOVA was done for end points in F2A for an effect of hour (Hours 4, 8, 12, 16, 20, 24). In experiment 2, data were analyzed by a one-way ANOVA for an effect of hour (Hours 4, 6, 8, 10, 12). Circulating FSH (experiment 2) was analyzed by the MIXED procedure with a repeated statement and a first order autoregressive structure to account for autocorrelation between sequential measurements. When the hour effect was significant or approached significance, the means were further compared by the Duncan multiple-range test. In addition, for experiment 1, paired t-tests were used within hours to compare F1C and F2C, and unpaired t-tests were used to compare F2C and F2A. The actual data and not the transformed data are presented as the mean ± SEM. A probability of P < 0.05 indicated that a difference was significant, and probabilities between P > 0.05 to P < 0.1 indicated that a difference approached significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For the comparison between F2C (controls) and F2A (F1 ablated at Hour 0), the diameter of F2A increased (P < 0.02) between Hours 8 and 12 and was higher (P < 0.05) than that for F2C at Hour 12 (Fig. 1). The diameter of F2A at Hour 12 (8.7 ± 0.2 mm) was similar (not significantly different) to the diameter of F1C at Hour 0 (8.5 ± 0.1 mm). The diameter of F2A increased progressively over Hours 8–24 as shown.
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Estradiol concentrations were greater (follicle effect, P < 0.02) in F1C than in F2C (Fig. 1) and were higher (P < 0.05) in F1C at each hour. Concentrations increased (P < 0.05) in F1C between Hours 0 and 12 and decreased (P < 0.05) in F2C between Hours 0 and 8. Concentrations were higher (P < 0.05) in F2A than in F2C at Hours 8 and 12 (follicle by hour interaction, P < 0.05). The increases in estradiol concentrations in F2A over Hours 4–24 are shown.
Averaged over hours, the concentrations of free IGF-I in the controls were higher (P < 0.05) in F1C than in F2C (Fig. 1). Concentrations in F1C were not different among hours, but a decrease in F2C between Hours 0 and 8 approached significance (P < 0.1). Concentrations at Hour 4 were lower (P < 0.05) in F2A than in F1C. Concentrations were higher in F2A than in F2C at Hour 12.
There were no significant differences involving F1C and F2C in the concentrations of activin A (Fig. 1). However, for the comparisons of F2C and F2A, the main effect of follicle (P < 0.05) and the interaction (P < 0.03) were significant over Hours 4, 8, and 12. In F2A, activin A concentration was higher (P < 0.05) at Hour 8 than at Hours 4 or 12 and then increased after Hour 12.
For progesterone concentrations, the hour effect (Hours 0–12) of F1C and F2C in the controls approached significance (P < 0.09) and for F2C versus F2A was significant (P < 0.02; Fig. 1). The hour effects seemed due primarily to an increase in concentrations in all 3 follicle types between Hours 4 and 8. There were no significant differences in androstenedione concentrations for any of the comparisons between Hours 0 to 12 (Fig. 1). Changes in concentrations of progesterone and androstenedione for F2A during Hours 4–24 are shown. There were no significant differences for any of the comparisons for inhibin A and inhibin B.
Experiment 2
The diameter of F2A (F1 ablated) at Hours 10 and 12 was greater (P < 0.05) than that at Hour 8 and similar to the diameter of F1 at Hour 0 (Table 1). The effect of hour on circulating concentrations of FSH between Hours -24 and 10 was significant (P < 0.02; Fig. 2). Concentrations decreased (P < 0.05) over Hours -24 to 0. A progressive increase occurred after Hour 2 so that the concentrations at Hour 6 were higher (P < 0.05) than at Hour 2. The concentrations of follicular-fluid factors in F2A for Hours 4, 6, 8, 10, and 12 and the results of the statistical analyses are shown (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mean diameter of F1 at the expected beginning of deviation (Hour 0) was 8.5 and 8.6 mm for the two experiments, similar to reported diameters at the observed beginning of deviation [1, 28]. The diameter of F2A (ablation of F1 at Hour 0) increased between Hours 8 and 12, resulting in greater diameter at Hour 12 than for F2C. The diameter of F2A at Hour 12 was similar to the diameter of F1C at Hour 0, indicating that F2A had reached a diameter characteristic of a largest follicle at the beginning of diameter deviation or preceding the manifestation of dominance. Therefore, a change in concentration of a follicular-fluid factor in F2A occurring within 12 h after ablation of F1 is interpreted as preceding the assumption of diameter-defined dominance by F2A. The diameters for F2A at Hours 4 and 6 seem high (Table 1), reflecting large diameters (9.0–9.3 mm) in a few heifers. These follicles may have represented future double-dominant follicles [29], but there was no provision in the protocol for removal of their data. Their presence partly accounted for the high means for free IGF-I and the low means for IGFBP-2 at Hours 4 and 6.
The estradiol result in control heifers is consistent with the results of previous studies [13, 17, 18]. In the present experiments, the increased follicular-fluid concentrations of estradiol in F2A after ablation of F1 occurred just before or simultaneously with increased diameter. These results indicated that estradiol concentrations in F2A increased before the experimental assumption of dominance and, on a temporal basis, may have been involved in increasing the gonadotropin responsiveness of F2A in its conversion to the status of a future dominant follicle.
Concentrations of free IGF-I in the control group of experiment 1 agree with previous studies in which concentrations of free IGF-I remained constant in F1 and decreased in F2 as the diameter of F1 increased from about 7.5 to 11.0 mm [12, 18]. Increased concentrations of free IGF-I in F2A occurred by Hour 10 or 12 or by the time the follicle reached a diameter characteristic of the beginning of deviation or before dominance of F2A would have been indicated by diameters. The free IGF-I concentrations in F2A at Hours 10 and 12 were similar to (not significantly different from) the concentrations in F1C at Hour 0. Although the increase by Hour 12 was significant only in experiment 2, the concentrations at Hour 12 in experiment 1 were greater for F2A than for F2C. The increase in free IGF-I concentrations after Hour 8 apparently provided F2A (future dominant follicle) with levels of IGF-I similar to those that encompass deviation in F1 of controls [13, 18]. Thus, both IGF-I and estradiol were available for increasing the gonadotropin responsiveness of F2A. Reciprocal relationships of the means occurred between free IGF-I and IGFBP-2 in F2A between Hours 4 and 12 after ablation of F1 in experiment 2. The free IGF-I/IGFBP-2 relationships apparently established a balance between these factors within F2A that was compatible with the conversion of F2A to a future dominant follicle.
The similarity in activin A concentrations in F1C and F2C during Hours 0 to 12 is consistent with the reported lack of a differential change among follicles encompassing deviation [18]. The reason for the lower concentrations in F2A than in F2C at Hour 4 is not known. Despite the reports that activin A does not play a role in deviation, an unexpected transient elevation occurred in F2A at Hour 8 in experiment 1. This observation became a hypothesis for experiment 2 and was supported by the results. Transient elevation in activin A may not have been detected in previous studies because of the long intervals between sampling [18, 30]. When the elevation occurred, estradiol and free IGF-I had not yet increased but were higher 2 h later. This strong temporal relationship indicates that the transient increase in activin A can be considered the first detected change in a follicular-fluid factor leading to an increase in production of estradiol and IGF-I in the conversion of F2A to the status of a future dominant follicle. In this regard, activin A stimulates aromatase activity and estradiol secretion in bovine [24] and ovine [31] granulosa cells in vitro. A role for activin A in the enhancement of aromatase activity in the future dominant follicle was indicated in a recent study in mares [32]; activin A began to increase differentially in the largest follicle before the beginning of diameter deviation. Before a short, transient activin A elevation can be considered an intrafollicular initiator of the sequential events leading to follicle dominance in cattle, several aspects will require demonstration or clarification, including 1) the presence of a similar elevation in F1 in controls before deviation between F1 and F2; 2) the manner in which follistatin, an activin-binding protein [7], is involved, especially considering that the activin A assay measured both follistatin-bound and free (bioactive) forms; 3) functional as well as temporal relationships among activin A, estradiol, and IGF-I; and 4) the stimulation of activin A production when circulating FSH concentrations decline to a critical level with only the future dominant follicle adequately developed for such a response. After the nadir following the transient elevation in activin A concentrations in F2A, the concentrations began to increase again so that higher concentrations were obtained by Hour 16. The reason and role for this latter increase in activin A in F2A are not known and will require further study.
Progesterone increased during Hours 4–8 in F1C and F2C of controls and in F2A after ablation of F1 with no differences among the three types of follicles. These results are consistent with the results of some reports [6, 18, 33, 34] and disagree with those of others [10, 11, 13]. More study will be required to clarify if differential changes in progesterone concentrations among follicles are temporally or functionally associated with deviation. The absence of an increase in androstenedione within 12 h after ablation of F1 is consistent with reports that androstenedione concentrations did not change differentially among follicles until well after apparent deviation would have begun [34, 35] and did not increase until the dominant follicle reached approximately >10 mm [13, 18]. Similarly, in experiment 1, concentrations in F2A were increased 24 h after ablation of F1 when the follicle was a mean 9.7 mm in diameter. Concentrations of inhibin A and inhibin B did not change in F2A of F1-ablated heifers during the first 12 h. This result is consistent with the reported lack of a differential effect among follicles encompassing deviation [18].
Several previous studies have shown that deviation begins only after circulating concentrations of FSH have declined and that the FSH concentrations continue to decline for 12–20 h after the observed or expected beginning of deviation [1]. The continued decline in FSH after the expected beginning of deviation is well established and therefore was not studied in control heifers. Concentrations of FSH increased in F1-ablated heifers rather than continuing to decrease. These results are consistent with reports that at the beginning of deviation, the largest follicle alone continues to support the FSH decline [1]. After ablation of F1 at Hour 0 in experiment 2, the mean circulating FSH concentrations were at a nadir at Hour 2 and then progressively increased until the end of blood sampling (Hour 10). The increase in circulating FSH reached significance 2 h before the transient activin A elevation and the beginning of an increase in concentrations of estradiol and free IGF-I in F2A. On a temporal basis, the increasing concentrations of activin A, estradiol, and IGF-I in F2A after ablation of F1 can be considered a response to the transient FSH increase. In this regard, an FSH stimulatory effect on activin A, estradiol, and IGF-I has been demonstrated in vitro [7, 16]. In addition, the stimulatory actions of FSH on estradiol and IGF-I may be mediated by activin A [7]. The relationships between FSH concentrations and the deviation between F2A and other follicles were not studied. In previous studies involving ablation of F1 at 8.5 mm (Hour 0), peak concentrations of FSH were reached at Hour 12, and concentrations returned to the preablation levels by Hours 24 or 36 [4, 5, 25].
In summary, the future largest subordinate follicle (F2) was converted to a future dominant follicle by ablation of the largest follicle (F1) at the expected beginning of deviation (F1, 8.5 mm). Circulating concentrations of FSH increased after ablation of F1 and were significantly elevated by 6 h, which preceded the concentration changes in follicular-fluid factors. The intrafollicular results for F2 indicated that the conversion of F2 to a future dominant follicle involved a transient elevation in activin A, an increase in concentrations of estradiol, an increase in IGF-I, and a decrease in IGFBP-2 but did not involve progesterone, androstenedione, inhibin A, and inhibin B. The activin A elevation occurred an average of 8 h after ablation of F1 or 2 h before the increased concentrations of estradiol and free IGF-I. These results indicated that the order of events in the conversion of a future subordinate follicle to a future dominant follicle was an increase in systemic FSH, a transient elevation in follicular-fluid activin A, and a simultaneous increase in follicular-fluid estradiol and apparent restoration of a growth-compatible balance in concentrations of free IGF-I and IGFBP-2.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Research supported by the University of Wisconsin, Madison, and by Equiservices Publishing and the Eutherian Foundation, Cross Plains, WI. 
2 Correspondence: O.J. Ginther, Department of Animal Health and Biomedical Sciences, 1656 Linden Dr., University of Wisconsin-Madison, Madison, WI 53706. FAX: 608 262 7420; ojg{at}ahabs.wisc.edu
Accepted: January 24, 2002.
Received: December 13, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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