| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Eutheria Foundation, Cross Plains, Wisconsin 53528 Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
activin, estradiol, follicle, follicle-stimulating hormone, growth factors
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The FSH surge that stimulates emergence of the ovulatory wave [3] begins to decline in circulating concentrations when the largest follicle reaches a diameter of about 13 mm [2, 4]. The FSH decline continues during the remaining common growth phase of the follicles in a wave and for several days after the beginning of deviation. The necessity for low concentrations of FSH for deviation is consistent with the formation of multiple dominant follicles following administration of FSH [5] or an anti-inhibin that raises the endogenous concentrations of FSH [6]. Multiple follicles of the common growth phase contribute to the FSH decline [4]. When circulating FSH concentrations decrease to a threshold level at the end of the common growth phase, only the most developed or largest follicle utilizes the low FSH concentrations and directs a continuing FSH decline [7]. Circulating LH may also be involved in deviation. Concentrations of LH are elevated at deviation in mares [2], and circulating concentrations of estradiol and total inhibin decreased when the concentration of LH was experimentally reduced [8].
Studies in mares and more detailed studies in cattle indicate that the largest follicle develops greater responsiveness to FSH and LH at the time of deviation, and therefore it alone is able to respond to the low gonadotropin concentrations (reviewed in [9]). The local factors that cause the apparent differential increase in responsiveness of the future dominant follicle to gonadotropins may be pivotal to the mechanism of follicle selection.
Earlier studies [7, 10] in mares demonstrated that estradiol increases selectively in the future dominant follicle before the beginning of deviation, indicating on a temporal basis that estradiol is a candidate for increasing the gonadotropin responsiveness of the follicle. In another study [11], various forms of inhibin were assayed in equine follicular fluid, but the diameter ranges seemed too great for relating changes in inhibin concentrations to the deviation phenomenon. However, a more recent study [12] focused on deviation and found that concentrations of follicular fluid estradiol, free insulin-like growth factor-1 (IGF-1), activin-A, and inhibin-A began to increase differentially in the largest follicle before the beginning of diameter deviation.
These in vivo results in mares are consistent with in vitro results in other species, especially cattle, that have demonstrated the following interrelationships among the four follicular fluid factors and gonadotropins: 1) estradiol increased the synthesis of IGF-1 and the sensitivity to FSH and LH and potentiated the expression of gonadotropin receptors in granulosa cells [13]; 2) IGF-1 increased the ability of FSH to stimulate estradiol secretion in granulosa cells [14, 15] but only at low doses of FSH [16]; 3) inhibin-A modulated gonadotropin responsiveness and steroidogenesis in granulosa and theca cells [17, 18]; 4) activin-A stimulated aromatase activity and estradiol secretion in granulosa cells [17]; and 5) FSH stimulated the production of estradiol, IGF-1, activin-A, and inhibin-A in granulosa cells [16, 19].
When the largest follicle (F1) was ablated at the expected beginning of deviation, the second-largest follicle (F2) became the dominant follicle in all of six mares, whereas F1 became dominant in all eight mares in which both F1 and F2 were retained [7]. The diameter of F2 and plasma FSH concentrations in F1-ablated mares increased within 1 day. Thus, a follicle that was destined to regress changed its status within 24 h after the future dominant follicle was removed. 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. This approach was used recently in cattle [20, 21], but has not been used in mares. The present experiment concerned the effects of ablating F1 at the expected beginning of deviation on the changes in diameter and concentrations of follicular fluid factors in F2 and F3 during the experimental assumption of dominance by F2 in mares. The temporal relationships between postablation changes in follicle characteristics and circulating FSH were also considered.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Nonlactating ponies of mixed breeding, aged 3–15 yr, and weighing 270–360 kg, were used during 48 estrous cycles in the last half of the ovulatory season (July–October 2001) in the northern hemisphere. The mares were kept outdoors and fed alfalfa and grass hay with free access to water and mineralized salt.
Follicles 
25 mm in diameter were monitored once a day by transrectal ultrasonography to establish the day of ovulation. Transvaginal ultrasonography was used to ablate all follicles 6 mm in diameter 10 days after ovulation as described [2]. This was done to eliminate follicles from previous waves to facilitate tracking individual follicles of the subsequent or new wave. Follicles of the new wave were scanned each day by transrectal ultrasonography as described [22] until the largest follicle reached 16 mm. Thereafter, the follicles were scanned every 12 h until the end of the experiment. The four largest follicles were tracked as described [23]. Follicles were measured with electronic calipers using the mean width and length from a frozen image.
Ablation of F1 was done at the expected beginning of deviation (Hour 0; F1, 22.5 mm [2]). Because of the 12-h interval between scanning sessions, ablation was done when F1 first reached >20.0 mm to obtain a range of actual diameters with an average of approximately 22.5 mm. Mares were not used if the difference in diameters between F1 and F2 at Hour 0 was >7.0 mm. This was done to increase the likelihood that F2 would be capable of becoming dominant after ablation of F1 [7]. Collection of follicular fluid of specified follicles was done by ultrasound-guided transvaginal aspiration of follicular contents as described [2]. The postablation conversion of the future largest subordinate follicle (F2) to a future dominant follicle was used to study systemic FSH and follicular changes temporally associated with the conversion. A change is defined as beginning at the examination preceding a significant difference.
Experimental Groups
Mares were randomized by replicate into six groups (n = 8 mares/group) as follows: 1) a control group, 2) a reference group, and 3) four ablation groups. In the control group, follicle diameters and systemic FSH concentrations were determined but follicular fluid was not collected. In the reference group, follicular fluid was collected from F1, F2, and F3 at Hour 0. In the F1-ablation groups, follicular fluid was collected from F2 and F3 at Hours 12, 24, 48, or 72. The largest and second largest remaining follicles at the hour of follicular fluid collection were defined as F2 and F3. For each group, the experiment ended at Hour 72 (controls) or at the hour of follicular fluid collection. Deviation between F2 and F3 in the F1-ablated groups was termed experimental deviation and was compared to deviation between F1 and F2 in controls.
The actual, as opposed to expected, hour of deviation was determined in two ways: 1) by estimating from the diameter data of individual mares when the two largest follicles began to differ in growth rates [2], using F1 and F2 in controls and F2 and F3 in F1-ablated mares in the 72-h group and 2) by determining when changes in mean diameters between F2 and F3 began to differ, using the reference group and the F1-ablated groups.
Blood sampling for FSH assay was begun when the largest follicle was 16 mm. Samples were collected every 12 h from the control group and the ablation group with follicular fluid collection at Hour 72 and from the reference and ablation groups at Hours 0, 12, 24, 48, or 72. Samples were collected by jugular venipuncture into heparinized tubes and immediately centrifuged at 1500 x g for 6 min. The plasma fraction was separated and stored at -20°C until the FSH assay was done.
Hormone Assays
Plasma FSH concentrations were measured by a double-antibody radioimmunoassay validated for mares [24] and recently modified [12] in our laboratory. Intraassay coefficients of variation (CV) and sensitivity were 6.8% and 0.54 ng/ml, respectively. For this and the other assays, sensitivity was calculated from two standard deviations above for enzyme-linked immunosorbent assays and two standard deviations below for radioimmunoassays, relative to the zero standard. Concentrations of follicular fluid factors were determined by procedures that have been described and validated for equine follicular fluid in this laboratory [12]. The intraassay and interassay CVs for quality control samples and the mean assay sensitivity, respectively, were as follows: free IGF-1, 1.2%, 5.6%, and 0.005 ng/ml; activin-A, 5.3%, 2.6%, and 40 pg/ml; inhibin-A, 6.3%, 13.2%, and 0.6 pg/ml; progesterone, 3.6%, 9.9%, and 0.02 ng/ml; and androstenedione, 2.9%, 5.4%, and 1.5 pg/ml. The intraassay CV and sensitivity for factors with no interassay CV were 4.5% and 3.0 pg/ml for estradiol and 6.0% and 0.3 ng/ml for insulin-like growth factor binding protein-2 (IGFBP-2).
Statistical Analyses
Data for each end point were challenged for extreme values with the Dixon outlier test [25] and for normality with the Kolmogorov-Smirnov test [26]. When the normality test was significant (P < 0.05), data were transformed by either natural logarithm or square root. Diameters of F1 and F2 in controls were compared with diameters of F2 and F3 in F1-ablated mares with follicular fluid collection at Hour 72, using a 2-by-2 factorial ANOVA; data were normalized to the beginning of deviation for each group. Plasma FSH concentrations were analyzed by the MIXED procedure with a repeated statement and a first-order autoregressive structure to account for autocorrelation between sequential measurements every 12 h. When the group effect or interaction was significant or approached significance, the follicle and FSH means were further compared within each hour by paired t-tests. Discrete data for follicle end points and circulating FSH at the hour of follicular fluid collection were analyzed by ANOVA. For follicle end points, the main effects of follicle (F2, F3), hour, and interaction were analyzed by a 2-by-5 factorial ANOVA. When a main effect or the interaction was significant, data were further examined for an hour effect for each follicle, followed by comparisons of means by unpaired t-tests. In addition, differences between F1 and F2 at Hour 0 and between F2 and F3 at each hour were examined by paired t-tests. The hour effect for FSH was analyzed by a one-way ANOVA. 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 and P < 0.1 indicated that a difference approached significance.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
Discrete data for concentrations of plasma FSH, diameters of follicles, and concentrations of follicular fluid factors from samples obtained at the hour of follicular fluid collection for studying experimental deviation in the F1-ablated groups are shown (Fig. 3). FSH concentrations did not change significantly between Hours 0 and 12, and progressively decreased during Hours 12 to 72 (P < 0.007). An interaction (P < 0.005) of follicle (F2, F3) by hour for diameters was attributable to an increase (P < 0.05) in diameter of both follicles between Hours 0 and 24, a continued increase (P < 0.05) in F2 after Hour 24, and no changes in F3 after Hour 24. There was no difference in mean diameter between F1 at Hour 0 (21.4 ± 0.2 mm) and F2 at Hour 24 (21.8 ± 0.6 mm).
|
The differences between F1 and F2 at Hour 0 were significant or approached significance for the concentrations of each of the six follicular fluid factors (Fig. 3). Follicular fluid estradiol concentrations for F2 and F3 for the F1-ablated groups showed a follicle effect that approached significance (P < 0.07), reflecting a higher average concentration in F2. Although there was no hour effect or interaction, concentrations between Hours 0 and 12 approached a decrease (P < 0.1) for F2 and decreased (P < 0.05) for F3. Concentrations increased (P < 0.05) for F2 and decreased (P < 0.05) for F3 between Hours 48 and 72, resulting in higher concentrations in F2 at Hour 72. Progesterone concentration was higher (P < 0.05) in F2 than in F3 at Hour 72, but there were no other significant differences (Fig. 3). There were no significant differences for androstenedione (data not shown), although a nonsignificant decrease in F2 and a nonsignificant increase in F3 between Hours 48 and 72 were associated with a difference (P < 0.003) between the follicles at Hour 72.
An interaction (P < 0.02) for follicular fluid concentrations of free IGF-1 was attributable to changes in F2 (P < 0.001) but not in F3 (Fig. 3). For F2, the concentrations decreased (P < 0.05) between Hours 0 and 12 and progressively increased (P < 0.05) over Hours 12 to 72, including a significant increase between Hours 12 and 24. The interaction for IGFBP-2 approached significance (P < 0.08; Fig. 3). Concentrations approached an increase (P < 0.1) in F2 between Hours 0 and 12 and a decrease (P < 0.05) between Hours 48 and 72. A difference (P < 0.05) between follicles at Hour 72 was attributable to the decrease in F2 and a nonsignificant increase in F3.
Changes in follicular fluid concentrations of activin-A occurred in F2 and were characterized by a progressive increase (P < 0.05) over Hours 12 to 72, resulting in higher (P < 0.04) or approaching higher (P < 0.06) concentrations in F2 than in F3 at Hours 48 and 72 (Fig. 3). There were no significant changes for F3. The mean follicular fluid concentrations of inhibin-A were higher (P < 0.04) for F2 than for F3, with a difference (P < 0.05) or an approaching difference (P < 0.1) between follicles at each hour. A decrease (P < 0.05) occurred in F2 between Hours 0 and 12 and an increase (P < 0.05) between Hours 48 and 72.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Circulating concentrations of FSH continued to decrease in the controls after the expected beginning of deviation, as previously reported [2, 7, 8]. In contrast, the concentrations did not decrease between Hours 0 and 12 in the F1-ablated mares. In a previous report, a significant FSH increase was obtained 24 h after ablation of F1 [7]. The functional two-way coupling between the largest follicle and circulating FSH was temporally close in the present experiment as previously reported for cattle [27]. This was indicated by the maintenance of FSH concentrations during the 24 h after F1 was ablated and the temporally associated continued growth of F2 and F3. This result indicated that F2 responded to a maintenance of FSH concentration, although the concentration could have been higher before Hour 12. The concentration of FSH was not different between deviation of F1 and F2 in controls and experimental deviation of F2 and F3 in the F1-ablated groups. Deviation may be initiated when FSH concentrations, as well as the diameter of the largest follicle, reach threshold values, but this was not studied critically. The initiation of deviation may involve other unknown mechanisms in addition to a close and sensitive follicle/FSH relationship. For example, the suggestion that the dominant follicle produces a factor that directly inhibits other follicles [18] cannot be discounted on the basis of the present results.
Temporal relationships in the changing diameters and concentrations of follicular fluid factors in F2 and F3 were used to identify the factors that were available in F2 for potential increased gonadotropin responsiveness in the conversion of F2 to dominant status. At the expected beginning of deviation (Hour 0), the concentrations of estradiol, progesterone, free IGF-1, activin-A, and inhibin-A became lower, and the concentration of IGFBP-2 became higher as the diameter ranking of the follicles (F1, F2, F3) decreased. The differences between F1 and F2 at Hour 0 were significant or approached significance for all six follicular fluid factors. Similar results were obtained for F1 and F2 in a previous study [12], except that the concentrations of progesterone and IGFBP-2 were not significantly different between F1 and F2. In F2 and sometimes in F3 of the present study, the concentrations of estradiol, free IGF-1, activin-A, and inhibin-A decreased or approached a significant decrease between Hours 0 and 12, whereas IGFBP-2 approached an increase. These changes may reflect regressive biochemical changes occurring in the two retained follicles after the beginning of deviation, but controls were not available for comparison.
After Hour 12 or during the conversion of F2 to dominant status following the removal of F1, the direction of concentration changes differed among follicular fluid factors. Most notable was the progressive increase in free IGF-1 in F2 but not in F3 between Hours 12 and 72 and the higher concentration in F2 than in F3 at the beginning of experimental deviation (Hour 24). The increased IGF-1 may have enhanced the gonadotropin responsiveness [16] of F2, thereby playing a role in experimental deviation. In this regard, in vitro studies in cattle support the concept that IGF-1 modulates the action of FSH on granulosa cells [14, 15], but similar studies apparently have not been done in mares. The selective free IGF-1 increase in F2 preceding the beginning of experimental deviation between F2 and F3 seems similar to the reported differential change between F1 and F2 before deviation [12]. The decrease in free IGF-1 between Hours 0 and 12 and the increase between Hours 48 and 72 were reciprocally related to the direction of change in IGFBP-2 at these hours, although the hour effect for IGFBP-2 in F2 was not significant. A similar reciprocal relation has been reported in cattle for deviation between F1 and F2 [28] and between F2 and F3 after ablation of F1 [20, 21].
Concentrations of inhibin-A did not change differentially between F2 and F3 in association with the beginning of experimental deviation. However, unlike any of the other factors, concentrations were consistently higher in F2 than in F3 during Hours 0 to 72 or throughout the experimental period. This may have imparted an advantage for F2 at the beginning of experimental deviation, despite the lack of a differential change in F2. In contrast to experimental deviation between F2 and F3, a recent study found that inhibin-A began to increase differentially in F1 before the beginning of deviation between F1 and F2 [12]. The physiologic role of intrafollicular inhibin-A in the future dominant follicle needs further study.
Estradiol did not begin to increase differentially in F2 until Hour 48, a day after the beginning of experimental deviation. This is consistent with a report that systemic estradiol continued at low concentrations for 2 or 3 days after ablation of F1 at the expected beginning of deviation [7]. In contrast to the delayed intrafollicular estradiol response until well after the beginning of experimental deviation, estradiol increases selectively in the future dominant follicle and in the circulation before the beginning of deviation between F1 and F2 [10]. Despite these reported temporal results, the present results indicated that a selective estradiol increase is not necessary for initiating experimental deviation. However, the estradiol concentrations were higher in F2 than in F3 at Hours 0 and 12 and therefore could have contributed to deviation, despite the absence of a differential change. The estradiol increase after Hour 48 (24 h after the beginning of experimental deviation) indicates that estradiol may play a role in the growth of the dominant follicle at a later stage. The results indicated on a temporal basis that progesterone and androstenedione were not selectively involved in the initiation of experimental deviation. During deviation between F1 and F2, progesterone increased in F1 simultaneously with the beginning of deviation [12]. The present results indicate that such a progesterone increase is not needed for deviation to occur. Progesterone began to increase in F2 and androstenedione began to increase in F3 at Hour 48 so that the concentrations in the two follicles were different at Hour 72.
Activin-A concentrations increased in F2 between Hours 12 and 72, but unlike free IGF-1, the increase between Hours 12 and 24 and the difference between F2 and F3 at Hour 24 were not significant. Thus, there was no temporal indication that activin-A contributed to a differential gonadotropin responsiveness preceding or at the beginning of experimental deviation. In contrast, an earlier report in mares indicated that activin-A increased in F1 before the beginning of deviation between F1 and F2 [12].
Some reservation in comparing the mechanisms of experimental and natural deviation is warranted. The observed differences between F2 and F3 at the time of F1 ablation could have been dissimilar to the differences between F1 and F2 at the corresponding time preceding natural deviation. In conclusion, however, ablation of the largest follicle (F1) at the expected beginning of deviation (Hour 0) maintained the FSH concentrations for 24 h in association with the continued growth of both the second (F2) and third (F3) largest follicles. Experimental deviation in diameter between F2 and F3 began at Hour 24 in F1-ablated mares at a time when systemic FSH concentration and F2 diameter were similar to systemic FSH concentration and F1 diameter at the beginning of deviation in control mares. However, the role of the diameter-to-FSH relationship in the initiation of deviation was not specifically studied. Concentrations of free IGF-1 increased differentially in F2 beginning at Hour 12, so that higher concentrations were available in F2 than in F3 by the beginning of experimental deviation at Hour 24. Inhibin-A was higher in F2 than in F3 throughout the experimental period and estradiol was higher at Hours 0 and 12, but differential changes between follicles were not detected by the time of deviation. Although the role of estradiol and inhibin-A is not clear, temporal results indicated that IGF-1 was involved in initiating experimental deviation.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by the Eutheria Foundation, Cross Plains, WI, and the University of Wisconsin, Madison. 
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
Accepted: April 15, 2002.
Received: February 6, 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M A Beg and O J Ginther Follicle selection in cattle and horses: role of intrafollicular factors. Reproduction, September 1, 2006; 132(3): 365 - 377. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O J Ginther, E L Gastal, M O Gastal, and M A Beg In vivo effects of pregnancy-associated plasma protein-A, activin-A and vascular endothelial growth factor on other follicular-fluid factors during follicle deviation in mares Reproduction, April 1, 2005; 129(4): 489 - 496. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E D Watson, S-E Bae, R Thomassen, S R M Thomson, K Woad, and D G Armstrong Insulin-like growth factors-I and -II and insulin-like growth factor-binding protein-2 in dominant equine follicles during spring transition and the ovulatory season Reproduction, September 1, 2004; 128(3): 321 - 329. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.J. Acosta, M.A. Beg, and O.J. Ginther Aberrant Blood Flow Area and Plasma Gonadotropin Concentrations During the Development of Dominant-Sized Transitional Anovulatory Follicles in Mares Biol Reprod, August 1, 2004; 71(2): 637 - 642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O.J. Ginther, E.L. Gastal, M.O. Gastal, and M.A. Beg Critical Role of Insulin-Like Growth Factor System in Follicle Selection and Dominance in Mares Biol Reprod, May 1, 2004; 70(5): 1374 - 1379. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O.J. Ginther, E.L. Gastal, M.O. Gastal, C.M. Checura, and M.A. Beg Dose-Response Study of Intrafollicular Injection of Insulin-Like Growth Factor-I on Follicular Fluid Factors and Follicle Dominance in Mares Biol Reprod, April 1, 2004; 70(4): 1063 - 1069. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O.J. Ginther, D.R. Bergfelt, M.A. Beg, C. Meira, and K. Kot In Vivo Effects of an Intrafollicular Injection of Insulin-Like Growth Factor 1 on the Mechanism of Follicle Deviation in Heifers and Mares Biol Reprod, January 1, 2004; 70(1): 99 - 105. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
