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Regular Article |
a Faculty of Veterinary Medicine, University College Dublin, Dublin 4, Ireland
b Faculty of Agriculture, University College Dublin, Dublin 4, Ireland
c School of Animal and Microbial Sciences, University of Reading, Reading RG6 6AJ, United Kingdom
d Molecular Reproductive Endocrinology Laboratory, Department of Animal Science, Michigan State University, East Lansing, Michigan 48824
ABSTRACT
Changes in follicular fluid (FF) concentrations of estradiol, inhibin forms, and insulin-like growth factor binding proteins (IGFBPs), percentage of apoptotic granulosa cells (%A), and follicular size for individual follicles in a growing cohort were determined throughout the first wave of follicular development during the bovine estrous cycle and related to FSH decline. Four groups of heifers (n = 31) were ovariectomized between Days 1.5 and 4.5 of the estrous cycle at 5 ± 1, 33 ± 2, 53 ± 1, and 84 ± 2 h after the periovulatory peak in FSH concentrations. Follicles
2.5 mm were dissected, measured, and FF aspirated. The five largest follicles were ranked based on their diameter (F1 to F5). Diameters of F1 to F5 were positively correlated with interval from FSH peak (r 0.6, P < 0.05). Five hours after the FSH peak, follicular diameter and FF concentrations of estradiol, inhibins, and IGFBPs were similar for F1 to F5. From 5 to 33 h, amounts of the six precursor inhibin forms (48 kDa) increased (P < 0.05) in F1 follicles. The IGFBPs in F1 follicles remained low at all time periods. At 33 h, amounts of IGFBP-4 and -5 were higher (P < 0.05) in F4 and F5 compared with F1 follicles. At 84 h, IGFBP-2, -4, and -5 were increased (P < 0.05) in F3, F4, and F5 compared with F1. At 5, 33, or 53 h, %A was not different between follicles in any size class. At 84 h %A was increased (P < 0.05) in follicles <6 mm in diameter. However, at that time, %A did not differ between the selected DF and the largest subordinate follicle. For individual heifers, the selected DF at 84 h was largest in size, highest in estradiol, and lowest in IGFBP-2 and -4. The F1 follicle had highest estradiol in 23 of 27 heifers irrespective of stage of the wave and lowest IGFBP-4 in 19 of 21 heifers from 33 h. We concluded that the earliest intrafollicular changes that differentiate a dominant-like follicle from the growing cohort are enhanced capacity to produce estradiol and maintenance of low levels of IGFBPs.
activin, estradiol, follicle, follicular development, follistatin, FSH, growth factors, inhibin, ovary
INTRODUCTION
The initial stages of folliculogenesis in cattle occur independent of gonadotropin stimulation. However, antral follicular development beyond 3–4 mm is an FSH-dependent process [1, 2] occurring in two or three waves during the estrous cycle [3–5]. Emergence of a follicular wave, identifiable by ultrasound examination as the first day a 4- to 5-mm follicle in a cohort of growing follicles is observed, is preceded by a transient increase in FSH concentrations [6, 7]. Within 12 h of the peak in FSH, a cohort of three to five growing follicles of 5–6 mm in diameter is detectable, and subsequent follicular selection proceeds during declining FSH concentrations [6]. This selection process culminates in the development of a single dominant follicle (DF), identifiable morphologically by its larger size and continued growth compared with smaller subordinate follicles [7, 8]. Subordinate follicles cease growth and eventually decrease in size with associated losses of gonadotropin binding sites in granulosa and thecal cells [9], limited capacity to produce estradiol [9], and onset of granulosa cell apoptosis coincides with atresia in ovine follicles [10]. The DF is characterized by increased estradiol synthesizing capacity [7, 11] and increased expression of mRNA for LH receptor in granulosa cells [12]. Differential expression and/or processing of intrafollicular growth factors and growth factor binding proteins by one follicle in a cohort of follicles has been hypothesized to be a mechanism whereby a single follicle is selected for continued growth and enhanced estradiol production during a period of declining FSH concentrations [13]. For example, DFs contain higher amounts of several forms of inhibins and lower intrafollicular amounts of low molecular weight insulin-like growth factor binding proteins (IGFBPs) than subordinate follicles [14, 15]. Such differences are likely to give the DF enhanced responsiveness to FSH and LH at a time when FSH concentrations are declining or have reached a nadir. In cattle, the question of whether a single follicle within a cohort during emergence of a follicular wave is uniquely differentiated, thus enabling its development to dominance, has not previously been addressed.
Based on ultrasound analysis, the future DF is the first follicle in a cohort to undergo emergence and it maintains this growth advantage until it reaches ovulatory size [8]. These findings suggest that follicles in a growing cohort are at different stages of differentiation, i.e., a hierarchy in developmental capacity exists within a growing cohort [16]. Sampling of follicular fluid in vivo from growing cohort follicles prior to selection of a DF, coupled with ultrasound measurement of follicular sizes, show that differences in follicular size determined by ultrasound are too small to identify reliably the future DF prior to its deviation in growth rate relative to the subordinates [17]. However, highest intrafollicular estradiol and lowest concentration of IGFBP-4 appear to be reliable predictors of follicular fate (i.e., dominance) 1.5 days after emergence of the follicular wave [18]. Nevertheless, it is not known when in relation to the initial FSH stimulus these biochemical differences first occur in a cohort of growing follicles during a follicular wave and whether these differences are maintained throughout wave development. Thus, in our study, heifers were ovariectomized at different stages of follicular growth after the transient rise in FSH that precedes the first follicle wave, to determine the alterations in the relationships between follicular diameter and intrafollicular growth factors, steroids, and apoptosis during the selection of a dominant follicle in a mono-ovulating species. In addition, sequential ovariectomy of heifers allows the timing of intrafollicular changes to be related to the clearly defined increase and subsequent decline in FSH that is associated with follicular growth and DF selection in cattle.
Currently, stage of follicular development is classified based on ultrasound-derived follicular size measurements at different times after follicular wave emergence or during the estrous cycle. However, this method is least accurate for follicles 
5 mm in diameter, especially between Days 1 and 3 of the estrous cycle, around the time of emergence of the first follicular wave [7]. In addition, this classification does not take into account the fact that the intrafollicular factors that may determine cohort follicle survival are dependent on the transient rise in serum FSH concentrations that precede each follicular wave. Therefore, an alternative method of evaluating follicular growth was designed based on interval from the peak of the first periovulatory increase in FSH and excision of follicles allowing direct measurement of follicular diameter. This procedure enables more accurate and physiological analyses of changes in differentiation of individual follicles in a cohort during a follicular wave. The aim of our present study was, therefore, to determine the associations between putative markers of follicular differentiation, growth, and atresia before, during, and after emergence of the first follicular wave relative to the periovulatory increase in FSH concentrations during the bovine estrous cycle.
MATERIALS AND METHODS
Animals
Thirty-three cross-bred heifers were randomly assigned to one of two experimental blocks: block 1 (n = 16), block 2 (n = 17). Each time-point for ovariectomy was represented in each block, and the study was completed in 2 weeks. All heifers were housed in straw-bedded pens. Grass-silage was available ad libitum, and each heifer received 2 kg of a 16% protein supplement daily. Estrous cycles were synchronized using Norgestomet ear implants (Crestar; Intervet Ireland Ltd., Dublin, Ireland) for 10 days with a single i.m. injection of a luteolytic dose of a prostaglandin F2
analogue (Prosolvin; Intervet Ireland) 3 days before implant removal. Observation for estrus commenced 24 h after implant removal and was performed every 3 h for 4 days or until the last heifer in the block exhibited estrus. Day of first detection of estrous behavior was designated as Day 0 of the subsequent cycle. All animal experimentation was performed in compliance with regulations set down by the BioMedical Centre, University College Dublin, and the Cruelty to Animals Act (Ireland) 1897.
Blood Sampling and Ultrasound Examination of the Ovaries
Blood samples were collected by jugular venipuncture at 3-h intervals from detected estrus until ovariectomy or at 3-h intervals for the first 48 h and then at 6-h intervals thereafter until ovariectomy. Samples were maintained at room temperature for 1 h, refrigerated at 4°C for 24 h, centrifuged at 1200 x g for 20 min, then decanted, and serum was stored at -20°C.
Ultrasound monitoring of growth of follicles 4 mm was performed daily using a realtime B-mode linear array ultrasound scanner with a 7.5-MHz probe (Dynamic Imaging Concept 500, Livingstone, UK). Using ultrasound measurement, the largest follicle in the cohort was identified as the dominant follicle if it was 1) a minimum diameter of 8.5 mm and 2) 1.5 mm larger than the next largest follicle in the cohort.
Ovariectomies, Follicular Fluid, and Granulosa Cell Recovery
Ovariectomies were performed by colpotomy [19] at different intervals after onset of estrus as follows: 36 h (to coincide with the approximate peak of the periovulatory FSH increase, n = 6 heifers), 60 h (approximate time of wave emergence, n = 6), 72 h (postemergence, n = 6), 84 h (predominance, n = 7), and 108 h (estimated first day of dominance, n = 7) after onset of estrus. However, after RIA of serum FSH, heifers were grouped based on when they were ovariectomized relative to the peak in serum FSH concentrations (see Table 1). Ovaries were placed in PBS. Each follicle 2.5 mm was excised from both ovaries for each heifer, and the diameter was measured under a dissecting microscope. Follicular fluid (FF) was aspirated from each follicle and stored at -20°C. Follicles were then bisected. Granulosa cells from each hemisphere were recovered by gently scraping cells from each follicular piece into PBS with a fine glass needle.
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Granulosa Cell Apoptosis
Percentage of granulosa cells with apoptotic DNA was determined using a modification of the method described by Blondin et al. [20]. Briefly, granulosa cells were centrifuged at 400 x g for 30 sec. The PBS was removed and cells were fixed by resuspension in 70% ethanol for a minimum of 20 h at 4°C. Fixed cells were spun at 400 x g for 4 min and ethanol removed. Cells were resuspended in 850 µl of PBS, vortexed, and passed up and down through a 25-gauge needle to ensure dispersion of cells. RNase A (0.1 mg/ml; Sigma Chemical Co., St. Louis, MO) and propidium iodide (50 µg/ml; Molecular Probes Inc., Eugene, OR) were added to the cells prior to incubation at 37°C for 30 min. Analysis of DNA content per cell was then performed with a Becton-Dickinson fluorescence-activated cell sorter flow cytometer. The percentage of granulosa cells per follicle that were apoptotic (%A = percentage of cells in the sub-G1 peak) was calculated from the resultant DNA histograms using the CellQuest program (Becton-Dickinson).
Hormone Assays
Steroid hormones Estradiol concentrations in FF were determined in samples diluted 1:100 with distilled water using a previously validated RIA [21]. Estradiol concentrations in serum were measured using the same assay following diethyl ether extraction of the samples. Mean intraassay (n = 6 samples) and interassay (n = 6 assays) coefficients of variation (CVs) for low (2.7 ± 0.1 pg/ml), medium (6.0 ± 0.5 pg/ml), and high (16.0 ± 0.6 pg/ml) diluted FF samples were; 7.7% and 12.3%, 7.2% and 19.2%, and 11.0% and 9.7%, respectively. Mean intraassay (n = 6 samples, n = 1 assay) CVs for low (1.7 ± 0.3 pg/ml), medium (3.0 ± 0.6 pg/ml), and high (5.1 ± 1.0 pg/ml) serum sample were 16.4%, 19.6%, and 18.8%, respectively.
Follicle-stimulating hormone Serum FSH concentrations were determined using a previously validated RIA [2]. Mean intraassay (n = 6 samples) and interassay (n = 5 assays) CVs for a low sample (15.1 ± 1.0 ng/ml) were 13.0% and 14.7%, respectively, and for a high sample (32.8 ± 1.9 ng/ml) were 10.0% and 13.1%. Sensitivity of the FSH assay was 2 ng/ml.
Ligand blot analysis of IGFBPs and individual molecular weight forms of inhibin Amounts of IGFBP-2, -3, -4, and -5 in FF from the five largest follicles per heifer (n = 27 heifers) were determined by ligand blot analysis as described by Jimenez-Krassel et al. [22]. Briefly, FF samples (25 µg protein/lane, 5 FF samples from 1 heifer per gel, n = 27 gels) from the five largest follicles per heifer were subjected to 15% SDS-PAGE for 100 min, prior to transfer onto Immobilon P membrane and incubation with 125I-labeled human recombinant (hr)-IGF-I (Bachem, Torrance, CA). One aliquot of the same pooled FF sample was analyzed on each gel as a quality control (QC). Intensity of IGFBP bands was determined using the Molecular Analyst Software for Bio-Rad GS 250 Molecular Imager after exposure of blots for 45 h to a Bio-Rad GS 250 Imaging Screen-B1 (Bio-Rad, Richmond, CA) as reported previously [22]. The CV for total density of the QC lane between the 27 gels was 32.2%. A representative ligand blot of FF from the five largest follicles in one heifer is shown in Figure 1.
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Amounts of the various molecular weight forms of inhibin were determined by immunoligand blot analysis in the presence of excess inhibin subunit antibody and 125I-labeled C1–26 gly·tyr using a previously validated procedure [23]. The CV for total density of the QC lane between the 27 gels was 21.8%. The sum of the densities for all inhibin forms per follicle will be referred to as total -inhibin. A representative immunoblot of FF from the five largest follicles in one heifer is shown in Figure 2.
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Activin-A and follistatin Total activin-A concentrations in FF were measured using a previously validated two-site ELISA [24]. Human recombinant activin-A (Genetech Inc., San Francisco, CA) was used as the standard. Intraassay and interassay coefficients of variation were 8.4% and 16.9%, respectively (n = 6 assays). Detection limit of the assay was 10 pg/well. Total follistatin concentrations in FF were measured by a previously validated ELISA [25] using hr follistatin as standard (National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD). Intraassay and interassay CVs were 7.1% and 10.0% respectively (n = 8 assays). Detection limit of the assay was 30 pg/well.
Statistical Analysis
Each pair of ovaries per heifer was grouped based on the interval from the peak of the increase in serum FSH concentrations to ovariectomy as follows: 0 to 21, 24 to 45, 48 to 69, and 72 to 93 h post the peak in FSH concentrations. Mean concentration of FSH at ovariectomy, concentration of FSH at ovariectomy as percentage of peak FSH concentration, and rate of decline in FSH concentration were compared between heifers ovariectomized during the different time intervals by ANOVA, with Fisher's least significance difference test as the posthoc comparison. P-values of <0.05 were interpreted as significant.
All follicles recovered from each heifer were initially classified by size as follows: 2.5 to 4.0, 4.5 to 6.0, 6.5 to 8.0, and 8.5 mm in diameter. To examine whether interval after the peak in serum FSH concentrations affected number and size of follicles 2.5 mm on the ovary, mean number of follicles in each size class was compared between the different time intervals and within each time interval between different follicular size classes. The average for each follicular size class per heifer was calculated (the heifer being the experimental unit) and means per size class were compared using ANOVA. Mean intrafollicular estradiol concentrations and %A in follicles in each size class were similarly compared between the different time intervals and within each time interval between follicular size classes. Follicular fluid estradiol concentrations were transformed to logarithmic values for statistical analysis though arithmetic means are presented in Table 3.
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To determine how intrafollicular factors changed among cohort follicles during differentiation into dominant and subordinate follicles, a separate follicle classification system was used. Specifically, the five largest follicles per heifer were ranked by size in descending order as being either F1, F2, F3, F4, or F5. In all animals F1 and F2 were identifiable by size criteria alone, but among the smaller follicles when follicles were similarly sized, the follicle with higher follicular estradiol was assigned the higher rank. Amounts of estradiol, IGFBPs, inhibins, activin-A, and follistatin were compared in follicles of similar rank between the different time intervals by ANOVA and within each time interval between follicles of different rank. Linear regression analysis was used to determine the relationship of size of the follicle at the different times of ovariectomy with %A and with follicular estradiol, IGFBPs, inhibins, follistatin, and activin-A concentrations.
RESULTS
Estrous Response and Ovulation
All 33 heifers were observed in estrus within 4 days of Norgestomet implant removal. Daily ultrasound examination showed that one heifer had not ovulated within 3 days of first detected estrus, and so this heifer was excluded from all further analyses. Ovulation was detected in all other heifers within 2 days of estrus.
Changes in Serum Concentrations of FSH
One heifer ovariectomized 60 h after onset of estrus had no periovulatory increase in FSH and had no follicles >2.0 mm. This heifer was excluded from all analyses. Mean interval from onset of estrus to peak of the periovulatory increase in FSH concentrations was 28 ± 2 h, which was not different (P > 0.05) between the groups of heifers. Concentration of FSH in serum from heifers (n = 7) ovariectomized on Day 4.5 of the estrous cycle are shown in Figure 3. Mean interval from the peak in serum FSH concentrations until ovariectomy in heifers ovariectomized at different times after the peak in FSH are shown in Table 1. Mean serum concentration of FSH at ovariectomy and percentage of the peak FSH concentration decreased in heifers ovariectomized at 33, 53, or 84 h (after the peak in FSH concentrations) compared with those ovariectomized at 5 h (Table 1; P < 0.05).
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Numbers and Diameters of Follicles Recovered after Ovariectomy
Number of 2.5- to 4.0- and 4.5- to 6.0-mm follicles per heifer did not change (P > 0.1) as the interval from the FSH peak increased (Table 2). By 53 h there was an increase (P < 0.05) in number of follicles 6.5 mm in size (6.5- to 8.0- and >8.5-mm size classes combined) compared with earlier time periods. In heifers ovariectomized 84 h after peak FSH concentrations, the number of follicles in the 8.5-mm size class increased (P < 0.05) compared with the 5- and 33-h time periods. When the five largest follicles (F1 to F5) per heifer were examined, mean diameter of F1 follicles increased (P < 0.05) from 4.8 ± 0.4 mm at 5 h to 10.8 ± 0.8 mm by 84 h (Fig. 4A). At 84 h, when a DF was identifiable by ultrasound examination, F1 was the only follicle 8.5 mm in each heifer, i.e., at 84 h F1 was the DF.
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Estradiol in Serum and FF
Serum concentrations of estradiol were at nadir (0.35 ± 0.05 pg/ml) at 33 h (Table 3). They increased (P < 0.05) to a maximum of 1.31 ± 0.16 pg/ml at 84 h.
Follicular estradiol concentrations of all follicles were relatively low at 5 h. In addition, there were no differences (P > 0.1) in estradiol concentration between follicles in the 2.5- to 4.0- or 4.5- to 6.0-mm size classes (Table 3). Follicles in the largest size class (8.5 mm) 53 h had higher (P < 0.05) concentrations of estradiol than follicles in all the smaller size classes. Similarly, estradiol was higher in F1 compared with F3, F4, and F5 follicles by 33 h and higher in F1 compared with all other follicles by 53 h after the peak of FSH (Fig. 4B). Follicle diameter was positively correlated with estradiol concentrations from 33 h (Table 4). At 84 h, the DF (F1) was the follicle in the cohort with highest concentration of estradiol in FF in all heifers (Table 5). Overall, F1 had the highest concentration of estradiol in 23 of 27 heifers irrespective of interval after the peak in FSH.
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Apoptosis of Granulosa Cells
Percentage of apoptotic granulosa cells did not differ (P > 0.1) between the different size-classes of follicles at 5, 33, or 53 h after the peak of FSH (Table 3). At 84 h, the F1 (DF) had lower (P < 0.05) %A than follicles 6.0 mm in diameter but similar %A compared with 6.5- to 8.0-mm follicles. Follicle diameter was negatively correlated with %A at 84 h and not correlated with %A during earlier time periods (Table 4). At 84 h, the DF (F1) was the follicle in the cohort with the lowest %A in 4 of 6 heifers (Table 5).
Insulin-Like Growth Factor Binding Proteins in FF
Four different IGFBPs were detectable in FF from follicles during the first follicle wave. They were identified based on approximate size: IGFBP-3 (40–46 kDa), IGFBP-2 (35 kDa), IGFBP-5 (30–32 kDa), and IGFBP-4 (doublet at 25 and 29 kDa, Fig. 1). Amounts of IGFBP-3 in FF did not differ among F1 to F5 follicles and were unaltered during the decline in FSH (data not shown). Amounts of IGFBP-2, -4, and -5 in FF were similar in F1 and F2 follicles at all times after the peak of FSH concentrations. In F3 and F5 follicles amounts of IGFBP-2, -4, and -5 increased (P < 0.05) between 53 and 84 h. In F4 follicles, amount of IGFBP-2 also increased (P < 0.05) between 53 and 84 h.
There were no differences in amounts of IGFBP-2, -4, or -5 among F1 to F5 follicles 5 h after the peak in FSH concentrations. By 84 h, amount of IGFBP-2 was 6–10-fold higher in F3, F4, and F5 follicles compared with the F1 (DF) (Fig. 5A). However, at 33 h, amounts of IGFBP-4 were increased (P < 0.05) in F4 and F5 follicles compared with F1 (Fig. 5B). Similarly, at 53 h, amounts of IGFBP-4 were 5-fold higher from F4 and F5 follicles compared with F1 follicles. At 84 h, amount of IGFBP-4 was higher in F3 and F5 follicles compared with F1 and F2 follicles (Fig. 5B). At 33 h amount of IGFBP-5 was higher in F4 and F5 follicles compared with F1 and F2, and at 84 h it was higher in F5 follicles compared with F1 and F2 (Fig. 5C).
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Follicle diameter was negatively correlated with intrafollicular concentrations of IGFBP-2, -4, and -5 at all times after the peak in FSH (Table 4). At 84 h, the F1 (DF) had the lowest amount of IGFBP-2 and -4 among the cohort follicles in all six heifers and it also had lowest amount of IGFBP-5 in five of six heifers (Table 5).
Inhibin Forms in FF
Eight different molecular weight forms of inhibin (precursors = >160, 110, 77, 58, 49, 48 kDa; fully processed = 34 kDa; free subunit = 29 kDa) were identified in FF of follicles in this study (Fig. 2). At 5 h, all follicular classes had similar amounts of each inhibin form. In F1 follicles, amounts of all the precursor forms of inhibin increased (P < 0.05) between 5 and 33 h (Fig. 6). At 33 h, F1 contained higher (P < 0.05) amounts of the six precursor inhibin forms than F4 and F5 follicles. Amounts of the 34- and 29-kDa inhibin forms in individual follicular classes (F1 to F5) were unaltered by interval after the peak in FSH. Follicle diameter was positively correlated (P < 0.05) with intrafollicular amounts of all six precursor inhibin forms at 33-, 53-, and 84-h time-points (Table 4). From 33 h onward, amounts of the >160-, 110-, 77-, and 48-kDa forms of inhibin were positively correlated with follicular estradiol concentrations, with tightest correlations at the 33-h time-point (33 h, r2 > 0.4; 53 h r2 > 0.3, 84 h r2 > 0.25). Correlations between the other forms of inhibin and follicular estradiol concentrations throughout the follicle wave were weaker (r2 < 0.25) or not significant (P > 0.05). At 84 h, the DF (F1) had highest total -inhibin in five of six heifers (Table 5).
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Follistatin and Activin-A in FF
Concentration of follistatin in F1 follicles decreased (P < 0.05) between 5 and 33 h and decreased further (P < 0.05) between 33 and 84 h (Fig. 7A). In F2 follicles, it increased between 5 and 33 h and then decreased between 33 and 53 h. In F3, F4, and F5 follicles, within each follicular class concentration of follistatin did not differ (P > 0.1) after the peak of FSH. At 5, 33, or 53 h, F1 to F5 follicles did not differ in concentration of follistatin. At 84 h, concentration of follistatin in the DF (F1) was lower (P < 0.05) than in F2, F3, F4, and F5 follicles. Follicle diameter and intrafollicular concentrations of follistatin were not correlated at 5, 33, or 53 h, but were negatively correlated (P < 0.05) at 84 h (Table 4). At 84 h, the DF (F1) had the lowest concentration of follistatin in all six heifers (Table 5).
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Activin-A increased (P < 0.05) in F1 follicles between 5 and 33 h (Fig. 7B). It increased (P < 0.05) in F3 follicles between 33 and 53 h but did not change further by 84 h. At 33 h, concentration of activin-A was higher (P < 0.05) in F1 than in F5. At 84 h, it was lower (P < 0.05) in the DF (F1) than in F2 but was similar to F3, F4, and F5. Size of follicle and intrafollicular concentrations of activin-A were positively correlated (P < 0.05) at 33 h (Table 4).
DISCUSSION
Growth of antral follicles beyond 3–4 mm in cattle is preceded by a transient increase in the circulating concentration of FSH [6, 7]. Previous studies examining follicular growth and steroidogenesis during the first follicular wave of the estrous cycle in heifers focused on the dominant follicle and on follicles recovered at a single time point prior to the attainment of dominance [7, 14]. This study is the first to examine changes in amounts of intrafollicular growth factors beginning near the peak of the periovulatory increase in FSH and continuing until the attainment of dominance by a single follicle. We show that within 33 h of the peak of FSH concentrations and prior to follicular dominance, changes in growth and differentiation occur within the five largest follicles of the emerged cohort. These changes are similar to those that occur at the end of the DF selection process. Specifically, the most significant findings of the study were that 1) lower molecular weight IGFBPs are suppressed in the largest follicles of the growing cohort despite the decline in FSH; 2) amounts of inhibin precursors increase initially in the largest follicles, but intrafollicular amounts of the large molecular weight forms do not change further with differentiation to dominance; 3) after selection, DFs contain highest estradiol concentration and and lowest amount of IGFBPs-2, -4, and follistatin; and 4) changes in follicular diameter, estradiol, and IGFBPs precede changes in apoptosis of granulosa cells of follicles destined for atresia.
The attainment of dominance by a single follicle is characterized by its largest size, highest intrafollicular estradiol concentrations, and continued growth despite low systemic FSH concentrations. A DF also acquires the capacity to suppress growth of other follicles, i.e., functional dominance [8]. Ginther et al. [26] reported that the future DF emerges earlier in the wave than future subordinates and that an earlier emerging follicle may have a growth advantage over later emerging follicles predisposing it to develop to dominance. In this study, we determined that from 33 h after the peak in FSH concentration, the largest follicle in the cohort also produced the most estradiol, in all but one heifer. This finding is consistent with the hypothesis that the unique differentiation of a follicle for continued growth and estradiol production, key characteristics of a dominant follicle, occurs during the period of cohort follicle growth and before morphological dominance defined by follicular size differences is established.
The decline in FSH concentrations results in the removal of several key intrafollicular survival factors, thereby increasing the risk of apoptotic death of granulosa cells and follicular atresia. In vivo studies using induced follicular atresia models in the ewe, such as suppression of FSH [10], or following withdrawal of FSH treatment [27], have led to the hypothesis that increased granulosa cell apoptosis is a very early event in follicular atresia [10]. In our study, the increase in percentage of apoptotic granulosa cells occurring in small follicles was found to be a late event in relation to the decline in FSH. The healthy estrogenic DF maintained a low level of apoptosis, but percentage of apoptotic granulosa cells was similar to that in the largest subordinate follicle, despite significant differences in size and estradiol production of the dominant and subordinate follicles. From these data, we conclude that apoptotic death of granulosa cells is a late event in follicular atresia, occurring after the cessation of follicular growth and estradiol production.
Intrafollicular factors such as the IGF family of proteins or inhibins have a role in controlling follicular development, through either regulation of systemic gonadotropins or local intraovarian modulation of the effects of the gonadotropins especially FSH [13]. Insulin-like growth factor-I synergizes with FSH in stimulating granulosa cell proliferation and steroidogenesis in vitro [28, 29], but concentrations of IGF-I are similar in follicular fluid from dominant and subordinate follicles [14, 30]. Lower molecular weight IGFBPs exert a negative influence on follicular development through their ability to sequester and negate the effects of IGF-I [31]. Here, we report that at 5 h after the peak of FSH, the majority of small follicles (5.5 mm) contain low amounts of IGFBP-2, -4, and -5. Previous work shows that bovine and rodent follicles recovered after endogenous or exogenous FSH stimulation contain low amounts of low molecular weight IGFBPs [14, 28]. In vitro evidence shows that in cattle expression of mRNA for IGFBP-2 is switched off following treatment with FSH [32]. However, in this study smaller follicles (F4 and F5) recovered after the peak of FSH contained lower concentrations of estradiol and increased IGFBP-4 and -5 compared with the larger cohort follicles. Throughout the follicular wave, the largest growing follicle in the cohort maintained low amounts of IGFBPs in follicular fluid. This finding provides strong evidence that maintenance of low intrafollicular levels of lower molecular weight IGFBPs is key to continued growth and onset of dominance of a single follicle.
The precise role of inhibins in follicular development is not clear. Previous research has shown that growing follicles contain increased total amounts of inhibin, whereas atretic DFs and subordinate follicles contain decreased amounts of the largest precursor forms (>160 and 110 kDa) and increased amounts of the fully processed 34-kDa form [14, 33]. In this study, amounts of the precursor forms were higher in F1 and F2 follicles than in F4 and F5 follicles from 33 h after the peak in FSH. Despite the divergent development of F1 and F2 into dominant and subordinate follicles, both maintain similar amounts of all inhibin forms throughout the decline in FSH in our study. However, the observation that by 33 h F1 follicles display sustained increases in all precursor forms, whereas F2 follicles only display significant increases in the >160- and 58-kDa forms of inhibin suggests a potentially important difference between F1 and F2 as early as 33 h. Furthermore, the failure of any forms of inhibin to change with time in F3–F5 follicles underscores the potential importance, for follicular selection, of these subtle differences in the patterns of increase in the >160- and 58-kDa forms in F1 and F2 follicles. The maintenance of amounts of the 34-kDa fully processed form in all follicles throughout the decline in FSH concentrations contrasts with the decrease in amounts of the precursor forms in F3, F4, and F5 follicles compared with F1 and F2 follicles. In atretic follicles as the amount of the precursor forms decrease the proportion of the 34-kDa form increases. Further studies are required to determine whether the proportional increase in the 34-kDa form of inhibin plays a role in initiation of atresia or is merely symptomatic of the atretic process.
In contrast to the early increases in follicular estradiol and inhibins during the wave, concentration of follistatin decreases in the largest follicle of the cohort. We report here for the first time that the selected dominant follicle contains the lowest concentration of follistatin of all follicles in the cohort. Concentrations of activin-A initially increase in the largest follicle in the cohort coincident with the early increase in precursor inhibins. Thus, increasing activin-A is a feature of early follicle selection, whereas decreasing follistatin continues in the largest follicle throughout the selection process. Follistatin is an activin-binding protein and alterations in follistatin may alter the amount of biologically active activin-A within the follicle. Consequently, decreasing follistatin in the largest follicle of the cohort may increase the amount of free activin-A within the follicle that could facilitate continued follicle growth despite the decline in FSH.
To summarize, selection of a dominant follicle from a pool of growing antral follicles is a dynamic process regulated by interactions of the gonadotropins and intraovarian factors. Based on our findings, we propose the following model for antral follicular growth leading to selection of a DF in cattle (Fig. 8). Following the increase in circulating FSH concentrations, a pool of follicles of 2.5–4 mm in diameter are stimulated to grow. At this time, the five largest follicles per heifer have similar amounts of estradiol, IGFBPs, inhibins, activin-A, follistatin, and percentage of apoptotic granulosa cells. By 33 h after the peak in FSH, the two largest follicles increase in size and continue to grow while FSH concentrations decline. In addition, these follicles gain an enhanced capacity to produce estradiol, inhibin precursors, and activin-A, but begin to loose the ability to produce follistatin while maintaining low amounts of IGFBP-2, -4, and -5 and a low percentage of apoptotic granulosa cells compared with the other follicles in the cohort. By 53 h after the peak in FSH, a dominant-like follicle can be distinguished in the cohort based on its largest size, highest estradiol, and lowest amount of IGFBPs, though functional dominance, i.e., suppression of growth of all other cohort follicles has not yet been established. At 84 h after the peak in FSH, the selected dominant follicle is the largest follicle in the cohort is approximately 10 mm in diameter and contains the highest amounts of estradiol and the lowest amount of IGFBPs and follistatin compared with the other follicles in the cohort. In contrast, the remaining follicles in the cohort (F2, F3, F4, and F5), though continuing to grow until 84 h after the peak in FSH, reach smaller maximum diameters of 5.5–8.5 mm. Thus, follicular growth is characterized by 1) increases in follicular diameter, estradiol, and at least initially in inhibin and activin-A production; 2) a decrease in follistatin; and 3) maintenance of low amounts of low molecular weight IGFBPs and a low percentage of apoptotic granulosa cells. Follicles in the wave destined to become atretic are characterized by loss of capacity to produce estradiol and enhanced production of low molecular weight IGFBPs. These changes not only precede atresia but also occur before the cessation of follicular growth, changes in intrafollicular inhibins, activin-A and follistatin, or increased granulosa cell apoptosis in these follicles. Based on these results we conclude that the earliest intrafollicular changes that distinguish a follicle destined to become dominant from the other follicles in a growing cohort are enhanced capacity to produce estradiol and maintenance of low levels of IGFBPs.
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ACKNOWLEDGMENTS
The authors gratefully acknowledge Dara Cooke and Pat Duffy for help with the animal work; Niamh Hynes and Tara Good for assistance and advice in carrying out assays; Prof. Finian Martin and Roddy Monks for assistance with flow cytometry; and Tony Harte and the staff at Lyons Estate Research Farm for the care of animals. The FSH antiserum (AFP-C5288113) and FSH for iodination (AFP-7571A) were obtained through Dr. A.F. Parlow (NHPP). They also acknowledge Dr. D. Bolt (U.S. Department of Agriculture, Beltsville, MD) for providing the FSH standard and Prof. N.P. Groome for providing reagents for activin-A and follistatin assays.
FOOTNOTES
First decision: 21 January 2000.
1 This work was supported by the Irish Department of Agriculture and Food Stimulus Fund and by a U.S. Department of Agriculture grant 94-37203-0720 and Research Excellence Funds to J.J.I. 
2 Correspondence. FAX: 353 1 660 0883; edward.austin{at}ucd.ie
3 Current address: University of Glasgow Veterinary School, Glasgow, UK.
Accepted: October 31, 2000.
Received: November 17, 1999.
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