ARTICLE |
Correspondence to: Jennifer Armstrong, 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 expression appears in some activated cell types. Because of dynamic changes in gonadotropes during the estrous cycle, this study was initiated to determine if fos might be expressed in gonadotropes before any period of activation. We detected c-fos and pituitary antigens in dissociated anterior pituitary cells by dual-labeling immunocytochemistry. The highest percentages of cells with fos protein were found in proestrous rat populations. In diestrous and proestrous populations, dual labeling showed that 6-9% of pituitary cells contained fos with adrenocorticotropin, thyroid-stimulating hormone, prolactin, or growth hormone antigens. In contrast, only 0.8-3% contained fos with luteinizing hormone (LH) or follicle-stimulating hormone (FSH) antigens. We then tested the hypothesis that gonadotropes might increase fos expression earlier in the cycle. In populations from metestrous rats, c-fos labeling was found in 45% of LH cells compared to only 23% of LH cells in the proestrous group. This suggests that proportionately more LH cells are being activated to produce fos early in the cycle. Perhaps fos is used in translation of LHß antigens or gonadotropin-releasing hormone (GnRH) receptor mRNAs. In contrast, less than 1% of all pituitary cells expressed fos with FSH at all stages of the cycle (only 6-12% of FSH cells). This differential expression suggests one mechanism behind the regulation of nonparallel storage and release of gonadotropin antigens. (J Histochem Cytochem 45:785-794, 1997)
Key Words: anterior pituitary, c-fos, immunocytochemistry, estrous cycle, gonadotrope, prolactin, rat, light microscopy
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Introduction |
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C-fos is an early expression proto-oncogene which is expressed at low levels in most cells. It is the cellular progenitor of viral-fos (v-fos), a retroviral DNA first isolated from a mouse sarcoma (
C-fos expression has been found in cells that are active along the hypothalamic-pituitary-reproductive axis.
Two groups have demonstrated c-fos expression in anterior pituitary gonadotropes. T3-1 gonadotrope cell line. Tests of second messengers showed that the expression of immediate-early response genes was dependent on calcium, protein kinase C (
In the above studies, the assumption was made that the changes in early gene expression occurred in gonad-otropes. This was supported by the evidence in the gonadotrope cell line (T3-1). However, there was no proof that the GnRH-induced c-fos expression in normal pituitary cells was due to expression in gonado-tropes. Dual-labeling cytochemistry was needed to identify the fos-expressing cells.
In view of the possibility that the GnRH-mediated changes in early gene expression could take place in gonadotropes, we initiated studies to determine if there were changes in fos proteins during the estrous cycle. If so, our second objective was to identify the cells that showed expression of the c-fos gene. Third, because gonadotropes are activated to translate gonadotropins and GnRH receptors during diestrus, we hypothesized that there might be an increase in fos expression during these stages. Our studies show differential expression of c-fos overall in anterior pituitary cells. However, the increase was not correlated with increases in gonadotropes. Nevertheless, LH antigen-bearing gonadotropes showed relatively high expression in metestrus, just before the production of key hormones and receptors.
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Materials and Methods |
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Collection and Dispersion of Pituitaries
To facilitate detection of c-fos and pituitary antigens in the same cells, we used pre-embedding dual-labeling methods on freshly dissociated pituitary cells that had been plated for 1 hr. Female Sprague-Dawley rats were acclimated for at least 7 days with food and water available ad libitum, in a regulated light-dark cycle (lights on at 0600 and off at 2000 hr). The rats were tested daily by vaginal smears and were used if they showed two consecutive complete estrous cycles.
At the time of sacrifice, the animals were removed and sacrificed by decapitation in a separate adjoining room within seconds of removal from their cage. The same animal handler performed these rapid procedures, which were approved by the Animal Care and Use Committee. The animals were not given any drugs before decapitation because of stress effects associated with the injection. All animals were sacrificed between 0900 and 1000 hr, unless otherwise noted. The animal care and use protocols were approved annually by the Institutional Review Committee.
The anterior pituitaries were rapidly removed after decapitation and placed in cold Dulbecco's modified Eagle's medium (DMEM; JRH Biosciences, Lenexa, KS) containing 0.3% BSA (Sigma Chemical; St Louis, MO), 1.8 g/500 ml HEPES (Sigma), and 24.65 ml/500 ml sodium bicarbonate (JRH Biosciences). Gentamicin (Sigma) was used at 1 µl/100 ml to prevent bacterial growth. After washing the pituitaries in fresh DMEM, they were cut into small pieces and placed in 0.3% trypsin (Sigma). The dissociation protocol was performed as reported previously (
One hour after plating the cells were fixed with 2% glu-taraldehyde (Polysciences; Warrington, PA) for 30 min, followed by washes with 0.1 M phosphate buffer containing 4.5% sucrose. Cells could then be stored in the refrigerator until use.
Immunocytochemistry for c-fos and Pituitary Antigens
Anterior pituitary cells were collected and dispersed as mentioned above. Coverslips were then labeled only for c-fos protein or dual labeled for c-fos protein and one of the six pituitary hormones. Rabbit polyclonal antibody Ab-2 to c-fos protein was purchased from Oncogene Science (Cambridge, MA) and was used at a dilution of 1:1200. Anti-bovine LHß was a gift from J.G. Pierce and was used at a dilution of 1:40 K. Anti-human FSHß was used at dilution of 1:10 K and was generously provided by the Pituitary Hormone Distribution Program (NIDDK), as was the anti-rat ß-thyrotropin, which was used at a dilution of 1:45 K. Anti-adrenocorticotropin was made in this laboratory from the 17-39 C-terminal fragment of ACTH (
In the single-labeling procedure for c-fos, the cells were washed twice with 0.05 M TBS, pretreated with 3% H2O2 in H2O, pretreated with 0.3% Triton X-100 (Sigma) in 0.05 M TBS, and then incubated with 1:1200 anti-fos for 30 min at 37C. The cells were then treated with biotinylated goat anti-rabbit IgG (1:100) (Vector Laboratories; Burlingame, CA) for 30 min at room temperature (RT) and then exposed to streptavidin-peroxidase (1:100) (Dako; Carpinteria, CA) for 30 min at RT. Then the cells were exposed to the peroxidase substrate, nickel-intensified black DAB (Sigma), for 6 min at RT. The DAB was made by dissolving one DAB tablet in 30 ml 0.05 M acetate buffer with 0.45 g nickel ammonium sulfate and 20 µl of H2O2. The mixture was filtered with Whatman paper. For single-labeling experiments, the cells on these coverslips were then dehydrated, dried, and the coverslips were attached to slides with Permount.
In the dual-labeling procedure, the cells were washed with 0.05 M TBS after DAB treatment and then incubated in the primary antisera (see above for types and dilutions) for 30 min at 37C. This was followed by an incubation in biotinylated goat anti-rabbit IgG (1:100) for 30 min at RT, followed by incubation with streptavidin-peroxidase (1:200) for 30 min at RT. The cells were then treated with amber DAB as in previous studies (
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 fos labeling, the diluted primary antibody was omitted or absorbed with 10 ng/ml fos protein (Oncogene Science). For dual labeling controls, the primary antisera were omitted as well. In addition, for the anterior pituitary hormones, the antisera had been pre-absorbed with each antigen as previously described (
Statistical Analysis
A single experiment utilized cells from one or two female rats in each stage of the estrous cycle. The experiments were repeated until data from at least six rats/stage of the cycle were collected and averaged. The cells were counted with an oil-immersion objective. The fields were scanned randomly across the slide to avoid overlap. The counts included the first labeled and unlabeled cells encountered in each field. Cell counts were performed until at least 100 cells had been counted for each coverslip. Each experiment had three coverslips. Significant differences were tested by ANOVA, followed by the Fischer's least significant difference (LSD) post hoc test to determine significant differences between individual groups. Significance was determined at p<0.05.
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Results |
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Changes in Percentages of c-fos in Anterior Pituitary Cells
Counts of cells immunolabeled for c-fos antigen alone showed differences in their expression when different stages of the cycle were compared. In populations from metestrous rats collected at 1000 hr, c-fos expression was found in 16% of the pituitary cells. However, populations from proestrous rats contained 26% cells with c-fos antigens. Percentages of fos-bearing cells in estrous rat populations were lower, at levels similar to those in the metestrous group. These data are shown in Figure 1. Figure 2 and Figure 3 show fields from metestrous and proestrous rats, respectively, illustrating the increased number and labeling density of fos reactive cells. The inset in Figure 3 shows a higher magnification of the single label for fos in the nucleus.
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Expression of c-fos in Specific Pituitary Cell Types
The single-labeling experiments suggested that the cells from proestrous rats contained the greatest number of fos-expressing cells. The pituitary at this stage of the cycle contains activated gonadotropes that are synthesizing receptors and gonadotropin antigens (
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The data in Figure 4 therefore show that in diestrous and proestrous rats, cells with ACTH, TSH, PRL, and GH antigens were most abundant among the fos-bearing cells. Gonadotropes bearing LH or FSH antigens were least abundant in the diestrous and proestrous groups. Note that the sum of the percentages of dual-labeled individual cell types (35.5-39%) does not add to the values reported as the overall percentages of c-fos-expressing cells (19-26%; Figure 1). This is because a number of these pituitary antigens are stored together, including ACTH and TSH, prolactin and GH, TSH and GH or PRL, and gonadotropins and ACTH or GH, and LH and FSH (reviewed in
Other stages of the cycle were examined to determine if there were changes in the expression of fos by gonadotropes. Figure 5 illustrates the changes in expression of fos by cells with LH antigens. In populations from metestrous rats, there were 8.8 ± 0.6% LH cells. The percentage expressing LH and fos proteins was 4 ± 0.6%, or 45% of LH cells. This is significantly higher than the expression in the proestrous cultures. In the proestrous group, there were 13 ± 0.9% LH cells, but only 3 ± 0.7% of pituitary cells expressed LH and fos, which represents only 23% of LH cells. Similarly, in estrous rats, there were only 7.5% ± 0.7% LH cells and 2.75 ± 0.8% with LH and fos. The proportion of LH cells expressing fos was relatively high, 36%, compared with the expression in proestrous cultures. Therefore, the level of expression in LH cells from estrous and metestrous rats suggests that a significant subset of these cells is being activated. Figure 6 and Figure 7 illustrate LH cells expressing fos from metestrous (Figure 6) and proestrous (Figure 7) rats. Note that the labeling for LH (seen as gray in these black-and-white photographs) is more intense in the cells from the proestrous group (Figure 7). This also illustrates the changes in intensity of stored LH as reported previously (
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In contrast, fos was seen with FSH in only 0.8-1.26% of pituitary cells throughout all stages of the estrous cycle (Figure 8). At most, these values represent only 5.7-12% of FSH antigen-bearing cells. Figure 9 shows an FSH cell dual labeled for fos and FSH taken from the proestrous group. The labeling for FSH is dense, which is typical of that seen in proestrus.
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Discussion |
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Fos Expression Along the Reproductive Axis
This study was stimulated by work that showed GnRH-mediated increases in immediate early response gene expression in the pituitary (
In the pituitary, T3-1). The work with the cell line was strong evidence that gonadotropes themselves can be stimulated by GnRH to produce the early response gene products. However, cytochemical proof would be needed before one can conclude that the GnRH stimulates fos production by normal gonado-tropes. It is possible that GnRH could have stimulated gonadotropes to produce a regulatory factor that would stimulate c-fos expression in another cell type.
The hypothesis in this study was that if GnRH stimulates fos expression in gonadotropes, then fos proteins might be highest in gonadotropes that are expressing peak receptivity for the neuropeptide (during the morning of proestrus) (
Our cytochemical studies showed that fos protein activity is highest in cells from proestrous rats. However, the dual-labeling evidence showed that only a few gonadotropes contributed to this high level. This raises a question about fos as a third messenger in activation of the gonadotrope during proestrus. Further work would be needed to determine if GnRH can stimulate fos expression by gonadotropes from proestrous rats.
The only significant expression by gonadotropes was seen in LH cells from metestrous rats. In this population, there were 8.8 ± 0.6% LH cells with 4 ± 0.6% also expressing fos. This is 45% of LH cells. Similarly, in estrous cultures, 36% of LH cells express fos proteins. This is significantly higher than the expression seen in the proestrous cultures, in which only 23% of LH cells express fos.
The expression of fos in normal LH cells that was first reported in the T3-1 gonadotrope cell line (
Cells with FSH antigens showed little evidence of fos expression during any of the times tested in this study. The low expression by FSH cells suggests that fos is mainly found in monohormonal LH cells. Such cells are abundant early in the cycle and may be considered "immature gonadotropes" (
Identity of the fos-expressing Cells in Diestrus and Proestrus
In light of the above data, it appears that most of the fos expression in cells from proestrous rats may be related to the activation of one or all of the other pituitary cell types. Fos protein was detected with ACTH, GH, PRL, or TSH in 6-9% of pituitary cell types during diestrus and proestrus. These percentages are significantly higher than those found when LH or FSH is detected. This broad distribution of fos agrees with results from other studies of its expression by pituitary cells. A number of studies have reported that corticotropes express c-fos and that this expression is modulated by corticotropin-releasing hormone (CRH). (
TSH cells also express fos proteins.
The neuropeptide secretagogue TRH will stimulate both c-jun and c-fos in GH- and PRL-secreting tumor cells (GH3 cells) (
Because many of the above studies used tumor cell lines, this is the first report of fos protein immunolabeling in all subsets of normal pituitary cells. Furthermore, our studies are the first to show differential expression in vitro when cells from different stages of the estrous cycle are compared. PRL and GH cells were among the most numerous fos-expressing cells. Their expression of fos may be related to luteotrophic functions of PRL cells (
Cautions in Interpretation of These Data
The working hypothesis in this study was tested with the use of dissociated whole pituitary cells that were freshly plated for 1 hr.
The expression of fos proteins may be brief in a given cell (or population) (
Another caveat should be noted. All of the c-fos-expressing cells in the proestrous rat groups might not have been identified with the dual-immunolabeling protocols. Some cells may express fos proteins and only the mRNA for the hormones in question. Future studies with dual in situ hybridization and c-fos antigen detection may identify cells that express hormone mRNAs, but not the products of translation. It is unlikely that these would include gonadotropes, because expression of gonadotropin mRNAs is relatively low early in proestrus (
Summary and Conclusions
In conclusion, our data suggest that fos expression may be associated with the activation of ACTH, TSH, GH, and PRL in cultured cells from diestrous and proestrous rats. It is associated with activation of 36-45% of LH cells in populations from metestrous or estrous rats. Its relative absence in FSH antigen-bearing cells suggests that it may be predominantly expressed in LH cells that are monohormonal. At this point there is no evidence that fos expression is associated with the activation of FSH protein synthesis normally seen during metestrus and early diestrus. However, further studies will be needed to determine if fos is a third messenger for FSH cells. These data point to a mechanism behind control of nonparallel synthesis and release of gonadotropins from cells that can potentially produce both.
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Acknowledgments |
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Supported by NIH R01 HD 15472 and by a developmental grant from the Sealy Smith Foundation.
We thank Ms Geda Unabia and Ms Diana Rougeau for excellent technical assistance during these studies. We also thank Dr J.G. Pierce and Dr A.F. Parlow for the antisera to LH and FSH, respectively. We thank the Hormone Distribution Program for antisera and antigens to pituitary hormones.
Received for publication October 21, 1996; accepted January 6, 1997.
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