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Endocrinology, doi:10.1210/en.2004-0980
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Endocrinology Vol. 146, No. 3 1128-1137
Copyright © 2005 by The Endocrine Society

Effects of Changing Gonadotropin-Releasing Hormone Pulse Frequency and Estrogen Treatment on Levels of Estradiol Receptor-{alpha} and Induction of Fos and Phosphorylated Cyclic Adenosine Monophosphate Response Element Binding Protein in Pituitary Gonadotropes: Studies in Hypothalamo-Pituitary Disconnected Ewes

Iain J. Clarke, Vicky A. Tobin, Sueli Pompolo and Alda Pereira

Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Professor Iain J. Clarke, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: iain.clarke{at}phimr.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen receptor-{alpha} (ER{alpha}) levels in gonadotropes are increased during the follicular phase of the ovine estrous cycle, a time of increased frequency of pulsatile secretion of GnRH and elevated plasma estrogen levels. In the present study, our first aim was to determine which of these factors causes the rise in the number of gonadotropes with ER{alpha}. Ovariectomized hypothalamo-pituitary disconnected ewes (n = 4–6) received the following treatments: 1) no treatment, 2) injection (im) of 50 µg estradiol benzoate (EB), 3) pulses (300 ng iv) of GnRH every 3 h, 4) GnRH treatment as in group 3 and EB treatment as in group 2, 5) increased frequency of GnRH pulses commencing 20 h before termination, and 6) GnRH treatment as in group 5 with EB treatment. These treatments had predictable effects on plasma LH levels. The number of gonadotropes in which ER{alpha} was present (by immunohistochemistry) was increased by either GnRH treatment or EB injection, but combined treatment had the greatest effect. Immunohistochemistry was also performed to detect phosphorylated cAMP response element binding protein (pCREB) and Fos protein in gonadotropes. The number of gonadotropes with Fos and with pCREB was increased only in group 6. We conclude that either estrogen or GnRH can up-regulate ER{alpha} in pituitary gonadotropes. On the other hand, during the period of positive feedback action of estrogen, the appearance of pCREB and Fos in gonadotropes requires the combined action of estrogen and increased frequency of GnRH input. This suggests convergence of signaling for GnRH and estrogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DECLINE IN plasma progesterone levels at the end of the luteal phase of the estrous cycle removes negative feedback on GnRH, allowing for increased frequency of pulsatile discharges of this neurohormone from the hypothalamus (1, 2). This, in turn, increases the frequency of pulsatile discharges of LH from the pituitary gonadotropes leading to growth of ovarian follicles and increased estrogen secretion (3, 4). The increase in circulating levels of estrogen induces a positive feedback effect on the secretion of GnRH and LH, which is a time-delayed mechanism (4). This involves actions of estrogen on cells in the brain as well as action on the pituitary gonadotropes, involving electrophysiological properties and mobilization of secretory granules (3, 5, 6, 7, 8). The specific molecular actions of estrogen that produce the GnRH/LH surge are not well understood, although this is the primary endocrine signal for ovulation. This estrogenic signal that initiates positive feedback occurs in the follicular phase of the normal estrous cycle (4).

Although there is a wealth of literature regarding the subcellular mechanisms for GnRH action in gonadotropes, there is relatively little information on the means by which estrogen acts on these cells. Estrogen regulates the expression of GnRH receptors in vivo (9, 10, 11) and in vitro (12), with an increase occurring during the follicular phase of the estrous cycle in the ewe (13, 14, 15, 16). Nevertheless, responsiveness of the gonadotropes to GnRH does not increase until close to the time of onset of the LH surge (1), suggesting that this increased GnRH receptor expression may be permissive of enhanced secretion at the time of the LH surge, but it is not the cause of a profound increase in responsiveness at the onset of the surge. LH-secreting granules migrate to the periphery of gonadotropes in preparation for the cyclic LH surge (17), which presumably reflects subcellular mechanisms activated by estrogen. This polarization of the granules also occurs in ovariectomized (OVX) ewes given a single im injection of estradiol benzoate (EB) (7).

A series of studies on rat pituitaries show that there is up-regulation of second messenger systems in the proestrous stage of the estrous cycle, suggesting that these factors may be related to the mechanism of positive feedback (18, 19, 20, 21, 22). Because these studies were done with hemipituitaries, however, and various cell types in the rat pituitary express ER{alpha} (23), it is difficult to ascribe the activities directly to the gonadotrope. One study in particular provides convincing evidence for the action of estrogen to up-regulate protein kinase C in rat pituitaries (18). These data suggest actions of estrogen that may either be genomic or nongenomic, and recent data (24) further indicate rapid effects via phosphorylation of MAPK and phosphorylation of cAMP response element binding protein (CREB). Short- and long-term effects of estrogen on CREB and related proteins, as well as CRE-DNA binding, are seen in various brain regions (25); the former could be caused by rapid nongenomic effects of estrogen (26, 27, 28). Measurement of phosphorylation of CREB is regarded as an index of cellular activation (24, 25). Fos is another marker of cellular response, and GnRH has been shown to increase the percentage of immuno-identified gonadotropes that produce Fos protein (29).

Whereas estrogen up-regulates GnRH receptor levels (see above), we showed that the number of gonadotropes that express estrogen receptor-{alpha} (ER{alpha}) is also increased in the follicular phase of the ovine estrous cycle (30). This could be a means of enhancing sensitivity to estrogen at this time. Early work in rat cells (31) suggested that GnRH could increase nuclear ER in pituitary cells. We hypothesized that the increased expression of ER{alpha} in gonadotropes in the follicular phase of the estrous cycle might be caused by increased frequency of GnRH input, increasing plasma levels of estrogen, or both. To investigate these possibilities, we employed our well characterized model of the OVX hypothalamo-pituitary disconnected (HPD) ewe, which allows the study of the pituitary gland without brain inputs (32, 33). Programmed replacement with pulsatile GnRH and estrogen allows the study of effects on the gonadotrope in vivo that can be correlated with subsequent postmortem analysis (34). We also examined the effects of either increased GnRH pulse frequency and/or estrogen treatment on the appearance of phosphorylated CREB (pCREB) and Fos in the gonadotropes. The treatment paradigm was such that the measurements were taken at the time when positive feedback actions of estrogen are operative (20 h after an im injection of EB) (33).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Ethics statement
All experimental procedures were conducted under a protocol approved by the Victorian Institute of Animal Science Animal Ethics Committee.

Sheep
Adult Corriedale female sheep used in this study were kept at the Prince Henry’s Institute facility at Werribee, Melbourne. The animals were bilaterally OVX at least 1 month before the experiment, and the HPD operation was performed as previously described (32). The latter procedure removes all neural inputs to the median eminence, but gonadotropin secretion can be restored by iv administration of GnRH in a pulsatile mode. Immediately after HPD, one external jugular vein was cannulated with a 12-gauge Teflon Dwellcath (Tuta Laboratories Australia Pty. Ltd., Bedford Park, South Australia, Australia) that was connected to a manometer line (Portex Ltd., Hythe, UK) for infusion of GnRH or vehicle (see below). These infusion lines were connected to a Braun Unita 1 infusion pump that could be programmed to deliver 2.25-ml pulses over 6 min as previously described (34). All animals received vaginal progesterone delivery devices (controlled intravaginal drug release) (Lyppard, Cheltenham, Victoria, Australia) for 5 d with GnRH or saline vehicle replacement as detailed below. These were removed at 1600 h on the fifth day and EBL or oil vehicle (1 ml peanut oil) was administered at 1600 h on the sixth day. The animals were killed for recovery of pituitary glands at 1200 h on the seventh day (20 h after EB or oil vehicle).

The animals were assigned to experimental groups as follows (summarized in Fig 1Go): 1) saline vehicle and oil vehicle (n = 5); 2) saline vehicle and injection (im) of 50 µg EB (Intervet, Baulkham Hills, New South Wales, Australia) (n = 5); 3) pulsatile replacement of GnRH by iv infusion of 300 ng every 3 h with oil vehicle (n = 6); 4) same GnRH regimen as group 3 with EB treatment (n = 4); 5) pulsatile replacement of GnRH by iv infusion of 300 ng every 3 h, switched to hourly pulses of 100 ng between 1600 h on d 6 until 0800 h on d 7, and then switched to 50-ng pulses every 0.5 h between 0800 h on d 7 until 1200 h on d 7 (time of termination), oil vehicle treatment (n = 4); and 6) same GnRH regimen as group 5 with EB treatment (n = 4).



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  		FIG. 1. Experimental design.

 
The dose of each GnRH pulse was varied in accordance with the interval of delivery, such that the animals received the same amount of GnRH per unit time. In this way, groups 3 and 4 received GnRH replacement at a low frequency, characteristic of that seen during the luteal phase of the estrous cycle (1, 35). Groups 5 and 6 received GnRH replacement where the pulsatile input increased in frequency, as seen in the follicular phase of the estrous cycle. Previous work has shown that the EB treatment of GnRH-replaced OVX-HPD ewes causes a biphasic response in terms of LH secretion, with a time frame similar to that seen in the OVX, hypothalamo-pituitary intact ewe (36, 37). A surge in LH secretion is seen in OVX-HPD ewes between 16 and 20 h after EB treatment, but this reaches only the magnitude of that seen in the hypothalamo-pituitary intact animal if the GnRH pulse frequency is increased to 10-min intervals or if a large bolus of GnRH is delivered at 16 h (36, 37).

Blood samples (5 ml) were taken as follows. On d 6, all animals were sampled over three GnRH pulses delivered at 0900, 1200, and 1500 h; samples were taken 5 min before the delivered pulse and at 7.5, 15, and 30 min after the pulses. For animals in groups 5 and 6, samples were collected over three pulses on d 6 and then over hourly pulses between 0500 and 0700 h and then over half-hourly pulses between 0800 and 1200 h. For sampling around half-hourly pulses on d 7, samples were taken 7.5, 15, and 25 min after the pulses. For groups 1–4, samples were collected at the same times as indicated for groups 5 and 6, even though GnRH or saline pulses were delivered at 3-hourly intervals in these animals; this enabled quantification of changes in total LH released over the last 3 h of the experiment.

The animals were overdosed with sodium pentobarbital (0.5 ml/kg body weight, iv Lethobarb, May and Baker Pty. Ltd., Melbourne, Australia), and the pituitaries were carefully removed from the sella turcica, cut into 3-mm sagittal slices, and placed in a 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) for 2 d at 4 C. The tissues were then placed in 30% sucrose in 0.1 M PB solution for 5 d at 4 C and then frozen on powdered dry ice and stored at –20 C.

Immunohistochemistry
For all immunohistochemical procedures, antibody incubations were carried out at room temperature unless otherwise specified and were followed by three 10-min washes in 0.05 M PBS. Pituitary sections (7 µm) were cut on a cryostat (Leica CM 1850) at –22 C, thaw-mounted onto Superfrost slides, and stored at –20 C until used for immunohistochemistry. A modified version of a recently published antigen retrieval method was used to detect ER{alpha} in ovine pituitary tissue (30). This was preceded by incubation with 3% H2O2 in methanol (30 min) to block endogenous peroxidase activity. Slides were then microwaved (four times for 5 min at 700 W) in 0.01 M citrate buffer (pH 6.0) and left to stand in the same buffer for another 20 min. For immunocytochemistry, the sections were incubated in blocking solution (10% normal donkey serum, 0.3% Triton X-100 in 0.1 M PB) for 30 min and then with ER{alpha} antibody (monoclonal ID5 raised against the N-terminal region of ER{alpha}, Dako, Carpinteria, CA) at a 1:8 dilution in blocking solution for 72 h at 4 C. The sections were then incubated (1 h) in biotinylated donkey antimouse IgG (Vector Laboratories, Burlingame CA) at a 1:400 dilution and then with Streptavidin horseradish peroxidase (STA-HRP) complex (Amersham, Piscataway, NJ) at a 1:500 dilution for 1 h. After washes in buffer, the sections were preincubated in NiDAB (0.5 mg/ml, 0.02% NH4NiSO4 and 0.026% CoCl2 in 0.1 M PB) for 10 min. This solution was removed and 0.15% H2O2 quickly added for approximately 4 min. The color reaction was monitored and halted by removing the NiDAB solution and washing in 0.1 M PB. Double labeling was then carried out to identify LH cells that also expressed ER{alpha}. Expression of LH was detected using an anti-LH polyclonal antibody (30) at 1/5000 and visualized with diaminobenzidine (DAB) to give a brown color. Briefly, after immunohistochemistry for ER{alpha}, sections were washed in 0.05 M PBS and incubated for 30 min in blocking solution containing normal goat serum, then with primary antibody for 48 h at 4 C, followed by incubation (1 h) in secondary antibody (biotinylated goat antirabbit, Vector) at 1/200 dilution. Sections were then incubated with STA-HRP complex for 1 h followed by DAB solution. The color reaction was as described above but without the addition of NH4NiSO4 and CoCl2.

To identify activated LH cells, a separate series of Fos/LH double-labeling and pCREB/LH double-labeling immunohistochemistry was carried out on two pituitary sections per animal per group. Polyclonal rabbit antibodies to Fos protein (AB5, Oncogene Research, Cambridge, MA; 1/1000 dilution) and pCREB (Upstate Biotechnology, Placid, NY; 1/500 dilution) were used as markers for early activation; both were visualized with NiDAB. Sections were washed in 0.05 M PBS followed by pretreatment with 3% H2O2 in methanol, washed in PBS, and then processed for antigen retrieval as described above. After washes in PBS, the sections were incubated with blocking solution containing normal goat serum for 30 min and then in primary antibody for 72 h at 4 C. This was followed by incubation (1 h) in biotinylated goat antirabbit IgG at 1/200 dilution and then in STA-HRP complex at 1/500 dilution for 1 h. After washes in PBS, sections were then incubated with NiDAB solution and color development carried out as above. Double labeling was achieved by incubation with polyclonal rabbit anti-LH antibody visualization with DAB as described above. The antibodies used for labeling Fos (38), pCREB (39), and ER{alpha} (40) have been characterized previously.

Analysis
Five regions (one in each of the four quadrants and one region in the center) of each coronal anterior pituitary section were examined under x60 magnification. At this magnification, the presence or absence of nuclear and cytoplasmic staining was unambiguous. In each region, 100 cells were counted to give a total of 500 cells per section. Single LH-positive cells and double-labeled LH/ER{alpha}, LH/Fos, and LH/pCREB cells were scored for each series of sections. Mean percent colocalization was then calculated for each experimental group.

LH RIA
Plasma LH levels were measured using a RIA as previously described (38). In six assays, sensitivity was 0.1 ng/ml, within-assay coefficient of variation was 3.5–9.8% between 3.3 and 6.1 ng/ml, and between-assay coefficient of variation was 12% at 12.7 ng/ml, 10% at 5.0 ng/ml, 7.3% at 6.6 ng/ml, and 6% at 23.3 ng/ml.

Statistical analysis
To determine statistically significant (P < 0.05) differences between means, area under curves (AUCs) of plasma LH concentration vs. time, and for the percentages of gonadotropes that colocalized either ER{alpha} or Fos or pCREB, two-way ANOVA was performed. Post hoc tests for differences between groups employed the method of least-significant differences. For analysis of percentage data, the arcsin transformation was used as recommended by Sokal and Rohlf (41). In the case where pretreatment values were compared with posttreatment values, paired Student’s t tests were used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma LH levels were undetectable in the sheep of groups 1 and 2. Analysis of effects of GnRH with either EB or oil treatment was therefore confined to groups 3–6.

Typical examples of the pattern of response to the different GnRH/EB treatments are seen in Fig. 2Go. LH pulse amplitudes in the animals given GnRH pulses every 3 h plus oil injection (group 3) decreased (not significant) slightly (14%) over the course of the experiment (Table 1Go), but the LH pulse amplitudes were reduced by 23% (P < 0.01) and broadened (Fig. 2Go) in animals given the same GnRH treatment with EB injection, (Table 1Go). When GnRH pulse frequency was increased (with oil treatment; group 5), the LH pulse amplitudes were reduced across the experiment with progressive increase in GnRH/LH pulse frequency (Table 1Go). In those animals that received EB with increased pulse frequency (group 6), the LH pulse amplitudes were not significantly reduced on hourly pulse frequency, because one animal showed a marked increase in LH pulse amplitude. When group 6 animals were given half-hourly pulses of GnRH, the LH pulse amplitudes were significantly (P < 0.01) reduced (Table 1Go).



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  		FIG. 2. Examples of plasma LH levels in individual OVX-HPD ewes receiving GnRH on a fixed 3-hourly pulse frequency or increased pulse frequency, with or without EB treatment. Black bars indicate the timing of GnRH pulses, which were either 300 ng (3-hourly) before EB/oil treatment or 50 ng (half-hourly) during the last 3 h of the animal treatments. Examples are given for animals from groups 3 (upper left), 4 (upper right), 5 (lower left), and 6 (lower right).

 

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  		TABLE 1. Effect of changing GnRH pulse frequency and effect of EB/oil on LH pulse amplitude in OVX-HPD ewes

 
AUCs of plasma LH concentration vs. time were analyzed for data obtained between 0900 and 1200 h on d 7, because all animals were sampled at the same clock times. As seen in Fig. 2Go, the LH pulse shapes were different in animals from groups 1–4, and mean AUC data for groups 4–6 are presented in Table 2Go. Plasma LH was undetectable in groups 1 and 2, so the analysis excluded these. The data show that EB treatment with increased GnRH pulse frequency (group 6) caused greater release of LH than all other treatments. In the partitioning of effects, the increased GnRH pulse frequency produced significantly (P < 0.01) higher values than the fixed-pulse regimen, and EB treatment produced greater values than oil treatment (P < 0.001). The interaction between the two was significant (P < 0.002), indicating that the combination of increased GnRH pulse frequency with EB treatment gave the greatest AUC values.


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  		TABLE 2. Area under plasma LH concentration vs. time curves between 0900 and 1200 h on d 7 (when groups 5 and 6 received half-hourly GnRH pulses)

 
Figure 3Go shows examples of gonadotrope immunostaining with double labeling for ER{alpha}, Fos, and pCREB. The percentage of gonadotropes that colocalized ER{alpha} is shown in Table 3Go. Both GnRH treatment and EB treatment increased the percentage of gonadotropes that colocalized ER{alpha} (group 1 vs. all other groups). With GnRH treatment alone (groups 2 and 3), the level of colocalization was the same with both pulse paradigms. Three-hourly GnRH pulses plus EB (group 5) had a greater effect than 3-hourly pulses alone (group 2). EB treatment alone (group 4) was more effective in elevating ER{alpha} in gonadotropes than GnRH given at the 3-hourly pulse frequency but not more effective than increased GnRH pulse frequency alone (group 3). Increased GnRH pulse frequency plus EB (group 6) was more effective than increased GnRH pulse frequency alone (group 3). The statistical analysis showed that there was a statistically significant (P < 0.001) interaction between the GnRH treatment and the EB treatment, which reflects the fact that EB treatment with increased GnRH pulse frequency gave a much bigger response than EB treatment with fixed GnRH pulse frequency.



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  		FIG. 3. Examples of the ER{alpha} (row 1), Fos (row 2), and pCREB (row 3) immunostaining in gonadotropes (detected with LH immunostaining). Boxed areas in the first and third columns are enlarged in the second and fourth columns. Open arrows show gonadotropes without dual staining, closed arrows show double labeling of gonadotropes with ER{alpha} (row 1), Fos (row 2), and pCREB. The arrowheads indicate ER{alpha} and Fos immunoreactive nuclei of pituitary cells that were not immunoreactive for LH.

 

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  		TABLE 3. Mean (± SEM) percentage of gonadotropes (LH immunopositive) that colocalized ER{alpha}, Fos, and pCREB as determined by immunostaining

 
Both the percentage of gonadotropes that colocalized Fos and the percentage that colocalized pCREB were increased only in the group that received increased GnRH pulse frequency and EB (group 6) (Table 3Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using an in vivo model, we have shown that both GnRH and estrogen increase the number of gonadotropes with immunohistochemically identifiable ER{alpha}. A complex interaction clearly exists between actions of the GnRH receptor and the ER that allows combined regulation of ER{alpha}, and this may explain the increase in ER{alpha} in gonadotropes that is seen in the follicular phase of the estrous cycle (30). Furthermore, we have shown that a combination of increased GnRH pulse frequency and estrogen is required to generate Fos and pCREB responses in these cells. Although earlier studies have shown that GnRH stimulates production of immediate early response genes in {alpha}T3-1 cells and gonadotropes (29, 42), and estrogen induction of Fos has been observed in the pituitary gland (see below), the combined effect on gonadotropes has not been observed previously. An earlier report (43) demonstrated that in vitro treatment of perifused pituitary cell aggregates with GnRH pulses increased the steady-state level of ER{alpha} mRNA with no increase in LHß mRNA. These data suggested that there may be convergence of signaling of GnRH and nuclear ER in pituitary gonadotropes, and the present data show that this is indeed the case.

The GnRH and estrogen treatments employed in our study caused predictable changes in the plasma levels of LH, highlighting the utility of the model. In particular, an increase in GnRH pulse frequency reduced LH pulse amplitude, as reported previously (44), and EB altered the shape of LH pulses at the time of predicted positive feedback, as shown previously (33). AUCs were the same for 3-hourly and hourly pulses, as predicted from earlier studies, showing the exactitude of frequency and amplitude relationships (45). The present study confirms that, in the OVX ewe without steroid treatment, the inverse relationship between GnRH pulse frequency and pulse amplitude is rigid and is a reflection of the releasable pool of LH within the cell (45). Although estrogen increased AUC units slightly when animals were given EB on a fixed-pulse frequency, most likely because of altered LH pulse shape, this was not statistically significant. The combination of increased pulse frequency and EB treatment elevated total secretion of LH, although surge-like secretion was not seen. This is because the frequency of GnRH input did not reach that seen during the EB-induced surge in the OVX hypothalamo-pituitary intact ewe (46). Higher-frequency GnRH input in combination with EB treatment does cause a full LH surge in this model (36, 37). The combination of increased GnRH pulse frequency and EB did significantly increase AUC, indicating that the animals were sampled at a time when positive feedback mechanisms were operative. This confirms the effect of estrogen to sensitize cells to respond to GnRH (33). These treatments allow dissection of subcellular events within the gonadotropes that relate to the positive feedback phenomenon.

We employed im injection of EB in oil as an effective means of eliciting the positive feedback phenomenon as previously shown in OVX ewes and in OVX-HPD ewes given pulsatile GnRH replacement (47). The positive feedback effect can also be elicited with iv injection of estradiol to OVX ewes (48), which raises plasma estrogen levels for a period of less than 3 h, and nuclear ER binding (measured by nuclear exchange assay) was elevated for up to 6 h. This indicates that the estrogen signal that initiates the positive feedback effect need only be transient and that a time delay is required before the surge in LH secretion occurs. More recently, others (49) have shown that treatment of OVX ewes with estrogen implants for 8 h will cause an LH surge 17–19 h after commencement of treatment. These authors reinforce the notion that the surge-inducing effect of estrogen involves activation (during which time, estrogen activates mechanisms that will cause a surge with a time delay) and transmission (which is not dependent upon the presence of estrogen). It was also shown in cultured rat pituitary cells by Kamel and Krey (50) that enhancement of LH release by estrogen continues beyond the time that estrogen is withdrawn. It is not known whether liganded ERs are required to be present at the time of the surge, but it is clear that the surge-generating action of estrogen occurs at a time before the surge. Presumably, the delay interval allows a sequence of intracellular signaling that is not well understood. In this paper, we show that activation of at least two transcription factors (Fos and pCREB) require increased pulsatile GnRH input as well as estrogen input. Additional use of this model with biochemical analysis of signaling systems will allow greater resolution of this unique phenomenon.

We previously showed that the number of gonadotropes expressing ER{alpha} is increased in the follicular phase of the estrous cycle (30), but it was not known whether this was because of increased estrogen levels and/or increased GnRH pulse frequency. The number of gonadotropes that also stain for ER{alpha}/ERß also increases at proestrus in the rat (51). The present work provides novel in vivo data to suggest that both factors are involved. EB treatment alone caused a significant increase in ER{alpha} as did 3-hourly pulses of GnRH, but the former was more effective. With increased GnRH pulse frequency in the absence of an EB effect, there was no significant increase in the number of gonadotropes in which immunostaining of ER{alpha} was seen, suggesting that this manipulation does not lead to an increase in ER{alpha}. Nevertheless, during the period when a positive feedback effect of EB is operative in our model (33), a combination of EB and increased GnRH pulse frequency increased the percentage of ER{alpha}-positive gonadotropes to the greatest extent. Thus, the combination is the most effective means of up-regulating ER{alpha} in this cell type. The follicular phase of the estrous cycle is characterized by increased GnRH/LH pulse frequency (1, 2, 35) as well as increasing levels of estrogen (17). Based on the data of the present study, the increase in gonadotropes that are immunopositive for ER{alpha} at this time (30) is most likely caused by a combination of these two factors.

Statistical analysis of the effect of GnRH pulse frequency and EB treatment on ER{alpha} in gonadotropes showed a highly significant interaction between these two parameters, which indicates convergence between estrogen action and GnRH action. Earlier work from various labs and in the ovine model (9) has clearly shown that estrogen up-regulates GnRH receptors, and the present data clearly show that GnRH up-regulates ER{alpha}. The effect of GnRH on ER{alpha} protein substantiates the results of Demay et al. (43) (see above). The additive effect could involve action of EB via ER{alpha} present in these cells even after 7 d of pituitary disconnection without replacement (see group 1 data in Table 3Go) or action by other means. There are few ERß receptors in the ovine pituitary gland (Scott, C., and I. J. Clarke, unpublished data), so the effect is most likely exclusive to mediation via ER{alpha}.

GnRH action involves a variety of second messenger systems, including the phospholipases C, D, and A2, protein kinase C, and MAPK pathways (20, 21, 22). Phospholipase hydrolysis of the phosphoinositides also plays a pivotal role, and the release of intracellular calcium is important for the secretory mechanism (20, 52). The cAMP/protein kinase A pathway is operative within gonadotropes, although it has been suggested to be more important for transcriptional regulation than the acute secretory response to GnRH (20). Many of these pathways have also been described in immortalized cell lines. Cell membrane ion channel events are a fundamental component of the secretory response to GnRH (52), and Rab3B appears to be involved (53). Ovine gonadotropes produce a wide range of soluble N-ethyl maleimide-sensitive factor attachment proteins/receptors (8), most likely involved in the exocytosis of gonadotropin-containing vesicles as well. Estrogen can act via ER to directly regulate transcription in gonadotropes (54), and rapid estrogen effects are also possible, involving membrane-associated ER and/or cytoplasmic ER interactions with signaling systems (24, 55, 56). Estrogen sensitization of gonadotropes is a well recognized phenomenon, allowing increased responsiveness to GnRH. This could involve an increase in GnRH receptor number, induced by estrogen but not (at least in the OVX-HPD ewe) by increased GnRH pulse frequency (9, 34). The present model will allow further subcellular dissection of the effects of the combination of estrogen and increased GnRH pulse frequency. A range of other intracellular responses are also seen in the gonadotrope at the time of the estrogen-induced surge in LH secretion (57) that could be the consequence of the genomic action of liganded ER complexes.

Estrogen can act directly on gonadotropes to regulate transcription of the gonadotropin subunits and the GnRH receptor. At least in the case of the ovine FSHß, the promoter region contains sites for the binding of progesterone receptor and activator protein 1 (AP-1) and a site for estrogen-dependent repression (58). The rat LHß gene has an imperfect palindromic sequence in the promoter region that confers estrogen responsiveness (59) allowing direct estrogen action. Similar information is lacking for the ovine LHß gene and the common {alpha}-subunit, but available evidence shows that estrogen negatively regulates the transcription of all of the gonadotropin subunit genes in the pituitary of the EB-treated OVX ewe over the time course relevant to this study (47). This study also showed that LHß gene expression was not reduced over the period of acute negative and positive feedback after EB treatment of OVX-HPD ewes that were receiving hourly GnRH pulses, presumably because GnRH stimulation was maintained, whereas it declined in the negative-feedback phase in the EB-treated OVX ewe. Steady-state mRNA levels for the GnRH receptor, the {alpha}-subunit, and LHß (but not FSHß) are increased in the follicular phase of the ovine estrous cycle (13). The human GnRH receptor promoter has a variety of cis-acting regulatory sequences including sites for CRE and AP-1 (60). Estrogen significantly up-regulates GnRH receptor transcription in EB-treated, OVX-HPD ewes receiving constant GnRH input, indicating direct pituitary action (34). Thus, genomic signaling of estrogen can regulate the transcription of various genes within the gonadotrope, and up-regulation of ER{alpha} by both estrogen and GnRH as shown in this study could facilitate the combined effects of the two hormones. As indicated above, effects on the secretion of LH probably involve a range of signaling systems.

It is also possible that convergence of estrogen and GnRH signaling occurs via membrane-associated ER and/or cytoplasmic ER interactions with signaling systems. In other words, estrogen can act to rapidly phosphorylate CREB and/or MAPK (24), and these could converge on GnRH signaling systems. Recent evidence suggests that ER may associate with calveolin to facilitate membrane binding and signaling via G proteins (61, 62). This can facilitate transactivation of the epidermal growth factor receptor by liganded ER (62). A similar scenario may exist for the interaction of ER{alpha} and the G protein-coupled GnRH receptor. Such convergence of postreceptor signaling might explain why the increase in GnRH pulse frequency and estrogen act together to activate the gonadotrope or whether effects are solely because of genomic action of liganded ER{alpha}. More work on this cell type is required before such a mechanism is delineated, and our in vivo HPD model offers a means by which this can be examined in nontransformed gonadotropes.

The appearance of Fos protein is generally regarded as a transient response to a specific activator of cells (63). GnRH produces a Fos response in gonadotropes, and although estrogen has been shown to do the same in a number of different types of cells, there are no published reports detailing the response in gonadotropes. A rapid (within 15 min) response of gonadotropes to GnRH has been seen using Fos as an index (29). An interesting feature of this cell type is that a prolonged Fos response may occur. Thus, Szijan et al. (64) found that a large (250 mg) sc injection of estrogen diundecylenate given to male rats produced elevated c-fos and c-myc mRNA levels in the pituitary gland that were sustained for 16–22 h. Because these authors did not define the cell type and various cell types possess ER in the rat (65), this may or may not relate to gonadotropes. In another study, using 1 µg estradiol given sc to OVX rats, Allen et al. (66) found that c-fos expression was elevated within 2 h and remained elevated for 48 h; this response was localized to lactotropes and folliculostellate cells. Sustained Fos/Fos-related antigen responses are also seen in the dopaminergic cells of the hypothalamus of OVX anestrous ewes chronically treated with estrogen (65). On the other hand, Fos responses to a single im injection of EB cause transient responses in brain cells of the OVX ewe (67). In the present study and with a fixed regimen of GnRH stimulation, the number of gonadotropes that were immunopositive for Fos was similar to untreated gonadotropes. There was no increase in the proportion of gonadotropes with Fos after increased frequency of GnRH input. A transient increase may have occurred earlier in response to the increased pulse frequency, but our design could not test for this. On a fixed GnRH pulse regimen, the number of gonadotropes immunostained for Fos was not increased by EB treatment 20 h earlier, but once again, there could have been an earlier transient response. The most significant feature of the present data is that the combination of increased GnRH pulse frequency and EB treatment significantly increased the number of gonadotropes displaying Fos-immunoreactive nuclei 20 h after the commencement of the combined treatment. This is the period when the positive-feedback effect of estrogen is operative (due to the combined effects of increased GnRH pulse frequency and estrogen), and this is clearly associated with activation of the cells, as indicated by the Fos responses. Because AP-1 binding sites are found in the GnRH receptor promoter (see above), this is at least one gene that could be affected by Fos production. As to how estrogen up-regulates GnRH receptor expression in this experimental model (see above), and during the estrous cycle (13), it is possible that this is via up-regulation of second messenger systems, as indicated above, and Fos.

As with Fos, the level of pCREB was increased only in animals that received increased frequency of GnRH pulses and EB. This could be effected by nongenomic action of estrogen, because rapid effects (within 15 min) of estrogen are seen in the brain for phosphorylation of CREB and MAPK (24). If this involves ER-dependent signaling that is rapid (24), it may involve interaction of the ligand with the receptor in the extranuclear compartment of the cell to phosphorylate signaling molecules directly (55). CREB may act as an integrating point for a range of intracellular signaling systems (68). As indicated above for Fos induction, it is possible that either estrogen or increased GnRH pulse frequency could have caused a transient effect to phosphorylate CREB, but at the time point studied, it was only the combination of the two that produced elevated pCREB production at 20 h. Although estrogen might rapidly phosphorylate CREB through nongenomic mechanisms, chronic estrogen treatment has also been shown to increase pCREB and other components of this system (25), so the increase that we have observed with a combination of increased GnRH pulse frequency could be important for both extranuclear activation of second messenger systems and for genomic effects to regulate transcription. As suggested by Zhou et al. (55), phosphorylation of CREB may allow integration of estrogen signaling and membrane receptor signaling. How this relates to the mechanism of positive feedback remains to be elucidated.

A series of studies by Nett and coworkers (69, 70) show that cAMP production is increased by GnRH treatment in ovine gonadotropes. GnRH stimulates parallel release of LH and cAMP (69). Dibutyryl cAMP was also able to stimulate LH release in their culture system (69). Later work confirmed that dibutyryl cAMP as well as phorbol ester (to stimulate protein kinase C) could stimulate LH and FSH release from ovine pituitary cells in primary culture, but a calcium ionophore selectively stimulated LH release (70). Interestingly, it has been reported that a CRE region is not found on the promoter of the ovine FSHß gene, and it is not known whether such a consensus sequence exists in the ovine LHß or common {alpha}-subunit gene promoters. As mentioned above, the human GnRH receptor promoter contains a CRE site, so pCREB could be relevant to the transcription of this gene. A range of other second messenger systems (which are activated by estrogen and GnRH; see above) (71) can phosphorylate CREB, leading to effects at the level of transcription.

In summary, we have used a unique in vivo model to elucidate combined effects of increased GnRH pulse frequency and estrogen injection on the levels of ER{alpha}, Fos, and pCREB in the gonadotropes of the ovine pituitary gland. The data show that the proportion of cells displaying immunoreactive ER{alpha} is increased by both increased GnRH pulse frequency and estrogen treatment, with interactive effects. This combined action explains the increased level of ER{alpha} seen during the follicular phase of the estrous cycle (30). In addition, the number of cells displaying Fos and pCREB immunoreactivity is increased by the combined treatment (increased GnRH pulse frequency and estrogen treatment) but not by either alone. It is also this combined treatment that brings about the greatest increase in sensitivity of gonadotropes to GnRH with respect to LH secretion. Again, this suggests an interaction between the signaling systems for GnRH receptors and ERs.


   Acknowledgments
 
We thank Alix Rao, Karen Briscoe, and Bruce Doughton for technical assistance and the National Institutes of Health for assay reagents.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia. V.A.T. received a Prince Henry’s Institute Boylan-Burke Fellowship.

Present address for V.A.T.: Division of Biomedical Sciences, University of Edinburgh Medical School, George Square, Edinburgh EH8 9XD, United Kingdom.

First Published Online November 24, 2004

Abbreviations: AP, Activator protein; CRE, cAMP response element; CREB, CRE-binding protein; DAB, diaminobenzidine; EB, estradiol benzoate; ER, estrogen receptor; HPD, hypathalamo-pituitary disconnected; OVX, ovariectomized; PB, phosphate buffer; pCREB, phosphorylated CREB; STA-HRP, streptavidin horseradish peroxidase.

Received August 3, 2004.

Accepted for publication November 15, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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