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Regular article |
a Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland
b Molecular Reproductive Endocrinology Laboratory, Department of Animal Science, Michigan State University, East Lansing, Michigan 48824
c School of Animal and Microbial Sciences, University of Reading, Reading RG6 2AJ, United Kingdom
ABSTRACT
A surgical procedure to aspirate follicular fluid concurrently from individual follicles from the same heifer was validated and used to determine if intrafollicular amounts of estradiol, progesterone, inhibins, activin-A, follistatins, and insulin-like growth factor binding proteins (IGFBP) differed for the future dominant compared with subordinate follicles during selection of the first wave dominant follicle. Heifers were subjected to surgery and aspiration of follicular fluid from the two or three largest follicles on Day 3 of the estrous cycle (~1.5 days after emergence). Ultrasound was used to determine the fate of each aspirated follicle after surgery. At aspiration, diameter of the future dominant and largest subordinate follicle was similar in heifers. However, estradiol was higher, whereas IGFBP-4 was lower in the future dominant compared with the largest or next largest subordinate follicles. Also, the future dominant follicle in most cohorts had the highest estradiol and lowest IGFBP-4 compared with future subordinate follicles. We concluded that: IGFBP-4 and estradiol may have key roles in determining the physiological fate of follicles during selection of the first wave dominant follicle in heifers, and that both are reliable markers to predict which follicle in a growing cohort of 5- to 8.5-mm follicles becomes dominant.
activin, follicle, follistatin, growth factors, inhibin
INTRODUCTION
Follicle stimulating hormone is the key hormone that regulates the regular occurrence of two or three follicle waves during the estrous cycle of heifers [1–4]. Each wave is about 7 to 10 days in duration. The first 3 days of a wave are characterized by a transient rise in basal serum FSH concentrations [5–7], a selection phase that results in a reduction in the number of growing follicles in the initial cohort from ~24 3-mm follicles to 6–7 follicles 
5 mm in diameter [1–3], and a decline in serum FSH concentrations coincident with onset of atresia of all but a single ~8-mm dominant follicle [3, 5–7]. The remainder of a nonovulatory wave (postselection) is characterized by growth and differentiation of a single dominant follicle to ovulatory size during a period of relatively low serum FSH concentrations [1–4]. Specifically, the dominant follicle has an enhanced capacity to produce estradiol as it reaches 8–9 mm in diameter [8, 9], an increased amount of FSH receptor or its mRNA in granulosal cells by 10 mm in diameter [5, 8, 10], and an enhanced number of LH receptor or its mRNA in thecal and granulosa cells by 11–12 mm in diameter [5, 10, 11]. Loss of dominance marks the end of a wave and is characterized by decreased production of estradiol, atresia or ovulation of the dominant follicle depending on stage of the estrous cycle the wave develops, and emergence of a new follicular wave [1–4]. These observations imply that, despite similarity in size of 3-mm follicles at the early stage of a wave, individual follicles in a cohort must respond differently to the same serum FSH stimulus. For example, only a single 3-mm follicle in a wave is induced by the FSH stimulus to produce enhanced amounts of estradiol, synthesize an increased number of FSH and LH receptors, and become dominant. Differential responsiveness of follicles to FSH may be regulated by intrafollicular factors such as estradiol, progesterone, inhibins, activin-A, follistatin, insulin-like growth factors (IGF), and insulin-like growth factor binding proteins (IGFBP). These factors are synthesized locally in follicles, regulated at least partially by FSH and may have important autocrine or paracrine roles in modulation of FSH action and hence follicular growth and function [12–15]. However, in vivo evidence linking any of the aforementioned factors to follicular selection is not available.
To identify potential intrafollicular factors involved in follicle selection, the present study had the following objectives: 1) to validate use of a surgical procedure to aspirate follicular fluid concurrently from multiple individual follicles from the same heifer, and 2) to use this procedure to determine if intrafollicular amounts of estradiol, progesterone, inhibins, activin-A, follistatin, and IGFBPs differed for the future dominant and subordinate follicles during the selection process of the first follicle wave in heifers. If amount of any of the aforementioned factors in the future dominant follicle differed significantly from future subordinate follicles, we reasoned that this factor(s) could have an important role in selection of a dominant follicle during each follicular wave and provide a useful biochemical marker to predict the physiological fate of follicles during the selection process.
MATERIALS AND METHODS
Animals and Treatments
Reproductively mature crossbred Charolais beef heifers (451.6 ± 4.7 kg) approximately 1.5 yr of age were used for these studies. Heifers had ad libitum access to water and grass silage supplemented daily with 2 kg of a 16% crude protein concentrate per heifer. Heifers received one or two i.m. injections of 15 mg luprostiol, a prostaglandin F2
analogue (PG; Prosolvin, Intervet UK Ltd., Cambridge, U.K.) to synchronize estrus (Day 0). Observations for estrus commenced 36 h after PG and were carried out four times daily. On the day of ovulation (determined by ultrasound), heifers were randomly allocated into two groups: untreated controls (n = 7; controls), or heifers to undergo surgery (n = 21). Each heifer in the surgery group was subjected to surgery on Day 3 of the estrous cycle, which was approximately 1 day after emergence of the first follicular wave as determined by ultrasound scanning. Before surgery, food was withdrawn for 24 h and water for 12 h. On the day of surgery, heifers were subjected to ultrasonography 3–7 h before surgery, and a map showing the relative location of all follicles >4 mm to each other was drawn for each ovary. Sizes of the two or three largest follicles were determined by ultrasound by taking two separate measurements of diameter in two different dimensions for each follicle. When diameters in each dimension were unequal for a follicle, they were averaged. Heifers received one i.v. injection of Planipart (0.3 mg clenbuterol, Boehringer Ingelheim Ltd., Bracknell, U.K.) to facilitate exteriorization of the genital tract, and 20 ml duphapen LA i.m. (Duphar Ireland Ltd., Dublin, Ireland). During anesthesia, a midline laparotomy was performed to expose the ovaries. No further ovarian manipulations were performed on six heifers that served as sham-operated controls (shams). For the remaining 15 heifers (treated), each of the two or three previously mapped follicles per pair of ovaries was located, and a tuberculin syringe with a 27-gauge needle was used to extract 15–20 µl follicular fluid (FF) from each follicle. The needle was inserted into each follicle approximately 2 mm below the interface of the surface of the follicle with the ovary to minimize leakage of FF after needle withdrawal. After follicle aspiration, blood was not detected in any follicle. Each FF sample was centrifuged and the supernatant placed into a separate container and immediately frozen. After surgery, ultrasound was used to determine fate (dominant or subordinate) of each aspirated and mapped follicle, as explained below.
All animal experimentation was in compliance with regulations set forth by the BioMedical Centre, University College Dublin, and the Cruelty to Animals Act (Ireland) 1897.
Ultrasound Examination of Ovaries and Definitions
Growth and regression of individual follicles >4 mm were monitored using a real-time, B-mode, linear array ultrasound scanner with a 7.5-MHz intrarectal transducer (Dynamic Imaging, Concept 500, Livingston, U.K.). Ovarian ultrasound examinations were performed daily beginning at the first estrus after the last injection of PG until the next estrus or from the first estrus after the last PG injection until the dominant follicle in the second wave of the same cycle was identified. Day of emergence of a follicular wave was defined as the first Day 1 follicle in a cohort reached 5 mm in diameter. However, because daily ultrasound did not precisely detect when emergence occurred in all heifers, day of emergence was arbitrarily assigned as follows: when ultrasound analysis showed that all follicles in a cohort in the first wave were <5 mm in diameter on one day, but at least one follicle in the cohort was >5 mm the next day, day of emergence was estimated to have occurred 0.5 days earlier. The first day of dominance was defined when all three of the following criteria were satisfied: 1) all subordinate follicles had stopped increasing in diameter, 2) the difference in size between the dominant follicle and the next largest (subordinate) follicle was >2 mm, and 3) the dominant follicle had achieved a minimum diameter of 8.5 mm.
Collection of Blood Samples
Blood samples were collected via jugular venipuncture (10 ml) either at 6- or 12-h intervals beginning at the first estrus after the last PG injection and ending at the next estrus or on the first day of dominance for the second-wave dominant follicle. Concentrations of FSH and LH were measured for each sample, whereas estradiol was measured in one sample per day for all heifers.
Assays
A monoclonal LH antibody (518B7 anti-LH; supplied by Dr. Jan Roser, University of California, Davis), ovine LH standard (NIAMDD-oLH-24, NIH, Bethesda, MD), and radioiodinated ovine LH tracer were used in a previously validated RIA [16] to determine serum LH concentrations. Sensitivity of the LH assay was 0.6 ng/ml. Intra- and interassay coefficients of variation (CV) for serum samples containing 2.2, 6.2, or 13.3 ng/ml LH were 15.8, 9.6, or 8.7% (n = 5–6 samples), and 18.8, 18.6, or 16.2% (n = 7–9 assays), respectively.
NIDDK-anti-oFSH antibody (AFP-C 5288113), ovine FSH tracer, and bovine FSH standard (USDA B1 bFSH) were used in a previously validated heterologous RIA to quantify serum FSH concentrations [17]. Sensitivity of this assay was 1.6 ng/ml. Intra- and interassay CV for serum samples containing 11.0, 14.6, or 30.1 ng/ml FSH were 16.8, 10.5, or 13.7% (n = 4–6 samples) and 10.5, 8.7, or 8.0% (n = 6–8 assays), respectively.
Concentrations of estradiol (E) in serum and FF were determined using a previously validated RIA [18]. Sensitivity of this assay was 0.4 pg/ml. Intra- and interassay CV for serum pools containing 0.8 or 4.0 pg/ml E were 11.3 or 11.5% (n = 2–4 samples), and 20.0 and 16.5% (n = 12 assays), respectively. All FF samples were analyzed in two assays. Intraassay and interassay CV for FF samples containing 0.33 pg/ml of E was 16.2% (n = 4 samples) and 8.8% (n = 2 assays), respectively.
Concentrations of progesterone (P) in FF were estimated using a previously validated RIA [19]. Sensitivity of this assay was 0.05 ng/ml. Intra- and interassay CV for samples of FF containing 0.4 ng/ml P averaged 13.0 (n = 4 samples) and 13.3% (n = 2 assays).
Ratio of E:P concentrations in FF was used to characterize follicles as estrogen-active (EA, E:P 1) or estrogen-inactive (EI, E:P < 1). The EA follicles are histologically healthy and growing, whereas EI follicles are atretic [5, 7].
Concentrations of inhibin-A in FF were measured in a single assay using a previously validated two-site immunoradiometric assay [20]. Each tube analyzed contained 0.1 µl FF. Detection limit was 0.02 ng/tube. Intraassay CV for samples containing 7.4 or 14.3 ng inhibin-A per µl FF was 0.9 (n = 3 samples) or 3.4 % (n = 4 samples).
Concentrations of total activin-A in FF (free activin plus follistatin-bound activin) were measured in a single assay using a two-site ELISA [21]. Sensitivity was 0.01 ng/well, and each well analyzed contained 0.05 µl FF. Intraassay CV for samples (n = 4) containing 5.3 ng activin-A per µl FF was 9.8%.
Concentrations of total follistatin in FF were measured in a single assay using a two-site ELISA [22]. Sensitivity was 0.01 ng/well, and each well analyzed contained 0.025 µl FF. Intraassay CV for samples (n = 4) containing 7.2 ng follistatin per µl FF was 17.8%.
For immunoblot analysis of the different molecular weight forms of inhibin, a sample (10 µg protein/lane, amount of protein determined by spectrophotometry) of FF from each follicle (n = 25 follicles) per heifer was allocated to one of five gels, with one aliquot of pooled bovine FF (bFF) added to each gel (quality control, QC). Samples were subjected to 12% SDS-PAGE [23] and then electrophoretically transferred to Immobilon P membranes (Millipore, Bedford, MA) [24]. Each membrane was blocked with 0.01% Blotto (Food Club, Skokie, IL) in TBS (50 mM Tris, 0.5 M NaCl), incubated with a mink anti-bovine c1–26gly-tyr antiserum (1:1000 in TTBS, 50 mM Tris, 0.5 M NaCl, 0.05% Tween-20), washed with TTBS, incubated with a rabbit anti-mink c1–26gly-tyr antiserum (1:25 000 in 6% bFF, TTBS), and rewashed in TTBS. Membranes were incubated with a 1:1000 dilution of goat anti-rabbit immunoglobulin linked to horseradish peroxidase (Amersham, Arlington Heights, IL), rewashed in TTBS, incubated with Amersham ECL Western blotting detection reagents (according to manufacturer's instructions), and exposed to Reflection film (Dupont, Boston, MA) for 22 h. Molecular weight was estimated using protein standards (Bio-Rad, Richmond, CA) after silver staining. Intensities of bands after immunoblotting were determined using the Molecular Analyst Software for Bio-Rad model GS-670 imaging densitometer. The CV for the QC lanes for 5 gels was 23%.
For immunoblot analysis of 26-kDa activin-A, a procedure similar to the one described above for inhibins was used. Activin-A was identified on blots with a human inhibin ßA82–114 monoclonal antibody [25]. The CV for the QC lanes for five gels was 25%.
For ligand blot analysis of IGF-1 binding proteins, recombinant human IGF-1 (5 µg; H-5555; Bachem, Torrance, CA) was iodinated using 1 mCi Na125I (NEZ-033H; New England Nuclear, Boston, MA) and 50 µg IODO-GEN (Pierce, Rockford, IL) for 10 min [26]. Iodinated IGF-1 was purified on a Sephadex G-25M column (PD-10, 5- x 1.6-cm prepacked column; Pharmacia, Piscataway, NJ) previously equilibrated with 25 ml of column buffer (0.01 M Na2HPO4, pH 7.2, 3% BSA), and aliquots were stored at 4°C until used. Specific activity was 17.8 µCi/µg protein. Radiolabeled IGF-1 was diluted to 200 000 cpm (0.1 µCi/ml in TTBS, 10 mM Tris, 0.15 M NaCl, 0.05% NaN3, 0.1% Tween-20, in 1% BSA) before ligand blot analysis. Each FF sample (10 µg of protein/lane; n = 25 follicles) per heifer, including a QC sample for each gel, as described above for the inhibin immunoblots, was subjected to 12% SDS-PAGE (n = 5 gels). After electrophoresis, proteins were transferred to Immobilon P membranes, air dried, rewet in methanol, equilibrated in water, washed in TBS (10 mM Tris, 0.15 M NaCl, 0.05% NaN3), and blocked in 1% Blotto in TBS. Membranes were subsequently incubated 18 h in radiolabeled IGF-1 at 4°C, washed in TTBS then TBS, placed in saran wrap, and exposed for 47 h in a Bio-Rad GS-250 Imaging Screen-BI. Intensities of bands after ligand blotting were determined using the Molecular Analyst Software for the Bio-Rad GS-250 Molecular Imager. The CV for the QC lane for five gels was 29%.
To verify that the 28-kDa band detected during ligand blot analysis of FF was IGFBP-4, 40 ng of a human recombinant nonglycosylated form of IGFBP-4 (hrIGFBP-4; Austral Biologicals, San Ramon, CA) and a pooled sample of FF (25 µg) were subjected to ligand blot analysis, as explained above. In addition, rhIGFBP-4 (20 ng) and FF (80 µg) were subjected to western blot analysis. After electrophoresis and transfer as explained for ligand blot analysis, Immobilon P membranes were incubated with 1% Blotto, washed, and incubated with an affinity-purified goat polyclonal antibody generated against a carboxy-terminal peptide fragment of rhIGFBP-4 per instructions provided by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). After incubation and washing, a signal was generated using an anti-goat polyclonal IgG linked to horseradish peroxidase (Santa Cruz Biotechnology, Inc.) followed by Super Signal West Dura Luminol & Chemiluminescent Solutions (Pierce, West Rockford, IL). Molecular weight of each band was determined as explained above.
Statistical Analysis
Six of the 15 heifers in the treated group were eliminated from the statistical analyses because the fate of each follicle that was aspirated at surgery could not be determined accurately during subsequent ultrasound analysis for two reasons: 1) multiple follicles in close proximity to each other during surgery made it difficult to determine by ultrasound which aspirated follicle became dominant (n = 5 heifers) after surgery, and 2) the future dominant follicle was not aspirated because the reproductive tract could not be exteriorized during surgery (n = 1 heifer).
The statistical analysis on the remaining nine heifers was in three parts. In part 1, all heifers in each group (control, n = 7; shams, n = 6; treated, n = 9) were used to determine if surgery and aspiration of FF from the two or three largest follicles disrupted growth of the dominant follicle, length of the estrous cycle, and serum concentrations of LH, FSH, and estradiol. In part 2, all heifers in the treated group (n = 9) were used to determine if intrafollicular concentrations of hormones or growth factors at the time of follicular aspiration differed for the follicle destined to become dominant compared with the largest or second largest subordinate follicle. In part 3, chi-square analysis was used to determine if the proportion of cohorts where the future dominant follicle in the cohort was also the largest, intermediate sized or smallest were statistically different (P < 0.05). Chi-square analysis was also used to determine if the proportion of cohorts where the future dominant follicle in the cohort also had the greatest, intermediate, or least intrafollicular concentration of steroid, growth factor, or binding protein were statistically different (P < 0.05). This analysis was done to determine if follicle size or intrafollicular amount of steroid, growth factor, or binding protein could be reliably used to predict which follicle in an emerging cohort on Day 3 of the estrous cycle would subsequently become dominant. All nine heifers in the treated group that had FF aspirated from the 2 (n = 1 heifer) or 3 (n = 8 heifers) largest follicles per pair of ovaries on Day 3 of the estrous cycle were used for this analysis. Note, however, as will be explained in detail in Table 3, some heifers could not be used in the chi-square analysis because at least two follicles in a cohort were either similar in size or had similar concentrations of steroids, growth factor, or binding protein.
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Repeated measures analysis of variance was used in part 1 to determine if FSH and LH serum concentrations differed among treatment groups. One-way ANOVA was used to determine if overall significant (P 
0.05) differences existed in the various parameters measured among the treatment groups (controls versus shams versus treated), or for comparisons of future dominant versus subordinate follicles in the treated group of heifers. Differences in individual means were determined by Bonferroni t-test. If a t-test showed significant differences between standard deviations, hormonal data were transformed to logarithmic values prior to statistical analysis, or a nonparametric test (Mann-Whitney test) was employed. However, data are presented as arithmetic means (mean ± SEM) for clarity.
Due to the small volume of FF aspirated per follicle, all hormone measurements could not be determined for each follicle, as will be indicated in each table or figure.
RESULTS
Part 1: Ultrasound Validation of the Surgical Model
Emergence of the first-wave dominant follicle occurred on similar days of the estrous cycle for the controls, shams, and treated groups of heifers (mean ± SEM, 1.9 ± 0.4, n = 7 heifers; 1.5 ± 0.2, n = 6; 1.5 ± 0.3, n = 9). Interval from first-wave emergence to surgery was similar for shams and treated groups (1.5 ± 0.2 versus 1.5 ± 0.3 days). The patterns of growth for the future dominant and two largest subordinate follicles for the control, sham, and treated groups of heifers were similar (Fig. 1, only data for treated group shown). Of the 15 different parameters compared among the treated, sham, and control groups of heifers during validation, only FSH differed significantly (Table 1 and Fig. 2). Specifically, serum FSH concentration was greater (P < 0.05) in controls on Day 1.75 compared with shams or the treated group of heifers, but this difference was not maintained (Fig. 2). After the first postovulatory rise in serum FSH concentrations, day of the estrous cycle for nadir (5.2 ± 0.3, 6.1 + 0.7 and 5.6 ± 0.4) or maximum (9.4 ± 0.6, 9.0 ±0.7, and 8.6 ± 0.4) serum FSH concentrations were similar for the controls, shams, and treated groups of heifers, respectively.
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Part 2: Comparison of Size and Intrafollicular Concentrations of Steroids, Growth Factors, and Binding Proteins in the Future Dominant and Subordinate Follicles of the First Wave
At the time of follicular aspiration (Day 3 of estrous cycle, ~1.5 days after emergence), deviation in growth patterns between the future dominant and the largest subordinate follicle had not occurred (Fig. 1); size of the future dominant follicle was similar compared with the largest subordinate follicle, but larger (P < 0.05) than the future second largest subordinate follicle (Fig. 1 and Table 2); and serum FSH concentrations were near nadir levels (Fig. 2). Intrafollicular concentrations of progesterone, inhibins, activin-A, follistatin, and IGFBP-2, -3, and -5 were similar among follicles. In contrast, intrafollicular concentration of estradiol and ratio of estradiol : progesterone were greater (P < 0.05), whereas amount of a 28-kDa form of IGFBP-4 was 15- and 11-fold lower (P < 0.05) in the future dominant compared with the future largest or second largest subordinate follicle. As shown in Figure 3, the 28-kDa band detected during ligand blot analysis of FF from dominant (DF) and subordinate follicles (Sub1, Sub2) was identified as the nonglycosylated form of IGFBP-4 based on ligand and Western blot analysis of recombinant human IGFBP-4 (rhBP-4) and pooled bovine FF (bFF).
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Part 3: Reliability of Predicting Which Follicle in a Cohort Is the Future Dominant Follicle Based on Follicle Size or Intrafollicular Biochemical Markers
Highest intrafollicular concentration of estradiol and lowest intrafollicular amount of IGFBP-4 were both reliable biochemical predictors of which follicle in a cohort of the two or three largest follicles on Day 3 of the estrous cycle would become the future dominant follicle. Specifically, the proportion of cohorts that had future dominant follicles with the greatest amount of estradiol compared with other follicles in the cohort differed (P < 0.05) both from the proportion of cohorts that had future dominant follicles with intermediate amounts of estradiol and from the proportion of cohorts that had a future dominant follicle with the least amount of estradiol (Table 3). In addition, the proportion of cohorts that had future dominant follicles with least amount of IGFBP-4 differed (P < 0.05) both from the proportion of cohorts that had a future dominant follicle with an intermediate amount of IGFBP-4 and from the proportion of cohorts that had a future dominant follicle with the greatest amount of IGFBP-4.
DISCUSSION
This is the first report of results of concurrent sampling of FF from multiple individual follicles in a growing cohort in the first wave of heifers, use of ultrasound to establish subsequent fate of each aspirated follicle (dominant or subordinate), and determination of the relationship between intrafollicular concentrations of steroids, growth factors, and binding proteins with the differential fate of follicles. Validation of our experimental approach demonstrated that 15–20 µl of FF could be removed concurrently from two or three follicles per pair of ovaries ranging from 5 to 8.5 mm in diameter on Day 3 of the estrous cycle. Our rigorous validation clearly illustrated that surgery and follicular aspiration did not disrupt subsequent growth, function, or turnover of dominant or subordinate follicles; gonadotropin secretion patterns (other than FSH on Day 1.75 of the estrous cycle); or estrous cycle length. The major limitation to this approach is reliability of using ultrasound to map location of follicles on each ovary prior to surgery and subsequent ultrasound identification of aspirated follicles 6 mm in diameter after surgery, especially when the aspirated follicles are on the same ovary, in close proximity to each other, and of similar size. However, this problem can be alleviated if ultrasound is used before surgery to screen out heifers with potentially problematic follicles.
Determination of diameter of the two or three largest follicles per pair of ovaries on Day 3 of the estrous cycle, coupled with measurement of putative intrafollicular growth factors, resulted in the several significant findings in our study. First of all, average size of the future dominant follicle was similar to the largest future subordinate follicle but larger than the second largest subordinate follicle. Second, despite similar intrafollicular levels of progesterone, inhibins, activin-A, follistatin, and IGFBP-2, -3, and -5 for all follicles in a growing cohort, concentrations of estradiol and estradiol:progesterone were higher, whereas IGFBP-4 was markedly lower in the future dominant compared with the largest or second largest future subordinate follicle. Finally, the highest intrafollicular concentration of estradiol and lowest IGFBP-4 were both reliable biochemical markers for predicting which follicle within a growing cohort of similar-sized follicles would become dominant.
Other approaches to sample individual follicles in vivo have been attempted in heifers. An in situ, transvaginal, ultrasound-guided needle was used to sample small amounts of FF from a single follicle (6 to 12 mm in diameter) per heifer during the first 4 days after emergence of the first wave in Holstein heifers [9]. In contrast to our results and others [7, 8, 27], their findings show that diameter and intrafollicular estradiol concentration are similar for the future dominant and largest subordinate follicle 1 and 2 days after follicular emergence, when follicles average 7 to 8 mm in diameter. While the reason for the different findings between laboratories is not clear, several possible explanations exist: 1) because follicular emergence and deviation vary among heifers [3], the variation in intrafollicular estradiol levels for the future dominant or subordinate follicles may be greater when FF samples are taken from a single follicle in each cohort of different heifers [9], compared with concurrent sampling of FF from the future dominant and subordinate follicles in each cohort of different heifers [7, 8, 27]; and(or) 2) precision of FF extraction from individual follicles may be greater using a needle and syringe after ovariectomy [7, 8, 27], or at surgery in unconscious cattle as in our study than with use of an ultrasound-guided transvaginal needle in conscious cattle [9].
Based on ultrasound monitoring of follicle growth and analysis of serum concentrations of gonadotropins in heifers, FF was aspirated from follicles approximately 1.5 days after emergence of the first wave, which was 36 h after the peak for the first postovulatory rise in serum FSH concentrations when serum FSH was approaching nadir values (Fig. 1). In addition, size of the future dominant and largest subordinate follicle were similar, and neither deviation in growth patterns between the dominant and largest subordinate follicle nor onset of follicular dominance had occurred. However, ultrasound analysis showed that the future second-largest subordinate follicle did not increase in size after surgery, and it was significantly smaller than the future dominant follicle. Because size and growth pattern of the future second-largest subordinate follicle in the treated group of heifers was similar to that for the control and sham groups of heifers in our study (data not shown), it is unlikely that aspiration of FF affected growth of the future second-largest subordinate follicle after surgery. Taken together, these findings implied that selection of the dominant follicle of the first wave was ongoing at the time of surgery and follicular aspiration in our study, and probably near completion. Consequently, our observation that only intrafollicular concentrations of estradiol and IGFBP-4 in the future dominant follicle differed significantly compared with the future subordinate follicles in a growing cohort of follicles in the first wave implies a potentially important physiological role for these two factors during follicular selection.
In our study, average intrafollicular concentration of estradiol was higher in the future dominant compared with subordinate follicles. In addition, comparisons within each cohort showed that the future dominant follicle had the highest concentration of estradiol in seven of nine cohorts in heifers. The reason two cohorts had future subordinate follicles with similar or higher concentrations of estradiol than the dominant follicle is unclear but may be related to use of a single measurement of estradiol in FF to assess estradiol-producing capacity of the follicle. For example, utero-ovarian venous sampling of blood from each ovary at hourly intervals in cattle indicates that estradiol production is highly variable [28]. In addition, measurement of estradiol concentrations in FF reflects the overall functional status (growing versus atretic, dominant versus subordinate) [7] of the follicle. Thus, follicular estradiol concentrations may not immediately reflect the maximum estradiol-producing capacity of a follicle for several reasons: limited availability of substrate could diminish capacity of the follicle to produce estradiol; FF may contain factors that limit estradiol production; and steroid binding proteins in FF may retard diffusion of estradiol from FF into the thecal capillary system, thus enhancing intrafollicular estradiol concentrations independent of the follicle's capacity to produce estradiol. Based on these aforementioned caveats, more direct measures of the estradiol-producing capacity of granulosal cells from individual follicles will be needed to confirm that estradiol-producing capacity is greater for the future dominant follicle during the selection process, as indicated by the results of our study. Nevertheless, FSH has a key role in induction of the aromatase enzyme system in bovine granulosal cells [29]. Thus, if the future dominant follicle is indeed more sensitive to the postovulatory FSH stimulus, as we hypothesized, it would be expected to have a greater capacity to produce estradiol and a higher intrafollicular concentration of estradiol, as demonstrated for the majority of future dominant follicles in our study. In support of our finding, previous studies report that intrafollicular concentration of estradiol is significantly higher in the largest follicle (mean = ~8 mm in diameter, presumed dominant) compared with the second largest follicle (mean = ~7 mm) on Day 3 of the estrous cycle or Day 2 of the first wave in cattle [7, 8, 27].
In our study, the future dominant follicle not only produced more estradiol but also had a markedly lower average amount of IGFBP-4 compared with future subordinate follicles. In addition, comparisons within each cohort showed that the future dominant follicle had the lowest amount of IGFBP-4 in seven of seven cohorts. This finding provides potential new insight into the follicle selection process, especially the biochemical mechanism explaining why the future dominant follicle has an enhanced capacity to produce estradiol during a period of relatively low serum gonadotropin concentrations. Use of knockout models in mice to examine the role of IGF-1 and FSH in folliculogenesis show that both are required for antral follicle development [30]. In addition, IGF-1 enhances both basal and FSH-induced estradiol synthesis by bovine granulosal cells [31] and in vivo in laboratory species [30]. Indeed, IGF-1 may be obligatory for FSH action [30, 32]. The IGFBPs are primary regulators of IGF action [33]. Specifically, relatively high amounts of IGFBP-4 are associated with follicular atresia in rats [34, 35], mice [36], and cattle [37] and polycystic ovarian syndrome in human ovaries [38]. In addition, IGFBP-4 has negative effects on various cell functions including FSH-induced estradiol production by human granulosal cells [39]. Consequently, IGFBP-4 appears to be a physiologically important negative regulator of FSH action. While the physiological role of IGFBP-4 in folliculogenesis in cattle is unknown, it is well established that dominant and subordinate follicles possess similar intrafollicular concentrations of IGF-1, but subordinate follicles have markedly higher amounts of the low molecular weight IGFBPs including IGFBP-2, -4, and -5 compared with dominant follicles [37, 40–43]. However, our study did not detect differences in IGFBP-2 or -5 when the follicle destined to become dominant was compared with future subordinate follicles approximately 2 days before the end of selection. This finding may indicate that intrafollicular production of IGFBP-4 is enhanced earlier in follicles destined to become atretic compared with the other small molecular weight IGFBPs. However, we cannot rule out the possibility that our immunoblot procedure was not sensitive enough to detect small intrafollicular differences in amounts of IGFBP-2 and -5 that may exist among growing follicles in a cohort in the first wave of follicle growth in heifers. Based on the aforementioned literature, low intrafollicular levels of IGFBP-4, as found in our study for future dominant follicles, would be expected to result in increased availability of bioactive IGFs. Increased availability of IGFs could either directly enhance estradiol production and(or) indirectly enhance FSH-induced estradiol production, as recently shown using bovine granulosal cells [31]. Follicle stimulating hormone has little effect on basal estradiol production by bovine granulosal cells isolated from medium-sized antral follicles compared with IGF-1 [31], and serum FSH concentrations in our study were relatively low when FF samples were aspirated. Based on these findings, we speculate that the enhanced intrafollicular estradiol concentrations in the future dominant follicle primarily resulted from a net increase in IGF bioactivity caused by the reduced intrafollicular amount of IGFBP-4. In addition, the relatively high intrafollicular levels of IGFBP-4 that were observed for future subordinate follicles in our study are characteristic of follicles destined to become atretic in the bovine, as already mentioned. Thus, despite ultrasound analysis in our study indicating that the two largest aspirated follicles in each cohort were in a growth phase at the time of surgery, the relatively high levels of IGFBP-4 may indicate impending apoptosis in the future subordinate follicles, which would explain their reduced capacity to produce estradiol and subsequent retarded growth rate compared with future dominant follicles.
While the biochemical explanation for the relatively reduced amounts of IGFBP-4 in future dominant follicles during the selection process is unknown, two explanations seem most plausible: 1) In cattle, IGFBP-4 mRNA is localized to thecal cells and responsive to LH treatment [37]. In addition, LH induces IGFBP-4 production by ovine thecal cells [44]. Thus, the capacity of LH to induce thecal synthesis of IGFBP-4 may have been diminished in future dominant compared with subordinate follicles in our study. 2) The IGFBP-4-specific protease activity is greater in FF of estrogen-dominant or growing follicles compared with presumably atretic follicles in humans [45], cattle [46], and pigs [47]. In addition, production of an IGFBP-4-specific protease is stimulated in human granulosal cells by FSH [48]. Thus, FSH-induced synthesis of an IGFBP-4-specific protease may be greater in the future dominant compared with subordinate follicles. Recently, human fibroblasts were shown to produce an IGF-dependent IGFBP-4 protease, also known as pregnancy-associated plasma protein-A, which is in high quantities in maternal circulation during human pregnancy [49]. Results of a recent study [50] indicate that the IGF-dependent IGFBP-4 protease produced by human fibroblasts may be identical to the IGFBP-4 protease activity in human, bovine and swine follicles [45–47]. Taken together, the increased intrafollicular concentrations of estradiol and the reduced amounts of IGFBP-4 in future dominant follicles compared with subordinate follicles strongly support the hypothesis that the follicle that becomes dominant in a growing cohort of similar-sized follicles is the one most responsive to FSH. It is also plausible that the future dominant follicle grows faster than all others in a cohort, as suggested by Ginther and colleagues [51], because it is the most responsive to FSH. Finally, our results show that future dominant follicles have distinct differences in intrafollicular levels of estradiol and IGFBP-4 during the last phase of the follicular selection process. Thus, it will be important to determine in future studies whether these same factors differ for future dominant follicles during an earlier stage of the selection process, when a larger cohort of smaller growing follicles exists.
In summary, the selective decrease in intrafollicular levels of IGFBP-4 and enhanced estradiol production in future dominant follicles compared with subordinate follicles, coupled with the absence of measurable alterations in progesterone, inhibins, activin-A, IGFBPs, and follistatin in dominant and subordinate follicles further support the important physiological role that IGFBP-4 and estradiol have in the follicular selection process. Based on results of our study and others, we conclude that IGFBP-4 and estradiol may have key roles in determining the physiological fate of follicles during the late phase of the selection process for dominant follicles in heifers, and that intrafollicular amounts of IGFBP-4 and estradiol are reliable biochemical markers for determining which follicle in a growing cohort of 5- to 8.5-mm follicles will become dominant.
FOOTNOTES
First decision: 22 December 1999.
1 Research supported by grants from USDA (90-27240-5508) and Research Excellence Funds from Michigan State University to J.J.I.; Research Stimulus Fund, Department of Agriculture and Food to J.F.R., and BBSRC (SO5760) to P.G.K. 
2 Correspondence. FAX: 353 16600883; jfroche{at}vetmed.ucd.ie
3 Current address: Department of Veterinary Preclinical Studies, University of Glasgow Veterinary School, Glasgow G61 1QH, U.K.
Accepted: April 24, 2000.
Received: November 16, 1999.
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) and LH (•) in control (A), sham (B), and treated (C) heifers. An arrow in B and C shows the time of surgery (S) for shams, and time of surgery and aspiration of FF from the two or three largest follicles per heifer in the treated group. Arrows in each panel indicate day of emergence (E) or first day of dominance (D). Each point represents the mean ± SEM value for two to nine heifers. Animal number varies because heifers were removed from the study once dominance was detected by ultrasound