Abstract
During a follicular wave in mares, the two largest follicles (F1 and F2) begin to deviate in diameter when F1 is a mean of 22.5 mm. The intrafollicular effects of pregnancy-associated plasma protein-A (PAPP-A), IGF-I, activin-A and vascular endothelial growth factor (VEGF) on other follicular-fluid factors during deviation were studied. In four treated groups (n = 7/group), a single dose of one of the four factors was injected into F2 when F1 was ≥20.0 mm (expected beginning of deviation). In a control group (n = 7), F2 was injected with vehicle. One day after treatment, a sample of follicular fluid was taken from F1 and F2 of the control group and from F2 of the treated groups and was assayed for free IGF-I, oestradiol, androstenedione, activin-A, inhibin-A, follistatin and VEGF. In the control group, the means for all end points were significantly greater in F1 than in F2, except that concentrations of androstenedione were lower in F1 than in F2. The treatment effects for F2 were significant as follows: PAPP-A increased the concentrations of free IGF-I, inhibin-A, follistatin and VEGF and decreased the concentrations of androstenedione; IGF-I increased the concentration of inhibin-A and decreased the concentration of androstenedione; activin-A decreased the concentrations of follistatin and androstenedione and increased the diameter of F2; and VEGF increased the concentration of IGF-I and decreased the concentration of androstenedione. These results support the hypotheses that during deviation in mares PAPP-A increases the follicular-fluid concentrations of free IGF-I, follistatin responds to changes in follicular-fluid concentrations of activin-A, and VEGF affects the concentrations of other follicular-fluid factors.
Introduction
Follicle deviation in mares involves a change in the differences in diameters between the future dominant follicle and the future subordinate follicles; the rate of diameter increase continues at an approximately constant rate in the dominant follicle and begins to diminish in the subordinate follicles (see Ginther et al. 2004a for review). Deviation begins when the future dominant follicle is a mean of 22.5 mm. Differences between the two largest follicles in the concentrations of follicular-fluid factors (steroids, growth factors, cytokines) are associated with diameter deviation and apparently underlie a greater responsiveness to gonadotrophins for the developing dominant follicle than for the other follicles. In a temporality study, follicular-fluid concentrations of free insulin-like growth factor-I (IGF-I), inhibin-A, activin-A and oestradiol differentially increased in the future dominant follicle compared with the future largest subordinate follicle before the beginning of diameter deviation, and concentrations of IGF-binding protein (BP)-2 began to increase in the subordinate follicles at the beginning of deviation (Donadeu & Ginther 2002).
When the largest follicle (F1) was ablated at the expected beginning of deviation, free IGF-I began to increase in the second-largest follicle (F2) before the beginning of experimental diameter deviation between the two largest retained follicles (Ginther et al. 2002) and was the first detected change among the potential intrafollicular gonadotrophin-enabling factors. Concentrations of free IGF-I began to increase differentially in the largest retained follicle 12 h before the beginning of the experimental diameter deviation, whereas oestradiol, inhibin-A and activin-A did not begin to increase until about 24 h after the beginning of deviation. The role of IGF-I was also studied by injecting a pharmacological dose of recombinant human (rh) IGF-I into F2 at the expected beginning of deviation (Ginther et al. 2004b,c). The IGF-I-injected F2 continued to grow, became dominant, and ovulated more frequently than the saline-injected F2. When a physiological dose of IGF-I was injected into F2, the concentrations of free IGF-I, inhibin-A, activin-A and vascular endothelial growth factor (VEGF) were higher 24 h later in the treated F2 than in the control F2 and were similar to the natural concentrations in F1. Concentrations of oestradiol were not affected. The role of the IGF system in follicle selection was also studied by injecting rhBP-3 into F1 at the expected beginning of deviation, with the expectation that BP-3 would reduce the available IGF-I (Ginther et al. 2004d). Follicular fluid was sampled 24 h later. The BP-3-treated F1 was more likely to stop growing than the saline-treated F1, and F2 frequently became the dominant follicle in the BP-3-injected group. In addition, injection of BP-3 into F1 decreased the follicular-fluid concentrations of free IGF-I, activin-A, inhibin-A, oestradiol and VEGF within 24 h.
The results from the studies involving experimental deviation, treatment of F2 with rhIGF-I, and treatment of F1 with rhBP-3 have led to the conclusion (Ginther et al. 2004d) that the IGF-I system plays a critical initiating role in follicle deviation and the development of follicle dominance in mares. The functional relationships of IGFs and BPs involve specific proteases that degrade the BPs and thus increase the bioavailability of IGF-I in the follicles (see Spicer 2004 for review). Pregnancy-associated plasma protein A (PAPP-A) is a protease that is involved in proteolysis of BPs in bovine, porcine and equine dominant follicles (Mazerbourg et al. 2001, Bridges et al. 2002, Monget et al. 2003, Rivera & Fortune 2003). In mares, a temporal or functional relationship of PAPP-A to follicle selection has not been reported. Apparently intrafollicular increases in oestradiol, activin-A and inhibin-A in the future dominant follicle are not a prerequisite to the initiation of diameter deviation in mares, even though these three factors increase differentially in the future dominant follicle before the beginning of natural deviation. These factors may, however, play a role in subsequent development of the dominant follicle.
Follistatin is a protein hormone present in follicular fluid of cattle (Austin et al. 2001) and women (Schneyer et al. 2000) and has activin-binding and to a lesser extent inhibin-binding activity (Knight 1996). Follistatin expression is higher in dominant follicles than in subordinate follicles in cattle (Singh & Adams 1998). In addition, immunization against follistatin is associated with an increase in follicle activity (Singh et al. 1999), and a reciprocal relationship occurs in follicular-fluid concentrations of activin and follistatin, apparently encompassing deviation (Austin et al. 2001). However, follistatin concentrations and temporal relationships during follicle deviation have not been reported for mares.
Concentrations of VEGF are higher in the dominant follicle than in the subordinate follicle of mares 1 day after the expected beginning of diameter deviation (Ginther et al. 2004c), but the earlier temporal relationships before deviation are not known. However, the stimulation of VEGF with a physiological dose of IGF-I (Ginther et al. 2004c) indicates that VEGF is a candidate for a role in development of the follicles during diameter deviation. In this regard, VEGF has been shown to stimulate mitosis of endothelial cells and to increase vascular permeability and angiogenesis in other species (see Redmer & Reynolds 1996, Martinez-Chequer et al. 2003 for reviews). However, direct or indirect effects of VEGF on other follicular-fluid factors apparently have not been studied in any species.
Although the results of the in vivo studies in mares have placed the IGF-I system in a primary position in an apparent cascade of intrafollicular events underlying follicle deviation, similar in vivo studies on the interrelationships of other follicular-fluid factors to one another have not been done in any species. Therefore, the present study was done to test three hypotheses in vivo in mares: (1) PAPP-A increases the follicular-fluid concentrations of free IGF-I, (2) intrafollicular follistatin responds to changes in the concentrations of activin-A, and (3) intrafollicular VEGF has an effect on concentrations of other follicular-fluid factors. In addition, the previously reported effects of IGF-I on activin-A, inhibin-A, androstenedione, oestradiol and VEGF were considered.
Materials and Methods
Mares and ultrasonography
Mares were handled in accordance with the Guide for Care and Use of Agricultural Animals in Agricultural Research (United States Department of Agriculture). A total of 53 mares was used in two experiments during the first portion of an ovulatory season that was induced with an artificially extended photoperiod, as described (Ginther 1992). The experiment was begun in March after the occurrence of two ovulatory periods. The mares were mixed breeds of ponies, 8–17 years of age, and weighed 300–450 kg. Each mare was used only once. The feeding programme and the equipment and techniques for transrectal and transvaginal ultrasound scanning and manipulation of ovaries have been described (Ginther 1995, Gastal et al. 1997). A new follicular wave was induced by ablation of all follicles ≥6 mm 10 days postovulation (Gastal et al. 1997). Ultrasound scanning of follicles was done every 24 h postablation in both experiments. A two-follicle model was prepared in the postablation follicular wave as described (Gastal et al. 1997), 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 F1 (largest) and F2 when F1 reached ≥20 mm (expected beginning of deviation; Day 0; Gastal et al. 1999a). Diameters of F1 and F2 were taken on Days 0 and 1, using the mean of vertical and horizontal measurements. Mares were not used if F2 was >4.0 mm smaller than F1 on Day 0, based on the report that an exaggerated difference in diameter between F1 and F2 reduced the likelihood that F2 would become dominant after F1 was ablated (Gastal et al. 1999b). A difference in diameter of >4.0 mm between F1 and F2 occurred in three mares. Transvaginal ultrasound targeting was used as described for intrafollicular treatment (Gastal et al. 1995, Ginther 1995) and for sampling follicular fluid (Gastal et al. 1999a). A designated intrafollicular treatment of F2 was done on Day 0, and a 200 μl sample of follicular fluid was taken on Day 1.
Experiment 1
The vehicle for intrafollicular injection into F2 was 50 μl PBS containing 0.1% BSA (RIA grade). Mares were randomized into one control group (saline vehicle injected into F2) and four treated groups (n = 7/group). The treated groups received an intrafollicular injection into F2 of one of the following: IGF-I (2.5 μg rhIGF-I; Genetech, Inc., San Francisco, CA, USA), PAPP-A (60 μg human (h) PAPP-A; Advanced Immunochemical, Inc., Long Beach, CA, USA), activin-A (20 μg rh-activin-A; R & D Systems, Inc., Minneapolis, MN, USA), and VEGF (2.5 μg rhVEGF; Bio-vision, Mountain View, CA, USA). The physiological dose of rhIGF-I (2.5 μg) was established in a previous dose–response study (Ginther et al. 2004c). Information on a physiological dose for the other injected factors (PAPP-A, activin-A and VEGF) is not available. Therefore, a calculated pharmacological dose was given. The dose for activin-A and VEGF was calculated as 10 times the expected content of the factor in F1 at the expected beginning of deviation in mares (Donadeu & Ginther 2002, Ginther et al. 2004c), similar to the approach used in the earlier studies with rhIGF-I (Ginther et al. 2004b,c). For PAPP-A, the dose was calculated on the basis of follicular-fluid concentration of PAPP-A in women (Stanger et al. 1985). On the day of sampling of follicular fluid (Day 1), both F1 and F2 were sampled in the control group and only F2 was sampled in the four treated groups. Samples were taken 24 h after treatment, based on the hour with the most consistent results in a previous study (Ginther et al. 2004c). End points were follicular-fluid concentrations of free IGF-I, BP-2, oestradiol, androstenedione, activin-A, inhibin-A, follistatin and VEGF. Follicle diameter of F2 also was considered, and a change in follicle diameter was calculated by subtracting the diameter on Day 0 from the diameter on Day 1. The change in diameter was also considered in order to account for variation in diameter on Day 0.
Experiment 2
This experiment was done to further explore two unexpected results of PAPP-A treatment of F2 in experiment 1: (i) a tendency for an increase in oestradiol concentrations and (ii) no confirmation of a previous report (Ginther et al. 2004c) that rhIGF-I increased the concentrations of activin-A. Mares were randomized into a control group and a PAPP-A group (n = 9/group). Follicles were treated and sampled as described for experiment 1. The end points were follicular-fluid concentrations of oestradiol and activin-A.
Hormone assays
Follicular-fluid samples were centrifuged (500 g for 10 min), decanted, and stored(−20 °C) until assay. Follicular-fluid samples were assayed for free IGF-I, BP-2, oestradiol, androstenedione, activin-A, inhibin-A and VEGF, using commercially available kits that have been modified and validated for use with equine follicular fluid in our laboratory (Ginther et al. 2004c,d). Intra-assay CV values for these hormones in both experiments were <9.0%. The sensitivities in experiment 1 were as follows: free IGF-I, 0.01 ng/ml; BP-2, 1.2 ng/ml; oestradiol, 2.3 pg/ml; androstenedione, 0.01 ng/ml; activin-A, 0.3 ng/ml; inhibin-A, 1.6 pg/ml; and VEGF, 0.6 ng/ml. In experiment 2, the sensitivities for oestradiol, free IGF-I and activin-A were 0.3 pg/ml, 0.01 ng/ml and 0.8 ng/ml respectively.
Concentrations of follistatin in the follicular fluid were determined with a sandwich ELISA kit (Catalog No. DNF00; R & D Systems). The kit was developed for use with human serum and follicular fluid and was adapted and validated for use with equine follicular fluid in our laboratory. The standards (16–0.5 ng/ml) were prepared by serial dilution after reconstitution of the supplied standard with assay diluent. The assay diluent also served as the zero standard. The colour intensity of the enzyme substrate was directly proportional to the concentration of follistatin. Serial dilutions (0.31–10 μl) of a pool of equine follicular fluid in a total volume of 100 μl of assay diluent resulted in a displacement curve that was similar to the standard curve. A working dilution of 1:40 was used for assaying the follicular-fluid samples; 2.5 μl of pooled follicular fluid resulted in an optical density (OD) that was central to the range of the standard curve. According to the manufacturer, there was no significant cross-reactivity of the assay with activin-A, inhibin-A and -B and other cytokines. The intra-assay CV for quality control samples was 1.4%, and the sensitivity was 0.1 ng/ml as determined by 2 s.d. above the mean OD of the zero standard.
Statistical analyses
The data for follicular factors and colour-Doppler end points were challenged for extreme values with Dixon’s outlier test (Kanji 1993) and outliers were removed. Normality was tested with the Kolmogorov–Smirnov test; when the normality test was significant (P < 0.05), data were transformed by either natural logarithm or square root. End points were analysed with a one-way ANOVA. A significant (P < 0.05) effect was further analysed by Duncan’s multiple range test to locate differences among the six means (F1 and F2 of the controls and F2 for each of the four treated groups). 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
Experiment 1
The concentrations of follicular-fluid factors on Day 1 in F1 and F2 of the control group and in F2 of the groups treated with rhIGF-I, hPAPP-A, rh-activin-A or rhVEGF and results of the statistical analyses are shown (Fig. 1). Means for each end point were significantly different among groups. Means in the control group were significantly different between F1 and F2 for all end points. The IGF-I treatment had a positive effect on the F2 concentrations of inhibin-A and follistatin and a negative effect on androstenedione; however, the effects on follistatin only approached significance. PAPP-A had a positive effect on free IGF-I, inhibin-A, follistatin and VEGF and a negative effect on BP-2 and androstenedione; a positive effect on oestradiol approached significance. Activin-A had a negative effect on BP-2, androstenedione and follistatin. VEGF had a positive effect on free IGF-I and a negative effect on BP-2 and androstenedione. The diameter of F2 on Day 1 and the increase in diameter between Days 0 and 1 were greater in the activin-A-treated group than in the control group. A summary of the effects of the four injected factors on other follicular-fluid factors is depicted (Fig. 2).
Experiment 2
The concentrations of oestradiol in the follicular fluid of F2 did not differ significantly between the controls (638 ± 109 ng/ml) and PAPP-A-treated (692 ± 191 ng/ml) groups. Treatment with PAPP-A did not alter the activin-A concentrations in F2 (data not shown).
Discussion
Although follicles were not examined after Day 1 in the present study, the intrafollicular injection and sampling procedures did not interfere with ovulation in a previous study (Ginther et al. 2004c), as indicated by ovulation of most F2 follicles treated with rhIGF-I. A transient decrease in diameter on the day after intrafollicular treatment or sampling did not appear to alter concentrations of follicular-fluid factors or subsequent expansion of the follicle (Gastal et al. 1999a, Ginther 2004b). Previous studies (Ginther et al. 2004b,c) indicated that Day 1 was an optimal time for detecting a response to treatment of F2 with rhIGF-I at Day 0. In both a previous study (Ginther et al. 2004c) and this study, 2.5 μg rhIGF-I can be considered a physiological dose in that it increased the concentration of free IGF-I in F2 to a level similar to the natural concentrations in F1. Reservation is indicated for the results of the PAPP-A, activin-A and VEGF treatments because a pharmacological dose was used. The basis for using a pharmacological dose in initial studies has been discussed (Ginther et al. 2004b). In this regard, intrafollicular effects may persist when a pharmacological but not a physiological dose is injected; injection of a pharmacological dose of IGF-I resulted in ovulation from F2 and injection of BP-3 resulted in regression of F1 (Ginther et al. 2004c,d).
The positive response of free IGF-I to treatment of F2 with PAPP-A supported Hypothesis 1 that PAPP-A increases the concentrations of free IGF-I during diameter deviation in mares. The free IGF-I concentrations were higher for F2 in the PAPP-A treated group than for F2 in the control group, but was not different from the concentrations in F1 of the control group. Based on in vitro studies (Mazerbourg et al. 2003), PAPP-A increased the concentrations of free IGF-I near the expected time of deviation in mares by releasing IGF-I from BPs. However, studies are needed on the temporal relationships between concentrations of free IGF-I and PAPP-A in association with deviation in untreated mares. The absence of a detected effect of rhIGF-I on BP-2 is consistent with the previous study (Ginther et al. 2004c). However, PAPP-A decreased the concentrations of BP-2 in F2 to a level similar to the concentrations in F1. This result is consistent with the degradation of BP-2 by PAPP-A in vitro (Mazerbourg et al. 2003, Gérard et al. 2004). The effects of PAPP-A on other follicular-fluid factors were probably exerted through the release of free IGF-I into the follicular fluid.
The positive effect of PAPP-A on oestradiol approached significance in experiment 1 and was an incentive for experiment 2; the tendency was not supported in experiment 2. Thus, the previous finding (Ginther et al. 2004c) that oestradiol did not respond immediately to IGF-I was confirmed. This cannot be attributed to lack of androstenedione; oestradiol does not increase in the second-largest follicle during natural deviation even though there is a large increase in androstenedione (Donadeu & Ginther 2002). Androstenedione is higher in F1 in cattle, but higher in F2 in mares; this species difference has been reviewed (Ginther et al. 2004b). Apparently an oestradiol increase is not an integral component of the deviation cascade in mares, despite its reported (Donadeu & Ginther 2002) simultaneous increase with free IGF-I in F1 during natural deviation. In contrast, in heifers the results of temporal (Beg et al. 2001, 2002) and functional (Beg et al. 2003, Ginther et al. 2004b) studies are consistent with an intrafollicular role of oestradiol in initiation of deviation, and oestradiol secretion responds immediately to in vivo IGF-I treatment (Ginther et al. 2004b).
Another profound difference between heifers and mares during follicle deviation is an increase in androstenedione in the developing dominant follicle in cattle (Beg et al. 2001, 2002), contrasting with an increase in the subordinate follicles in mares (Donadeu & Ginther 2002). Similarly, intrafollicular treatment of F2 with rhIGF-I stimulated an increase in androstenedione in F2 in cattle (Ginther et al. 2004b), and in the previous studies (Ginther et al. 2004b,c) and the present experiment 1, IGF-I prevented the increase in androstenedione that would have occurred in F2 in mares. Furthermore, in the present study in mares, treatment of F2 with PAPP-A, activin-A and VEGF also prevented the increase in androstenedione. Apparently, the androstenedione increase in F2 during deviation in mares occurs when several intrafollicular factors are at low concentrations, but clarifying the cause-and-effect relationships will require focused study.
Activin-A, inhibin-A and follistatin will be considered together, given that the intrafollicular ratios of activin:follistatin and activin:inhibin are potential regulators of folliculogenesis (Glister et al. 2001). However, in the present experiments all three factors were assayed but only activin-A was injected; we were unable to locate a source of inhibin-A. The positive effect of IGF-I and PAPP-A on inhibin-A agrees with the previous results of IGF-I intrafollicular administration (Ginther et al. 2004b,c). A difference between the present and previous studies was the absence of an effect of IGF-I and PAPP-A on activin-A concentrations in the present experiment 1 vs a positive effect of IGF-I in the previous experiment (Ginther et al. 2004c). The disagreement on the activin-A response was another incentive for experiment 2, which also did not indicate an effect of PAPP-A on activin-A. The reason for the lack of agreement with the reported study is not known; the source of the injected rhIGF-I, the activin-A assay kits, and the dose of rhIGF-I were the same for all experiments. Regardless, we can no longer support the previous conclusion (Ginther et al. 2004c) that the predeviation increase in activin-A that occurs concurrently with natural increases in free IGF-I (Donadeu & Ginther 2002) is attributable to IGF-I.
Follistatin has activin- and to a lesser extent inhibin-binding activity (Knight 1996). In the present study in mares, follistatin concentrations were reduced approximately 22-fold in the activin-treated group, supporting Hypothesis 2. An effect of PAPP-A but not rhIGF-I treatment on follistatin is attributable to a physiological dose of IGF-I and a pharmacological dose of PAPP-A that stimulated a greater than physiological concentration of IGF-I. Apparently most of the follistatin became bound to activin, and the assay detects only the free form of follistatin. Many in vitro studies on the relationships of activin-A, follistatin and inhibin-A and other follicular factors have been done in cattle and other species (Glister et al. 2001, 2003), but not in mares. The effect of PAPP-A treatment on follistatin was positive and approached being positive for IGF-I treatment. The reason for this result is not known; the response of follistatin to intrafollicular administration of other factors and the temporal relationship to deviation have not been examined previously. However, IGF-I increased follistatin secretion from bovine granulosa cells in vitro (Glister et al. 2001). Considering the profound differences between mares and cattle in the temporal relationships among follicular-fluid factors during natural and experimental deviation and in response to in vivo IGF-I treatment (see Ginther et al. 2004a for review), the non-equine in vitro studies could be misleading; specific studies are needed using equine theca and granulosa cells.
An unexpected finding was the reduction in BP-2 by activin-A treatment, as well as by PAPP-A and VEGF. In contrast, a previous report (Cataldo et al. 1998) indicated an increase in BP-2 from human granulosa cells when exposed to activin; however, the cells were from women who were treated with human chorionic gonadotrophin. The reason for an activin- and VEGF-induced reduction in BP-2 in the present study is not known and needs further study. Another unexpected finding was the positive effect of activin-A, but not the other factors, on the diameter of F2 at Day 1. The diameter measurements considered only the antrum and a possible oedematous effect on the wall was not evaluated. An effect of activin-A on diameter requires confirmation. The greater F2 diameter occurred in the absence of any positive effects of activin-A treatment on IGF-I or other factors. In this regard, in vitro studies have indicated that activin induces proliferation and differentiation of rat and human granulosa cells (Knight & Glister 2001). In addition, targeted deletion of α-subunit gene in mice, which results in overproduction of activin, is associated with uncontrolled proliferation of granulosa cells (Matzuk et al. 1992). Conversely, in knock-out mice lacking activin IIB receptor, follicle development was arrested at an early stage, consistent with a key role for activin in granulosa cell proliferation and differentiation (Nishimori & Matzuk 1996). Thus, although unexpected, a positive activin-A effect on follicle diameter in mares is consistent with reported studies in humans, mice and rats.
The present finding that PAPP-A, presumably through IGF-I, stimulated the production of VEGF in mares agrees with the reported effects of a physiological dose of IGF-I in a similar study (Ginther et al. 2004c). However, the effect of IGF-I treatment on VEGF concentrations only approached significance in the present study. Positive in vivo results, however, are consistent with the reports that IGF-I increases secretion of VEGF in cultures of granulosa cells in cattle (Schams et al. 2001) and monkeys (Martinez-Chequer et al. 2003).
Treatment of F2 with VEGF stimulated production of free IGF-I, although the effect was slight, and had a negative effect on androstenedione, supporting Hypothesis 3; therefore, VEGF may have a direct or indirect role in the interrelationships among follicular-fluid factors during deviation. The increase in IGF-I and a decrease in androstenedione from treatment with VEGF presumably contributed to the health of F2; IGF-I increases and androstenedione decreases in the growing dominant follicle in mares (Donadeu & Ginther 2002). In this regard, VEGF ligand and its receptor mRNA are coexpressed in bovine ovarian granulosa cells and expression of both the ligand and the receptor increased in healthy follicles, and VEGF treatment enhanced the survival of granulosa cells in vitro (Greenaway et al. 2004). In prepubertal gilts, treatment with direct injection of VEGF gene fragments into ovaries had a follicle-stimulating effect (Shimizu et al. 2003). Conversely, systemic treatment with an anti-VEGF decreased follicle activity in monkeys (Wulff et al. 2002), and VEGF expression has been linked to angiogenesis in the equine corpus luteum (Al-zi’abi et al. 2003). Unlike activin-A, however, VEGF did not stimulate follicle growth in mares in experiment 1. The role of VEGF in follicle deviation or selection has not been studied specifically in any species. However, follicular-fluid concentrations of VEGF have been shown to increase in cattle (Berisha et al. 2000) and pigs (Barboni et al. 2000) as follicle diameter increases and VEGF is higher in the follicular fluid of F1 than in F2 1 day after the expected beginning of deviation in mares (Ginther et al. 2004c). The latter finding was confirmed in the present experiment 1. On a temporal basis, VEGF could play a role in follicle deviation or selection, but further studies are required that include groups from before the beginning of deviation.
In summary, a single dose of rhIGF-I, hPAPP-A, rh-activin-A or rhVEGF was injected into the second-largest follicle (F2) in mares at the expected beginning of deviation, and the follicular fluid was sampled 1 day later. Results supported the hypothesis that PAPP-A increases the concentrations of free IGF-I during deviation in mares. Activin-A decreased the concentrations of follistatin, demonstrating an intrafollicular relationship between these two factors. Treatment with VEGF slightly increased the concentrations of IGF-I and reduced the concentrations of androstenedione, indicating that VEGF affects other follicular-fluid factors during deviation either directly or indirectly.
Mean±s.e.m. concentrations of follicular-fluid factors and other follicle end points for the largest follicle (F1) and the second-largest follicle (F2) of the control group and F2 of the treated groups on Day 1 after treatment of F2 at the expected beginning of deviation (Day 0; n = 7/group). Vascularity refers to percentage of follicle wall with colour-Doppler signals. Diameter change refers to the diameter at Day 0 subtracted from the diameter at Day 1. Differences among groups were significant for each end point (P < 0.003 to P < 0.0001). abc = means within an end point without a common lower-case letter are different (P < 0.05). Experiment 1.
Citation: Reproduction 129, 4; 10.1530/rep.1.00555
Schematic summary of the positive (+), negative (−) or no effect (=) of an injected factor (circled) on the concentrations of other factors. The arrow indicates the direction of an effect. PAPP-A effects on other factors presumably are exerted through IGF-I. The positive indicated effects of PAPP-A on follistatin and VEGF were significant, but the corresponding effects of IGF-I only approached significance. Experiment 1.
Citation: Reproduction 129, 4; 10.1530/rep.1.00555
We thank Susan Jensen and Matt Utt for technical assistance. This work was supported by the Eutheria Foundation (Cross Plains, WI, USA); projects P2-OG-04 and P6-OG-04. The authors thank Genetech, Inc. (San Francisco, CA, USA) for a gift of recombinant human IGF-I, and Pharmacia & Upjohn Company (Kalamazoo, MI, USA) for a gift of Lutalyse. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
Al-zi’abi MO, Watson ED & Fraser HM2003 Angiogenesis and vascular endothelial growth factor expression in the equine corpus luteum. Reproduction 125 259–270.
Austin EJ, Mihm M, Evans ACO, Knight PG, Ireland JLH & Ireland JJet al.2001 Alterations in intrafollicular regulatory factors and apoptosis during selection of follicles in the first follicular wave of the bovine estrous cycle. Biology of Reproduction 64 839–848.
Barboni B, Turriani M, Galeati G, Spinaci M, Bacci ML & Forni Met al.2000 Vascular endothelial growth factor production in growing pig antral follicles. Biology of Reproduction 63 858–864.
Beg MA, Bergfelt DR, Kot K, Wiltbank MC & Ginther OJ2001 Follicular-fluid factors and granulosa-cell gene expression associated with follicle deviation in cattle. Biology of Reproduction 64 432–441.
Beg MA, Bergfelt DR, Kot K & Ginther OJ2002 Follicle selection in cattle: dynamics of follicular-fluid factors during development of follicle dominance. Biology of Reproduction 66 120–126.
Beg MA, Meira C, Bergfelt DR & Ginther OJ2003 Role of oestradiol in growth of follicles and follicle deviation in heifers. Reproduction 125 847–854.
Berisha B, Schams D, Kosmann M & Amselgruber W2000 Expression and localisation of vascular endothelial growth factor and basic fibroblast growth factor during the final growth of bovine ovarian follicles. Journal of Endocrinology 167 371–382.
Bridges TS, Davidson TR, Chamberlain CS, Geisert RD & Spicer LJ2002 Changes in follicular fluid steroids, insulin-like growth factors (IGF) and IGF-binding protein concentration, and proteolytic activity during equine follicular development. Journal of Animal Science 80 179–190.
Cataldo NA, Fujimoto VY & Jaffe RB1998 Interferon-γ and activin A promote insulin-like growth factor-binding protein-2 and -4 accumulation by human luteinizing granulosa cells, and interferon-γ promotes their apoptosis. Journal of Clinical Endocrinology and Metabolism 83 179–186.
Donadeu FX & Ginther OJ2002 Changes in concentrations of follicular-fluid factors during follicle selection in mares. Biology of Reproduction 66 1111–1118.
Gastal EL, Kot K & Ginther OJ1995 Ultrasound-guided intrafollicular treatment in mares. Theriogenology 44 1027–1037.
Gastal EL, Gastal MO, Bergfelt DR & Ginther OJ1997 Role of diameter differences among follicles in selection of a future dominant follicle in mares. Biology of Reproduction 57 1320–1327.
Gastal EL, Gastal MO, Wiltbank MC & Ginther OJ1999a Follicle deviation and intrafollicular and systemic estradiol concentrations in mares. Biology of Reproduction 61 31–39.
Gastal EL, Gastal MO & Ginther OJ1999b Experimental assumption of dominance by a smaller follicle and associated hormonal changes in mares. Biology of Reproduction 61 724–730.
Gérard N, Delpuech T, Oxvig C, Overgaard MT & Monget P2004 Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles. Journal of Endocrinology 182 457–466.
Ginther OJ1992 Reproductive Biology of the Mare, Basic and Applied Aspects, edn 2, pp 173–290. Cross Plains, WI, USA: Equiservices Publishing.
Ginther OJ1995 Ultrasonic Imaging and Animal Reproduction: Book 2, Horses, pp 44–72. Cross Plains, WI, USA: Equiservices Publishing.
Ginther OJ, Meira C, Beg MA & Bergfelt DR2002 Follicle and endocrine dynamics during experimental follicle deviation in mares. Biology of Reproduction 67 862–867.
Ginther OJ, Beg MA, Gastal MO & Gastal EL2004a Follicle dynamics and selection in mares. Animal Reproduction 1 46–64.
Ginther OJ, Bergfelt DR, Beg MA, Meira C & Kot K2004b In vivo effects of an intrafollicular injection of insulin-like growth factor 1 on the mechanism of follicle deviation in heifers and mares. Biology of Reproduction 70 99–105.
Ginther OJ, Gastal EL, Gastal MO, Checura CM & Beg MA2004c Dose–response study of intrafollicular injection of insulin-like growth factor-1 on follicular-fluid factors and follicle dominance in mares. Biology of Reproduction 70 1063–1069.
Ginther OJ, Gastal EL, Gastal MO & Beg MA2004d Critical role of insulin-like growth factor system in follicle selection and dominance in mares. Biology of Reproduction 70 1374–1379.
Glister C, Tannetta DS, Groome NP & Knight PG2001 Interactions between follicle-stimulating hormone and growth factors in modulating secretion of steroids and inhibin-related peptides by non-luteinized bovine granulosa cells. Biology of Reproduction 65 1020–1028.
Glister C, Groome NP & Knight PG2003 Oocyte-mediated suppression of follicle-stimulating hormone- and insulin-like growth factor-induced secretion of steroids and inhibin-related proteins by bovine granulosa cells in vitro: possible role of transforming growth factor α. Biology of Reproduction 68 758–765.
Greenaway J, Connor K, Pedersen HG, Coomber BL, Lamarre J & Petrik J2004 Vascular endothelial growth factor and its receptor, Flk-1/KDR, are cytoprotective in the extravascular compartment of the ovarian follicle. Endocrinology 145 2896–2905.
Kanji GK1993 Statistical Tests. London: Sage Publications.
Knight PG1996 Roles of inhibins, activins, and follistatin in the female reproductive system. Frontiers in Neuroendocrinology 17 476–509.
Knight PG & Glister C2001 Potential local regulatory functions of inhibins, activins and follistatin in the ovary. Reproduction 121 503–512.
Martinez-Chequer JC, Stouffer RL, Hazzard TM, Patton PE & Molskness TA2003 Insulin-like growth factors-1 and -2, but not hypoxia, synergize with gonadotropin hormone to promote vascular endothelial growth factor-A secretion by monkey granulosa cells from preovulatory follicles. Biology of Reproduction 68 1112–1118.
Matzuk M, Ginegold M, Su J, Hsuch A & Bradley A1992 α inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature 360 313–319.
Mazerbourg S, Overgaard MT, Oxvig C, Christiansen M, Conover CA & Laurendeau Iet al.2001 Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding protein-4 proteolytic degradation and mRNA expression during follicular development. Endocrinology 142 5243–5253.
Mazerbourg S, Bondy CA, Zhou J & Monget P2003 The insulin-like growth factor system: a key determinant role in the growth and selection of ovarian follicles? A comparative species study. Reproduction in Domestic Animals 38 247–258.
Monget P, Mazerbourg S, Delpuech T, Maurel M-C, Manière S & Zapf Jet al.2003 Pregnancy-associated plasma protein-A is involved in insulin-like growth factor binding protein-2 (IGFBP-2) proteolytic degradation in bovine and porcine preovulatory follicles: identification of cleavage site and characterization of IGFBP-2 degradation. Biology of Reproduction 68 77–86.
Nishimori K & Matzuk MM1996 Transgenic mice in the analysis of reproductive development and function. Reviews in Reproduction 1 203–212.
Redmer DA & Reynolds LP1996 Angiogenesis in the ovary. Reviews in Reproduction 1 182–192.
Rivera GM & Fortune JE2003 Selection of the dominant follicle and insulin-like growth factor (IGF)-binding proteins: evidence that pregnancy-associated plasma protein A contributes to proteolysis of IGF-binding protein 5 in bovine follicular fluid. Endocrinology 144 437–446.
Schams D, Kosmann B, Berisha B, Amselgruber WM & Miyamoto A2001 Stimulatory and synergistic effects of luteinising hormone and insulin like growth factor 1 on the secretion of vascular endothelial growth factor and progesterone of cultured bovine granulosa cells. Experimental and Clinical Endocrinology of Diabetes 109 155–162.
Schneyer AL, Fujiwara T, Fox J, Welt CK, Adams J & Messerlian GMet al.2000 Dynamic changes in the intrafollicular inhibin/activin/follistatin axis during human follicular development: relationship to circulating hormone concentrations. Journal of Clinical Endocrinology and Metabolism 85 3319–3330.
Shimizu T, Jiang J-Y, Iijima K, Miyabayashi K, Ogawa Y & Sasada Het al.2003 Induction of follicular development by direct single injection of vascular endothelial growth factor gene fragments into the ovary of miniature gilts. Biology of Reproduction 69 1388–1393.
Singh J & Adams GP1998 Immunohistochemical distribution of follistatin in dominant and subordinate follicles and the corpus luteum of cattle. Biology of Reproduction 59 561–570.
Singh J, Brogliatti GM, Christensen CR & Adams GP1999 Active immunization against follistatin and its effect on FSH, follicle development and superovulation in heifers. Theriogenology 52 49–66.
Spicer LJ2004 Proteolytic degradation of insulin-like growth factor binding proteins by ovarian follicles: a control mechanism for selection of dominant follicles. Biology of Reproduction 70 1223–1230.
Stanger JD, Yovich JL, Grudzinskas JG & Bolton AE1985 Relation between pregnancy-associated plasma protein A (PAPP-A) in human peri-ovulatory follicle fluid and the collection and fertilization of human ova in vitro. British Journal of Obstetrics and Gynaecology 92 786–792.
Wulff C, Wilson H, Wiegand SJ, Rudge JS & Fraser HM2002 Prevention of thecal angiogenesis, antral follicular growth, and ovulation in the primate by treatment with vascular endothelial growth factor trap R1R2. Endocrinology 143 2797–2807.
