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Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, Texas 77555
Address all correspondence and requests for reprints to: Jennifer L. Armstrong, Department of Anatomy and Neurosciences, MRB 10-104, 303 University Boulevard, University of Texas Medical Branch, Galveston, Texas 77555. E-mail: armstrong{at}mbian.utmb.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EGF may regulate pituitary cells via paracrine or autocrine mechanisms. Previously, we demonstrated that EGF messenger RNA (mRNA) is produced by subsets of all of the pituitary cell types, including gonadotropes (7). Another group has shown that subsets of all pituitary cells will release EGF in reverse hemolytic plaque assays (8, 9). In a study of immature pituitary cells, Mouihate et al. (10) showed that most (72%) of the EGF-secreting cells were LH antigen-bearing gonadotropes and that the secretion of EGF could be enhanced by 10 nM GnRH. Collectively, these data suggest that EGF is a paracrine or autocrine mediator that may be regulated by key reproductive hormones. To further support the hypothesis for autocrine mechanisms, Mouihate and Lestage reported data that showed that most (73%) of the EGF secreting cells also express EGF receptors (11).
Recent studies in this laboratory have characterized the distribution of GnRH or EGF receptors and changes in their expression during the estrous cycle (7, 12, 13, 14, 15). Dual-labeling studies showed that subsets of all pituitary cell types expressed GnRH or EGF receptors (7, 12, 13, 14, 15, 16). Furthermore, the changes in EGF receptor expression by gonadotropes were similar to those reported previously for GnRH (13, 14, 15, 16). There is an increase in gonadotropes that bind EGF (12) or GnRH (13, 14, 15, 16) from metestrus to the morning of proestrus. The increase in GnRH receptive cells agreed with results from radioreceptor assays (reviewed in Refs. 13–16). After the proestrous peak, there is a decline in EGF or GnRH receptor expression by gonadotropes to reach a nadir by estrus (12, 13, 14, 15, 16).
These parallel changes suggested that common regulatory factors might control expression of both sets of receptors. We have already shown that the increase in GnRH receptors could be partially mimicked by treating cells from metestrous or diestrous rats with estradiol for 18–24 h (13). It could be completely mimicked by treating diestrous rat pituitary cells with activin (15). Similarly, the decline seen during the afternoon of proestrus could be mimicked by estradiol (13) or inhibin (16) pretreatment of cells taken during the morning of proestrus (13).
Thus, we hypothesized that the same regulatory factors might be involved in the expression of EGF receptors. The present studies applied the same experimental design used for the studies of GnRH receptors (13, 14, 15) to learn if estradiol or activin would affect expression of EGF receptors. The results of the first set of experiments showed that neither estradiol nor activin stimulated expression of EGF receptors by gonadotropes from metestrous or diestrous rats. This study presents these results along with evidence that EGF itself may be one of the regulators for increased expression of EGF receptors by gonadotropes.
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Materials and Methods |
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Collection and growth of pituitary cells
The anterior pituitary glands were collected in the morning and
rapidly placed in cold DMEM (JRH Biosciences, Lenexa, KS) containing
0.3% BSA (Sigma Chemical Co., St. Louis, MO), 1.8 g/500 ml HEPES
(Sigma), and 24.65 ml/500 ml sodium bicarbonate (JRH Biosciences). In
order to prevent bacterial growth, 1 µl/100 ml gentamicin (Sigma) was
used. The dissociation protocol was performed as reported previously
(7). Cells were tested for viability by the trypan-blue dye exclusion
test. The protocol produced 2–3 million cells/pituitary that were 98%
viable. Cells were suspended in DMEM containing 0.005 mg/ml insulin
(Sigma), 0.05 mg/ml transferrin (Sigma), and 0.001 mM
sodium selenite (Johnson Matthey Chemical Ltd., New York, NY).
Experimental design
Group 1: This group of experiments was designed to test the
hypothesis that estradiol or activin might be among the regulatory
factors that affect the expression of EGFR by gonadotropes. In
addition, because estradiol or activin might work indirectly, by first
increasing GnRH receptors, we added experiments that included exposure
to GnRH during the last 4 h of treatment.
Cells were plated on glass coverslips (Thomas Scientific, Suredesboro, NJ) that had been coated with poly-D-lysine (Sigma) in a 24-well tray at a density of approximately 40,000–50,000 cells/50 µl/well. They were placed at 37 C for 1 h and then fed with the same DME containing 10% FBS (JRH Sciences). The cells were grown overnight (18 h) in this media with or without 100 pM ß-estradiol 3-benzoate (Sigma). A second set of cultures were grown with or without 60 ng/ml activin (Genentech, San Francisco, CA). After the overnight plating, one half of each set of cultures was stimulated with 1 nM GnRH (Peninsula Laboratories, Inc., Belmont, CA) for 4 h at 37 C.
Group 2: This experiment was designed after the results from the study of activin, estradiol and GnRH showed that none of the regulatory hormones stimulated increases in EGF receptor expression by gonadotropes. However, serum-containing media alone increased percentages of cells with EGF and gonadotropins in estrous and metestrous rats. Therefore, the second group of experiments was designed to test the hypothesis that EGF or other factors in serum might promote the expression of EGF receptors. Cells from metestrous rats were grown 18 h in DME with or without serum. Another group of cultures also received media with serum and a 1:100 dilution of anti-EGF (rabbit antimouse, Upstate Biotechnology Inc., Lake Placid, NY). Finally, one-half of the cultures grown in serum-free media received 10 ng/ml EGF during the culture period. This dose was chosen following dose-response tests. The cultures were then fixed in 2% glutaraldehyde for 30 minutes followed by washing for 1 h with 4.5% sucrose. Cells were then stored in the refrigerator until used for immunocytochemistry.
Immunocytochemistry
Single and dual-immunolabeling with EGF receptor and LH or FSH
was performed on the coverslips, as previously described (7, 12, 13, 14, 15).
Briefly, cells were exposed to a 1:1000 dilution of anti-EGFR overnight
at room temperature. The antibody binding sites were detected by the
DAKO (Carpinteria, CA) rapid immunoperoxidase kit using a biotinylated
horse antimouse IgG, streptavidin peroxidase, and nickel-intensified
diaminobenzidine. This gave a blue-black reaction product. The
pituitary gonadotropins were each detected in separate protocols. The
cells were incubated for 2 h at 37 degrees with 1:30 K antibovine
LH-beta or 1:10 K antihuman FSHß. Heterologous antisera were used to
prevent reactions from contaminating antibodies against 
-subunits,
which are unique when different species are compared. The gonadotropin
antibodies were detected with the DAKO rapid kits which utilized a
biotinylated goat antirabbit IgG, streptavidin peroxidase and
orange-amber diaminobenzidine as the detection systems.
The EGF receptor antibody was a monoclonal antibody (E3138, Sigma) raised against the intracellular domain of the EGF receptor. Specificity tests and controls have been reported previously (7). These were done both by preabsorption of the antiserum with the intracellular domain fragment as well as tests of reactivity of the antiserum in a dot blot assay. Preabsorption of the anti-EGFR antibody abolished labeling.
Anti-bovine LHß was a gift from Dr. J. G. Pierce and antihuman FSHß was provided by Dr. A. F. Parlowe and the Pituitary Hormone Distribution Program (NIH). Both antisera have been tested repeatedly in tests that involved preabsorption of the antisera with either LH or FSH. The homologous antigens neutralized labeling with the antibody. Cross-absorbing each antiserum with the other gonadotropin had no affect on immunolabeling.
Analysis of immunolabeled cell populations
One to two rats per stage of the estrous cycle were collected
for each experimental group. The experiments were repeated until 6–9
rats were collected. Therefore, each experiment was repeated at least
three times. The final averages reported are of data from each of the
repeated experiments (n = 3–5).
Data points for each experiment were collected by counting cells plated on three coverslips. The counts were done at 40x. Fields were randomly selected and the first 100–250 cells encountered were counted and analyzed. The raw counts of cells with single or dual labels were inserted into an Excel template which automatically calculated the percentage of cells labeled for each antigen as well as the percentage of dual labeled cells. This allowed us to constantly monitor the dual labeling to ensure that it did not interfere with the detection of either antigen. The percentages from the three coverslips provided an average for that experiment. These values were then averaged to produce the final data point (n = 3–5 experiments). To determine significance in a group of experiments, 1x ANOVA or 2x ANOVA tests were performed. If the f value was significant (P < 0.05), individual differences were then detected by the Fisher’s least significant difference (LSD) post-hoc test.
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Results |
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In the present studies, the percentages of proestrous pituitary cells
with LH or FSH and EGFR remained the same over the 18-h culture period.
However, the serum containing media alone caused a 1.8- to 2.25-fold
increase in percentages of metestrous or estrous cells with EGFR and
LH. Similarly, serum-containing media increased percentages of cells
with FSH and EGFR 2 fold in estrous, metestrous or diestrous rats.
[Note: As previously reported (12), 1 h after culture, LH cells
with EGFR were 2.66 ± 0.6% (metestrus), 2.7 ± 0.6%
(estrus), and 5.9 ± 0.9% (diestrus) of pituitary cells, and FSH
cells with EGFR were 4 ± 0.6% (metestrus), 3.8 ± 0.8%
(estrus), and 3.8 ± 0.6% (diestrus) of pituitary cells.]
Because of the changes during the culture period, the 18-h cultures
showed no significant differences in expression of EGF receptors by
gonadotropes when the different stages of the cycle were compared
(Figs. 1
and 2). Also, analysis of the percentages of
LH or FSH cells showed that the 18-h culture period did not increase
percentages of gonadotropes. They were within the range reported in
previous studies (17, 18, 19, 20, 21).
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and 2 also show that GnRH did not affect percentages of
pituitary cells with LH and EGFR if it was given during the last 4
h of culture. It did stimulate percentages of cells with FSH and EGFR
in estrous rats (Fig. 2) when compared with 1 h cultures (12).
However, the values were not different from those in the control
cultures.
Figures 3 and 4 show the effects of estradiol on
expression of EGFR by LH or FSH gonadotropes. Estradiol had no
stimulatory effects on percentages of pituitary cells with EGFR and
gonadotropins during any stage of the cycle. This is in striking
contrast to estradiol’s stimulatory effects on expression of GnRH
receptors by cells from metestrous or diestrous rats (13). Furthermore,
in spite of the fact that cells with GnRH receptors are elevated
following estradiol treatment of metestrous and diestrous populations
(13, 14, 15), GnRH treatment during the last 4 h of culture did not
stimulate more LH or FSH gonadotropes with EGF receptors. Finally,
parallel tests of activin modeled after recently published studies (15)
showed that it also did not stimulate expression of EGF receptors when
added with or without GnRH (data not shown). Two-way ANOVA tests showed
no significant differences among activin treated and untreated groups
(P < 0.05) in all stages of the cycle studied.
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shows that estradiol decreased the percentages of
pituitary cells with LH and EGFR in proestrous rats. This is consistent
with its known effects on GnRH receptors in this same group (13). It
did not affect percentages of cells with FSH and EGFR in proestrous
rats (Fig. 4). However, estradiol caused a significant reduction in
cells with FSH and EGFR in diestrous rats (Fig. 4).
Regulatory effects of EGF on expression of EGFR by gonadotropes
Because of the media effects on gonadotropes early in the cycle
(Figs. 1 and 2), the second group of experiments focused on metestrous
rat populations. The experiments were designed to test the hypothesis
that EGF might be one of the regulatory factors responsible for
up-regulation of EGFR by gonadotropes.
Figure 5 shows that removal of serum from the media did not block the
increase in percentage of cells with LH and EGFR over the 18-h culture
period. As shown in the previous experiments (Fig. 1), serum caused a
2.4-fold increase in LH cells with EGFR when compared with the 1-h
cultures (2.66 ± 0.6% of pituitary cells have LH and EGFR in the
1 h cultures, 12). However, Fig. 5
shows that, even if serum was removed, there was still a 2.2-fold
increase in percentages of pituitary cells with LH antigens and EGFR
after 18 h of culture.
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Finally, Fig. 5 also shows that adding EGF to metestrous cells for
18 h increased pituitary cells with EGFR and LH or FSH 1.4- to
1.5-fold. The resulting percentages are similar to those seen during
peak expression periods in proestrous rats (Figs. 1 and 2). Counts of
immunolabeled LH and FSH cells showed that this change did not coincide
with increased percentages of LH- or FSH-bearing gonadotropes.
Figures 6–8 illustrate the effects of EGF on EGF receptor expression
by the population of LH cells from metestrous rats. Figure 6 and 7 are
lower magnifications to show the changes overall in the cells
populations. The insets show examples of both single and
dual-labeled cells. The most striking morphological change seen in the
EGF treated cells is the extension of processes. Even the normally
round or ovoid gonadotropes have extended processes and become more
stellate. Higher magnifications are shown in Fig. 8, a and b, to illustrate the dual
labeling more clearly. The EGF receptor labeling is in patches or a
linear pattern on the cells (seen as dense blue-black patches in
color), whereas the labeling for the antigens is more diffuse and gray
(seen as orange-amber in color).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because estradiol and activin appear to be primary stimulators of the expression of GnRH receptors, these studies used the same experimental design that showed estradiol (13) and activin (15) up-regulation of GnRH receptors during metestrus and diestrus. Estradiol pretreatment also mimicked the decreases in GnRH receptive cells seen during proestrus (13). Experiments with GnRH were performed (during the last 4 h of the experiment) to learn if the changes in EGF receptor expression might be secondary to the up- or down-regulation of GnRH receptors.
The results of the first group of experiments showed that the up-regulation of EGF receptors in gonadotropes appears to be mediated by factors other than those that increase expression of GnRH receptors. Neither activin nor estradiol increased percentages of pituitary cells with LH or FSH and EGFR. GnRH also had no stimulatory effect in most experimental groups, even if cells were stimulated to promote expression of GnRH receptors. The one group that showed a GnRH-mediated change were the FSH cells from estrous cell populations. GnRH stimulated an increase in percentages of cells with FSH and EGFR above the values seen after only 1 h (12). The values were not greater than those after 18 h in media alone, however.
The estradiol mediated decline in percentages of pituitary cells with LH and EGFR seen in proestrous populations was similar to that of GnRH receptors. In recent studies of inhibin-treated populations, we have also shown that inhibin reduces the expression of GnRH receptors by proestrous rat gonadotropes (16). Future studies are needed to test possible effects of inhibin on gonadotrope expression of EGF receptors.
In contrast, estradiol had no effects on percentages of cells with FSH and EGFR in proestrous populations. However, estradiol did reduce percentages of pituitary cells with FSH and EGFR in diestrous rats. These data are difficult to explain in light of the general positive feedback effects of estradiol seen normally during diestrus (13).
Differential expression of EGFR by LH and FSH cells
A common theme found in this and the previous (7, 12) studies is
the nonparallel expression of EGFR when LH and FSH gonadotropes are
compared. In the first study (7), we showed that cold stress increased
expression of EGFR by FSH cells only. It had no effect on LH cells. The
more recent studies (12) showed a similar increase in expression of
EGFR in antigen-bearing LH or FSH cells from estrous to proestrous.
However, the increase in percentages of cells with FSH and EGFR
occurred later than that with LH and EGFR. Furthermore, when LHß and
FSHß mRNAs were detected by in situ hybridization in metestrous
populations, proportionately more cells with EGFR and LHß mRNA were
found. (Fifty percent of total metestrous LH cells contained EGFR
compared to only 25% of total FSH cells.) Collectively, these data
suggested that EGFR may be produced earlier in the cycle by LH cells.
Furthermore, we suggested that EGFR was produced initially by a
population of monohormonal LH cells (12).
The present studies find more examples of non-parallel expression of EGFR by LH and FSH gonadotropes. In diestrous populations, serum containing media increased the percentages of pituitary cells with FSH and EGFR while having no effects on cells with LH and EGFR. Removal of serum or adding anti-EGF sera to the media abolished the serum-mediated increase in EGFR expression by metestrous FSH cells, whereas it had little effect on EGFR expression by metestrous LH cells. These data suggest that different factors in serum may regulate increases in EGFR in FSH cells. Finally, as stated in the previous section, estradiol also differentially regulated EGFR in LH and FSH cells. It had potent effects on expression of EGFR by LH cells in proestrous rats and FSH cells in diestrous populations.
Collectively, these data point to EGFR expression by a subset of monohormonal gonadotropes. These represent 25–50% of the gonadotrope population depending on the stage of the cycle. They are also more numerous in estrus and metestrus (17, 18, 19, 20, 21). Often these cells are small and considered developmentally immature. However, sometimes they can be found among the largest gonadotropes (21). The presence of EGFR may be a marker for monohormonal gonadotropes that are either immature or subserving a different function in the population. They may have different sensitivities to regulatory factors, like estradiol or EGF. Further tests are needed to learn their significance. Finally, we recognize that other factors, in addition to EGF could be involved in stimulating the expression of EGF receptors during early maturation. This could include factors produced by pituitary cells themselves. Further studies would be needed to identify them.
Functions for EGF in gonadotrope regulation
The changes in expression of EGF receptors during the cycle
suggests that they may be used by gonadotropes to modulate or mediate
functional responses. There is evidence in the literature that EGF may
be both a differentiating factor and a secretagogue for gonadotropes.
Przylipiak et al. (22) reported that EGF enhanced the
secretion of LH in dispersed monolayers of pituitary cells as well as
in superfusion columns. The stimulatory effects were both dose and time
dependent. Miyake et al. (5) found an increase in LH
secretion when EGF was administered in a sequential double chamber
perifusion system. They used the medial basal hypothalamus in one
chamber in series with another chamber containing anterior pituitary
quarters. The group reported increased LH release when EGF was added
with estradiol.
As stated in the introduction, EGF may actually function via autocrine or paracrine mechanisms. Studies by Mouihate et al. reported EGF release by gonadotropes and other cell types in a number of physiological states related to the reproductive system (11). Three sets of findings suggest that EGF may work via autocrine mechanisms. First, in lactating rats, nearly 3/4ths of EGF-secreting cells also expressed EGF receptors. Second, in immature female rats, over 70% of EGF secreting cells were LH gonadotropes (9). Third, GnRH stimulated more EGF secretion from individual cells which points once again to a gonadotrope source.
Mouihate et al. (8) also reported differences in EGF secretion with the stage of the cycle (8) that correlate with changes in expression of EGFR in our studies (12). Cells from proestrous rats secreted higher amounts of EGF than those from metestrous rats. However, unlike our studies of EGFR, estradiol stimulated increases in the metestrous group to levels similar to those from proestrous populations (8).
These data provide clues to an EGF regulatory circuit. This may begin with an increase in estradiol receptors during diestrus (23, 24). This is followed by estradiol stimulation of the production of GnRH receptors (reviewed in Ref.13). Both estradiol and GnRH may stimulate secretion of EGF (8, 9). Evidence from the present study suggests that EGF itself may increase expression of EGF receptors. Thus, estradiol and GnRH may work indirectly through EGF to elevate EGF receptors.
To summarize, these data suggest that EGF may stimulate the production of its own receptor by gonadotropes early in the cycle. GnRH appears to have little if any direct effect on expression of the EGF receptors by either LH or FSH cells. Estradiol has a negative feedback effect on EGF receptor expression by proestrous LH gonadotropes. The fact that estradiol can inhibit expression of EGFR during proestrus points to it as a potential regulatory hormone for the receptor later in the cycle. However, the same feedback effects are not seen on FSH gonadotropes. This supports the hypothesis that the target cells are monohormonal LH cells.
Nonparallel changes in the expression of EGF receptors by LH and FSH cells seen in this and the previous studies (7, 12) suggest expression of EGFR by monohormonal subtypes that have different periods of sensitivity to EGF stimulation or estradiol regulation. In past studies, the monohormonal subtypes have been characterized as small and relatively immature (17, 18, 19, 20, 21). They may comprise a population of gonadotropes that are being readied for a future cycle. Alternatively, they may serve a different function on the population. Perhaps the EGFR modulate their development or responsiveness to secretagogues.
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Acknowledgments |
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Footnotes |
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Received June 6, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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messenger ribonucleic acids and their
receptors in the rat anterior pituitary: localization and regulation.
Endocrinology 136:2284–2293[Abstract]
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