| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706
ABSTRACT
The circulating concentrations of LH were reduced by administration of 50 mg of progesterone every 8 h for 72 h, beginning when the largest follicle was 6.0 mm (experiment 1; n = 10). Progesterone treatment prevented the transient increase in LH that accompanied deviation (partitioning into dominant and subordinate categories) in control heifers (n = 10). The reduced LH concentrations were associated with reduced growth of the largest follicle, beginning a mean of 31 h after deviation, but did not alter the time of deviation or the growth and regression of the second-largest follicle. In experiment 2, 0 mg (controls) or 50 mg of progesterone was given every 8 h for three injections, beginning when the largest follicle was 7.0 mm (predeviation group) or 9.0 mm (postdeviation group; n = 8 for each of the four groups). Blood samples from the jugular vein and follicular-fluid samples from the two largest follicles were taken 8 h after the last treatment when the largest follicle was a mean of 8.7 mm in the predeviation group and 10.8 mm in the postdeviation group. In the controls, follicular-fluid concentrations of estradiol and free insulin-like growth factor (IGF)-1 in the largest follicle and IGF binding protein (IGFBP)-2 in the second-largest follicle were higher (P < 0.05) in the postdeviation group than in the predeviation group. Progesterone treatment lowered (P < 0.006) the circulating LH concentrations to a similar extent in both groups. In the predeviation group, progesterone treatment did not have a significant effect on any of the characteristics of the largest follicle. In the postdeviation group, the largest follicle of the progesterone-treated heifers had significant reductions in diameter and in follicular-fluid concentrations of estradiol and free IGF-1. Follicular-fluid concentrations of immunoreactive inhibin were not different for any of the comparisons. The results supported the hypothesis that LH has a positive effect on diameter of the largest follicle but not until after the beginning of diameter deviation. In addition, the results indicated that LH is involved in the production of estradiol by the largest follicle and that free IGF-1 concentrations increase in the largest follicle during deviation.
estradiol, follicle, follicle-stimulating hormone, follicular development, growth factors, luteinizing hormone
INTRODUCTION
Each follicular wave in cattle results from stimulation by an FSH surge [1]. After the surge reaches a peak, the FSH concentrations decline over several days while the follicles grow from about 4.0 to 8.5 mm [2, 3]. On average, the follicles grow at a similar rate (parallel growing phase) and then partition into a dominant and multiple subordinate follicles (diameter deviation). Deviation is indicated by a continuation of the growth rate of the largest follicle and a decline or cessation of growth rates of the other follicles. The beginning of deviation is defined as occurring at the examination preceding the examination in which the diameter difference between the two largest follicles has increased [2]. The beginning of deviation occurs when the largest follicle reaches a mean of approximately 8.5 mm, based on several studies that used an 8-h interval between measurements of follicular diameters [2–5]. Despite variation among individuals, the future dominant follicle emerges earlier, on average, than the future largest subordinate follicle. When a group of waves is normalized to the beginning of deviation or when the largest follicle is 8.5 mm, the mean difference in diameter between the two follicles during the parallel growing phase is about 0.5 mm or equivalent to a growing period of approximately 8 h [2, 4]. Near the beginning of diameter deviation, the largest follicle becomes established as the dominant follicle apparently before the next largest follicle reaches a similar diameter. Thus, the deviation mechanism is established within 8 h, while FSH is still declining to basal concentrations [3].
An FSH-follicle coupling hypothesis states that the essence of follicle selection is a close two-way functional coupling between the follicles and changing FSH concentrations [6]. During the parallel growing phase, both the future dominant and subordinate follicles contribute to the declining portion of the FSH surge [7] yet remain closely dependent on the decreasing concentrations of FSH [6]. At the beginning of diameter deviation (end of parallel phase), the largest follicle assumes the role for FSH-follicle coupling by continuing the decline in circulating FSH concentrations and requiring the reduced concentrations for its continued growth [7]. Deviation results from differences among follicles in the extent of FSH dependency, according to the developmental stage of the follicles. The more-developed largest follicle, unlike the smaller follicles, is able to tolerate the self-imposed reduced FSH concentrations.
Functional studies indicate that an increased secretion of estradiol by the largest follicle at the beginning of diameter deviation accounts for the continuing decline in FSH concentrations in association with deviation [8]. Other factors found in follicular fluid may also play a role in the control or utilization of the gonadotropins, including insulin-like growth factors (IGFs) and inhibin [9]. In cattle, differential increases in diameter between the two largest follicles (diameter deviation) begin simultaneously with the beginning of differential increases in estradiol in the follicular fluid [10] and in the blood plasma [8]. However, in ponies estradiol increases preceded diameter deviation by a mean of 1 day. The estradiol changes were expressed by an increase in estradiol concentrations in the follicular fluid [11] and estrogen-like ultrasound echotextural changes of the wall of the future dominant follicle [12]. These changes were most pronounced in the future dominant follicle.
The role of LH in the deviation phenomenon has not been clarified. A transient increase in circulating LH encompasses the time of diameter deviation in cattle [3–5] and ponies [13]. In ponies, the effect of LH on diameter deviation has been studied by experimental reduction of circulating LH well before expected deviation. Reduction of LH did not alter the day of deviation or the growth and regression profiles of the largest subordinate follicle [14, 15]. The LH reduction interfered with the growth and FSH-suppressing function of the largest follicle, but depressed growth was not detected until 2 days after the expected beginning of deviation. These results suggested that LH is not involved in the initiation of diameter deviation in ponies but is utilized for growth of the dominant follicle after deviation. In cattle, several reports are consistent with a role for LH at the beginning of diameter deviation or shortly thereafter [16]. Chronic treatment of cattle with a GnRH agonist suppressed the pulsatile secretion of LH, and the largest follicle did not grow beyond 7 to 9 mm [17]. Expression for LH receptors appeared in the granulosa cells between 2 and 4 days after wave emergence [18, 19], and this time period encompasses the expected time of deviation [4]. It has been well demonstrated that the life span of an established dominant follicle can be extended by an increased LH pulse frequency [20, 21], but apparently there are no studies that directly implicate LH in diameter deviation.
The present experiments in cattle tested the hypothesis that LH manifests its effect on growth of the largest follicle after diameter deviation has been established (experiments 1 and 2). Changes in the concentrations of estradiol, progesterone, immunoreactive inhibin (inh-ir), free IGF-1, and IGF binding protein-2 (IGFBP-2) in the follicular fluid of the two largest follicles at the expected beginning of deviation and after deviation and the relationship between these follicular-fluid factors in the largest follicle and circulating LH concentrations were also examined (experiment 2).
MATERIALS AND METHODS
Animals and Ultrasound Scanning
The experiments were conducted during the wave that emerges in the periovulatory period (wave 1). The animals were Holstein heifers between 24 and 36 mo of age. The feeding program and the prostaglandin (PG)F2
protocol for inducing luteolysis to schedule ovulation have been described [10]. An ultrasound scanner (Aloka SSD-500V Micrus; Aloka Co., Wallingford, CT) equipped with a 7.5-MHz linear-array intrarectal transducer was used to monitor the ovaries and measure the follicles as described [3, 22]. Scanning was done every 24 h beginning on the day of induced luteolysis and continuing until the largest follicle of a new wave reached a diameter of 5.0 mm (experiment 1) or 6.0 mm (experiment 2). Thereafter, scanning was done every 8 h. Circulating concentrations of LH were reduced by administration of progesterone, as described for ponies [14]. The dose of progesterone was injected i.m. every 8 h in a vehicle of l ml of safflower oil. The vials of progesterone were color-coded so that the ultrasound operator was not aware of the dose for each heifer. The selection of doses was based on our previous study in cattle [23] in which 75 or 150 mg was given every 12 h. The 75-mg dose resulted in systemic progesterone concentrations characteristic of mid-diestrus and did not alter the FSH concentrations.
Experiment 1
Heifers were randomized into three groups (n = 10/group) to receive either 0, 50, or 100 mg of progesterone. Both the 50-mg and 100-mg doses were used because of inadequate information on the required dose for reducing LH in heifers. The 0-mg group (controls) was given vehicle only. Progesterone was given every 8 h for 10 injections beginning when the largest follicle was 6.0 mm (Hour 0). Scanning of follicles and collection of blood samples were done just before each injection and continued every 8 h until Hour 96 and then daily for two additional examinations (Hours 120 and 144). Data were analyzed and plotted from the time of first treatment to Hour 144. Expected hour of deviation was taken as the examination when the largest follicle reached 8.5 mm, based on previous studies [3, 5, 7].
Experiment 2
Heifers were randomized into four groups (n = 8/group) in a 2 x 2 design. Heifers received doses of either 0 mg (controls) or 50 mg of progesterone, as described for experiment 1. Treatments began when the largest follicle first reached 7.0 mm or 9.0 mm (Hour 0). A 7.0-mm largest follicle was used for Hour 0 so that this group would be approximately at the beginning of deviation (largest follicle, 8.5 mm) at Hour 24, based on a mean follicular growth rate of 1.5 mm/day [4]. Thus, the heifers first treated when the largest follicle was 7.0 mm and 9.0 mm were defined as the predeviation and postdeviation groups, respectively. Progesterone treatments were done at Hours 0, 8, and 16, and blood and follicular-fluid samples were taken at Hour 24. The blood sample was collected from the jugular vein, and the follicular fluid was aspirated from the largest follicle. In addition, follicular fluid of the second-largest follicle was aspirated in the control heifers. The follicular-fluid collections were made by ultrasound-guided transvaginal aspiration with a 20-gauge needle, as described [22]. Blood plasma was assayed for FSH and LH, and the follicular fluid was assayed for estradiol, progesterone, inh-ir, free IGF-1, and IGFBP-2.
Hormone Assays
Blood samples were collected into heparinized tubes, and the plasma was separated by centrifuging, decanted into storage vials, and frozen (-20°C) until assay. Plasma concentrations of FSH and LH were determined, using a validated RIA for cattle [24–26]. Details on the methodology as used in this laboratory have been reported [1, 3]. Mean assay sensitivity was calculated as 2 SD below the mean counts per minute of maximum binding. The sensitivity was 0.01 ng/ml for FSH and 0.06 ng/ml for LH in experiment 1 (n = 3 assays) and 0.01 ng/ml for FSH and 0.12 ng/ml for LH in experiment 2 (n = 1 assay). The within-assay and between-assay coefficients of variation (CVs) for experiment 1 were 3.8% and 15.1%, respectively, for FSH and 6.4% and 16.3% for LH. The within-assay CVs for experiment 2 were 7.3% for FSH and 12.0% for LH as determined from a pool of bovine plasma collected during diestrus.
Follicular-fluid samples were cooled in an ice bath, centrifuged at 500 x g for 10 min to remove cells, and stored at -20°C until assay. Follicular-fluid concentrations of estradiol were determined using modifications of an RIA kit (Double Antibody Estradiol; Diagnostic Products Corp., Los Angeles, CA) that has been validated for use in cattle [27, 28]. Follicular-fluid samples were diluted 1:1000 in assay buffer. Assay sensitivity was 1.4 pg/ml (n = 1 assay), as determined by 2 SD below the mean counts per minute of maximum binding. Quality-control samples were prepared from a pool of bovine follicular fluid collected from all follicles visible from the surface of slaughterhouse ovaries. The within-assay CVs were 6.8% and 10.3% for low and high quality-control samples.
Follicular-fluid concentrations of progesterone were determined using a competitive ELISA described for use in cattle [18, 29]. A protein-based assay buffer containing 1% (v/v) steroid-reduced bovine follicular fluid was used to prepare the standards (0.16 to 10 ng/ml), and buffer alone was used for dilution of follicular fluid and quality-control samples. Serial dilutions (0.25 to 5 µl) of a pool of bovine follicular fluid in a total volume of 100 µl resulted in a displacement curve that was similar to the linear portion of the standard curve. A working dilution of 1:100 was used for the experimental follicular-fluid samples because 1 µl of pooled follicular fluid resulted in a percent binding that was central to the range of the standard curve. Mean assay sensitivity was 0.012 ng/ml (n = 2 assays) as calculated from 2 SD below the mean optical density (OD) of the maximum binding. The within-assay and between-assay CVs were 10.4% and 5.2% for low-quality control samples, respectively, and 6.0% and 2.3% for high-quality control samples.
Concentrations of inh-ir in follicular fluid were determined using an RIA kit (Institute of Reproduction and Development; Monash Medical Center, Clayton, Victoria, Australia). The kit included inhibin as a 32-kDa fraction of bovine follicular fluid for iodination and anti-inhibin (#1989) that was generated against a 31-kDa fraction of bovine follicular fluid. A recombinant preparation of the 32-kDa form of bovine inhibin (IP-1095; Peninsula Laboratories Europe Ltd., St. Helens, Merseyside, England) was used as the reference standard. Procedures for the assay were similar to those previously described [30], except that the iodination was done using Iodogen for 3 min [31]. Serial dilutions of standard (2.5 to 250 ng/ml) and a pool of bovine follicular fluid (0.025 to 0.4 µl) in a total volume of 100 µl resulted in displacement curves that were similar. A working dilution of 1:4000 was used for experimental follicular-fluid samples as 0.025 µl resulted in a percent binding that was central to the range of the standard curve. As previously described [30], the assay cross-reacts with the full-length forms of the -subunit as well as the various intact forms of inhibin. The assay sensitivity was 1.9 ng/ml (n = 1 assay) as determined by 2 SD below the mean counts per minute of the maximum binding. The within-assay CV was 14.1%.
Several techniques have been reported for determining the concentrations of putative free IGF-1 in serum and follicular fluid in human and laboratory species but apparently not in the large domestic species. Follicular-fluid concentrations of free IGF-I were determined using a sandwich-type ELISA kit (DSL 10-9400; Diagnostic Systems Laboratories, Inc., Webster, TX) that uses a specific antibody to capture free IGF-1 from the fluid and another specific antibody to detect the IGF-1. The kit was developed for use with human serum but was adapted and validated for use with bovine follicular fluid in our laboratory. The color intensity of the enzyme substrate was directly proportional to the free IGF-1 concentration. The standards (recombinant human IGF-1; 0.06 to 2.7 ng/ml) and quality-control samples supplied with the kit were reconstituted with distilled water. Follicular-fluid samples were diluted with the protein-based zero standard containing BSA supplied in the kit. Serial dilutions (3 to 25 µl) of a pool of bovine follicular fluid in a total volume of 50 µl resulted in a displacement curve that was similar to the standard curve. A working dilution of 1:10 was used for the experimental follicular-fluid samples as 5 µl resulted in an OD that was central to the range of the standard curve. According to the manufacturer, the cross-reactivity of this assay with IGF-2, insulin, proinsulin, and growth hormone was not detectable at 0.2 µg/tube. Mean assay sensitivity was 0.018 ng/ml (n = 2 assays) as determined by 2 SD above the mean OD of the zero standard. The within-assay and between-assay CVs were 3.1% and 1.7% for low quality-control samples, respectively, and 7.1% and 4.4% for high quality-control samples, respectively.
Follicular-fluid concentrations of IGFBP-2 were determined using a double antibody RIA kit (DSL-7100; Diagnostic Systems Laboratories). The RIA approach contrasts with previous studies that used qualitative or semiquantitative ligand blot assays [32]. The kit was developed for use with human serum but was adapted and validated for use with bovine follicular fluid in our laboratory. The standards (2.5 to 100 ng/ml) and quality-control samples supplied with the kit were reconstituted with distilled water. Follicular-fluid samples were diluted with the protein-based zero standard supplied in the kit. Serial dilutions (2 to 8 µl) of a pool of bovine follicular fluid in a total volume of 200 µl resulted in a displacement curve that was similar to the standard curve. A working dilution of 1:50 was used for the experimental follicular-fluid samples because 4 µl resulted in a percentage binding that was central to the range of the standard curve. According to the manufacturer, the cross-reactivity of this assay with IGFBP-3, -4, -5, and -6 was not detectable at 0.5 µg/tube and IGF-1 and IGF-2 were not detectable at 1 µg/tube. The assay sensitivity was 0.317 ng/ml (n = 1 assay) as determined by 2 SD below the mean counts per minute of the maximum binding. The within-assay CVs were 11.2% and 15.6% for low and high quality-control samples, respectively.
Statistical Analyses
The concentrations of FSH in experiment 1 were converted to percent changes from Hour 0. Percentages were used because of a disparity between the 0-mg and 50-mg group means at Hour 0. It was thought that the disparity, although not significant at Hour 0, could contribute to significant differences at other hours. In this regard, the FSH values within heifers most often (60% of the time between Hours 0 and 32) were significantly correlated with later values. For example, the correlation in FSH concentrations between Hours 0 and 32 in controls was r = 0.7 (P < 0.04). For percent data, Hour 0 was not included in the statistical analyses because of the absence of variation. For sequential follicular and hormonal data (experiment 1), split-plot ANOVA were used for determining main effects of group and hour and the interaction. Variation due to sequential data was accounted for by using heifer as the error term to test the effect of group. If a significant effect of hour or an interaction of group-by-hour was indicated, Duncan's multiple-range tests or Student's t-tests were used to locate mean differences among groups within hours and among hours within groups. A significant group-by-hour interaction for gonadotropins for the three groups (0, 50, and 100 mg) was further examined by comparing two groups at a time in separate split-plot ANOVA. In experiment 2, end points for the control heifers were analyzed by a 2 x 2 factorial ANOVA, followed by t-tests. The factors were follicles (largest and second-largest at Hour 24) and groups (predeviation and postdeviation). When a significant effect was obtained, three separate analyses were made with t-tests for the four data sets as follows: 1) between the largest and second-largest follicle within the predeviation group, 2) between follicles within the postdeviation group, and 3) between the largest follicles of each group. Pearson correlation analyses were done among diameter and each of the follicular-fluid factors of the two largest follicles in the control heifers separately within the predeviation and postdeviation groups. A separate 2 x 2 analysis was used for the largest follicle for treatment (control and progesterone) and group (predeviation and postdeviation). The observations for LH, progesterone, and free IGF-1 concentrations were not normally distributed and were analyzed by a ranking procedure (Kruskal-Wallis test). Data are presented as the mean ± SEM unless otherwise indicated. Significance was indicated by a probability of P < 0.05.
RESULTS
Experiment 1
Actual diameter of the largest follicle at Hour 0 (first progesterone treatment) was 6.5 ± 0.1 mm. Diameter of the two largest follicles at the expected time of deviation (largest follicle, 8.5 mm or larger) was 8.6 ± 0.1 mm and 7.5 ± 0.3 mm, respectively, for the controls and 8.6 ± 0.1 mm and 7.3 ± 0.2 mm for the 50-mg group. Double dominant follicles (>11.0 mm) developed in one heifer (50-mg group). To include this heifer in the analyses, the largest dominant follicle was used for the largest follicle and the third-largest follicle was used for the second-largest follicle. In the 0-mg and 50-mg groups, the first follicle to attain 6.0 mm became the first follicle to reach 8.5 mm in 74% of the heifers. The mean times of expected deviation in the 0-mg (controls) and 50-mg groups were Hours 33.6 ± 4.7 and 33.6 ± 3.7, respectively. The characteristics of deviation were not assessed in the 100-mg group because a dominant follicle (>11.0 mm) did not develop in five of eight heifers, and three heifers did not develop a follicle of 8.5 mm. The mean maximum diameter of the largest follicle was greater (P < 0.01) in the 0-mg group (15.3 ± 0.4 mm) than in the 50-mg group (13.5 ± 0.6 mm), or 100-mg group (10.4 ± 1.0 mm). A new follicular wave developed at a mean of Hour 110 in the five heifers that did not develop a dominant follicle in the 100-mg group. None of the heifers in the other two groups developed a new wave by the end of the experiment at Hour 144.
The interaction of group by hour was significant for diameter of the largest follicle (P < 0.001) and the second-largest follicle (P < 0.05; Fig. 1). The interaction for the largest follicle reflected reduced (P < 0.05) diameter by Hour 64 in the 50-mg group, compared to the control group, and reduced (P < 0.05) diameter beginning at Hour 32 in the 100-mg group, compared to each of the other two groups. The interaction for the second-largest follicle was attributable primarily to reduced (P < 0.05) diameter beginning at Hour 16 in the 100-mg group and continuing until the end of the experiment. For the second-largest follicle, there were no significant differences between the control and 50-mg groups.
|
The group-by-hour interaction was significant (P < 0.001) for concentrations of LH (Fig. 1). The interaction resulted, in part, from reduced (P < 0.05) concentrations over Hours 16 to 72 in the 50-mg and 100-mg groups, compared to the control group. There were no significant differences between the 50-mg and 100-mg groups when analyzed separately. However, an analysis within dose indicated that LH concentrations decreased more rapidly in the 100-mg group (decrease by Hour 8, P < 0.05) than within the 50-mg dose (decrease by Hour 32, P < 0.05). Within the controls, LH concentrations increased (P < 0.05) between Hours 0 and 32 and decreased (P < 0.05) by Hour 64. There was also an interaction (P < 0.001) for percent change in concentrations of FSH (Fig. 1). A separate analysis for the control and 50-mg groups did not show a significant group effect or an interaction. For the control and 100-mg groups, an interaction was attributable to a greater (P < 0.05) percent decrease in FSH by Hour 8 and an increase by Hour 72 in the 100-mg group. The actual mean concentrations at Hour 0 were similar between the control and 100-mg groups, and therefore the data for these two groups were also analyzed using the original values and not the percent conversion. With actual data, the FSH concentrations in the 100-mg group were less (P < 0.05) at Hours 8 (0.14 ± 0.02 ng/ml) and 16 (0.10 ± 0.01 ng/ml) than in the control group (0.20 ± 0.02 ng/ml and 0.18 ± 0.03 ng/ml); the concentrations were higher (P < 0.05) in the 100-mg group than in the control group for Hours 56 to 120.
Experiment 2
Actual diameter of the largest follicle at Hour 0 was 7.3 ± 0.1 mm and 9.2 ± 0.1 mm for the predeviation and postdeviation groups, respectively. The largest follicle at Hour 0 was the largest follicle at Hour 24 in 82% and 94% of the heifers in the two groups, respectively. Results of the hormonal assays of follicular-fluid and the statistical analyses for the two largest follicles at Hour 24 in control heifers are shown (Table 1). There were group-by-follicle interactions for follicle diameter (P < 0.04) and concentrations of estradiol (P < 0.0001), progesterone (P < 0.02), free IGF-1 (P < 0.006), and IGFBP-2 (P < 0.03). The interactions were primarily attributable to the postdeviation group; means were highest for diameter of the largest follicle, concentrations of estradiol, progesterone, and free IGF-1 in the largest follicle, and concentrations of IGFBP-2 in the second-largest follicle. Combined for the two follicles in the control heifers of the predeviation group, there was a significant and negative correlation between free IGF-1 and IGFBP-2; none of the other correlations were significant. In the postdeviation group, diameter, estradiol, and free IGF-1 were significantly and positively correlated with each other, and IGFBP-2 was significantly and negatively correlated with each of the other three end points.
|
Plasma concentrations of LH at Hour 24 showed a main effect (P < 0.006) of treatment (control versus progesterone-treated heifers; Table 2). Averaged over the predeviation and postdeviation groups, the concentrations were lower for the treated heifers (0.33 ± 0.05 ng/ml) than for the controls (0.47 ± 0.05 ng/ml). The main effects of group and the interaction were not significant. There were no significant main effects or an interaction for concentrations of FSH (Table 2). For the end points involving the largest follicle, the group effect (predeviation versus postdeviation) or the group-by-treatment interaction for the largest follicle were significant for diameter (P < 0.0001) and for concentrations of estradiol (P < 0.0001), progesterone (P < 0.04), and free IGF-1 (P < 0.05). Neither the interaction nor the main effects were significant for inh-ir and IGFBP-2 (Table 2). Within the predeviation group, none of the end-point means were significantly different between the control and treated heifers. However, one of the estradiol observations (350 ng/ml) in the treated group was an extreme value, according to the Dixon's outlier test. When this observation was removed, the estradiol concentrations were significantly lowered by progesterone treatment in the predeviation group. Within the postdeviation group, the means for the largest follicle were lower in the treated heifers for diameter of follicle (P < 0.04) and follicular-fluid concentrations of estradiol (P < 0.0001) and progesterone (P < 0.006); a reduction in concentrations of free IGF-1 approached significance (P < 0.06).
|
DISCUSSION
The diameters of the largest and second-largest follicles at the expected time of deviation in both experiments were close to reported values for observed deviation [2–5]. The first follicle to attain 6.0 mm (experiment 1) and 7.0 mm (experiment 2) became the first follicle to reach 8.5 mm in 74% and 82% of the heifers, respectively. These percentages agree with previous reports [33, 34]. Blood samples for gonadotropin assays were collected at 8-h intervals on the basis of previous reports. A study of FSH and LH pulses [5] did not reveal any significant pulsatility characteristics for either gonadotropin in association with follicle emergence or deviation that were not expressed by significant changes in mean concentrations obtained from blood sampling at 8-h intervals [4]. Although the effects of progesterone are attributed to altered concentrations of gonadotropins in these experiments, a direct effect of progesterone on the follicles is not ruled out.
The 50-mg doses of progesterone reduced the circulating concentrations of LH in both experiments, as expected on the basis of previous studies in pony mares [14, 15]. These results in cattle are also consistent with several reports of a negative effect of circulating concentrations of progesterone on LH pulse frequency [23]. The LH concentrations were lower than in the controls by Hour 16 (experiment 1) and in both the predeviation and postdeviation groups at Hour 24 (experiment 2). In experiment 1, both the 50-mg and 100-mg doses seemed equally effective in reducing LH. However, the LH mean at first treatment (Hour 0) was higher in the 100-mg group than in the 50-mg group. The apparent difference at Hour 0 likely contributed to the enigma of no significance in the analysis for the two groups, despite a more rapid decrease in the 100-mg group (within 8 h) than in the 50-mg group (within 32 h) when each group was analyzed separately. The elevated LH concentrations that encompassed the expected time of deviation in the controls agrees with previous reports of a transient elevation at this time in heifers [4, 5, 28] and mares [13, 35]. The slight but significant LH increase in heifers before diameter deviation followed by a significant decrease after deviation will be termed a deviation LH surge for differentiation from the preovulatory LH surge. Both the 50-mg and 100-mg doses of progesterone prevented the deviation LH surge.
The progesterone-induced reduction in LH well before deviation (experiment 1) and after treatment in both the predeviation and postdeviation groups (experiment 2) provided tests for the hypothesis that the effect of LH on follicle diameter is not apparent until after deviation has begun. In experiment 1, a reduction in diameter of the largest follicle for the 50-mg dose was not detected until Hour 64 or a mean of 31 h after the beginning of expected diameter deviation. The inhibitory effect on diameter of the largest follicle also was expressed by reduced maximum diameter of the largest follicle. The 50-mg dose did not alter the hour when the largest follicle reached 8.5 mm (expected deviation) and did not alter the growth and regression profile of the second-largest follicle. In experiment 2, beginning treatment when the largest follicle was 7.0 mm (predeviation) did not reduce the diameter of the largest follicle 24 h later, even though LH concentrations were reduced. The same treatment protocol beginning when the largest follicle was 9.0 mm (postdeviation) did reduce the diameter of the largest follicle. Thus, the results of both experiments supported the hypothesis that circulating LH was utilized for postdeviation growth of the largest follicle but was not involved in the manifestation of diameter deviation.
In experiment 1, the 100-mg dose of progesterone had an inhibitory effect on both the largest and second-largest follicles, whereas the 50-mg dose significantly affected only the largest follicle. The rapid reduction (within 8 h) in concentrations of FSH after administration of the 100-mg dose likely accounts, at least partly, for the different follicular effect of the two doses. Decreased growth of the smaller follicle began by Hour 16 or 8 h after the FSH decrease. The rapid inhibitory effect of reduced FSH concentrations on follicle growth supports the report [7] of a close functional coupling between the two events, as demonstrated after FSH reduction by administration of a proteinaceous fraction of follicular fluid [7, 28] or estradiol [8]. Although the FSH effects were not different significantly between the 50-mg and 0-mg doses, they also did not differ significantly between the 50-mg and 100-mg doses. The means for the 50-mg dose during the first 16 h were located approximately midway between those of the 0-mg and 100-mg doses. Despite the absence of a significant difference from controls, therefore, the lack of an effect of the 50-mg dose on FSH concentrations is considered equivocal. In this regard, FSH concentrations were not affected when 75- and 150-mg doses of progesterone were given to heifers every 12 h during early diestrus [23]; the diameter of the largest follicle was reduced, but LH concentrations were not determined. Lack of a direct effect of exogenous progesterone on FSH concentrations has also been shown in other studies in cattle and sheep [23].
Concentrations of FSH increased well before the end of treatment with the 100-mg dose. Concentrations of FSH increased after Hour 48 for the 50-mg dose but not significantly. With the 100-mg dose, the FSH concentrations seemed to begin increasing after Hour 16 and were significantly higher than in the controls by the time the last treatment was given. The early development of a new FSH surge with the 100-mg dose accounts for the early emergence of a new follicular wave in this group. The functional capability of the dominant follicle in the 50-mg group was indicated by the continued suppression of FSH and a resulting delay in the emergence of a new follicular wave. In contrast to FSH concentrations, the LH concentrations did not increase until after the end of treatment. An immediate decrease in circulating FSH followed by an increase before the end of a treatment series also occurred during treatment with estradiol [8] and a proteinaceous fraction of follicular fluid [28]. The immediate FSH depression followed by a rebound apparently involves the hypothalamic-pituitary area, but specific study is needed on the mechanisms that would account for both an initial depression and a subsequent rebound before the end of treatment.
In control heifers of experiment 2, follicular-fluid estradiol concentrations were greater in the largest follicle than in the second-largest follicle at the approximate beginning of deviation, and concentrations were much greater in the largest follicle after the beginning of deviation. These results agree with the results of an in vivo follicular-fluid sampling study [10]. In the sampling study, diameter deviation and the changes in estradiol concentrations occurred on the same day. In the present study, the difference in follicular-fluid estradiol concentrations between the two follicles was much greater postdeviation than at the approximate beginning of deviation. The estradiol comparisons between the two largest follicles (controls, experiment 2) and the continuing depression of FSH concentrations after deviation (controls, experiment 1) are consistent with the conclusions from functional studies [8]; the largest follicle releases increased estradiol at the beginning of diameter deviation, and the increased systemic estradiol is involved in the continuing depression of FSH to below the requirement of the smaller follicles. The largest follicle is able to utilize the depressed concentrations of FSH [7].
The reduction of LH by three injections of progesterone before the beginning of deviation did not significantly alter the estradiol concentrations of the largest follicle. However, estradiol concentrations were reduced in the progesterone-treated group after a statistical outlier was removed. In the postdeviation group, the same treatment protocol, with a similar negative effect on circulating LH concentrations, was associated with an almost sevenfold reduction in the estradiol concentrations of the largest follicle. These results suggested that LH was required at the beginning of deviation for estradiol production and indicated that LH was required after deviation for estradiol production as well as growth of the largest follicle. Although the initial stimulation of estrogen production by growing follicles has been attributed to FSH [36], a role of LH in follicular production of estrogens also has been well established [9]. These reviewers noted that LH facilitates estrogen production by acting on theca cells to produce androgens that are aromatized to estrogens by granulosa cells. Also, when the granulosa cells acquire sufficient LH receptors, LH becomes an additional stimulus to cAMP-regulated processes of the granulosa, thereby further stimulating steroidogenic enzymes in the pathway to estrogen production.
Progesterone concentrations in the follicular fluid changed in the same direction as estradiol concentrations and were positively correlated with estradiol concentrations. The changes in progesterone concentrations may reflect the increased steroidogenic activity of the follicles in association with estradiol production. Concentrations of immunoreactive inhibin in the follicular fluid were not different between the two largest follicles of control heifers in either the predeviation or postdeviation groups. In addition, the concentrations in the largest follicle at these two times were not altered by the experimental reduction in circulating LH concentrations. The findings in the control heifers agree with the report that the total amount of inhibin and all of the inhibin forms in the follicular fluid of the dominant follicle in cattle were not significantly different between 3 and 6 days after estrus [37]. For comparison to the published findings, Hour 24 for the predeviation and postdeviation groups was approximately equivalent to 3.5 and 5.5 days after estrus, respectively. It has been reported [38] that bioactive inhibin concentrations during the same approximate period of the estrous cycle were higher in dominant follicles than in atretic follicles, defined according to intrafollicular ratios of estrogen:progesterone; however, the atretic follicles apparently were from a previous wave. In the present study, the inhibin assay did not distinguish between the bioactive dimeric forms of inhibin and the monomeric and other forms of inhibin. Despite the lack of specificity of the assay used herein, the results indicated that intrafollicular concentrations of immunoreactive inhibin were not altered by experimentally reducing the systemic LH concentrations. The present results did not suggest that inhibin was the factor that caused diameter deviation.
In the present study, putative free IGF-1 was measured in bovine follicular fluid, and concentrations were expressed relative to a standard of recombinant human IGF-1. Most of the IGF-1 is bound to IGFBPs, and therefore free or unbound IGF-1 is only a small portion of the total IGF-1 in the body [39]. Other studies measured total IGF-1 and approximated the concentration of the free IGF-1 as 1–5% of the total IGF-1 [40]. In this regard, the mean concentrations of free IGF-1 in the present study (6 to 12 ng/ml) are about 4.7% of mean concentrations of the total IGF-1 reported in other studies (90 to 293 ng/ml [36, 41]). The postdeviation intrafollicular concentrations of free IGF-1 in the control heifers of experiment 2 were higher in the largest follicle than in the second-largest follicle. In the predeviation group, the concentrations were not different between the two largest follicles. These findings agree with a report of higher total IGF-1 concentrations in the follicular-fluid of large estrogen-active follicles (
8 mm) than in large estrogen-inactive follicles (8 mm) or pools of follicular fluid from medium (5–7 mm) and small (<5 mm) follicles [42]. Furthermore, intrafollicular concentrations of total IGF-1 were higher in dominant follicles (mean, 11.9 mm) than in smaller follicles (mean, 6.6 mm) from earlier in the wave [36]. Conversely, no difference was found in total IGF-1 levels in cattle between dominant (mean 11.9 mm), large (mean, 8.5 mm), or small follicles (<6 mm) of wave 1 [41]. However, comparing the findings for free IGF-1 as determined by the assay used herein with published findings for total IGF-1 may have limited value. The insulin-like growth factors and their binding proteins play a role in folliculogenesis in cattle and other species through paracrine-autocrine regulation [43]. In cattle, intraovarian stromal infusion of IGF-1 was associated with an increase in the diameter of largest follicle [44], and cattle selected for twinning had elevated levels of total IGF-1 in follicular fluid of the two largest follicles [45]. In vitro studies have shown that IGF-1 stimulates mitosis of bovine granulosa cells [46] and theca cells [47, 48] and increases estradiol, progesterone, and androgen synthesis [48]. The reported and present findings suggest a pivotal role for IGF-1 in diameter deviation by facilitating increased estradiol production [48] by the largest or developing dominant follicle [8]. The estradiol reduces the circulating FSH concentrations to below the requirements of the smaller follicles [8]. The largest follicle is able to thrive despite the self-imposed low FSH concentrations [7], perhaps because follicular factors, such as estradiol and IGF-1, increase the sensitivity of the granulosa cells to FSH [49]. Experimental reduction of LH after deviation affected the largest follicle not only by reduced diameter and follicular-fluid concentrations of estradiol, but also by reduced concentrations of IGF-1. These results indicated that LH was required after deviation for function as well as for maximum growth of the largest follicle. This conclusion is consistent with a report that progesterone treatment of cattle that had a maintained dominant follicle resulted in decreased diameter of the follicle and decreased intrafollicular concentrations of estradiol, testosterone, and total IGF-1 [50]. Furthermore, in vitro studies have shown that LH increases the production of total IGF-1 from porcine granulosa cells [51] and that IGF-1 increases the number of LH receptors of bovine theca cells [47].
Although only one of at least three IGF-1 binding proteins with a proposed role in folliculogenesis (IGFBP-2,-4, -5 [42]) was considered in the present study, a reciprocal relationship between free IGF-1 and IGFBP-2 was found. There were higher levels of IGFBP-2 in the second-largest follicle than in the largest follicle in the controls of the postdeviation group and a negative correlation combined for the two follicles between concentrations of free IGF-1 and IGFBP-2. These results are consistent with reports that concentrations of IGFBP-2, -4, -5 in follicular fluid were lower or undetectable in dominant follicles and higher in subordinate follicles [36, 42, 52]. It has been reported that IGFBPs in follicular fluid may exert a pivotal role in bioavailability of IGF-1 by selectively binding to the IGF-1 and negating its potential tropic activity [53]. Low concentrations of binding proteins may increase the concentrations of free IGF-1 in dominant follicles [54]. The present findings of low IGFBP-2 and high free IGF-1 in the largest follicle and the reciprocal relationship in the second-largest follicle apparently is the first direct support for this concept; previous studies did not measure free IGF-1.
In conclusion, administration of progesterone for 72 h beginning when the largest follicle was 6.0 mm reduced the circulating concentrations of LH until the end of treatment. Reduced concentrations of LH beginning about 17 h before expected follicle deviation did not affect diameter deviation, as indicated by time of occurrence and diameter characteristics. However, the diameter of the largest follicle was reduced, beginning a mean of 31 h after the beginning of diameter deviation. Reducing LH concentrations by three injections of progesterone at 8-h intervals before the expected beginning of deviation did not affect diameter of the two largest follicle or the concentration of any of the follicular-fluid factors in the largest follicle. Reducing LH concentration by treatment when the largest follicle was 9.0 mm (after the beginning of deviation) resulted in reduced diameter of the largest follicle and reduced follicular-fluid concentrations of estradiol and free IGF-1. Results indicated that LH has a role in the growth and function of the largest follicle after the beginning of deviation.
ACKNOWLEDGMENTS
The authors thank the Pharmacia and Upjohn Company for a gift of lutalyse, the U.S. Department of Agriculture Animal Health Program for providing antigens and the primary antisera for the gonadotropin assays, and L.J. Kulick and S.C. Jensen for technical assistance.
FOOTNOTES
1 Research supported by the University of Wisconsin, Madison, U.S. Department of Agriculture grant 99-35203-7669, and Equiservices Publishing and the Eutherian Foundation, Cross Plains, WI. 
2 Correspondence: O.J. Ginther, Department of Animal Health and Biomedical Sciences, 1656 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706. FAX: 608 262 7420; ojg{at}ahabs.wisc.edu
Accepted: August 22, 2000.
Received: June 5, 2000.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  D. M.W. Barrett, R. Duggavathi, K. L. Davies, P. M. Bartlewski, E. T. Bagu, and N. C. Rawlings Differential Effects of Various Estradiol-17beta Treatments on Follicle-Stimulating Hormone Peaks, Luteinizing Hormone Pulses, Basal Gonadotropin Concentrations, and Antral Follicle and Luteal Development in Cyclic Ewes Biol Reprod, August 1, 2007; 77(2): 252 - 262. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M.W. Barrett, P. M. Bartlewski, R. Duggavathi, K. L. Davies, and N. C. Rawlings Suppression of Follicle Wave Emergence in Cyclic Ewes by Supraphysiologic Concentrations of Estradiol-17beta and Induction with a Physiologic Dose of Exogenous Ovine Follicle-Stimulating Hormone Biol Reprod, October 1, 2006; 75(4): 633 - 641. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Lopez, D. Z. Caraviello, L. D. Satter, P. M. Fricke, and M. C. Wiltbank Relationship Between Level of Milk Production and Multiple Ovulations in Lactating Dairy Cows J Dairy Sci, August 1, 2005; 88(8): 2783 - 2793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
H. Lopez, R. Sartori, and M. C. Wiltbank Reproductive Hormones and Follicular Growth During Development of One or Multiple Dominant Follicles in Cattle Biol Reprod, April 1, 2005; 72(4): 788 - 795. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kobayashi, F. Jimenez-Krassel, Q. Li, J. Yao, R. Huang, J. J. Ireland, P. M. Coussens, and G. W. Smith Evidence that Cocaine- and Amphetamine-Regulated Transcript Is a Novel Intraovarian Regulator of Follicular Atresia Endocrinology, November 1, 2004; 145(11): 5373 - 5383. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Shirasuna, H. Asaoka, T. J. Acosta, M. P.B. Wijayagunawardane, M. Ohtani, M. Hayashi, M. Matsui, and A. Miyamoto Real-Time Relationships in Intraluteal Release among Prostaglandin F2{alpha}, Endothelin-1, and Angiotensin II During Spontaneous Luteolysis in the Cow Biol Reprod, November 1, 2004; 71(5): 1706 - 1711. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.J. Acosta, M.A. Beg, and O.J. Ginther Aberrant Blood Flow Area and Plasma Gonadotropin Concentrations During the Development of Dominant-Sized Transitional Anovulatory Follicles in Mares Biol Reprod, August 1, 2004; 71(2): 637 - 642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Ohtani, S Takase, M P B Wijayagunawardane, M Tetsuka, and A Miyamoto Local interaction of prostaglandin F2{alpha} with endothelin-1 and tumor necrosis factor-{alpha} on the release of progesterone and oxytocin in ovine corpora lutea in vivo: a possible implication for a luteolytic cascade Reproduction, January 1, 2004; 127(1): 117 - 124. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Sisco, L. J. Hagemann, A. N. Shelling, and P. L. Pfeffer Isolation of Genes Differentially Expressed in Dominant and Subordinate Bovine Follicles Endocrinology, September 1, 2003; 144(9): 3904 - 3913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Baerwald, G. P. Adams, and R. A. Pierson Characterization of Ovarian Follicular Wave Dynamics in Women Biol Reprod, September 1, 2003; 69(3): 1023 - 1031. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Levy, M. Gordin, M. F. Smith, O. U. Bolden-Tiller, and R. Meidan Hormonal Regulation and Cell-Specific Expression of Endothelin-Converting Enzyme 1 Isoforms in Bovine Ovarian Endothelial and Steroidogenic Cells Biol Reprod, April 1, 2003; 68(4): 1361 - 1368. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
