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Ovary |
a Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706 Eutheria 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, follicular development, growth factors, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent reviewers [1, 4, 5] seem to agree that enhanced differential FSH and LH sensitivity or responsiveness of the future dominant follicle is an integral component of follicle selection or deviation. In cattle, increased responsiveness of the future dominant follicle to gonadotropins apparently becomes established rapidly (<8 h), before the second-largest follicle can reach a similar developmental stage [1]. Estradiol and insulin-like growth factor-1 (IGF-1) are the most likely candidates for an intrafollicular role in increasing the gonadotropin responsiveness of the developing dominant follicle [1]. In heifers, concentrations of estradiol in follicular fluid begin to increase differentially in the future dominant follicle versus other follicles before [2] or at [3, 6] the beginning of deviation. Similarly, an increase in circulating estradiol levels began at the beginning of diameter deviation and was attributable to the largest follicle [7]. Based on in vitro studies, estradiol enhances aromatase activity, increases the sensitivity of granulosa cells to FSH and LH [8], potentiates granulosa cell expression of gonadotropin receptors [8], and increases the synthesis of IGF-1 from granulosa cells [9]. In studies that encompassed deviation, concentrations of free IGF-1 did not change in the largest follicle but they decreased in the second-largest follicle [2, 3], suggesting that continued IGF-1 availability in the largest follicle was needed for its development of dominance. In vitro studies indicate that IGFs stimulate the mitosis of cultured theca and granulosa cells [10, 11], increase the synthesis of androgens and estradiol [10, 12], and modulate gonadotropin action on granulosa and theca cells [1, 11]. Comparisons of follicular-fluid concentrations of androstenedione, testosterone, progesterone, inhibin-A, inhibin-B, and total inhibin between the future dominant and subordinate follicles have not suggested on a temporal basis that these factors are involved in the deviation mechanism in cattle [2, 3, 13].
Results of a recent study [13] using ablation of the largest follicle (F1) at the expected beginning of deviation (8.5 mm) and sampling follicular fluid every 2 or 4 h indicated temporally that activin-A was involved in experimental deviation or the conversion of the second-largest follicle (F2) to dominant status. Activin-A concentrations in F2 increased and decreased during a span of 4–12 h after F1 ablation. An increase in estradiol and free IGF-1 began in F2 at the peak of the transient activin-A elevation. In this regard, activin-A stimulates aromatase activity and estradiol secretion in cattle [14] and sheep [15] granulosa cells in vitro and begins to increase differentially in the largest follicle before the beginning of diameter deviation in horses [16]. Although an activin-A elevation in F1 was temporally associated with experimental deviation in cattle [13], it is not known whether a similar elevation occurs before natural deviation or whether the elevation occurs only in F1.
The production of follicular fluid factors by F2 has been studied, but with indirect reference points such as diameter of the associated F1 [2, 3] or number of hours or days following a given follicular or hormonal event [17]. Specified diameters of F2 would occur at different times relative to various diameters of the associated F1 or various intervals from an event, thereby masking the independent activity of F2 as its diameter changes.
The present experiment tested two hypotheses: 1) that concentrations of estradiol and free IGF-1 simultaneously become higher in the future dominant follicle than in the associated future largest subordinate follicle by the beginning of diameter deviation, and 2) that a transient elevation in activin-A is present in the future dominant follicle but not in the associated future largest subordinate follicle at the beginning of the estradiol and IGF-1 changes. In addition, a novel aspect concerned the changing production of estradiol, free IGF-1, and activin-A by F2 when F2 diameters were considered independently of F1 diameters.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiment was performed during the wave that emerges during the periovulatory period (wave 1). We used Holstein heifers between 24 and 36 mo of age weighing 490–680 kg. The feeding program and the PGF2
(Lutalyse, Pharmacia Co., Kalamazoo, MI) protocol for inducing luteolysis to schedule ovulation, and the equipment and techniques for transrectal ultrasound scanning of ovaries and follicle measurements have been described elsewhere [18]. Transrectal ultrasonic scanning was performed every 24 h beginning on the day of induced luteolysis at mid-diestrus and continued until the largest follicle of the new wave reached 6.5 mm. Thereafter, scanning was performed at 8-h intervals until F1 reached a randomly designated diameter. The designated diameters of F1 for assignment by replicate to diameter groups were 7.4–8.5 mm at increments of 0.1 mm (12 groups, 6 heifers per group). It was sometimes necessary to assign a heifer to an alternate group or to scan at a shorter interval (1–7 h) in order to complete a replicate.
The two largest follicles (F1 and F2) at the time of group assignment were each measured in two diameters at right angles, and the mean was used as the diameter. In addition, the mean diameter was taken from two separately obtained and frozen images. This was done to assess the repeatability of the mean diameter measurements. This information was used at the end of the group assignments to reallocate the groups if the difference in mean diameter measurements between the two frozen images was greater than the 0.1 mm increment between groups. The growth rate of F1 during the 24 h before follicular fluid collection was calculated so that the results could be expressed in hours as well as diameters.
In addition to the evaluation of F2 associated with the F1 diameter groups, the F2 data were grouped using F2 diameters as the reference independent of F1 diameter groups. To minimize obscuring the interpretations, F2 data were not taken from the F1 8.4- and 8.5-mm groups because of potential negative effects on F2 as diameter deviation approached. The effects of the independent diameter groups for F2 on the three hormonal endpoints (estradiol, free IGF-1, and activin-A) were studied within F2 using F2 diameter groups and between F1 and F2 at common diameter groups.
Follicular Fluid and Hormone Assays
Follicular fluid was collected from F1 and F2 by ultrasound-guided transvaginal aspiration of follicle contents until the antrum collapsed as described [2]. Two heifers were replaced because follicular fluid was not successfully collected. The follicular fluid was centrifuged at 500 x g for 10 min, decanted, and stored at -20°C. Follicular fluid concentrations of estradiol [19], free IGF-I [3], and total activin-A [2] were determined by procedures that have been described and validated for bovine follicular fluid in this laboratory. The intraassay and interassay coefficients of variation (CV) for quality control samples and the mean assay sensitivity, respectively, were as follows: estradiol, 8.1% (intraassay CV only) and 0.3 pg/ml; free IGF-1, 1.3%, 6.5%, and 0.03 ng/ml; and activin-A, 6.3%, 7.4%, and 0.1 ng/ml.
Statistical Analyses
Data for follicular end points were challenged for extreme values with the Dixon outlier test [20] and were tested for normality with the Kolmogorov-Smirnov test [21]. When the normality test was significant (P < 0.05), data were transformed by either natural logarithm or square root. Associated data for F1 and F2 were analyzed by diameter-group factorial ANOVA. The means were further compared among diameter groups by unpaired t-tests and between follicles within a group by paired t-tests. When an end point was not significantly different between follicles or for the interaction, the data were combined for F1 and F2. The beginning of diameter deviation was defined as occurring in the group preceding the group with a significant mean change in differences between F1 and F2. Diameter groups of F2 also were analyzed separately by using F2 diameters independent of F1 diameters. A two (F1, F2) by four (common diameters between F1 and F2) factorial ANOVA was used to compare the two follicles at the same diameters. The actual data rather than the transformed data are presented as means ± 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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Comparisons of F1 and F2 Using F2 Associated with Each F1 Diameter Group
For diameters of F1 and the associated F2, both main effects (P < 0.0001) and the interaction (P < 0.002) were significant, and the diameter-group effect was significant for F1 (P < 0.0001) and for F2 (P < 0.05; Fig. 1). A decrease in diameter of F2 between the 8.2- and 8.4-mm groups approached significance (P < 0.07). The differences in diameter between F1 and F2 first increased (P < 0.02) between the 8.2- and 8.4-mm groups.
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For comparison of estradiol concentrations between F1 and the associated F2, the main effects of follicle (P < 0.0001), diameter group (P < 0.02), and interaction (P < 0.02) were significant (Fig. 1). When follicles were analyzed separately there was a significant difference among groups for F1 (P < 0.006) but not for the associated F2. Estradiol concentrations in F1 increased (P < 0.05) between the 8.0- and 8.4-mm groups and were higher (P < 0.04) in F1 than in F2 in the 8.2- and 8.4-mm groups. The difference in concentrations between F1 and F2 was greater (i.e., it approached significance, P < 0.07) in the 8.4-mm group than in the 8.0-mm group.
An interaction of follicle and diameter group (P < 0.04) existed for the comparison of free IGF-1 concentrations between F1 and the associated F2 (Fig. 1). The interaction resulted primarily from higher free IGF-1 concentrations in F1 than in F2 in the 7.6-mm (P < 0.004), 8.2-mm (P < 0.06), and 8.4-mm groups (P < 0.02) but not in the other diameter groups. The concentrations in F1 increased (P < 0.01) between the 7.4- and 7.6-mm groups and decreased (P < 0.03) between the 7.6- and 7.8-mm groups. Although concentrations in the associated F2 did not change significantly, a decrease between the 8.0- and 8.2-mm diameter groups approached significance (P < 0.06).
The main effects and interaction of activin-A concentrations in F1 and the associated F2 were not significant. When concentrations in F1 and F2 were combined, there was an increase (P < 0.04) between the 7.6- and 8.2-mm groups (Fig. 1). Concentrations then decreased (P < 0.05) between the 8.2- and 8.4-mm groups.
Comparisons of F1 and F2 Using Independent Diameter Groups for Each Follicle
When F2 was grouped independently, diameter groups of 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0 mm were obtained (Fig. 2). Independent common diameter groups for F1 and F2 were obtained for 7.4, 7.6, 7.8, and 8.0 mm. For the independent F2 groups, the estradiol concentrations were different (P < 0.02) among groups (Fig. 2). In the common F1 and F2 diameter groups (7.4, 7.6, 7.8, 8.0 mm) there was a main effect of group (P < 0.003), reflecting an estradiol increase (P < 0.05) between the 7.6- and 7.8-mm groups averaged over the two follicles, but not an effect of follicle (F1 versus F2) or an interaction.
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The differences in free IGF-1 concentrations among the independent F2 groups approached significance (P < 0.06); concentrations increased (P < 0.003) between 7.0 and 7.2 mm and decreased (P < 0.03) between 7.2 and 7.4 mm (Fig. 2). When free IGF-1 concentrations were compared between F1 and F2 for the common independent diameter groups, there was an interaction of F1 and F2 by diameter group that approached significance (P < 0.08; Fig. 2); concentrations were greater (P < 0.01) in F1 than in F2 when the common diameter was 7.6 mm. The differences in activin-A concentrations among the independent F2 diameter groups or between F1 and F2 at the common diameters were not significant (Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Follicular fluid concentrations of estradiol, free IGF-1, and activin-A were previously shown [2, 3, 13] to change differentially between follicles near deviation and were therefore the focus of the present study; inhibins and steroids other than estradiol have not been temporally implicated in deviation in cattle. Previous studies of sequential changes in follicular fluid factors during follicle selection or deviation used intersampling intervals of 12 h [2] or the equivalent of 16 h [2, 3]; the latter studies used an F1 diameter increase of 1.0 mm between groups. In a recent study, ablation of F1 at the expected beginning of deviation (8.5 mm) was used as a reference point and the conversion of the associated F2 to dominant status was used to assess the follicular fluid changes preceding experimental deviation [13]; a mean fluctuation in activin-A concentrations, as indicated by a significant increase followed by a significant decrease, occurred within 8 h. On this basis, the 0.2-mm increment between groups in the present study favored the detection of fluctuations. An incremental diameter increase of 0.2 mm occurred during a mean equivalent of 4 h. This estimate was calculated from a determined growth rate of 1.2 mm during the 24 h before sampling.
Diameter deviation began in the 8.2-mm group. This was shown by a diameter difference between F1 and the associated F2 that was greater in the 8.4-mm group than in the 8.2-mm group, indicating the first differential growth between F1 and F2. The beginning of deviation at 8.2 mm seems close to the reported means of 8.5 mm [1] and during a diameter range of 8.0–8.9 mm [2] or 8.0–8.4 mm [3]. The present data may be more precise because follicle growth between sampling was less (increments of 0.2 mm versus means of 0.5 mm [1, 3] or 1.0 mm [2, 3]).
The hypothesis supported the concept that estradiol and free IGF-1 simultaneously (within 4 h) become higher in concentration in the follicular fluid of F1 than in the associated F2 by the beginning of diameter deviation. Both factors established a differential elevation in F1 versus F2 beginning in the 8.0-mm group, or 0.2 mm (equivalent to 4 h) before the beginning of deviation. For estradiol, the differential increase in F1 before the beginning of diameter deviation was indicated by a difference between follicles that first reached significance in the 8.2-mm group. Grouping of F2 according to F2 diameters independent of the association with the F1 groups provided additional perspective on the relationship between the two follicles. The factorial analysis of estradiol concentrations for the common F1 and F2 diameter groups (7.4, 7.6, 7.8, and 8.0 mm) did not indicate a difference due to follicle status and indicated that estradiol concentrations began to increase in both follicles when they reached 7.6 mm. Significant differences in estradiol among the independent F2 groups, but not among the F2 follicles associated with the F1 groups, are attributable to distribution of the independent F2 diameters among the F1 groups. For example, data for each of four independent F2 groups (7.0, 7.2, 7.4, and 7.6 mm) were initially distributed over four or five F1 groups, thereby masking the changes in follicular fluid factors during the increasing diameter of F2. Thus, it appears that more estradiol is secreted into the follicular fluid independently by each follicle on the basis of its diameter or developmental stage, followed by a differential increase in F1 in association with the developing deviation mechanism.
The interfollicle relationships in the concentrations of free IGF-1 were more complex than for estradiol. The changes immediately encompassing diameter deviation were represented by a continuing concentration in F1 and a tendency for decreasing concentration in the associated F2 rather than by an estradiol-like differential increase in F1. The results for free IGF-1 as well as for estradiol are consistent with the direction of concentration changes in F1 and the associated F2 previously reported [2, 3] for bovine follicular fluid. Unlike estradiol, mean fluctuations in free IGF-1 (a significant increase followed by a significant decrease) were detected in both follicles when each follicle was grouped independently by diameter. A fluctuation occurred during an equivalent of 8 h or during the 7.0- to 7.4-mm F2 diameter groups and the 7.4- to 7.8-mm F1 diameter groups. The 7.0–7.4 range of F2 groups provided information that was not available for F1. When common diameter groups were available for the two follicles (7.4, 7.6, 7.8, and 8.0 mm), the free IGF-1 concentration changes were not similar between follicles, as shown by a fluctuation between the 7.4- to 7.8-mm F1 diameter groups but not for the corresponding independent F2 diameter groups. Fluctuations in concentrations of follicular fluid factors apparently have not been previously reported. The controlling mechanisms are unknown. The intrafollicular fluctuations were unexpected and were not included in the hypotheses. They are therefore considered as observations and require confirmation.
The patterns of activin-A changes in the follicular fluid of the two follicles were unlike the changes for either estradiol or free IGF-1. The activin-A hypothesis was only partly supported. A transient elevation in activin-A was present at the beginning of the differential changes between F1 and the associated F2 in estradiol and free IGF-1 and preceding diameter deviation. However, there was no indication that the elevation occurred differentially in F1 versus F2 and was not detected until F1 and F2 data were combined. In addition, the mean increase in activin-A concentrations was gradual, encompassing the equivalent of 16 h compared with 4 or 8 h in the study [13] involving conversion of F2 to dominant status during experimental deviation. No significant changes were found in F2 when F2 follicle diameters were grouped independently of F1. These results suggest that a systemic factor contributed to the regulation of the activin-A increase regardless of follicle status because it occurred simultaneously in the two follicles when F2 was associated with the F1 diameter groups. This does not negate a role for activin-A in deviation; speculatively, only F1 may have reached a developmental stage that allowed F1 to respond to the activin-A elevation, thereby selectively affecting the F1 estradiol and free IGF-1 concentrations. The manner in which follistatin [11] and other [23] activin-binding proteins are involved in this potential role of activin-A needs investigation, especially because the activin-A assay measured both bound and free (bioactive) forms.
In conclusion, diameter deviation began when F1 was 8.2–8.3 mm (i.e., the 8.2-mm group) as shown by a diameter difference between F1 and the associated F2 in the 8.4-mm group that was greater than in the 8.2-mm group. Follicular fluid estradiol and free IGF-1 concentrations both began to change differentially between F1 and the associated F2 in the 8.0-mm group; estradiol increased in F1 and free IGF-1 tended to decrease in F2. Concentrations of activin-A did not change differentially between follicles. However, a transient elevation in activin-A combined for the associated F1 and F2 encompassed the beginning of the differential concentration changes between follicles in estradiol and free IGF-1. When F1 and F2 were grouped according to the independent diameters of each follicle, estradiol increased in both follicles beginning at 7.6 mm. Intrafollicular fluctuations in concentrations of free IGF-1 (a significant increase followed by a significant decrease) were found in the independently grouped follicles. The mean fluctuations were completed in an equivalent of 8 h.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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
3 C.M. is on leave from the Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science-UNESP, Botucatu, SP, Brazil
Received: 19 June 2002.
First decision: 10 July 2002.
Accepted: 12 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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