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From the Department of Anatomy (G.V.C.), University of Arkansas for Medical Science, Little Rock, Arkansas 72205-7199; and the Department of Anatomy and Neuroscience (G.U.), University of Texas Medical Branch, Galveston, Texas 77555
Address all correspondence and requests for reprints to: Gwen V. Childs, Ph.D., Department of Anatomy, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 510, Little Rock, Arkansas 77205. E-mail: childsgwenv{at}uams.edu
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Abstract |
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Introduction |
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A number of studies have been initiated to learn more about the function of EGF receptors in specific target cells in the pituitary. Recent studies by Leblanc et al. (7) showed that EGF potentiates the responsiveness of gonadotropes to GnRH. These workers suggested that EGF might unmask cryptic GnRH receptors. It is also possible that EGF might recruit additional cells that express GnRH receptors either by differentiation (8) or by a mitogenic effect. EGF may work in partnership with GnRH to effect these changes in the population. Recently, Chaidarun et al. (9) reported evidence for EGF-stimulated tritiated thymidine uptake in sheep pituitary cultures that were over 50% gonadotropes. EGF had no significant effect on gonadotropin subunit gene expression in this population.
Mitotic activity may help add gonadotropes to the proestrous cell population (1, 2, 3, 4). Hunt and Hunt have reported that female rats renew their cell populations 2x faster than male rats (10). Furthermore, there is increased uptake of tritiated thymidine during estrus when compared with other stages of the cycle. This coincides with the period of highest expression of EGFR by pituitary cells (5). However, are these mitotic cells differentiated gonadotropes? In the study by Chaidarun et al. (9), the mitotic cells could have come from the nongonadotropes, which comprised 40% of the cell cultures. In fact, in earlier studies by Sakuma et al. (11), the most numerous mitotic cells were labeled for PRL or GH. Finally, our recent dual labeling evidence showed that only a small percentage of the EGF target cells in metestrous and estrous populations (10–15%) could actually be identified immunocytochemically as gonadotropes (5).
Collectively, these previous studies suggest that EGF may act on undifferentiated cells that have the potential to become gonadotropes. The present studies were designed to test this hypothesis. The objective of the studies in this presentation was to learn whether EGF was mitogenic for differentiated gonadotropes. The studies used populations of GnRH-responsive gonadotropes enriched to >90% by centrifugal elutriation testing EGF’s stimulatory effects on responses in MTT cell growth/cell death assays. Because the MTT assays also detect effects on cell survival, we added cytochemical tests of bromodeoxyuridine (BrDU) uptake during DNA synthesis. After allowing BrDU incorporation into the nucleus, the cells were fixed and dual-labeled for BrDU and gonadotropin ß subunit antigens. Mitogenic effects of exposure to EGF and/or GnRH were compared with those seen following exposure to activin, another known mitogen for gonadotropes (12).
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Materials and Methods |
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Rats were killed by decapitation within seconds of removal from their cages. The pituitaries were then removed, placed in defined medium and then dissociated, as previously described (1, 2, 3, 4, 5). For the MTT cell growth/cell death assays (13), gonadotropes were isolated from this population by centrifugal elutriation. For the detection of BrDU uptake (13), mixed populations from diestrous female rats were plated and used within a day of culture.
Enrichment of gonadotropes
The method was similar to that originally developed for
pituitary corticotropes (13, 14). In that study, the
pituitary cells were first separated by size into three fractions by
centrifugal elutriation. Then, target cells were enlarged by CRH, after
which they were separated from other cell types by re-elutriation. In
the present study, however the protocol involved stimulation with 1
nM GnRH to enlarge the gonadotropes. Pituitary cells from
male rats or female rats in diestrus or proestrus were used for these
experiments. Diestrus and proestrus stages were chosen because they
contained gonadotropes with the highest concentration of receptors for
GnRH or EGF. During the initial elutriation, cells were loaded at 8
ml/min, while the centrifuge was running at 1940–1950 rpm. After a
50-ml load fraction was obtained (which contains mostly red blood cells
and the smallest pituitary cells), three additional 50-ml fractions
were collected at successively higher flow rates: 15 ml/min, 25 ml/min,
and 35 ml/min. Each increment in flow rate eluted larger cells. The
centrifuge speed remained at 1940–1950 until the final fraction (35
ml/min) was collected when it was slowed to a stop. This helped push
the largest cells into the fraction collected at 35 ml/min. The
elutriation chamber was then removed and the remaining fluid (which
contains the largest pituitary cells) was collected. The initial
loading fraction was pooled with the smallest cell fraction collected
at 15 ml/min and was called Fraction 1. The fraction collected at 25
ml/min was Fraction 2. The cells in the chamber were pooled with the
fraction collected at 35 ml/min and was called Fraction 3. Each of the
three fractions was then resuspended in minimum essential medium
containing supplements. Routinely, this defined DMEM (high glucose-DME)
included: 2.5 µg/500 ml HEPES buffer, 0.3% BSA, 5 µg/ml insulin,
30 nM sodium selenite, 50 µg/ml transferrin, and 4.2
µg/ml fibronectin (13, 14). The fractions were then
stimulated with 1 nM GnRH for 3 h at 37 C. Samples of
each fraction were placed in a hemocytometer and at least 100 cells
were measured with a Bioquant Image Analysis system before and after
GnRH stimulation. Area and diameter measurements were plotted on a
distribution curve as described in previous studies (13, 14). There was significant enlargement by a subpopulation of
cells in each fraction following GnRH stimulation. If GnRH was not in
the media, no enlargement of cells was evident.
After the 3 h GnRH stimulation, each fraction was re-eluted as follows at its original flow rate. Then the larger cells were eluted at a new flow rate that was 10 ml/min faster than the original. This produced a fraction containing the enlarged gonadotropes. These were pooled and plated in 96-well trays with the defined media described above and in previous studies (13, 14). Some aliquots were also plated in 24-well trays for fixation and immunolabeling for pituitary hormones (4, 5, 6, 8, 15, 16).
MTT cell growth/cell death assays
These assays detect changes in cell numbers. The studies began
by testing two different populations of male rats with GnRH and EGF
only. Then, studies were continued on three additional populations of
male rats and five populations of female rats. The assays were run, as
previously described (13) on the enriched gonadotrope
fractions plated in 96-well trays. The cells were grown in defined
media (described above) containing 0–10 nM EGF, 10–50
ng/ml activin, or 0–1 nM GnRH for 3–4 days. Combinations
of these peptides were also tested. To prevent protease action, 100
kallikrein inhibitor units of aprotinin were added to the diluent for
the secretagogues After the 3–4 day culture period, the media were
removed and replaced with DME without BSA (because it interfered with
the MTT reaction). Previous tests of cell loss showed less than 10,000
cells lost in the combined media collected from 80–96 wells
(13). An aliquot of the MTT solution (Chemicon Inc., El
Segundo, CA) was then added to each well for 4 h. It had been
dissolved in PBS for at least 12 h before use and was kept no
longer than 1 month. Blue crystals developed over the living
mitochondria in the cells, and live cells were thereby detected. The
crystals were then dissolved in the extraction buffer (supplied with
the kit) and the Optical density was read in a microplate reader at a
test wavelength of 570 and a reference wavelength of 630. Tests of this
assay repeatedly showed a linear relationship between cell number and
optical density readings. Four to six wells were sampled per
experimental group in a given experiment. Averages are reported ±
SD. The control wells were compared with the EGF, GnRH, or
activin-treated wells in that group. Differences between experimental
groups as well as between experimental groups and the control,
untreated wells were detected by ANOVA. If the F value was significant
(P < 0.05), individual differences were detected by
Fisher’s least significant difference test.
BrDU detection of DNA synthesis
In a separate group of experiments, mixed cultures of diestrous
rat pituitary cells were plated in 24-well trays on glass coverslips.
The BrDU experiments were run as previously described
(13). Mixed cultures were grown in defined media
containing 0–1 nM GnRH or 0–10 ng/ml EGF for 3 days. Some
cultures were exposed to both peptides. During the last hour of
incubation, 110 µM/liter BrDU was added to the cultures.
The solution was then removed and the cells fixed in 4%
paraformaldehyde for 2 h at 4 C. They were then washed in PBS
containing 4.5% sucrose followed by treatment with 0.05 M
Tris buffered saline, the diluent for subsequent solutions. To promote
penetration, the cells were treated for 15 min with 4 N
hydrochloric acid (HCl) at room temperature. This was followed by two
washes in 0.1 M sodium tetraborate.
The immunolabeling protocol for BrDU was described in a previous publication (13). The peroxidase reaction product was nickel intensified diaminobenzidine. After the first labeling protocol, pituitary hormones were detected by immunolabeling as described in previous publications (4, 8, 13). The reaction product was orange-amber diaminobenzidine. The percentages of cells with BrDU labeling were calculated after counts of at least 150 cells/coverslip (3–4 coverslips/group). Experiments were repeated three times. To detect changes in mitotic gonadotropes, percentages of dual- and single-labeled cells were also calculated. Numbers were placed on a spread sheet that automatically checked the calculations of the individually labeled cells to verify the reaction conditions. Any deviation from the values obtained with single labeling protocols would suggest that the dual labeling protocol had artefactually masked or added to the reaction for one or both products. Results from the three experiments were averaged and ANOVA was run to detect any significant differences. If the F value showed significance at P < 0.05, Fisher’s least significant difference test was run to detect which values were different.
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Results |
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shows fields from proestrous rat pituitary cells dual labeled for LH
and/or FSH. The labeling pattern shows variability in storage of these
two gonadotropins from cell to cell.
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). In female rats,
there were more cells with GH or PRL antigens. In both populations,
there was evidence for considerable storage overlap with gonadotropins.
Most of the population (>90%) were labeled for gonadotropins, leaving
only 7–9% as nongonadotrope containing cells (based on
immunolabeling). However, if one adds the percentages of other
hormone-bearing cell types seen in Table 1, the storage overlap
becomes obvious, because the sum of the percentages is higher than
7–9%. In some cases (GH and PRL, for example), both hormones could be
stored in the same gonadotrope. Finally, in the studies of the male,
the populations were labeled for EGF receptors (EGFR) and 90–93% of
these enriched gonadotropes expressed EGFR.
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illustrates the average of data from the last groups of experiments
that included activin. Cells were grown 3–4 days in 1–10 ng/ml EGF
with or without 0.5–1 nM GnRH or 60 ng/ml activin.
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shows that EGF alone or 1 nM GnRH stimulated
increased numbers of gonadotropes. The effects of EGF and GnRH were not
additive, however. Activin also stimulates cell growth in the enriched
gonadotrope population. However, the effects of activin did not add to
those of either EGF or GnRH. All values from the treated groups are
significantly higher than the DME vehicle control. However, there were
no gender differences, nor were there differences between experimental
groups.
Uptake of bromodeoxyuridine
The cell growth/cell death assay detects changes in cell number
that could be interpreted as a mitogenic effect, a stabilization of the
culture, and/or enhanced cell survival. Therefore, to verify the
results of the MTT assays, we also detected DNA synthesis
cytochemically by exposing cells to bromodeoxyuridine (BrDU) followed
by immunolabeling for gonadotropin ß subunit antigens
(13). Dual labeling was then performed to identify the
cells that showed signs of DNA synthesis. These studies were limited to
tests of EGF or GnRH because previous work had shown effects of activin
on FSH cells (12).
These studies were done in mixed cultures of pituitary cells to rule
out the possibility that EGF and GnRH effects were unique to the
enriched population. Figure 3 illustrates
the stimulatory effects of EGF and GnRH on the percentages of total
pituitary cells that expressed BrDU labeling. EGF and GnRH did not have
additive effects (data not shown).
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, the data show
that 8% of LH- or FSH-bearing gonadotropes exhibited BrDU uptake in
the control cultures compared with over 24% after EGF or GnRH
treatment. When these values were expressed as a percentage of the
entire pituitary cell population (Fig. 5), 1–1.5% of the diestrous
pituitary cell population were labeled for BrDU and LH or FSH in
control cultures. Significant increases in BrDU-labeled gonadotropes
were seen following exposure to GnRH or EGF. The dual labeling data are
illustrated in Figs. 6 and 7, which show FSH cells labeled for BrDU
from cultures stimulated by GnRH (Fig. 6) or EGF (Fig. 7). The
morphology of EGF treated cultures showed two unique features. First,
clusters or colonies of cells were frequently found, often containing
several BrDU-labeled cells. Some of these clusters were labeled for
gonadotropins. Second, more stellate cells with long processes
appeared, indicating an effect on differentiation.
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Discussion |
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The studies in the present report support the hypothesis that another effect of EGF may be to stimulate gonadotrope proliferation. Evidence for mitogenic effects of EGF, activin and GnRH were seen in enriched populations of gonadotropes exposed to the MTT cell growth/cell death assay that detects increased cell numbers (compared with control cultures). Because this can also reflect enhanced cell survival, cytochemical assays for BrDU uptake were run to confirm the mitogenic effects.
The MTT and cytochemical assays agreed that GnRH and EGF are mitogenic. Because they stimulated more BrDU uptake in gonadotropes that were immunolabeled, this suggests that some of their actions are on differentiated gonadotropes. Because BrDU uptake was assayed after only 1 h exposure, it is likely that the target cell population had included cells that were actively translating gonadotropins. This does not rule out an effect on differentiation as well, however. Finally, because the stimulatory effects were seen in both enriched and mixed populations of pituitary cells (from diestrous rats), these data suggest that they were not unique to the special population of enriched cells or the particular end-point assay used.
The effects of EGF and GnRH were not additive. This may not be surprising in the enriched gonadotrope population because most of the cells contained EGF and GnRH receptors. However, the effects on the mixed population were somewhat surprising because EGF receptors are found on subsets of a number of cell types (6) and GnRH has a more limited distribution (8). We suggest that these factors may be mitogenic only on subsets of pituitary cells that are able to divide. The normal population of dividing cells in the pituitary may contain a subset of the cells with EGF or GnRH receptors. When the two factors are added, their target subsets may overlap sufficiently to show no additive effects. For example, we showed recently that EGF stimulated c-fos expression by subsets of FSH, ACTH, and GH cells, even though its receptors are found on other cell types (16). We have found GnRH receptors on a subset of GH, FSH, and LH cells (8), but GnRH stimulates c-fos expression by only GH or ACTH cells (16). Thus, we suggest that the appearance of GnRH or EGF receptors on a set of pituitary cells does not mean that the receptors mediate all events in all of the potential target cells. Furthermore, some of the common mitogenic effects seen after GnRH or EGF exposure may reflect common target cells.
Another reason for the lack of additive effects may relate to paracrine interactions. GnRH may stimulate the release of regulatory factors that in turn stimulate other cell types to divide. This could include EGF or its receptor (6) or other paracrine factors that are mitogenic. If GnRH stimulates EGF release, then adding exogenous EGF may be redundant and no additive effects would be expected.
These studies confirm and add to recent work by Chaidarun et
al. (9) who reported that 1 nM
EGF stimulated a 200% increase in tritiated thymidine uptake by sheep
pituitary cultures. In these sheep cultures, gonadotropes predominated
representing 50–75% depending on the gonadotropin being detected.
Some of the uptake could have been due to mitotic activities by
nongonadotropes. Our studies of enriched and cytochemically identified
gonadotropes show that a subset of well differentiated gonadotropes
(Figs. 6 and 7) may indeed be among the targets for EGF or GnRH
mitogenic actions.
EGF as a regulator for gonadotropes
As gonadotropes are prepared for their proestrous surge activity,
a number of important changes are seen in the population (1, 2, 3, 4, 15, 16, 17, 18). EGF may be a regulator for a number of these changes.
Early in the cycle (metestrus), EGF receptors appear on about 25% of
the LH cells, identified by mRNA for ß subunits (5) and
increase later in the cycle to 40–50% of immunolabeled gonadotropes,
reaching a peak early in proestrus. Interestingly, the enriched
gonadotrope population appears to have more cells with EGFR (over
90%), which suggests that either the culture conditions or the GnRH
during elutriation may have up-regulated the EGF receptor.
During diestrus, a subset of the expanded gonadotrope population is multipotential, sharing somatotropic and gonadotropic phenotypes. Late in diestrus, gonadotropes also express GH-releasing hormone (GHRH) receptors (15). There is a corresponding peak in GH cells that contain LH and FSH mRNAs (4) and GnRH receptors (8). As gonadotropes translate gonadotropins during diestrus, they also increase their expression of GH mRNA (17) to reach a peak of 50% of LH and FSH gonadotropes by late proestrus. EGF may play a role in regulating differentiation or function in this multipotential cell type. As stated above, our recent work shows that it stimulates c-fos protein expression in FSH cells and GH cells from proestrous animals (16).
Another step in the differentiation process is the synthesis of estradiol receptors (19) and GnRH receptors (15, 20, 21, 22, 23, 24), which is enhanced by estradiol and activin. Peak expression of GnRH receptors is seen on the afternoon of diestrus or the morning of proestrus. Studies by Leblanc et al. (7) suggest that EGF may also play a role in unmasking cryptic GnRH receptors. EGF may facilitate the expression of GnRH receptivity, during late diestrus and early proestrus, because this is also the time when gonadotropes express more EGF receptors as well. Finally, the findings showing that EGF and GnRH mitogenic effects are not additive suggest that EGF is not dependent on a set of GnRH receptors for its mitogenic effects.
Multiple regulators of mitoses in gonadotropes
The present studies have added two mitogens for gonadotropes to
the list, which includes activin (12) and estrogens
(25, 26). Activin has also shown mitogenic activity
in vitro, stimulating more immunolabeled FSH cells
(12). With respect to estradiol, McArdle et al.
(25) showed that estradiol caused a dose-dependent
increase in [3H] thymidine incorporation and
cell number in the 
-subunit producing cell line, T3–1 cells.
These cells are believed to be like immature gonadotropes in that they
produce GnRH receptors and -subunits, but not ß-subunits.
Estradiol’s effects were at the expense of GnRH receptor numbers.
However, it also caused an increase in GnRH stimulated inositol
phosphate accumulation, suggesting that it had increased the efficiency
of the response (fewer GnRH receptors were needed). More recent studies
by this same group (26) showed that estrogen actually
regulates the length of time in each stage of the cell cycle for these
T3–1 cells. Estradiol increased the proportion of cells in the S
and G2/M phases of the cell cycle at the expense of those in the G1/G0
stages. It also reduced the doubling time from 73 h to 39 h
(26). These data suggested that estrogen may either be
recruiting quiescent cells into the G1 phase, or driving cells through
G1 and or the G1/S transition. A similar finding was reported by
Chaidarun et al. (9), in studies of sheep
pituitary cells. Estradiol produced a 33% increase in tritiated
thymidine uptake as it reduced mRNA for gonadotropins. This suggested
that estradiol had shifted the stage of the secretory cycle as it
stimulated proliferation. Finally, with respect to GnRH, the current
studies cannot rule out the possibility that GnRH works through EGF to
stimulate proliferation because there are cells that contain EGF in the
pituitary. Most of the EGF is found in GH cells, which suggests that it
might serve as a paracrine regulator (26, 27).
To summarize, these studies have shown one more potential function for EGF receptors on pituitary gonadotropes. In addition to the regulation of EGF receptors and c-fos activity, EGF is mitogenic, stimulating significant increases in DNA synthesis from a baseline level of 8% to nearly 25% of LH or FSH gonadotropes. EGF and GnRH thus adds to several known mitogens for gonadotropes which include activin (12) and estradiol (25, 26).
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Acknowledgments |
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Footnotes |
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Received June 19, 2000.
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T3–1 cells.
Mol Cell Endocrinol 87:95–103[CrossRef][Medline]
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