ARTICLE

Differential Expression of c-fos In Vitro by All Anterior Pituitary Cell Types During the Estrous Cycle: Enhanced Expression by Luteinizing Hormone but Not by Follicle-stimulating Hormone Cells

Correspondence to: Jennifer Armstrong, Dept. of Anatomy and Neurosciences, MRB 10-104, 303 University Blvd., U. of Texas Medical Branch, Galveston, TX 77555.

	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

	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 (Bishop 1983 ). C-fos is homologous to v-fos; both are found in the nucleus and are able to induce transformation, but the two genes differ at the COOH terminal (Kruijer et al. 1985 ). C-fos, along with c-jun and jun-B genes, is called a primary or immediate early response gene. These genes encode for transcriptional regulatory proteins (third messengers) that control secondary (late) response genes. C-fos can be activated by various second messenger signals, including calcium, protein kinase C, and cAMP. Specifically, when activated, c-fos encodes for a nuclear protein fos that dimerizes with the phosphorylated Jun protein to form the active gene regulatory protein called AP-1. AP-1 then stimulates transcription by binding to specific sites on DNA (Rauscher et al. 1988 ). Expression of c-fos antigens occurs once the cell has been stimulated. Changes that can occur in cell function include mitosis, synthesis of proteins, and secretion of proteins. The cell becomes cancerous if these immediate-early response genes are overstimulated or damaged.

C-fos expression has been found in cells that are active along the hypothalamic-pituitary-reproductive axis. Hoffman et al. 1990 found c-fos expression in gonadotropin-releasing hormone (GnRH) neurons that had been stimulated by both estrogen and progesterone. Expression of the oncogene indicated that the GnRH neuron was activated. Similarly, Lee et al. 1990 found that GnRH neurons expressed c-fos during the proestrous surge. The expression of c-fos was not seen during any other stage of the cycle, as would be expected because GnRH neurons are most active at proestrus during the preovulatory surge.

Two groups have demonstrated c-fos expression in anterior pituitary gonadotropes. Padmanabhan et al. 1995 showed increases in c-fos and c-jun mRNA levels correlated with increases in mRNA for GnRH receptors in cycling female sheep. In addition, c-fos remained elevated from Day 8 before estrus until the onset of estrus. Cesnjaj et al. 1994 reported GnRH-stimulated changes in c-fos in primary pituitary cell cultures from randomly cycling female rats. They also reported c-fos, c-jun, and jun-B activation in the 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 (Cesnjaj et al. 1994 ), and phospholipase D (Cesnjaj et al. 1995 ).

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.

	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 (Childs et al. 1987 ). The protocol normally yielded 2-3 million cells/pituitary that were 98% viable, as tested by the trypan blue exclusion test. Cells were suspended in DMEM containing .005 mg/ml insulin (Sigma), 0.05 mg/ml transferrin (Sigma), and 30 mM sodium selenite (Johnson Matthey Chemical; New York, NY). Cells were then plated onto poly-D-lysine (Sigma)-coated glass coverslips (AH Thomas Scientific; Suredesboro, NJ) in 24-well trays at a density of approximately 40,000-50,000 cells/50 µl/well.

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 (Moriarty and Halmi 1972 ) and was used at a dilution of 1:30 K. Rabbit anti-rat PRL, used at a dilution of 1:40 K, and rabbit anti-rat GH, used at a dilution of 1:35 K, were purchased from Chemicon (Temecula, CA).

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 (Childs et al. 1994a ).

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 (Childs et al. 1994b ). All controls showed no labeling for the antigen in question.

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.

	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.

View larger version (52K): [in this window] [in a new window]   Figure 1. Percentage of total pituitary cells labeled for fos proteins during the estrous cycle one hour after plating. *, significantly different from the values in the metestrous group (p<0.05).

View larger version (59K): [in this window] [in a new window]   Figure 2. Field showing single labeling for fos protein from a metestrous group of rats. Arrow indicates fos nuclear staining. U, control cell without fos label. Bar = 10 µm. Figure 3. Field showing labeling for fos in cells from proestrous rats. Arrow indicates intense nuclear staining for fos protein. U, control cell. (Inset) A higher magnification of cells expressing nuclear fos protein. Bar = 10 µm.

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 (Childs et al. 1994b ; Lloyd and Childs 1988 ). However, dual labeling was needed to determine if gonadotropes contributed to the fos-expressing cells. We focused on two of the stages, diestrus and proestrus, to determine which cells expressed the fos protein. During diestrus, fos was localized in subsets of all of the hormone-bearing cells. The percentages of pituitary cells with fos and each of the hormones are as follows: 7.6 ± 1.4% ACTH; 6.8 ± 1.2% PRL; 8.4 ± 1.4% TSH; 8.6 ± 1.8% GH; 0.8 ± 0.2% FSH; and 3.3 ± 0.5% LH. In the group of cells from proestrous rats, the percentages of each type of dual-labeled cells were similar, although the percentages of cells bearing fos proteins and PRL were 9.4 ± 1.7%. Statistical analyses showed that this apparent increase was not significantly higher than percentages of such cells from diestrous rats. Figure 4 illustrates these findings.

View larger version (45K): [in this window] [in a new window]   Figure 4. Counts of cells dual labeled for fos and ACTH, PRL, TSH, GH, FSH, or LH antigens. Cell populations were taken from diestrous and proestrous rats. No significant differences in the percentages of cells were noted when the two stages of the cycle were compared.

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 Childs et al. 1994a ). Therefore, some of the c-fos-expressing cells may be multihormonal and may store two or more of the above hormones.

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 (Childs et al. 1987 ).

View larger version (56K): [in this window] [in a new window]   Figure 5. Percentages of pituitary cells dual labeled for LHß antigens and fos taken from groups throughout the estrous cycle. The overall percentages of dual-labeled cells did not change. However, the relative proportion of LH cells that expressed fos was significantly higher in the metestrous group compared to other stages of the cycle (see text). In all cases, the percentages of cells dual labeled for LH and fos were greater than those seen in the FSH cell population.

View larger version (108K): [in this window] [in a new window]   Figure 6. Field showing a cell dual labeled for fos and LHß, taken from a metestrous rat. Intense black label for fos is indicated by the arrow pointing to the nucleus. The orange label for the LH protein is shown in these black-and-white photographs by paler gray labeling of the cytoplasm. F, a cell that contains fos but no LH; U, control cell. Bar = 5 µm.

View larger version (52K): [in this window] [in a new window]   Figure 7. Cell from a proestrous rat showing dual labeling for fos and LHß. Labeling for the LH antigens is denser in the proestrous group. U, control cell. Bar = 5 µm.

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.

View larger version (29K): [in this window] [in a new window]   Figure 8. Percentages of pituitary cells dual labeled for fos and FSHß throughout the estrous cycle. No significant differences in the percentages were detected. These percentages represent only 5.7-12% of FSH antigen-bearing cells, which is relatively low compared with all other cell types.

View larger version (61K): [in this window] [in a new window]   Figure 9. Field showing cells dual labeled for fos and FSH antigens. Arrow indicates fos antigen nuclear label; paler gray label indicates FSH antigens. U, control cell. Bar = 5 µm.

	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 (Cesnjaj et al. 1994 ). It was designed to ascertain if fos protein expression varied with the stage of the estrous cycle. It was also designed to identify the cell types involved. Recent studies of GnRH neurons and gonadotropes showed that steroids and neuropeptides will activate expression of c-fos in cells along the reproductive axis. Hoffman et al. 1990 found c-fos expression in GnRH neurons that had been stimulated by both estrogen and progesterone. Similarly, GnRH neurons expressed c-fos during the proestrous surge (Lee et al. 1990 ).

In the pituitary, Padmanabhan et al. 1995 reported enhanced c-fos expression just before estrous in cycling sheep. Cesnjaj et al. 1994 , Cesnjaj et al. 1995 discovered that GnRH and its second messengers stimulated c-fos expression in primary cultures of gonadotropes from (randomly cycling) female rats. GnRH also stimulated expression of c-fos, c-jun, and jun-B mRNAs in a gonadotrope cell line (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) (Childs et al. 1994b ; Lloyd and Childs 1988 ). In designing these experiments, however, additional times were tested as potential points at which gonadotropes may be activated. For example, gonadotropes translate gonadotropin mRNA (Childs et al. 1992a , Childs et al. 1992b , Childs et al. 1994a ), and produce receptors for estradiol (Kikuta et al. 1993 ) and GnRH (Lloyd and Childs 1988 ) during diestrus. Peak transcriptional activity for gonadotropin ß-subunit mRNA occurs during the LH surge late in proestrus (for LHß mRNA) or during the FSH rise in early estrus (for FSHß mRNA) (reviewed in Childs et al. 1994a ).

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 (Cesnjaj et al. 1994 ) is therefore confirmed. However, metestrus represents a nadir for GnRH receptivity. Therefore, the factors that regulate this early expression are not known. Perhaps the fos expression by metestrous or estrous LH gonadotropes reflects its involvement in the synthesis of LHß proteins (Childs et al. 1994a ), and/or estradiol (Kikuta et al. 1993 ) or GnRH receptors (Childs et al. 1994b ; Lloyd and Childs 1988 ). With respect to the last possibility, our studies agree with Padmanabhan et al. 1995 , who correlated the time of enhanced expression of c-fos in sheep with enhanced production of GnRH receptor mRNA.

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" (Childs et al. 1992a , Childs et al. 1994a ). It is possible that FSH cells will express fos protein just before peak transcription of FSHß mRNA (midnight to 0400 hr of estrus), or after specific stimulation.

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). (Bou-tillier et al. 1995 ; Ruzicka and Akil 1995 ; Autelitano and Sheppard 1993 ). Glucocorticoids will repress the CRH-induced increase in fos (Autelitano 1994 ). Several groups have reported stress-induced increases in c-fos in the anterior pituitary (Pan et al. 1994 ; Senba et al. 1994 ; Handa et al. 1993 ). Kjaer et al. 1994 showed that histamine activated c-fos expression in association with ACTH release.

TSH cells also express fos proteins. Kim et al. 1993 showed that AP-1 and Pit 1 transcription factors are both needed to induce expression of the human TSH-ß gene. Luo and Jackson 1995 used double-label in situ hybridization to detect the neuropeptide thyrotropin-releasing hormone (TRH) and c-fos in a subset of cells in the anterior pituitary gland. Previously, May et al. 1987 from our laboratory had shown that TRH can be found in cells with TSH, LH, or ACTH antigens.

The neuropeptide secretagogue TRH will stimulate both c-jun and c-fos in GH- and PRL-secreting tumor cells (GH₃ cells) (Passegue et al. 1994 , Passegue et al. 1995a , Passegue et al. 1995b ; Li et al. 1994 ; Carr et al. 1993 ). Todisco et al. 1994 reported that somatostatin inhibited both c-fos expression and AP-1 binding in GH₃ cells. Chernavsky et al. 1993 showed that in normal rats c-fos expression was induced by factors that stimulated PRL synthesis or secretion (estrogens and haloperidol).

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 (Ueda et al. 1985 ; Smith et al. 1976 ) or gonadotropic functions of growth hormone cells (Childs et al. 1994a , Childs et al. 1994b ). Prolactin has been shown to decrease estrogen biosynthesis before proestrus (Tsai-Morris et al. 1983 ; Uilenbroek et al. 1982 ) and can also shorten a 5-day estrous cycle to a 4-day estrous cycle (Boehm et al. 1984 ).

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. Cesnjaj et al. 1994 had reported that c-fos mRNA was stable in culture in serum-free media for 6 hr. Then, expression declined unless serum was added to the media. The whole-cell approach used in the present study facilitated the detection of fos and hormone antigens in the same cells and prevented artifacts that are sometimes associated with analysis of dual labeling in thick paraffin or frozen sections.

The expression of fos proteins may be brief in a given cell (or population) (Auger and Blaustein 1995 ; Morishita et al. 1995 ; Van Der Beek et al. 1994 ; Cesnjaj et al. 1993 ; Sagar et al. 1991 ). Therefore, plating time was limited to the minimum required to allow the cells to adhere to the coverslips. In spite of these precautions, these data should be interpreted cautiously. The in vitro approach might have altered differential expression of the fos antigens and may not reflect changes in expression in vivo. Subsequent studies of c-fos expression in tissue sections will be needed to correlate any changes in vivo seen during the estrous cycle. If the culture conditions altered fos expression, it should be noted that cells from proestrous rats were most receptive to the activation.

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 (Childs et al. 1994a ).

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.

	Acknowledgments

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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