Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print April 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028498
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/2/637    most recent
biolreprod.104.028498v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Acosta, T.J.
Right arrow Articles by Ginther, O.J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Acosta, T.J.
Right arrow Articles by Ginther, O.J.
Agricola
Right arrow Articles by Acosta, T.J.
Right arrow Articles by Ginther, O.J.
BIOLOGY OF REPRODUCTION 71, 637–642 (2004)
DOI: 10.1095/biolreprod.104.028498
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Aberrant Blood Flow Area and Plasma Gonadotropin Concentrations During the Development of Dominant-Sized Transitional Anovulatory Follicles in Mares1

T.J. Acosta3, M.A. Beg, and O.J. Ginther2

Eutheria Foundation, Cross Plains, Wisconsin 53528


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Color Doppler transrectal ultrasound was used to evaluate blood flow area in the wall of dominant anovulatory follicles versus ovulatory follicles in mares during the transition between anovulatory and ovulatory seasons. Daily examinations were done in 11 control mares toward the end of the anovulatory season. In 13 separate mares, follicular fluid was collected from 30-mm follicles, and blood flow areas from control mares were used as a basis for designating the sampled follicle as either anovulatory or ovulatory. Blood flow area in the controls ranged from 0.18 to 0.35 cm2 in six mares on the day of a 30-mm anovulatory follicle and from 0.25 to 0.86 cm2 in 11 mares on the day of a 30-mm ovulatory follicle; the ranges did not overlap except for one follicle. In the controls, mean blood flow area was lower (P < 0.05) in the anovulatory group than in the ovulatory group for each day beginning with the first Doppler examination at 25 mm. For plasma LH in controls, an effect of follicle group (P < 0.0001) and an interaction (P < 0.0001) of group by day reflected lower (P < 0.05) concentrations in the anovulatory group on Days –6, –2, and 5–8 (Day 0 = 30-mm follicle). For plasma FSH, an interaction (P < 0.0001) reflected higher (P < 0.05) concentrations in the anovulatory group on Days –3 and 1–4. More (P < 0.05) statistically identified FSH surges occurred in the anovulatory group during Days –7 to 8. In the sampled mares, follicular-fluid concentrations of estradiol, free insulin-like growth factor-1, inhibin-A, and vascular endothelial growth factor were lower (P < 0.05) in 30-mm designated anovulatory follicles than in 30-mm designated ovulatory follicles. Results were interpreted as follows: 1) The future anovulatory dominant-sized follicle developed under an LH deficiency, 2) the LH deficiency led to reductions in blood flow area and in concentrations of follicular-fluid factors, and 3) the reduction in follicle production of FSH suppressors resulted in higher plasma FSH concentrations.

follicular development, growth factors, ovary, seasonal reproduction, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mares have anovulatory and ovulatory seasons (for review, see [1]). The transition between the anovulatory and ovulatory seasons occurs in the spring and often is characterized by the formation of one or several sequential anovulatory dominant (diameter, ≥30 mm) follicles until a dominant ovulatory follicle terminates the anovulatory season. The number of mares developing dominant anovulatory follicles during the transitional period varies among reports, from 15 of 15 horses [2] and 9 of 10 ponies [3] to three of eight ponies [4]. Some of the anovulatory follicles reach a diameter that is not distinguishable from the maximum diameter of an ovulatory follicle. In this regard, 7 of 15 horse mares developed one to three follicular waves in which the dominant anovulatory follicle was 38 mm or greater in diameter and comparable to that of the preovulatory follicle [2].

The systemic hormonal aspects of the development of seasonal anovulatory dominant follicles versus ovulatory follicles have been considered in several studies. The wave-stimulating FSH surge is similar for both anovulatory and ovulatory waves regardless of whether the anovulatory wave develops a dominant follicle [4–6]. Circulating estradiol concentrations are very low during the anovulatory season [4], and a detectable increase does not occur until the end of the season, during development of the first ovulatory wave [7–9]. Intrafollicular production of estradiol by dominant-sized follicles, as indicated by tissue culture and follicular-fluid assays, is minimal during early transition and increases during late transition [7, 10]. However, in a recent study [5], circulating estradiol concentrations did not increase during the last anovulatory wave and, on a temporal basis, did not play a role in FSH reduction during deviation in anovulatory waves. Circulating LH concentrations are low during the anovulatory season and increase a few days before the first ovulation [4, 11, 12]. Administration of estradiol during transition increases the production of LH [13, 14]. However, the increase in LH at the end of the anovulatory season occurs even in the absence of the ovaries [8, 11, 15], and the initial portion of the LH surge before the first ovulation is attributable to seasonal effects rather than to estradiol [5]. These results indicate that the LH surge associated with the first ovulation of the year is initially a function of season followed by a positive effect of estradiol.

The growth of a dominant follicle, whether anovulatory or ovulatory, is associated with a follicular wave and a deviation process wherein the developing dominant follicle continues to grow and the other follicles regress (subordinate follicles [5]). The characteristics of deviation are similar between seasonal anovulatory follicular waves with dominant follicles and seasonal ovulatory waves. Diameter deviation in mares has been attributed to increased responsiveness of the developing dominant follicle to gonadotropins in association with the increased production of enabling follicular-fluid factors (for review, see [16]). The temporal associations among follicular-fluid factors (estradiol, free insulin-like growth factor [IGF]-I, activin-A, and inhibin-A) have been studied in association with deviation during the ovulatory season in mares [17]. Results of other studies during ovulatory follicular waves [18–21] also indicate that the role of enabling follicular-fluid factors in deviation should be considered in the anovulatory versus ovulatory destiny of dominant follicles during the transitional period.

A difficulty in clarifying the endocrine aspects of the dominant anovulatory follicles during transition into the ovulatory season has been the inability to adequately distinguish between the anovulatory and ovulatory follicles except in retrospect. Attempts to identify an anovulatory follicle on the basis of companion follicles [10], uterine echotexture [2, 22], or time of year [7, 23] do not seem to be satisfactory. Estrous behavior can occur during the anovulatory season even in the absence of the ovaries (for review, see [1]), and its absence is not a useful indicator of an anovulatory follicle.

The concentrations of follicular-fluid estradiol and inhibin-A [7] as well as the morphology and angiogenesis [23] of anovulatory dominant follicles obtained during the transitional period have been compared to those of dominant follicles obtained during the ovulatory season; however, these studies involved surgical collection of an ovary with a dominant follicle, precluding determination of whether the follicle would have ovulated. The follicular fluid collected during the transitional period had lower concentrations of estradiol, progesterone, and inhibin-A than fluid from preovulatory follicles. In addition, the follicles from the transitional period were visibly less vascularized and had fewer blood vessels, less proliferative activity, and lower vascular endothelial growth factor (VEGF) protein expression as determined by immunostaining.

Color Doppler ultrasonography (for review, see [24]) has been used for noninvasive hemodynamic studies of preovulatory follicles in humans [25] and cows [26]. In humans, blood flow determinations of individual preovulatory follicles provide an index of the intrafollicular environment and may be used to predict the developmental competence of the oocyte [27]. In cows, transrectal color Doppler ultrasonography demonstrated a clear difference in the vascularity of the wall of preovulatory follicles compared with that of anovulatory (atretic) follicles [26]. The color Doppler approach has potential for investigating the vasculature of seasonal anovulatory dominant follicles in mares and in identifying dominant follicles that will fail to ovulate.

The present study was done 1) to test the hypothesis in vivo that low blood flow area of the follicle wall is temporally associated with the development of future dominant-sized anovulatory follicles, 2) to determine if color Doppler ultrasonography would distinguish between anovulatory versus ovulatory dominant follicles, 3) to determine if the low concentrations of follicular-fluid factors that are temporally associated with development of the largest subordinate follicle versus the dominant follicle during deviation are also associated temporally with the development of dominant anovulatory versus ovulatory follicles during the transitional period, and 4) to determine the temporal relationships of circulating gonadotropins to the early development of follicles that are destined to become anovulatory versus those destined to become ovulatory.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Ultrasound Scanning

Animals were handled in accordance with the Guide for Care and Use of Agricultural Animals in Agricultural Research. A total of 28 nonlactating pony mares of mixed breeding (age, 9–17 yr; weight, 290–430 kg) were used. The experiment began on March 24, 2003 in the northern hemisphere (latitude, 43°N) during the transitional period between the anovulatory and ovulatory seasons. The mares were kept under natural light. The feeding program and the equipment and techniques for transrectal and transvaginal ultrasound scanning and manipulation of equine ovaries have been described previously [28, 29]. A color Doppler ultrasound scanner (Aloka SD-2000; Aloka America, Wallingford, CT) equipped with a 7.5-MHz linear-array transducer (UST-5821-7.5) was used for determinations of diameter and blood flow area.

Follicle measurements were recorded and blood samples taken daily from the day the first 25-mm follicle developed and continued until ovulation. Doppler assessment of blood flow area was begun when each growing follicle reached 25 mm or larger in diameter. Each animal was randomized into the control group or follicular-fluid sampled group when a follicle reached 30.0 mm or larger in diameter. Because of the 1-day interval between measurements, actual diameter varied from 30 to 32 mm, but the follicle is referred to as a 30-mm follicle and was considered to be a dominant follicle based only on its diameter [29]. In the control mares, the daily examinations continued until the follicle regressed to less than 20 mm (anovulatory follicle) or ovulated (ovulatory follicle).

In the sampled mares, follicles were measured daily for diameter until the largest follicle was 30 mm. A single collection of 500 µl of follicular fluid was done on the day the follicle was 30 mm. The follicular fluid was collected with a double-channel device (inner needle, 25 gauge; outer needle, 20 gauge), using transvaginal ultrasound-guidance as described previously [29, 30]. If another 30-mm follicle developed, it was assessed and sampled in a similar fashion. However, an anovulatory follicle was not used if it appeared to be from the same follicular wave as an ovulatory follicle. Also, double ovulatory follicles were not used. Each of these events occurred in only one mare. For each dominant follicle in the sampled mares, diameter measurements and samples of blood and follicular fluid were collected only on the day that a follicle was 30 mm. However, diameter was measured on the following day to assess the extent of diameter decrease associated with sampling [20, 30]. The mares were monitored daily postsampling for development of another 30-mm follicle and for detection of ovulation.

For the ultrasonographic Doppler blood flow determinations, the flow mode was activated, and the blood flow area within the follicle wall was quantified from the color images (for review, see [24]). Areas of color represent regions with a flow velocity higher than 10 mm/sec. All scans were performed at a pulse-repetition frequency of 6 Hz. Identical color gain settings were used for all scans. The blood flow area was evaluated in a vertical plane at the maximum diameter of the follicle. The distance between the transducer face and follicle was minimized to reduce signal attenuation. The angle of insonation was not calculated because of the small diameter of vessels, but care was taken to obtain the maximum color intensity. Scan records (real-time images) were stored using a digital video camera (Handycam Camcorder; Sony Electronics, Inc., San Diego, CA) for blood flow area analyses in the laboratory. The tapes were viewed on the monitor of a computer. Using iMovie software, the images were captured and saved as joint photographic experts group (JPEG) format files. The blood flow areas in the follicle wall were measured by outlining a belt circumscribing the anechoic antral cavity as described for the bovine preovulatory follicle [26]. The colored flow area was changed to black and white using Adobe PhotoShop 5.5 software (Adobe Systems, San Jose, CA). The flow area was quantified using an NIH Image software (Version 1.62; National Institutes of Health, Bethesda, MD). The sum of blood flow areas in each circumscribed band was calculated and used as the daily value.

Dominant Anovulatory and Ovulatory Follicles and Associated End Points

The control mares were used to study the relationships of 30-mm follicles with known outcomes (anovulation or ovulation) to blood flow areas and systemic gonadotropin concentrations. Data were normalized to the day the follicle was 30 mm (Day 0). The dominant-sized anovulatory follicle that developed before the ovulatory follicle in each mare was used for the statistical comparisons of anovulatory and ovulatory waves. The data set was truncated because of variation among mares in the number of days with observations before and after Day 0. After truncation, the diameter, blood flow area, FSH concentrations, and LH concentrations were analyzed from Days –4 to 7, –2 to 7, –7 to 8, and –12 to 8, respectively. The control mares on Day 0 were also used to establish the blood flow area value that was most likely to indicate an anovulatory or ovulatory outcome for 30-mm follicles. The resulting value for blood flow area was 0.29 cm2 on the basis of the midway point between the highest value for a 30-mm anovulatory follicle (0.32 cm2) and the lowest value for a 30-mm ovulatory follicle (0.25 cm2).

In the sampled mares, all follicles at 30 mm (Day 0) were designated as anovulatory dominant follicles or ovulatory dominant follicles on the basis of the value for blood flow area that was used to indicate follicle outcome based on the outcome in controls. Follicles with a blood flow area of less than 0.29 cm2 were designated as anovulatory follicles, and follicles with a blood flow area of greater than 0.29 cm2 were designated as ovulatory follicles. Designated rather than actual outcomes (regression or ovulation) were used to avoid potential errors from an effect of follicular-fluid sampling on the outcome of the follicles [20]. The effect of sampling on the subsequent outcome of the follicles was assessed by comparing, first, the diameter change between the day of sampling and the next day for designated ovulatory follicles that subsequently did versus those that did not ovulate and, second, the maximum diameter of the actual ovulatory follicle between the control and the sampled mares. The blood flow area, concentrations of plasma LH and FSH, and concentrations of follicular-fluid factors on Day 0 in the sampled mares were compared between the last designated anovulatory 30-mm follicle (anovulatory group) and the subsequent designated or actual ovulatory follicle (ovulatory group). That is, only one dominant anovulatory and ovulatory follicle from each mare was used for each point. The end points for follicular-fluid factors were concentrations of estradiol, androstenedione, progesterone, free IGF-I, activin-A, inhibin-A, and VEGF.

Hormone Assays

Blood and follicular-fluid samples were centrifuged (1500 x g for 10 min), decanted, and stored (–20°C) until assay. Plasma samples were assayed for FSH concentrations by a radioimmunoassay as validated previously [11] and modified [17] in our laboratory and for LH concentrations by a radioimmunoassay as validated previously [31] and modified [17] in our laboratory for equine plasma. The intra- and interassay coefficients of variation (CVs) and mean assay sensitivity were 13.9%, 5.7%, and 0.4 ng/ ml, respectively, for FSH and 8.4%, 9.4%, and 0.1 ng/ml, respectively, for LH. The follicular-fluid samples were assayed for estradiol, androstenedione, free IGF-I, activin-A, and inhibin-A by commercially available kits and for progesterone by an in-house assay; all assays were validated in our laboratory for equine follicular fluid [17]. The concentrations of VEGF in follicular-fluid samples were determined by a commercially available kit that was validated in our laboratory for equine follicular fluid [20]. The intraassay CV and assay sensitivity, respectively, were as follows: estradiol, 5.4%, 0.74 pg/ml; progesterone, 1.2%, 0.04 ng/ml; androstenedione, 3.3%, 0.01 ng/ml; free IGF-I, 3.2%, 0.01 ng/ml; activin-A, 4.3%, 0.03 ng/ml; inhibin-A, 1.9%, 0.06 pg/ml; and VEGF, 10.4%, 1.2 ng/ml.

Statistical Analysis

In the control mares, plasma FSH and LH concentrations, follicle diameter, and blood flow area were analyzed to determine the main effect of follicle group (anovulatory vs. ovulatory) and day as well as the interaction of group by day. An SAS mixed procedure was used with a repeated statement to account for autocorrelation between sequential measurements and taking mare within follicle group as the random effect (Version 8.2; SAS Institute, Cary, NC). If a significant interaction was detected, unpaired t-tests were used to locate differences between follicle groups within a day. Surges of FSH in the control mares were differentiated from variation using the values for the ascending and descending portion of the suspected surge as described previously [32]. An identified surge was defined by a CV that was at least three times greater than the intraassay CV [4]. In the sampled mares, differences at Day 0 between the designated dominant anovulatory group and the designated or actual ovulatory group for plasma LH and FSH and for follicular-fluid end points were tested by analysis of variance. A probability of P ≤ 0.05 indicated that the difference was significant, and a probability of P > 0.05 to P < 0.1 indicated that the difference approached significance. Data are presented as the mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Three control mares were removed from the present study because of development of a hemorrhagic anovulatory follicle, an ovarian cyst, and a double ovulation, respectively. One sampled mare was removed because of a sampling problem. As a result, 11 control mares and 13 sampled mares were used. Dominant anovulatory follicles developed in 6 of 11 control mares, and two mares had two or three anovulatory follicles. On the basis of the values for blood flow area in the controls, 7 of 13 sampled mares developed a 30-mm follicle that was designated as anovulatory. In addition, the 13 sampled mares developed a 30-mm follicle that either ovulated (7/13) or was designated as ovulatory (6/13). For the designated ovulatory follicles in the sampled mares, the decrease in diameter between Days 0 and 1 was greater (P < 0.0001) for the follicles that regressed (–8.2 ± 1.4 mm) than for the follicles that ovulated (–0.9 ± 1.1 mm). Maximum diameter of the ovulating follicle was greater (P < 0.003) in control mares (45.2 ± 1.3 mm) than in sampled mares (38.7 ± 1.5 mm). No significant difference was found in blood flow areas at 30 mm between designated ovulatory follicles (0.53 ± 0.06 cm2) and actual ovulatory follicles (0.56 ± 0.03 cm2).

In the 11 control mares, a main effect of follicle group (dominant anovulatory and dominant ovulatory; P < 0.0001) and an interaction (P < 0.0001) of group by day was found for follicle diameter, follicle blood flow area, plasma LH concentration, and plasma FSH concentration (Fig. 1). The diameter of the anovulatory follicles was smaller (P < 0.05) than that for the ovulatory follicles on each day beginning on Day 1. Blood flow area of the follicle wall was less (P < 0.05) for the anovulatory group for each day beginning when the follicles were 25 mm or greater (first day examined). Plasma concentrations of LH were lower (P < 0.05) for the anovulatory group on Days –6, –2, and 5–8, and they approached being lower (P < 0.07 to P < 0.09) on Days –7, –5, –4, –1, 3, and 4. Concentrations of FSH were greater (P < 0.05) for the anovulatory group on Days –3 and 0–3. More (P < 0.0003) FSH surges were identified per mare over Days –7 to 8 in the anovulatory group than in the ovulatory group (Fig. 1). No differences were found between anovulatory and ovulatory groups in the width of the base of the FSH surge (4.8 ± 0.4 versus 4.5 ± 1.2 days) or in the peak concentrations of FSH (17.2 ±1.6 versus 13.3 ± 2.4 ng/ ml).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 1. Mean ± SEM for follicle diameter, blood flow area of the follicle wall, and plasma gonadotropin concentrations in control mares for dominant (30-mm) anovulatory (n = 6) and ovulatory (n = 11) groups. The interaction of group by day was significant (P < 0.0001) for each end point. The asterisks indicate days when the difference between follicle groups was significant (P < 0.05). The indicated number of FSH surges per mare was greater (P < 0.0003) for the anovulatory group than for the ovulatory group

In the sampled mares, the plasma concentrations of LH and FSH and the concentrations of follicular-fluid factors in the designated anovulatory follicle or in the designated or actual ovulatory follicle on the day the follicle was 30 mm (Day 0) are shown (Table 1). Plasma concentrations of FSH on Day 0 were higher (P < 0.03) for the anovulatory group than for the ovulatory group. Follicular-fluid concentrations were lower in the anovulatory group than in the ovulatory group for estradiol (P < 0.001), free IGF-I (P < 0.0004), inhibin-A (P < 0.04), and VEGF (P < 0.001).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Mean ± SEM for blood flow area of the follicle wall and con centrations of plasma gonadotropins and follicular-fluid factors in 30-mm designated dominant anovulatory and ovulatory follicles during the tran sition from anovulatory to ovulatory seasons


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The color Doppler ultrasound technology distinguished between future anovulatory and ovulatory dominant follicles with reasonable accuracy. In the 11 control mares, the mean blood flow area was approximately twofold greater for the 30-mm ovulatory follicles than for the 30-mm anovulatory follicles. The lowest value in the ovulatory group was higher than each value in the anovulatory group with one exception. On each of the following 7 days, each value in the ovulatory group was higher than each value in the anovulatory group. On the day the follicle reached 35 mm, the values ranged from 0.48 to 0.81 cm2 in the ovulatory group and from 0.12 to 0.28 cm2 in the anovulatory group. Consideration of the ovulatory potential of 35-mm follicles during the transitional period is important on breeding farms, because at that time a breeding decision may be needed. The lower blood flow areas in dominant-sized anovulatory follicles in the present study is consistent with the report that presumed anovulatory dominant follicles removed during the transitional period had underdeveloped vascularization [23].

It became apparent during the experiment that the outcome (anovulation or ovulation) of sampled follicles might be altered by the sampling procedure; the diameter decreases seemed to be greater than those during previous experiments [20, 30] and were attributable, at least in part, to a fluid sample that was 2.5-fold greater than samples in previous studies. The greater postsampling diameter decrease between Days 0 and 1 in designated ovulatory follicles that regressed compared with that in designated ovulatory follicles that ovulated confirmed that follicle outcome was altered by the sampling procedure. A negative effect of sampling was also indicated by the smaller preovulatory diameter for sampled than for nonsampled follicles. Therefore, a calculated blood flow value was used for designation of anovulatory versus ovulatory sampled follicles based on the extent of blood flow areas in 30-mm follicles in the control mares. This approach was effective, as indicated by 1) the similarity in number of control mares with anovulatory follicles and number of sampled mares with designated anovulatory follicles (6/11 and 7/13, respectively), 2) fewer anovulatory follicles in the control mares (0.8 per mare) than nonovulatory follicles in the sampled mares (1.5 per mare; sum of designated anovulatory and designated ovulatory follicles that did not ovulate), and 3) no significant difference in blood flow area between designated and actual ovulatory follicles at 30 mm. Judging the future expected outcome of sampled dominant follicles on the basis of blood flow areas in controls circumvented, at least in part, the negative effect of sampling.

Approximately half (54%) of each of the control and sampled groups developed 30-mm anovulatory follicles or designated anovulatory follicles, respectively, during the transitional period between the anovulatory and ovulatory seasons. This percentage seems to be low compared to some reports [2, 3], but it is comparable to the incidence previously reported for a similar pony herd [4]. The 30-mm follicle in the actual and designated anovulatory waves occurred an average of 9.8 days before the 30-mm follicle in the actual or designated ovulatory waves. This interval between waves is similar to reported intervals [2, 3]. The lower blood flow area in the anovulatory group on the first day of comparison (25-mm follicle) indicated that, on average, the destiny of the follicle was developing close to the expected diameter at the beginning of deviation for both the last anovulatory and ovulatory waves in a similar herd (23.7 mm [5]).

Concentrations of LH were lower in the anovulatory group during the week before a 30-mm follicle was present. During this week, the differences between the two groups were significant on two of the days and approached significance on four other days. Before Day –7, LH was similar between the two follicle groups. Thus, the anovulatory follicle apparently was exposed to less LH beginning when the follicle was approximately 15 mm based on extrapolation from Figure 1 and published data [28]. Anovulatory dominant follicles, therefore, may be a consequence of LH deficiency during early development of the follicle.

A progressive decrease in concentrations of FSH in the wave-stimulating FSH surge occurred in the controls over approximately 6 days before development of a 30-mm ovulatory follicle, as previously described (for review, see [33]). The greater concentrations of FSH and more frequent occurrences of statistically detected FSH surges in the controls with anovulatory waves were striking. On average, the largest follicle in the anovulatory group was 20 mm on the day of the peak of the first FSH surge. These results indicated that the follicles of anovulatory waves had impaired FSH-suppressing ability well before the dominant follicle reached 30 mm.

In the planned analyses using one follicle of each type per sampled mare, follicular-fluid concentrations of estradiol, free IGF-I, inhibin-A, and VEGF were lower in the 30-mm designated anovulatory follicle than in the designated or actual ovulatory follicle. When all sampled follicles were considered (anovulatory, n = 10; ovulatory, n = 24), progesterone also was significantly lower, and activin-A and plasma LH approached being lower. The estradiol and inhibin-A results are consistent with reported concentrations in the follicular fluid of dominant follicles during the transitional period [7]. The lower concentration of IGF-I in the present study is especially noteworthy because of its role in diameter deviation and in the production of other follicular-fluid factors, including inhibin-A and VEGF [19, 20]. The lower concentrations of several follicular-fluid factors and the smaller blood flow area indicated a generally impaired status of the anovulatory follicle rather than the impairment of a single system.

Regarding the temporal relationship between low concentrations of LH and follicular-fluid factors in the present study, experimentally reducing circulating LH concentrations beginning 4 days before diameter deviation in mares resulted in reduced circulating concentrations of estradiol and immunoreactive inhibin [34]. In heifers, experimental reduction in LH decreased the intrafollicular concentrations of free IGF-I as well as estradiol [35, 36]. Several studies in monkeys and cattle have demonstrated that in vivo or in vitro treatment with LH results in increased VEGF production by granulosa cells in culture [37–39] and that IGF-I acts synergistically with LH in promoting LH stimulation of VEGF [38]. The temporal relationships between deficient plasma LH and deficient follicular-fluid factors in the present study during anovulatory waves is also consistent with the results of GnRH and pituitary extract studies. Administration of GnRH [40, 41] or a pituitary extract [42] to mares during the anovulatory season stimulated growth and ovulation of follicles. The treatments not only stimulated growth of small follicles but also seasonal anovulatory follicles that were 25 mm or greater. Based on the results of the present study, stimulation of the larger follicles may have represented a rescue of follicles that were already impaired, but this assumption requires specific study.

Our interpretation of the results (and current working hypothesis) is that future dominant anovulatory follicles develop in an LH-deficient environment. As a result, the developing follicle is low in certain follicular-fluid factors, notably estradiol, IGF-I, inhibin-A, and VEGF. Estradiol and inhibin-A are FSH suppressors [16], and their limited production accounts for an increase in plasma FSH beginning when the future dominant anovulatory follicle is approximately 20 mm. The low IGF-I and estradiol can be expected to retard follicle development, and the low VEGF can be expected to retard follicle angiogenesis.

During the growth of follicles from 20 to 30 mm, the anovulatory follicles expanded at the same rate as the ovulatory follicles. This occurred despite the reduction in LH beginning when the follicle was approximately 15 mm, the considerable reduction in blood flow areas in the wall of the anovulatory 25- to 30-mm follicles, and the lower concentrations of follicular-fluid factors at 30 mm. Even after 30 mm was reached, the anovulatory follicles that continued to expand appeared to do so at a rate similar to growth of the ovulatory follicles. These observations indicate that the growth or expansion rate of dominant follicles was not, in itself, an indicator of the status or health of the follicle. Apparently, expansion occurred at a given rate, without regard to the extent of the diminished plasma LH, vascularity, and concentrations of the follicular-fluid factors. Alternatively, the increased FSH during early follicle development of anovulatory follicles may compensate for impaired blood flow or concentrations of follicular-fluid factors.

In summary, dominant-sized anovulatory follicles were distinguished from dominant ovulatory follicles by daily blood flow area determinations in control mares and by sampling of follicular fluid at 30 mm in separate mares. In the control mares, blood flow area was less for dominant anovulatory follicles than for ovulatory follicles by the time blood flow determinations began at 25 mm. Concentrations of FSH encompassing a 30-mm follicle were greater in the anovulatory group, indicating a deficiency in the FSH-suppressing abilities of the follicles. In the sampled mares, follicular-fluid concentrations in 30-mm anovulatory follicles were lower than those in 30-mm ovulatory follicles for estradiol, IGF-I, inhibin-A, and VEGF. The future dominant anovulatory follicle developed during low LH concentrations, beginning when the follicle was approximately 15 mm. Based on the temporal increase in FSH, functional impairment of the future dominant anovulatory follicles began when the follicle was approximately 20 mm.


   ACKNOWLEDGMENTS
 
The present study was supported by the Eutheria Foundation and Equiservices Publishing. The authors thank Dr. A.F. Parlow for equine gonadotropin RIA reagents, M. Acosta and S. Jensen for technical assistance, and D. Acqua and S. Roman of Aloka America for help in acquiring the color Doppler equipment.


   FOOTNOTES
 
1 Supported by the Eutheria Foundation and Equiservices Publishing, Cross Plains, Wisconsin. Project P1-TA-03. Back

2 Correspondence: O.J. Ginther, Department of Animal Health and Biomedical Sciences, 1656 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706. FAX: 608 262 7420; ginther{at}svm.vetmed.wisc.edu Back

3 Current address: Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama University, Tsushima Naka, Okayama 700-8530, Japan Back

Received: 13 February 2004.

First decision: 3 March 2004.

Accepted: 1 April 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ginther OJ. Reproductive Biology of the Mare: Basic and Applied Aspects, 2nd edition. Cross Plains, WI: Equiservices Publishing; 1992:95–97, 135–172
  2. Ginther OJ. Folliculogenesis during the transitional period and early ovulatory season in mares. J Reprod Fertil 1990 90:311-320[Abstract]
  3. Watson ED, Heald M, Tsigos A, Leask R, Steele M, Groome NP, Riley SC. Plasma FSH, inhibin A, and inhibin isoforms containing pro- and {delta}C during winter anestrus, spring transition, and the breeding season in mares. Reproduction 2002 123:535-542[Abstract]
  4. Donadeu FX, Ginther OJ. Follicular waves and circulating concentrations of gonadotrophins, inhibin, and estradiol during the anovulatory season in mares. Reproduction 2002 124:875-885[Abstract]
  5. Donadeu FX, Ginther OJ. Interrelationships of estradiol, inhibin, and gonadotropins during follicle deviation in pony mares. Theriogenology 2004 61:1395-1405[CrossRef][Medline]
  6. Donadeu FX, Ginther OJ. Interactions of follicular factors and season in the regulation of circulating concentrations of gonadotrophins in mares. Reproduction 2003 125:743-750[Abstract]
  7. Watson ED, Thomassen R, Steele M, Heald M, Leask R, Groome NP, Riley SC. Concentrations of inhibin, progesterone, and estradiol in fluid from dominant and subordinate follicles from mares during spring transition and the breeding season. Anim Reprod Sci 2002 74: : 55-67[CrossRef][Medline]
  8. Peltier MR, Robinson G, Sharp DC. Effects of melatonin implants in pony mares. 2. Long-term effects. Theriogenology 1996 49:1125-1142[CrossRef]
  9. Seamans KW, Sharp DC. Changes in equine follicular aromatase activity during transition from winter anestrus. J Reprod Fertil Suppl 1982 32:225-233[Medline]
  10. Davis SD, Sharp DC. Intrafollicular and peripheral steroid characteristics during vernal transition in the pony mare. J Reprod Fertil Suppl 1991 44:333-340[Medline]
  11. Freedman LJ, Garcia MC, Ginther OJ. Influence of photoperiod and ovaries on seasonal reproductive activity in mares. Biol Reprod 1979; 20:567-574[Abstract]
  12. Fitzgerald BP, Affleck KJ, Barrows SP, Murdoch WL, Barker KB, Loy RG. Changes in LH pulse frequency and amplitude in intact mares during the transition into the breeding season. J Reprod Fertil 1987 79:485-493[Abstract]
  13. Sharp DC, Grubaugh WR, Weithenauer J, Davis SD, Wilcox CJ. Effects of steroid administration on pituitary luteinizing hormone and follicle-stimulating hormone in ovariectomized pony mares in the early spring: pituitary responsiveness to gonadotropin-releasing hormone and pituitary gonadotropin content. Biol Reprod 1991 44:983-990[Abstract]
  14. Sharp DC, Wolfe MW, Cleaver BD, Nilson J. Effects of estradiol-17ß administration on steady-state messenger ribonucleic acid (mRNA) encoding equine {alpha} and LH/CG ß subunits in pituitaries of ovariectomized pony mares. Theriogenology 2001 55:1083-1093[CrossRef][Medline]
  15. Affleck KJ, Conboy HS, Fitzgerald BP. A negative feedback role for the ovaries of the mare on tonic LH secretion before the first ovulation of the breeding season. J Reprod Fertil Suppl 1991 44:241-247[Medline]
  16. Ginther OJ, Beg MA, Donadeu FX, Bergfelt DR. Mechanism of follicle deviation in monovular farm species. Anim Reprod Sci 2003 78 239-257
  17. Donadeu FX, Ginther OJ. Changes in concentrations of follicular-fluid factors during follicle selection in mares. Biol Reprod 2002 66:1111-1118[Abstract/Free Full Text]
  18. Ginther OJ, Meira C, Beg MA, Bergfelt DR. Follicle and endocrine dynamics during experimental follicle deviation in mares. Biol Reprod 2002 67:862-867[Abstract/Free Full Text]
  19. Ginther OJ, Bergfelt DR, Beg MA, Meira C, Kot K. In vivo effects of an intrafollicular injection of insulin-like growth factor 1 on the mechanism of follicle deviation in heifers and mares. Biol Reprod 2004 70:99-105[Abstract/Free Full Text]
  20. Ginther OJ, Gastal EL, Gastal MO, Checura CM, Beg MA. Dose-response study of intrafollicular injection of insulin-like growth factor-1 on follicular-fluid factors and follicle dominance in mares. Biol Reprod 2004 70:1063-1069[Abstract/Free Full Text]
  21. Ginther OJ, Gastal EL, Gastal MO, Beg MA. Critical role of insulin-like growth factor system in follicle selection and dominance in mares. Biol Reprod 2004 70:1374-1379[Abstract/Free Full Text]
  22. Watson ED, Thomassen R, Nikolakopoulos , Association of uterine edema with follicle waves around the onset of the breeding season in pony mares. Theriogenology 2003 59:1181-1187[CrossRef][Medline]
  23. Watson ED, Al-zi'abi MO. Characterization of morphology and angiogenesis in follicles of mares during spring transition and the breeding season. Reproduction 2002 124:227-234[Abstract]
  24. Maulik D. Principles of Doppler signal processing and hemodynamic analysis. In: Copel JA, Reed KI (eds.), Doppler Ultrasound in Obstetrics and Gynecology. New York: Raven Press; 1995:1–18
  25. Brannstrom M, Zackrisson U, Hagstrom H-G, Josefsson B, Hellberg P, Granberg S, Collins WP, Bourne T. Preovulatory changes of blood flow in different regions of the human follicle. Fertil Steril 1998 69: : 435-442[CrossRef][Medline]
  26. Acosta TJ, Hayashi KG, Ohtani M, Miyamoto A. Local changes in blood flow within the preovulatory follicle wall and early corpus luteum in the cow. Reproduction 2003 125:759-767[Abstract]
  27. Coulam CB, Goodman C, Rinehart JS. Color Doppler indices of follicular blood flow as predictors of pregnancy after in-vitro fertilization and embryo transfer. Hum Reprod 1999 14:1979-1982[Abstract/Free Full Text]
  28. Gastal EL, Gastal MO, Bergfelt DR, Ginther OJ. Role of diameter differences among follicles in selection of a future dominant follicle in mares. Biol Reprod 1997 57:1320-1327[Abstract]
  29. Ginther OJ. Ultrasonic Imaging and Animal Reproduction: Book 2, Horses. Cross Plains, WI: Equiservices Publishing; 1995:66–72
  30. Gastal EL, Gastal MO, Wiltbank MC, Ginther OJ. Follicle deviation and intrafollicular and systemic estradiol concentrations in mares. Biol Reprod 1999 61:31-39[Abstract/Free Full Text]
  31. Whitmore HL, Wentworth BC, Ginther OJ. Circulating concentrations of luteinizing hormone during estrous cycle of mares as determined by radioimmunoassay. Am J Vet Res 1973 34:631-636[Medline]
  32. Fitzgerald BP, l'Anson H, Legan SJ, Loy RG. Changes in patterns of luteinizing hormone secretion before and after the first ovulation in the postpartum mare. Biol Reprod 1985 33:316-323[Abstract]
  33. Ginther OJ. Selection of the dominant follicle in cattle and horses. Anim Reprod Sci 2000 60:61-79
  34. Bergfelt DR, Gastal EL, Ginther OJ. Response of estradiol and inhibin to experimentally reduced luteinizing hormone during follicle deviation in mares. Biol Reprod 2001 65:426-432[Abstract/Free Full Text]
  35. Ginther OJ, Bergfelt DR, Beg MA, Kot K. Follicle selection in cattle: role of luteinizing hormone. Biol Reprod 2001 64:197-205[Abstract/Free Full Text]
  36. Ginther OJ, Bergfelt DR, Beg MA, Kot K. Effect of LH on circulating estradiol and follicular fluid factors during follicle deviation in cattle. Reproduction 2001 122:103-110[Abstract]
  37. Christenson LK, Stouffer RL. Follicle-stimulating hormone and luteinizing hormone/chorionic gonadotropin stimulation of vascular endothelial growth factor production by macaque granulosa cells from pre- and periovulatory follicles. J Clin Endocrinol Metab 1997 82: : 2135-2142[Abstract/Free Full Text]
  38. Martinez-Chequer JC, Stouffer RL, Hazzard TM, Patton PE, Molskness TA. 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. Biol Reprod 2003 68:1112-1118[Abstract/Free Full Text]
  39. Schams D, Kosmann B, Berisha B, Amselgruber WM, Miyamoto A. Stimulatory and synergistic effects of luteinizing hormone and insulin like growth factor 1 on the secretion of vascular endothelial growth factor and progesterone of cultured bovine granulosa cells. Exp Clin Endocrinol Diabetes 2001 109:155-162[CrossRef][Medline]
  40. Ginther OJ, Bergfelt DR. Effect of GnRH treatment during the anovulatory season on multiple ovulation rate and on follicular development during the ensuing pregnancy in mares. J Reprod Fertil 1990; 88:119-126[Abstract]
  41. Johnson AL. Gonadotropin-releasing hormone treatment induces follicular growth and ovulation in seasonally anestrous mares. Biol Reprod 1987 36:1199-1206[Abstract]
  42. Douglas RH, Nuti L, Ginther OJ. Induction of ovulation and multiple ovulation in seasonally anovulatory mares with equine pituitary fractions. Theriogenology 1974 2:133-142[CrossRef][Medline]



This article has been cited by other articles:


Home page
 ReproductionHome page
E L Gastal, M O Gastal, and O J Ginther
Relationships of changes in B-mode echotexture and colour-Doppler signals in the wall of the preovulatory follicle to changes in systemic oestradiol concentrations and the effects of human chorionic gonadotrophin in mares.
Reproduction, April 1, 2006; 131(4): 699 - 709.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/2/637    most recent
biolreprod.104.028498v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Acosta, T.J.
Right arrow Articles by Ginther, O.J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Acosta, T.J.
Right arrow Articles by Ginther, O.J.
Agricola
Right arrow Articles by Acosta, T.J.
Right arrow Articles by Ginther, O.J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS