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a Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706
b Veterinary Medicine, São Paulo State University, Araç;t1tuba, SP 16050-680, Brazil
c Department of Animal Science, Federal University of Viçosa, Viçosa, MG 36570-000, Brazil
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6 mm were ablated at Day 10 (Day 0 = ovulation) to initiate a new follicular wave; prostaglandin F2
was given to induce luteolysis, and progesterone was given from Days 10 to 24. The low dose did not significantly alter any of the ovarian or gonadotropin end points. The high dose reduced (P < 0.05) the ablation-induced FSH concentrations on Day 11. Maximum diameter of the largest follicle (17.2 ± 0.6 mm) and the second-largest follicle (15.5 ± 0.9 mm) in the high-dose group was less (P < 0.04) than the diameter of the second-largest follicle in the controls (20.0 ± 1.0 mm) at the beginning of deviation (Day 16.7 ± 0.4). Thus, the growth of the two largest follicles was reduced by the high dose, presumably through depression of FSH, so that the follicles did not attain a diameter characteristic of deviation in the controls. The intermediate dose did not affect FSH concentrations. However, the LH concentrations increased in the control, low, and intermediate groups, but then decreased (P < 0.05) in the intermediate group to pretreatment levels. The LH decrease in the intermediate group occurred 2 days before deviation in the controls. The maximum diameter of the largest follicle was less (P < 0.0001) in the intermediate group (27.3 ± 1.8 mm) than in the controls (38.9 ± 1.5 mm), but the maximum diameter of the second-largest follicle was not different between the two groups (19.0 ± 1.1 vs. 20.3 ± 1.0 mm). Thus, the onset of deviation, as assessed by the second-largest follicle, was not delayed by the decrease in LH. Diameter of the largest follicle by Day 18 in the intermediate group (23.1 ± 1.6 mm) was less (P < 0.05) than in the controls (28.0 ± 1.0 mm). These results suggest that circulating LH was not involved in the initiation of dominance (inhibition of other follicles by the largest follicle) but was required for the continued growth of the largest follicle after or concurrently with its initial expression of dominance.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30 mm, whereas a dominant follicle does not develop during minor waves. With use of a two-follicle model, the future dominant follicle was found to emerge at 6 mm at a mean of 1 day earlier than the future subordinate follicle [10]. The two follicles grew similarly until the larger follicle reached 21–23 mm at an average of 6 days after emergence of the future dominant follicle. Thereafter, the growth rates began to differ between the two follicles; this process has been called deviation [10, 11]. At deviation, the larger follicle apparently inhibited the smaller follicle [1] before it could reach a similar diameter an average of 1 day later [10]. The larger follicle usually became dominant and continued to grow to a preovulatory diameter of 30 mm, whereas the other follicle regressed. These findings indicated that the follicle destined to become dominant has a size advantage and is the first to reach a critical diameter. Similar results have been reported in cattle [3, 11], except for the differences between species in follicle diameters. Surges in circulating concentrations of FSH and LH are more synchronous during the luteal phase than during the follicular phase in mares [12, 13]. In the two-follicle model, concentrations of FSH increased after the day the larger follicle was 6 mm, peaked 3 days later when the larger follicle was 13 mm, and then declined to reach near-minimal levels a few days later at the time of follicle deviation [10]. Until the day of the FSH peak, which occurred during the progesterone decline, FSH and LH were closely associated and then became dissociated, with a decrease in FSH and an increase in LH. The surge in FSH that occurs in association with the preovulatory LH surge in cattle [3] does not occur in mares [7]. Follicle deviation occurred during increased LH and decreased FSH concentrations, and either or both hormonal conditions may be necessary for deviation to occur. Recently, the effects of the largest follicle on systemic estradiol and FSH concentrations were studied by ablation of the larger follicle at the expected beginning of deviation, using the two-follicle model [1]. The beginning of an increase in systemic concentrations of estradiol and a continued decrease in FSH were attributable entirely to the future dominant follicle; there was no indication that the smaller follicle was involved.
In mares with a prolonged progesterone phase (maintained corpus luteum), dissociation in directional change between FSH and LH concentrations did not occur [10]. Exogenous progesterone inhibited ovulation of the largest follicle and depressed circulating LH concentrations but not consistently; however, synthetic progestins are effective for applied control of the time of ovulation [7]. In cattle, exogenous progesterone inhibited the dominant follicle in a dose-dependent manner when the follicle was exposed during its growing phase, but suppression of FSH concentrations was not detected [14].
In the present experiment, the effects of progesterone on FSH and LH concentrations, as well as the resulting relationships between the changes in concentrations of FSH and LH and follicle development and deviation, were studied. The hypothesis was that a high dose of progesterone can negatively affect the maximum diameters of the largest follicle by depressing LH concentrations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nonlactating pony mares (n = 28) between 3 and 14 yr of age and weighing 200 to 415 kg were used from August to October. The mares were kept in partially sheltered outdoor paddocks and were maintained on alfalfa/grass hay with access to water and trace-mineralized salt. The ultrasound scanner was equipped with a 5-MHz linear-array transrectal transducer (Tokyo Keiki LS300; Products Group International, Lyons, CO) for examinations of the ovaries and uterus. All mares were in the ovulatory season as indicated by detection of ovulation and the formation of a corpus luteum by ultrasonography [15]. Ultrasonic examinations of the ovaries and uterus were done daily or every other day as described previously [15]. Mares were not used if they had indications of ovarian or uterine abnormalities. Before the start of the study, mares were synchronized with an injection of prostaglandin F2 (PGF2; 5 mg, i.m.; Lutalyse; Pharmacia & Upjohn Co., Kalamazoo, MI). Follicles 30 mm in diameter were monitored daily until the pretreatment ovulation (Day 0).
On Day 8, mares were randomized into experimental groups. On Day 10, all follicles 6 mm were ablated in all mares in each of the treatment groups. Immediately thereafter, mares of all groups received two i.m. injections of PGF2 (5 mg, 12 h apart) to induce luteolysis. Mares in the progesterone groups were given the assigned daily dose in 3 ml of safflower oil by injection of one half of the daily dose every 12 h from Day 10 until ovulation or Day 24. Injections were given i.m. on either side of the neck at alternate times in both experiments. Mares (n = 7 per group) were treated with the following doses of progesterone per day: 1) 0 mg (controls), 2) 30 mg (low dose), 3) 100 mg (intermediate dose), and 4) 300 mg (high dose).
Follicle Ablation
All follicles 6 mm were measured by transrectal ultrasonography using the height and width of the antrum at the maximal area prior to the ablations. Follicle ablation on Day 10 was done to facilitate the tracking of individual follicles of the new or postablation wave. Follicle ablations were done transvaginally by ultrasound-guided follicle entry, using an ultrasound scanner (Aloka SSD-500V; Aloka, Wallingford, CT) equipped with a 5-MHz convex-array transvaginal transducer (Aloka UST974V-5) as described previously [10, 15]. Briefly, 10 days after ovulation, the bladder was emptied, and all follicles ( 6 mm) were ablated by aspiration of follicular contents through a 17-gauge needle using a vacuum pump (250–300 mm Hg). Follicle ablation was defined by collapse of the antrum after evacuation of follicular contents. Ablated follicles that apparently refilled and grew to a diameter 15 mm were ablated again.
Ovarian Changes Postablation
Growing follicles ( 4 mm) of the new or postablation wave were tracked daily to maintain individual identity [15]. The relative locations of follicles, corpus luteum, and follicle-ablation sites (echoic areas) were used as references for identifying and tracking follicles. Diameter of follicles 4 mm and the corpus luteum was determined daily from the average of height and width for a single frozen image of the apparent maximal cross-sectional area for each structure [15]. Each follicle was measured until the largest follicle reached 15 mm. After the largest follicle was 15 mm, the diameter of the four largest follicles was measured daily until ovulation or Day 25 using three frozen images (six measurements per follicle). The four largest follicles were assessed to ensure including the two follicles that later reached the greatest maximum diameters. Beginning on Day 25 (day after last treatment), mares that did not have a 30-mm follicle and had not ovulated were scanned every other day until a 30-mm follicle appeared, and daily thereafter until the largest follicle ovulated or regressed as indicated by four successive days of decreasing diameters.
Definitions
The day of emergence of the postablation follicles was defined as the day before the follicle first exceeded 6 mm. Follicles that emerged on the same day or on successive days with no more than one elapsed day between them were considered to be part of the same wave. When 2 days elapsed, the follicles were considered to be part of a different wave. The beginning of deviation between the future dominant follicle (one that grew to a diameter of 30 mm) and largest subordinate follicle in individual waves was defined as the day at the beginning of the greatest difference in growth rates between the two largest follicles at or before the day when the largest subordinate follicle reached maximum diameter or an apparent diameter plateau [10, 11]. If no follicle reached 30 mm, deviation was recorded as not having occurred [9, 10]. Parallel changes in the same direction (i.e., increase or decrease) on a given day in systemic concentrations of LH and FSH were defined as an association between the two gonadotropins. Differences in gonadotropin dissociation among groups were objectively assessed by examining the day-to-day mean profiles of the LH:FSH ratios (LH concentration divided by FSH concentration on each day for each mare).
Blood Sampling and Hormone Assay
Blood samples were collected twice a day (10 ml every 12 h), beginning on Day 8 and ending on the day of the next posttreatment ovulation or Day 25. Samples were collected by jugular venipuncture into heparinized tubes and separated in a refrigerated centrifuge (4°C). Plasma was decanted into polyethylene vials and stored frozen (-20°C) until assay.
Circulating concentrations of FSH [16], LH [17], and progesterone [18] were determined using RIAs previously validated in this laboratory for this species. The intra- and interassay coefficients of variation and the sensitivity for the FSH, LH, and progesterone assays were as follows: FSH, 5.6%, 9.8%, and 0.2 ng/ml; LH, 3.3%, 7.5%, and 0.5 ng/ml; and progesterone, 3.4%, 5.2%, and 0.07 ng/ml.
Statistical Analyses
The sequential nature of the data for the ovarian and hormonal end points was examined using a split-plot ANOVA [19]. Main effects of group and day and the group-by-day interaction were determined. Variation due to the sequential nature of the data was accounted for by using the mare-by-group interaction as the whole-plot error term to test the effect of group. When a significant effect of group, day, or an interaction of group by day was detected, Duncan's multiple-range tests were used to locate mean differences among groups within days. Daily changes in hormone concentrations from Day 8 and follicle diameter from Day 11 were compared among groups until the last day in which the data from all mares in each group could be included as determined by the first day of an ovulation. ANOVA was used for determining a group effect for single-point measurements (e.g., day of follicle emergence, maximum follicle diameter, and follicle growth rate), and Duncan's multiple-range tests were used to locate mean differences between groups. Proportional data were examined using chi-square analysis for determining group effects. Data are presented as the mean ± SEM unless otherwise indicated. Significance was indicated by a probability of P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The low dose (30 mg/day) of progesterone did not significantly alter any of the ovarian end points, including day of ovulation and growth of follicles, despite the more than 3-fold increase in mean circulating progesterone concentrations as compared to the control value (0.8 vs. 2.6 ng/ml). The progressively increasing length of the interovulatory interval for the intermediate (100 mg) and high (300 mg) doses reflected the mean increase in circulating progesterone concentrations (6.4 vs. 11.9 ng/ml) and is attributed to interference with the day of ovulation. Only 1 of 14 mares in these two groups ovulated from the first postablation follicular wave. The ovulations occurred from a subsequent wave, and the preovulatory diameter of the follicles was not affected. Progestin regimens have been used in mares to inhibit ovulation and to synchronize ovulations after withdrawal of the progestin [7].
This is apparently the first study to critically examine the effects of progesterone on follicles early in the follicular wave from follicle emergence until after the expected day of follicle deviation. The intermediate and high doses of progesterone did not alter the day of follicle emergence, but they negatively affected the diameter attained by the follicles. The intermediate and high doses of progesterone interfered with growth of the largest follicle, and the interference was greater for the high dose as indicated by the following: 1) reduced maximum diameter (means, 27.3 vs. 17.2 mm for intermediate and high dose, respectively); 2) reduced growth rates over Days 11–22 (2.0 vs. 0.9 mm/day); and 3) significant interaction for the growth profiles, which was attributable to smaller diameters beginning on Day 18 in the intermediate-dose group and Day 15 in the high-dose group. In addition, the high dose, but not the intermediate dose, negatively affected the second-largest follicle as indicated by the reduced maximum diameter and growth rate. The maximum-diameter difference between the two largest follicles was less for the high dose than for the intermediate dose. These results represent the negative effect on only the largest follicle for the intermediate dose and on both follicles for the high dose. The two follicles were inhibited to a similar extent for the high dose.
The absence of deviation in growth rates between the two largest follicles in the intermediate- and high-dose groups, except in two mares in the intermediate group, reflects the inhibitory effect of progesterone on the follicles. The beginning of deviation in the control and low-dose mares occurred on mean Day 17, and the first day of a significant reduction in diameter of the largest follicle in the intermediate group was Day 18. The absence of deviation in 5 of 7 mares in the intermediate group was based on failure of the largest follicle to attain 30 mm [9, 10]. The two mares with deviation in the intermediate group had dominant follicles that reached 31 and 33 mm; in the remaining mares in this group, the largest follicle reached 18–29 mm. In cattle [14], exogenous progesterone was given early in a wave, similar to the procedure in the present study in mares. The mean maximum diameter of the dominant follicle was reduced in the treated group (12.7 ± 0.9 mm) compared to the control group (15.3 ± 0.7 mm). The inhibitory effects on growth of the largest follicle were not significant until 3 days after ovulation, which would be near the expected day [11] of deviation in cattle. Thus, the cattle results are consistent with the present mare results in terms of initiation of growth suppression of the largest follicle at the approximate time of deviation, although the day of deviation was not determined in the cattle study.
Circulating concentrations of FSH and LH were not altered significantly by the low dose of progesterone, accounting for the absence of detected effects on the follicles. The high dose of progesterone resulted in an initial depression in FSH concentrations on Days 11.0 and 11.5 at a time when FSH was increasing in the other groups. After Day 15, the concentrations of FSH began to fluctuate significantly for both the intermediate and high doses as indicated by transient and periodic peaks in the means toward the end of treatment. The effect of this FSH response on follicular dynamics was not apparent. In a study in ovariectomized mares, treatment with progesterone increased the FSH secretion [21]. In the present study, the low dose of progesterone beginning on Day 10 was associated with an increase in FSH concentrations over Days 12–17, but this effect was not significant according to multiple-range tests. Other studies on the effects of exogenous progesterone on FSH concentrations produced negative or equivocal results [7, 22]. With regard to conflicting reported results, the present results demonstrated that the effect of progesterone on FSH is influenced by dose.
The results for both the intermediate and high doses supported the hypothesis that elevated progesterone concentrations would depress LH concentrations and thereby interfere with development of the largest follicle. The depressing effect of exogenous progesterone on circulating LH has been previously reported for ovariectomized mares [23, 24] and ovarian-intact mares late in diestrus [22]; progesterone prevented the increase in LH following luteolysis. On the basis of the results of a study of pulsatile release of LH from the pituitary in mares [25], it was proposed that endogenous progesterone results in slower frequency of episodic GnRH release and lower circulating concentrations of LH without altering the FSH output. Similar GnRH-induced gonadotropin responses have been reported for steroid-treated ovariectomized mares [26].
The LH:FSH ratio in the low-dose group was reduced after Day 16.5 but apparently did not affect follicle growth. The higher LH:FSH ratios in the controls and low-dose groups as compared to the intermediate- and high-dose groups were primarily a result of the start of dissociation between LH and FSH concentrations beginning a few days before deviation. For the intermediate and high doses of progesterone, the close association in directions of change between FSH and LH concentrations continued throughout progesterone treatment. The association is indicated by the unchanging LH:FSH ratios. A similar association occurred in a mare with a maintained corpus luteum [10]. Thus, dissociation between FSH and LH did not occur during prolonged natural and experimental progestational states.
The intermediate and high doses of progesterone altered the gonadotropin concentrations differently, in a manner that would account for the different effects on the follicles. The reduction in growth rates of both the largest and second-largest follicles resulted in smaller diameters by Days 15 and 16, respectively, in the high-dose group and is attributable to the initial FSH depression on Day 11. The initial depression in FSH concentrations did not prevent emergence of follicles but apparently did prevent the follicles from reaching the diameters characteristic of deviation; neither the largest follicle nor the second-largest follicle in the high-dose group attained the diameters of the corresponding follicles at the beginning of deviation in the controls. However, the concentration of LH did not increase after the high dose was initiated as it did for the other three groups, and the effect of reduced LH cannot be separated from the effect of reduced FSH.
At the time LH concentrations began to increase in the control and low-dose groups, an initial increase in LH also occurred in the intermediate group. Later (beginning on Day 15), the concentrations in the intermediate group were significantly less than in the controls; the concentrations returned to the pretreatment concentrations. This effect on LH likely accounts for the depression in growth of the largest follicle but not the second-largest follicle in the intermediate group. The depressed LH concentrations in the intermediate group reached significance 2 days before the mean day of deviation in controls. The maximum diameter and growth pattern of the second-largest follicle in the intermediate group were not different from these end points in the controls. These results indicate that circulating LH concentrations were not involved in the cessation or reduction in growth rate of the second-largest follicle at the beginning of deviation. That is, the deviation mechanism apparently was activated at the normal time and at normal follicle diameters in the intermediate group, based on the second-largest follicle, despite the low LH concentrations. Results of a recent follicle-ablation study [1] indicated that the largest follicle establishes dominance in mares by reducing the concentrations of circulating FSH below the requirement of the other follicles. In the present study, deviation occurred on mean Days 16.7 to 17.3 in the groups with deviation, whereas significant depressed growth of the largest follicle occurred on mean Day 18 in the intermediate-dose group. These results indicate that LH was needed for continued growth of the largest follicle, during the time when the smaller follicles were being inhibited. The results do not clarify, however, the precise temporal relationship between the following two apparent components of the equine deviation mechanism: 1) the inhibition of the smaller follicles by the FSH-suppressing action of the larger follicle [1] and 2) the change in gonadotropin dependency of the largest follicle from FSH to LH. The effects of progesterone on the largest follicle in association with depressed LH concentrations seem similar to the results of a study in cattle, involving LH depression by chronic delivery of a GnRH agonist [27]. Concentrations of LH fell to apparent basal levels, and the largest follicle did not grow beyond 7–9 mm, which was near the expected time of deviation in other studies in cattle [11, 28]. However, follicle growth profiles were not given for the cattle study, and therefore it is not known whether the follicle inhibition associated with reduced LH began before, during, or after the beginning of follicle deviation.
A temporal indication for a role of LH in the continued growth of the largest follicle after the beginning of deviation is that a transient elevation in LH concentrations usually encompasses the day of deviation in mares [1, 10] as well as in cattle [21]. Although estradiol concentrations were not assessed in the present study, reports on systemic and intrafollicular estradiol concentrations [29], as well as the differential estradiol output of the largest follicle versus the second-largest follicle at the expected beginning of deviation [1], have indicated that estradiol is a candidate for involvement in the deviation mechanism in mares. A possible role of estradiol may be the facilitation of a change in gonadotropin dependence from FSH to LH in the largest follicle, allowing it to continue growing despite the depressed FSH. Speculation on the potential manner in which estradiol could be involved in the utilization of LH has been reviewed for cattle [3].
In conclusion, prolonged administration of a high dose of progesterone (300 mg/day) on Days 10–24 inhibited the circulating concentrations of FSH on the approximate day of emergence of the largest follicle of the postablation follicular wave at 6 mm. Growth rate of the two largest follicles was reduced so that neither follicle reached the diameter characteristic of deviation in nontreated mares; the two follicles were similarly affected. Prolonged administration of an intermediate dose (100 mg/day) of progesterone did not alter the postablation changes in FSH concentrations but reduced the LH concentrations by Day 15, which was 2 days before the mean day of follicle deviation in the controls. The diameter profile of the second-largest follicle was equivalent between the intermediate group and the controls, suggesting that the onset of the deviation mechanism was not altered by the reduced LH. However, growth of the largest follicle was depressed by Day 18 in the intermediate group, suggesting a requirement for LH in the continued growth of the dominant follicle after or concurrently with its initial expression of dominance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: O.J. Ginther, Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1656 Linden Drive, Madison, WI 53706. FAX: 608 262 7420; ojg{at}ahabs.wisc.edu
Accepted: July 14, 1999.
Received: May 19, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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