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Biology of Reproduction 66, 1104-1110 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Loss of Luteinizing Hormone Surges Induced by Chronic Estradiol Is Associated with Decreased Activation of Gonadotropin-Releasing Hormone Neurons1

Houng-Wei Tsaia, and Sandra J. Legan2,a

a Department of Physiology, University of Kentucky, Lexington, Kentucky 40536


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic exposure of young ovariectomized rats to elevated circulating estradiol causes loss of steroid-induced LH surges. Such LH surges are associated with cFos-induced activation of GnRH neurons; therefore, we hypothesized that chronic estradiol treatment abolishes LH surges by decreasing activation of GnRH neurons. Regularly cycling rats were ovariectomized and immediately received an estradiol implant or remained untreated. Three days or 2 or 4 wk later, the estradiol-treated rats received vehicle or progesterone at 1200 h, and 7 hourly blood samples were collected for RIA of LH. Thereafter, all rats were perfused, and the brains were examined for immunocytochemical localization of cFos and GnRH. The GnRH neurons from untreated ovariectomized rats rarely expressed cFos. As reported, LH surges induced by 3 days of estradiol treatment were associated with a 30% increase in cFos-containing GnRH neurons, and progesterone enhanced both the amplitude of LH surges and the proportion of cFos-immunopositive GnRH neurons. As hypothesized, the abolition of LH surges caused by 2 or more weeks of estradiol was paralleled by a reduction in the percentage of cFos-containing GnRH neurons, and this effect was delayed by progesterone. These results suggest that chronic estradiol abolishes steroid-induced LH surges in part by inactivating GnRH neurons.

aging, estradiol, GnRH, hypothalamus, LH, progesterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In rats, the preovulatory LH surge is triggered by the positive feedback action of estradiol [1] in conjunction with a circadian signal [2, 3]. In addition, the concomitant rise in circulating progesterone levels during the surge on proestrous afternoon enhances the action of estradiol by increasing the amplitude of the LH surge [4]. The final target for these various inputs is a subpopulation of GnRH neurons, the majority of which are located in the basal forebrain at the level of the organum vasculosum of the lamina terminalis (OVLT) and the rostral preoptic area [5–7]. Using cFos, an early immediate gene product, as a marker for increased neuronal activity, about 40% of GnRH neurons express cFos, i.e., are activated, only during spontaneous proestrous or steroid-induced LH surges [8–10]. Administration of pentobarbital, which blocks the LH surge, also blocks cFos expression in GnRH neurons [8]. In contrast, enhancement of the LH surge by administration of progesterone is associated with an increase in the proportion of cFos-containing GnRH neurons [9]. Thus, the amplitude of the LH surge is highly correlated with the number of GnRH neurons containing cFos, suggesting that cFos is a reliable marker for monitoring GnRH activation.

The first overt sign of reproductive aging in female rats occurs around 8–12 mo of age, when the preovulatory LH surge becomes attenuated and delayed [11–13]. It remains to be determined what causes the attenuation of LH surges in middle age, although it probably results from a decrease in response of the hypothalamic-pituitary axis to the positive feedback action of estradiol. Within the next month or two, estrous cycles become irregular in length, with prolonged periods of estrus [14] during which circulating estradiol levels are elevated. These prolonged periods of elevated estradiol reflect the persistence of ovarian follicles in the absence of an adequate LH surge. Soon thereafter, around 9–11 mo of age, female rats cease cycling altogether. This state of acyclicity is termed persistent estrus (PE) and is characterized by persistent vaginal cornification, which is indicative of sustained follicular development and estradiol secretion but lack of LH surges and ovulation [15, 16]. Even though circulating estradiol levels are chronically elevated to 30–40 pg/ml in PE rats [16, 17], these rats cannot respond to the positive feedback action of estradiol with an LH surge [18, 19]. During the first month of PE (early PE), however, LH surges can still be elicited by progesterone [19, 20]. Eventually, even the ability of progesterone to induce LH surges in estradiol-primed PE females disappears [19]. When rats age further, they become pseudopregnant, or persistently diestrous, and the ability of estradiol and progesterone to induce LH surges returns [17]. Based on this finding, it has been suggested that the extinction of LH surges in PE rats may not be totally caused by aging [17, 21].

This possibility is supported by several reports indicating that exposure of young ovariectomized females to elevated estradiol levels for 9–12 days abolishes the positive feedback action of estradiol [22–24]. We recently reported that there is also a subsequent loss of the ability of progesterone to induce LH surges in young ovariectomized rats exposed to a chronic elevation in estradiol, just as occurs in PE rats [25].

Based on the foregoing considerations, we hypothesized that chronic exposure to estradiol causes loss of the LH surge-inducing actions of estradiol and progesterone in part by decreasing activation of the GnRH neurons. Because cFos can serve as a marker to monitor GnRH neuronal activation, the following experiment was designed to test the hypothesis that in young ovariectomized rats, chronic exposure to estradiol causes a gradual decrease in the numbers of GnRH neurons expressing cFos. Further, this decrease parallels the gradual decrease in amplitude and occurrence of LH surges in response to estradiol alone or with progesterone.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Young (2-mo-old) female Sprague-Dawley rats (Zivic-Miller Laboratories, Zelienople, PA) were maintained under constant conditions of temperature (22–24°C) and photoperiod (14L:10D, lights-on at 0500 h). Food and water were provided ad libitum. Vaginal smears were monitored 6 days/wk for 3–4 wk to determine estrous cyclicity. Only females that showed at least two consecutive 4-day estrous cycles were used. All experimental protocols were approved by the University Internal Animal Care and Use Committee and were performed according to the NIH guidelines.

Experimental Design

To determine whether chronic exposure to elevated circulating estradiol concentrations, which causes a progressive loss of the LH surge-inducing actions of estradiol and progesterone, is paralleled by a gradual inactivation of GnRH neurons, nine groups of 4–6 regularly cycling rats (n = 43) were bilaterally ovariectomized under anesthesia (Day 0). At the time of ovariectomy (OVX), they averaged 93.6 ± 0.8 days of age (mean ± SEM) and weighed 268.4 ± 3.8 g. Immediately thereafter, 6 of the groups (n = 30, OVX+E2) received a Silastic capsule (Dow Corning Co., Midland, MI) containing crystalline estradiol-17ß (Sigma Chemical Co., St. Louis, MO) constructed as described previously [26]. The remaining 3 groups served as ovariectomized controls (n = 13, OVX). Such estradiol implants have been reported to generate plasma estradiol concentrations of about 75 pg/ml [26]. At 0.5, 2, or 4 wk after ovariectomy, plasma LH concentrations were assessed in samples obtained from one group of OVX rats (n = 4, 5, and 4, respectively) and two groups of OVX+E2 rats (n = 4–6 per group). By the time of sampling, the rats in the 3 age groups were between 3 and 4 mo of age, but we did not control for age because the LH surges induced in response to estradiol alone or with progesterone are identical in 3- and 5-mo-old rats [25]. Blood samples (0.2 ml) were obtained hourly from 1200 to 1700 h via a right atrial cannula that had been inserted under anesthesia on the previous day [27]. The plasma samples were separated and stored at -20°C until determination of LH concentrations. To compare the LH surges in response to estradiol alone or with progesterone, at 1200 h on each sampling day after the first blood sample was obtained, one group of OVX+E2 rats received an s.c. injection of corn oil vehicle (0.1 ml/100 g body weight) (n = 5, 5, and 4 at 0.5, 2, and 4 wk, respectively), and the other group received progesterone (0.5 mg/0.1 ml/100 g body weight) (n = 5, 5, and 6 at 0.5, 2, and 4 wk, respectively). For immunocytochemical localization of cFos and GnRH, at the end of sampling all rats were deeply anesthetized and perfused transcardially between 1700 and 1800 h. They were perfused first with Dulbecco PBS (0.05 M, pH 7.0; Gibco BRL, Grand Island, NY) followed by phosphate-buffered 4% paraformaldehyde (0.05 M, pH 7.3–7.4; Fisher, Fair Lawn, NJ) containing 7.5% picric acid (Sigma). The brains were removed, blocked, and postfixed in the latter fixative at 4°C overnight and then infiltrated with 25% sucrose in PBS (0.05 M, pH 7.0). Brains were subsequently cut on a cryostat into 10 series of 40-µm frontal sections from the septal nuclei through the mammillary bodies. The sections were stored in cryoprotectant [28] at -20°C. One of the sets containing the OVLT was subsequently processed by immunocytochemistry for dual localization of cFos and GnRH.

Immunocytochemistry

GnRH and cFos were localized by means of a previously described dual immunoperoxidase method [8, 10, 29] modified as follows. Brain sections were rinsed in Tris-buffered saline (TBS, 0.05 M, pH 7.4; Sigma) and then incubated in rabbit polyclonal anti-human cFos (1:10 000; Santa Cruz Biotechnology, Santa Cruz, CA) in TBS containing 0.4% Triton X-100 (Sigma) and 10% normal goat serum (Vector Laboratories, Burlingame, CA) at 4°C for 48 h. The cFos antibody was raised against a peptide mapping within a highly conserved domain (amino acids 128–153) of human cFos and cross-reacts with cFos, Fos B, Fra-1, and Fra-2. After thorough rinsing in TBS, the sections were incubated in 1:600 goat anti-rabbit biotinylated IgG (Vector) in TBS containing 0.4% Triton X-100 at room temperature for 1 h. After rinsing in TBS, the sections were incubated in 1:45 avidin/biotinylated peroxidase solution (Vectastain Elite ABC Kits; Vector) for 1 h at room temperature. The sections were then rinsed in 0.175 M sodium acetate buffer (pH 6.5). Nickel (0.025 M nickel sulfate)-intensified diaminobenzidine (0.02% DAB; Sigma) was used as the chromogen to yield a blue-black reaction product. After rinsing in TBS to stop the reaction, the tissue sections were incubated in 1:10 000 rabbit polyclonal anti-GnRH (LR1; a kind gift from Dr. Robert Benoit) in TBS containing 0.4% Triton X-100 and 10% normal goat serum at 4°C for 48 h. The procedure for localization of GnRH was essentially the same as that described above, except that DAB was used to generate a brown reaction product. Sections were mounted, air dried, and coverslipped. All sections for each rat were examined under a light microscope (Eclipse E600; Nikon, Morton Grove, IL), and GnRH cells with and without cFos were counted at a magnification of 200–400x. All cell counts were performed without knowledge of the animal's treatment group.

Radioimmunoassay

Plasma LH levels were determined in 20-µl and 5-µl aliquots by means of an RIA described previously [30]. CSU 120 (kindly provided by 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 (Covance Laboratories, Vienna, VA) was diluted to approximately 14 000 cpm/100 µl in assay buffer. The tubes were incubated at 4°C for 48 h between the addition of first antibody, radiolabeled LH, and second antibody. Plasma LH concentrations are reported in terms of nanograms of RP-3 (National Hormone and Pituitary Program, NIH, Washington, DC) per milliliter. The sensitivity of the assay (100% - 2 SD of maximum binding) averaged 0.02 ng/tube, and the interassay coefficient of variation was 14.1% for an intact serum pool that inhibited binding of labeled hormone to 76.4% of maximal binding.

Statistical Analysis

Ages and body weights were compared among groups by separate two-way ANOVAs (SigmaStat; SPSS Science, Chicago, IL) to reveal the main effects of estradiol implantation and duration of treatment. An LH surge was defined as an increase in plasma LH concentrations 2 SD above the mean value at 1200 h for at least two consecutive samples [31]. Only results from the groups in which more than two rats had LH surges were used in the analyses of LH levels. Peak LH levels were compared by two-way ANOVA to reveal the main effects of duration of estradiol treatment and progesterone administration and their interaction. The total number of GnRH cells and the number and percentage (raw data) of cFos-containing GnRH neurons in the 2 sections nearest the OVLT were compared by two-way ANOVA to reveal any main effect of the duration of estradiol implantation and progesterone treatments. A confidence level of P < 0.05 was considered significant, and if there was a significant interaction, a Tukey test was used for post hoc comparisons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Body Weight and Age of Rats

By the time of sampling, the 4-wk rats (122.2 ± 1.7 days, n = 14) were older than the rats in the other 2 groups (0.5 wk: 96.1 ± 1.7 days, n = 14; 2 wk: 107.8 ± 1.6 days, n = 15) (P < 0.001). In addition, the average weight of rats in the 4-wk group (309.0 ± 8.3 g) was greater than that in the other 2 groups (0.5 wk: 272.5 ± 8.3 g; 2 wk: 277.2 ± 7.7 g) (P = 0.006). Further, the untreated ovariectomized rats (299.5 ± 7.8 g, n = 13) weighed more than those receiving estradiol (273.0 ± 5.1 g, n = 30, P = 0.007).

Distribution of GnRH Neurons in the Rat Brain

Figure 1 depicts the typical nuclear staining of cFos in GnRH neurons in a representative section from the OVLT of a progesterone-treated rat that was pretreated with estradiol for 0.5 wk, as compared to the absence of cFos staining in a section from a rat ovariectomized 4 wk earlier. As reported previously [5–7], the majority of GnRH neurons and of cFos-containing neurons are localized near the OVLT, and this distribution was not affected by any of our treatments (Fig. 2, left). Therefore, in all subsequent analyses only counts from the 2 sections nearest the OVLT were analyzed. Generally, expression of cFos in GnRH neurons was correlated with the amplitude of LH surges. Thus, in untreated ovariectomized rats, the GnRH neurons were virtually devoid of cFos staining regardless of the duration after ovariectomy (Fig. 2, upper). In contrast, induction of an LH surge by estradiol alone was associated with an induction of cFos in GnRH neurons (Fig. 2, middle), and enhancement of the LH surge by progesterone was associated with a further increase in the expression of cFos in GnRH neurons (Fig. 2, lower).



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  		FIG. 1. Immunolabeled cFos and GnRH during an LH surge induced by progesterone injection in a rat pretreated with estradiol for for 0.5 wk (left panel) and in the absence of an LH surge in an untreated rat ovariectomized 4 wk earlier (right panel) (x400). After steroid treatment, many more GnRH neurons (gray cytoplasm) express cFos (black nuclei). These sections were also stained for dopamine ß-hydroxylase (black fibers)



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  		FIG. 2. Anatomical distribution of all GnRH neurons (open bars, left), the cFos-containing GnRH neurons (closed bars, left), and plasma LH levels (right) in a representative rat from each of three treatment groups. Results from a control rat 2 wk after ovariectomy (OVX) are depicted in the upper panel. The middle panel depicts results from a rat receiving an oil injection (Oil) after being pretreated with estradiol (OVX+E2) for 3 days (0.5 wk). Results from a rat injected with progesterone (P) after 2 wk of pretreatment with estradiol (OVX+E2) are shown in the bottom panel

Effect of Ovariectomy and Steroid Treatments on Plasma LH Concentrations

As expected, in the untreated ovariectomized controls (OVX), there were no LH surges (Fig. 3, upper), and basal concentrations of plasma LH remained relatively constant between 1200 and 1700 h in all 3 groups (Fig. 4, upper). Basal LH concentrations underwent a typical "castration" response and increased with time after ovariectomy, averaging 2.52 ± 0.54 ng/ml, 8.79 ± 0.96 ng/ml, and 14.85 ± 1.38 ng/ml at 0.5, 2, and 4 wk postovariectomy, respectively (P < 0.001). In all 6 groups of estradiol-treated rats, basal LH concentrations, i.e., those at 1200 h, were undetectable.



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  		FIG. 3. Suppression of the incidence of steroid-induced LH surges by chronic exposure of young ovariectomized rats to elevated estradiol levels (OVX+E2). Results from untreated ovariectomized controls (OVX) are shown in the upper panel. In the estradiol-pretreated rats, either corn oil (Oil, middle) or progesterone (P, lower) was injected s.c. at 1200 h on the day of sampling



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  		FIG. 4. Suppression of the amplitude of steroid-induced LH surges by chronic exposure of young ovariectomized rats to elevated levels of estradiol. Mean (± SEM) plasma LH concentrations in untreated ovariectomized rats (OVX) are shown in the upper panel. Plasma LH levels in the rats that were treated with estradiol for 0.5, 2, or 4 wk (OVX+E2) are shown in the lower 2 panels. On the day of sampling, either corn oil (Oil, middle) or progesterone (P, lower) was injected s.c. at 1200 h. In the estradiol-treated groups in which more than one rat surged, only the LH levels from the rats that surged are included in the mean

With regard to the phasic mode of LH secretion, as we reported previously [25], increasing the period of exposure to sustained elevations in circulating estradiol led to a gradual decrease in the incidence and amplitude of estradiol-induced LH surges (P < 0.001). Thus, although all rats receiving oil injections (OVX+E2) surged 0.5 wk after estradiol treatment, only 1 rat exhibited an attenuated LH surge after 2 wk, and LH surges were abolished after 4 wk of estradiol treatment (Figs. 3 and 4, middle). Progesterone treatment (OVX+E2+P) enhanced the LH surge-inducing action of estradiol (P < 0.001) and thereby delayed the disappearance of LH surges induced by chronic estradiol. Thus, in 5 of 5 rats 0.5 wk after receiving estradiol implants, injection of progesterone at 1200 h elicited robust LH surges that began at 1400 h and attained a peak of 20.29 ± 4.37 ng/ml at 1600 h (Figs. 3 and 4, lower). Two weeks later, 3 of 5 rats still displayed LH surges in response to progesterone, reaching a peak of 16.68 ± 3.79 ng/ml at 1700 h. However, after 4 wk of exposure to chronically increased estradiol levels, the action of progesterone was severely diminished, and only 1 rat responded with a reduced LH surge (peak of 7.74 ng/ml at 1600 h).

Effects of Ovariectomy and Chronic Exposure to Elevated Estradiol on cFos Expression in GnRH Neurons

The total number of GnRH neurons in the 2 sections nearest the OVLT was not altered with time after ovariectomy (P > 0.05), and the GnRH neurons were virtually devoid of cFos at all times after ovariectomy (Fig. 5, upper).



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  		FIG. 5. Increased duration of estradiol exposure in rats decreases the number of GnRH neurons showing cFos at the level of the OVLT. Neither ovariectomy (OVX, upper) nor chronic exposure to estradiol (OVX+E2, middle and lower) affected the total number of GnRH cells at the level of the OVLT (open bars). Expression of cFos was virtually absent in untreated controls (closed bars, upper). However, prolonged elevation of estradiol levels decreased the number of cFos-containing GnRH cells (closed bars, middle and lower) in vehicle-treated rats (Oil, middle) more rapidly than in progesterone-treated rats (P, lower). Asterisks indicate a significant difference from the respective 0.5-wk mean

The total number of GnRH neurons in the 2 sections closest to the OVLT remained constant among all 6 steroid-treated groups regardless of duration of exposure to chronic estradiol with or without progesterone (P = 0.90; Fig. 5, open bars, middle and lower). In the OVX+E2 rats, increasing duration of exposure to estradiol decreased the number of GnRH neurons expressing cFos (Fig. 5, closed bars, middle). Thus, the proportion of GnRH neurons immunopositive for cFos was also decreased by chronic exposure to estradiol from 37.0% ± 6.8% after 0.5 wk of treatment to 13.5% ± 5.5% and 2.0% ± 0.9% at 2 and 4 wk after estradiol, respectively (P < 0.001; Fig. 6, open bars). Among the 5 rats receiving estradiol for 2 wk, 29.2% of GnRH neurons were immunopositive for cFos in the one rat that surged. In the remaining 4 rats that did not surge, the proportions of cFos-containing GnRH neurons were 0%, 2.2%, 15.9%, and 20.0% (mean ± SEM, 9.5% ± 5.0%).



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  		FIG. 6. Progesterone (P, closed bars) delays suppression of the proportion of cFos-containing GnRH neurons caused by exposure of young ovariectomized rats to chronically elevated estradiol levels (OVX+E2). Results from vehicle-treated controls (Oil) are depicted by the open bars. Asterisks indicate a significant difference from the respective 0.5-wk mean

In the OVX+E2+P rats, progesterone enhanced the activation of GnRH neurons by estradiol treatment (P < 0.001), and the number of cFos-immunopositive GnRH neurons was gradually reduced with time after estradiol (P < 0.001) in parallel with the progressive attenuation of the amplitude and incidence of LH surges (Fig. 5, compare closed bars, middle and lower). Thus, cFos was found in 63.1% ± 8.5%, 50.3% ± 10.2%, and 20.6% ± 2.3% of GnRH neurons after 0.5, 2, and 4 wk of exposure to an elevation in chronic estradiol, respectively, reflecting progesterone's action in delaying the estradiol-induced abolition of LH surges (Fig. 6, closed bars). Among the 5 OVX+E2+P rats treated with estradiol for 2 wk, 66.5% ± 3.0% of GnRH neurons contained cFos in the 3 rats that surged. In the remaining 2 rats, 31.3% and 20.7% of GnRH neurons expressed cFos. In the OVX+E2+P rats treated with estradiol for 4 wk, the only rat that surged in response to progesterone displayed colocalization of cFos in 24.6% of GnRH neurons, whereas 10.9%, 17.2%, 22.4%, 23.1%, and 25.6% (mean ± SEM, 19.8% ± 2.6%) of GnRH neurons contained cFos in the 5 rats that did not surge.

When results from oil and progesterone-treated rats are grouped across time, there is a strong correlation between the percentage of cFos-containing GnRH neurons and amplitude of the LH surges (r2 = 0.726, P < 0.001; Fig. 7).



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  		FIG. 7. Correlation between maximum plasma LH levels and cFos expression in GnRH neurons in young ovariectomized rats 0.5, 2, or 4 wk after estradiol treatment. A significant correlation (P < 0.05) exists between peak plasma LH concentrations and the percentage of cFos-expressing GnRH cells (r2 = 0.749) ({circ}, oil-treated rats; , progesterone-treated rats)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In agreement with previous findings [22, 24, 25], these results demonstrate that the positive feedback action of estradiol in inducing LH surges in ovariectomized rats disappears within 2 wk after exposure to chronically elevated estradiol levels. The results herein also concur with an earlier report that about 23% of GnRH neurons contain cFos during the LH surge in estradiol benzoate-treated ovariectomized rats and that enhancement of the LH surge by administration of progesterone is associated with an increase in the proportion of cFos-containing GnRH neurons to about 42% [9]. Based on these findings, we hypothesized that the mechanism whereby a chronic increase in estradiol levels abolishes LH surges within 2 wk is mediated in part by a progressive decrease in the proportion of GnRH neurons immunopositive for cFos. Our results support this hypothesis and demonstrate for the first time that the gradual loss of estradiol- or progesterone-induced LH surges in young ovariectomized rats that are exposed to chronically elevated circulating levels of estradiol is accompanied by a gradual decrease in the proportion of cFos-labeled GnRH neurons.

In several previous studies, the GnRH neurons were devoid of cFos at times during the estrous cycle when plasma LH concentrations were basal [8, 32] or at the expected time of the LH peak in rats that did not surge, e.g., in pentobarbital-blocked proestrous rats [8], and in PE rats [20]. In another study, about 12% of all GnRH neurons still contained cFos on the morning of proestrus, and about 25% of GnRH neurons in the OVLT were immunopositive for cFos between 1300 and 1430 h at proestrus in young rats [33]. The discrepancies among these reports may be due to the specificity and/or sensitivity of the different antibodies employed. In proestrous rats treated with the {alpha}-adrenergic receptor antagonist phenoxybenzamine to block the LH surge, the proportion of GnRH neurons that colocalized with cFos ranged from 0% to about 30% [34]. We found that anywhere from 0% to about 30% of GnRH neurons still expressed cFos after steroid-induced LH surges were abolished by chronic estrogen treatment of young rats. Thus, pharmacologic suppression of the LH surge by two different approaches was unable to inhibit activation of all GnRH neurons.

The presence of cFos in 0%–30% of GnRH neurons in the absence of LH surges may reflect the activation of one or more subpopulations of GnRH neurons by an incomplete neural signal. Thus, after 4 wk of chronic estradiol treatment, the neural signal may not be totally suppressed, whereas with longer exposure to estradiol, as occurs in middle-aged PE rats [20], the signal becomes completely abolished. Alternatively, the neural signal to the GnRH neurons is mediated by a number of different neurotransmitters and some of them are not completely suppressed after 4 wk of estradiol treatment. Numerous inputs, both stimulatory and inhibitory, control GnRH release [35–38], and a plethora of anatomical and pharmacological evidence indicates that there are a number of different subpopulations of GnRH neurons [39–44]. Thus, when a given subpopulation of GnRH neurons is blocked, e.g., the neurons activated by estradiol, the action of any residual inputs such as progesterone may be reflected in part by cFos activation of the remaining GnRH neurons.

In untreated ovariectomized rats, mean plasma LH concentrations increase with time after ovariectomy [45, 46], reflecting an increase in both LH pulse frequency and amplitude [47–49]. Although mean portal plasma GnRH concentrations increase after ovariectomy [50–52], it remains to be determined whether this increase reflects an increase in GnRH pulse frequency or amplitude, or both. In this regard, there is a well-established postovariectomy increase in LH pulse frequency in rats [50, 53]. Therefore, it is highly likely that an increase in GnRH pulse frequency also occurs after ovariectomy in rats, which drives the increase in LH pulse frequency and mean LH levels. Although it might be postulated that such postovariectomy increases in GnRH and thus LH release are associated with increased cFos activation of GnRH neurons, the foregoing results demonstrate that this is not the case. These data suggest that the negative feedback action of estradiol is not mediated by increasing cFos in GnRH neurons, or if it is, the increase was not detected by the methods employed herein. The latter possibility would be likely if, for example, only a small proportion of GnRH neurons is synchronously activated to elicit each pulse. Another possibility is that cFos is only elevated for a relatively brief period of time once a neural system is activated.

The rats that had been treated with increased estradiol for 4 wk were older than the rats in the other groups at the time of sampling. We are confident that this age difference did not cause loss of LH surges because we have previously demonstrated that both 3- and 5-mo-old rats respond to the positive feedback stimulation of estradiol and progesterone with similar LH surges [25].

The sequential loss of the LH surge-inducing actions of estradiol and progesterone in chronically estradiol-treated young rats is similar to that observed in middle-aged PE rats [19]. In middle age, intact cycling female rats show attenuated LH surges at proestrus that are associated with a decreased percentage of GnRH neurons containing cFos [32, 33]. When rats age further and become PE, the resultant loss of spontaneous LH surges is accompanied by a virtual absence of cFos in GnRH neurons [20]. Further, reinstatement of LH surges and estrous cycles in PE rats by administration of progesterone every 4–5 days is also accompanied by an increase in the proportion of GnRH neurons that are immunopositive for cFos [20]. These results suggest that the mechanisms causing loss of the positive feedback action of estradiol in inducing LH surges with increasing age are similar to those causing abolition of LH surges after a chronic elevation in circulating estradiol levels, in that they are also mediated by a gradual decrease in activation of cFos in GnRH neurons. Our results support the hypothesis proposed previously [17, 54, 55], that the chronic elevation in estradiol levels associated with irregular cycles and PE in middle-aged rats may cause the loss of LH surges observed with age.

Chronic exposure of young female rats to estradiol abolishes the LH surge-inducing actions of estradiol and progesterone in association with a gradual abolition of the activation of cFos in the GnRH neurons. The parallel disappearances of LH surges and cFos induction in GnRH neurons are similar to those that occur during aging. Thus, young rats chronically treated with estradiol might serve as an appropriate model for studying the mechanism whereby LH surges disappear during aging.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Drs. Terry Nett and Robert Benoit for providing the anti-rat LH and anti-rat GnRH antibodies, respectively. We also thank Mr. Hunter Callahan for his expert assistance with the LH RIA.


   FOOTNOTES
 
First decision: 8 November 2001.

1 This work was supported by NIH grants R01-AG13454 (S.J.L.), T32-AG00242, and F31-MH12289 (H.-W.T.). Back

2 Correspondence: Sandra Legan, Department of Physiology, University of Kentucky, 800 Rose Street, Lexington, KY 40536-0298. FAX: 859 323 1070; sjlegan{at}uky.edu Back

Accepted: November 8, 2001.

Received: September 26, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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