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Ovary |
Eutheria Foundation, Cross Plains, Wisconsin 53528
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20 mm) of the largest follicle (F1). Mares in IGF-I groups were given a dose of 500 µg (experiment 1) or 250, 25, or 2.5 µg (experiment 2). Ablation of F1 at Hour 24 was done in experiment 1, but not in experiment 2. The 500- and 250-µg doses stimulated growth, leading to ovulation of F2 in 10 of 10 and 4 of 5 mares in the two experiments, respectively, compared to 4 of 12 and 0 of 5 in saline-injected controls. These doses prevented (P < 0.05) the increase in IGF binding protein-2 and androstenedione that occurred in F2 of controls and increased (P < 0.05) the concentrations of activin-A, inhibin-A, and vascular endothelial growth factor (VEGF). The 500-µg dose stimulated higher (P < 0.05) concentrations of estradiol, but not until Hour 48, whereas the lower doses were ineffective. In experiment 2, free IGF-I concentrations in F2 at Hour 24 decreased progressively as the dose decreased so that concentrations for the 2.5-µg dose were higher (P < 0.05) than in F2 of controls and similar (not significantly different) to endogenous concentrations in F1. Correspondingly, concentrations of androstenedione in F2 at Hour 24 were lower (P < 0.05) and concentrations of activin-A, inhibin-A, and VEGF were higher (P < 0.05) after treatment of F2 with the 2.5-µg dose than in F2 of controls and were similar to concentrations in F1. Hence, a physiologic intrafollicular dose of IGF-I did not stimulate estradiol production but reduced the production of androstenedione and stimulated the production of activin-A, inhibin-A, and VEGF during follicle selection in mares.
cytokines, estradiol, follicular development, growth factors, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, the in vivo effects of IGF-I on follicular fluid factors were studied in heifers and mares [4]. The second-largest follicle (F2) was injected with recombinant human (rh) IGF-I at the expected beginning of deviation, and follicular fluid samples were taken at various hours during the 24-h posttreatment period. In heifers, IGF-I treatment of F2 increased the follicular fluid concentrations of estradiol and androstenedione after 3 h. In mares, IGF-I treatment of F2 resulted in a decrease in androstenedione and an increase in activin-A and inhibin-A at 24 h. These effects of IGF-I on follicular fluid factors were consistent with reported temporal relationships of follicular fluid factors between the largest follicle (F1) and F2 during deviation [3], but no indication was found that IGF-I stimulated estradiol production in mares during the 24 h after treatment. In a study of the experimental assumption of dominance by F2 after ablation of F1, free IGF-I began to increase in the future dominant follicle (i.e., F2) 12 h before the beginning of experimental deviation, whereas estradiol did not begin to increase in F2 until 36 h after the beginning of the free IGF-I increase [5]. Therefore, in the experiment on the effects of IGF-I injection [4], the last sampling at 24 h may have been too early after IGF-I treatment to detect an estradiol increase.
In mares, diameter deviation is indicated morphologically not only by the differential growth rate between the developing dominant and subordinate follicles but also by an expanded nonechoic ultrasonic layer within the wall of the dominant follicle. The echotextural changes distinguished the future dominant follicle from the future largest subordinate follicle approximately 1 day earlier than the beginning of diameter deviation [6]. The echogenic changes were attributed to increased vascularization. In other species, the increased vascularization of the theca interna is associated with angiogenic factors, such as vascular endothelial growth factor (VEGF; for reviews, see [7, 8]). Recent studies have suggested that IGF-I may be involved in VEGF-mediated vascularization of the follicle; in cattle [9] and monkeys [8], IGFs stimulate the secretion of VEGF from cultured granulosa cells. It has been suggested that angiogenic factors may play a role in follicle selection [10–12]. However, to our knowledge, the position of angiogenic factors, such as VEGF, in the cascade of events leading to follicle deviation has not been studied in any species.
In the present study, a single injection of rhIGF-I was made into F2 to determine, first, if a pharmacologic dose increases the follicular fluid concentrations of estradiol within 48 h and affects the circulating concentrations of FSH and, second, if a physiologic dose that simulates the IGF-I concentrations in the companion F1 will alter the concentrations of follicular fluid factors, including VEGF, in F2.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals were handled in accordance with the Guide for Care and Use of Agricultural Animals in Agricultural Research. Seventy-three mares were used in two experiments during the ovulatory season (May–September, Northern Hemisphere). The mares were mixed breeds of ponies, were 10 to 17 yr of age, and weighed 300 to 450 kg. The feeding program and the equipment and techniques for transrectal and transvaginal ultrasound scanning and manipulation of ovaries have been described previously [13, 14]. A new follicular wave was induced by ablation of all 6-mm follicles 10 days postovulation, also as described previously [13]. Ultrasound scanning of follicles was done every 24 h postablation in both experiments, except that mares in experiment 1 were also scanned 12 h after treatment. A two-follicle model was prepared in the postablation follicular wave as described previously [13] so that continuous follicle identity was not complicated by many follicles. All follicles of the postablation wave, except the two largest, were ablated when the largest follicle reached 15 mm. The two retained follicles were designated as F1 and F2 when F1 reached 20 mm (the expected beginning of deviation; Hour 0). Transvaginal ultrasound targeting was used as described previously for treatment of a designated follicle in mares [14, 15] and for sampling of follicular fluid [16]. Sequential sampling of follicles was done in each mare at designated hours. Diameters of F1 and F2 were recorded before follicle treatment or sampling and every 24 h until Hour 96. Thereafter, only the fate (ovulation or regression) of the two follicles was determined.
Experiment 1
Mares were randomized into a saline group (injection of physiologic saline) and an IGF-I group (n = 12/group). At Hour 0 (F1 20 mm), F2 was injected with either 50 µl of physiologic saline or 50 µl of rhIGF-I (10 µg/µl; total dose, 500 µg; Genetech, Inc., San Francisco, CA). A sample of follicular fluid (200 µl) was taken from F2 sequentially from each mare at Hours 12, 24, and 48. End points for F2 were follicular fluid concentrations of free IGF-I, IGFBP-2, estradiol, progesterone, androstenedione, activin-A, inhibin-A, and VEGF; follicle diameter; and the occurrence of ovulation. At Hour 24, F1 was ablated to determine if follicle growth and the rate of ovulation for F2 or experimental assumption of dominance by F2 would be different between groups. Blood samples (20 ml) for assay of FSH were taken at Hours 0, 12, 24, 48, and 72 from the jugular vein.
Experiment 2
Mares were randomized into a control group (no puncture of F2 until sampling), a saline group, and three IGF-I groups (n = 7–12/group). At Hour 0, F2 of the saline group was given 25 µl of physiologic saline containing 0.1% BSA (RIA grade; Sigma Chemical Co., St. Louis, MO). In the mares of the three IGF-I groups, F2 was given a single 25-µl injection containing rhIGF-I doses of 250 µg (high), 25 µg (medium), or 2.5 µg (low). To prepare the IGF-I doses, dilution was done with saline containing 0.1% BSA. A sample of follicular fluid (200 µl) was taken from F1 and from F2 sequentially at Hours 24 and 48. Sampling of F1 was done to determine if the induced concentrations of follicular fluid factors in the IGF-I-treated F2 were similar to those in the nontreated F1 and whether treatment of F2 affected the follicular fluid factors of F1. The end points for F1 and F2 were the same as for F2 in experiment 1.
The effects of follicle treatment and sampling and the resulting extent of fluid loss (diameter decrease) on the future ability of the follicle to develop and ovulate were examined. This was done by comparing the extent of the diameter decrease during the 24 h after a sampling of F1 with the ovulation rate of the groups in which the nontreated F1 was expected to have a high ovulation rate (control and saline groups). The greatest 24-h decrease in diameter after sampling F1 at either Hour 24 or Hour 48 was used as the value for each mare. The intention was to develop a definition of excessive fluid loss for replacement of mares.
Hormone Assays
Blood and follicular fluid samples were centrifuged (500 x g for 10 min), decanted, and stored (-20°C) until assay. Plasma samples were assayed for FSH by radioimmunoassay as validated previously [17] and modified [3] in our laboratory. The intraassay coefficient of variation (CV) was 10.6%, and the sensitivity was 0.6 ng/ml. Follicular fluid samples were assayed for free IGF-I, IGFBP-2, estradiol, progesterone, androstenedione, activin-A, and inhibin-A using commercially available kits that have been modified and validated for use with equine follicular fluid in our laboratory [3]. Intra- and interassay CVs for these hormones and experiments were less than 9.9%. The sensitivities for the two experiments, respectively, were as follows: free IGF-I, 0.03 and 0.01 ng/ml; IGFBP-2, 0.8 and 0.3 ng/ml; estradiol, 0.2 and 0.3 pg/ml; progesterone, 0.02 and 0.08 ng/ml; androstenedione, 0.01 and 0.03 ng/ml; activin-A, 0.02 and 0.1 ng/ml; and inhibin-A, 1.6 and 3.8 pg/ml.
Concentrations of VEGF in the follicular fluid were determined using a competitive ELISA kit (product no. 412710; Neogen Corporation, Lexington, KY). The kit was developed for use with human serum, plasma, and other biological fluids and was adapted and validated for use with equine follicular fluid in our laboratory. The supplied standards (50.00–0.78 ng/ml) were reconstituted with assay diluent number 2, which also served as the zero standard. The color intensity of the enzyme substrate was inversely proportional to the concentration. Serial dilutions (1.56–100 µl) of a pool of equine follicular fluid in a total volume of 100 µl of assay diluent number 2 resulted in a displacement curve that was similar to the standard curve. A working dilution of 1:5 was used for the follicular fluid samples, because 20 µl resulted in an optical density that was central to the range of the standard curve. According to the manufacturer, the cross-reactivity of the assay with other cytokines and World Health Organization cytokine standards is less than 0.5%. The intra- and interassay CVs, respectively, for quality-control samples were 1.6% and 5.3% for experiment 1 and 10.2% and 5.3% for experiment 2. The sensitivity was 0.6 and 0.7 ng/ml for experiments 1 and 2, respectively, as determined by 2 SD below the mean optical density of the zero standard.
Statistical Analyses
The follicular and hormonal data were challenged for extreme values with the Dixon outlier test [18] and for normality with the Kolmogorov-Smirnov test. The concentrations of FSH were converted to the percentage change from Hour 0 because of a wide disparity among mares in FSH concentrations throughout the experimental period. When the normality test was significant (P < 0.05), data were transformed by either natural logarithm or square root. End points were analyzed to determine the effects of group, hour, and interaction using a mixed linear model with a repeated statement to account for the autocorrelation between sequential measurements (SAS Institute, Inc., Cary, NC). If a significant (P < 0.05) effect of group or a group-by-hour interaction was detected, unpaired t-tests were used to locate the mean differences between groups within an hour, and paired t-tests were used between hours within a group. The difference in frequency of ovulation among groups was analyzed by a chi-square test. A probability of P = 0.05 indicated that a difference was significant, and probabilities of between P > 0.05 and P = 0.1 indicated that a difference approached significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two mares were removed from the IGF-I group because of failure to obtain a follicular fluid sample, resulting in 12 and 10 mares in the saline and IGF-I groups, respectively. The ovulation rate for F2 was greater (P < 0.001) in the IGF-I group (10 of 10) than in the saline group (4 of 12). The group-by-hour interaction for diameter of F2 during Hours -96 to 96 was significant, reflecting primarily greater diameters at Hours 24–96 in the IGF-I group (Fig. 1). The interaction of percentage change from Hour 0 in FSH concentrations was significant, reflecting a greater percentage increase in the saline group at Hours 48 and 72 (Fig. 1).
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Differences in concentrations of follicular fluid factors between the saline and IGF-I groups over Hours 12, 24, and 48 (main effect of group) were significant for all factors (Fig. 2). The group-by-hour interaction was significant for free IGF-I, IGFBP-2, estradiol, androstenedione, activin-A, and inhibin-A; only the two main effects were significant or approached significance for progesterone and VEGF. The interactions reflected primarily greater concentrations of free IGF-I and inhibin-A and lower concentrations of androstenedione at Hours 24 and 48 in the IGF-I group and progressively increasing concentrations of IGFBP-2 in the saline group and activin-A in the IGF-I group. In the IGF-I group, estradiol increased, but only between Hours 24 and 48, and progesterone was greater at all hours. Concentrations of VEGF decreased in the saline group over Hours 12–48 and were higher in the IGF-I group at Hours 24 and 48.
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Experiment 2
In the control and saline groups, ovulation from F1 occurred in 17 of 22 mares. The mean maximum decrease in F1 diameter per mare during the 24 h after a follicular fluid sampling was less (P < 0.0001) in the F1-ovulatory mares (-0.4 ± 0.8 mm, mean ± SEM) than in the F1-nonovulatory mares (-7.8 ± 0.8 mm, mean ± SEM). The 24-h diameter decrease was greater in every F1-nonovulatory mare than in any F1-ovulatory mare, with one exception in an F1-ovulatory mare. For this reason, a diameter decrease in F1 or F2 during the 24 h after an injection or sampling that exceeded the minimal decrease (5.7 mm) in the F1-nonovulatory mares was defined as an indicator of excessive fluid loss. Mares in all groups with excessive fluid loss were replaced and not used in the statistical analyses. Totaled over all groups, a diameter decrease of more than 5.7 mm occurred in more mares when the sampled follicles (F1 and F2) were in the same ovary (13 of 24 mares; 54%) than when the two follicles were in opposite ovaries (7 of 25; 28%); the difference between groups approached significance (P < 0.06). After replacement of mares because of excessive fluid loss, injection or sampling problems, and persistent corpus luteum, the number of mares in the groups was as follows: control, six; saline, five; high-dose IGF-I, five; medium-dose IGF-I, five; and low-dose IGF-I, six.
The ovulation rate from F2 was different (P < 0.03) among groups (control, zero of six; saline, zero of five; high-dose IGF-I, four of five; medium-dose IGF-I, zero of five; low-dose IGF-I, zero of six). In the control, saline, medium-dose IGF-I, and low-dose IGF-I groups, all mares ovulated from F1. In the high-dose IGF-I group, one mare had a small F2 (15.4 mm) at the time of treatment and ovulated from only F1, two ovulated from both F1 and F2, and two ovulated from only F2. The diameter of F2 showed a group-by-hour interaction (P < 0.002) over Hours -96 to 96, but F1 did not (Fig. 3). The F2 diameter at Hour 24 was smaller in the saline group than in the control (P < 0.02) or high-dose IGF-I (P < 0.03) groups. At Hour 48, the diameter of F2 was greater (P < 0.05) in the high-dose IGF-I group than in the other groups.
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For each follicular fluid factor, the concentrations in F1 did not differ among the five groups, and the group-by-hour interaction was not significant. The F1 data for the five groups were combined to examine the differences for F1 between Hours 24 and 48 and as an F1 control for the F2 groups. Similarly, no differences were observed between the F2 control and saline groups for any F2 follicular fluid factor, and F2 data for the two groups were combined to serve as F2 controls. Thus, comparisons were made among F1-control, F2-control, and the high-, medium-, and low-dose IGF-I groups as shown (Fig. 4). The group-by-hour interaction was significant (P < 0.03) for free IGF-I, IGFBP-2, estradiol, androstenedione, and VEGF. The main effects of group and hour were significant (P < 0.03) for progesterone, activin-A, and inhibin-A. Within the IGF-I groups, concentrations of free IGF-I decreased (P < 0.0004) as the dose decreased within both hours. For Hour 48, concentrations of inhibin-A decreased (P < 0.03) as the IGF-I dose decreased, whereas androstenedione and IGFBP-2 increased (P < 0.009) as the IGF-I dose decreased. At Hour 24, concentrations of androstenedione, activin-A, inhibin-A, and VEGF in F2 of the low-dose IGF-I group were not significantly different from the concentrations in the F1 controls. At Hour 24, free IGF-I concentration in F2 for the high- and medium-dose IGF-I groups was higher (P < 0.05) than in the F1 controls. Androstenedione concentration was lower (P < 0.05) in F2 of the high- and medium-dose IGF-I groups than in the F1 controls. Differences (P < 0.05) between hours for each group and groups that differed (P < 0.05) from the F2 controls within each hour are shown (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A comparison of follicular fluid factors between F1 and F2 at Hours 24 and 48 for follicles that were not injected with IGF-I (controls) was available in experiment 2. Higher concentrations of free IGF-I, estradiol, progesterone, activin-A, and inhibin-A and lower concentrations of IGFBP-2 and androstenedione were found in F1 compared with F2 at both hours. All these results are consistent with those of a previous study regarding temporal relationships among follicular fluid factors in mares [3].
The highest doses for a single injection of rhIGF-I (500 and 250 µg) in the two experiments were pharmacologic; the 250-µg dose resulted in free IGF-I concentrations in F2 at Hour 24 that were 14-fold higher than those in the nontreated F1 at Hour 24 (89.3 vs. 6.2 ng/ml). The basis for using a pharmacologic dose in these initial studies has been discussed previously [4]. In experiment 2, the free IGF-I concentrations in F2 at Hour 24 decreased progressively as the IGF-I dose decreased so that the concentrations for the low dose (2.5 µg) were higher than those in F2 of the controls but similar to the endogenous concentrations in F1. Thus, the 2.5-µg dose resulted in a concentration of free IGF-I in F2 that can be considered physiologic for F1 at that hour. Concentrations also decreased at Hour 48 as the dose decreased so that the concentrations for the low dose at Hour 48 were equivalent to those in F2 of the controls.
Increased growth and ovulation rate (100%) for F2 after treatment with 500 µg of rhIGF-I at the expected beginning of deviation in experiment 1 confirmed the results of a previous study [4]. In experiment 2, ovulation occurred from F2 in most mares (80%) in the high-dose (250 µg) IGF-I group compared to none in the control group (no follicle puncture), saline-treated, and medium-dose (25 µg) and low-dose (2.5 µg) IGF-I groups. The stimulation of F2 growth and ovulation by single pharmacologic doses of rhIGF-I (250 and 500 µg), but not by a single physiologic dose (2.5 µg), is attributable to the prolonged versus transient stimulatory effects. In experiment 2, follicular fluid sampling was done at Hours 24 and 48 from F1 as well as from F2, and ovulation in the high-dose IGF-I group occurred from both follicles in 40% of mares. The single mare in the high-dose IGF-I group that did not ovulate from F2 had the smallest diameter (15.4 mm) of F2 at the time of treatment, compared to diameters of more than 18.5 mm for the mares that ovulated from F2. Apparently, a follicle must be at a certain stage of development or diameter before it will respond to IGF-I treatment, but this hypothesis requires specific study.
The effect of the experimental procedures on circulating concentrations of FSH was examined in experiment 1. Ablation of F1 at Hour 24 in the saline group resulted in an immediate increase in FSH, similar to a reported increase following ablation of F1 at Hour 0 [5]. Treatment of F2 with rhIGF-I at Hour 0 prevented the FSH increase following ablation of F1. Thus, the IGF-I stimulation of F2 was demonstrated by the depression in systemic FSH, similar to what occurs when F1 is intact [5]. The decrease in FSH may have been in response to the increase in inhibin-A concentrations in F2 by Hour 24, as discussed below.
In experiment 1, concentrations of IGFBP-2 did not increase in the IGF-I group as they did in the saline group. In experiment 2, concentrations of IGFBP-2 in F2 at Hour 48 progressively increased as the dose of IGF-I decreased. In this regard, a reciprocal relationship between follicular fluid concentrations of free IGF-I and IGFBP-2 has been shown in mares during natural [3] and experimental [5] deviation.
Concentration of follicular fluid estradiol was not altered at Hour 12 or 24 in experiment 1, similar to the results of a previous study [4], but was higher at Hour 48. In experiment 2, the estradiol concentrations were not altered by exogenous IGF-I at Hours 24 or 48. The difference between experiments may be related to the higher rhIGF-I dose in experiment 1 (500 µg) compared with experiment 2 (250 µg) or to the continued high concentrations of free IGF-I in experiment 1 compared to the decrease between Hours 24 and 48 in experiment 2. The delay in an estradiol response to the 500-µg dose of IGF-I until Hour 48 in experiment 1 is consistent with the results of ablating F1 at the expected beginning of natural deviation, thereby causing F2 to convert to a dominant follicle [5]. In that study, estradiol did not begin to increase in F2 until 35 h after the beginning of the free IGF-I increase, and in the present study (experiment 1), the estradiol increase began between 24 and 48 h following the injection of 500 µg of IGF-I. Despite the reported increase of estradiol in plasma and follicular fluid before the beginning of natural diameter deviation [3, 16], our interpretation of the present results is that granulosa cell estradiol production is not immediately responsive to IGF-I and does not play a critical intrafollicular role in initiating the deviation mechanism in mares. In contrast, the results of temporality [19] and functional [4, 20] studies in heifers are consistent with an intrafollicular role of estradiol in the initiation of deviation, and estradiol secretion responds immediately to in vivo IGF-I treatment [4].
An increase in follicular fluid progesterone was the first detected change in an ovarian steroid hormone following treatment with 500 µg of rhIGF-I in a previous study [4] and in experiment 1. In experiment 2, an effect on progesterone was not detected. However, follicular fluid samples were not taken until Hour 24, and changes could have occurred earlier. The higher progesterone concentrations in F1 compared with nontreated F2 at Hours 24 and 48 could indicate that progesterone is a part of the deviation mechanism. However, in a temporality study [3], progesterone did not differentially increase in the developing dominant follicle until after increases occurred in other factors.
The higher concentrations of androstenedione in F2 of controls at Hours 24 and 48 was consistent for both experiments in the present study, a previous functional study [4], and a temporality study [3]. Various aspects of the increase in androstenedione in subordinate follicles in mares, as opposed to a decrease in heifers, have been discussed previously [4]. A close, reciprocal relationship was observed between free IGF-I and androstenedione concentrations in experiment 2. Higher concentrations of free IGF-I over the five groups were associated with correspondingly lower concentrations of androstenedione. Both free IGF-I and androstenedione concentrations in F2 in the low-dose IGF-I group were similar to the concentrations in the F1 controls, indicating that physiologic levels of free IGF-I in F1 account for the prevailing concentrations of androstenedione in F1. At Hour 48, the progressively increasing concentrations of androstenedione in F2 as the IGF-I dose decreased were accounted for by the progressively decreasing concentrations of free IGF-I.
Both activin-A and inhibin-A concentrations were higher in F2 of the IGF-I-treated follicles than in the controls, confirming the results of a previous study [4]. These results are also compatible with those of a temporality study [3] that demonstrated a differential increase in these factors in the future dominant follicle versus the future largest subordinate follicle at the time of a differential increase in free IGF-I. However, an increase in IGF-I occurred before an increase in activin-A and inhibin-A during experimental assumption of dominance by F2 [5]. In addition, in other species, IGF-I stimulated the production of both factors in vitro [21]. Results of the reported studies, together with those of the present study, have demonstrated both temporally and functionally (in vivo) that IGF-I stimulates increases in activin-A and inhibin-A in the developing dominant follicle of mares. At Hour 24 in the low-dose IGF-I group, concentrations of free IGF-I, activin-A, and inhibin-A were at greater concentrations than in the F2 controls, but concentrations of all three factors were similar to concentrations in the F1 controls. These results indicate that endogenous IGF-I in F1 regulates the production of activin-A and inhibin-A in F1.
The VEGF results are of special interest. This factor apparently has not been previously assayed in equine follicular fluid, although mRNA expression of VEGF in the equine corpus luteum has been reported [22]. The role of VEGF in follicle deviation or selection has not been studied specifically in any species, although follicular fluid concentrations have been shown to increase in cattle [10] and pigs [23] as the follicle diameter increases. In the F1 and F2 controls of experiment 2, VEGF was higher in the follicular fluid of F1 than in F2 at both Hour 24 and Hour 48. On a temporal basis, this finding suggests that VEGF could play a role in follicle deviation or selection, but further studies that include groups from before the beginning of deviation are required. The present finding that IGF-I stimulated the production of VEGF in mares in vivo in both experiments is consistent with the reports that VEGF secretion increases in cultures of granulosa cells of cattle [9] and monkeys [8] when exposed to IGF-I. The lowest IGF-I dose stimulated a VEGF increase in F2 at Hour 24 so that the concentrations of both VEGF and free IGF-I in F2 were similar to the concentrations in the F1 controls at this time.
In summary, a single injection of a pharmacologic dose (250 or 500 µg) of rhIGF-I into F2 at the expected beginning of follicle deviation (Hour 0) increased the production of activin-A, inhibin-A, and VEGF and stimulated growth of a dominant follicle, leading to ovulation. Estradiol and progesterone were less responsive to IGF-I than other follicular fluid factors. Injection of progressively lower doses of rhIGF-I into F2 resulted in increasingly higher concentrations of androstenedione. The concentrations of free IGF-I, androstenedione, activin-A, inhibin-A, and VEGF in F2 at Hour 24 after treatment of F2 with a physiologic dose (2.5 µg) of IGF-I were not significantly different from concentrations in F1, indicating that these factors are regulated in F1 by endogenous IGF-I. Our interpretation of the results is that IGF-I alters the concentrations of other follicular fluid factors during follicle selection in mares.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: O.J. Ginther, Animal Health and Biomedical Sciences, 1656 Linden Drive, University of Wisconsin, Madison WI 53706. FAX: 608 262 7420; ginther{at}svm.vetmed.wisc.edu
Received: 28 October 2003.
First decision: 21 November 2003.
Accepted: 25 November 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Biol Reprod 2003 68:758-765This article has been cited by other articles:
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  L K Doyle, C O Hogg, E D Watson, and F X Donadeu Seasonal effects on the response of ovarian follicles to IGF1 in mares Reproduction, November 1, 2008; 136(5): 589 - 598. [Abstract] [Full Text] [PDF]
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M A Beg and O J Ginther Follicle selection in cattle and horses: role of intrafollicular factors. Reproduction, September 1, 2006; 132(3): 365 - 377. [Abstract] [Full Text] [PDF]
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O J Ginther, E L Gastal, M O Gastal, and M A Beg In vivo effects of pregnancy-associated plasma protein-A, activin-A and vascular endothelial growth factor on other follicular-fluid factors during follicle deviation in mares Reproduction, April 1, 2005; 129(4): 489 - 496. [Abstract] [Full Text] [PDF]
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 T.J. Acosta, E.L. Gastal, M.O. Gastal, M.A. Beg, and O.J. Ginther Differential Blood Flow Changes Between the Future Dominant and Subordinate Follicles Precede Diameter Changes During Follicle Selection in Mares Biol Reprod, August 1, 2004; 71(2): 502 - 507. [Abstract] [Full Text] [PDF]
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T.J. Acosta, M.A. Beg, and O.J. Ginther Aberrant Blood Flow Area and Plasma Gonadotropin Concentrations During the Development of Dominant-Sized Transitional Anovulatory Follicles in Mares Biol Reprod, August 1, 2004; 71(2): 637 - 642. [Abstract] [Full Text] [PDF]
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