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Biology of Reproduction 65, 426-432 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Response of Estradiol and Inhibin to Experimentally Reduced Luteinizing Hormone During Follicle Deviation in Mares1

D.R. Bergfelta, E.L. Gastalb, and O.J. Ginther

a Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706 b Department of Veterinary Science, Federal University of Viçosa, Viçosa, MG 36571-000, Brazil

ABSTRACT

The increase in LH concentrations at the time of the decrease in FSH concentrations during follicle deviation in mares was studied to determine the role of LH in the production of estradiol and immunoreactive inhibin (ir-inhibin). Ten days after ovulation, all follicles >=6 mm were ablated, prostaglandin F2{alpha} was given, and either 0 mg (control group, n = 15) or 100 mg of progesterone in safflower oil (treated group, n = 16) was given daily for 14 days, encompassing the day of diameter deviation. The follicular and hormonal data were normalized to the expected day of the beginning of diameter deviation when the largest follicle first reached >=20 mm (Day 0). The experimentally induced decrease in LH concentrations during follicle deviation beginning on Day -4 delayed and stunted the increase in circulating concentrations of ir-inhibin and estradiol beginning on Days -3 and -1, respectively, but did not alter the predeviation FSH surge and the initiation of diameter deviation between the two largest follicles. Combined for both groups, the interval to the expected day of deviation was 16.6 days after ovulation when the largest follicle was a mean of 21.6 mm. After deviation, the largest follicle started to regress in the treated group beginning on Day 1 and was associated with decreased concentrations of ir-inhibin and estradiol, and increased concentrations of FSH. The negative influence of the dominant follicle on the postdeviation decrease in FSH observed in the control group was alleviated and concentrations resurged in the treated group. Apparently this is the first in vivo evidence that the increase in LH that precedes follicle deviation has a positive effect in supporting the production of inhibin during diameter deviation. It was concluded that the increase in LH concentrations before diameter deviation played a role in the production of estradiol and inhibin by the largest follicle during deviation.

estradiol, follicle, follicle-stimulating hormone, inhibin, luteinizing hormone

INTRODUCTION

Changes in circulating concentrations of gonadotropins (FSH and LH), estradiol, and immunoreactive inhibin (ir-inhibin) have been studied throughout the estrous cycle in mares [1–4] but not together in relation to each other during follicular wave development, especially in association with follicle diameter deviation. Comparative aspects of follicle selection in horses and cattle have been reviewed recently [5]. Averaged over the control groups of several studies done in mares [6–9], the two largest follicles of a midcycle ablation-induced wave increased in diameter at a common rate until the largest follicle reached a mean of 22.5 mm. Thereafter, the growth rate of the now dominant follicle continued unabated while that of the now subordinate follicle began to decrease. The end of the common growth phase involving the future dominant and subordinate follicles marked the beginning of follicle diameter deviation. Associated followed by disassociated changes in circulating concentrations of FSH and LH have been detected during the common growth phase [6, 10]. Concomitant with follicular wave emergence, both gonadotropins increased until the largest follicle reached about 13 mm; thereafter, FSH began to decrease while LH continued to increase [6]. The directional change in the gonadotropins (decreasing FSH and increasing LH) began approximately 3 days before follicle deviation and continued until the periovulatory period.

Decreasing FSH concentrations during the follicular phase are associated with increasing circulating concentrations of estradiol and inhibin [1–4] that are known FSH suppressants in the mare [11]. Studies using follicular-fluid sampling in vivo [9] and follicle ablation [8] have indicated that the future dominant follicle alone was responsible for the increase in systemic estradiol near the beginning of deviation and, consequently, contributed to the continuing decrease in FSH after deviation. Similar results have been reported in heifers using follicular-fluid sampling in vivo [12], follicle ablation [13], and estradiol [14] and follicular fluid [15] treatments at the expected time of follicle deviation (largest follicle, >=8.5 mm). Other follicular-fluid factors (e.g., inhibin) may have also contributed to the FSH decrease during deviation in both species; however, this has not been clarified.

Temporally, the increase in concentrations of LH before the beginning of follicle deviation in mares suggested that LH may be directly involved in initiating deviation [6]. In this regard, hastening [16] or delaying [7, 16] the LH increase relative to the beginning of deviation did not alter the mean diameter increase of the largest and second-largest follicles before deviation (i.e., the beginning of diameter deviation was not altered). After deviation, however, diameter of the largest follicle decreased in association with the experimental reduction in LH concentrations. Conversely, the diameter changes of the second-largest follicles were not different between groups. Hence, the growth of the largest or dominant follicle was not interrupted by the reduced LH until approximately 2 days after the beginning of observed deviation. Similar results have been reported in cattle following a progesterone-induced decrease in LH before deviation [17, 18]. Thus, it appears that the LH increase before follicle deviation in both species is not directly involved in initiating diameter deviation but is necessary to support continued growth of the dominant follicle after deviation.

Although increasing concentrations of LH before the time of follicle deviation may not be necessary to initiate diameter deviation, other intrafollicular events may require elevated LH to prepare the follicles for their future direction. As reported in heifers [17, 18], experimental reduction of systemic LH concentrations before follicle deviation resulted in reduced intrafollicular concentrations of estradiol and free insulin-like growth factor-1 (IGF-1) but not ir-inhibin of the two largest follicles at the expected beginning of deviation. With regard to the latter, no significant changes in follicular-fluid concentrations of dimeric inhibin-A in the two largest follicles were detected encompassing the expected time of deviation in cows [19]. In cattle, therefore, the future dominant follicle utilizes the increasing portion of the LH surge that precedes deviation for the production of estradiol near the time of diameter deviation, whereas the role of LH in the production of inhibin is inconclusive. In horses, the role of LH in association with estradiol and inhibin production during follicle deviation is not known.

In the present study, the temporal relationships among circulating concentrations of FSH, LH, estradiol, and ir-inhibin encompassing the expected day of diameter deviation between the 2 largest follicles of the preovulatory wave were examined within and between groups. In the treated group, exogenous progesterone was used to experimentally alter circulating concentrations of LH to study the functional role of LH during diameter deviation. It was hypothesized that experimentally reduced LH concentrations before the expected day of diameter deviation will 1) inhibit the increase in circulating concentrations of estradiol, and consequently, interfere with the FSH decline during deviation, and 2) have no effect on circulating concentrations of ir-inhibin.

MATERIALS AND METHODS

Experimental Design and Treatment

The follicular and gonadotropin data were combined from two earlier studies done in our laboratory [7, 16]. In the present study, however, plasma samples from the previous studies were assayed for estradiol and ir-inhibin, which were not assayed in the earlier studies. Furthermore, the results were focused on the day of deviation when the difference in growth rates between the two largest follicles was expected to be initiated. The expected day of deviation was used as a reference point when the largest follicle first reached >=20 mm [6–9].

Thirty-one of 34 mares from the prostaglandin (PG) control and the progesterone-treated groups of the earlier studies were used in the present study. In each of the three animals not considered, the largest follicle did not reach >18 mm in one control mare, and the second-largest follicle did not reach >7 mm in a control and treated mare. The handling and management of the mares in each group was similar between studies, and therefore, the animals of respective groups were combined. Details of the animals, treatments, and data collection are documented in the earlier reports. Briefly, contents of all follicles >=6 mm were aspirated (i.e., functionally ablated [13]) using transvaginal ultrasonography 10 days after ovulation in the two groups. Immediately after follicle ablation, both groups received two intramuscular injections of PGF2{alpha} (5 mg, 12 h apart) and either 0 (control group, n = 15) or 100 mg (treated group, n = 16) of progesterone in safflower oil daily for 14 days. Transrectal ultrasonography and collection of jugular blood samples were done daily throughout the treatment period. Changes in diameter of all growing follicles >=6 mm of the ablation-induced wave were determined from examination to examination while maintaining individual identity. However, only the largest and second-largest follicles were examined in the present study.

Hormonal Assays

Circulating concentrations of FSH and LH were measured using RIA validated in this laboratory for this species. Assay variation and sensitivities for the gonadotropin results have been reported [7, 16].

Circulating concentrations of estradiol were measured using an RIA kit (Double Antibody Estradiol; Diagnostic Products Corporation, Los Angeles, CA) validated for use with bovine plasma [20] and adapted for use with equine plasma in our laboratory [8]. The intra- and interassay coefficients of variation (CVs) were 15.5% and 18.5%, respectively, as determined from a pool of plasma collected during estrus (n = 5 assays). Mean assay sensitivity was 0.18 pg/ml.

Circulating concentrations of ir-inhibin were measured using an RIA kit (Institute of Reproduction and Development, Monash Medical Center, Clayton, Victoria, Australia). The kit included inhibin as a 32-kDa fraction of bovine follicular fluid for iodination and anti-inhibin (Pool B, 1989) generated against a 31-kDa fraction of bovine follicular fluid. According to the supplier, the antibody recognizes the larger precursor and dimeric forms of inhibin, as well as the full length {alpha}-subunit and pro-{alpha}-C. In this regard, ir-inhibin refers to the bioactive forms of inhibin possessing FSH-suppressing activity (dimeric, {alpha}ßA and {alpha}ßB) as well as nonbioactive forms of inhibin without FSH-suppressing activity (monomeric, {alpha}-subunit [21]). Assay procedures were similar to those previously described using mare plasma [2], except that iodination was done using Iodogen [22] and that a recombinant 32-kDa form of bovine inhibin (IP-1095; Peninsula Laboratories Europe Ltd., St. Helens, Merseyside, England) was used for the preparation of reference standards. Serial dilution of the standard (5–250 ng/ml) and two different pools of plasma collected from mares during estrus and diestrus (25, 50, 75, and 100 µl) resulted in three displacement curves in which the slopes were similar. The intra- and interassay CVs were 8.1% and 9.8%, respectively. Determinations were made from a pool of plasma collected during estrus (n = 3 assays). Mean assay sensitivity was 6.24 ng/ml.

Data Handling and Statistical Analyses

The expected day of follicle deviation was defined as the examination when the largest follicle first reached >=20 mm and was designated Day 0. Diameters of the largest and second-largest follicles and hormonal concentrations were adjusted to Day 0 for each animal. As a result of the adjustment, the follicular and hormonal data were staggered and the number of observations for each day decreased as the number of days decreased or increased from Day 0. Therefore, the follicular data were truncated from Days -5 to 5 and the hormonal data from Days -7 to 6. As a result, the daily observations were maximal and remained consistent during the period of analysis encompassing follicle deviation. The follicular and hormonal data were examined for normality using the Kolmogorov-Smirnov test, and suspected outliers were challenged using Dixon's Outlier test [23]. The hormonal but not the follicular data lacked normality; therefore, the FSH, LH, ir-inhibin, and estradiol concentrations were transformed logarithmically to adjust for normality. Outlying observations were detected only for estradiol in both the control and treated groups. Out of a total of 411 observations, 20 observations (<5%) were determined as extreme and excluded from further analyses. Overall analysis of the follicular and hormonal data was done using the Statistical Analysis Systems Mixed procedure with a repeated statement and a first order autoregressive structure to account for the autocorrelation between sequential measurements. Main effects of group (control and treated) and day and the group-by-day interaction were determined. When a significant main effect or interaction was detected, Duncan multiple range tests were used to detect significant differences between days within a group and unpaired t-tests were used to detect significant differences between groups within a day. Unpaired t-tests were also used to detect differences between groups involving single-point measurements. The observed data and not the transformed data are presented as the mean ± SEM. A probability of P < 0.05 indicated that a difference was significant and probabilities between P >= 0.06 to P <= 0.1 indicated that a difference approached significance.

RESULTS

There were no significant differences between the control and treated groups for the following end points: 1) interval from ovulation to the expected day of deviation, 16.5 ± 0.3 and 16.6 ± 0.4 days; 2) diameter of the largest follicle on the day of expected deviation, 21.9 ± 0.3 and 21.2 ± 0.3 mm; 3) diameter of the second-largest follicle on the day of expected deviation, 18.7 ± 0.5 and 17.7 ± 0.6 mm; and 4) diameter difference between the largest and second-largest follicles on the day of expected deviation, 3.2 ± 0.6 and 3.6 ± 0.7 mm.

Day-to-day changes in diameter of the largest and second-largest follicles and in circulating concentrations of FSH, LH, ir-inhibin, and estradiol during the treatment period encompassing follicle deviation (Day 0) are shown with the results of the statistical analyses (Fig. 1). Prior to Day 0, mean diameter of the largest and second-largest follicles increased (P < 0.05) in both the control and treated groups in a similar manner. The group-by-day interaction for the largest follicle was attributed to a continued mean increase in diameter in the control group and a reduced increase in the treated group after Day 0. Diameter of the largest follicle was greater (P < 0.05) in the control than in the treated group beginning on Day 1. For the second-largest follicle, the mean diameter changes were similar between groups.



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  		FIG. 1. Mean (±SEM) day-to-day diameters of the two largest follicles and circulating concentrations of FSH, LH, ir-inhibin, and estradiol in the control and treated groups encompassing Day 0 when the largest follicle first reached >=20 mm. The results of the statistical analyses are shown for each end point (G, group effect; D, day effect; GD, interaction). A star indicates a difference (P < 0.05) between group means within a day. For each day after the last star, means are different (P < 0.05) between groups. Means with different letters indicate differences (P < 0.05) within the control (abcd) and treated (xyz) groups

In both the control and treated groups, an FSH surge was detected before the expected beginning of follicle deviation as indicated by a mean increase (P < 0.05) and decrease (P < 0.05) in concentrations encompassing peak concentrations on Day -3 (Fig. 1). The group-by-day interaction for FSH was attributed to a mean increase (P < 0.05) in concentrations in the treated group and a continued decrease (P < 0.05) to basal concentrations in the control group after Day 0. Mean FSH concentrations were higher (P < 0.05) in the treated group than in the control group beginning on Day 2.

Mean concentrations of LH increased (P < 0.05) before the expected day of deviation in both the control and treated groups (Fig. 1). The group-by-day interaction for LH was attributed to the following: 1) a more rapid increase in the control group before deviation, resulting in higher (P < 0.05) mean concentrations in the control than in the treated group beginning on Day -4, and 2) elevated but unchangeable (P > 0.05) mean concentrations in the control group after Day -2 compared to a decrease (P < 0.05) in concentrations in the treated group after Day -3.

Mean concentrations of ir-inhibin increased (P < 0.05) beginning after Day -4 in the control group and after Day -3 in the treated group (Fig. 1). Compared to the control group, the delayed increase in ir-inhibin in the treated group resulted in lower (P < 0.05) mean concentrations on Days -3, -1, 1, and thereafter. On Days -2 and 0, concentrations tended (P < 0.09) to be different between groups. The group-by-day interaction was attributed primarily to a mean decrease (P < 0.05) in ir-inhibin concentrations in the treated group beginning after Day 1 while concentrations in the control group remained elevated but unchangeable (P > 0.05).

Mean concentrations of estradiol in both the control and treated groups were near basal concentrations prior to Day -2 (Fig. 1). Thereafter, concentrations increased (P < 0.05) by Day 0 in the control group and by Day 2 in the treated group. Mean concentrations were higher (P < 0.05) in the control group than in the treated group beginning on Day -1. The group-by-day interaction was attributed to a continued mean increase in estradiol in the control group after Day 0 while concentrations in the treated group did not change (P > 0.05) after Day 2.

DISCUSSION

Exogenous progesterone was used experimentally to suppress the increase in circulating concentrations of LH that occurs during the late luteal and follicular phases in the mare to study the role of LH in the production of estradiol and inhibin during follicle diameter deviation. The dose of progesterone (100 mg/day) has been reported [7, 16] to reduce circulating concentrations of LH without affecting FSH concentrations; a higher dose (300 mg/day) affected both gonadotropins [7]. The beginning of the difference in growth rates between the two largest follicles (diameter deviation) of an ablation-induced wave was used as a reference point for normalizing the follicular and hormonal data to focus on the temporal changes within and between treatment groups. In both the control and progesterone-treated mares, aspiration of follicle contents as a method of ablation was done 10 days after ovulation to facilitate maintaining individual identity of growing follicles of the new preovulatory wave without the mingling and interference of regressing follicles of the previous wave. Although the extent of removal or destruction of the follicle components by the aspiration procedure is not known, the follicles were functionally ablated as indicated by subsequent follicular and hormonal responses reported herein as well as in other studies involving similar follicle aspiration procedures in horses [6, 24, 25] and cattle [13, 26, 27].

The expected day of the beginning of diameter deviation between the two largest follicles of the ablation-induced wave was assigned to the day when the largest follicle first reached >=20 mm and was designated Day 0. The target diameter of the largest follicle was chosen from the results of previous studies done in ponies [6, 7, 9, 16] involving 73 major waves in which the beginning of observed deviation occurred when the largest follicle was a mean of 22.5 mm with a mean difference in diameter between the largest and second-largest follicles of 3.1 mm. In the present study, diameter of the largest follicle (combined mean, 21.6 mm) and diameter difference between the largest and second-largest follicles (combined mean, 3.4 mm) at the expected time of deviation were comparable to the previous studies and, therefore, supported using the day when the largest follicle reached >=20 mm as an approximation to the beginning of diameter deviation. The experimental suppression of LH beginning before follicle deviation did not alter the expected day of diameter deviation in the present study or the observed day of deviation reported in the previous studies [7, 16]. Correspondingly, in heifers, the expected hour of follicle deviation was not altered by the progesterone-induced decrease in LH concentrations before deviation [17, 18].

The increase and decrease in FSH concentrations of the ablation-induced surge and development of the two largest follicles were similar between groups before the expected day of deviation. After deviation, however, there was a continued increase in diameter of the largest follicle and a decrease in concentrations of FSH in the control group, compared to a decrease in the largest follicle and an increase in FSH in the treated group. In the control group, the relationship between FSH and follicular development before and after the expected day of deviation was similar to that reported during wave emergence and the observed day of deviation in a previous study [6]. In the treated group, regression of the largest follicle after deviation was associated with the emergence of other follicular waves during the period of progesterone treatment and has been addressed in the previous studies [7, 16]. Not considered in the previous studies, however, was the postdeviation resurgence in FSH and its relationship with the demise of the largest follicle. In this regard, the cessation of growth of the largest follicle and the concomitant resurgence in FSH in the treated group after the expected day of deviation indicated that the functional capacity of the larger follicle to continue to support the FSH decrease deteriorated in response to the experimentally reduced LH concentrations during deviation.

Estrogen and inhibin are follicular factors that possess FSH-suppressing properties as reviewed for the mare [11] and other species [28, 29]. In the control group, an increase in circulating concentrations of estradiol began 1–2 days before the expected day of follicle deviation (Day 0) and was significantly higher on Day 0 when the larger follicle reached a mean of 21.9 mm. After deviation, estradiol concentrations markedly increased in association with the continued growth of the larger follicle and corresponding decline in FSH. In a previous study [9], follicular-fluid concentrations of estradiol in the largest follicle and in the systemic circulation increased beginning 1 day before the expected day of follicle deviation; intrafollicular concentrations in the largest follicle were greater compared to the second-largest follicle on the day of deviation. In another study [8], there was no increase in systemic estradiol concentrations and a transient increase in FSH concentrations after ablation of the largest but not the second-largest follicle on the expected day of deviation. Similar hormonal events were not observed when the largest follicle was left intact and the second-largest follicle was ablated (i.e., estradiol increased and FSH decreased). In cattle, when the largest follicle was ablated at the expected hour of follicle deviation, the ablation-induced FSH surge was delayed by exogenous estradiol [14]. In the present study, the nature of the reduced but significant increase in circulating concentrations of estradiol by Day 2 in the treated group is not known but may have reflected a residual increase as a result of the initial development of the largest follicle in response to the reduced but significant increase in LH before Day 0 and the resurgence in FSH thereafter. After Day 2, however, the dramatic difference in estradiol concentrations between groups was attributed to the reduced versus the continued development of the largest follicle in the treated versus the control group. There was no difference in the diameter profile of the second-largest follicle within or between groups encompassing follicle deviation. On a temporal basis, the present results provide additional evidence that the future dominant follicle is primarily responsible for the increase in systemic estradiol when it approaches the day of deviation (largest follicle, >20 mm) and contributes to the corresponding decrease in FSH to nadir concentrations after deviation.

Circulating concentrations of ir-inhibin progressively increased beginning 3–4 days before the expected day of deviation (Day 0) in the control group. The ir-inhibin increase occurred in association with an increase in diameter of the two largest follicles and coincided with the beginning of the decrease in concentrations of FSH after Day -3 when the larger follicle reached a mean of 12.1 mm. The increase in ir-inhibin continued progressively until Day 1. Thereafter, no significant changes were detected, but ir-inhibin concentrations remained elevated as an apparent mean plateau while growth of the larger follicle and the corresponding decline in FSH continued. In a previous study in mares [6], the decreasing portion of the FSH surge began when the largest follicle attained approximately 13 mm, which is comparable to the results herein. The follicle source of inhibin in the mare has been shown using bioassay [30], immunoassay [1], and Western blot analysis [31] of follicular fluid. Indirectly, inhibin-like activity in equine follicular fluid has been demonstrated by the postablation increase in FSH concentrations [6, 24, 25] and the post-treatment decrease in FSH using a proteinaceous fraction of follicular fluid in intact [32] and ovariectomized [33] mares. In addition, inhibin-like activity was greater in the follicular fluid of viable follicles than in atretic follicles during the estrous cycle [30] and was greater in preovulatory-sized follicles than in follicles collected during late diestrus/early estrus [33]. In regard to the latter study, it has recently been reported that the inhibin {alpha}-, ßA-, and the ßB-subunits were detected in equine granulosa cells of follicles >30 mm [3] during the late follicular phase. The bioactive or FSH-suppressing forms of dimeric inhibin ({alpha}ßA and {alpha}ßB) have apparently not been detected in follicles <30 mm; however, it has been reported that the inhibin {alpha}-subunit was greater in granulosa cells of 15–19 mm than in 10- to 14-mm follicles [34] and was the only monomeric form of inhibin in follicles <10 mm [3]. Although the absence of a negative effect of the {alpha}-subunit alone on FSH has not been demonstrated in horses as it has been in cattle [21], a simultaneous increase in ir-inhibin and FSH was detected before the largest follicle reached 13 mm, suggesting that the form of inhibin produced by follicles <13 lacked FSH-suppressing activity [35]. The extent of the contribution of various follicles to the overall increase in circulating concentrations of inhibin during follicular wave development is not known; however, it has been reported that a slower FSH decrease was associated with a slower ir-inhibin increase when one follicle >10 mm was retained after ablation compared to three or all follicles [35]. In cattle, multiple follicles >5 mm contribute to the initial FSH decline during wave development [36]. Temporally, the present study provides new information that an FSH-suppressing form of inhibin was produced beginning when follicles reached >12 mm and that the quantity was sufficient to raise circulating concentrations and initiate the FSH decline beginning about 3 days before diameter deviation.

In the control group, the increase in circulating concentrations of ir-inhibin occurred 2–3 days before an increase in circulating concentrations of estradiol was detected, suggesting that the initial phase of the FSH decrease before follicle deviation primarily involved inhibin and that the final phase of the FSH decrease after deviation involved both inhibin and estrogen. Compared to the present study, the negative effect of inhibin on FSH before and after follicle deviation has been reported during comparable stages of the estrous cycle in mares [37, 38]. Concentrations of FSH increased after passive immunization against the native form of inhibin starting at mid-diestrus [37] or against the {alpha}-subunit starting when the largest follicle of the preovulatory wave reached 20 mm [38]. The form of dimeric inhibin that is involved in the suppression of FSH during follicle deviation in the mare is not known, but in women systemic and follicular fluid concentrations of inhibin-A were elevated during the later portion of the follicular phase compared to low or unchanged concentrations of inhibin-B [39]. The role of the inhibin family in follicle selection has recently been compared among horses, cattle, and humans [40]. Although the role of estrogen and inhibin during deviation has not been clarified, it has been reported [41] that estrogen and an inhibin-like factor of follicular fluid alone as well as the synergistic effect of both hormones together suppressed circulating concentrations of FSH in ovariectomized mares. Perhaps therefore, the continued suppression of FSH in the control group after deviation reflected the combined effect of estradiol and inhibin, and the resurgence in FSH in the treated group during the corresponding time reflected the reduced ability of the dominant follicle to sustain the production of sufficient FSH-suppressing factors. The concept that low FSH concentrations are necessary to facilitate diameter deviation in association with follicle selection in monovular species has been reviewed [5, 40].

The role of LH in the production of estrogen involving the theca and granulosa cells is well established and has been reviewed for many species [42]; however, its role in estrogen production during follicle deviation in the mare has not been previously documented. In the present study, circulating concentrations of estradiol were significantly lower in the treated group than in the control group before the expected day of follicle deviation (Day 0), indicating that the steroidogenic capacity of the largest follicle was disrupted. In the treated versus the control groups, LH concentrations were lower beginning on Day -4 and preceded the reduced concentrations of estradiol beginning on Day -1. In the mare, the positive effect of LH on follicle estrogen production has been shown in vitro using pieces of the follicular wall [43]. Apparently, the greatest LH-induced production of estradiol occurred in follicles collected during late diestrus, which corresponded to 14 days after ovulation. In the present study, the expected day of follicle deviation combined for both groups was a mean of 16.6 days after ovulation. In another more recent study [34], LH receptor protein content of equine granulosa cells was significantly greater in 15- to 19-mm than 10- to 14-mm follicles, and aromatase content was significantly greater in 20- to 24-mm than 15- to 19-mm follicles. Although the granulosa cells were apparently collected without regard to follicle identity or status, the results suggested that granulosa cell responsiveness to LH increased before aromatase increased. In cattle, mRNA expression of the LH receptor in granulosa cells of the largest follicle but not the second-largest follicle increased approximately 8 h before an increase in follicular-fluid estradiol concentrations of the largest follicle at the expected hour of deviation [19]; there was no detectable change in follicular-fluid estradiol of the second-largest follicle. In addition, the experimental suppression of the postovulatory LH surge that encompasses the beginning of follicle deviation in cattle resulted in a decrease in follicular-fluid concentrations of estradiol as well as free IGF-1 of the two largest follicles at the expected beginning of diameter deviation [17, 18]. It has been reported in species other than the mare [44] that progesterone can have an inhibitory effect on aromatase activity; however, the results are not conclusive regarding a direct negative effect of progesterone on estrogen production. In the present study, therefore, the reduced production of estradiol in the treated group beginning shortly before follicle deviation was primarily attributed to the reduced availability of LH. The hypothesis that experimentally reduced LH concentrations before the expected day of follicle deviation will inhibit the systemic increase in estradiol concentrations during diameter deviation was supported.

Compared to the numerous studies demonstrating the positive role of FSH in the follicle production of inhibin, the role of LH in the production of inhibin has been limited to in vitro studies using tissue from laboratory animals [45]. In the present study, the interrelationship between LH and inhibin is supported by the following temporal events: 1) In both groups, an increase in LH preceded the beginning of the increase in ir-inhibin by 3 or 4 days. 2) In the control group, the beginning of an apparent mean LH plateau preceded the beginning of an apparent mean ir-inhibin plateau by 3 days. 3) In the treated group, the beginning of the decrease in LH preceded the beginning of the decrease in ir-inhibin by 4 days. From cultured rat granulosa cells, FSH as well as LH stimulated inhibin protein ({alpha}- and ßA-subunits) and mRNA production apparently through the cAMP pathway [45]; thus indicating that the stimulatory action of both gonadotropins on inhibin production is linked by a common intracellular mechanism. In this regard, FSH [46] and LH [34, 46] receptor proteins have been detected in equine granulosa cells beginning when follicles attained >5 mm during the follicular phase, suggesting that both gonadotropins have a role in inhibin production in this species. Although a direct negative effect of progesterone on inhibin production cannot be discounted, the hypothesis that experimentally reduced LH would not alter circulating concentrations of ir-inhibin was not supported. Instead, the temporal relationships in the present study provide apparently the first in vivo evidence that the increase in LH that preceded follicle deviation has a functional role in supporting the production of inhibin.

In conclusion, the experimentally induced decrease in LH concentrations during follicle deviation in mares delayed and stunted the increase in circulating concentrations of ir-inhibin and estradiol before the expected day of deviation but did not alter the predeviation FSH surge and the initiation of diameter deviation between the two largest follicles. The day after deviation, the largest follicle started to regress and coincided with decreased concentrations of ir-inhibin and estradiol, and increased concentrations of FSH. Apparently, the negative influence of the dominant follicle on the postdeviation decrease in FSH observed in the control group was alleviated and concentrations resurged in the treated group. Thus, the increase in LH concentrations before diameter deviation played a functional role in the production of estradiol and inhibin by the largest follicle during deviation. Furthermore, the follicular and hormonal temporal relationships within and between groups encompassing follicle diameter deviation suggested that the initial phase of the FSH decline before deviation involved inhibin beginning when the largest follicle reached >12 mm and that the continuing FSH decline after the beginning of deviation apparently involved both inhibin and estrogen beginning when the future dominant follicle approached the day of deviation (largest follicle, >20 mm).

ACKNOWLEDGMENTS

The authors thank M.O. Gastal and S.C. Jensen for technical assistance, the Pharmacia and Upjohn Company for the gift of Lutalyse, and A.F. Parlow of the National Hormone and Pituitary Program for providing the anti-human FSH.

FOOTNOTES

First decision: 29 January 2001.

1 Research supported by the University of Wisconsin, Madison, and by the Eutherian Foundation, Cross Plains, WI. Back

2 Correspondence: D.R. Bergfelt, Department of Animal Health and Biomedical Sciences, 1656 Linden Drive, University of Wisconsin, Madison, WI 53706. FAX: 608 262 7420; drb{at}ahabs.wisc.edu Back

Accepted: March 20, 2001.

Received: December 19, 2000.

REFERENCES

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