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Endocrinology, doi:10.1210/en.2007-0986
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Endocrinology Vol. 148, No. 12 5794-5802
Copyright © 2007 by The Endocrine Society

Rapid in Vivo Effects of Estradiol-17ß in Ovine Pituitary Gonadotropes Are Displayed by Phosphorylation of Extracellularly Regulated Kinase, Serine/Threonine Kinase, and 3',5'-Cyclic Adenosine 5'-Monophosphate-Responsive Element-Binding Protein

Javed Iqbal, Olivier Latchoumanin and Iain J. Clarke

Department of Physiology, Monash University, Clayton, Victoria 3800, Australia

Address all correspondence and requests for reprints to: Prof. Iain J. Clarke, Department of Physiology, Monash University, P.O. Box 13 F, Clayton, Victoria 3880, Australia. E-mail: Iain.Clarke{at}med.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have determined the time course of phosphorylation of MAPK/ERK, cAMP-responsive element-binding protein (CREB), and serine/threonine kinase (Akt) in ovine pituitary gonadotropes after in vivo injection (iv) of either 25 µg estradiol-17ß (E17ß) or vehicle. In ovariectomized ewes, E17ß increased the number of gonadotropes expressing phosphorylated (p)ERK-1/2 and pCREB immunoreactivity (-IR) within 90 min, as assessed by immunohistochemistry. By Western blot, we also showed that pERK-1/2, pCREB, and pAkt (ser 473) proteins were up-regulated by E17ß. In ovariectomized, hypothalamo-pituitary-disconnected animals, gonadotrope function was restored with hourly GnRH pulses (iv), and E17ß injection (iv) reduced LH response within 1 h. Immunohistochemistry showed that E17ß increased pERK-1/2-IR in gonadotropes within 15 min and peak response at 60 min. The number of cells immunoreactive for pCREB was greater in E17ß-treated animals than in vehicle-injected controls at 60 and 90 min. Western blot revealed a pERK-1/2 response within 15 min and pCREB response at 30 min. Up-regulation of pAkt occurred within 60 min of E17ß treatment. Thus, rapid effects of E17ß on gonadotropes involve phosphorylation of second messenger proteins with a time course that may relate to the rapid negative feedback effect to reduce responsiveness to GnRH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LIGANDED ESTROGEN RECEPTORS (ER) act as transcription factors that bind to promoter regions of genomic DNA to regulate transcription of a range of genes in various tissues (1). These genomic events involve phosphorylation of ER proteins, coactivators, and cosuppressors to enhance ER-mediated transcription (2). Such effects of estrogens may be transduced by ER{alpha}/ß or other means of transduction via the ER-related receptor (3), but rapid effects of estrogen have also been proposed through action on the cell membrane involving G protein-coupled membrane protein receptor 30 (GPR30) (4) or some other mechanism (5). Significant evidence has accumulated to show that rapid effects of estrogens occur in a variety of cell types including breast cancer cell lines (6, 7), endothelial cell lines (8, 9), pituitary cell lines (10), and many other cells such as adipocytes (11). Thus, the concept that estrogens have rapid effects on cellular activity unexplained by genomic mechanisms has gained support (10). This is manifest in the phosphorylation of MAPK/ERK and cAMP-responsive element-binding protein (CREB) in the central nervous system within 15 min (12).

Various means by which estrogens cause rapid signaling have been proposed. For example, studies from the laboratory of Kelly and colleagues (13, 14) have proposed that estrogens bind to the plasma membrane to cause rapid mobilization of intracellular calcium and activation of membranous potassium channels through G protein-coupled receptors within seconds to minutes, culminating in rapidly elicited cellular events. These effects include rapid activation (phosphorylation) of second messengers leading to altered gene transcription. Alternatively, Levin (15) proposes that rapid (nongenomic) cellular effects of estrogens, such as activation of phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase (Akt), and MAPK (ERK), are mediated by a subpopulation of conventional intracellular ER{alpha} that localize to the plasma membrane. Recent studies have shown similarities between the membrane-associated and nuclear ER and the mechanisms of membrane translocation of the steroid receptors, suggesting that they are the same, providing support for the model of Levin (5, 16). Alternatively, there is evidence that GPR30 is involved in the induction of ERK activation by estrogen in breast cancer cells (17), supporting the notion that this receptor is responsible for rapid effects (17). Although localization of GPR30 to the plasma membrane and its up-regulation after estrogen treatment is clearly demonstrable (18), a role for this receptor in the rapid effect remains controversial (5). Certainly the involvement of GPR30 is consistent with the model proposed by Kelly and colleagues (13, 14), being that rapid estrogen effects occur through G protein-coupled receptors (GPCR)/channels on the plasma membrane.

Despite the popular and now well-accepted notion that rapid estrogen action is functional in a range of cell types, as demonstrated by both in vitro and in vivo models (see above), there is relatively little information on this phenomenon in normal cells in tissues that are estrogen responsive. Several in vivo studies have shown that estrogens cause rapid changes in the neuronal excitability (19) and induction of phosphorylation of second messenger molecules in the brain (20). Pituitary gonadotropes represent a useful model for analysis of estrogen action in vivo (21). Whereas GnRH is the main tropic stimulus to these cells, acting via a specific GPCR (22), the action of GnRH is modulated by a range of factors including estradiol-17ß (E17ß) (23) and other paracrine/endocrine effectors (24). The second messenger signaling mechanisms for GnRH receptor-mediated function have been extensively studied, but the way that E17ß and GnRH action might be coordinated is not well understood. In particular, there is no information on how GnRH and E17ß might act in concert to activate subcellular processes during periods of negative and positive feedback. Although an increase in GnRH secretion is the primary stimulus for the preovulatory surge in the LH secretion, this is preceded by a negative feedback effect of E17ß (23). Recent work indicates that the acute negative feedback effect of E17ß in pituitary gonadotropes involves rapid signaling (25). We now detail the second messenger signaling systems that are activated in ovine gonadotropes after E17ß injection. We hypothesized that this biphasic pattern of secretory response (negative feedback followed by positive feedback) is such that rapid effects may relate to either or both of the feedback phenomena. The latter (positive) response involves E17ß action on the pituitary gonadotropes to sensitize the cells to the action of GnRH as well as brain action to increase the secretion of GnRH.

Direct action of E17ß on ovine pituitary gonadotropes may be studied in vivo using the hypothalamo-pituitary-disconnected (HPD) model, which removes all hypophysiotropic input from the hypothalamus (26). The function of gonadotropes in HPD sheep is then restored by iv delivery of GnRH in a pulsatile mode (26). With invariant GnRH replacement, a consistent baseline pattern of pulsatile gonadotropin secretion is achieved and the action of other factors on the gonadotropes may be analyzed without the confounding factor of changing hypothalamic secretion of GnRH (due to brain action of factors such as sex steroids). Using this model, direct pituitary action of sex steroids has been demonstrated, but the cellular mechanisms by which estrogen modulates gonadotropin (LH and FSH) secretion have not been elucidated. In the present study, we have used the HPD model to examine rapid effects of E17ß in vivo by measurement of the time course of phosphorylation of second messenger proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and ethics
Adult Corriedale female sheep were ovariectomized (OVX) at least 1 month before the commencement of the studies to remove sex steroids. Food and water were ad libitum, and the sheep were exposed to normal ambient temperature and lighting. The experimental procedures were conducted under a protocol approved by the Animal Ethics Committee, Monash University.

Experiment 1: rapid E17ß effects in gonadotropes in OVX ewes
This study was performed to ascertain whether E17ß causes phosphorylation of MAPK/ERK-1/2, Akt, and CREB in ovine gonadotropes. OVX ewes were used, and the response to E17ß over 90 min was quantified. The animals were housed in individual pens for 1 wk to acclimatize to the surroundings and minimize stress responses to treatment. They were administered progesterone using controlled intravaginal drug release (Lyppard, Cheltenham, Victoria, Australia). One day before the experiment, an external jugular vein was cannulated with a 12-gauge Teflon Dwellcath (Tuta Laboratories Australia Pty. Ltd., Bedford Park, South Australia, Australia). Blood samples (5 ml) were taken at 15-min intervals from 90 min before iv injection of E17ß (25 µg in 10% ethanol in water) or vehicle until 90 min after injection. Blood samples (5 ml) were centrifuged for 10 min at 4 C and plasma obtained for determination of LH levels. The animals were then euthanized by overdose of sodium pentobarbital (Lethobarb, 0.5 ml/kg body weight iv; May and Baker Pty. Ltd., Melbourne, Australia) for the collection of pituitary glands for subsequent immunohistochemistry and Western blot analysis. The pituitaries were removed from the sella turcica and cut in half. One half was snap-frozen in liquid nitrogen for protein extraction and Western blot analysis, and the other was cut into 2- to 3-mm-thick slices and placed in 4% paraformaldehyde solution in 0.1 M phosphate buffer (PB) (pH 7.4) for 24 h at 4 C on a shaker. The tissues for histology were then sunk in 30% sucrose in PB for 24 h at 4 C and frozen on powdered dry ice for storage at –20 C. Tissue sections (7 µm) were cut on a cryostat at –20 C, thaw-mounted onto precoated Superfrost slides, dried overnight, and stored at –20 C until processed for immunohistochemistry. Western blotting and assay of LH in plasma were performed as indicated below. In this study, effects of E17ß could be mediated directly at the level of the gonadotrope or indirectly via altered secretion of GnRH.

Experiment 2: time course of the direct pituitary action of E17ß in gonadotropes in vivo
The experiment used OVX-HPD animals that received GnRH replacement as outlined in Fig. 1Go, so that direct pituitary action of E17ß could be determined. Because experiment 1 showed phosphorylation within 90 min in hypothalamo-pituitary-intact animals, this study examined the time course of rapid induction of phosphorylation of ERK-1/2, CREB, and Akt by E17ß with a constant background of GnRH input. The HPD operation was performed on OVX ewes, and GnRH pulses were administered to restore the LH secretion as previously described (23, 26). External jugular veins were cannulated as above, and one cannula in each animal was connected to an automated pump by a manometer line (Portex Ltd., Hythe, UK) so that GnRH could be delivered in a pulsatile mode. The pump was programmed to deliver 250 ng GnRH in 2.25 ml over 6 min every 2 h as previously described (23). After 1 wk of the GnRH replacement, all animals also received a controlled intravaginal drug release (as above) for 5 d. These were removed 24 h before the experiment, and pumps were set to hourly mode of GnRH delivery. On the experimental day, at 0700 h, pumps were disconnected and GnRH was delivered by hand at 0800, 0900, 1000, 1100, and 1200 h in 5 ml to give greater accuracy of delivery over a shorter period of time. At 1200 h, animals also received an E17ß or vehicle injection (iv) as in experiment 1 (above). Blood samples (5 ml) were collected relative to the onset of GnRH pulses, as indicated in Fig. 1Go to measure LH responses to GnRH. The animals (n = 3 per group) were euthanized 15, 30, 60, and 90 min after E17ß or vehicle injection for the recovery of pituitaries (as above). Because the experiment involved 24 animals, it was logistically not possible to treat all at once, and the experiment was conducted so that animals at each time point (E17ß-treated and vehicle controls) were treated contemporaneously.


Figure 1
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  		FIG. 1. Design of experiment 2. CIDR, Controlled intravaginal drug release.

 
Double-labeling immunocytochemistry
All incubations were at room temperature unless otherwise specified and were followed by three 10-min washes in 0.05 M PBS. A modified version of an antigen retrieval method was used for histological detection of phosphorylated (p)ERK-1/2 and pCREB immunoreactivity (IR) in ovine pituitary tissue (23). This was preceded by incubation with 3% H2O2 in 50% methanol (15–20 min) to block endogenous peroxidase activity. Slides were then microwaved (twice for 5 min at 700 W) in 0.01 M citrate buffer (pH 6.0) and allowed to stand in the same buffer for another 20 min. For immunocytochemistry, the sections were incubated in blocking solution (10% normal goat serum, 0.3% Triton X-100 in 0.1 M PB) for 30 min and then with either rabbit monoclonal anti-pERK-1/2 (1:200) or rabbit polyclonal anti-pCREB (1:500) (Cell Signaling Technology, Danvers, MA) in blocking solution for 72 h at 4 C. The sections were then incubated for 1 h in biotinylated goat antirabbit IgG (Vector Laboratories, Burlingame, CA) at a 1:250 dilution and then with streptavidin horseradish peroxidase complex (Amersham Biosciences, Piscataway, NJ) at a 1:500 dilution for 1 h. For color reaction, the sections were preincubated in a chromogen solution containing 3,3'-diaminobenzidine (0.5 mg/ml), 0.02% ammonium nickel sulfate, and 0.026% cobalt chloride in 0.1 M PB for 10 min. This solution was removed and 0.15% H2O2 added for approximately 4 min. The color reaction was monitored and stopped by washing in 0.1 M PB. This procedure gave black staining in the nucleus of cells positive for pERK-1/2 and pCREB. Double labeling was then carried out to determine localization of pERK-1/2 and pCREB-IR to gonadotropes using mouse monoclonal anti-LH antibody (23) at 1:5000 and visualized with 3,3'-diaminobenzidine to give a brown color in the cytoplasm of cells. The antibodies used for labeling pERK-1/2 and pCREB were characterized using omission of the primary and secondary antibodies and have been used previously (12, 23). Negative controls were run in each series of immunohistochemistry.

For analysis of immunohistochemical staining, five regions (one in each of the four quadrants and one region in the center) of three coronal sections for each animal were examined under a x40 objective, and cells were counted in an area of 0.35 mm2 determined using a grid in the eyepiece as reported previously (27). At this magnification, the presence or absence of nuclear and cytoplasmic staining was unambiguous. In each region, single-labeled and double-labeled cells were counted, and the percentage colocalization of LH with each phosphorylated second messenger was then calculated for each experimental group.

Western blotting
Frozen pituitary tissues were suspended in lysis buffer (0.1 M PBS) containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, a protease inhibitor cocktail (Complete; Roche Diagnostics, Indianapolis, IN), and phosphatase inhibitor (1 mM sodium vanadate, 0.05 µM okadaic acid, 1 mM phenylmethylsulfonyl fluoride) for homogenization (five times for 15 sec each, continuous pulses at 20% speed). Supernatants were recovered by centrifugation at 12,000 rpm for 5 min at 4 C. Final protein concentrations were determined with protein assay (Bio-Rad Laboratories, Richmond, CA), and samples were stored at –80 C for Western blot analysis. Extracts from tissues were boiled for 3 min in SDS sample buffer (0.0625 M Tris-HCl, pH 6.8) containing 50 mM 1,4-dithiothreitol, 1% SDS, 10% glycerol, and 1% bromophenol blue Electrophoresis was performed with 10% SDS-polyacrylamide gels in separation buffer (100 mM Tris, 760 mM glycine, and 1% SDS) and the proteins were transferred to Immobilon-P Transfer membranes (Millipore, Bedford, MA) in transfer buffer (100 mM Tris, 150 mM glycine, and 0.2% ethanol). The membranes were incubated in blocking buffer (5% nonfat milk diluted in Tris-buffered saline containing 0.05% Tween 20) for 60 min. We probed these membranes with polyclonal antibodies for pERK-1/2, total ERK, pCREB, total CREB, pAkt (Ser743), pAkt (Thr308), and total Akt (Cell Signaling Technology) at a dilution of 1:1000 in blocking buffer for 16 h at 4 C. A secondary antibody conjugated to horseradish peroxidase (Antibodies Australia, Melbourne, Australia) was applied for 1 h at room temperature and detection was accomplished using enhanced chemiluminescence reagent (Amersham). For reprobing, the membranes were stripped in 0.2% SDS, 0.1 M NaCl, and 0.1 M glycine/HCl (pH 2) for 1 h at room temperature and reequilibrated in Tris-buffered saline containing 0.05% Tween 20. Full Range Rainbow Markers (GE Healthcare, Chalfont, UK) were used as molecular weight markers. The films were scanned with a Canon scanner (Akimachi, Kunisaki-shi, Japan), using a Multigauge software program (Fujifilm Corp., Minato-ku, Tokyo, Japan) to determine band densities. An area of the band was kept constant for each scan and made relative to background. The protein extracts from animals at each time point were analyzed separately, and the vehicle acted as control of the same time point. For this reason, the data for each time point were normalized so that vehicle treatment was taken as 100%, and estrogen effects were examined in relation to this. Such a design and data analysis takes into account any effects that GnRH has on second messenger induction (28) at each time point.

LH RIA
Plasma LH levels were measured using a RIA as previously described (29), with a sensitivity of 0.1–0.3 ng/ml. Between-assay coefficients of variation (CV) were 9.1% at 5.9 ng/ml, 5.1% at 3.3 ng/ml, and 9.0% at 13.3 ng/ml. Interassay CV was less than 10% between 0.2 and 16.4 ng/ml, and maximal-precision intraassay CV was 3.6–8.9% from 2.1–3.1 ng/ml.

Statistical analysis
The data were analyzed using ANOVA, and a least significant differences method was used as a post hoc test to compare the means. The data are presented as means (±SEM), with Western blot data shown as means (±SEM) of arbitrary units normalized to 100% of control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: effect of E17ß in OVX ewes
E17ß treatment reduced (P < 0.001) mean plasma LH secretion within 60–90 min in OVX ewes, whereas vehicle injection did not (Fig. 2Go). Examples of gonadotropes that were immunohistochemically labeled for pERK-1/2 or pCREB are shown in Fig. 3Go. E17ß increased (P < 0.001) the percentage of gonadotropes that were immunostained for pERK-1/2 and pCREB (Fig. 4Go). To further define the effects of E17ß, Western blot analysis was performed, and results are presented in Fig. 5Go. E17ß increased (P < 0.05) pERK-1/2 (Fig. 5Go, A and C), pCREB (Fig. 5Go, B and C), and pAkt (Ser473) (Fig. 5Go, D and E) over levels found in vehicle-treated animals. E17ß treatment did not alter the level of nonphosphorylated proteins, nor did it increase phosphorylation of Akt (Thr308) (Fig. 5Go, D and E).


Figure 2
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  		FIG. 2. Mean (±SEM) plasma LH (ng/ml) levels in OVX ewes (n = 4 per group) before and after iv injection of E17ß ({blacksquare}) or vehicle (V) ({square}). Data are presented for 30-min periods. a vs. b, P < 0.05 within E17ß-treated animals; c vs. b P < 0.01.

 

Figure 3
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  		FIG. 3. Histological detection of rapid induction of pERK-1/2 and pCREB-IR in gonadotropes of OVX ewes by E17ß. Photomicrographs show examples of immunolabeling for pERK-1/2 (A and B) and pCREB (C and D) 90 min after iv injection of either E17ß (A and C) or vehicle (V) (C and D). Brown labeling identifies gonadotropes by LH immunohistochemistry. Red arrows indicate pERK-1/2 or pCREB labeling of nuclei (black), and black arrows identify gonadotropes that are not labeled for pERK-1/2 or pCREB. Scale bar, 20 µm.

 

Figure 4
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  		FIG. 4. Mean (±SEM) percentage of gonadotropes in OVX ewes (n = 4 per group) that were also immunolabeled for either pERK-1/2 or pCREB-IR 90 min after iv injection of either E17ß ({blacksquare}) or vehicle ({square}). ***, P < 0.001, compared with vehicle.

 

Figure 5
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  		FIG. 5. Rapid effects of E17ß or vehicle (V) on the phosphorylation of second messenger proteins in ovine pituitary gonadotropes of OVX ewes 90 min after iv injection. Examples of phosphorylation are presented in the upper panels; ERK-1/2 (panel A), CREB (panel B), Ser473 Akt (panel C), and Thr308 Akt (panel D). Panels E and F present mean (±SEM) data (n = 4 per group), expressed as arbitrary units where control is 100%. *, P < 0.05.

 
Experiment 2: effect of E17ß in OVX-HPD ewes receiving pulsatile GnRH replacement
Pulsatile replacement of GnRH for 2 wk restored the plasma LH levels to a stable baseline (Fig. 6Go, A–D). Mean (±SEM) plasma LH pulse amplitudes before E17ß or vehicle were 2.8 ± 1 and 2.4 ± 0.6 ng/ml, respectively. As shown in Fig. 6DGo, iv injection of 25 µg E17ß eliminated the response to GnRH in the group of animals that were sampled 90 min later, which demonstrates the negative feedback effect in this model. Animals sampled at earlier time points did not receive a pulse of GnRH after E17ß/vehicle treatment and before tissue collection, so the effect on response to GnRH after treatment could not be determined in these animals.


Figure 6
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  		FIG. 6. Effects of iv injection of E17ß or vehicle (V) on plasma LH levels in OVX-HPD animals (n = 3 per group) receiving hourly injections of GnRH (arrows). The animals were euthanized for pituitary collection 15, 30, 60, or 90 min after injection of E17ß or V (panels A–D, respectively). Acute suppression of LH response to GnRH was seen in animals that received a GnRH pulse 60 min after injection and euthanized at 90 min (panel D). SED, Standard error of difference.

 
Examples of immunohistochemical detection of pERK-1/2 in pituitary gonadotropes in GnRH-pulsed, OVX-HPD animals after E17ß or vehicle treatment are shown in Fig. 7AGo. E17ß injection increased pERK-1/2 within 15 min with a peak response at 60 min (Fig. 7BGo). E17ß also induced pCREB (Fig. 8Go, A and B) with a significant (P < 0.05) increase after 60 min of injection and peak response at 90 min (P < 0.01).


Figure 7
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  		FIG. 7. Induction of pERK-1/2 by E17ß as measured by immunohistochemistry in pituitary gonadotropes of GnRH-pulsed, OVX-HPD animals. A, Examples of pERK-1/2-IR (red arrows; black nuclear staining) in gonadotropes (brown staining) 15 and 30 min after iv injection of either E17ß or vehicle (V). Black arrows show single-labeled gonadotropes (brown staining). Scale bar in A, 20 µm. B, Mean (±SEM) percentage of gonadotropes labeled for pERK-1/2 after E17ß ({blacksquare}) or vehicle ({square}) treatment. ***, P < 0.001, compared with vehicle within time points.

 

Figure 8
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  		FIG. 8. Induction of pCREB by E17ß measured by immunohistochemistry in pituitary gonadotropes of GnRH-pulsed, OVX-HPD animals. A, Examples of pCREB labeling (black nuclear staining; red arrows) within the gonadotropes (brown staining) 60 min after iv injection of either E17ß or vehicle (V). Black arrows show single label gonadotropes (brown staining only). Scale bar in A, 20 µm. B, Mean (±SEM) percentage of gonadotropes labeled for pCREB after E17ß ({blacksquare}) or vehicle ({square}) treatment. *, P < 0.05; **, P < 0.01, compared with compared with vehicle within time points.

 
Western blot analysis of protein extracts from pituitaries collected 15–90 min after E17ß or vehicle injection indicated rapid E17ß induced up-regulation of protein levels for pERK-1/2, pCREB, and pAkt in a time-dependent manner. pERK-1/2 protein was increased within 15 min (P < 0.01) of E17ß injection (Fig. 9Go, A and B), reaching peak levels within 30 min (P < 0.001). pCREB levels were increased (P < 0.001) within 30 min of E17ß injection (Fig. 10Go, A and B). E17ß up-regulated pAkt both at Ser473 and Thr308 positions after 60 min (P < 0.05) of E17ß injection (Fig. 11Go).


Figure 9
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  		FIG. 9. Effect of E17ß injection (iv) or vehicle on phosphorylation of ERK-1/2 in a time-dependent manner in the pituitaries of GnRH-pulsed, OVX-HPD ewes (n = 3 per group). A, Examples of pERK-1/2 protein levels after iv injection of either E17ß or vehicle (V); B, mean (±SEM) data expressed as arbitrary units normalized to 100% of control (vehicle) at each time point after treatment with either E17ß ({blacksquare}) or vehicle ({square}). **, P < 0.01; ***, P < 0.001, compared with vehicle at the same time point.

 

Figure 10
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  		FIG. 10. Effect of E17ß injection (iv) or vehicle on phosphorylation of CREB in pituitaries of GnRH-pulsed, OVX-HPD ewes (n = 3 per group). A, Examples of pCREB protein levels after iv injection of either E17ß or vehicle (V); B, mean (±SEM) data expressed as arbitrary units normalized to 100% of control (vehicle) at each time point after treatment with either E17ß ({blacksquare}) or vehicle ({square}). ***, P < 0.001, compared with vehicle at the same time point.

 

Figure 11
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  		FIG. 11. Effect of iv injection of E17ß or vehicle (V) on phosphorylation of Ser473 Akt and Thr308 Akt in a time-dependent manner in the pituitaries of OVX-HPD ewes receiving GnRH replacement (3 per group). Panel A shows examples of Western blots at each time point, and panels B and C show mean (±SEM) data for Ser473 Akt and Thr308 Akt, respectively, expressed as arbitrary units normalized to 100% of control (vehicle) at each time point after treatment with either E17ß or V. *, P < 0.05, compared with vehicle controls of the same time point.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated rapid effects of E17ß in ovine gonadotropes in vivo. The GnRH-replaced, OVX-HPD model has utility for such studies for the following reasons: 1) effects of E17ß on the brain that may modify GnRH secretion (and thus gonadotropin secretion) are circumvented (23, 26), 2) most pituitary cells of the HPD animal are either dormant or operate in a basal secretory mode (with the exception of lactotropes) (30) and activation of the gonadotropes by pulsatile GnRH replacement is specific to this cell type, and 3) ER{alpha} expression in the adult ovine pituitary gland is substantially confined to the gonadotropes (31) and there is minimal expression of ERß in the ovine pituitary (32), so action of E17ß to up-regulate signaling molecules is localized to this cell type. We show that E17ß treatment of GnRH-pulsed, OVX-HPD ewes specifically induces phosphorylation of second messengers in gonadotropes (Figs. 3Go, 7Go, and 8Go), even though the gland consists of a range of cell types. This has allowed us to examine rapid effects of E17ß in an in vivo model in a way that has not been shown previously. A small proportion of nongonadotrope cells show pCREB-IR, but this background is minimal, and no pERK-IR is seen in cells from non-E17ß-treated animals.

One particular feature of our in vivo model is that we have studied E17ß action upon a background of constant pulsatile input of GnRH. This is important because the gonadotropes normally function in response to pulsatile GnRH input from the hypothalamus, via the hypothalamo-hypophyseal portal system. Without such pulsatile GnRH input, LH secretion ceases (26), and constant GnRH input does not support secretion (33). On the other hand, FSH secretion is not as rigidly linked to pulsatile input of GnRH (34, 35), and passive secretion may occur. Because GnRH activates ERK and other second messengers (36, 37), we have examined E17ß action in relation to this. Phosphorylated second messenger levels would be expected to vary over time in relation to GnRH stimulus in the control (vehicle-treated) OVX-HPD animals. It was not appropriate to document GnRH-stimulated changes in phosphorylation of ERK, CREB, and Akt across time because all of the animals were not treated contemporaneously, and the sample number was too large to run a single Western blot analysis. The overlay of E17ß treatment showed phosphorylation of second messengers significantly above levels seen in GnRH-pulsed animals treated with vehicle at particular time points.

Experiment 1 showed that E17ß led to phosphorylation of ERK-1/2, CREB, and Akt (Ser473) within 90 min, so we conducted experiment 2 within this timeframe. The immunohistochemical data show this effect is specific to gonadotropes in OVX ewes (Fig. 3Go). There are only rare examples of the phosphorylation of these proteins in other cell types after vehicle or E17ß treatment. Consistent with this, Western blotting showed up-regulation of pERK-1/2, pCREB, and pAkt protein levels (pAkt could not be detected by immunohistochemistry). Because the effects in experiment 1 could have been due to action of E17ß on GnRH secretion as well as direct pituitary action, we performed experiment 2 to interrogate the pituitary site of action. In the GnRH-pulsed, OVX-HPD model, we demonstrated specific effects of E17ß to phosphorylate second messengers within 15 min in vivo. E17ß increased pERK-1/2 within 15 min, reaching a peak response at 1 h. Our immunohistochemical data showed virtually exclusive localization of pERK-1/2 to gonadotropes and localization to the cell nuclei. Thus, translocation of pERK-1/2 from cytoplasm to the nucleus with E17ß treatment could activate downstream transcription factors, such as CREB (38). Up-regulation of pCREB occurred within 30 min and persisted for at least 90 min. We found that E17ß induced pAkt at position Ser473 but not Thr308 in OVX ewes. In GnRH-driven OVX-HPD ewes, E17ß induced phosphorylation at both sites within 60 min. The difference between the two models is that GnRH input to the gonadotropes is maintained in the latter, so combined action of GnRH and E17ß may lead to phosphorylation at Thr308. This point requires further investigation with an experiment that is specifically designed to test interaction between the actions of the two hormones.

Liganded ER acts as a nuclear transcription factor to regulate gene transcription (39, 40), but there are no estrogen response elements on the gonadotropin {alpha}-subunit or the LH ß-subunit promoters, so transcriptional control of these genes is most likely controlled by signaling via second messengers (41). In addition to classical genomic effects, accumulating evidence indicates that estrogen also exerts rapid (nonclassical/nongenomic) actions and activation of the second messengers in a variety of models (see introductory section). Debate continues on the mechanism for this rapid effect, with one proposal being that liganded ER{alpha} acts on the internal face of the plasma membrane (5, 15) and another invoking a mechanism via calcium channels and cAMP-activated second messengers (13, 14). The recently identified novel receptor GPR30 is proposed to act as an ER on the membrane to mediate the rapid effects of E17ß (18, 42), but this is not consistent with the model proposed by Levin and associates (5). Recent studies in CHO cells have described the mechanism of steroid receptor translocation to the plasma membrane (16). It is not known whether the E17ß-induced rapid signaling in the ovine pituitary gonadotropes involves translocation of classical ER to the plasma membrane, but whatever the mechanism, it is clear that the rapid effect is operative in these cells and that additional studies are warranted.

We have shown previously that treatment of ovine gonadotropes in vitro with E17ß for 16–20 h modifies the electrophysiological response to GnRH, including a change in the firing pattern (43, 44) and altered function of the rectifying K channels (45). Furthermore, using the OVX-HPD model, we examined effects of E17ß on the expression of gonadotropin genes across the short-term negative feedback effect and the sequential positive feedback effect of estrogen seen under invariant pulsatile GnRH input (46). In these earlier studies, E17ß did not alter expression of LHß subunit but reduced {alpha}-subunit and FSH-ß mRNA levels during the acute negative-feedback period (8 h after E17ß) and throughout the ensuing positive-feedback period (20 h after E17ß) of response. Another effect of E17ß is to mobilize the secretory vesicles to the membrane of the cell in preparation for the positive-feedback phenomenon (47) and whether this involves rapid signaling or a genomic action remains to be determined. Throughout the normal estrous cycle, an increase of gonadotropin synthesis is seen in the follicular phase of the estrous cycle, in preparation for the preovulatory surge (48), which could be due to changes in GnRH secretion as well as E17ß effects on the gonadotropes. A cyclic increase in ER{alpha} expression occurs in the follicular phase of the cycle (31), which would optimize E17ß signaling. Interestingly, the increase in ER{alpha} expression appears to depend upon increasing frequency of GnRH input as well as E17ß action (31). Thus, expression of Fos (indicating a generalized cellular response) is seen only when GnRH pulse input is increased in combination with E17ß input and not with a change in one parameter (increased GnRH frequency) or the other (E17ß treatment with no change in GnRH pulse frequency) (23). This further emphasizes that function in these cells requires complex interaction between the secretagogue (GnRH) acting via a GPCR and E17ß, acting through either membranous receptors or ER{alpha} (or both). Other factors such as gonadotropin-inhibitory hormone (49) may also be involved in the fully integrated output of gonadotropes. The extent to which these phenomena are the result of rapid effects of E17ß remains to be elucidated (31) and is the subject of further investigation in our laboratory.

E17ß treatment of OVX ewes or GnRH-pulsed OVX-HPD ewes causes a rapid reduction in response to GnRH and reduction in mean plasma LH levels (25, 34) as confirmed in the present studies. E17ß also affects the expression of GnRH receptor, but the short-term response is up-regulation, even when the response to GnRH is reduced by negative feedback of the steroid (50). Thus, it seems unlikely that the short-term negative-feedback effect of E17ß involves changes in function of the GnRH receptor. It is now clear that the negative feedback effect of E17ß involves rapid signaling to reduce LH output. Other studies in the OVX sheep show that E17ß injection suppresses LH secretion within 20 min, which is considered to be too rapid to be a response mediated by a genomic mechanism (25). We saw a similar trend in the suppression of LH secretion after E17ß injection, which was clearly evident in animals sampled at 90 min because this group of animal received an additional pulse of GnRH after 60 min of E17ß injection. This suggests that the cytosolic ER may also be affecting the second messengers. The effect to cause negative feedback may be replicated by an agonist specific for ER{alpha} (46).

Results of the present study support the notion that short-term negative feedback involves rapid and nongenomic E17ß action (25) and extend this to indicate the induction of pERK-1/2, pCREB, and pAkt within this timeframe. This may involve modulation of calcium flux, leading to a cascade of second messenger response (13, 14) and/or the GPRC30-initiated rapid effects of E17ß (18, 42). E17ß causes the phosphorylation of Akt, which is downstream of the PI3K pathway (51, 52), so it may be reasonably assumed that there is also an effect on mobilization of intracellular free calcium (13). Earlier in vitro studies in which ovine pituitary gonadotropes were treated for 16–20 h showed E17ß transiently increased calcium currents leading to increased intracellular calcium levels (53). Studies of hippocampal neurons have indicated that E17ß treatment causes calcium influx and increased dendritic and nuclear calcium levels (54) via the L-type calcium channels, which requires PI3K activation of the protein kinase C pathway (55). Others showed that E17ß rapidly reduced calcium current (L-type current) in rat neostriatal neurons, presumably through membrane receptor mechanisms (56). It is possible, therefore, that E17ß-induced rapid effects operate differently in different systems and/or depending upon the timeframe. If the rapid effect in gonadotropes is specific to the negative feedback on gonadotropin secretion, it is most likely that this would be associated with a reduction in intracellular free calcium response to GnRH, but this requires in vitro studies to carefully examine the calcium flux/electrophysiology in single cells.

In summary, we have used a novel in vivo approach to study rapid effects of E17ß on pituitary gonadotropes. Our results demonstrate E17ß-induced activation of MAPK pathways by rapid phosphorylation of second messengers (ERK-1/2) within 15 min, which may then lead to phosphorylation of downstream signaling molecules such as CREB (at 30 min) and possible transcriptional regulation in these cells. These results support a direct role for E17ß in the acute negative-feedback effect (acute suppression) on LH secretion under physiological conditions. The other possible mechanism of E17ß-induced acute suppression of LH secretion could involve the PI3K-Akt pathway leading to regulation of intracellular calcium in gonadotropes. Induction of pAkt, however, occurs at a later time point (60 min) than induction of pERK and pCREB.


   Acknowledgments
 
We thank Bruce Doughton, Lynda Morrish, Jessica Thomas, Alix Rao, and Alda Pereira for technical assistance. We thank Dr. A. Parlow of the National Hormone and Peptide Program for LH assay reagents.


   Footnotes
 
Olivier Latchoumanin was supported by a Prize of the Bettencourt-Schueller Foundation, France. This work was supported by the Australian Research Council, Australia.

Disclosure Statement: All of the authors have nothing to disclose.

First Published Online September 6, 2007

Abbreviations: Akt, Serine/threonine kinase; CREB, cAMP-responsive element-binding protein; CV, coefficient of variation; E17ß, estradiol-17ß; ER, estrogen receptor; GPCR, G protein-coupled receptor; GPR30, G protein-coupled membrane protein receptor 30; HPD, hypothalamo-pituitary disconnection; IR, immunoreactivity; OVX, ovariectomized; p, phosphorylated; PB, phosphate buffer; PI3K, phosphatidylinositol 3-kinase.

Received July 18, 2007.

Accepted for publication August 29, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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