Changes in Expression of Epidermal Growth Factor Receptors by ...

0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 5 Printed in U.S.A.

Changes in Expression of Epidermal Growth Factor Receptors by Anterior Pituitary Cells during the Estrous Cycle: Cyclic Expression by Gonadotropes* JENNIFER ARMSTRONG

AND

GWEN V. CHILDS

Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, Texas 77555-1043 ABSTRACT Epidermal growth factor (EGF) stimulates gonadotropin secretion, suggesting that it may regulate gonadotrope functions. These responses may be modulated by changes in expression of EGF receptors (EGFR), especially during the estrous cycle. To test this hypothesis, EGFR and pituitary hormones were detected by dual immunocytochemistry. Pituitary cells from metestrous rats contained 41 6 4% cells labeled for EGFR. This peak was followed by a decline to 17.6 6 2% of cells from proestrous rats. The percentages of metestrous pituitary cells with EGFR and each hormone were: PRL, 11.8 6 1; ACTH, 9.9 6 1.8%; GH, 8.2 6 0.6%; TSH, 6.3 6 0.8%; FSH, 4 6 0.6%; and LH, 2.6 6 0.6%. The relatively low percentages of gonadotropes may have reflected the low expression of LH or FSH antigens during metestrus. Dual labeling for EGFR and LHb or FSHb messenger RNAs (mRNAs) showed a significant increase in the percentages of

pituitary cells with LHb mRNA and EGFR (to 5.7% of pituitary cells), but there were no increases in the EGF target cells bearing FSHb mRNA. When gonadotropin antigens were detected in EGF target cells during other stages of the cycle, there was an increase to reach a peak of 6.6 –7% of pituitary cells by the morning of proestrus (or 40 –50% of gonadotropes). To summarize, EGFR are seen in few gonadotropes during the metestrous peak, although more LH cells (but not FSH cells) can be identified by their content of LHb mRNA. This suggests that EGFR is expressed initially in monohormonal LH gonadotropes. The peak expression of EGFR by gonadotropes during diestrus and proestrus suggests that EGF may be involved in the development of the gonadotropes as they approach surge secretory activity. It also may help stimulate the transcription of new gonadotropin b-subunit mRNA seen late in proestrus, early in estrus. (Endocrinology 138: 1903–1908, 1997)

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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

PIDERMAL growth factor (EGF) was discovered in the mouse submaxillary gland. It induces epidermal thickening and keratinization (1). Mouse EGF is a 53-amino acid peptide that contains three disulfide bonds. EGF binds to its receptor to transduce its action via tyrosine kinase activity (2). EGF acts to stimulate DNA synthesis and regulate cell proliferation in mouse fibroblasts, human fibroblasts, glia, mammary epithelia, keratinocytes, vascular endothelia, rabbit chondrocytes, bovine granulosa, and corneal endothelial cells (3). 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

Received November 1, 1996. 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: [email protected]. * This research was supported by NIH Grant R01-HD-15472.

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EGFR EXPRESSION DURING THE ESTROUS CYCLE

antigen-bearing gonadotropes appearing in proestrus. However, relatively high expression is seen in cells with LHb mRNA in metestrus. Materials and Methods Collection and dispersion of pituitaries Female Sprague-Dawley rats were housed three per cage under artificial illumination between 0600 and 1800 h and given food and water ad libitum. They were acclimated for 1 week before the start of daily vaginal smearing, performed to determine the stage of the cycle. The females underwent at least two complete estrous cycles before they were used. The Institutional Review Committee approved the animal care and use protocol annually. 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 ml/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 mm 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 mlzwell. 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-b-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 LHb (diluted to 1:30K) was a gift from J. G. Pierce, and antihuman FSHb (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).

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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.

Results Changes in EGFR with the estrous cycle

When cells expressing EGFR were counted, there was a distinct cyclic trend in the labeling patterns. The percentages of pituitary cells with EGFR protein was highest (41%) in cells from metestrous rats. The lowest percentages were found in cells from proestrous rats (17.6%). Estrous cell population had 29% EGF target cells. Figure 1 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. Identity of pituitary cells that express EGFR

We chose the peak expression period (metestrus) to identify the target cells by their content of pituitary antigens. As reported for the male rat (12), the dual-labeled fields showed that EGFR antigens were found in all pituitary cell types (Fig. 3). The percentages of pituitary cells with both EGFR and each of the pituitary hormones are listed: 11.8 6 1.3%, PRL; 9.9 6 1.8%, ACTH; 8.2 6 0.6%, GH; and 6.3 6 0.8%, TSH. In contrast, few antigen-bearing gonadotropes contributed to this peak. Only 2.6 6 0.6% of pituitary cells contained LHb

In situ hybridization with biotinylated oligonucleotide probes to LHb or FSHb mRNA The in situ hybridization protocol was performed on pituitary cells from metestrous female rats using oligonucleotide probes to LHb or FSHb 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

FIG. 1. Percentage of total anterior pituitary cells labeled for EGFR antigens throughout the estrous cycle. Star, Significantly different from the values in the proestrous group (P , 0.05).


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FIG. 2. Field showing single label for EGFR taken from metestrous (2a) or proestrous (2b) groups of female rats. Black arrow, EGFR label; U, unlabeled cell. Magnification, 14433; bar, 10 mm.

antigens and EGFR, and 4 6 0.6% of pituitary cells contained FSHb antigens and EGFR. Are metestrous EGF target cells immature gonadotropes?

During metestrus, gonadotropin antigen expression is at a nadir (less than 8% of the cell population) (10). However, gonadotropin mRNA expression is relatively high early in

metestrous, dropping to a nadir during diestrous II (14). Therefore, perhaps some of the EGF target cells seen in metestrous populations might be identified by their content of LH or FSH b-subunit mRNAs. This was tested in dual-labeling studies that detected the mRNA first by in situ hybridization and the EGFR antigens, by immunocytochemistry.

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If LH cells were detected by their b-subunit mRNA, more of them expressed EGFR. Recall that only 2.6% of pituitary cells contained EGFR and LHb antigens (Fig. 3). However, nearly 2.53 more (5.7%) LHb mRNA-bearing cells express the EGFR. Furthermore, whereas 4% of pituitary cells contained EGFR and FSHb antigens during metestrus, only 3.3% contained EGFR and FSHb mRNA. Figure 4 illustrates dual labeling for LHb mRNA or FSHb mRNA and EGFR.

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Changes in dual-labeled cells for EGFR and LHb or FSHb antigens with the estrous cycle

Dual immunocytochemical labeling for EGFR and gonadotropin b-subunit antigens was then performed on cells from all stages of the cycle, to learn whether the expression by gonadotropes changed as they approached proestrous surge secretory activity. Figure 5 illustrates the changes in the percentages of cells labeled for EGFR and either LHb or FSHb antigens. Peak expression of EGFR by LHb or FSHb antigenbearing gonadotropes is seen in the proestrous cell population to 6 –7% of the pituitary cells. Furthermore, the increase in LHb antigen-bearing cells occurs earlier than that for FSHb antigen-bearing cells. Figure 6 illustrates dual labeling for EGFR by LHb and FSHb 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. Discussion

FIG. 3. Percentage of total pituitary cells labeled for EGFR and each of the pituitary hormones, including ACTH, PRL, TSH, GH, LH, or FSH. The cell populations were taken from metestrous rats during the peak period of expression of EGFR in the anterior pituitary.

FIG. 4. Field showing a cell from a metestrous rat dual labeled for LHb mRNA (4a) or FSHb mRNA (4b) and EGFR antigens. The arrow towards the center of the cell indicates black label for the mRNA and the lighter grey on the edge of the cell indicates orange EGFR label. Magnification, 34333; bar, 5 mm.

Recent studies during the past 2 decades show evidence for EGF as a secretagogue for LH and FSH secretion. In the earliest studies, rat anterior pituitary glands were placed in series with the hypothalamus in a perifusion chamber, and EGF stimulated the release of LH (6). When pituitary glands were directly stimulated with EGF in the presence of estradiol, there also was a significant increase in LH release (6). Rat pituitary cell cultures stimulated directly with EGF also showed an increase in LH expression (7). 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

FIG. 5. Counts of cells dual labeled for LHb or FSHb antigens and EGFR throughout the estrous cycle. Star, Significantly different from metestrous and estrous values (LSD test).


FIG. 6. Field showing cell dual labeled for LHb (6a) or FSHb (6b) and EGFR antigens. The cell was taken from populations of metestrous cells. Black label for EGFR is indicated by the black arrow and the orange label for gonadotropins is shown as grey throughout the cell. Magnification, 25393; bar, 5 mm.

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 b-subunit mRNAs into protein.

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These cells may be identified by their content of mRNAs for b-subunits. In situ hybridization with metestrous cells was therefore performed to learn whether there were cells with EGFR and LHb or FSHb mRNA. Counts of these populations showed an increase in cells with EGFR and LHb 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 FSHb 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 LHb antigens occurred during diestrus and proestrus. Furthermore, the highest number of FSHb 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 LHb or FSHb 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 LHb or FSHb 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 LHb mRNA in nearly 6% of the pituitary population. However, only about 3% of the pituitary cells express EGFR with FSHb 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 LHb antigens and EGFR to reach a peak of 6% (or about 40% of LHb antigen-bearing cells). In contrast, the increase in the percentages of cells with FSHb antigens and EGFR occurs later (proestrus). This later expression could reflect synthesis of FSHb 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 LHb antigen-bearing gonadotropes. The later expression of FSHb 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

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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 LHb 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. Acknowledgments The authors wish to thank Ms. Geda Unabia and Ms. Diana Rougeau for their technical assistance during these studies. We also would like to thank Drs. A. F. Parlow and J. G. Pierce for the antisera to FSH and LH, respectively. We thank the Hormone Distribution Program for antigens and antisera to pituitary hormones.

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Epidermal growth factor stimulates secretion of rat pituitary luteinizing hormone in vitro. Acta Endocrinol (Copenh) 108:175–178 Przylipiak A, Kiesel L, Rabe T, Helm K, Przylipiak M, Runnebaum B 1988 Epidermal growth factor stimulates luteinizing hormone and arachidonic acid release in rat pituitary cells. Mol Cell Endocrinol 57:157–162 Radford H, Panaretto B, Avenell J, Turnbull K 1987 Effect of mouse epidermal growth factor on plasma concentrations of FSH, LH and progesterone and on oestrus, ovulation and ovulation rate in Merino ewes. J Reprod Fertil 80:383–393 Childs G, Unabia G, Tibolt R, Lloyd J 1987 Cytological factors that support nonparallel secretion of luteinizing hormone and follicle-stimulating hormone during the estrous cycle. Endocrinology 121:1801–1813 Childs G, Unabia G, Lee B, Lloyd J 1992 Maturation of follicle-stimulating hormone gonadotropes during the rat estrous cycle. Endocrinology 131:29 –36 Moriarty GV, Halmi 1972 Electron microscopic study of the adrenocorticotropin-producing cell with the use of unlabeled antibodies and the soluble peroxidase-antiperoxidase complex. J Histochem Cytochem 20:590 – 603 Fan X, Childs GV 1995 Epidermal growth factor and transforming growth factor-a messenger ribonucleic acids and their receptors in the rat anterior pituitary: localization and regulation. Endocrinology 136:2284 –2293 Childs GV, Unabia G, Miller B 1994 Cytochemical detection of gonadotropinreleasing hormone-binding sites on rat pituitary cells with luteinizing hormone, follicle-stimulating hormone, and growth hormone antigens during diestrous up-regulation. Endocrinology 134:1943–1951 Childs G, Unabia G, Rougeau D 1994 Cells that express luteinizing hormone (LH) and follicle-stimulating hormone (FSH) b-subunit messenger ribonucleic acids during the estrous cycle: the major contributors contain LHb, FSHb, and/or growth hormone. Endocrinology 134:990 –997 Shaw G, Jorgensen G, Tweedale M, Waters M 1985 Effect of epidermal growth factor on reproductive function of ewes. J Endocrinol 107:429 – 436 Childs G, Patterson J, Unabia G, Rougeau D, Wu P 1991 Epidermal growth factor enhances ACTH secretion and expression of POMC mRNA by corticotropes in mixed and enriched cultures. Mol Cell Neurosci 2:2885–2895 Altschuler L, Parisi M, Cageao L, Chiocchio S, Fernandez-Pol J, Zaninovich A 1993 Epidermal growth factor stimulates thyrotropin secretion in the rat. Neuroendocrinology 57:23–27 Aanestad M, Rotnes J, Torjesen P, Haug E, Sand O, Bjoro T 1993 Epidermal growth factor stimulates the prolactin synthesis and secretion in rat pituitary cells in culture (GH4C1 cells) by increasing the intracellular concentration of free calcium. Acta Endocrinol (Copenh) 128:361–366 Felix R, Meza U, Cota G 1995 Induction of classical lactotropes by epidermal growth factor in rat pituitary cell cultures. Endocrinology 136:939 –946