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Eutheria Foundation, Cross Plains, Wisconsin 53528
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6 mm were ablated on Day 7, and every 2 days thereafter, newly emerging follicles were also ablated. Estradiol concentrations decreased (P < 0.01) similarly in the controls and the follicle-ablation group between Days 7 and 11 and by Day 15 began to increase in the controls and continued to decrease in the follicle-ablation group. Concentrations of progesterone were not affected by follicle ablation, but diameter of the corpus luteum was greater (P < 0.05) by Day 21 in the follicle-ablation group; these results indicated that the follicles were involved in morphologic luteolysis, but not in functional luteolysis. Concentrations of LH were higher (P < 0.05) on Days 15 and 16 in the follicle-ablation group than in the controls, indicating an initial negative effect of follicles on LH. Immunoreactive inhibin and estradiol decreased (P < 0.0001) and FSH and LH increased (P < 0.05) within 1 or 2 days after ovariectomy; these changes occurred more slowly in the follicle-ablation group. The maximum value for an FSH surge in each control mare was below the lower 95% confidence limit in the ovariectomy group. Maximum concentration for the periovulatory LH surge in the controls was not different from the mean maximum LH concentrations in the ovariectomy group. Our interpretation is that the gonadotropin surges resulted from changes in the magnitude of the negative effects of ovarian hormones on the positive effects of extraovarian control. There was no indication of a positive ovarian effect on either FSH or LH.
estradiol, follicle, follicle-stimulating hormone, gonadotropins, inhibin, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A proteinaceous fraction of follicular fluid (inhibin source) suppresses circulating concentrations of FSH in mares [3, 4]. Immunoreactive (ir) inhibin concentrations begin to increase before the beginning of the declining portion of the wave-stimulating FSH surge [5, 6]. Similar results were obtained for inhibin-A [7], indicating that inhibin-A is at least one of the active forms that depresses FSH before and during deviation. The initial 2 days of the decline in the FSH surge is caused by inhibin alone, based on temporal relationships among FSH, ir-inhibin, and estradiol [5, 8]. Circulating estradiol begins to increase near the beginning of deviation [6, 9], but the relative contribution of estradiol to the continued decline in the FSH surge is not known. The relationship of ovarian hormones to systemic FSH concentrations was studied every 4 days in an ovariectomy experiment [10]. Daily mean concentrations of FSH were higher in ovariectomized mares than the daily mean concentrations during the interovulatory interval in controls. However, concentrations in ovariectomized mares have not been compared with FSH surges, using the peak concentration in individual surges.
A periovulatory LH surge develops progressively over approximately a week, beginning near deviation and extending for an average of 1 day after ovulation; the LH surge then gradually recedes over several days (reviewed in [11]). A temporal negative relationship exists between systemic changes in concentrations of LH and progesterone. Progesterone secretion by the new corpus luteum begins at ovulation before the beginning of the postovulatory reduction in LH. Toward the end of diestrus, progesterone begins to decrease about 2 days before an increase in LH. In addition to these temporal relationships, a negative effect of progesterone on LH also has been shown, using exogenous progesterone [6, 12, 13]. Treatment with progesterone during early development of the ovulatory follicular wave reduced the circulating concentrations of LH. In addition to the negative effect of progesterone on LH, a negative effect of follicles has been demonstrated by an LH increase after ablation of the largest follicle at the beginning of deviation [14]. The LH increase may have resulted from loss of estradiol but a negative effect of estradiol on LH has not been demonstrated directly. However, on a temporal basis, a later positive effect of estradiol on LH is consistent with the simultaneous preovulatory increase in both hormones and a positive effect of estradiol treatment on LH concentrations in ovariectomized mares and after luteolysis in intact mares (reviewed in [11]). There are indications, therefore, that estradiol may affect LH concentrations either negatively or positively at different times during the development of the ovulatory follicular wave. Concentrations of LH increased following ovariectomy 14 days after ovulation but not to the maximum concentrations of a periovulatory LH surge (reviewed in [2]). However, the interval from ovariectomy to the time of ovulation in control mares may have been too short for LH to reach maximum concentrations after ovariectomy. Further comparisons of control and ovariectomized mares are needed to clarify whether the ovaries have a positive effect on the periovulatory LH surge.
Estrogen has been implicated in triggering luteal regression in several species (reviewed in [11]). For example, destruction of follicles in sheep causes luteal maintenance, and administration of estradiol late in diestrus causes luteal regression. In mares, the effect of the follicles or estradiol on luteal life has not been clarified. An extension of luteal life by exogenous estrogens was reported for an earlier study, but this effect was not confirmed in subsequent studies.
In the present study, the temporal and functional interrelationships among circulating gonadotropins (FSH, LH) and ovarian hormones (estradiol, progesterone, inhibin) were considered in conjunction with ovariectomy or repeated follicle ablation to prevent development of follicles to >10 mm. Progesterone concentrations and luteal diameters were compared between controls and the follicle-ablation group to test the hypothesis that the follicles and estradiol play a role in luteolysis. Maximum FSH concentrations were compared between controls and an ovariectomy group to test the hypothesis that changes in circulating concentrations of FSH reflect changes in a continuous negative influence of the ovaries, even during the maximum concentrations of individual FSH surges. Concentrations of LH and estradiol were compared among control, follicle-ablation, and ovariectomy groups to test the hypothesis that follicles and estradiol have an initial negative effect and a later positive effect on circulating concentrations of LH during the periovulatory LH surge.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mares were handled according to the Guide for Care and Use of Agricultural Animals in Agricultural Research and Teaching. A total of 18 mares was used during the middle of an ovulatory season (June–July, Northern Hemisphere). The mares were mixed breeds of ponies, 5–6 yr of age, and weighed 300–420 kg. The feeding program and the equipment and techniques for transrectal and transvaginal ultrasound scanning of ovaries, including determination of ovulation and measurement of follicles and the corpus luteum, have been described [15, 16]. Mares with two ovulations or hemorrhagic-anovulatory follicles [16] were not used.
Experimental Groups
The first day of the experiment was 7 days after ovulation (Day 7) and the last day was Day 27. Mares were randomized into controls, follicle ablation, and ovariectomy groups (n = 6/group). Day 7 was chosen for initiating the experimental procedures (follicle ablation, ovariectomy) by considering the day when LH and estradiol reductions were expected to approach baseline but before the peak of the wave-stimulating FSH surge for the ovulatory wave. The mares were acclimated and trained for frequent handling and ultrasound procedures. Follicle ablations were done while the mare was standing in a simple box chute. A low dose of a short-acting tranquilizer was used, as described [15]. Potential sources of stress from herding through lanes, standing in the chute, and transrectal ultrasound examination were present for both the control and follicle-ablation groups. Follicles were ablated by transvaginal ultrasound-guided aspiration of follicle contents, as described [15, 16]. Follicles 6 mm were ablated on Day 7, and thereafter, every 2 days throughout the experiment, newly emerging follicles 6 mm were ablated. It was expected that ablations every 2 days would prevent growth of new follicles to >10 mm, based on previous study of the postablation growth of new follicles [15]. For ovariectomy, sedation and analgesia were induced [17] and ovaries were removed with a chain écraseur through a colpotomy [18], as described. The diameter of the largest follicle and corpus luteum in the controls and diameter of all follicles 6 mm and the corpus luteum in the follicle-ablation group were recorded every 2 days. When the largest follicle reached 28 mm in the controls, the ovaries were examined daily to determine the day of ovulation at the end of the experimental interovulatory interval. The expected beginning of deviation in the controls was assigned to the day the largest follicle was closest to 22.5 mm, based on previous studies [2].
Daily jugular blood samples were collected on Days 7–27 into heparinized tubes. Blood samples were centrifuged (1500 x g for 10 min) and decanted, and plasma samples were stored (–20°C) until assayed. Assays for progesterone, FSH, and LH were done on the daily samples collected over Days 7–27. Estradiol was assayed for samples collected every 2 days. Assay of ir-inhibin was done daily only for Days 7–17 because of a shortage of antiserum.
Hormone Assays
Plasma samples were assayed for FSH and LH by radioimmunoassay as validated and described for mares in our laboratory [19]. The intra- and interassay coefficients of variation (CV) and mean sensitivity, respectively, were 2.5%, 6.3%, and 1.0 ng/ml for FSH and 5.1%, 10.3%, and 0.2 ng/ ml for LH. The concentrations of ir-inhibin were measured by a double-antibody radioimmunoassay kit (Institute of Reproduction and Development, Monash Medical Center, Clayton, Australia) as validated and described in our laboratory for mare plasma [5]. The antibody recognizes dimeric forms of inhibin, as well as free 
subunits [20]. Nevertheless, ir-inhibin is a good indicator of FSH inhibitory activity in mares, as indicated by the consistent reciprocal relationship between circulating ir-inhibin and FSH concentrations during follicular wave development [5, 6]. Furthermore, during the last half of the anovulatory season and early ovulatory season, the profiles for circulating ir-inhibin [21] were similar to those for inhibin A [7]. The intraassay CV and assay sensitivity for ir-inhibin were 6.1% and 1.4 ng/ml, respectively.
Progesterone concentrations were determined in plasma extracted with petroleum ether and assayed with a competitive ELISA as reported for cattle [22]. The color intensity of the enzyme substrate is inversely proportional to progesterone concentration. To validate the assay for use with mare plasma, the unknown plasma samples (200 µl) were ether extracted and the dried extract was dissolved in 500 µl of ELISA assay buffer. Progesterone concentrations were determined in 100 µl of dissolved ether extract in duplicate wells for each sample. Serial volumes of a pool of diestrus mare plasma (100–400 µl) processed similarly as the unknown samples resulted in a displacement curve that was similar to the standard curve. The intra- and interassay CVs for assaying the experimental samples were 7.2% and 13.6%, respectively, and the sensitivity was 0.03 ng/ ml. Plasma concentrations of estradiol were measured by a double-antibody radioimmunoassay kit (Double Antibody Estradiol; Diagnostic Products Corporation, Los Angeles, CA) as described for mares in our laboratory [14], except for the following modifications. Standards (1.0–100 pg/ml) were prepared in steroid-free (charcoal-extracted) equine plasma. The standards and plasma samples (400 µl) were extracted simultaneously with diethyl ether before the assay. The intraassay CV and sensitivity were 8.9% and 0.55 pg/ml, respectively.
Statistical Analyses
Data for each hormone were challenged for extreme values with the Dixon outlier test [23] and for normality with the Kolmogorov-Smirnov test [24]. Data for estradiol, progesterone, and LH were not normally distributed and were transformed to natural logarithms before analysis. The difference in concentrations of ir-inhibin between the controls and the other two groups on Day 7 (first day of experiment) approached significance (P < 0.09). It was thought that a disparity between groups on Day 7 could contribute to significant differences on later days. In this regard, significant autocorrelation in hormone concentrations among days has been shown in heifers [25]. In the present study, ir-inhibin concentrations within mares in the ovariectomy group were highly correlated among days, based on Pearson correlations. For example, the concentrations on Day 7 were correlated with Day 10 (day with lowest mean; r = 0.93) and Day 12 (day with highest subsequent mean; r = 0.97). Because of the Day 7 disparity and the subsequent autocorrelations, data for ir-inhibin were converted to percentage change from the concentrations on Day 7, and the percentage change was compared among the three groups. For the three-group analyses, sequential hormone data or percentages were examined using the MIXED procedure of SAS with a repeated measures statement and a first-order autoregressive structure to account for the autocorrelation between measurements (SAS Institute Inc., Cary, NC). Main effects of group and day and their interaction were determined. When an interaction was significant or approached significance, the Tukey test for multiple comparisons among the three groups was used for each day.
Two-group analyses also were used. For estradiol, the ovariectomy group was not included because concentrations were below assay sensitivity in four of six mares the day after ovariectomy and remained low throughout the experiment. In addition, only the two groups were needed to test the hypotheses of follicle involvement in luteolysis and a negative effect of estradiol on LH. For ir-inhibin, the actual concentrations were analyzed for the follicle-ablation and ovariectomy groups; the controls were excluded because of the disparity among means on Day 7. Data were analyzed as for the three-group analyses, except that an interaction was further considered by a Student unpaired t-test within each day. Selected increases or decreases in concentrations between days within a hormone were examined by Student paired t-tests. For luteal diameters, a value of 10 mm (smallest recorded diameter) was used for illustrative purposes when a luteal structure was no longer detected, and a ranking test was used to analyze the diameter differences between the controls and the follicle-ablation group. Chi-square analysis was used to compare the number of mares with detected luteal structures between groups within each day. The rate of increase in FSH concentration between Days 7 and 8 in the ovariectomy group was compared with the rate of increase between the day before and the day of the maximum value in the FSH surge that seemed associated with follicle emergence in the controls; a 2 x 2 ANOVA was used. 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. Data are presented as the mean ± SEM, unless otherwise indicated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Diameter of largest follicle for the controls and follicle-ablation group showed significant interaction as well as main effects (P < 0.0001); the mean day of expected deviation (Day 14.7 ± 0.5) and ovulation (Day 23.2 ± 0.5) for the controls are indicated (Fig. 2). Diameter of the largest follicle in the follicle-ablation group showed a significant (P < 0.0001) day effect. Diameter decreased (P < 0.05) between Days 7 and 9 and diameter of the largest newly emerging follicle decreased (P < 0.05) between Days 9–27. Number of new follicles 6 mm in the follicle-ablation group were different (P < 0.0001) among days. Number of follicles decreased between Days 7 and 9 and continued to decrease (P < 0.05) between Days 9 and 27. At least one 6-mm follicle was present at 58% of the ablation sessions, and this result was consistent among mares (50–67%). The diameter of the largest follicle and number of follicles 6 mm per day averaged over Days 9–27 in the follicle-ablation group were 7.6 ± 0.3 mm and 2.9 ± 0.3 follicles, respectively.
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There was a group-by-day interaction (P < 0.0001) for the controls and follicle-ablation group for estradiol (Fig. 3). Concentrations decreased (P < 0.01) in both groups between Days 7 and 11. Concentrations continued to decrease in the follicle-ablation group with a decrease (P < 0.05) as late as Days 19–27. In the controls, concentrations increased (P < 0.04) between Days 15 and 17. Estradiol concentrations decreased precipitously after ovariectomy and were below assay sensitivity in four of six mares on Day 9.
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The main effects (group, day) and interaction were significant (P < 0.0001) for progesterone concentrations (Fig. 4). The concentrations decreased (P < 0.0003) between Days 7 and 8 and were lower (P < 0.05) in the ovariectomy group than in the other two groups on Days 8–14. There were no differences among the three groups on Days 15– 23. Concentrations in the controls increased (P < 0.05) between Days 23 and 27. Diameter of the corpus luteum showed a day effect (P < 0.0001), and the group effect approached significance (P < 0.1; Fig. 4). Diameter was greater (P < 0.05) in the follicle-ablation group than in the controls on Days 21, 23, and 25. The first day that the luteal structure was no longer detected was later (P < 0.0006) in the follicle-ablation group (Day 27 in all mares) than in the controls (Day 22.7 ± 1.4). On the mean day of ovulation (Day 23), the structure was detected in only two of six controls compared with six of six follicle-ablation mares (P < 0.02). Nondetection of the luteal structure (<10 mm diameter) in the controls did not occur until about 6 days after progesterone had decreased to minimal concentrations.
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The group and day effects for the three groups over Days 7–17 and the interaction for percentage change in ir-inhibin were significant (P < 0.003; Fig. 5). Percentage reduction from Day 7 was greater (P < 0.05) in the ovariectomy group than in the controls on each day and was greater (P < 0.05) in the follicle-ablation group on each day from Days 12–17. Percentage reduction was greater (P < 0.05) in the ovariectomy group than in the other two groups on Days 8 and 9. In the two-group comparison between the follicle-ablation and ovariectomy groups, the interaction was significant (P < 0.0001). Concentrations decreased between Days 7 and 8 in the follicle-ablation group (P < 0. 03) and in the ovariectomy group (P < 0. 001). Concentrations were lower in the ovariectomy group on Day 9 (P < 0.04) and Day 10 (P < 0.02) and increased (P < 0.0001) between Days 10 and 11.
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Each main effect (group, day) and the interaction for circulating concentrations of FSH were significant (P < 0.0001; Fig. 6). Concentrations increased (P < 0.02) dramatically between Days 7 and 8 in the ovariectomy group and more slowly (Days 7 and 12; P < 0.002) in the follicle-ablation group. Concentrations were greater (P < 0.05) each day in the ovariectomy group than in the other two groups from Day 8 (day after ovariectomy) until Day 19, except on Days 9 and 11. Concentrations were greater (P < 0.05) each day in the follicle-ablation group than in the controls from Day 14 to Day 27. The rate of increase in FSH between Days 11 and 17 was not different between the ovariectomy group (1.6 ± 0.5 ng/ml/day) and the follicle-ablation group (1.7 ± 0.5 ng/ml/day). Mean FSH concentrations in the ovariectomy group reached maximum on Day 18; the confidence limits for the means on Days 18– 27 are shown (Fig. 7). The maximum value during Days 7–27 for the apparent wave-stimulating FSH surge in each mare in the controls was below the lower mean confidence limit in the ovariectomy group on Days 18–27. The maximum value for FSH surges (29.2 ± 3.1 ng/ml; P < 0.0004) and the day of occurrence (Day 9.0 ± 0.6; P < 0.0006) in the controls were less than in the ovariectomy group (64.0 ± 6.6 ng/ml; Day 21.0 ± 2.6). The increase in FSH concentrations for the day preceding the maximum value in controls and on the day following ovariectomy are shown (Fig. 8); the day effect was significant (P < 0.0009), but the group effect and the interaction were not.
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The day effect and the interaction for concentrations of LH were significant (P < 0.0001; Fig. 6). Concentrations were greater (P < 0.05) in the ovariectomy group than in the controls and follicle-ablation group on each of Days 11–15, 17, and 18. There were no differences between the controls and follicle-ablation group on any day in the three-group analysis. The first increase (P < 0.05) in LH concentrations occurred between Days 13 and 15 in the follicle-ablation group and between Days 7 and 9 in the ovariectomy group. Mean concentrations of LH in the control group were at a maximum on Day 24, and there were no significant differences among the three groups on Day 24. However, variability in maximum values among individual controls was extensive, as indicated by two, two, and two values above, within, and below the confidence limits for the ovariectomy group (not shown). When the LH analysis was limited to the controls and follicle-ablation group, the interaction approached significance (P < 0.1); concentrations in the follicle-ablation group were greater (P < 0.05) than in the controls on Days 15, 16, and 20 and approached being greater (P < 0.09) on Day 25.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6.0 mm. In the present experiment, the follicle-ablation procedure allowed testing of the hypotheses, as indicated by significantly greater concentrations of FSH in the ablation group than in the controls by Day 14 and prevention in the ablation group of the rise in estradiol that began on Day 15 in the controls.
Changes in diameter of the largest follicle and corpus luteum and circulating concentrations of FSH, LH, ir-inhibin, estradiol, and progesterone and the temporal interrelationships among the ovarian structures and hormones in the control mares are consistent with literature reports (see Introduction). The small diameter increase of the largest follicle in the controls on Days 7–11 can be accounted for by obscurement of the growing follicles of the ovulatory wave by follicles from previous waves [26]. Periodic ablation sessions were effective in preventing the growth of newly emerging follicles to >10 mm; only five of 50 follicles grew to >10 mm by the day of an ablation session. The decrease between Days 9 and 27 in diameter of largest follicle, number of follicles, and concentrations of estradiol in the follicle-ablation group occurred despite an associated continuing increase in FSH. This result indicated that follicles were being ablated faster than they were replaced. Although the number of follicles emerging in the controls during the experimental period was not determined, the number of follicles ablated during Days 9–23 (mean, 26) in the ablation group seems to exceed the reported mean number 6 mm that developed naturally between Day 10 and ovulation in a previous study (16 follicles [27]).
Estradiol concentrations were similar between the controls and follicle-ablation group on Days 7–13; an expected more precipitous decrease in the follicle-ablation group did not occur. Suggested interpretations are 1) follicles <10 mm produced the estradiol in both the controls and follicle-ablation group; 2) the granulosa cells in the follicle-ablation group on Day 7 continued to function for several days; and 3) the estradiol at this time originated from a nonfollicular, but ovarian, source. In regard to the latter interpretation, midcycle equine corpora lutea produce estradiol in vitro [28] and secrete estradiol in vivo during pregnancy in response to eCG [29]. Further study will be needed to determine the estradiol source before Day 13 under these experimental conditions as well as in control mares.
Progesterone concentrations were similar for the controls and follicle-ablation group until the increase in the controls beginning at Day 23 or the mean day of ovulation and therefore did not support the hypothesis that follicles and circulating concentrations of estradiol play a role in luteolysis in mares. In addition, luteolysis in the controls, as indicated by a progesterone decrease, was underway before the occurrence of a significant increase in circulating estradiol. However, morphologic regression (luteal diameter decrease) occurred more slowly in the controls than functional regression (progesterone decrease). Although the characteristics of the circulating progesterone decrease [30] and luteal diameter decrease (based on ultrasound; [31]) have been reported independently, this is apparently the first report of the asynchronous decrease in the two events in mares, but similar asynchronous regression has been reported for heifers [32]. In the present study, the slower rate of morphologic regression in the follicle-ablation group than in the controls partly supported the hypothesis by indicating on a temporal basis that this portion of luteolysis does involve the follicles and on a temporal basis may have involved estradiol secretion of the developing dominant follicle. Thus, the results indicated that luteolysis in mares has a morphologic component that involves the follicles, perhaps through estradiol production, as well as a reported functional component that involves endometrial prostaglandin F2 production [11].
The ir-inhibin results in the controls are consistent with the reports [5, 6] of a gradual increase in concentrations from 1 or 2 days before the largest follicle reached an estimated 13 mm until the expected beginning of deviation, followed by a plateau. The greater decrease in ir-inhibin after ovariectomy than after follicle ablation is consistent with the secretion of inhibin by follicles <10 mm in diameter. An average of about three follicles growing to 7.6 mm (largest follicle) during 2-day intervals in the follicle-ablation group resulted in ir-inhibin concentrations that initially were above those of the ovariectomy group. These results are consistent with the report [5] that, following ablation of all follicles 6 mm, inhibin began to increase when the largest new follicle was a mean of 7.7 mm. Nonfollicular ovarian sources could have been involved in the inhibin secretion in the present study, as suggested for estradiol, but apparently there is no literature support for this alternative. The marked mean decrease on Days 7–8 and the rebound between Days 10 and 11 occurred in each ovariectomized mare. The rebound raised the concentrations to a level similar to the concentrations in the follicle-ablation group. The concentrations then remained similar for the follicle-ablation and ovariectomy groups until the last day of assay (Day 17). The dramatic increase following the decrease in ir-inhibin concentrations in the ovariectomy group is perplexing and if confirmed will require elucidation of the underlying mechanisms.
The irregularity in the mean FSH concentrations in the controls on Days 7–12 is attributable to the occurrence of the peak of an FSH surge on different days among mares. The increase in FSH in the controls beginning at the mean day of ovulation is consistent with postovulation emergence of anovulatory follicular waves [26]. The dramatic FSH increase during the day after ovariectomy is consistent with the dramatic ir-inhibin decrease and the discharge of stored FSH. The subsequent slower increase in FSH concentrations between approximately Days 8–17 likely reflected synthesis and release until maximum output was reached on Day 18. The similar rate of increase in FSH secretion during Days 11–17 in the follicle-ablation and ovariectomy groups occurred during the days that ir-inhibin concentrations were stabilized and similar between groups. The FSH increase therefore is attributable to an extraovarian effect.
The hypothesis of a continuous negative influence of the ovaries, even during the maximal concentrations of an FSH surge, was supported. The 95% confidence limit below the mean for FSH in the ovariectomy group during the plateau in mean concentrations was used to evaluate the surges in controls. The maximal concentration of the FSH surge was below the confidence limit in each of the six mares and was well below in five of six. As a reservation, an FSH surge was based on daily sampling. However, daily sampling has provided FSH concentration profiles that have been compatible consistently with the time of emergence and deviation of follicular waves, including surges induced after ablation of follicles (reviewed in [2]). Pulses of FSH, which can be adequately characterized by sampling at 4-h intervals [33], are superimposed on the surges that are detected with daily sampling. The pulses, therefore, are a major source of the variation in daily samples [33] and may have caused underestimation of the maximal value for individuals in the present study. This consideration does not negate the support for the hypothesis but, encouraged by the present results, does indicate a need for further study with more frequent sampling. An observation that is consistent with a strong negative effect of ovarian hormones is that the daily rate of increase in FSH after removal of the negative suppression of the ovaries was similar to the rate of increase for the day preceding the peak of an FSH surge. Thus, the inclining portion of the FSH surge appears to represent release from a negative ovarian effect rather than from the stimulation of a positive ovarian effect.
In contrast with the wave-stimulating FSH surge, the periovulatory LH surge is more closely characterized by daily sampling [33]. The pattern of the LH increase following ovariectomy was similar to the FSH increase. However, in the follicle-ablation group, the pattern of LH increase following the first ablation session on Day 7 was dissimilar from the FSH increase; LH concentrations for the week after Day 7 were similar to those in controls. The suppression of LH at this time can be attributed to a negative effect of progesterone in both the controls and follicle-ablation groups, whereas the rapid increase in the ovariectomy group can be accounted for by the loss of progesterone. The temporal progesterone/LH relationship was demonstrated within the controls and represented the reported negative effect of progesterone on LH (see Introduction).
Concentrations of LH were lower in the controls on Days 15 and 16 than in the follicle-ablation group. Thereby, the portion of the hypothesis of an initial negative effect of the follicles on the LH surge was supported. However, the interaction that justified the within-day comparisons only approached significance, indicating a need for further study. The first significantly lower LH concentration in the controls than in the ablated group (Day 15) occurred before estradiol increased in the controls (after Day 15). The negative effect of the follicles on LH, therefore, was not temporally attributable to increased estradiol and also will require additional study. The last day of a temporal indication of a negative effect of the follicles or estradiol on LH was Day 20. Thus, for a few days before the maximum concentrations of LH in the controls, neither a negative nor positive effect of the follicles or estradiol was evident in the comparison of LH concentrations between the controls and follicle-ablation group. That is, the portion of the hypothesis on a later positive effect of the follicles or estradiol on LH was not supported. In this regard, the absence of a positive effect of estradiol on LH is consistent with increasing LH concentrations in controls from 2 days before through 1 day after ovulation when estradiol was decreasing. The mean maximal LH concentrations in controls were not different from the mean concentrations in ovariectomized mares, also indicating the absence of a positive contribution of the ovaries to the magnitude of the LH surge. However, reservation is indicated by the wide variability in the maximum value among individual controls. In addition, the lack of support for a positive effect of estradiol on LH contrasts with previous results of an LH increase after estradiol treatment (see Introduction). Further study will be needed.
The FSH and LH results indicated that the ovarian control of gonadotropin surges is negative throughout the surges, except that no negative ovarian effects were detected for the maximum concentrations of the periovulatory LH surge. This conclusion encourages the concept that changes in circulating concentrations of FSH and LH during an estrous cycle reflect changes in a balance between a continuous positive impact of extraovarian sources and a continuous but changing negative effect of the ovaries. The nature of the extraovarian control will require study but involves the environment, as indicated by long-term ovariectomy studies; FSH and LH concentrations in ovariectomized mares are low during the anovulatory season and high during the ovulatory season (reviewed in [1]).
In summary, preventing the growth of follicles to 10 mm by ablation of newly emerging follicles every 2 days indicated that the follicles 10 mm were not involved in functional luteolysis, as indicated by systemic progesterone concentrations. However, involvement in morphologic regression of the luteal structure was indicated by a slower decrease in luteal diameter in the follicle-ablation group than in controls. Comparisons among control, follicle-ablation, and ovariectomy groups were interpretable on the basis of a negative effect of ovarian hormones on systemic gonadotropin concentrations, except that negative effects were not apparent at the maximum concentration of the periovulatory LH surge. The negative effect on FSH was attributable to inhibin with a later possible contribution of estradiol. The negative effect on LH was attributable to progesterone and estradiol. There was no indication of gonadotropin regulation by positive feedback effects of ovarian hormones. The novel concept that ovarian hormones give cyclicity to both gonadotropins in mares entirely by counteracting the positive effects of extraovarian influences will require confirmation and further elucidation, and the nature of extraovarian influences will also require study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: O.J. Ginther, Animal Health and Biomedical Sciences, 1656 Linden Drive, University of Wisconsin, Madison WI 53706. FAX: 608 262 7420; ginther{at}svm.vetmed.wisc.edu
Received: 21 January 2005.
First decision: 17 March 2005.
Accepted: 12 April 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C, FSH, and estradiol-17ß during estrous cycle in mares and their relationship with follicular growth. Endocrine 2004 25:7-14[Medline]
M. The late induction of prostaglandin G/H synthase-2 in equine preovulatory follicles supports its role as a determinant of the ovulatory process. Endocrinology 1997 138:4427-4434This article has been cited by other articles:
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  O.J Ginther, M.D Utt, M.A Beg, E.L Gastal, and M.O Gastal Negative Effect of Estradiol on Luteinizing Hormone Throughout the Ovulatory Luteinizing Hormone Surge in Mares Biol Reprod, September 1, 2007; 77(3): 543 - 550. [Abstract] [Full Text] [PDF]
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O.J. Ginther, E.L. Gastal, M.O. Gastal, M.A.R. Siddiqui, and M.A. Beg Relationships of Follicle Versus Oocyte Maturity to Ultrasound Morphology, Blood Flow, and Hormone Concentrations of the Preovulatory Follicle in Mares Biol Reprod, August 1, 2007; 77(2): 202 - 208. [Abstract] [Full Text] [PDF]
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 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]
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