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BOR - Papers in Press, published online ahead of print January 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026195
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BIOLOGY OF REPRODUCTION 70, 1374–1379 (2004)
DOI: 10.1095/biolreprod.103.026195
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Critical Role of Insulin-Like Growth Factor System in Follicle Selection and Dominance in Mares1

O.J. Ginther2, E.L. Gastal, M.O. Gastal, and M.A. Beg

Eutheria Foundation, Cross Plains, Wisconsin 53528


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of the insulin-like growth factor (IGF) system in the deviation in growth rates among follicles (follicle selection) was studied in mares using an IGF binding protein (BP) to reduce the follicular-fluid concentrations of IGFs. The future dominant follicle (F1) was treated by intrafollicular injection at the expected beginning of deviation (F1 >= 20 mm; Day 0). The experimental groups were control (no injection, n = 8), vehicle (injection of vehicle; n = 6), and BP (injection of 250 µg of recombinant human IGFBP-3; n = 6). A sample of follicular fluid was taken from F1 on Day 1 in all groups. Compared with the control group, IGFBP-3 reduced (P < 0.05) the follicular-fluid concentration of free IGF-1 by 90%; lowered (P < 0.05) the concentrations of estradiol, activin-A, inhibin-A, and vascular endothelial growth factor; and increased (P < 0.05) the concentration of androstenedione. The diameter of F1 decreased and the diameter of F2 increased after Day 0 in the BP group, compared with the control and vehicle groups. A greater (P < 0.05) increase in circulating concentrations of FSH between Days 0 and 1 occurred in the BP group than in the other groups and accounted for the increased growth of F2. Dominance and ovulation from F1 occurred from fewer (P < 0.03) mares in the BP group (1 of 6) than from the control and vehicle groups combined (11 of 14); the remaining mares in the BP group ovulated from F2. Results indicated that the IGF system has a critical intrafollicular role in the differential changes in concentrations of follicular-fluid factors between the future dominant and subordinate follicles, leading to the development of follicle dominance (selection) and ovulation in mares.

cytokines, follicular development, growth factors, ovary, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mares, an ovulatory wave of follicles emerges midway during an interovulatory interval. The follicles grow in a common-growth phase for several days until the largest follicle reaches about 22.5 mm. Deviation then begins and is characterized by a continued growth rate of the dominant follicle and reduced growth rate of the subordinate follicles [for reviews, see 1, 2]. Differences between the two largest follicles in the rate of production of follicular-fluid factors (steroids, growth factors, cytokines) are temporally associated with diameter deviation and apparently underlie a greater gonadotropin responsiveness of the developing dominant follicle than the other follicles [3]. The follicular- fluid factors that are candidates for a role in deviation in mares are activin-A, inhibin-A, estradiol, and components of the insulin-like growth factor (IGF) system [for a review, see 2]. The components of the IGF system in mammals are IGF-1 and -2 and their receptors (IGF type-1 and -2), a superfamily that includes six high-affinity IGF binding proteins (IGFBP-1, -2, -3, -4, -5, and -6) and several low- affinity IGFBP-related proteins (IGFBP-rP), and IGFBP proteases (kallikreins, cathepsins, and matrix metalloproteinases) [4–8]. The proteases degrade IGFBPs and thereby regulate the bioavailability and actions of IGFs.

In mares [3], as in cattle [9], follicular-fluid concentration of free IGF-1 becomes higher in the future dominant follicle than in other follicles before diameter deviation begins. Follicular-fluid concentrations of free IGF-2 in mares are not known, although total IGF-2 concentrations were not different among follicles of various diameters [10]. In cattle, higher concentration of free IGF-1 in the largest follicle near the beginning of deviation is temporally associated with greater proteolytic activity that degrades IGFBP- 4 and -5 [11, 12]. Concomitantly, the concentrations of IGFBP-4, IGFBP-5 [11], and IGFBP-2 [9] are greater in the second-largest follicle. Proteolytic degradation of IGFBP-2 in bovine preovulatory follicles has also been demonstrated; the proteolytic activity was enhanced by IGFs [13]. In mares, as in cattle, IGFBP-2, -3, -4, and -5 have been reported in follicular fluid [10, 14]. In addition, equine follicular fluid contains 1–3 high molecular weight (90–135 kDa) IGFBPs, which are thought to be the nondissociated complexes of IGFBP-3 [10, 14]. Also, IGFBP- 2 and -5 have been shown to be produced by equine granulosa cells in culture [15]. In mares, proteolytic degradation of IGFBP-2 [8], IGFBP-4 [16], and IGFBP-5 [10] has been reported for the dominant follicle during the follicular phase. Only IGFBP-2 has been studied with reference to deviation; concentrations increased in the subordinate follicles at the beginning of diameter deviation [3]. On a temporal basis, these results indicate that the IGF system plays a role in the differential growth rates of the two largest follicles during deviation in mares, as well as in cattle.

Recently, the in vivo effects of IGF-1 on other follicular- fluid factors and on follicle destiny (dominant versus subordinate) were studied in mares [17, 18]. The second-largest follicle (F2) was treated by direct injection of IGF-1 at the expected beginning of deviation, and follicular-fluid samples were taken from F2 sequentially from each mare at various hours. High doses of IGF-1 (250 and 500 µg) increased the concentrations of activin-A, inhibin-A, and vascular endothelial growth factor (VEGF) 24 h after treatment; prevented the increase in IGFBP-2 and androstenedione that occurred in F2 controls; and stimulated growth and ovulation of F2. Decreasing intrafollicular IGF-1 doses of 250 µg, 25 µg, and 2.5 µg resulted in progressively decreasing free IGF-1 concentrations in F2 at 24 h [18]. Concentrations in F2 for the low dose (2.5 µg) were higher than in F2 controls and similar to endogenous concentrations in F1 controls. Correspondingly, concentrations of androstenedione were lower and concentrations of activin-A, inhibin-A, and VEGF were higher in F2 of the low-dose IGF-1 group than in F2 of the controls and were similar to concentrations in F1. These results indicated that IGF-1 at physiologic F1 concentrations stimulates secretion of activin-A, inhibin-A, and VEGF and prevents an increase in androstenedione. Some or all of these local IGF-1 effects are likely involved in the manifestation of follicle dominance in mares. These studies used IGF-1 treatment of F2 at the expected beginning of deviation. The opposite in vivo approach of experimentally decreasing concentrations of free IGF-1 in F1 to study deviation has not been used in any species.

The present experiment was done to test the hypothesis that the IGF system has a critical role in the changes in concentrations of follicular-fluid factors that lead to selection and growth of the dominant follicle in mares. The hypothesis was tested by injecting IGFBP-3 into the future dominant follicle at the beginning of deviation. It was expected that IGFBP-3 would bind the IGFs in the follicle, resulting in interference with the concentrations of follicular-fluid factors and with growth and development of the follicle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Ultrasonography

Animals were handled in accordance with the Guide for Care and Use of Agricultural Animals in Agricultural Research. The experiment was done during August–September (Northern Hemisphere), which is the later portion of the ovulatory season. The mares were mixed breeds of ponies, 5–15 yr of age, and weighed 250–480 kg. The feeding program and the equipment and techniques for transrectal and transvaginal ultrasound scanning and manipulation of ovaries have been described [19–21]. A new follicular wave was induced by ablation of all follicles >=6 mm 10 days postovulation as described [19]. Follicle ultrasound scanning was done every 24 h postablation. The number of follicles in the postablation follicular wave was reduced to two as validated and described [19], so that continuous follicle identity was not complicated by large numbers of follicles. The two retained follicles were designated F1 and F2 when F1 reached >=20 mm (expected beginning of deviation with F1 as the future dominant follicle; Day 0). Treatment of a designated follicle [20] and sampling follicular fluid at defined intervals [21] were done by transvaginal ultrasound targeting as described for mares [22]. Diameters of F1 and F2 were recorded every day until Day 4. Thereafter, only the fate (ovulation, regression) of the two follicles was determined.

Mares were randomized into a control group (no treatment or puncture of F1), vehicle group, and a BP (binding-protein) group (n = 8/group). On Day 0, a single intrafollicular treatment was given to F1 of mares in the vehicle and BP groups. The injection in the BP group consisted of 250 µg of recombinant human (rh) IGFBP-3 (Catalogue No. 01-204; Upstate Inc., Lake Placed, NY) in 70 µl of vehicle (100 mM sodium acetate, 210 mM sodium chloride in 10 mM acetic acid). The injection in the vehicle group consisted of 70 µl of vehicle only. A sample of follicular fluid (200 µl) was taken from F1 at Day 1 in all mares. End points were follicular-fluid concentrations of IGFBP-3, IGFBP-2, free IGF-1, estradiol, progesterone, androstenedione, activin-A, inhibin-A, and VEGF; diameters of F1 and F2; and identification of the ovulatory follicle. Blood samples (10 ml) were taken at Days 0, 1, 2, 3, and 4 from the jugular vein.

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 [23] and modified [3] in our laboratory. The intra-assay coefficient of variation (CV) was 8.6%, and the sensitivity was 3.2 ng/ml. Follicular-fluid samples were assayed for IGFBP-2, free IGF-1, estradiol, progesterone, androstenedione, activin- A, inhibin-A, and VEGF using commercially available kits that were modified and validated for use with equine follicular fluid in our laboratory [3, 18]. The intra-assay coefficient of variation and sensitivity, respectively, were as follows: IGFBP-2, 11.6%, 0.3 ng/ml; free IGF-1, 2.7%, 0.01 ng/ml; estradiol, 7.9%, 0.3 pg/ml; progesterone, 6.0%, 0.08 ng/ml; androstenedione, 5.3%, 0.03 ng/ml; activin-A, 4.2%, 0.1 ng/ml; inhibin-A, 5.1%, 3.8 pg/ml; and VEGF, 10.2%, 0.01 ng/ml.

Follicular fluid concentrations of IGFBP-3 were determined using a two-site immunoradiometric assay kit (Diagnostic Systems Laboratories, Inc., Webster, TX). The kit was developed for use with human serum or plasma and was adapted and validated for use with equine follicular fluid in our laboratory. The standards (2–100 ng/ml) were reconstituted with distilled water, and follicular-fluid samples were diluted with the protein- based zero standard supplied with the kit. Serial dilutions (0.5–8.0 µl) of a pool of equine follicular fluid resulted in a displacement curve that was similar to the standard curve. A working dilution of 1:20 was used for the experimental follicular-fluid samples because 2.5 µl resulted in percentage binding that was central to the range of the standard curve. According to the manufacturer, the cross reactivity with other IGFBPs and IGFs was not detectable at 1.0 µg/tube. The cross reactivity to equine IGFBP-3 is not known because of unavailability of equine IGFBP-3. However, there is about 80% homology in the amino acid sequence of IGFBP-3 across species [4]. The cross reactivity of rh-IGFBP-3 in the assay was 25%, and therefore the indicated concentrations of IGFBP-3 in the BP group (Table 1) are likely about 75% below actual values. All experimental samples were assayed in a single assay. The intra-assay coefficient of variation for quality control samples was 4.0% and the sensitivity was 0.1 ng/ml, as determined by 2 SD above the mean percentage binding of the zero standard.


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  		TABLE 1. Concentrations (mean ± SEM) of follicular-fluid factors at Day 1 after no treatment (control group), injection of vehicle (vehicle group), or injection of rh-IGFBP-3 (BP group) into F1 at the expected beginning of deviation (F1 >= 20 mm; Day 0)

Statistical Analyses

The follicular and hormonal data were challenged for extreme values with the Dixon outlier test [24] and for normality with the Kolmogorov- Smirnov test. When the normality test was significant (P < 0.05), data were transformed by either natural logarithm or square root. The concentrations of FSH were converted to percentage change from Day 0. Percentages were used because of a wide disparity among mares in FSH concentrations throughout the experimental period. Follicle diameter and FSH concentrations were analyzed to determine the effects of group, day, and the interaction using a SAS Mixed Procedure with a repeated statement to account for the autocorrelation between sequential measurements (8.2 version; SAS Institute Inc., Cary, NC). If a significant (P < 0.05) effect of group or a group-by-day 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 days within a group. Concentrations of follicular-fluid factors were analyzed by a one-way ANOVA; when a significant (P < 0.05) group effect was obtained, differences among groups were further examined by unpaired t-tests. Frequency of ovulation was analyzed by Chi-square test. A probability of P < 0.05 indicated that a difference was significant, and probabilities between P > 0.05 and P <= 0.1 indicated that a difference approached significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Two mares in each of the vehicle and BP groups were removed because of injection difficulties, resulting in 8, 6, and 6 mares in the control, vehicle, and BP groups, respectively. All mares ovulated from either F1 or F2 or both F1 and F2. The proportion of mares with an ovulation from F1 was different (P < 0.03) among groups (control, 7 of 8; vehicle, 4 of 6; BP, 1 of 6). The one BP-treated mare that ovulated from F1 formed a dominant (33 mm) anovulatory follicle from F2; the remaining mares in the BP group ovulated from F2. Both main effects (group, day) and the interaction of group by day was significant for diameter of both F1 and F2 (Fig. 1). The interaction for F1 reflected transiently reduced diameter in the vehicle group between Days 0 and 1 followed by a resurgence, reduced diameter in the control group between Days 1 and 2 followed by resurgence, and continued reduced diameter in the BP group after Day 0. At Day 1, the diameter in the control group was greater (P < 0.05) than for the other two groups, whereas by Day 3, diameter in the BP group was smaller (P < 0.05) than in the control group and approached being smaller (P < 0.07) than in the vehicle group. The interaction for F2 reflected continued diameter increase for the BP group after Day 0, compared with a reduced diameter for the other two groups. The diameter of F2 for the BP group was greater (P < 0.05) than for the vehicle group on Day 1 and was greater (P < 0.05) than for the control and vehicle groups within each day from Day 2 to Day 4. The difference between the control and vehicle groups was not significant on any day. The interaction for percentage change in circulating FSH concentrations from Day 0 was significant (Fig. 2). The positive percentage change between Days 0 and 1 in the BP group was different from the negative changes in the control (P < 0.01) and vehicle (P < 0.03) groups. For concentrations of follicular-fluid factors at Day 1, the means and probabilities for a difference among the three groups and the probabilities for a difference between any two groups are shown (Table 1). Concentrations in the BP group were lower than in the vehicle group for free IGF-1 (P < 0.02), estradiol (P < 0.02), activin-A (P < 0.03), and inhibin-A (P < 0.005) and higher (P < 0.04) for androstenedione. Lower concentrations of VEGF in the BP group than in the vehicle group approached significance (P < 0.1).



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  		FIG. 1. Mean (±SEM) diameter of F1 and F2 for 4 days before and after treatment of F1 at the expected beginning of deviation (Day 0). Mares of the BP group were given a single injection of rh-IGFBP-3. The interaction probability for group (G) by day (D) is shown for each follicle



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  		FIG. 2. Mean (±SEM) for the percentage change in FSH concentrations from treatment of F1 at the expected beginning of deviation (Day 0) to Days 1, 2, 3, or 4. Mares of the BP group were given a single injection of rh-IGFBP-3. The interaction probability for group (G) by day (D) is shown. The asterisk indicates a difference (P < 0.05) between groups


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The intrafollicular route for injection of substances for testing hypotheses involving follicles has been used in mares for treatment with eCG [20] and IGF-1 [17, 18]. A transient diameter decrease for the injected F1, presumably from loss of some follicular fluid, occurred during the day after the first follicle puncture in the vehicle group (vehicle injection) and control group (sampling). The growth rate after the transient delay seemed similar to the growth rate before Day 0. A transient reduction in growth rate during the first day after puncture of a follicle with subsequent recovery has been described and discussed [17, 21].

The IGFBP-3 is the predominant IGFBP in serum and follicular fluid and binds most of the IGFs present in follicular fluid [4, 6, 8]. The IGFBP-3 is present in a high molecular weight ternary complex of IGF and acid-labile subunit protein, with a greater half-life than either free IGFs or IGFBP-3 alone. Even though IGFBP-2, -4, and -5 may have more relevance to deviation, rh-IGFBP-3 was chosen for intrafollicular injection for the following reasons: 1) upon injection, IGFBP-3 immediately (within 2 min in rats [25]) becomes associated with the high molecular weight complex and thus was expected to remain in the follicle for a prolonged time; 2) at equal concentrations, IGFBP-3 had a 50% higher inhibition of the binding of IGF-1 to its receptor than IGFBP-2 [26]; 3) equine preovulatory follicular fluid has proteolytic activity that degrades IGFBP-2, -4, and -5, but not IGFBP-3 [8]; and 4) rh-IGFBP3 is readily available in milligram quantities. Intrafollicular injection of IGFBPs for in vivo study of the role of the IGF system in deviation has not been done previously. However, ovarian intrabursal administration of IGFBP-3 has been used to inhibit ovulation in gonadotropin-treated rats [27].

The 250-µg intrafollicular dose of rh-IGFBP-3 represents 10 times the follicular fluid content of IGFBP-3, as calculated on the basis of concentration of IGFBP-2 [3] and the ratio of IGFBP-2 to IGFBP-3 [14] near the beginning of deviation in mares. The concentration of IGFBP-3 in F1 at Day 1 following injection at Day 0 (BP group) was more than 100% higher than in the control and vehicle groups. In addition, the values in the BP group likely were about 75% below actual values because of the low cross reactivity of rh-IGFBP-3 in the assay. There were no differences among the three groups in the concentrations of IGFBP-2 in F1. The high concentrations of IGFBP-3 in F1 of the BP group may have interfered with the normal reciprocal relationship, wherein low concentrations of free IGF-1 are temporally associated with high concentrations of IGFBP- 2 [3, 18, 28].

The F1 follicular-fluid concentrations of free IGF-1 were reduced 90–93% in the BP group compared with control and vehicle groups. In addition to the reduction in free IGF- 1, concentrations of estradiol, inhibin-A, activin-A, and VEGF were lower and androstenedione was higher. The concentrations of all of these factors in F1 of the BP group seemed to approximate concentrations in F2 of the controls of a previous experiment [18]. The duration of these effects of IGFBP-3 beyond Day 1 is not known. However, the report of an association of injected IGFBP-3 with a high molecular weight complex [25] is compatible with a prolonged effect of the single treatment. In addition, a prolonged negative effect of IGFBP-3 treatment on the development of F1 was indicated by the smaller diameter of F1 in the BP group than in the vehicle group that approached significance and the reduced F1 ovulation rate in the BP group (17%), compared with the combined control and vehicle groups (80%). Thus, the results supported the hypothesis that the IGF system has a critical role in changes in concentrations of follicular-fluid factors that lead to selection and growth of the dominant follicle in mares.

The negative effect of IGFBP-3 treatment on F1 was associated with a continuation in the growth rate of F2 as it assumed the role of dominance, leading to ovulation of F2 in 5 of 6 mares. Thus, the IGFBP-3 treatment of F1 at the expected beginning of deviation was associated with conversion of F2 to dominant status, similar to the result of ablating F1 at this time [28, 29]. Similarly, both IGFBP- 3 treatment and ablation of F1 were associated with an increase in FSH during the first day. The transient FSH increase is consistent with the continued growth and development of dominance of F2 under both experimental conditions.

In regard to the lowered concentrations of estradiol on the day after an injection of IGFBP-3, in vitro studies with bovine granulosa cells have shown that IGFBP-3 inhibited the IGF-1 induced, but not the gonadotropin induced, increase in estradiol by 80%, and the inhibition was overcome by increasing the concentrations of IGF-1 [26]. Other in vitro studies in rats and humans have shown that IGFBP- 3 reduced the IGF-1-induced estradiol production from granulosa cells [for reviews, see 6, 26], further supporting our findings in this in vivo experiment in mares. Paradoxically, increasing the free IGF-1 concentrations of F2 at Day 0 by an intrafollicular injection of IGF-1 in either pharmacologic or physiologic doses did not increase or alter estradiol concentrations 1 day after treatment in mares [17, 18]. The reason for the apparent enigma in results of the IGFBP-3 versus IGF-1 treatments is not known. Nevertheless, the present findings do not appear to negate our previous interpretation [18] that stimulation of estradiol production from the granulosa cells by the endogenous IGF-1 in F1 does not play a critical role in the deviation mechanism in mares. In contrast, intrafollicular injection of IGF- 1 had a positive effect on estradiol production in cattle, as shown by an immediate increase in follicular-fluid estradiol concentrations [17].

The absence of a detected negative effect of IGFBP-3 on progesterone concentrations in F1 is consistent with the absence of a detected positive effect of injecting a physiologic dose of IGF-1 into F2 [18]. In pigs, rats, and cattle, IGFBP-3 inhibited the IGF-1-induced progesterone production in granulosa cells in vitro [for reviews, see 6, 26]. However, a role for progesterone during follicle deviation needs further study. In the present study, follicular-fluid samples were not taken until 24 h after treatment and a transient effect on progesterone changes could have occurred earlier.

The reason for the intermediate concentrations of androstenedione in the vehicle group is not known. The previous study [18] did not find an effect of intrafollicular saline injection on any follicular-fluid factor. In the present study, androstenedione concentrations in F1 were greater in the BP group than in the control and vehicle groups. This result is consistent with the reports of low concentration of androstenedione in the dominant follicle and high concentration in subordinate follicles in mares [3] and a decrease in androstenedione when IGF-1 was injected into F2 [17, 18]. In contrast with mares, concentrations of androstenedione in cattle are high in the dominant follicle and low in subordinate follicles [9], and an injection of IGF-1 into F2 stimulates androstenedione production [17]. In addition, in vitro studies with bovine thecal cells indicated that IGFBP- 3 inhibited the IGF-1 stimulation of androstenedione production [30]. The mechanisms accounting for the profound species difference in androstenedione concentrations in the two largest follicles will require specific study.

The lower concentrations of activin-A and inhibin-A in the BP group than in the control and vehicle groups are attributable to the IGFBP-3-induced reduction of free IGF- 1. This study, as well as a temporal study of activin-A and inhibin-A between F1 and F2 [3] and in vivo studies that found an increase in both factors in F2 after IGF-1 administration [17, 18], all support the conclusion that IGF-1 stimulates the production of activin-A and inhibin-A in F1. In addition, in vitro studies in cattle [31, 32] have shown that IGF-1 alone or in combination with FSH increased the secretion of activin-A and inhibin-A from granulosa cells. Activin-A and inhibin-A, as well as IGF-1, can be expected to increase the responsiveness of the developing dominant follicle to gonadotropins during follicle selection in mares [for a review, see 2].

Lower concentrations of VEGF in F1 of the BP group than in the vehicle group only approached significance and is a reservation in interpretation of the results. The lower concentrations of VEGF in F1 of the BP group than in the control group is consistent with an increase in VEGF in F2 after treatment with IGF-1 [18] and also with in vitro reports of stimulation of VEGF by IGF-1 in monkey and cattle granulosa cells [33, 34]. The in vivo results in mares [18] and in vitro and follicular-fluid assay results in other species [for a review, see 35] indicate that VEGF and other angiogenic factors may play a role in follicle selection or deviation. In mares, VEGF concentrations were higher in F1 than in F2 1 day after the expected beginning of deviation [18]. However, the position of angiogenic factors, such as VEGF, in the cascade of events leading to deviation is not known for any species. In this regard, diameter deviation is indicated morphologically in mares, not only by differential growth rate between the developing dominant and subordinate follicles, but also by an expanded anechoic ultrasonic layer within the wall of the dominant follicle; the echotextural changes distinguished the future dominant follicle from the future largest subordinate follicle about 1 day earlier than the beginning of diameter deviation [36]. The echogenic changes were attributed to increased vascularization; in other species, increased vascularization of the theca interna is associated with angiogenic factors, such as VEGF [for reviews, see 35, 37]. Thus, there are indirect indications that angiogenic factors are involved in deviation, but specific study is required with consideration of the production of such factors differentially among follicles beginning before diameter deviation.

In addition to the IGF-mediated actions of IGFBPs, recent studies have described IGF-independent actions of IGFBP-3 [for reviews, see 4, 5]. However, on the basis of in vivo studies with IGF-1 [17, 18], temporal studies on the IGF system in follicular fluid of mares [3], and in vitro studies on rat and bovine granulosa cells [for reviews, see 6, 26], we conclude that the results of this experiment were due to sequestration of endogenous IGF-1 by the injected IGFBP-3, resulting in a decrease in follicular-fluid factors and reduced follicle growth and loss of dominance.

In summary, IGFBP-3 was injected into the largest follicle (F1; BP group) at the expected beginning of deviation between F1 and F2 (Day 0). At Day 1, concentrations of follicular-fluid factors in the treated F1 were lower in free IGF-1, estradiol, activin-A, inhibin-A, and VEGF and higher in androstenedione than in controls. Dominance and ovulation occurred from primarily F2 in the BP group and from F1 in the control and vehicle groups. It is concluded that the IGF system has a critical role in the differential changes in concentrations of follicular-fluid factors between the future dominant and subordinate follicles and thereby in diameter deviation and follicle selection in mares.


   ACKNOWLEDGMENTS
 
The authors thank Pharmacia and Upjohn Company (Kalamazoo, MI) for a gift of lutalyse, Dr A.F. Parlow for equine FSH RIA reagents, and Luciano Silva and Susan Jensen for technical assistance.


   FOOTNOTES
 
1 Supported by the Eutheria Foundation, Cross Plains, WI. Project P4-OG- 03. E.L. and M.O. Gastal are on leave from the Departments of Veterinary and Animal Science, respectively, Federal University of Viçosa, Viçosa, Brazil. Back

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 Back

Received: 3 December 2003.

First decision: 18 December 2003.

Accepted: 7 January 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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