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BOR - Papers in Press, published online ahead of print June 6, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.061705
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BIOLOGY OF REPRODUCTION 77, 543–550 (2007)
DOI: 10.1095/biolreprod.107.061705
© 2007 by the Society for the Study of Reproduction, Inc.

Negative Effect of Estradiol on Luteinizing Hormone Throughout the Ovulatory Luteinizing Hormone Surge in Mares1

O.J Ginther 2 3 4, M.D Utt 3, M.A Beg 4, E.L Gastal 4, and M.O Gastal 3

Eutheria Foundation,3 Cross Plains, Wisconsin 53528 Department of Pathobiological Sciences,4 University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT

The negative effect of estradiol-17ß (E2) on LH, based on exogenous E2 treatments, and the reciprocal effect of LH on endogenous E2, based on hCG treatments, were studied throughout the ovulatory follicular wave during a total of 103 equine estrous cycles in seven experiments. An initial study developed E2 treatment protocols that approximated physiologic E2 concentrations during the estrous cycle. On Day 13 (ovulation = Day 0), when basal concentrations of E2 and LH precede the ovulatory surges, exogenous E2 significantly depressed LH concentrations to below basal levels. Ablation of all follicles ≥10 mm when the largest was ≥20 mm resulted in an increase in percentage change in LH concentration within 8 h that was greater (P < 0.03) than for controls or E2-treated/follicle-ablated mares. Significant decreases in LH occurred when E2 was given when the largest follicle was either ≥25 mm, ≥28 mm, ≥35 mm, or near ovulation. Treatment with 200 or 2000 IU of hCG did not affect E2 concentrations during the initial portion of the LH surge (largest follicle, ≥25 mm), but 2000 IU significantly depressed E2 concentrations before ovulation (largest follicle, ≥35 mm). Results indicated a continuous negative effect of E2 on LH throughout the ovulatory follicular wave and may be related to the long LH surge and the long follicular phase in mares. Results also indicated that a reciprocal negative effect of LH on E2 does not develop until the E2 surge reaches a peak.

estradiol, follicle, human chorionic gonadotropin, luteinizing hormone, mares, ovulation

INTRODUCTION

The length of the luteal phase during the equine estrous cycle or the interval from ovulation to a progesterone decrease to <2 ng/ml is approximately 16 days [reviewed in 1]. The length of the follicular phase is 7 days, with ovulation occurring at a follicle diameter of approximately 40 mm. The ovulatory follicular wave begins several days before the end of the luteal phase, with multiple follicles growing at a similar rate during a common growth phase. Near the end of the luteal phase, the largest follicle reaches 22–24 mm, and diameter deviation begins. Deviation is recognized retrospectively by continued growth of one follicle and reduced growth or regression of the remaining follicles. Circulating concentrations of estradiol (E2; [2]) and LH [3] begin to increase 1 or 2 days before the beginning of deviation. The E2 concentrations increase to a peak 2 days before ovulation when the preovulatory follicle is about 35 mm [3, 4]. The LH concentration increases at a slow rate (first segment of the LH surge) until the peak of the E2 surge [4]. During a second segment of the LH surge, concentrations increase at a more rapid rate until reaching maximum the day after ovulation. Thus, the rapid increase in LH is temporally associated with decreasing E2 (Fig. 1).


Figure 01
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  		FIG. 1 Means (±SEM) from published reports [3, 9] to illustrate the days and follicle diameters when experiments were initiated (Ex2 = experiment 2 and so on). Luteolysis indicates the beginning of a 2-day rapid decrease in progesterone. Days were selected on the basis of relative endogenous concentrations between estradiol and LH during the ovulatory follicular wave.

A decrease in progesterone and an increase in LH begin simultaneously [4]. A negative effect of progesterone on LH is indicated by this temporal relationship and by studies using exogenous progesterone. Treatment with progesterone during early development of the ovulatory follicular wave reduces the circulating concentrations of LH [5–7]. 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 [8]. Periodic ablation of new follicles when they reach ≥10 mm results in greater LH concentrations throughout the first segment of the LH surge [9]. An LH-inhibiting factor lost by follicle ablation may be E2, but this has not been studied directly.

Depending on the stage of the estrous/menstrual cycle, E2 exerts either a positive or a negative effect on the hypothalamic-pituitary axis to regulate the synthesis of LH [10]. In regard to reported positive effects of E2 on plasma LH, the preovulatory increase in E2 is believed to trigger release of the LH surge in species with a rapidly developing surge of short duration (e.g., cattle [11, 12]). The LH surge is essential for the synthesis of E2 and ovulation [11]. The LH surge is blocked when an E2 antiserum is given [13]. In sheep, a positive regulatory effect of E2 on LH has been demonstrated by several experimental approaches [14]. In mares, it has been concluded that the LH surge that induces ovulation is caused by the positive feedback action of the preceding estradiol surge, as in other species [15]. The initiation of the ovulatory season in mares has also been proposed to involve a positive effect of E2 on LH [16] and has been attributed to the stimulation of GnRH secretion [17–19]. In an in vitro study with anterior pituitary cells, E2 did not stimulate LH release directly but increased the GnRH-stimulated release of LH [15]. However, in vivo treatment with E2 did not affect the LH response to GnRH [20, 21]. An injection of E2 near the beginning of estrus resulted in an increase in LH concentration [22]. Daily treatment with a massive dose of E2 (10 mg) for about a month beginning at ovulation did not affect LH during the luteal phase; however, the authors concluded that E2 had a positive effect on LH secretion for up to 2 wk after luteolysis [23]. In a similar study [24], LH was elevated at the ovulation following the initiation of treatment, but it was stated that this may have resulted from a positive effect of E2 or an extended follicular phase.

Ovariectomy of mares was an early approach to studies on the interaction of extraovarian and ovarian factors in the regulation of LH [25]. Concentrations of LH follow a seasonal pattern in the absence of the ovaries. High concentrations during the equivalent of the ovulatory season represent the influence of extraovarian control. Exogenous E2 has a positive effect on the LH concentrations in ovariectomized mares [26–28]. The LH concentrations in ovariectomized mares during the ovulatory season seem similar to concentrations during estrus [9, 25, 29, 30]. The apparent additional increase in LH in ovarian-intact mares at the postovulatory peak of the LH surge may be a function of a positive effect of the estradiol surge. However, an adequate comparison has not been done between LH concentrations in ovariectomized mares and the concentrations at the peak of the LH surge in ovarian-intact mares.

A negative effect of E2 on LH has been demonstrated in mice and sheep [14]. In sheep, E2 treatment inhibited the LH pulse amplitude and decreased the expression of LH mRNA in the pituitary. In mares, exogenous E2 reduced the circulating concentrations of LH at the expected beginning of deviation [31]. In ovariectomized mares, a single injection of E2 induced a decrease in LH concentrations over the first few hours, an increase between 8 and 12 h, and then a decrease [32]. In summary, published reports have not resolved whether the effect of E2 on LH is positive, negative, or both during the equine estrous cycle. The direct reciprocal effect of LH on E2 also is not clear, although a recent study showed that hCG treatment during the preovulatory period caused an immediate reduction in E2 and cessation of growth of the preovulatory follicle [33].

The purposes of the present series of experiments were as follows: 1) to develop dose and protocol regimens of E2 that would approximate physiologic circulating concentrations of E2 during the estrous cycle, 2) to determine whether physiologic doses of E2 had a negative or positive effect on LH concentrations preceding and during various portions of the ovulation-inducing LH surge, and 3) to determine if the LH-like activity of hCG had a positive or negative effect on endogenous E2 during the slow and rapid segments of the LH surge.

MATERIALS AND METHODS

Mares were handled according to the Guide for Care and Use of Agricultural Animals in Agricultural Research and Teaching. A total of 103 estrous cycles in 57 mares was used within the ovulatory season (May–September, Northern Hemisphere) in seven experiments. The mares were mixed breeds of large ponies and apparent pony-horse crosses, aged 4–16 yr, and weighed 313–454 kg. The feeding program and the equipment and techniques for transrectal ultrasound scanning of ovaries, including determination of ovulation and measurement of follicles, have been described [5, 34]. Mares with two ovulations or hemorrhagic anovulatory follicles [35] were not used. Estradiol-17ß (E2) was prepared by dissolving 50 mg in 10 ml of benzyl alcohol and adding 40 ml of safflower oil, resulting in a stock solution of 1 mg/ml, as described [36]. Each dose described in the individual experiments was given i.m. in 2 ml (experiment 1) or 1 ml (remaining experiments) of vehicle (safflower oil) by diluting the stock solution.

The experiments were initiated on selected days of the ovulatory follicular wave (Day 0 = ovulation) to encompass the common follicle growth phase (before follicle deviation), deviation, postdeviation, and the preovulatory period, as shown (Fig. 1). The beginning of deviation is defined by continued growth rate of the largest follicle and the onset of reduced growth rate or regression of the second-largest follicle. Thus, the E2/LH relationship was studied when the two hormones were at basal concentrations (experiment 2), near the expected day of deviation or when the concentrations of the two hormones were expected to begin to increase (experiment 3), during sequential portions of the initial segment of the LH surge when the rate of LH increase is slower (experiments 3–5), near the expected peak of the E2 surge or the beginning of the rapid segment of the LH surge (experiment 6), and during the E2 decrease and LH increase near the occurrence of ovulation (experiment 7; [4, 9]).

Experiment 1

Effect of various doses of E2 on circulating concentrations of E2 was examined to aid in estimating an optimal dose and protocol for the subsequent experiments. Each dose of E2 was given as a single injection. The doses were 0, 0.05, 0.1, 0.5, and 1.0 mg (n = 3 mares/group). The E2 was given on a day when endogenous concentrations of E2 were expected to be minimal (Days 10–13; [9]). A blood sample was collected from a jugular vein at Hours 0 (just before E2 injection), 0.25, 0.50, 1, 2, 3, 4, 5, 6, 7, 8, 12, and 24 for assay of E2. The endogenous concentration of E2 on Days 10–13 was determined from the Hour 0 samples from the 15 mares. A sample was also obtained from each mare on a single day during Days 19–23. These days were expected to encompass the upper portion of the preovulatory E2 surge (Fig. 1). In addition, a sample was taken when the largest follicle was ≥35 mm, to represent the day of the expected peak of the preovulatory E2 surge.

Experiment 2

The effect of exogenous E2 on concentrations of LH on Day 13 was studied in a control (vehicle) and an E2-treated group (n = 4/group). The E2 was given at a dose of 0.2 mg at Hour 0 and 0.1 mg at Hours 4 and 8. Blood samples were taken for E2 and LH assay every 4 h from Hours 0 to 24, except that E2 was not assayed at Hours 16 and 20.

Experiment 3

The effect of the follicles and E2 on concentrations of LH was studied beginning near the expected onset of deviation (largest follicle, ≥20mm; actual diameter, 21.3 ± 0.3). Three groups (n = 4/group) were used: controls, follicles ablated, and follicles ablated plus E2 administered. Transvaginal ultrasound-guided aspiration of follicle contents was done for ablation, as described [37]. All follicles ≥6 mm were ablated at Day 10 so that the follicles to be ablated near the beginning of deviation were growing follicles of a new wave. When the largest follicle of the new wave was ≥20 mm, all follicles ≥10 mm were ablated so that the subsequent endogenous E2 would be at minimal concentrations, based on a previous study [9]. The dose of E2 was 0.2 mg at Hour 0 and 0.1 mg every 4 h thereafter until Hour 20. Blood samples for LH assay were obtained every 4 h during Hours 0–24.

Experiment 4

The effect of E2 on LH and the effect of hCG on E2 were studied when the largest follicle was ≥25 mm (actual diameter, 26.5 ± 0.2 mm). The hCG was expected to have an LH-like effect [38]. The following six groups (n = 6/group) were used: 1) control (no injections), 2) vehicle (safflower oil), 3) low dose of E2, 4) high dose of E2, 5) low dose of hCG, and 6) high dose of hCG. Treatment with E2 was done at Hours 0, 4, and 8, using a low dose of 0.2, 0.1, and 0.1 mg and a high dose of 2.0, 1.0, and 1.0 mg, respectively. The low dose of E2 was intended to simulate concentrations during the upper portion of an E2 surge. The high dose was similar to reported doses and was intended to provide E2 levels that greatly exceeded physiologic concentrations (see Introduction). The low dose of hCG was 200 IU, and the high dose was 2000 IU. The low dose has been used for frequent administration in immunologic studies, and the high dose induces ovulation [38]. The hCG was given as a single i.v. injection. Blood samples were collected at Hours 0, 4, 8, and 12 for E2 assay in all groups and for LH assay in the groups treated with E2.

Experiment 5

The effect of exogenous E2 on concentrations of LH was studied when the largest follicle was ≥28 mm (actual diameter, 30.5 ± 0.8 mm) in control and E2 treated groups (n = 4/group). The E2 was given at a dose of 0.2 mg at Hour 0 and 0.1 mg at Hours 4 and 8. Blood samples were taken every 4 h from Hours 0 to 24. Concentrations of LH were assayed for all hours, but E2 was assayed only for Hour 12.

Experiment 6

The effect of a single injection of hCG (2000 IU, i.v.) on plasma E2 concentrations was studied when the largest follicle was ≥35 mm (actual diameter, 36.3 ± 2.0 mm), using control and hCG groups (n = 6/group). Blood samples were taken at Hours 0, 4, 8, 12, 24, and 36 for assay of E2. The interval from treatment to ovulation was determined by ultrasound examination every 12 h.

Experiment 7

The effect of exogenous E2 on concentrations of LH was studied just before or after ovulation (actual mean, 6 h before Day 0). Control and E2 groups were used (n = 6/group). The E2 was given at a dose of 0.2 mg at Hour 0 and 0.1 mg every hour thereafter until Hour 8. Hour 0 was determined from the examinations at 12-h intervals, using serration of the granulosa to indicate impending ovulation within a few hours [39]. When serration was not detected, ovulation was used as Hour 0. Blood samples for E2 and LH assay were obtained every 12 h after the follicle was ≥35 mm so that samples were available at Hours –36, –24, and –12, and samples also were taken at Hours 0, 4, 8, 12, 24, 36, and 48.

Hormone Assays

Blood samples were collected into heparinized tubes and centrifuged (1500 x g for 10 min), and the plasma was decanted and stored (–20°C) until assay. Plasma concentrations of estradiol were measured by a double-antibody radioimmunoassay kit (Double Antibody Estradiol, Diagnostic Products Corporation, Los Angeles, CA), as described and validated in our laboratory for mare plasma [9]. The intra-assay and interassay coefficients of variation (CV) and mean sensitivity ranged between 8.7 and 13.4%, 6.7 and 15.2%, and 0.1 and 0.2 pg/ml, respectively. Plasma samples were assayed for LH by radioimmunoassay as validated and described for mares in our laboratory [31]. The intra- and interassay CVs and mean sensitivity were 4.7–6.1%, 4.5%, and 0.1–0.3 ng/ml, respectively. Cross-reactivity of hCG in the LH assay was 0.04%.

Statistical Analyses

Sequential data were analyzed by the SAS MIXED procedure to determine the main effects of group and hour and their interaction, using a repeated statement with spatial power to account for autocorrelation between measurements (version 8.2; SAS Institute Inc., Cary, NC). When the differences among groups at Hour 0 were significant or approached significance, data were converted to percentage change from Hour 0; Hour 0 was excluded from the analysis. Percentage change was used because of the demonstrated [9] measurable repeatability in hormone concentrations over time within mares. When an interaction was obtained, Student unpaired and paired t-tests were used to compare two means between and within groups, respectively. Duncan multiple range tests were used to compare more than two means within an hour when an interaction was obtained and to compare the means averaged over all hours when a group effect was obtained. A probability of P ≤ 0.05 indicated that a difference was significant, and a probability between P > 0.05 and P ≤ 0.1 indicated that significance was approached. Data are presented as the mean ± SEM unless otherwise specified.

RESULTS

Experiment 1

The endogenous E2 concentration before treatment on Days 10–13 was 0.8 ± 0.2 pg/ml; the concentration in each mare was <1.0 pg/ml, except for 2 of 15 samples. The endogenous E2 concentration on Days 19–23 was 4.5 ± 0.5 pg/ml, and the concentration when the largest follicle was ≥35 mm was 7.0 ± 0.8 pg/ml. The E2 concentrations for the five groups for Hours 0–24 are shown (Fig. 2). The concentrations of E2 increased (P < 0.05–P < 0.0001) by Hour 0.25 for all doses except for the 0 dose. The first hour when the E2 concentration for the 0.05- and 0.1-mg doses was no longer greater than for Hour 0 was Hour 5. The first hour that at least one of the postinjection E2 concentrations decreased to <1.0 pg/ml was Hours 7, 5, 12, and 24 for the doses 0.05, 0.1, 0.5, and 1.0 mg, respectively.


Figure 02
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  		FIG. 2 Mean circulating concentrations of estradiol (E2) when exogenous E2 was given in a single dose for five groups of mares (n = 3/group). The injection was given at Hour 0 during expected minimal concentrations of endogenous E2 (Days 10–13). The inset is an enlargement of the responses to doses of 0.0, 0.05, and 0.1 mg for Hours 0–8. Concentrations increased (P < 0.05–P < 0.0001) by Hour 0.25 for all doses, except for the 0 dose. By Hour 5, E2 for the 0.05- and 0.1-mg doses was no longer greater than for Hour 0. Experiment 1.

Experiment 2

Both main effects (group and hour) and the interaction were significant (P < 0.001) for E2 concentrations because of greater concentrations in the E2 group after Hour 0 (Fig. 3). The concentrations decreased (P < 0.008) between Hours 8 and 12 in the E2 group. Concentrations of LH were lower (group effect; P < 0.03) in the E2 group, and the group-by-hour interaction was significant (P < 0.05), primarily because of lower (P < 0.05) concentrations in the E2 group at Hours 4–16.


Figure 03
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  		FIG. 3 Mean (±SEM) circulating concentrations of estradiol (E2) and LH in controls and in mares treated with the indicated doses of E2 (n = 4/group). Hour 0 is on Day 13 after ovulation. Asterisks indicate significant main effects (G = group; H = hour) and the interaction (GH): * P < 0.05; *** P < 0.001. Experiment 2.

Experiment 3

For the LH analyses of percentage change from Hour 0 over Hours 4–24, the main effects of group (P < 0.05) and hour (P < 0.0001) were significant, but the interaction was not significant (Fig. 4). The group effect was attributable to greater increase (P < 0.05) in LH percentage change from Hour 0 in the follicles-ablated/vehicle group (74.8 ± 12.0%) than in the follicles-ablated/E2-treated group (29.6 ± 9.1%) and in the controls (7.4 ± 7.6%). Percentage change was first greater (P < 0.03) in the follicles-ablated/vehicle group than in the other two groups at Hour 8.


Figure 04
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  		FIG. 4 Mean (±SEM) for percentage change in circulating concentrations of LH from Hour 0 in controls and in mares with all follicles ≥10 mm ablated when the largest follicle was ≥20 mm (Hour 0) and given either estradiol (E2; doses indicated) or vehicle (n = 4/group). Asterisks indicate significant main effects (G = group; H = hour); the interaction was not significant. * P < 0.05; *** P < 0.001. ab = groups with a different superscript are different (main effect of group; P < 0.05). Experiment 3.

Experiment 4

Concentration of E2 at Hour 0 combined for all groups was 1.3 ± 0.1 pg/ml. The control and vehicle groups were not significantly different and were combined as one control group (n = 12) for the E2 data analysis. Concentrations of E2 increased by Hour 4 to 4.1 ± 1.1 pg/ml and 18.5 ± 3.1 pg/ml in the low and high E2-treated groups, respectively. There were no significant differences in E2 concentrations among the two hCG groups and control group (Fig. 5). The group effect for percentage change in LH from Hour 0 in the three groups (vehicle, low E2, and high E2) was significant (P < 0.0001), but the hour effect and interaction were not (Fig. 5). Averaged over Hours 4, 8, and 12, the group effect represented a higher (P < 0.05) concentration in the vehicle group than in the two E2 groups and lower (P < 0.05) concentration in the high-E2 group than in the low-E2 group. Length of the interovulatory interval was not different among the five groups (means, 22.8 ± 0.4–23.7 ± 0.9 days).


Figure 05
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  		FIG. 5 Mean (±SEM) circulating concentrations of estradiol (E2) in mares treated with E2 as shown or with a single injection of hCG (low dose = 200 IU; high dose = 2000 IU) and percentage change in LH from Hour 0 in the control and E2-treated groups (n = 6/group). Hour 0 was on the day the largest follicle reached ≥25 mm. Concentrations of E2 were not different among the controls and the two hCG groups. Asterisks indicate a significant effect of group (G) and an interaction of group and hour (GH). *** P < 0.001. ab = groups with a different superscript are different (main effect of group; P < 0.05). Experiment 4.

Experiment 5

Concentration of E2 at Hour 12 was greater (P < 0.02) in the E2-treated group (6.4 ± 1.0 pg/ml) than in the controls (3.1 ± 0.9 pg/ml). For the LH analysis of percentage change from Hour 0, the main effect of group for Hours 4–24 was significant (P < 0.04), resulting from a decreasing percentage change in the E2-treated group (Fig. 6).


Figure 06
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  		FIG. 6 Mean (±SEM) for percentage change from Hour 0 in circulating concentrations of LH in controls and in mares treated with estradiol (E2) at the indicated doses (n = 4/group). Hour 0 is on the day the largest follicle was ≥28 mm. The asterisk indicates a significant (P < 0.05) effect of group (G) over Hours 4–24. Experiment 5.

Experiment 6

Plasma concentrations of E2 showed a main effect of hour (P < 0.03) and a group-by-hour interaction (P < 0.03; Fig. 7). The interaction was attributable primarily to lower E2 concentrations in the hCG group at Hours 12 (approached significance, P < 0.07), 24 (P < 0.01), and 36 (P < 0.03). The increase in diameter of the largest follicle between Hours 0 and 12 was less (P < 0.03) for the hCG group (1.0 ± 0.5 mm) than for the controls (2.5 ± 0.5 mm). The interval from treatment to ovulation was shorter (P < 0.008) in the hCG group (1.4 ± 0.1 days) than in the controls (2.5 ± 0.4 days).


Figure 07
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  		FIG. 7 Mean (±SEM) circulating concentrations of estradiol in controls and in mares treated with a single injection of 2000 IU of hCG on the day the largest follicle was ≥35 mm (Hour 0; n = 6/group). The asterisk indicates a significant (P < 0.05) main effect of hour (H). The difference between groups is significant at Hours 24 (P < 0.01) and 36 (P < 0.03). Experiment 6.

Experiment 7

Plasma E2 concentrations showed significant main effects and a group-by-hour interaction (P < 0.0001; Fig. 8). The interaction resulted from high concentrations at Hours 4, 8, and 12 in the E2-treated group. For LH, the hour effect (P < 0.0001) and the interaction (P < 0.01) were significant. Concentration approached being lower (P < 0.1) in the treated group than in the controls at Hour 8 and was lower (P < 0.05) at Hour 12. The reference points for Hour 0 were based on serration of the granulosa (n = 6) and ovulation (n = 6). There was no difference in E2 and LH concentrations between the two reference points. All mares with serration for Hour 0 ovulated by the next examination 12 h later.


Figure 08
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  		FIG. 8 Mean (±SEM) circulating concentrations of estradiol (E2) and LH in controls and in mares treated with E2, as shown (n = 6 mares/group). Hour 0 is on the half day when impending ovulation (n = 6) or ovulation (n = 6) was detected. Asterisks indicate significant main effects (G = group; H = hour) and an interaction (GH). ** P < 0.01; *** P < 0.001. For LH, the difference between groups is significant (P < 0.05) at Hour 12. Experiment 7.

DISCUSSION

Previous studies in mares that were smaller or similar in body weight to those of these experiments used dosages of 0.5 mg [22], 1 mg [21, 26, 32, 40], 5 mg [41], or 10 mg [23, 24, 42]. In experiment 1, the 1-mg dose raised the concentrations during the first hour to 36–47 pg/ml or four to six times higher than expected concentrations at the peak of an E2 surge. The concentrations exceeded reported levels at the mean peak of a surge for 7 h and decreased to approximately pretreatment level at 24 h. Thus, the previous studies used supradoses that resulted in E2 levels that greatly exceeded maximal physiologic concentrations during the estrous cycle. In this regard, a supradose of E2 (2 mg followed by 1 mg) in experiment 4 produced a greater reduction in LH than a more physiologic dose (0.2 mg followed by 0.1 mg), illustrating the impact of dose. In experiment 1, the mean endogenous concentration of E2 for Days 10–13 represented the basal levels; the highest concentration in an individual was 1.8 pg/ml. The lowest dose of E2 (0.05 mg) raised the concentrations to >2 pg/ml in all mares at the first posttreatment examination (Hour 0.25) and maintained a mean of approximately 2 pg/ml for 4 h. The 0.1-mg dose increased the concentrations by Hour 0.5 to a level comparable to the 7-pg/ml endogenous concentrations at the expected peak of the preovulatory E2 surge. In experiment 2, an initial dose of 0.2 mg followed by 0.1 mg every 4 h during expected basal endogenous concentrations maintained the E2 concentrations to approximately 4 pg/ml, comparable to the endogenous concentrations during the upper portion of the preovulatory E2 surge in experiment 1 (4.5 pg/ml). Repeating the 0.1-mg dose every hour (experiment 7) resulted in continuous concentrations comparable to the level at the expected peak of the E2 surge. These comparisons indicate that the E2 doses and regimens used in these experiments were appropriate for study of the functional relationships between E2 and LH.

Basal or minimal concentrations of both E2 and LH during the equine estrous cycle precede the initial increase associated with the E2 and LH surges during the ovulatory follicular wave. At Day 13, luteolysis has not yet begun [4], and the major portion of the LH depression during the basal E2 and LH concentrations can be attributed to a negative effect of progesterone (see Introduction). However, increasing the E2 concentrations depressed the LH concentrations to below basal levels, indicating that a negative effect of E2 also contributes to the basal concentrations of LH. This is consistent with the report that administration of E2 plus progesterone to ovariectomized mares had a greater negative effect on LH than E2 alone [26]. The basal levels of E2 on Days 10–13 in the present experiments may have represented the output primarily of viable follicles ≤10 mm. Ablation of follicles >10 mm every 2 days beginning on Day 7 was followed by a gradual reduction in E2 to basal levels over the next few days; thereafter, E2 increased in the control group but not in the ablation group [9].

Ablation of all follicles ≥10 mm when the largest was ≥20 mm or near the expected beginning of deviation resulted in an increase in LH within 8 h. Administration of E2 prevented the postablation LH increase. Administration of E2 in follicle-intact mares when the largest follicle was ≥25 and ≥28 mm decreased the circulating LH concentrations. In a study in which only the two largest follicles of the wave were maintained, ablation of the largest follicle at ≥20 mm resulted in reduced circulating E2 and elevated LH [8]. Ablation of all follicles ≥10 mm every 2 d beginning on Day 7 resulted in higher LH than in controls during the slow segment of the LH surge [9]; this conclusion required reservation because the statistical interaction that justified the within-day comparisons only approached significance. The present results implicate the loss of E2 and not other follicular factors in accounting for the postablation increases in LH in the previous studies. Our interpretation is that E2 has a continuous negative effect on LH concentrations throughout the initial slowly increasing segment of the LH surge from before the beginning of follicle deviation to the peak of the E2 surge or approximately 2 d before ovulation.

Results of temporal studies [3, 4, 9] are consistent with the concept that the rapid increase in LH after the peak of the E2 surge results from the decrease in E2 concentrations and thereby a gradual loss of a negative effect of E2. A novel finding in these experiments was that the decreasing E2 continued to exert a negative effect on LH during the second rapidly increasing segment of the LH surge. This was indicated by a transient decrease in LH when E2 was given near ovulation or about 1 d before the LH surge was expected to reach maximum concentrations.

Reconciling the negative effect of E2 on LH found in these studies with the many reports of a positive effect (see Introduction) is a concern. It was noted earlier that the previous studies used supradoses of E2 that ranged from approximately 5 to 100 times greater than the doses used in the present experiments. Previous studies that used a single or daily treatment with collection of blood samples 24 h after each treatment may have missed a negative effect by the infrequent sampling. In this regard, one study in mares found a decrease in LH during the first few hours after treatment and continuing until 8 h; at 12 h, the LH was higher than pretreatment levels, demonstrating a transient rebounding effect [32]. In cattle, LH concentrations were reduced for 6 h after E2 treatment but surged by 24 h [43]. The rebounding effect may be characteristic of supradoses of E2 and may account for a positive effect for samples first collected 24 h posttreatment. In this regard, the physiologic doses used in the present studies did not produce a rebounding effect that exceeded the control concentrations by Hour 24 (experiments 2 and 5) or Hour 48 (experiment 7). However, the negative effect of E2 in the present experiments from multiple treatments over 8 h does not eliminate the possibility that a positive feedback requires continually increasing E2 over many days. These observations and speculations indicate that specific study would be needed to adequately account for the divergent results between the present and previous studies. The role of the negative effect of E2 on LH in mares during both the slow and the rapid segments of the LH surge or from before the beginning of deviation to ovulation is speculative. After the loss of the negative effect of progesterone (see Introduction), seasonal (extraovarian) factors may account for the subsequent LH increase. The negative effect of E2 on LH serves to dampen the seasonally induced prolonged inclining portion of the LH surge during the long follicular phase (e.g., 7 days). Thereby, the preovulatory and ovulatory functions of LH are delayed until the dominant follicle reaches an appropriate diameter. In other species (e.g., cattle), the follicular phase and the LH surge encompass a relatively shorter portion of the estrous cycle.

Neither a negative nor a positive effect of LH on E2 during the initial slow segment of the LH surge was indicated by the results of experiment 4, based on treatment with a dose of hCG that induces ovulation in the presence of a preovulatory follicle. However, results of experiment 6 indicated that LH has a reciprocal negative effect on E2 during the rapid increase of the second segment of the LH surge. This is consistent with the temporal association between the increased rate of LH output and the decrease in E2 [4]. On the day that E2 was expected to reach peak concentrations (largest follicle, ≥35 mm), an injection of hCG caused an E2 decrease within 8 h after treatment, a smaller follicle diameter increase at 12 h, and an induction of ovulation in 24 h (1 mare) or 36 h (five mares; experiment 6). These results confirm those of a previous study [33]. Thus, hCG or LH did not have ovulatory capability or a negative effect on E2 during the slow segment of the LH surge or, specifically, when the largest follicle was ≥25 mm (actual diameter, 26.5 mm) but acquired these effects by the time the follicle reached ≥35 mm or approximately 2 d before ovulation. An increase in LH receptor expression by the time the follicle reaches ≥35 mm may account for the negative effect of LH on E2; an increase in LH receptors in equine follicles as they mature to ≥35 mm has been reported [44]. The negative preovulatory effect of LH on systemic E2 is consistent with a report that hCG induces an intrafollicular increase in 17ß-hydroxysteroid dehydrogenase in mares [45]; these enzymes have the potential of reducing circulatory concentrations of E2 associated with the LH surge. Furthermore, granulosa cells from mature dominant equine follicles, as opposed to growing follicles, had lower levels of steroidogenic enzymes [46]. A study [47] in cattle indicated that treatment with hCG was associated with a decrease in the expression of mRNA for aromatase enzyme in granulosa cells of dominant follicles and a decrease in the E2:progesterone ratio in follicular fluid. Thus, the hCG used in experiment 6 may have blocked E2 production by downregulation of aromatase and other steroidogenic enzymes. Apparently, the dominant follicle acquires this capacity when it reaches ≥35 mm, accounting for the subsequent decrease in E2.

In conclusion, a negative effect of physiologic doses of E2 on LH was demonstrated during basal concentrations of LH and throughout the subsequent LH surge until ovulation. These findings are contrary to many previous conclusions that E2 has a positive effect on the ovulatory LH surge in mares. A reciprocal negative effect of LH on E2, examined by hCG treatment, did not occur during the initial portion of the LH surge but developed during preovulation when the largest follicle reached ≥35 mm.

ACKNOWLEDGMENTS

The authors thank Dee Cooper for assistance with the figures, text, and statistical analyses.

FOOTNOTES

1Supported by the Eutheria Foundation (Cross Plains, WI); Project P1-P7-OG-05. Back

Correspondence: 2O.J. Ginther, Department of Pathobiological Sciences, School of Veterinary Medicine, 1656 Linden Dr., University of Wisconsin, Madison, WI 53706. FAX: 608 262 7420; e-mail: ginther{at}svm.vetmed.wisc.edu

Received: 20 March 2007.

First decision: 1 May 2007.

Accepted: 5 June 2007.

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