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BOR - Papers in Press, published online ahead of print March 16, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.040329
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BIOLOGY OF REPRODUCTION 73, 115–122 (2005)
DOI: 10.1095/biolreprod.105.040329
© 2005 by the Society for the Study of Reproduction, Inc.

A Nongenomic Action of 17ß-Estradiol as the Mechanism Underlying the Acute Suppression of Secretion of Luteinizing Hormone1

J. Alejandro Arreguin-Arevalo , and Terry M. Nett 2 

Department of Biomedical Science, Colorado State University, Fort Collins, Colorado 80523


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of the present study was to determine the ability of 17ß-estradiol (E2) and conjugated forms of E2 (E2 conjugated to BSA [E2-BSA] and a novel conjugate, E2 conjugated to a small peptide [E2-PEP]) to prevent the GnRH-induced secretion of LH and to determine the role of estradiol receptors (ERs) and ER subtypes (ER{alpha}, also known as ESR1, and ERß, also known as ESR2) in the mediation of the acute action of E2 in primary cultures of ovine pituitary cells. Preincubation of cells for 15 min with E2, E2-BSA, or E2-PEP prevented the GnRH-induced secretion of LH (P < 0.01). Treatment of cells with nonestrogenic steroid hormones did not affect secretion of LH when given alone, nor did these steroids impair the E2-induced inhibition of LH secretion (P > 0.1). Likewise, treatment of cells with the ER-antagonists tamoxifen, hydroxytamoxifen, or ICI 182 780 did not affect (P > 0.1) secretion of LH when given alone but did prevent (P < 0.01) the inhibition by E2 and the E2-conjugates on GnRH-induced secretion of LH. When cells were treated with subtype-selective ER agonists, the ER{alpha} agonist (propylpyrazole-triol), but not the ERß agonist (diarylpropionitrile), decreased (P < 0.01) the GnRH-induced secretion of LH. In conclusion, the rapidity by which E2 prevented GnRH-induced release of LH in ovine pituitary cells suggests that this inhibition is mediated via a nongenomic action of E2. The inhibition of GnRH-induced secretion of LH proved to be steroid specific and mediated by ERs. It may occur specifically through ER{alpha}. The fact that E2-BSA or E2-PEP mimicked the action of E2 suggests that this effect was mediated by an ER associated with the plasma membrane.

anterior pituitary, estradiol, estradiol receptor, gonadotropin-releasing hormone, luteinizing hormone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many aspects of the positive feedback of 17ß-estradiol (E2) on LH secretion have been determined [1–4]; however, the mechanisms underlying the rapid decrease in LH secretion immediately following administration of E2 remain unknown. In vivo studies indicate that the decrease in LH secretion induced by E2 occurs, at least in part, at the level of the pituitary gland by impairing the GnRH-induced release of LH [5–7]. Moreover, it has been proposed that the short time frame (within an hour) required for E2 to decrease LH secretion observed in ovariectomized ewes is not compatible with the classic genomic mechanism of steroid hormone action [5].

Estradiol conjugated to a carrier molecule, such as BSA, has been used successfully to mimic the acute (within minutes) actions of E2 in vitro [8–14], which implicates a nongenomic action of E2. Mounting evidence supports a role of E2-binding proteins located on the plasma membrane in mediating the acute actions of E2 [15–17]. Several E2-binding proteins may be involved in mediating the acute actions of E2, including the primary estradiol-receptor (ER) subtypes, ER{alpha} (also known as ESR1) and ERß (also known as ESR2) [8, 18–21]; E2-binding to membrane proteins not related to the classic ER [22–25], such as ion channels [26– 28]; and sex hormone-binding globulin [29, 30]. Furthermore, ER{alpha} and ERß have been detected on the plasma membrane of estrogen-responsive cells [8, 18–21], and a differential [8, 9, 31, 32], synergistic [33, 34], or antagonistic [8, 35] action of ER{alpha} and ERß regulating cell function has been demonstrated in some cellular systems. Classically, antiestrogens, which bind to both ER{alpha} and ERß [36, 37] and generally prevent both the long-term [3, 38– 41] and acute actions of E2 [10, 13, 42–46], have been used to substantiate the role of ERs in mediating the actions of E2. Recently, a new generation of E2 agonists that selectively activate ER{alpha} or ERß has been developed; these agonists promise to be important tools for identifying the role of specific ER subtypes in mediating actions of E2 [39, 47– 51]. The present study was conducted to examine the ability of E2 and conjugated forms of E2 to rapidly prevent the GnRH-induced secretion of LH and to determine which ER subtype is responsible for mediating the acute action of E2 in ovine pituitary cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Conjugation of E2 to an Amino Acid Sequence

Six-keto-17ß-estradiol-6-carboxy methyl oxime (E2-6-CMO; Steraloids, Inc., Newport, RI) was conjugated to the amino group of serine of a 15-amino acid sequence (N-terminus-SGGEVVVDQPMERLY-C-terminus; PEP; Macromolecular Resources, Colorado State University, Fort Collins, CO). The E2-6-CMO was chosen to prepare a conjugate, because derivatization at carbon 6 of E2 does not prevent interaction of receptors with the hydroxyl radicals at positions 3 and 17 [52–54]. The reactions were performed at 10°C and pH 8 using N,N-dimethylformamide (DMF) as solvent [55] as follows: 4 mmol of E2-6-CMO dissolved in 0.5 ml of DMF were allowed to react for 10 min with 4 mmol of tri-N-butylamine, then 4 mmol of isobutyl chloroformate was added and the reaction continued for 20 min. Finally, 2 mmol of PEP were dissolved in DMF and mixed with the other reagents, and the reaction was continued for an additional 60 min before termination by the addition of approximately 10 µl of 0.1 M acetic acid.

Purification of E2-PEP Conjugate

Conjugation reactions were added to a 50-ml Sephadex LH20 column (Sigma, St. Louis, MO) using methanol as solvent. Twenty-five fractions (2 ml each) were collected. Presence of reactants was determined by optical densitometry at a wavelength of 280 nm. Identification of the reactants in the eluate was determined by Mass Spectrum Analysis (Macromolecular Resources) using two different procedures: Matrix Assisted Laser Desorption Ionization Time of Flight for compounds with an Mr of greater than 1000 (free PEP and E2-PEP conjugate), and electrospray mass spectrometry for compounds with an Mr of less than 500 (free E2-6-CMO). Conjugation efficiency was calculated based on the amount of unconjugated E2-6-CMO after separation of reactants by chromatography. The weight of unconjugated E2-6-CMO was estimated by plotting the absorbency values against those from known amounts of E2-6-CMO.

The E2-BSA (~30 molecules of E2 attached to each molecule of BSA) was purchased from Steraloids. To remove free E2, approximately 20 mg of E2-BSA were dissolved in 2 ml of PBS in a 16- x 150-mm glass tube. Five milliliters of diethylether were added, and the contents of the tubes were vortexed for 1 min and then frozen in dry-ice methanol. The organic fraction was poured into a 12- x 75-mm glass tube and evaporated under nitrogen in a warm heating block. Dried extracts were reconstituted in PBS containing 0.1% gel, incubated at 4°C overnight, and vortexed before quantification of E2 by radioimmunoassay. The extraction procedure was repeated six times for each sample, and each extract was kept separate and assayed for free E2 [56].

Preparation of Media and Stock Solutions

Pituitary dissociation medium consisted of 137 mM NaCl, 5 mM KCl, and 25 mM Hepes (U.S. Biochemical Corp., Cleveland, OH), pH 7.3, plus an enzymatic cocktail containing 1.0 mg/ml of collagenase (type II), 1.0 mg/ml of hyaluronidase (type V), and 0.02 mg/ml of deoxyribonuclease. Enzymes were freshly prepared immediately before dissociation. Culture medium consisted of Dulbecco modified Eagle medium (DMEM; Sigma-Aldrich, Inc., Saint Louis, MO) supplemented with 10% ovariectomized ewe serum, 500 mg/ml of streptomycin sulfate, 313 mg/ml of potassium penicillin G, and 2.2 g/L of NaHCO3. Dissociation and culture medium, as well as the enzymatic cocktail, were sterilized by filtration through 0.2-µm Millipore membranes (Fisher Science LLC, Denver, CO). Stock solutions of GnRH (in saline solution plus 0.1% BSA), E2-BSA, or E2-PEP (both in saline solution) were stored at –20°C in small aliquots. Steroid hormones were purchased from Sigma-Aldrich, and antagonists and agonists of ERs were purchased from Tocris Cookson, Inc. (Ellisville, MO). Estradiol, progesterone (P4), testosterone (T), hydrocortisone (HC), 17{alpha}-estradiol ({alpha}E2), tamoxifen (Tx), 4-OH-tamoxifen (HTx), ICI 182 780 (ICI), ER {alpha}-selective agonist (propylpyrazole-triol [PPT]), and ER ß-selective agonist (diarylpropionitrile [DPN]) were freshly dissolved in ethanol on the day of treatment of pituitary cells.

Dissociation and Incubation of Pituitary Cells

All procedures involving animals were approved by the Colorado State University Animal Care and Use Committee and complied with National Institutes of Health (NIH) guidelines. Anterior pituitary glands were collected during the breeding season following anesthesia of ewes with sodium pentobarbital and exsanguination. Tissues were removed and immediately placed in ice-cold dissociation medium. Anterior pituitary tissue from ovariectomized ewes was dispersed as described by Adams et al. [57] with the omission of trypsin digestion. Briefly, tissue was sectioned (thickness, 0.5 mm) with a Stadie-Riggs tissue slicer (Thomas Scientific, Swedesboro, NJ) and washed five times with dissociation medium without enzymes. Tissue was incubated in dissociation medium containing the enzymatic cocktail at 37°C in a Dubnoff metabolic shaker (GCA/Precision Scientific, Winchester, VA) for 90 min, and every 30 min, the cell suspension was passed through a Pasteur pipette. After dissociation, the cell suspension was washed (400 x g, 4 min) five times with dissociation medium without enzymes, resuspended in DMEM, and plated at 2 x 105 cells/well in 24-well tissue culture plates (Becton Dickinson Co., Franklin Lake, NJ). Cells were incubated for 2 days at 37°C under an atmosphere of 95% air:5% CO2. Cell viability was evaluated immediately after tissue dissociation and before administration of treatments by incubating the cells with 1% trypan blue for 3–4 min.

The amount of LH released from primary cultures of ovine pituitary cells [58] and the amount of free E2 extracted from E2-BSA [59] were quantified by a double-antibody radioimmunoassay. The reference standard for LH was NIH-OLH-S24. Triplicate standard curves were included in each assay, and samples were analyzed in duplicate at 50 and 100 µl sample/tube for LH and E2, respectively. Intra- and interassay coefficients of variation for LH were 3% and 7%, respectively, and the minimum detectable dose of LH averaged 28 pg. Intra- and interassay coefficients of variation for E2 were 5% and 10%, respectively, and the minimum detectable dose of E2 averaged 0.5 pg.

Experimental Procedure

The experiments were performed to evaluate the acute effects of conjugated or unconjugated forms of E2 on GnRH-induced release of LH, the steroid specificity of E2 actions, and the involvement of ERs as well as to determine which ER subtype is responsible for mediating the acute effect of E2 in primary cultures of ovine pituitary cells. After 2 days of incubation, anterior pituitary cells were washed twice with culture medium, and treatments were applied in 1 ml of medium. Cells (2 x 105 cells/ well, four wells per treatment, and three replicates [pituitaries] per experiment) were preincubated for 15–60 min with the corresponding treatment. After preincubation, cells were washed once with medium and incubated for 15 min with culture medium (negative control) or the previous treatment plus 2 nM GnRH. The dose of GnRH used in the present study is within the minimum dose that induces maximal release of LH in culture ovine pituitary cells as reported previously [57]. Culture medium was collected, centrifuged at 400 x g for 5 min to remove cells, transferred (~90% of medium) to a 12- x 75-mm plastic culture tube, and stored at –20°C for quantification of LH. When ethanol was used as solvent, its final concentration in the culture medium was never more than 0.1%, and the addition of 0.1% of ethanol to the GnRH-treated cells did not affect the amount of LH released.

Experiment 1 To evaluate the effect of E2, E2-BSA, and E2-PEP on GnRH-induced release of LH, anterior pituitary cells were preincubated for 60 min with 0, 0.01, 0.1, 1, 10, or 100 nM E2, E2-BSA, or E2-PEP. After preincubation, cells were washed and incubated for 15 min with culture medium or the previous treatment of E2, E2-BSA, or E2-PEP plus 2 nM GnRH.

Experiment 2 The effect of preincubation time on the ability of conjugated or unconjugated forms of E2 to decrease the amount of LH released during a subsequent 15-min GnRH challenge was analyzed by preincubating the cells for 15, 30, or 60 min with 0–100 nM E2, E2-BSA, or E2-PEP. The experiment was conducted in two parts. In the first, dispersed pituitary cells were preincubated for 30–60 min (control group); in the second part, cells were incubated for 15–60 min (control group).

Experiment 3 To evaluate the effect of nonestrogenic steroid hormones and an E2 stereoisomer ({alpha}E2) on GnRH-induced release of LH, cells were preincubated for 15 min with 0 or 1 nM E2 or 100 nM P4, T, HC, or {alpha}E2 with and without 1 nM E2. After preincubation, cells were treated with GnRH as described above.

Experiment 4 To study the ability of E2 antagonists to prevent the inhibitory action of E2 on the release of LH induced by GnRH, cells were preincubated for 15 min with 0 or 1 nM E2 or 100 nM Tx, HTx, or ICI with and without 1 nM E2, E2-BSA, or E2-PEP. After preincubation, cells were treated with GnRH as described above.

Experiment 5 The ability of specific ER ({alpha} and ß) agonists to mimic the action of E2 on secretion of LH was determined by preincubating the cells for 15 min with 0, 0.01, 0.1, 1, 10, or 100 nM PPT or DPN. After preincubation, cells were treated with GnRH as described above.

Data Analysis

Data were subjected to ANOVA using the general linear model of SAS [60] in a completely randomized design. When a significant F-value occurred, means were separated using Fisher least significant difference adjusted by the Tukey procedure. Data are presented as mean ± SEM throughout.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chromatography of the reactants subjected to conjugation revealed two major peaks in fractions collected from the Sephadex LH20 column (Fig. 1). Mass Spectrum Analysis revealed two main compounds in fractions 9–11, one with an Mr of 1679, corresponding to unconjugated PEP, and another with an Mr of 2020, corresponding to E2-PEP conjugate. The main compound detected in fractions 13 and 14 had an Mr of 359, corresponding to unconjugated E2-6-CMO. Efficiency of the individual conjugations ranged from 20% to 100% (n = 10) and averaged 44% ± 22%.



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  		FIG. 1. Two peaks were detected by optical densitometry in fractions collected after Sephadex LH20 column chromatography of the reaction mixture. Mass Spectrum Analysis revealed two compounds in fractions 9–11, one with a molecular weight of 1679, corresponding to free PEP, and another with a molecular weight of 2020, corresponding to E2-PEP. The major compound detected in fractions 13 and 14, with a molecular weight of 359, corresponded to free E2-6-CMO

Extraction with diethyl ether removed 0.7% (15.12 ± 4.02 µg of E2, n = 3 independent extractions of conjugate) of total expected E2 (at a 30:1 ratio) present in 20 mg of E2-BSA powder. The amount of free E2 represented less than 1% of the weight of the conjugate.

The dissociation procedure yielded 76.5 ± 0.41 x 106 cells per pituitary (n = 12). Viability of pituitary cells after tissue dissociation was always greater than 95% (96.4% ± 0.6%, n = 12). After incubation for 2 days, cell viability was always greater than 80% (86.2% ± 1.1%, n = 12).

For experiment 1, incubation of anterior pituitary cells with 2 nM GnRH for 15 min increased the release of LH compared with that in untreated cells (53.11 ± 2.46 versus 3.3 ± 2.01 ng/ml, P < 0.01). Treatment with E2 (Fig. 2A), E2-BSA (Fig. 2B), or E2-PEP (Fig. 2C) inhibited (P < 0.01) GnRH-induced release of LH in a dose-dependent manner. For E2-treated cells, 0.1 nM E2 decreased release of LH compared to cells treated with GnRH only, but 10 nM E2 was required to abolish completely the GnRH-induced release of LH (Fig. 2A). Similarly, 0.1 nM E2-BSA (Fig. 2B) or E2-PEP (Fig. 2C) decreased that release of LH compared to that in cells treated with GnRH only, and as with E2, 10 nM of each conjugate was required to abolish completely the GnRH-induced release of LH.



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  		FIG. 2. Cells (2 x 105 cells/well) were preincubated for 60 min with culture medium and E2 (A), E2-BSA (B), or E2-PEP (C) and then washed and incubated for 15 min with the previous treatment plus 2 nM GnRH. Data represent the average of three pituitaries and three or four wells per treatment per pituitary and are presented as the mean ± SEM. Bars with unlike letters differ (P < 0.01)

In experiment 2, preincubation time (30 vs. 60 min [Fig. 3A] or 15 vs. 60 [Fig. 3B]) did not alter the inhibitory effect of E2, E2-BSA, or E2-PEP on GnRH-induced release of LH. No significant interactions between incubation time and dosage (0.01, 0.1, 1, 10, and 100 nM; P > 0.1) or between treatment (E2, E2-BSA, or E2-PEP) and dosage (P > 0.1) were found. Therefore, treatments were pooled by dosage, and the interaction of treatment with preincubation time was analyzed.



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  		FIG. 3. Cells (2 x 105 cells per well) were preincubated for 30 or 60 min (A) or for 15 or 60 min (B) with culture medium and E2, E2-BSA, or E2-PEP and then washed and incubated for 15 min with the previous treatment plus 2 nM GnRH. Because no significant interactions between incubation time and dosage (P > 0.1) or between treatment and dosage (P > 0.1) were found, treatments were pooled. Comparisons were made within dosage. Data represent the average of three pituitaries and three or four wells per treatment per pituitary and are presented as the mean ± SEM. Bars with unlike letters differ (P < 0.01)

In experiments 3–5, incubation of anterior pituitary cells with 2 nM GnRH for 15 min increased the release of LH compared with that in untreated cells (33.5 ± 2.6 vs. 18.76 ± 2.6 ng/ml, P < 0.01). In these cells, treatment with steroid hormones (100 nM P4, T, HC, or {alpha}E2) did not affect the GnRH-induced release of LH (Fig. 4A). Moreover, when pituitary cells were coincubated with these steroids plus E2, they did not (P > 0.1) alter the inhibition of GnRH-induced release of LH caused by 1 nM of E2 (Fig. 4B). Similarly, treatment of pituitary cells with E2 antagonists did not (P > 0.1) influence the GnRH-induced release of LH (Fig. 5A). However, ER antagonists prevented the inhibition of GnRH-induced release of LH induced by 1 nM E2 (P < 0.01) (Fig. 5B), E2-BSA (P < 0.1) (Fig. 5C), or E2-PEP (P < 0.1) (Fig. 5D). The selective ER{alpha}-agonist PPT decreased (P < 0.01) the release of LH in response to a GnRH challenge, but only at a concentration of 100 nM (Fig. 6A), whereas the selective ERß-agonist DPN did not decrease GnRH-induced release of LH at the concentrations tested (Fig. 6B).



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  		FIG. 4. A. Cells (2 x 105 cells/well) were preincubated for 15 min with culture medium and 1 nM E2 or 100 nM of one of the following steroids: P4, T, HC, or {alpha}E2. Culture medium was aspirated and cells incubated for 15 min with culture medium containing the previous treatment plus 2 nM GnRH or culture medium. B. Cells were treated as described in A, but 1 nM E2 was added to cells treated with other steroids during the 15-min challenge with GnRH. Data represent the average of three pituitaries and three or four wells per treatment and are presented as the mean ± SEM. Comparisons were among controls versus treatments per treatment per pituitary. Bars with unlike letters differ (P < 0.01)



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  		FIG. 5. A. Cells (2 x 105 cells/well) were preincubated for 15 min with culture medium and 1 nM E2 or 100 nM of one of the following E2 antagonists: Tx, HTx, or ICI. Culture medium was aspirated and cells incubated for 15 min with culture medium containing the previous treatment plus 2 nM GnRH or culture medium. B. Cells were treated as described in A, but 1 nM E2 was added to cells treated with antagonists during the 15-min challenge with GnRH. C. Cells were treated as described in B, but E2-BSA replaced E2. D. Cells were treated as described in B, but E2-PEP replaced E2. Data represent the average of three pituitaries and three or four wells per treatment per pituitary and are presented as the mean ± SEM. Comparisons were among controls versus treatments. Bars with unlike letters differ (P < 0.01)



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  		FIG. 6. Cells (2 x 105 cells/well) were preincubated for 15 min with the ER{alpha} selective-agonist PPT (A) or the ERß selective-agonist DPN (B) from 0 to 100 nM. Culture medium was aspirated and cells incubated for 15 min with culture medium containing the previous treatment plus 2 nM GnRH or culture medium in the control group. Data represent the average of three pituitaries and three or four wells per treatment per pituitary and are presented as the mean ± SEM. Comparisons were among controls versus treatments. Bars with unlike letters differ (P < 0.01)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Conjugation of E2-6-CMO to a 15-amino acid sequence yielded a biologically active, conjugated form of E2 free of unconjugated E2. The 1:1 ratio of E2 to carrier molecule, together with the presence of a tyrosine residue at the C-terminal position in the PEP, makes this novel E2 conjugate suitable for biological studies. It provides a preferable alternative to E2-BSA, because only one molecule of E2 can be linked to the PEP. Thus, when performing kinetic analyses, only one molecule of ER can interact with the E2-PEP. In the case of E2-BSA, at least theoretically, it may be possible for several ERs to bind to a single E2-BSA because of the multiple molecules of E2 attached to each BSA. This would complicate its use for any kinetic analysis.

Steroid-BSA conjugates have been reported to contain unbound or adsorbed steroid in the range of 1–4% [55, 61] of the weight of the conjugate. In the present study, less than 1% of the weight of the conjugate was removed as free E2 from the commercial E2-BSA by solvent extraction. The amount of free E2 remaining in the E2-BSA conjugate after the sixth extraction was substantially less than the concentration needed to elicit a physiological response.

In the first experiment, preincubation of pituitary cells for 60 min with E2 resulted in a dose-dependent inhibition of GnRH-induced release of LH. The 0.1 nM E2 resulted in a significant decrease (P < 0.01) in GnRH-induced release of LH, but 10 nM E2 was required to suppress completely the release of LH. This finding is consistent with other reports in which E2 concentrations in the range of 0.1–10 nM have been used successfully to modulate signaling pathways in hypothalamus [43], endothelial cells [19], adipocytes [12], neurons [14], and a pituitary tumor cell line [62, 63]. Similarly, 10 nM of either E2-BSA or E2-PEP completely prevented GnRH-induced release of LH. Likewise, 1–10 nM E2-BSA has been used successfully to mimic acute actions of E2 in vitro [8–14, 18].

Because of the limited number of dispersed cells obtained from ovine pituitaries, evaluation of the effect of preincubation time (15, 30, or 60 min) on inhibition of LH secretion induced by free or conjugated E2 was performed in separate experiments. A 60-min preincubation time was used in each experiment to serve as a basis for comparison. Preincubation of pituitary cells with E2 or its conjugated forms for 15, 30, or 60 min induced a similar dose-dependent inhibition of GnRH-induced release of LH. However, the amount of LH released by GnRH in pituitaries used to compare 15 min versus 60 min of preincubation time was approximately 25% the amount of LH released by GnRH in previous experiments (compare positive controls in Fig. 3, A and B). In these less responsive pituitaries, basal levels of LH were threefold higher than the basal levels of LH detected in the more sensitive cells. Although a lack of or reduced responsiveness to physiological secretogogues (thought to result from enzyme-induced receptor damage) is commonly observed in enzymatically dissociated secretory cells [64, 65], it has been demonstrated that ovine pituitary cells recover their ability to secrete LH in response to GnRH from 16 to 24 h after enzymatic dispersion [57]. In the present study, cells were cultured for 48 h before challenge with GnRH; therefore, potential damage to GnRH receptors may not be the overriding cause for the low sensitivity to GnRH. Whatever the reason for the low sensitivity to GnRH, it is reasonable to assume that the high basal levels of LH decreased the amount of LH stored in pituitary cells, resulting in poor release of LH in response to GnRH.

As mentioned above, preincubation of pituitary cells with E2 or its conjugated forms for 15, 30, or 60 min induced a similar dose-dependent inhibition of GnRH-induced release of LH. Therefore, it appears that only one molecule of E2 on the E2-BSA conjugate interacts with ER. In several studies, treatment with E2 or E2-BSA over a wide range of intervals (1–40 min) modulated a variety of signaling pathways, including cGMP production [44], activation of extracellular signal-related kinase (ERK) [9, 12], nitric oxide synthase (NOS) [18], and Akt [10], among others. Therefore, our data support the concept that in ovine pituitary cells, E2 acutely suppresses the GnRH-induced release of LH by a nongenomic mechanism. In this regard, several studies have indicated that E2-BSA, distinct from E2, did not stimulate reporter activity in cells transfected with ERE-luciferase reporter constructs [8, 11, 45, 46], further suggesting that E2-BSA acts through a nongenomic mechanism. Moreover, the novel E2-PEP proved to be active biologically and to mimic the acute suppression of GnRH-induced secretion of LH by E2.

As in the last group of experiments, the sensitivity of pituitary cells to a GnRH challenge also was lower and accompanied by higher basal levels of LH compared with the more responsive cells used in the first experiment. Treatment of pituitary cells with E2, E2-BSA, or E2-PEP did not induce the smooth, dose-dependent inhibition of GnRH-induced release of LH observed in more sensitive cells; instead, a sudden decrease in LH release to the media was detected at 1 nM unconjugated or 10 nM conjugated E2, respectively. As expected, incubation of these pituitary cells with a 100-fold excess of P4, T, HC, or the stereoisomer {alpha}E2 did not mimic the acute inhibition of E2 on GnRH-induced release of LH. This suggests that inhibition of GnRH-induced release of LH is a specific action of E2 and agrees with previous results in which other steroid hormones failed to mimic the acute actions of E2 on activation of NOS [43, 66] and several protein kinases [9, 12, 14, 46, 67, 68] as well as cAMP production [69]. Furthermore, in the present study, addition of a 100-fold excess of the steroid hormones tested did not alter the inhibition by E2 of the GnRH-induced release of LH, further supporting the specificity of this effect. Similarly, in most studies {alpha}E2 did not activate signaling pathways acutely stimulated by E2 [9, 12, 19, 67, 69–71].

A 100-fold excess of antiestrogens completely prevented the inhibition of GnRH-induced secretion of LH by E2, providing further support for the hypothesis that the acute action of E2 occurs via ERs; numerous others have reported that Tx, HTx, and ICI prevented the acute action of E2 on modulation of signaling pathways, including activation of NOS [10, 13, 18, 42, 44, 66, 71, 72], mitogen-activated protein (MAP) kinase cascades [8, 9, 11, 12, 41, 73–77], and tyrosine kinases [73, 76] as well as phosphorylation of transcription factors [9, 12, 76, 78]. The fact that antiestrogens did not cause an acute effect on cellular response when administered alone also agrees with previous reports [42, 66, 70] and further supports the idea that binding of ERs to these compounds prevents their occupancy by E2, resulting in a rapid antagonistic action. Likewise, in the present study, antiestrogens prevented the inhibition of GnRH-induced release of LH by conjugated forms of E2, suggesting a plasma membrane location of ERs. An antagonistic action of Tx and ICI on the mimetic action of E2-BSA in signaling pathways rapidly activated by E2 have been reported. For example, the rapid release of nitric oxide by E2-BSA from median-eminence fragments was prevented by Tx [43]; moreover, the E2-BSA-induced phosphorylation of MAP kinases was prevented by ICI [9, 11, 12].

It has been proposed that in some cells, acute actions of E2 in cellular function may be mediated by a binding protein that is different from the common ER{alpha} and ERß [25, 79, 80]. In this scenario, ICI appears to behave atypically, and this has been interpreted to suggest interaction with a novel binding protein [14, 25, 81]. For example, in immature neural cells, ICI mimicked the acute actions of E2 on cAMP/protein kinase A activation [81] and phosphorylation of ERK 1/2 [14], whereas in neural cells from mice lacking ER, ICI did not antagonize the acute actions of E2 on ERK phosphorylation [80] or the E2-induced potentiation of kainate currents [81]. Interestingly, when ICI behaved atypically, {alpha}E2 also mimicked actions of E2 [14, 81].

The role of ER subtypes in mediating inhibition of GnRH-induced release of LH by E2 was further evaluated by using selective agonists for ER{alpha} or ERß. Our data agree with previous observations implicating ER{alpha} in the mediation of the acute inhibition induced by E2. The selective-agonists PPT and DPN selectively recruit coactivators via ER{alpha} or ERß, respectively [49]; however, their ability to mimic acute actions of E2 has just begun to be investigated. In a recent study, PPT acutely mimicked the vasodilatory action of E2 in mesenteric arteries; however, when DPN was given at pharmacological concentrations, it also had a lesser, but significant, effect on vasodilation [82]. Because there appeared to be a slight effect of DPN on the inhibition of GnRH-induced LH secretion at the highest concentration tested, similar findings might have occurred in the present study if we had used even higher concentrations of DPN.

In conclusion, the rapidity by which E2 prevented GnRH-induced release of LH in cultured ovine pituitary cells supports the concept that this effect is mediated by a nongenomic action of E2. The inhibitory action of E2, E2-BSA, or E2-PEP in secretion of LH proved to be steroid specific and equally sensitive to blockade by E2 antagonists. These data indicate that ERs mediate the acute inhibitory action of E2 on secretion of LH. The presumed impermeability of conjugated forms of E2 suggests a plasma membrane location of ERs mediating the rapid inhibition of LH secretion. Moreover, the use of selective ER agonists indicates that acute inhibition of GnRH-induced release of LH by E2 may occur via ER{alpha}.


   FOOTNOTES
 
1 Supported by a grant from the Colorado State University Agricultural Experiments Station. The academic program of J.A.A.-A. was supported by the National Council for Science and Technology (CONACYT-Mexico) and the National Institute for Research in Forestry, Agriculture, and Livestock (INIFAP-Mexico). Back

2 Correspondence: Terry M. Nett, Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Science, 3801 W Rampart Rd., Fort Collins, CO 80523-1683. FAX: 970 491 3557; terry.nett{at}colostate.edu Back

Received: 26 January 2005.

First decision: 17 February 2005.

Accepted: 8 March 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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