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Regular Article |
a Department of Physiology, University of Kentucky, Lexington, Kentucky 40536
ABSTRACT
This study was designed to test the hypothesis that the loss of LH surges in response to the stimulatory actions of estradiol and progesterone in middle-aged, persistent-estrous (PE) rats may be caused by chronic elevations in circulating estradiol. Five groups of regularly cycling young rats received an s.c. estradiol implant immediately after ovariectomy (Day 0). For determination of LH surges, blood samples were collected hourly between 1200–1900 h from each of the five groups at one of the following times: 3 days, or 1, 2, 4, or 8 wk later. On the next day, either progesterone (0.5 mg/100 g BW) or corn oil was injected s.c. at 1200 h, and samples were obtained as before. Incidence and amplitude of estradiol-induced LH surges decreased during the first 2 wk of estradiol treatment, after which no surges occurred. Progesterone enhanced the incidence and amplitude of estradiol-induced LH surges thus delaying their disappearance. These results support our hypothesis and demonstrate that the stimulatory actions of estradiol and progesterone on the LH surge sequentially diminish with time after exposure to estradiol in young rats. Thus, young rats chronically treated with estradiol may be a useful model for studying the mechanisms whereby LH surges are abolished in middle age during the hyperestrogenic state of PE.
aging, estradiol, LH surges, progesterone
INTRODUCTION
Among the first signs of reproductive aging in female rats are the attenuation and delayed onset of the proestrous LH surges that occur during middle age (9–10 mo) even in the presence of regular estrous cycles [1–5]. These modulations of the amplitude and timing of LH surges reflect age-related changes in the two mechanisms that control the LH surge in rodents, namely the response to the positive feedback action of estradiol and the circadian timing signal, respectively [6]. As rats age further, many of them experience a period of irregular cyclicity that is followed by an anovulatory state of persistent estrus (PE). During PE, follicular development continues to occur, as manifested by a chronic elevation in circulating estradiol concentrations to 30–40 pg/ml [7, 8], which in turn causes persistent vaginal cornification [7, 9]. By the time PE begins, the mechanisms mediating estradiol-induced LH surges are lost because the positive feedback action of estradiol no longer elicits an LH surge after ovariectomy unless followed by administration of progesterone [10]. However, even progesterone no longer induces LH surges after about 90 days of PE [10]. As rats age even further, the ability of progesterone to elicit LH surges in estradiol-primed rats returns, and the animals show repetitive pseudopregnancy [7]. This finding suggests that extinction of LH surges in PE rats is not totally caused by aging. This conclusion is supported by the observation that the stimulatory feedback action of estradiol in combination with progesterone on LH surges is functional in acutely ovariectomized repeat pseudopregnant rats but not in age-matched PE females [8].
Loss of the LH-surge-inducing action of estradiol is not unique to middle-aged PE rats. Indeed, exposure of young ovariectomized rats to estradiol causes a similar loss of LH surges within 2 wk [11–13]. It is not known, however, whether response to the stimulatory action of progesterone in such rats is also lost, and if so, when. Based on these findings and on the previous considerations, it has been hypothesized that in aging PE rats, chronic exposure to the increased estradiol levels associated with acyclicity, rather than aging per se, causes the loss of steroid-induced LH surges [8, 14, 15]. Therefore, the following study was designed to investigate whether chronic exposure of young ovariectomized rats to elevated circulating estradiol levels would mimic the sequential loss of the stimulatory actions of estradiol and progesterone on LH surges that is observed in PE rats.
MATERIALS AND METHODS
Animals
Young (2-mo-old) female Sprague-Dawley rats (Zivic-Miller Laboratories Inc., Zelienople, PA) were maintained under constant conditions of temperature (22–24°C) and photoperiod (14L:10D, lights-on 0500 h). Food and water were provided ad libitum. Daily vaginal smears were monitored for 3–4 wk to determine estrous cyclicity. Only females that showed at least two consecutive 4-day estrous cycles were used.
Experimental Design
To investigate the effect of chronic estradiol treatment on the positive feedback action of estradiol and the LH surge-enhancing effect of progesterone, 51 regularly cycling rats were bilaterally ovariectomized (Day 0). Rats weighing from 201–370 g (mean ± SEM, 271.3 ± 5.2 g) were ovariectomized between the ages of 85 and 119 days (averaging 94.1 ± 1.3 days). Neither the age nor body weight (BW) were different among the groups at time of ovariectomy. Immediately thereafter, a 5-mm Silastic capsule (Dow Corning Co., Midland, MI) containing crystalline estradiol-17ß (Sigma Chemical Co., St. Louis, MO), prepared as described previously [16], was inserted s.c. Such implants have previously been reported to generate sustained plasma estradiol concentrations averaging about 75 pg/ml [16]. The rats were randomly divided into five groups that were tested on Day 3 (0.5 wk), or 1, 2, 4, or 8 wk after estradiol treatment (n = 11, 11, 12, 9, and 8, respectively). Blood samples (0.2 ml) were obtained hourly between 1200–1900 h for 2 days via a right atrial cannula that was inserted on the day before sampling. On the second day of sampling, either progesterone (0.5 mg/100 g BW, n = 6, 6, 7, 5, and 4) or corn oil vehicle (0.1 ml/100 g BW, n = 5, 5, 5, 4, and 4) was injected s.c. at 1200 h. The plasmas were separated and stored at -20°C until determination of LH concentrations by RIA.
At the time of sampling, the rats receiving estradiol for 8 wk were about 1–2 mo older than the animals in the other groups. Therefore, to determine whether this age difference contributes to the diminution of LH surges, 3-mo-old (96.1 ± 5.7 days, n = 15) and 5-mo-old (134.8 ± 9.3 days, n = 5) regularly cycling rats were ovariectomized and immediately treated with estradiol implants (Day 0). They were cannulated on Day 2, and at 1200 h on Day 3 they received an s.c. injection of either corn oil (n = 8 and 2, respectively) or progesterone (n = 7 and 3, respectively). Blood samples were obtained hourly between 1200–1700 h, and the plasmas were processed as described previously.
Radioimmunoassay
Plasma LH levels were determined in three aliquots, a single 20-µl and duplicate 5-µl aliquots, by means of a previously described rat LH assay [17] with the following modifications. CSU 120 (Dr. Terry Nett, Colorado State University, Fort Collins, CO) was used as the first antibody at a working dilution of 1:10 000. Iodinated rat LH (12 000 cpm/100 µl buffer) was obtained from Covance Laboratories, Inc. (Vienna, VA), and second antibody, goat anti-rabbit IgG, was obtained from Linco Research Inc. (St. Charles, MO). The tubes were incubated at 4°C for 48 h following the addition of each reagent. Plasma LH concentrations are reported in terms of ng RP-3 per ml, provided by the National Hormone and Pituitary Program. The sensitivity of the assay (100% minus 2 SD of maximum binding) averaged 0.03 ng/tube, and the interassay coefficient of variation was 8.3% for an intact serum pool that inhibited binding of labeled hormone to 79.5% of maximal binding.
Data Analyses
Both age and BWs were compared separately among groups by one-way ANOVA. An LH surge was defined as an increase in plasma LH concentrations of 2 SD above the mean value at 1200 h (1.54 ± 0.52 ng/ml) for at least two consecutive samples [18]. The numbers of rats with LH surges from each group were compared by chi-square tests. Only results from the groups in which more than two rats had LH surges were used in the statistical analyses. Peak LH levels on sampling Day 1 and 2 were compared by one-way and two-way ANOVA, respectively, to reveal the main effects of duration of estradiol treatment, progesterone administration, and their interaction. If there was a significant interaction (P < 0.05), post hoc comparisons were made using Tukey's test.
RESULTS
Age and BW of Rats
At the time of sampling, rats sampled after 8 wk of treatment were older than those in all other groups, and the animals sampled after 2 and 4 wk of exposure to estradiol were older than those in the 3-day and 1-wk groups (Fig. 1, upper panel). The only difference among groups in BW was between the heaviest and lightest groups, i.e., the rats treated for 8 wk weighed more than the 1-wk rats (Fig. 1, lower panel).
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Response to the Positive Feedback Action of Estradiol on Sampling Day 1
The longer the duration of exposure to estradiol, the lower the incidence and amplitude of LH surges (Fig. 2). Thus, 3 days (0.5 wk), and 1 and 2 wk after estradiol treatment, 9 of 11 (82%), 5 of 11 (45%), and 2 of 12 (17%) rats displayed an LH surge (Fig. 2, left panel). The peak levels of the LH surges averaged 15.24 ± 3.27 (3-day), 6.62 ± 2.33 (1-wk), and 4.96 ± 0.23 (2-wk) ng/ml, which occurred at 1600, 1700, and 1700 h, respectively (Fig. 2, right panel). There were no LH surges in response to estradiol alone after 4 or 8 wk of treatment. Mean plasma LH concentrations in the rats that did not surge remained undetectable throughout the sampling period (data not shown).
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Enhancement of the Positive Feedback Action of Estradiol by Progesterone on Sampling Day 2
In those rats receiving corn oil vehicle on the second day of sampling (Fig. 3, upper panels), an LH surge occurred in 80% (four of five) of rats after 4 days (0.5 wk) of exposure to elevated estradiol levels. Two of these rats displayed LH surges on both sampling days, reflecting the circadian timing signal that can trigger daily LH surges. The mean peak level of the LH surges on Day 4 was 7.25 ± 2.47 ng/ml (n = 4) at 1700 h (Fig. 3, upper right panel). After 1 wk of pretreatment with estradiol, only one of five oil-treated rats had an LH surge on the second day that peaked at 8.04 ng/ml at 1600 h. This rat had not exhibited an LH surge on the previous day. No LH surges were observed on the second day of sampling in the ovariectomized rats exposed to elevated estradiol levels for 2 or more weeks. In oil- or progesterone-treated rats that did not surge, plasma LH concentrations remained undetectable throughout the sampling period (data not shown).
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In contrast to the virtual disappearance of the response to estradiol alone by 2 wk after treatment, the LH surge-enhancing action of progesterone remained vigorous in most rats bearing estradiol implants for 4 days, and 1 and 2 wk (Fig. 3, lower panels). Thus, an LH surge occurred in six of six, five of six, and five of seven progesterone-treated rats after 4 days, and 1 and 2 wk of estradiol treatment, respectively. The mean peak levels of progesterone-enhanced LH surges 4 days (16.83 ± 2.39 ng/ml) and 1 wk (12.56 ± 1.51 ng/ml) after exposure to elevated estradiol levels were greater than those in the oil-treated rats similarly primed with estradiol (P < 0.05, compare Fig. 3 upper and lower right panels). However, this surge-enhancing action of progesterone was also greatly diminished with longer durations of estradiol exposure. Namely, after 4 or 8 wk of exposure to increased estradiol levels, only one rat in each group displayed an LH surge after progesterone injection. Thus, exposure of young ovariectomized rats to chronic estradiol results in sequential loss of the positive feedback action of estradiol within the first 2 wk, and of the surge-enhancing effect of progesterone during the next 2 wk. These results indicate that the surge-enhancing action of progesterone delays the loss of LH surges that result from chronic exposure to elevated levels of estradiol (Fig. 4).
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Comparison of Steroid-Induced LH Surges in 3- and 5-Month-Old Female Rats
To determine whether the lack of LH surges in those rats receiving estradiol for 8 wk was due in part to their age, the LH surge-inducing action of a 3-day estradiol treatment alone or with progesterone was compared between 3- and 5-mo-old rats. Three days after ovariectomy and estradiol implantation, all rats in both age groups displayed LH surges, with no difference in peak levels (3 mo, 7.38 ± 1.00 ng/ml at 1700 h; and 5-mo, 9.89 ± 2.60 ng/ml at 1600 h) (Fig. 5, upper panel). In addition, the LH surge-enhancing action of progesterone also elicited identical LH surges in both age groups (3-mo peak, 28.63 ± 6.90 ng/ml; and 5-mo, 26.97 ± 7.78 ng/ml) (Fig. 5, lower panel). These results demonstrate that there is no age difference up to 5 mo of age in the neuroendocrine mechanism whereby estradiol, alone or with progesterone, induces an LH surge.
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DISCUSSION
The foregoing results are consistent with previous findings that a chronic increase in circulating estradiol levels elicits repetitive daily LH surges for at least 10 days in young ovariectomized rats [11, 19]. Such prolonged exposure to an elevation in estradiol is associated with a decline in the positive feedback action of estradiol on LH surges. This diminution of LH release is not only reflected in a gradual decrease in amplitude of the LH surges [11–13, 20] but also as we have observed, in the proportion of rats showing LH surges. In some previous reports, the LH surges were abolished within 10 days [11, 13]. In other studies, LH surges were still observed 14–15 days after estradiol treatments; however, the amplitudes of these LH surges were greatly decreased, suggesting their imminent disappearance [12, 20]. In our study, LH surges were abolished in most rats by 14 days and in all rats by 28 days after estradiol treatment. This variability in the time of extinction of the positive feedback action of estradiol among studies may be due to differences in the strains of rats, doses of estradiol, sampling frequency, or interval between ovariectomy and estradiol treatment. In spite of these differences, the foregoing observations indicate that chronic exposure to elevated estradiol levels abolishes daily LH surges within approximately 2 wk.
It has been well established that progesterone can enhance the positive feedback action of estradiol by increasing the amplitude of LH surges in estrogen-primed rats [18, 21–23]. The results presented herein demonstrate that progesterone also increases the proportion of ovariectomized rats showing LH surges within 4 or 8 days after estradiol treatment. Even after 2 wk of exposure to increased circulating estradiol levels, when the positive feedback action of estradiol alone is too weak to elicit any detectable rise in plasma LH, the enhancing action of progesterone is still manifested by the occurrence of low-amplitude LH surges. However, after 4 wk of estradiol treatment, most rats are completely refractory to induction of LH surges by estradiol either alone or with progesterone and remain so for up to 8 wk. These findings demonstrate for the first time that exposure of young rats to chronic estradiol causes a sequential loss of the stimulatory actions of estradiol first, followed by progesterone.
In addition to increasing the amplitude of LH surges, progesterone, when administered at 0900 h, advances LH surges by several hours in both intact proestrous [22] and ovariectomized estrogen-primed rats [14]. However, in the present study, progesterone injection at 1200 h did not advance the time of onset of LH surges. This finding is consistent with those reported previously, in which progesterone was administered at noon [21] or later [23]. Thus, the time of treatment determines whether or not progesterone advances LH surges.
The rats receiving estradiol for 8 wk (at the age of about 5 mo) were about 1–2 mo older at the time of sampling than the animals in the other groups. However, their lack of response was not an effect of age because both 3- and 5-mo-old young rats have similar LH surges in response to the positive feedback stimulation of estradiol alone or with progesterone. Thus, in young rats treated for up to 8 wk, it is the duration of exposure to an increase in circulating estradiol rather than age that causes loss of the stimulatory actions of estradiol and progesterone on LH surges.
The underlying mechanisms mediating the estradiol-induced loss of the stimulatory actions of estradiol and progesterone on LH surges in chronically treated rats are still unclear. One possibility is that the daily LH surges cause gradual depletion of pituitary LH stores or pituitary response or sensitivity to GnRH. It has been previously reported that even after chronic estradiol treatment has abolished LH surges, an i.v. injection of synthetic GnRH (50 ng/100 g BW) increases plasma levels of LH equally in both young and middle-aged PE rats [14]. To date, however, no dose response study has been performed under conditions of chronic versus acute estradiol treatment. An alternative mechanism whereby chronic estradiol abolishes LH surges might reside in the brain. Another possibility is that the high levels of circulating prolactin that are generally associated with chronic administration of estradiol inhibit LH release [13]. However, chronic administration of the dopamine agonist, bromocriptine, to rats that have been exposed to elevated estradiol levels for 2 wk only partially restores LH surges [13]. This finding indicates that other as yet undetermined factors may also play important roles in the mechanism whereby chronic estradiol suppresses LH surges.
A similar sequential loss of the stimulatory actions of estradiol and progesterone on LH surges in the presence of chronically elevated estradiol levels (30 pg/ml) also occurs naturally during aging. In PE middle-aged rats, estradiol alone no longer induces LH surges [24]. During the early phase of PE (30–40 days), however, progesterone can still enhance the effect of estradiol and thereby elicit LH surges [10]. After a prolonged duration of PE, estradiol cannot trigger LH surges, either alone or with progesterone [8, 10, 14, 24]. Thus, the neuroendocrine responses of the LH surge mechanism to estradiol and progesterone in early PE and long-term PE rats are similar to those in young rats after 2 or 4 wk of exposure to estradiol, respectively. It is important to note, however, that in PE rats, circulating levels of estradiol average 30 pg/ml [7, 8]. These levels are about 50% lower than those reported in young rats after insertion of estradiol implants identical to those used in this study [16]. Finally, the difference in age between intact PE rats and chronically estradiol-treated young rats must be considered before drawing any conclusions about PE rats from results obtained in young rats.
In summary, the current results, in conjunction with previous findings, suggest that the sustained increase in circulating estradiol that occurs with aging may cause a sequential loss of the stimulatory actions of estradiol and progesterone on the LH surge in middle-aged rats. Further, the same mechanism that mediates the estradiol-induced loss of LH surges in young rats may cause loss of cyclicity in middle age. If this is the case, young rats chronically treated with estradiol would serve as an ideal model for investigating the mechanism whereby LH surges disappear during aging.
ACKNOWLEDGMENTS
We gratefully acknowledge Dr. Terry Nett for kindly providing the anti-rat LH antibody and Mr. Hunter Callahan for his expert assistance with the LH RIA.
FOOTNOTES
First decision: 3 August 2000.
1 Supported by NIH grants R01-AG13454 (S.J.L.), T32-AG00242, and F31-MH12289 (H.-W.T.). 
2 Correspondence: Sandra Legan, Department of Physiology, University of Kentucky, 800 Rose Street, Lexington, KY 40536-0298. FAX: 859 323 1070; sjlegan{at}pop.uky.edu
Accepted: October 3, 2000.
Received: July 10, 2000.
REFERENCES
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