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Regular Article |
a Department of Animal Health and Biomedical Science, University of Wisconsin, Madison, Wisconsin 53706
b The Eutherian Foundation, Cross Plains, Wisconsin 53528
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5 mm were ablated 10 days after ovulation, followed by follicular fluid collection from the three largest follicles (F1, F2, and F3) when F1 of the new wave reached a diameter of 8.0–11.9, 12.0–15.9, 16.0–19.9, 20.0–23.9, 24.0–27.9, or 28.0–31.9 mm (n = 4–8 mares/range). Diameter deviation between F1 and F2 began during the 20.0- to 23.9-mm range, as indicated by a greater difference in diameter between the two follicles at the 24.0- to 27.9-mm range than at the 20.0- to 23.9-mm range. Androstenedione concentrations increased in F1, F2, and F3 between the 16.0- to 19.9- and 20.0- to 23.9-mm ranges. In contrast, estradiol, free insulin-like growth factor (IGF)-1, activin-A, and inhibin-A concentrations increased only in F1 beginning at the 16.0- to 19.9-mm range. As a result, the concentrations of all four factors were higher in F1 than in F2 and F3 at all the later ranges, including the 20.0- to 23.9-mm range (beginning of diameter deviation). Concentrations of progesterone differentially increased in F1, concentrations of androstenedione and IGF-binding protein (IGFBP)-2 increased only in F2 and F3, and concentrations of inhibin-B differentially decreased in F2 and F3 simultaneous with the beginning of deviation. Concentrations of FSH, LH, pro-
C inhibin, and total inhibin did not change differentially among follicles. Results indicated that, on a temporal basis, estradiol, free IGF-1, activin-A, and inhibin-A may have played a role in the initiation of follicle deviation. In addition, these four factors as well as progesterone, androstenedione, IGFBP-2, and inhibin-B may have been involved in the subsequent differential development of the follicles.
activin, estradiol, follicle, follicular development, growth factors, inhibin, progesterone, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A surge in circulating FSH concentrations stimulates the initiation of a follicular wave [1]. When the largest follicle reaches a mean of 13 mm, a decline in circulating FSH concentrations begins, apparently as a result of the secretion of inhibin by the growing follicles [3]. Beginning at the time of deviation, only the largest follicle continues to contribute to the decline in circulating FSH concentrations, apparently through increased secretion of estradiol [4, 5] and continued secretion of inhibin [3]. In cattle, circulating FSH at the time of deviation has decreased to concentrations less than the requirements of the future subordinate follicles, but not of the future dominant follicle, which is able to continue growing in spite of low concentrations [6]. In mares, temporal relationships suggest a similar differential sensitivity of the follicles to FSH in association with deviation, but to our knowledge, this has not been demonstrated directly. In both cattle [7] and mares [8], an increase in circulating LH and a functional dependence of the largest follicle on LH at the time of deviation have been demonstrated. In cattle [2] and, apparently, in mares [9], the granulosa cells of the future dominant follicle acquire LH receptors just before the beginning of diameter deviation.
Candidates for a role in increasing the responsiveness of the future dominant follicle to pituitary gonadotropins include estradiol, insulin-like growth factors (IGFs), and activin/inhibin peptides. Estradiol has been shown to promote expression of FSH and LH receptors [10, 11] and to enhance aromatase activity [12] and IGF-1 synthesis [13] from granulosa cells. In cattle, temporal relationships indicate that the increase in sensitivity of the largest follicle to low concentrations of FSH in association with deviation [6] is a function, at least in part, of estradiol [14]. A similar potential exists in mares, because the intrafollicular concentrations of estradiol increase specifically in the future dominant follicle before diameter deviation begins [5].
The actions of FSH and LH on ovarian cells from a variety of species are enhanced by IGF-1 (reviewed in [15]). The IGF-1 stimulates granulosa cell mitosis, progesterone production, LH-induced androgen production, and estradiol production. Follicular IGF-binding proteins (IGFBPs) decrease the bioavailability of IGF-1, thus inhibiting IGF-1 actions on the ovary [15]. In cattle, concentrations of unbound or free IGF-1 were maintained in the largest follicle but decreased in the second-largest follicle during a period encompassing deviation, whereas the concentrations of IGFBP-2 did not change in the largest follicle and increased in the smaller follicles, respectively, after the beginning of deviation [2]. Differential changes in concentrations of other IGFBPs in the largest versus smaller follicles at an apparently comparable period have also been reported in cattle [16]. Total IGF-1 [17] and IGFBPs [18] have been measured in equine follicular fluid, but to our knowledge, their relationships to the development of follicular waves have not been studied.
Activins and inhibins also modulate the action of pituitary gonadotropins on granulosa and theca cells (reviewed in [19]). In cattle, no significant differential changes were detected in activin-A, total inhibin, inhibin-A, and inhibin-B concentrations among follicles encompassing deviation [2, 20]. In women, follicular fluid concentrations of inhibin-A, but not of inhibin-B or activin-A, increased as both diameter and maturity of the follicles increased [21]. To our knowledge, the associations between follicular waves and local concentrations of activins and inhibins have not been studied in mares.
This study was done in mares during follicular wave development to characterize the temporal changes and relationships in follicular fluid concentrations of gonadotropins (FSH and LH), steroids (estradiol, androstenedione, testosterone, and progesterone), IGF-system components (free IGF-1 and IGFBP-2), and activin/inhibin peptides (activin-A, inhibin-A, inhibin-B, pro-C inhibin, and total inhibin). Interpretation of the results was based on the premise that factors changing in concentrations differentially among follicles before the beginning of diameter deviation may play a role in the initiation of deviation. Factors that change differentially among follicles at or after the beginning of deviation are less likely to be involved in the initiation of deviation but may be involved in the subsequent differential development of the follicles.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nonlactating pony mares of mixed breeding, 5–17 years old and weighing 230–460 kg, were used during September and October 2000 in the northern hemisphere. Mares were kept in an outdoor paddock with access to alfalfa/grass hay, water, and mineralized salt. Before the experiment, follicles >25 mm were monitored daily by ultrasound to establish the day of ovulation.
All follicles 5 mm were ablated 10 days after ovulation by ultrasound-guided transvaginal aspiration of follicular contents as described previously [22]. This was done to initiate the development of a new wave so that follicle identities would not be obscured by follicles from a previous wave. Aspirated follicles that refilled with fluid to 10 mm were reaspirated. The four largest follicles of the new wave were measured transrectally once a day using an ultrasound scanner equipped with a 5-MHz, linear-array transducer. Follicle measurements were done with the electronic calipers of the ultrasound screen by taking the average of width and length from a frozen image [23]. Mares were randomized into six groups (eight replicates) in which the follicular fluid of the three largest follicles (termed F1, F2, and F3 in descending diameter) was collected when the largest follicle of the new wave first reached a diameter of 8.0–11.9, 12.0–15.9, 16.0–19.9, 20.0–23.9, 24.0–27.9, and 28.0–31.9 mm, respectively. The mean diameter just before collection of fluid was taken from two frozen images on the ultrasound screen. Follicular contents were aspirated transvaginally through a 17-gauge needle attached to a syringe as described previously [22]. Aspirations were done manually, by slowly drawing the fluid into a syringe until the antral cavity was emptied, as observed on the monitor screen. Aspiration of follicular fluid took 2–10 sec, depending on the follicle size. Collected fluid was immediately centrifuged at 2500 rpm for 5 min to remove cells and debris, and fluid was stored at -20°C. Ovaries were periodically scanned after follicular fluid collection to detect ovulation. Mares in which follicular fluid collection was not followed by the development of an ovulatory follicle from either the extant or a subsequent follicular wave were considered to have entered the anovulatory season and were removed from the experiment; this was done to ensure that the mares were in the ovulatory season at the time of fluid collection.
The mean beginning of diameter deviation was based on the first significant increase in the difference in diameter between F1 and F2 and on the diameter differences within follicles among ranges. A mean change in the concentrations of a follicular fluid factor between diameter ranges that was different among follicles was defined as a differential change. The temporal relationship between the beginning of diameter deviation and differential changes in the concentrations of each follicular fluid factor was determined by examination for differential changes among follicles beginning at the diameter range before and at the beginning of deviation.
Hormone Assays
All assays were validated in our laboratory for use with equine follicular fluid. Validations were done by ensuring parallelism between the curve obtained from assaying serial dilutions of pooled equine follicular fluid and the corresponding standard curve. Sample dilutions were used in each assay that resulted in a percentage binding for RIA or optical density for ELISA that was central to the range of the standard curve. The RIA sensitivity was calculated by subtracting two standard deviations from the mean maximum percentage binding. For ELISA, two standard deviations were added to the optical density for zero-standards. In each instance, sensitivity was averaged over all assays.
A double-antibody RIA previously used for mare plasma in our laboratory [24] was used to measure FSH concentrations in follicular fluid with the following modifications: Iodination was done with iodogen [25], and antiserum and tracer were added to samples in a single step, followed by incubation at room temperature for 24 h and subsequent addition of precipitating antibody and 6% polyethylene glycol (2:5 [v/v] ratio) 3 h before centrifugation and counting. Highly purified equine antigen (FSH, AFP-5022B; A.F. Parlow, National Hormone & Pituitary Program, Torrance, CA) was used for iodination and standards, and rabbit anti-equine FSH (AFP-2062096) diluted 1:20 000 in normal rabbit serum (NRS) was used as primary antibody. Cross-reactivity of the antibody with eLH is 2.5% according to the supplier. The LH concentrations were measured by an equine double-antibody RIA developed in our laboratory [26] with the same modifications as those for the FSH assay. A highly purified equine antigen (LH, AFP-5130A) and anti-equine LH (AFP-240580) diluted 1:160 000 in NRS were used. According to the supplier, the antibody shows less than 10% cross-reactivity with eCG and eFSH. Intraassay coefficients of variation (CVs) were 6.7% for FSH and 3.3% for LH, and assay sensitivities were 1.51 and 0.05 ng/ml, respectively.
Estradiol concentrations were determined by a double-antibody RIA kit (Diagnostic Products Corporation, Los Angeles, CA) without extraction as described for our laboratory [4]. The samples were diluted 1:6000 in assay buffer (PBS with 0.1% gelatin). According to the manufacturer, the assay has low cross-reactivity with estrone (12.5%). Intraassay CV and assay sensitivity were 7.3% and 2.79 pg/ml, respectively.
Concentrations of androstenedione were measured by a commercial double-antibody RIA kit (Diagnostic Systems Laboratories, Inc., Webster, TX) developed for use with human samples and adapted for use with equine follicular fluid in our laboratory. Assay buffer (PBS with 0.1% gelatin) was used to prepare androstenedione (Sigma Chemical Co., St. Louis, MO) standards (0.078–20 ng/ml) and to dilute samples (1:500) before assay. Manufacturer information indicates that the antiserum has minimal cross-reactivity with other steroids (<0.35%). Intraassay CV and assay sensitivity were 2.2% and 4.78 ng/ml, respectively.
Testosterone concentrations were determined by a double-antibody RIA (Diagnostic Systems Laboratories) previously used for bovine samples in our laboratory [2] and adapted for use with equine samples. A 1:75 sample dilution in assay buffer (PBS with 0.1% gelatin) was used. Low levels of antisera cross-reactivity occur with 5-dihydrotestosterone (6.6%), 5-androstane-3ß,17ß-diol (2.2%), 11-oxotestosterone (1.8%), and a few other steroids (<1%) according to the manufacturer. Intraassay CV was 2.8%, and assay sensitivity was 0.61 ng/ml.
Concentrations of progesterone were measured by a competitive ELISA as described previously [27] and adapted for use in our laboratory for mare follicular fluid. A protein-based assay buffer was used to prepare standards (0.3–10 ng/ml) and to dilute samples (1:100). Intra- and interassay CVs were 8.3% and 10.1%, respectively, and the assay sensitivity was 71.60 pg/ml.
Concentrations of total (i.e., immunoreactive) inhibin in follicular fluid were measured by an RIA kit (Institute of Reproduction and Development, Monash Medical Center, Clayton, Victoria, Australia) as described for our laboratory [3]. The antibody recognizes dimeric inhibin as well as free subunit forms [28]. Intraassay CV was 8.1%, and the assay sensitivity was 3.66 ng/ml.
Concentrations of inhibin-A and inhibin-B were measured by two different solid-phase sandwich ELISAs (Oxford Bio-Innovation Ltd., Oxfordshire, U.K.) as described previously [2, 20] and adapted for use with equine samples. Follicular fluid dilutions of 1:1000 and 1:100 in fetal calf serum (zero-standard) were used for inhibin-A and inhibin-B, respectively. According to the manufacturer, cross-reactivity with other inhibins, activins, and follistatin is minimal for the two assays. Intra- and interassay CVs were 7.0% and 17.2%, respectively, for inhibin-A and 0.9% and 16.1%, respectively, for inhibin-B, and assay sensitivities were 3.05 and 18.96 pg/ml, respectively.
Concentrations of pro-C inhibin were measured by a solid-phase sandwich ELISA (Oxford Bio-Innovation). The assay was developed for use with human samples and has been previously used with mare plasma [29]. The assay uses an immobilized monoclonal antibody against the pro region of the inhibin subunit and a second monoclonal antibody coupled to alkaline phosphatase against the C region. The assay shows <0.1% cross-reactivity with inhibin-A, inhibin-B, activin-A, activin-B, and follistatin according to the manufacturer. A working dilution of samples to 1:10 000 in the zero-standard provided by the manufacturer was used. Intra- and interassay CVs and assay sensitivity were 6.8%, 2.5%, and 0.3 pg/ml, respectively.
Activin-A concentrations were measured by a solid-phase sandwich ELISA (Oxford Bio-Innovation) previously used in our laboratory for bovine follicular fluid samples [20] and adapted for use with equine samples. A sample dilution of 1:250 in 5% BSA was used. Assay cross-reactivity with other activins, inhibin, and follistatin is minimal (< 0.5%) as indicated by the manufacturer. Intra- and interassay CVs were 7.3% and 7.1%, respectively, and assay sensitivity was 39.57 pg/ml.
Concentrations of free IGF-1 were quantified by a sandwich-type ELISA (Diagnostic Systems Laboratories) as previously described for cattle in our laboratory [2] and adapted for use with mare follicular fluid. A working dilution of 1:15 in the BSA-based zero-standard provided by the manufacturer was used. Antisera cross-reactivity with IGF-2, insulin, or IGFBPs was not found by the manufacturer. Intra- and interassay CVs and assay sensitivity were 2.6%, 9.7%, and 37.04 pg/ml, respectively.
The IGFBP-2 concentrations were measured by a double-antibody RIA kit (Diagnostic Systems Laboratories) as described for cattle [2] and adapted for use with equine samples in our laboratory. A follicular fluid dilution of 1:85 in the protein-based zero-standard supplied with the kit was used. As indicated by the manufacturer, the IGFBP-2 antibody does not show detectable cross-reactivity with IGBPs -3, -4, -5, and -6 at 0.5 µg/tube and with rhGH, IGF-1, and IGF-2 at 1 µg/tube. Intraassay CV was 10.1%, and assay sensitivity was 0.16 ng/ml.
Statistical Analyses
Data for each end point were tested for normality with a Kolmogorov-Smirnov test [30] and normalized by log-transformation when the probability level was P < 0.01. For each end point, extreme values were tested (P < 0.05) using the extreme standardized deviate [31] after adjusting for normality. After removing outliers, hormonal data were analyzed by the SAS MIXED procedure as a split-plot design taking the animal within diameter range as the random effect [32]. Main effects of diameter range and follicle and the interaction were determined. Means were compared within diameter range or follicle when the corresponding main effect or the interaction was significant (P < 0.05). Comparisons within a follicle between two diameter ranges beginning at the range before or at the beginning of diameter deviation were tested by unpaired t-tests. All other comparisons were done using Tukey-Kramer tests [30]. A probability of P < 0.05 was considered to be significant, and probabilities between P > 0.05 and P < 0.1 were considered to be approaching significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mean follicle diameters and follicular fluid concentrations of FSH relative to diameter ranges of F1 are shown in Figure 1; adequate follicular fluid was not available for assay of FSH in F2 and F3 at the 8.0- to 11.9-mm range. Diameter of the three follicles increased initially, but only F1 continued to increase (P < 0.05) beyond the 20.0- to 23.9-mm range. The first increase (P < 0.03) in the diameter difference between F1 and F2 occurred between the 20.0- to 23.9- and 24.0- to 27.9-mm ranges. Intrafollicular concentrations of FSH were similar in the three follicles; a significant effect of diameter range was due to a progressive decrease in FSH concentrations between the 12.0- to 15.9- and 28.0- to 31.9-mm ranges. Main effects (P > 0.1) of diameter range and follicle or an interaction were not detected in intrafollicular concentrations of LH (data not shown).
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Concentrations of follicular fluid estradiol (Fig. 2) increased (P < 0.03) only in F1 between the 16.0- to 19.9- and 20.0- to 23.9-mm ranges, and concentrations were higher (P < 0.05) in F1 than in F2 and F3 at the 20.0- to 23.9-mm and subsequent ranges. Androstenedione concentrations increased (P < 0.03) in each of the three follicles between the 16.0- to 19.9- and 20.0- to 23.9-mm ranges. Concentrations further increased (P < 0.02) in F2 and F3 between the 20.0- to 23.9- and 28.0- to 31.9-mm ranges. Differences between F1 and each of F2 and F3 in intrafollicular testosterone concentrations approached significance (P < 0.1) at the 28.0- to 31.9-mm range. Concentrations of progesterone differentially increased in F1 (P < 0.04) between the 20.0- to 23.9- and 24.0- to 27.9-mm ranges, resulting in higher (P < 0.0007) concentrations in F1 than in F2 and F3 at the 24.0- to 27.9-mm range.
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Concentrations of IGF-1, IGFBP-2, and activin-A are shown in Figure 3. An increase in concentrations of IGF-1 in F1 approached significance (P < 0.08) between the 16.0- to 19.9- and 24.0- to 27.9-mm ranges. Concentrations in F2 and F3 decreased (P < 0.04) between the 20.0- to 23.9- and 28.0- to 31.9-mm ranges. Concentrations were higher (P < 0.05) in F1 than in F2 and F3 at the 20.0- to 23.9-mm and subsequent ranges. The IGFBP-2 concentrations increased (P < 0.0009) in F2 and F3, but not in F1, between the 20.0- to 23.9- and 28.0- to 31.9-mm ranges. Activin-A concentrations in F1 differentially increased (P < 0.05) between the 16.0- to 19.9- and 20.0- to 23.9-mm ranges. Concentrations were higher (P < 0.04) in F1 than in F2 and F3 at the 20.0- to 23.9- and 24.0- to 27.9-mm ranges. Differences in concentrations between F1 and F2 approached significance (P < 0.09) at the 28.0- to 31.9-mm range.
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An increase in inhibin-A follicular fluid concentrations (Fig. 4) in F1, but not in F2 and F3, between the 16.0- to 19.9- and 24.0- to 27.9-mm ranges approached significance (P < 0.08). Inhibin-A concentrations decreased in F3 (P < 0.004) from the 20.0- to 23.9- to the 28.0- to 31.9-mm ranges, whereas a decrease in F2 approached significance (P < 0.08). Concentrations were higher (P < 0.04) in F1 than in F2 and F3 at the 20.0- to 23.9-mm and subsequent ranges. Inhibin-B concentrations decreased (P < 0.007) in each of F2 and F3 between the 20.0- to 23.9- and 28.0- to 31.9-mm ranges, accompanied by lower (P < 0.007) concentrations in F2 and F3 than in F1 at the 24.0- to 27.9-mm range. The differences in pro-C concentrations among diameter ranges approached significance (P < 0.09). Concentrations appeared to increase over the ranges of 8.0–11.9 to 24.0–27.9 mm, except for a high mean with a large standard error in F3 at the 12.0- to 15.9-mm range, and then decreased. No significant differences were observed for concentrations of total inhibin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The beginning of diameter deviation between the two largest follicles occurred when the largest follicle reached 20.0–23.9 mm; the diameter difference between F1 and F2 first increased between the 20.0- to 23.9 and 24.0- to 27.9-mm ranges. In addition, F1 continued to grow after the 20.0- to 23.9-mm range, whereas F2 and F3 did not (no subsequent differences in mean diameter). This is consistent with previous studies in which observed deviation began when the mean diameter of the largest follicle was 21–23 mm [4, 22].
Intrafollicular FSH and LH profiles were similar to reported circulating profiles during a follicular wave [3, 22]. Previous studies have considered FSH and LH concentrations in the follicular fluid of cattle [33], humans [34], and horses [35], but not in a manner that can be interpreted with reference to development of a follicular wave or diameter deviation. In the present study, no differences were found among follicles, suggesting that differential follicular fluid concentrations of gonadotropins are not involved in follicle selection in mares.
A differential increase in estradiol concentrations among follicles began in F1 before it attained 20.0–23.9 mm or one diameter range before the beginning of diameter deviation. This finding confirms the previous finding of an increase in follicular and circulating estradiol concentrations in mares starting a mean of 1 day before the beginning of deviation [5]. In cattle, an increase in estradiol concentrations in the largest follicle also occurred in temporal association with deviation [2, 36]. Thus, on a temporal basis, estradiol may be involved with an increased responsiveness of the future dominant follicle in mares to the low circulating concentrations of FSH at deviation, as has been previously reported in cattle [6, 14].
The similar concentrations of androgens (androstenedione and testosterone) in all three follicles before the 20.0- to 23.9-mm range indicated that differential androgen concentrations are not involved in the beginning of follicle deviation in mares. Equine follicular fluid contained higher concentrations of androstenedione than testosterone, in agreement with previous results [35] and the finding that androstenedione is the major steroid secreted by equine theca cells [37]. Concentrations of androstenedione increased in all three follicles before diameter deviation, coinciding with the beginning of the estradiol increase in F1. This result suggests that inadequate androgen was not a factor in the limited estrogen synthesis in the smaller follicles by the time that deviation began. Alternatively, a selective stimulation of aromatase activity may have occurred in F1 before the beginning of deviation. Androgen production in mares occurs mostly in theca cells under stimulation by LH [37], and LH concentrations are increased at a time encompassing deviation in mares [4, 22]. A mare eliminated from the experiment on the basis of high statistically outlying LH concentrations in all three follicles also had higher (5- to 20-fold) intrafollicular androstenedione concentrations than those for any other experimental mare. The increased mean concentrations of androgens in F2 and F3, but not in F1, at the largest diameter range are consistent with the finding, in other species, that atretic follicles have higher ratios of androgen to estrogen than are found in healthy follicles [12].
An increase in the production of progesterone by F1 began simultaneously with the beginning of diameter deviation at the 20.0- to 23.9-mm range. Because the differential changes in progesterone did not precede the beginning of diameter deviation, whether progesterone contributed to the initiation of deviation is not clear. In mares, progesterone is secreted mainly by granulosa cells under FSH and LH stimulation [37]. Increased progesterone concentrations after deviation may have served as a substrate for the continued secretion of estradiol by the dominant follicle.
Concentrations of free, rather than of total, IGF-1 were measured in the present study to assess intrafollicular changes in biologically active IGF-1 [2]. The temporal availability of IGF-1 as a factor involved in deviation was indicated by an apparent differential increase in the concentrations in F1 after the 16.0- to 19.9-mm range and by the higher concentrations in F1 than in each of the smaller follicles at the 20.0- to 23.9-mm and subsequent ranges. Concentrations of free IGF-1 began to decrease in F2 and F3 at the beginning of deviation, which is similar to results obtained in cattle [2]. The IGF-1 amplifies the action of FSH on granulosa cells [38], and the higher concentrations in F1 during the present study indicated that IGF-1 may facilitate the action of FSH on F1 at the beginning of deviation and during subsequent development of the dominant follicle. Increased IGF-1 concentrations were temporally associated with high progesterone and estradiol concentrations in F1. The IGF-1 stimulates progesterone and LH-induced thecal androgen production and, in some species, estradiol production in vitro [15].
The present results suggest that a decrease in free IGF-1 concentrations in F2 and F3 after the beginning of deviation was attributable, at least in part, to an increase in concentrations of IGFBP-2. This is in agreement with results in cattle [2]. Concentrations of IGFBP-2, but also of other low molecular weight IGFBPs (presumably IGFBP-4 and -5), have been reported to be higher in subordinate equine follicles than in dominant follicles [18]. The role of IGFBP-4 and -5 in the decrease of free IGF-1 concentrations in the smaller follicles of mares during deviation needs to be studied.
To our knowledge, activin-A concentrations have not been previously reported in horses. Activin-A began to increase differentially in F1 simultaneously with estradiol concentrations before diameter deviation. An increase in follicular fluid concentrations of activin-A with follicle size has been reported in goats [39]. In contrast, no differences in activin-A concentrations were reported among follicles encompassing the time of deviation in cattle [2, 40] or with increasing follicle size in humans [41]. Activin-A stimulates aromatase activity and estradiol secretion in granulosa cells of cattle, rats, and primates [19]. The present results suggest that activin-A may play a role in the enhancement of aromatase activity in F1 preceding the beginning of deviation in mares. The assay used in the present and in previous studies measured total activin-A concentrations, which include both follistatin-bound and free (i.e., bioactive) activin-A [19]. Future studies on the changes in free activin relative to total activin concentrations in equine follicular fluid are needed.
An apparent differential F1 increase in inhibin-A after the 16.0- to 19.9-mm range and higher concentrations in F1 than in F2 and F3 at the subsequent ranges suggested that inhibin-A may play a role in the initiation of follicle deviation and the subsequent differential growth of the dominant follicle. Inhibin-A and inhibin-B decreased in F2 and F3 after the 20.0- to 23.9-mm range, which corresponds to decreased growth of these follicles. The differential changes in dimeric inhibin (inhibin-A and inhibin-B) among follicles seem to be similar to those recently reported in women [21]: Inhibin-A concentrations increased with follicle diameter and were lower in nondominant than in dominant follicles, whereas intrafollicular inhibin-B concentrations did not change with increasing follicle diameter or maturity. However, whether changes in women occur relative to presumptive [42] follicle deviation is not known. In contrast, in cattle, differential changes among follicles were not found in concentrations of inhibin-A or inhibin-B encompassing deviation [20]. Inhibin-A is a known stimulator of LH-induced androgen production in several species [19]. In this regard, increased follicular levels of inhibin-A in F1 at and after the beginning of deviation may serve to ensure that adequate androgen is available as substrate for production of estradiol by the dominant follicle in mares. The subunit precursor (pro-C) was much more abundant than dimeric inhibin in follicular fluid. Granulosa cells in other species synthesize an excess of over ß subunits [43]. The subunit precursors can antagonize the actions of FSH on granulosa cells by binding to the FSH receptor [44]. In view of the present results, a role of pro-C inhibin in follicle selection in mares is unlikely.
No significant differences were observed among follicles or diameter ranges for total intrafollicular inhibin concentrations. This result agrees with that of a previous report [45] of no differences in total inhibin concentrations among equine follicles <15, 15–30, and >30 mm and with results in cattle [2]. The assay for total inhibin measured concentrations of dimeric inhibin as well as free subunit forms [28]. Several subunit variants are found in equine follicular fluid [46]. The present results suggest that, relative to inhibin-A and inhibin-B, pro-C accounted for most of the inhibin measured by the total inhibin assay.
In conclusion, diameter deviation began at the 20.0- to 23.9-mm range, as indicated by an increase in the differences in diameter between F1 and F2. Between the 16.0- to 19.9- and 20.0- to 23.9-mm ranges, androstenedione increased simultaneously in the three follicles, whereas estradiol increased differentially in F1. These results suggest that androgen availability was not a factor in the reduced estradiol production of the smaller follicles after the 16.0- to 19.9-mm range. In addition to estradiol, free IGF-1, activin-A, and inhibin-A concentrations increased differentially in F1 beginning at the 16.0- to 19.9-mm range or one diameter range before the beginning of diameter deviation. Concentrations of progesterone and inhibin-B differentially increased or remained elevated in F1, and concentrations of androstenedione and IGFBP-2 increased in F2 and F3 beginning at the 20.0- to 23.9-mm range. Concentrations of FSH, LH, pro-C inhibin, and total inhibin did not change differentially among follicles. On a temporal basis, estradiol, free IGF-1, activin-A, and inhibin-A may have played a role in initiating diameter deviation. In addition, these four factors as well as progesterone, androstenedione, IGFBP-2, and inhibin-B may have been involved in the differential development of the follicles after the beginning of deviation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the University of Wisconsin, Madison, and by Equiservices Publishing and The Eutherian Foundation, Cross Plains, Wisconsin. 
2 Correspondence: F.X. Donadeu, Department of Animal Health and Biomedical Sciences, 1656 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706. FAX: 608 262 7420; donadeu{at}ahabs.wisc.edu
Accepted: November 9, 2001.
Received: October 4, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inhibin, and aromatase in relation to follicular growth, estrous cycle stage, and oocyte competence for in vitro maturation in the mare. Biol Reprod 1999; 60:1120-1127C at the time of ovulation in the mare. Am J Physiol 1999; 277:E870-E875-inhibin modulate follicle-stimulating hormone receptor binding and biological activity. Endocrinology 1991; 129:1987-1999[Abstract]This article has been cited by other articles:
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  O.J. Ginther, E.L. Gastal, M.O. Gastal, M.A.R. Siddiqui, and M.A. Beg Relationships of Follicle Versus Oocyte Maturity to Ultrasound Morphology, Blood Flow, and Hormone Concentrations of the Preovulatory Follicle in Mares Biol Reprod, August 1, 2007; 77(2): 202 - 208. [Abstract] [Full Text] [PDF]
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 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]
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O J Ginther, M A Beg, E L Gastal, M O Gastal, A R Baerwald, and R A Pierson Systemic concentrations of hormones during the development of follicular waves in mares and women: a comparative study Reproduction, September 1, 2005; 130(3): 379 - 388. [Abstract] [Full Text] [PDF]
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O.J. Ginther, E.L. Gastal, M.O. Gastal, and M.A. Beg Regulation of Circulating Gonadotropins by the Negative Effects of Ovarian Hormones in Mares Biol Reprod, August 1, 2005; 73(2): 315 - 323. [Abstract] [Full Text] [PDF]
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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]
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B. Nicholas, R. Alberio, A.A. Fouladi-Nashta, and R. Webb Relationship Between Low-Molecular-Weight Insulin-Like Growth Factor-Binding Proteins, Caspase-3 Activity, and Oocyte Quality Biol Reprod, April 1, 2005; 72(4): 796 - 804. [Abstract] [Full Text] [PDF]
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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]
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T.J. Acosta, E.L. Gastal, M.O. Gastal, M.A. Beg, and O.J. Ginther Differential Blood Flow Changes Between the Future Dominant and Subordinate Follicles Precede Diameter Changes During Follicle Selection in Mares Biol Reprod, August 1, 2004; 71(2): 502 - 507. [Abstract] [Full Text] [PDF]
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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]
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O.J. Ginther, E.L. Gastal, M.O. Gastal, and M.A. Beg Critical Role of Insulin-Like Growth Factor System in Follicle Selection and Dominance in Mares Biol Reprod, May 1, 2004; 70(5): 1374 - 1379. [Abstract] [Full Text] [PDF]
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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]
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O.J. Ginther, D.R. Bergfelt, M.A. Beg, C. Meira, and K. Kot In Vivo Effects of an Intrafollicular Injection of Insulin-Like Growth Factor 1 on the Mechanism of Follicle Deviation in Heifers and Mares Biol Reprod, January 1, 2004; 70(1): 99 - 105. [Abstract] [Full Text] [PDF]
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O.J. Ginther, M.A. Beg, K. Kot, C. Meira, and D.R. Bergfelt Associated and Independent Comparisons Between the Two Largest Follicles Preceding Follicle Deviation in Cattle Biol Reprod, February 1, 2003; 68(2): 524 - 529. [Abstract] [Full Text] [PDF]
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O.J. Ginther, C. Meira, M.A. Beg, and D.R. Bergfelt Follicle and Endocrine Dynamics During Experimental Follicle Deviation in Mares Biol Reprod, September 1, 2002; 67(3): 862 - 867. [Abstract] [Full Text] [PDF]
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O.J. Ginther, M.A. Beg, D.R. Bergfelt, and K. Kot Activin A, Estradiol, and Free Insulin-Like Growth Factor I in Follicular Fluid Preceding the Experimental Assumption of Follicle Dominance in Cattle Biol Reprod, July 1, 2002; 67(1): 14 - 19. [Abstract] [Full Text] [PDF]
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