ARTICLE |
Correspondence to: Gwen V. Childs, Dept. of Anatomy and Neurosciences, MRB 10-104, 303 University Blvd., U. of Texas Medical Branch, Galveston, TX 77555.
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Summary |
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C-fos is an early expression oncogene that can be stimulated by a variety of regulators. It is expressed by subsets of all pituitary cells, with increased expression seen in proestrous rats. However, in freshly dispersed pituitary cells studied during different stages of the cycle, there is limited expression of fos by luteinizing hormone (LH) cells and little basal expression by cells with follicle-stimulating hormone (FSH) antigens. Proestrus is a time during which pituitary gonadotropes express peak levels of receptors for gonadotropin-releasing hormone (GnRH) and epidermal growth factor (EGF). We hypothesized that if GnRH or EGF stimulated fos activity in gonadotropes they would be most effective during the peak expression of their receptors. Anterior pituitaries were removed, cut into small pieces, and stimulated for 30 min. Total RNA was then collected and analyzed by Northern analysis. Both EGF and GnRH caused an increase in c-fos mRNA levels in the anterior pituitary gland compared with unstimulated pituitary glands assayed immediately after removal from the pituitary. However, the stimulatory effects were no greater than those seen with medium alone. This suggested that fos expression could be stimulated by local factors either in the pituitary or the medium itself. The second phase of the study focused on pituitary cells plated for 1 hr and then stimulated with EGF and GnRH for 15 min. Dual immunocytochemistry was done to learn which cell types expressed the fos proteins. After 15 min, EGF and GnRH both increased the percentages of fos-bearing cells above levels seen in medium alone. EGF stimulated fos proteins in subsets of FSH, adrenocorticotropin (ACTH), and growth hormone (GH) cells. GnRH increased fos proteins in subsets of ACTH and GH cells. These results suggest that EGF and GnRH may regulate fos expression, but not necessarily in gonadotropes. They also highlight the need for carefully timed experiments because endogenous factors in the pituitary itself may stimulate immediate early gene expression. (J Histochem Cytochem 46:935943, 1998)
Key Words: epidermal growth factor, gonadotropin-releasing, hormone, gonadotrope, immunocytochemistry
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Introduction |
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C-fos and c-jun are early expression proto-oncogenes that are expressed in low levels in most cells. They are called primary or immediate early response genes. The fos and jun proteins join to form the AP-1 protein. This protein can then bind to the AP-1 binding site on DNA to induce transcription of various genes (
Gonadotropin-releasing hormone (GnRH) is an important regulator of fos activity in both the brain and the pituitary (T3-1 gonadotrope cell line. Recent studies in our laboratory confirm that c-fos is expressed by gonadotropes, particularly those bearing luteinizing hormone-ß (LHß) antigens or mRNA (
Another regulatory factor for early gene expression is epidermal growth factor (EGF). . This group showed stimulation of c-fos mRNA within 30 min, and fos protein was induced within 1 hr.
We and other workers have shown that EGF may play an important role in the stimulation of gonadotropes during the estrous cycle (
Furthermore, expression of EGFR could be regulated by EGF itself, which is a product of subsets of pituitary cells including gonadotropes and growth hormone (GH) cells (
EGF's functions in the gonadotrope population appear to involve differentiation events (such as production of EGF receptors;
To test these hypotheses, pituitary cells were taken at the time in the cycle when EGF or GnRH receptors were at a peak (proestrous AM). Dual immunocytochemistry was employed to determine the identity of the cells with fos proteins and each of the pituitary hormones. Northern analysis was utilized to determine if either EGF or GnRH stimulated an increase in c-fos mRNA in pituitaries taken from proestrous rats. This article reports the results of this investigation.
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Materials and Methods |
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Animals and Collection of Anterior Pituitary Glands
Female SpragueDawley rats (HarlanSprague Dawley; Houston, TX) weighing 250300 g were housed three per cage under artificial illumination between 0600 and 1800 hr. The rats were given food and water ad libitum. They were acclimated for 710 days before the start of daily vaginal smearing, which was performed to determine the stage of the cycle. The females underwent at least two complete normal 4- or 5-day estrous cycles before they were used. The animal care and use protocol was approved annually by the Institutional Review Committee. All animals were sacrificed between 0900 and 1000 hr. The animals were removed from their housing room and sacrificed by decapitation in a separate, adjoining room within seconds of removal from the cage. The same animal handler performed these rapid procedures, which were approved by the Animal Care and Use Committee.
Anterior pituitary glands were dissected from the animal and rapidly placed in cold Dulbecco's modified Eagle's medium (DMEM) (JRH Biosciences; Lenexa, KS) containing 0.3% bovine serum albumin (BSA; Sigma, St Louis, MO), 1.8 g/500 ml HEPES (Sigma), and 24.65 ml/500 ml sodium bicarbonate (JRH Biosciences). To prevent bacterial growth, 1 µl/100 ml gentamicin (Sigma) was used.
Collection of Total RNA
For the assays of c-fos mRNA, the pituitaries were washed three times with DMEM and then chopped into smaller pieces. The anterior pituitary pieces were then stimulated for 30 min with 10 ng/ml EGF (Upstate Biotechnology; Lake Placid, NY), 100 nM GnRH (Peninsula Laboratories; Belmont, CA), or DMEM alone. The DMEM used at this time contained identical reagents as listed above as well as 1 µg/50 ml ascorbic acid (Sigma). In addition, some RNA extractions utilized DMEM with these additives: 0.005 mg/ml insulin, 0.05 mg/ml transferrin, and 0.001 mM sodium selenite. Stimulation occurred at 37C in a CO2-controlled environment. Total RNA was isolated by the single-step method (
Northern Analysis
Northern blot analysis was carried out as previously described (
Total RNA (20 µg) collected from the anterior pituitary was separated on a 1% agarose gel (Gibco). The RNA was transferred from the gel onto a Nytran membrane (Schliecher & Schuell Maximum Strength Nytran Plus; Keene, NH). Once the transfer to the Nytran membrane was complete, the membrane was UV-crosslinked (Ultra Lum UVC 515 Ultraviolet Multlinker), dried for 1 hr, and prepared for prehybridization. The prehybridization and hybridization buffer consisted of 2 x Denhardt's solution (Sigma), 50% formamide, 0.5% SDS, 5 x SSC, salmon sperm DNA, and DEPCH2O.
The DNA probe was labeled using a Prime It-II Random Primer Labeling Kit (Stratagene; La Jolla, CA). Briefly, the c-fos DNA insert (a generous gift from Dr. Tom Curran, St. Jude Children's Research Hospital, Memphis TN) was mixed with DEPCH2O and random primers. Unincorporated nucleotides were removed by chromatography on NucTrap columns (Stratagene). Salmon sperm DNA was mixed into the solution and the radioactivity of the probe was quantified. The probe was added to the prehybridization container and the membrane was hybridized overnight at 42C.
The following day the membrane was washed with 1 x SSC and 0.1% SDS in DEPCH2O at 42C and then placed in the autoradiography cassette with two intensifying screens and film (Sigma) at -70C. The film was developed the next day.
Dissociation of Pituitary Glands
For the plated cultures, pituitary glands were dissociated enzymatically and mechanically as described previously (
Immunocytochemical Studies
Anterior pituitary cells were collected and dispersed as mentioned above. Coverslips were single labeled for c-fos protein or dual labeled for c-fos protein and one of the six pituitary hormones. Single and dual immunochemical techniques have been described previously (
Control labeling protocols were performed for both the single label for c-fos protein and the dual label with c-fos protein and pituitary hormones. For both primary antisera, the antibodies were omitted from the diluent buffer and the coverslips were incubated in buffer alone. In addition, the antisera had been preabsorbed with each antigen as previously described (
Statistical Analysis
One or two rats were collected at a time for each experimental group until there were 68 rats/group. All rats were taken on the morning of proestrus because of the peak expression of EGF receptors (
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Results |
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Regulation of c-fos mRNA by EGF and GnRH
The Northern analyses were done on pituitary pieces that were freshly removed from the animals as well as those that had been incubated in DMEM alone or DMEM containing GnRH or EGF for 30 min. C-fos mRNA was undetectable in pituitary pieces immediately after removal. However, a 30-min incubation in defined medium with or without 10 ng/ml EGF or 100 nM GnRH increased c-fos mRNA levels. There were no significant differences when the effects of medium alone, EGF, or GnRH were compared (data not shown). The defined medium contained the following additives: insulin, transferrin, sodium selenite, and bovine serum albumin (0.25%). The BSA was needed because at least two of the pituitary cell types will not respond to secretagogues without it. To test possible effects of insulin, transferrin, or sodium selenite, all additives except the BSA were removed. Incubation in the remaining medium continued to stimulate the same increment in expression of c-fos mRNA. Neither EGF nor GnRH increased expression beyond that of the medium alone (data not shown).
Fos Stimulation by EGF and GnRH in Total Pituitary Cell Populations
The dissociation protocol took 1 hr and the resulting pituitary cultures were plated for an additional hour. Therefore, in view of the endogenous increases in fos activity seen in the previous group of experiments, it was not surprising to detect fos proteins in these 2-hr cultures. After 1-hr plating in defined medium, 26.3% of pituitary cells expressed fos proteins.
After an additional 1530 min in DMEM alone, the values did not change. Figure 1A illustrates a field showing baseline fos expression in several pituitary cells after 1-hr plating and an additional 15 min in medium alone. Note that the density of the labeling varies from cell to cell.
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Figure 2 shows the changes seen after EGF or GnRH exposure. After only 15 min of incubation in EGF or GnRH, the percentages of pituitary cells with fos proteins were increased significantly to 34.2% and 31.8%, respectively. No further stimulation was seen after 30 min. These values are significantly higher than those seen for the DMEM controls. Figure 1B and Figure 1C illustrate the increased percentages of cells labeled for fos in EGF- (Figure 1B) or GnRH- (Figure 1C) treated cultures.
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Fos Stimulation Within Specific Cell Types in the Pituitary Gland
The dual labeling identified the specific cell types affected by EGF or GnRH. EGF treatment significantly increased fos expression in cells bearing the following antigens: FSH (1.23.7% of pituitary cells), ACTH (1.75.3% of pituitary cells), or GH (6.313.6% of pituitary cells) (Figure 3). EGF treatment had no effect on fos-immunolabeled cells that also contained LH, TSH, or prolactin antigens.
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Figure 3 also shows that GnRH treatment significantly increased fos expression within each of the following antigen-bearing cells: ACTH (1.84.4% of pituitary cells) and GH (6.310.4% of pituitary cells). The other four pituitary cell types did not increase fos expression compared with the control levels. Surprisingly, GnRH stimulation had no effect on gonadotropes.
Figure 4 illustrates these data. Figure 4A and Figure 4B show different magnifications of nonstimulated pituitary cells. A cluster of cells from Figure 4A is shown, at higher magnification, in Figure 4B. The cluster includes orange-labeled growth hormone cells (GH). Note that fos expression is seen in the nucleus of one of the GH cells in this cluster. The remaining GH cells do not appear to express fos, although fos is expressed by non-GH cells in the field (Figure 4A and Figure 4B).
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Figure 4CD show fos expression after stimulation by EGF. The lower magnification shown in Figure 4E illustrates two clusters, each of which shows a GH cell with fos expression. Cluster 1 is magnified in Figure 4C and cluster 2 is magnified in Figure 4D to better visualize the dual labeled GH cells.
Finally, Figure 4F and Figure 4G show the effects of EGF on FSH gonadotropes. A cluster of cells is shown in Figure 4F which was taken from a control (DMEM only) population. One of the FSH cells expresses fos proteins. After EGF stimulation, Figure 4G illustrates the increased expression of fos by FSH cells. The FSH labeling is also denser as a result of EGF stimulation.
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Discussion |
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Our recent cytochemical studies showed that fos protein activity is highest in cells from proestrous rats. However, the dual labeling evidence indicated that most of the fos-bearing cells in this population are not gonadotropes (
Activation of c-fos occurs in a very short time frame, as shown by these and other experiments (
Endogenous Regulation of c-fos mRNA Expression
The present studies raised questions about the events that occur as the pituitaries are removed from the animal. This affects interpretation of data from assays of fos mRNA or proteins. When we assayed pituitary pieces immediately after removal, no c-fos mRNA was detected. However, after 30 min of incubation in medium alone, c-fos mRNA was activated. Neither GnRH nor EGF stimulated c-fos mRNA levels above the background levels seen with this defined medium. Similarly, additional experiments in medium without insulin, sodium selenite, and transferrin also showed that medium alone continued to be a stimulant for c-fos expression. Therefore, under these experimental conditions we were unable to separate possible stimulatory effects of GnRH and EGF on c-fos mRNA from effects seen after removal from the pituitary and incubation in a defined medium.
These data suggest that c-fos mRNA is stimulated by local factors, possibly in response to the stress associated with pituitary removal from the body. Locally produced EGF or other growth factors by pituitary cells themselves are candidates for regulation of this response. Therefore, to show fos stimulation by any growth factor, one must define the basal conditions carefully and recognize that exogenous stimulation may be superimposed on endogenous stimulation by resident growth factors. The pituitary will undoubtedly respond to its removal from the influence of the hypothalamus, and early gene expression may mediate that response.
Identity of the fos-producing Cells
The findings from the Northern analysis suggested that one must be cautious about interpreting data from any in vitro studies. Nevertheless, the studies by
In the studies by
Therefore, we knew that GnRH could stimulate fos. The finding that it was stimulatory in populations of a gonadotrope cell line led to the hypothesis that it stimulated fos production in normal gonadotropes as well. Therefore, one objective of our in vitro studies was to test this hypothesis and determine which cell types might be responding to GnRH with fos production. Furthermore, since GnRH and EGF receptors peak at the same time (proestrous AM), we hypothesized that the gonadotropes may be acutely sensitive to effects of EGF or GnRH early in proestrus. This would be an ideal time to test EGF's effects on fos proteins.
In these experiments, we also wanted to reduce the time between removal from the animal and fixation to that needed for dissociation (1 hr) and plating (1 hr), to reduce the time during which endogenous factors may be stimulating fos. When we compared cells fixed at the end of the plating period with those fixed after the 15 min needed for the stimulation, medium alone did not produce increases in fos proteins as detected in immunocytochemical tests. Therefore, a baseline was established above which the effects of stimulatory factors could be detected. Both GnRH- and EGF-treated cultures showed 3040% increases in the percentages of cells expressing fos proteins overall in the pituitary (over those produced by medium alone) (Figure 1).
However, our studies showed some surprising results about the identity of the fos-producing cells. GnRH stimulated fos expression in ACTH, GH, and prolactin cells but did not stimulate fos production in proestrous gonadotropes. EGF stimulated fos expression in FSH cells, ACTH cells, and GH cells, cells known to possess EGF receptors (
Effects of GnRH on fos Proteins
There is no doubt that gonadotropes express the c-fos early response gene and that GnRH stimulates expression of c-fos mRNA or fos proteins in the anterior pituitary (
The GnRH-mediated increase in fos expression appears to be in either GH cells (which may become transitional gonadotropes;
GnRH receptors are not found on corticotropes. However, GnRH actions could also be mediated indirectly by paracrine interactions. Further evidence for paracrine interactions modulated by GnRH can be seen in studies by
Finally, one possible paracrine factor that might mediate the GnRH action on these cells is angiotensin II. Gonadotropes have been shown to release angiotensin II (
Effects of EGF on fos Proteins
As stated above, corticotropes do not have GnRH receptors. However, they do have receptors for EGF (
In addition to its effects on corticotropes, EGF increases fos expression in GH cells and FSH cells. The increase in percentages of FSH cells with fos brings the final values to levels similar to those expressed by LH cells. Therefore, it is possible that EGF is selectively stimulating fos expression by LH cells, leading to translation of FSH (monohormonal LH cells are becoming bihormonal). This hypothesis is supported by the cytochemical data showing increased labeling for FSH in EGF-treated cells (Figure 4G). Alternatively, EGF could be acting on a population of monohormonal FSH cells to prepare them for the proestrous or estrous surge secretory activity.
Summary and Conclusions
To summarize, both EGF and GnRH may regulate immediate early gene expression in key pituitary cells, either directly or indirectly via paracrine factors. The dual cytochemical evidence showed the identity of the target cells and the fact that multiple cell types may be stimulated. Whereas this study showed that the in vitro studies are valuable in that they enable one to test individual secretagogues or growth factors in a system that removes the cells from other regulators (such as steroids or other serum influences), they must also be interpreted with caution. The Northern blot analyses of fos mRNA in the anterior pituitary gland assayed within 30 min of removal from the gland showed significant increases in expression of c-fos in medium containing virtually no exogenous growth factors. Therefore, local paracrine or autocrine regulators may also play a role in regulating early gene expression. This background regulation must be considered in designing the in vitro experiments.
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Acknowledgments |
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Supported by NSF IBN 9724066 and NIH R01 HD 33915.
We thank Ms Diana Rougeau and Geda Unabia for excellent technical assistance with this work. We also thank Drs Gregg Nagle and Xuemo Fan for help with the Northern Analysis.
Received for publication December 29, 1997; accepted April 15, 1998.
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