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Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, Texas 77555-1043
Address all correspondence and requests for reprints to: Jennifer Armstrong, Department of Anatomy and Neurosciences, MRB 10–104, 303 University Boulevard, University of Texas Medical Branch, Galveston, Texas 77555-1043. E-mail: armstrong{at}mbian.utmb.edu
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Abstract |
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Introduction |
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EGF plays an important role in the female reproductive system and specifically acts on the hypothalamic-pituitary axis in the cycling female rat. Its sites of action may include both the pituitary and the hypothalamus. GnRH release is mediated by EGF (4). EGF receptor (EGFR) messenger RNA (mRNA) levels increase in the hypothalamus at the onset of puberty, which suggests a regulatory role in the maturation of the axis (5).
In the anterior pituitary gland, EGF stimulates release of LH after perifusion in tandem with hypothalamic tissue (6). Thus, EGF regulated the secretion of gonadotropins by stimulating hypothalamic neurons. However, their tests showed that it may also increase LH secretion by increasing pituitary responsiveness to estradiol (6). EGF also has direct effects on stimulating LH release in cultured pituitary cells (7). Cycles were not distinguished in this study. The evidence suggests that EGF may stimulate the release of the gonadotropins indirectly by acting on the hypothalamus and directly, by actions on the pituitary.
It would be beneficial to determine whether EGF mediates the release of both LH and FSH differentially throughout the estrous cycle. Few researchers have examined the effects of EGF on cycling animals. EGF has been shown to have differential effects on release of both gonadotropins in cycling Merino ewes (8). The effects varied with the stage of the cycle, and similar effects might also be seen in cycling rats.
If EGF helps to modulate gonadotrope function, then EGFR levels also may be changing throughout the estrous cycle. The receptors may be synthesized and then undergo receptor-mediated endocytosis and lysis or recycling once their role in gonadotrope activation is completed. We hypothesize that the pituitary EGFR may increase in a pattern that allows stimulation of gonadotrope function during the cycle. The rationale for this hypothesis comes from studies in the hypothalamus. Specifically, EGFR mRNA levels increased in the medial basal hypothalamus at the initiation of puberty, decreased during the morning of the first proestrus, and increased again during the afternoon of first proestrus, at the time of the gonadotropin surge (5). In this study, we tested the hypothesis that EGFR levels may change throughout the estrous cycle. We used dual immunolabeling for EGFR protein and each of the pituitary hormone antigens to detect and identify EGF target cells. We report cyclic changes in EGFR expression in the pituitary with peak expression in antigen-bearing gonadotropes appearing in proestrus. However, relatively high expression is seen in cells with LHß mRNA in metestrus.
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Materials and Methods |
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After the rats were killed by decapitation, the anterior pituitaries were rapidly removed and 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 Chemical), and 24.65 mg/500 ml sodium bicarbonate (JRH Biosciences). To prevent bacterial growth, 1 µl/100 ml gentamicin (Sigma Chemical) was used. The dissociation protocol was performed as reported previously (9, 10). Cells were tested for viability by the trypan-blue exclusion test. Normally, the protocol yielded 2,000,000–3,000,000 cells/pituitary that were 98% viable. The cells were plated in DME containing 0.005 mg/50 ml insulin (Sigma Chemical), 0.05 mg/50 ml transferrin (Sigma Chemical), and 0.03 µM sodium selenite (Johnson Matthey Chemical Ltd., New York, NY). Cells were plated onto glass coverslips (A. H. Thomas Scientific, Swedesboro, NJ) that had been coated with poly-D-lysine (Sigma Chemical) in 24-well trays at a density of approximately 40,000–50,000 cells/50 µl·well.
After 1 h of plating, the cells were fixed with 2% glutaraldehyde (Polysciences, Inc., Warrington, PA) for 30 min and then washed in 4.5% sucrose in 0.1 M phosphate buffer. The cells were then stored in the refrigerator until needed.
Immunocytochemistry with EGFR and pituitary hormones
Anterior pituitaries from each stage of the cycle were collected
and dispersed as mentioned above. Parallel groups of cells were labeled
for EGFR only or dual labeled for EGFR and each of the pituitary
hormones. The EGFR antibody used was a mouse monoclonal antibody, E3138
(Sigma Chemical), raised specifically against the intracellular domain
of the EGFR.
Anti-ACTH from 17–39 C terminal fragment of ACTH was made in this lab (11) and used at a dilution of 1:30K. Rat anti-ß-TSH was a gift from the NIDDK-NIH and used at a dilution of 1:45K. Rabbit antirat PRL and rabbit antirat GH were both purchased from Chemicon (Temecula, CA). Anti-PRL was used at a dilution of 1:40K and anti-GH was diluted to 1:35K. Antibovine LHß (diluted to 1:30K) was a gift from J. G. Pierce, and antihuman FSHß (diluted to 1:10K) was generously provided by the Pituitary Hormone Distribution Program (NIDDK). The labeling protocol was used as previously described (12), with a few modifications. After the color reaction with nickel intensified diaminobenzidine to label the EGFR antigen, cells were washed in 0.05 M acetate buffer and 0.05 M Tris-buffered saline, followed by pretreatment with a blocking solution that contained 0.05 M Tris-buffered saline and 5% normal goat serum. The coverslips were then incubated for 2 h at 37 C in each of the pituitary hormone antibodies. The remaining protocol was the same as described previously (12).
Control labeling procedures were performed for both single- and double-labeling techniques. In both, the primary antisera were left out, and the coverslips were incubated with only the diluent buffer. Also preabsorption tests were done previously in this lab for EGFR antibodies (12) and for the pituitary hormone antibodies (13).
In situ hybridization with biotinylated oligonucleotide probes to
LHß or FSHß mRNA
The in situ hybridization protocol was performed on
pituitary cells from metestrous female rats using oligonucleotide
probes to LHß or FSHß mRNA. The cells were then immunolabeled for
EGFR proteins. The dual-labeling hybridization procedure used has been
described previously (14), with the following modifications.
Biotinylated antisense oligonucleotide probes were purchased from DNA
International (Lake Oswego, OR). The cells were plated and grown for
24 h in DMEM and 10% FBS (JRH Biosciences). The following day,
they were fixed for 30 min in 2% glutaraldehyde. After detection with
1:100 streptavidin peroxidase and nickel intensified diaminobenzidine,
the label for the mRNAs was black. The coverslips were then colabeled
for EGFR antigen by immunoperoxidase methods that resulted in an orange
label.
Statistical analysis
Each experiment sampled cells from one or two female rats in a
given stage. The experiments were repeated until cells from at least
six rats/stage of the cycle were collected. The data from each
experiment were averaged. Significant differences were tested by ANOVA,
followed by the LSD post hoc test. Significance was
determined at P less than 0.05.
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Results |
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graphs these data. Figure 2 shows
fields labeled for EGFR in single-labeling protocols. They compare
labeling in the metestrous (Fig. 2a) and proestrous (Fig. 2b) rat cell
populations.
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). The
percentages of pituitary cells with both EGFR and each of the pituitary
hormones are listed: 11.8 ± 1.3%, PRL; 9.9 ± 1.8%, ACTH;
8.2 ± 0.6%, GH; and 6.3 ± 0.8%, TSH. In contrast, few
antigen-bearing gonadotropes contributed to this peak. Only 2.6 ±
0.6% of pituitary cells contained LHß antigens and EGFR, and 4
± 0.6% of pituitary cells contained FSHß antigens and EGFR.
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If LH cells were detected by their ß-subunit mRNA, more of them
expressed EGFR. Recall that only 2.6% of pituitary cells contained
EGFR and LHß antigens (Fig. 3
). However, nearly 2.5x more (5.7%)
LHß mRNA-bearing cells express the EGFR. Furthermore, whereas 4% of
pituitary cells contained EGFR and FSHß antigens during metestrus,
only 3.3% contained EGFR and FSHß mRNA. Figure 4
illustrates dual labeling for LHß mRNA or FSHß mRNA and EGFR.
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illustrates the changes in the percentages of cells labeled for EGFR
and either LHß or FSHß antigens. Peak expression of EGFR by LHß
or FSHß antigen-bearing gonadotropes is seen in the proestrous cell
population to 6–7% of the pituitary cells. Furthermore, the increase
in LHß antigen-bearing cells occurs earlier than that for FSHß
antigen-bearing cells. Figure 6 illustrates dual
labeling for EGFR by LHß and FSHß gonadotropes from metestrous and
diestrous rats, respectively. The EGFR is localized mainly in the
periphery of the cell, whereas the LH or FSH immunocytochemical label
is found in the center.
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Discussion |
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When the effects of EGF were tested on ewes, it was discovered that EGF stimulated higher levels of LH and FSH while inhibiting the estrous surge of LH and FSH (15). EGF also stimulated high gonadotropin secretion, similar to the pattern seen after ovariectomy (15). In another study, Merino ewes, infused with EGF, showed an increase in both LH and FSH secretion (8).
Collectively, these findings suggest that EGF may be involved in the modulation of gonadotropin surge secretory activity or the differentiation of gonadotropes. If EGF is involved in the regulation of secretion of gonadotropes during the estrous cycle, we hypothesized that expression of EGFR may change with the dynamic changes in the gonadotrope population (9, 10, 13).
The single-labeling protocols showed a clear cyclic change in EGFR expression in the pituitary. Cells from metestrous rats contained the most EGF-receptive cells. This would suggest that EGF may be involved in early events in the cycle. We used dual immunocytochemistry to identify the cell types contributing to the peak expression of EGFR and learned that all pituitary cells were involved. This agrees with studies of EGF affects on all pituitary cell types. We have shown EGF-mediated increases in ACTH secretion and expression of POMC mRNA (16). Pituitary glands perifused with EGF also show increases in the release of TSH (17). GH4C1 tumor cells treated with EGF increase their PRL release (18). In addition, when GH3 cell cultures and neonatal pituitary cells were treated with EGF at the end of treatment, classical lactotropes were dramatically increased in numbers (19). Thus, the abundant EGF target cells in metestrous cell populations may reflect cyclic EGF stimulation of cell types other than the gonadotropes. Finally, these studies also agree with our recent reports of the distribution of EGFR in pituitary cells from male rats (12).
When dual labeling was used to identify gonadotropes bearing EGFR in the metestrous rat population, only a few antigen-bearing gonadotropes contributed to the peak expression. This may reflect the fact that the antigen-bearing gonadotropes are scarce in metestrous rats. Perhaps the cells expressing EGFR are new gonadotropes that are beginning to translate gonadotropin ß-subunit mRNAs into protein. These cells may be identified by their content of mRNAs for ß-subunits. In situ hybridization with metestrous cells was therefore performed to learn whether there were cells with EGFR and LHß or FSHß mRNA. Counts of these populations showed an increase in cells with EGFR and LHß mRNA, which suggests that EGFR may indeed be expressed in the developing LH gonadotrope. However, there was no increase in expression of EGFR by cells bearing FSHß mRNA.
When other stages of the cycle were examined for the presence of EGFR in gonadotropes, the highest expression of cells labeled for both EGFR and LHß antigens occurred during diestrus and proestrus. Furthermore, the highest number of FSHß antigen-bearing cells with EGFR was seen in proestrous rat populations. This is a time when the gonadotropes are increasing their gonadotropin stores (9, 10, 13). This results in a 2- to 3-fold increase in the percentage of gonadotropes bearing LHß or FSHß antigens by the morning of proestrus (9, 10, 13). The observed increases could therefore reflect the ongoing process of differentiation in gonadotropes, which renders the cells detectable by immunocytochemistry (for LHß or FSHß antigens). The new data in this study suggest that this differentiation process also includes the production of EGFR proteins by at least half of the gonadotrope population.
Our studies also suggest that EGF target LH and FSH gonadotropes do not develop in parallel. Early in the cycle (metestrus), EGFR is found with LHß mRNA in nearly 6% of the pituitary population. However, only about 3% of the pituitary cells express EGFR with FSHß mRNA. This suggests that initially, EGFR may be expressed by a monohormonal subset of gonadotropes that is translating LH antigens.
This hypothesis is strengthened by the analysis of diestrous and proestrous populations, which show a steady increase in the percentages of pituitary cells with LHß antigens and EGFR to reach a peak of 6% (or about 40% of LHß antigen-bearing cells). In contrast, the increase in the percentages of cells with FSHß antigens and EGFR occurs later (proestrus). This later expression could reflect synthesis of FSHß antigens by LH cells, resulting in the production of the bihormonal gonadotrope subtype. It also could reflect nonparallel contributions by monohormonal FSH cells.
Thus, expression of EGFR may be an early event in the maturation or differentiation of LHß antigen-bearing gonadotropes. The later expression of FSHß antigens by EGF target cells (seen during proestrus) may reflect the transition from a monohormonal LH cell to a bihormonal cell containing both LH and FSH stores. Collectively, these data suggest that expression of EGFR may be one marker for the maturing gonadotrope as it develops to a point where it can support the proestrous surge secretion. This confirms recent studies showing EGF effects on LH and FSH secretion and points to possible roles for EGF as a modulator during surge secretory activity. Also, EGF may stimulate synthesis of new mRNAs, which occurs during the surge. Further work is needed to define the exact roles of this growth factor. Also, further work will identify regulatory factors for EGFR expression in gonadotropes.
In conclusion, the pituitary cells change their expression of EGFR during the estrous cycle. EGF stimulates gonadotropes during the cycle; so, it follows that expression of its receptor by gonadotropes also might change. We found that EGFR levels increase in metestrous populations to support EGF stimulation later in the cycle. Yet, antigen-bearing gonadotropes are in the minority in this population. EGFRs do appear in immature gonadotropes that can be identified only by their content of LHß mRNA. EGFR expression then increases within LH gonadotropes during diestrus and FSH gonadotropes, during proestrus. This increase coincides with the peak expression of gonadotropin antigens. These data suggest a role for EGF in both the early expression of gonadotrope function and the later secretory functions.
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Acknowledgments |
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Footnotes |
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Received November 1, 1996.
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References |
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in the release of luteinizing hormone-releasing hormone from the
developing female hypothalamus. Proc Natl Acad Sci USA 87:9698–9702 messenger ribonucleic acids and their
receptors in the rat anterior pituitary: localization and regulation.
Endocrinology 136:2284–2293[Abstract]
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