Development of gonadotropes may involve cyclic transdifferentiation of growth hormone cells.

The cyclic rise in expression of anterior pituitary gonadotropins coincides with the appearance of cells sharing gonadotropic and somatotropic phenotypes. To learn more about possible factors that regulate the origin of this cell type, we studied the time of appearance of cells that co-expressed growth hormone (GH) and gonadotropins and estrogen receptors during the estrous cycle and compared this timing with known changes in regulatory hormones or their receptors. The first event in this cell population is an increase in expression of estrogen receptor (ER)beta by GH cells from estrus to metestrus suggesting that estrogen may mediate this early change. Expression of GH mRNA rises rapidly from metestrus to mid-cycle. The rise is seen first in GH cells and then in cells with luteinizing hormone (LH) antigens. These data suggest that, early in the cycle, cells bearing GH and growth hormone releasing hormone (GHRH) receptors begin to produce LH and gonadotropin releasing hormone (GnRH) receptors. Early in proestrus, there is an increase in cells with GH and follicle-stimulating hormone (FSH) suggesting that this set of multipotential cells develops later than GH-LH cells. This fits with earlier studies showing the later rise in expression of FSH mRNA. Collectively these data suggest that the anterior pituitary contains a subset of GH cells that have the capacity to respond to multiple releasing hormones and support more than one system.

The use of counterflow centrifugation to enrich gonadotropes and somatotropes.

Counterflow centrifugation produces populations of gonadotropes or growth hormone (GH) cells enriched to 90% in a Beckman elutriator. The pituitary populations are first separated by size into three fractions applying different flow rates, stimulated with either gonadotropin-releasing hormone (GnRH) to enlarge the gonadotropes or growth hormone-releasing hormone (GHRH) to enlarge the somatotropes for 3 hr. The fractions are re-eluted, first at the original flow rates and then at higher flow rates to separate enlarged gonadotropes or somatotropes. Most other cell types are reduced to less than 5%. However, co-storage of GH and gonadotropin antigens is seen in either population. Enriched gonadotropes or somatotropes can be used in studies of proliferation, autocrine or paracrine regulation, or ion channel functions.(J Histochem Cytochem 49:663-664, 2001)

Differential expression of estradiol receptors alpha and beta by gonadotropes during the estrous cycle.

This study focused on expression of estradiol receptors (ER) during the estrous cycle. Labeling for ERalpha or beta antigens and luteinizing hormone (LH) or follicle-stimulating hormone (FSH) beta-subunits was done on freshly dispersed pituitary cells. The lowest expression of ERalpha and beta was seen in estrus (23% and 12%, respectively). Expression increased to 42-54% of pituitary cells by diestrus. In males, cells with ERalpha or beta were 37% or 20% of the population, respectively. ERalpha or beta and gonadotropin antigens were in 6-9% of pituitary cells from male rats. Early in the cycle (estrus and metestrus), less than 5% of pituitary cells expressed ERalpha or beta with gonadotropins. These values doubled to reach a peak of 10% during proestrus (just before ovulation). These data show that a rise in expression of both ERalpha and ERbeta is a part of preovulatory differentiation of pituitary gonadotropes.(J Histochem Cytochem 49:665-666, 2001)

Sites of epidermal growth factor synthesis and action in the pituitary: paracrine and autocrine interactions.

1. Epidermal growth factor (EGF) is produced by growth hormone (GH) cells and gonadotropes in normal pituitary cell populations. The studies were initiated to determine whether EGF is a paracrine or autocrine regulator of gonadotrope function. 2. The first group of studies tested for the presence of EGF receptors in gonadotropes from cycling female rats by immunolabelling. Expression varied with the stage of the cycle. At the highest point (metoestrus), only a few EGF target cells are gonadotropes, identified by their content of luteinizing hormone (LH)-beta mRNA. Expression by gonadotropes then increased to reach a peak of 50% of cells during pro-oestrus. 3. Studies investigating the regulation of expression of EGF receptor (R) showed that all culture conditions (in media with or without serum) and EGF itself both stimulated expression of the receptor by gonadotropes in populations from oestrus or metoestrus rats. Gonadotropin-releasing hormone (GnRH) also stimulated EGFR expression in follicle-stimulating hormone (FSH) gonadotropes from oestrus animals. Additional tests of expression of immediate early genes (c-fos) showed that, after 15 min, EGF stimulated expression in cells with FSH antigens. 4. Epidermal growth factor also stimulated gonadotrope proliferation, as detected by the MTT cell growth/cell death assays and bromodeoxyuridine uptake by gonadotropes during the S phase (DNA synthesis) of the cell cycle. 5. Epidermal growth factor and GnRH both stimulated a significant increase in the percentage of mitotic gonadotropes. Epidermal growth factor may be an autocrine or a paracrine growth factor to maintain and develop the gonadotrope population and EGF may also be involved in early differentiation events that prepare cells to support the LH surge.

Epidermal growth factor and gonadotropin-releasing hormone stimulate proliferation of enriched population of gonadotropes.

Recent studies of epidermal growth factor (EGF) receptors on gonadotropes show that they appear early in the estrous cycle on immature gonadotropes, most of which could be identified by LH messenger RNA only. As diestrous gonadotropes translate the messenger RNAs, the percentages of LH and FSH cells with EGF receptors increase to reach a peak during proestrus. To learn more about the function of EGF in gonadotrope regulation, parallel studies of its mitogenic potential were conducted. To test this in a cell growth assay, we initially developed a protocol for enrichment of gonadotropes by counterflow centrifugation (elutriation). Analysis of immunolabeled cells in the enriched fraction showed that the population contained 90-95% cells with LH and/or FSH antigens. Less than 4% have TSH or PRL antigens, and less than 7% have ACTH antigens. About 15% of the enriched population expressed GH antigens in male rats and nearly 30% of the population express GH in females. This agrees with the known hormone storage overlap between these cells, especially in proestrous female rats. The MTT cell growth/cell death assay was then used to test the mitogenic potential of EGF, GnRH, and activin. This assay showed a linear relationship between plated cell numbers and optical density of the media after the MTT reaction was run. The enriched gonadotropes were plated in 96-microwell trays and grown for 3-4 days in the presence of defined media alone (no serum), or defined media containing 0.5-10 ng/ml EGF, 0.5-1 nM GnRH, 60 ng/ml activin or two of these factors. In all of the 12 experiments, each of the factors stimulated a 3- to 10-fold increase in optical density values, depending on the dose of the stimulating factor. The effects of any two factors were not additive. Because the MTT assays do not discriminate between mitogenic effects and enhanced cell survival, a second group of tests was run with mixed cultures of pituitary cells from diestrous female rats. These cells were cultured in the same combinations of EGF with and without GnRH for 3 h. During the last hour of culture, they were exposed to bromodeoxyuridine (BrDU) to identify cells that were synthesizing DNA. Cells in the S phase were thereby detected with dual immunocytochemical labeling for nuclear BrDU and gonadotropins. The analysis of dual labeled cells showed a 3-fold increase in percentage of LH or FSH cells with BrDU labeled nuclei following EGF or GnRH stimulation. The effects of the two growth factors were not additive. Collectively, these data confirm previous studies showing mitogenic functions for activin and now add EGF and GnRH as mitogens for gonadotropes.

Growth hormone cells as co-gonadotropes: partners in the regulation of the reproductive system.

Through unique receptors, growth hormone (GH) stimulates ovarian follicles and Leydig cells, working alone or synergistically with luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The source of GH might include a unique cell type that expresses mRNA encoding gonadotropin and GH and the antigens themselves, together with gonadotropin-releasing hormone (GnRH) and GH-releasing hormone (GHRH) receptors. This multifunctional cell might provide a cocktail of hormones needed to effect optimal gonadotropic activity.

Differential expression of growth hormone messenger ribonucleic acid by somatotropes and gonadotropes in male and cycling female rats.

Past studies have reported the appearance of cells sharing phenotypic characteristics of gonadotropes and GH cells. During diestrus and early proestrus, a subset of somatotropes (40-60%) expressed both GH antigens and gonadotropin (LH-beta, LHbeta, or FSH-beta) messenger RNAs (mRNAs) or GnRH receptors. More recently, we reported that subsets of gonadotropes identified by LHbeta or FSHbeta antigens expressed GH- releasing hormone (GHRH) binding sites. The present studies were designed to learn if these putative multipotential cells also expressed GH mRNA. Biotinylated sense and antisense oligonucleotide probes were developed and cytochemical in situ hybridization tests were optimized for the detection of GH mRNA with GH, LHbeta, and FSHbeta antigens. RNase protection assays were developed with a complementary RNA probe that detected a 380-bp region at the 5' end of the GH mRNA. Both the in situ hybridization and RNase protection assays detected changes in expression of GH mRNA during the estrous cycle with the lowest expression occurring during metestrus and peak expression occurring on the morning of proestrus. Cell counts confirmed the results of the RNase protection assays showing that increases in mRNA levels seen from metestrus to proestrus reflected increased percentages of GH mRNA-bearing cells. In addition, densitometric analyses demonstrated that the higher GH mRNA levels assayed from diestrus to proestrus reflected increased area and density of label per cell. Both types of assays showed sex differences in expression of GH mRNA; male rat cell populations had higher values than female rats in metestrus, diestrus, or estrus. However, percentages of GH cells in male rats were equal to those from proestrous female rats and levels of GH mRNA were lower in male rats than proestrous females. Dual labeling experiments showed that, in male rats and diestrous, proestrous, or estrous females, GH mRNA was expressed in over 70% of GH cells. Expression of GH mRNA was also found in 50-57% of cells with LHbeta or FSHbeta antigens in the same groups. The lowest expression was seen in the metestrous groups (30-40% of GH cells or gonadotropes expressed GH mRNA). Expression of GH mRNA was first increased from metestrus to diestrous largely in GH cells, and slightly in cells with LHbeta antigens. Further increases were seen in GH and LH cells by the morning of proestrus. In contrast, FSH gonadotropes did not show an increased expression of GH mRNA until the morning of proestrus (reaching the same peak reached by LH cells). These data confirm the working hypothesis that a multihormonal cell type develops during diestrus to support both the somatotrope and gonadotrope populations. Collectively, our studies suggest that this multihormonal cell may function to help support the regulatory functions of the gonadotrope during the periovulatory period. In addition, the appearance of significant levels of expression of GH mRNA by male rat gonadotropes suggests that this multihormonal cell may play a role in regulation of the male reproductive system as well.

Differential expression of gonadotropin and prolactin antigens by GHRH target cells from male and female rats.

There is a 2- to 3-fold increase in luteinizing hormone-beta (LHbeta) or follicle-stimulating hormone-beta (FSHbeta) antigen-bearing gonadotropes during diestrus in preparation for the peak LH or FSH secretory activity. This coincides with an increase in cells bearing LHbeta or FSHbeta mRNA. Similarly, there is a 3- to 4-fold increase in the percentage of cells that bind GnRH. In 1994, we reported that this augmentation in gonadotropes may come partially from subsets of somatotropes that transitionally express LHbeta or FSHbeta mRNA and GnRH-binding sites. The next phase of the study focused on questions relating to the somatotropes themselves. Do these putative somatogonadotropes retain a somatotrope phenotype? As a part of ongoing studies that address this question, a biotinylated analog of GHRH was produced, separated by HPLC and characterized for its ability to elicit the release of GH as well as bind to pituitary target cells. The biotinylated analog (Bio-GHRH) was detected cytochemically by the avidin-peroxidase complex technique. It could be displaced by competition with 100-1000 nM GHRH but not corticotropin-releasing hormone or GnRH. In cells from male rats exposed to 1 nM Bio-GHRH, 28+/-6% (mean+/-s.d) of pituitary cells exhibited label for Bio-GHRH (compared with 0.8+/-0.6% in the controls). There were no differences in percentages of GHRH target cells in populations from proestrous (28+/-5%) and estrous (25+/-5%) rats. Maximal percentages of labeled cells were seen following addition of 1 nM analog for 10 min. In dual-labeled fields, GHRH target cells contained all major pituitary hormones, but their expression of ACTH and TRH was very low (less than 3% of the pituitary cell population) and the expression of prolactin (PRL) and gonadotropins varied with the sex and stage of the animal. In all experimental groups, 78-80% of Bio-GHRH-reactive cells contained GH (80-91% of GH cells). In male rats, 33+/-6% of GHRH target cells contained PRL (37+/-9% of PRL cells) and less than 20% of these GHRH-receptive cells contained gonadotropins (23+/-1% of LH and 31+/-9% of FSH cells). In contrast, expression of PRL and gonadotropins was found in over half of the GHRH target cells from proestrous female rats (55+/-10% contained PRL; 56+/-8% contained FSHbeta; and 66+/-1% contained LHbeta). This reflected GHRH binding by 71+/-2% PRL cells, 85+/-5% of LH cells and 83+/-9% of FSH cells. In estrous female rats, the hormonal storage patterns in GHRH target cells were similar to those in the male rat. Because the overall percentages of cells with Bio-GHRH or GH label do not vary among the three groups, the differences seen in the proestrous group reflect internal changes within a single group of somatotropes that retain their GHRH receptor phenotype. Hence, these data correlate with earlier findings that showed that somatotropes may be converted to transitional gonadotropes just before proestrus secretory activity. The LH and FSH antigen content of the GHRH target cells from proestrous rats demonstrates that the LHbeta and FSHbeta mRNAs are indeed translated. Furthermore, the increased expression of PRL antigens by these cells signifies that these convertible somatotropes may also be somatomammotropes.

Expression of the L-type Ca(2+) channel in AtT-20 cells is regulated by cyclic AMP.

Activation of adenylyl cyclase by corticotropin-releasing hormone (CRH) stimulates secretion of adrenocorticotropin (ACTH) in rat anterior pituitary corticotropes and in the murine AtT-20 cell line. The stimulation of secretion is mediated by cAMP and is largely dependent on Ca(2+) influx through voltage-gated L-type Ca(2+) channels. To investigate whether CRH and cAMP also increase expression of the L-type Ca(2+) channel in AtT-20 cells, an RNase protection assay was used to measure the alpha(1C) mRNA that encodes the pore-forming subunit of the L-type Ca(2+) channel. The alpha(1C) mRNA level was measured by autoradiographic densitometry and normalized to the beta-actin mRNA level in the same sample. The alpha(1C) mRNA was not changed by 24-hour treatment with CRH (10-500 nM). A 24-hour treatment with 1 mM 8Br-cAMP significantly increased the alpha(1C) mRNA by 40% over its control. The stimulatory effect was blocked by 2 microM actinomycin D and was, therefore, dependent on gene transcription. The measured half-life of the alpha(1C) mRNA, after inhibition of transcription, was 4.7 +/- 0.3 h in control and 5.2 +/- 0.6 h in the presence of 8Br-cAMP. Thus the 8Br-cAMP- induced increase in alpha(1C) mRNA could be due to an increase in alpha(1C) gene transcription or to a transcriptionally regulated increase in a protein that helps to stabilize alpha(1C) mRNA. Finally, to determine if the increased mRNA was followed by an increase in production of L-type Ca(2+) channels, the binding of [(3)H]PN200-110 to Ca(2+) channel proteins was assayed in AtT-20 membrane fragments. 8Br-cAMP increased [(3)H]PN200-110 binding sites by 32% (B(max) 36.0 +/- 1.2 fmol/mg protein in control vs. 47.4 +/- 3.2 fmol/mg protein in 8Br-cAMP-treated cells) but did not change the K(d). These studies show that both alpha(1C) mRNA and L-type Ca(2+) channel protein are increased in AtT-20 cells by cAMP.

Cold stress and corticotropin-releasing hormone induced changes in messenger ribonucleic acid for the alpha(1)-subunit of the L-type Ca(2+) channel in the rat anterior pituitary and enriched populations of corticotropes.

In response to stress, adrenocorticotropin (ACTH) is secreted from anterior pituitary corticotropes. Corticotropin-releasing hormone (CRH) is a potent stimulator of ACTH secretion. The CRH stimulation of secretion is mediated by cAMP and is largely dependent on Ca(2+) influx through voltage-gated L-type Ca(2+) channels. This study was designed to investigate whether the expression of L-type Ca(2+) channels in the rat anterior pituitary and in corticotropes is regulated by acute stress and CRH. RNase protection assays were used to quantify alpha(1C) mRNA of the L-type Ca(2+) channel. The alpha(1C) mRNA levels from stressed rats increased by 31% in anterior pituitaries of rats after 30 min of exposure to cold stress. Neither 60 min cold stress nor 30 min restraint stress had an effect on alpha(1C) mRNA levels. When alpha(1C) mRNA was detected by in situ hybridization in a population of corticotropes enriched to 90%, 0.5 nM CRH (3 h) stimulated a 36% increase in the average area of label/cell and a 10% increase in the average density of label. Our results suggest that (1) the expression of alpha(1C) subunit mRNA of L-type Ca(2+) channels is increased in the rat anterior pituitary with a stress-specific response that might reflect an increase both in thyrotropes and corticotropes (both are known to be stimulated by cold stress), and (2) the CRH-mediated increase in alpha(1C) mRNA expression in individual rat corticotropes, in vitro, supports the hypothesis that some of the increase in vivo is due to changes in corticotropes.

Regulation of c-fos expression by EGF and GnRH in specific anterior pituitary cells from proestrous female rats.

C-fos is an early expression oncogene that can be stimulated by a variety of regulators. It is expressed by subsets of all pituitary cells, with increased expression seen in proestrous rats. However, in freshly dispersed pituitary cells studied during different stages of the cycle, there is limited expression of fos by luteinizing hormone (LH) cells and little basal expression by cells with follicle-stimulating hormone (FSH) antigens. Proestrus is a time during which pituitary gonadotropes express peak levels of receptors for gonadotropin-releasing hormone (GnRH) and epidermal growth factor (EGF). We hypothesized that if GnRH or EGF stimulated fos activity in gonadotropes they would be most effective during the peak expression of their receptors. Anterior pituitaries were removed, cut into small pieces, and stimulated for 30 min. Total RNA was then collected and analyzed by Northern analysis. Both EGF and GnRH caused an increase in c-fos mRNA levels in the anterior pituitary gland compared with unstimulated pituitary glands assayed immediately after removal from the pituitary. However, the stimulatory effects were no greater than those seen with medium alone. This suggested that fos expression could be stimulated by local factors either in the pituitary or the medium itself. The second phase of the study focused on pituitary cells plated for 1 hr and then stimulated with EGF and GnRH for 15 min. Dual immunocytochemistry was done to learn which cell types expressed the fos proteins. After 15 min, EGF and GnRH both increased the percentages of fos-bearing cells above levels seen in medium alone. EGF stimulated fos proteins in subsets of FSH, adrenocorticotropin (ACTH), and growth hormone (GH) cells. GnRH increased fos proteins in subsets of ACTH and GH cells. These results suggest that EGF and GnRH may regulate fos expression, but not necessarily in gonadotropes. They also highlight the need for carefully timed experiments because endogenous factors in the pituitary itself may stimulate immediate early gene expression. (J Histochem Cytochem 46:935-943, 1998)

Regulation of expression of epidermal growth factor receptors in gonadotropes by epidermal growth factor and estradiol: studies in cycling female rats.

Changes in expression of epidermal growth factor (EGF) receptors by gonadotropes parallel those of GnRH receptors. Gonadotropes increase their expression of EGF receptors (EGFR) during diestrus to reach a peak on the morning of proestrus. This is followed by a decline in expression to reach a nadir by estrus. We hypothesized that regulatory factors that stimulate changes in GnRH receptors might mediate the same changes in EGFR. To test this hypothesis, pituitary cells were collected from cycling rats and grown overnight in media with or without serum, 100 pM estradiol, or 60 ng/ml activin. On the next day, some of the cultures were further stimulated with 1 nM GnRH (4 h). The cells were then dual-labeled for EGFR and LHbeta or FSHbeta antigens and analyzed for their content of EGFR and gonadotropins. Neither activin nor estradiol increased percentages of cells with gonadotropin antigens and EGFR. Estradiol decreased percentages of cells with EGFR and LH in proestrous rats and those with EGFR and FSH in diestrous rats. The estradiol-mediated decline in EGFR expression during proestrus is similar to that seen when GnRH receptors are studied. Serum containing media alone increased percentages of LH and FSH cells with EGFR in populations from estrous or metestrous rats. Therefore, further experiments were conducted to learn if serum factors or EGF might be a regulator. Removal of serum from the growth media did not prevent the increase in percentages of LH cells with EGFR over the 18-h growth period. However, removal of serum did prevent the increased percentages of FSH cells with EGFR. Similarly, adding 1:100 anti-EGF to the serum containing media did not affect expression of EGFR by LH cells. However, it did cause a 27% decrease in percentages of FSH cells with EGFR. Finally, when 10 ng/ml EGF was added to metestrous populations in serum-free media there was a 1.4-1.5-fold increase in percentages of LH or FSH cells with EGFR. Collectively, these studies show that EGF receptors are not stimulated in gonadotropes by the same hormones that up-regulate GnRH receptors. Furthermore, EGF itself may be among the factors that up regulate EGFR in gonadotropes. EGF receptors may be down-regulated by estradiol during proestrus, but the effect is limited to LH cells. Finally, EGF's differential effects on LH and FSH cells suggests that it may selectively act on monohormonal gonadotropes. EGF receptors may be a marker for a unique subset of developing gonadotropes.

Cytochemical studies of multifunctional gonadotropes.

Studies have focused on the roles of the gonadotrope subsets defined by cytochemical and morphological tools. The evidence points to groups of gonadotropes that may be stimulated to mature and secrete to support surge activity. We postulate that these gonadotropes stem from the medium-sized subset. Other gonadotropes may more involved with maintenance functions. Perhaps these come from the larger cell pools. Monohormonal gonadotropes may play unique roles, such as FSH secretion early in estrus. Some may be immature, others may be regulatory and play both paracrine or autocrine roles in the pituitary cell population. We also recognize that one of the limitations of the current-day cytochemical techniques is that it does not define the entire gonadotrope population in any given two-label protocol. Nevertheless, based on past cytochemical studies, assumptions are made about the extent to which the cells express both hormones or behave in a uniform manner. These assumptions have led researchers to focus on one subset of the gonadotrope population. In their attempts to simplify the population to be studied, they may have eliminated important regulatory, secretory, or monohormonal gonadotropes from the pool. The approach is valid, as long as they recognize that they are studying a subset of a complex and dynamic population.

Corticotropin releasing hormone inhibits an inwardly rectifying potassium current in rat corticotropes.

1. The perforated-patch-clamp technique was used to identify an inwardly rectifying K+ current (IK(IR)) in cultured rat anterior pituitary cells highly enriched in corticotropes. IK(IR) was rapidly activating and highly selective for K+. The K+ conductance was approximately proportional to the square root of the extracellular K+ concentration. 2. IK(IR) was blocked in a voltage-dependent manner by external Ba2+ and Cs+, slightly attenuated by 5 mM 4-aminopyridine (15% inhibition) and insensitive to 10 mM tetraethylammonium, 2 mM Ca2+, 1 mM Cd2+ and 50 microM La3+. 3. In physiological saline, 100 microM Ba2+, which inhibits 86% of IK(IR) at the cell resting potential, depolarized cells by 6.1 +/- 0.7 mV from a mean resting potential of -59.6 +/- 0.8 mV. 4. Corticotropin releasing hormone (CRH), which activates adenylyl cyclase and stimulates adrenocorticotropic hormone (ACTH) secretion from corticotropes, inhibited IK(IR) by 25% and depolarized the cells by 10.2 +/- 1.0 mV. Dibutyryl cAMP ((Bu)2cAMP) mimicked these effects. 5. The membrane depolarization evoked by Ba2+ or CRH increased the cell firing frequency. Comparison of cells exhibiting a membrane potential of approximately -50 mV revealed that spike frequency in the presence of CRH (109 +/- 7 spikes (5 min)-1) was greater than in control (60 +/- 5 spikes (5 min)-1) or Ba(2+)-treated (77 +/- 15 spikes (5 min)-1) corticotropes. 6. The data suggest that IK(IR) contributes to maintenance of the resting membrane potential of rat corticotropes. Inhibition of IK(IR) plays a role in, but does not account for all of, the membrane depolarization and enhancement of firing frequency evoked by CRH.

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.

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 percentage 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 LH beta 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 non-parallel storage and release of gonadotropin antigens.

Changes in expression of epidermal growth factor receptors by anterior pituitary cells during the estrous cycle: cyclic expression by gonadotropes.

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 +/- 4% cells labeled for EGFR. This peak was followed by a decline to 17.6 +/- 2% of cells from proestrous rats. The percentages of metestrous pituitary cells with EGFR and each hormone were: PRL, 11.8 +/- 1; ACTH, 9.9 +/- 1.8%; GH, 8.2 +/- 0.6%; TSH, 6.3 +/- 0.8%; FSH, 4 +/- 0.6%; and LH, 2.6 +/- 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 LHbeta or FSHbeta messenger RNAs (mRNAs) showed a significant increase in the percentages of pituitary cells with LHbeta mRNA and EGFR (to 5.7% of pituitary cells), but there were no increases in the EGF target cells bearing FSHbeta 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 LHbeta 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 beta-subunit mRNA seen late in proestrus, early in estrus.

Differential effects of inhibin on gonadotropin stores and gonadotropin-releasing hormone binding to pituitary cells from cycling female rats.

Numerous studies of rat pituitaries have reported that inhibin suppresses the synthesis and release of FSH and decreases the release of LH. The latter effect seems to be related to the down-regulation of receptors for GnRH. The studies reported here identified cellular changes behind the inhibitory effects of inhibin on gonadotropes to learn whether its effects are mediated by changes in subtypes of gonadotropes. Cell populations from diestrous day 2 and proestrous (morning) rats were collected, dispersed to single cell populations, and plated in medium containing either recombinant 32-kDa inhibin or porcine follicular fluid for 24 h. GnRH binding was detected by exposing the cells to a biotinylated analog (Bio-GnRH) for 10 min before fixation, followed by avidin-peroxidase labeling protocols to detect the biotin on the analog. In parallel fields, the cells were further identified by immunolabeling for LH or FSH beta-subunits or for GH with a different colored reaction product. The most striking changes were seen in cells from proestrous rats. Inhibin reduced the percentages of Bio-GnRH target cells in the population by 60% and the area and density of Bio-GnRH label on the remaining cells. Inhibin reduced the percentages of FSH cells by 30% and caused nearly a 60% reduction in the binding of Bio-GnRH by this cell type (from 83% of FSH cells to 32% of FSH cells). Inhibin also reduced the area of FSH cells and the density of FSH stores. Inhibin's effects on LH cells were limited to a reduction in the area of the cells and the density of LH stores, but not the number of LH cells. In addition, it reduced the percentages of LH cells with Bio-GnRH receptors from 84% to 40%. When cells with GH were analyzed, inhibin had no effect on their percentages, areas, or GH stores. In populations from proestrous rats, inhibin reduced the percentages of GH cells with Bio-GnRH binding from 38% to 21%. These data suggest that inhibin's target cell is the abundant multihormonal gonadotrope that contains LH, FSH, and GH and predominates during proestrus. Inhibin's effects are most severe on FSH cells, which suggests that it may either selectively affect FSH synthesis and stores in bihormonal gonadotropes and/or affect monohormonal FSH cells. Thus, mechanisms behind its inhibitory effects include 1) a reduction in the percentage of Bio-GnRH target cells, 2) a reduction in the area of Bio-GnRH-binding sites on individual cells, and 3) a reduction in the stores of FSH and the percentages of FSH cells. These last effects are consistent with known reductions in FSH synthesis. The effects of inhibin on LH secretion may be secondary to the effects on Bio-GnRH receptors in bihormonal gonadotropes.

Cytochemical studies of the effects of activin on gonadotropin-releasing hormone (GnRH) binding by pituitary gonadotropes and growth hormone cells.

Activin stimulates the synthesis and secretion of follicle-stimulating hormone (FSH). It inhibits the synthesis and release of growth hormone (GH). It acts on gonadotropes by stimulating the synthesis of gonadotropin-releasing hormone (GnRH) receptors. To test activin's effects on GnRH target cells, pituitary cells from diestrous or proestrous rats were exposed to media with and without 60 ng/ml activin for 24 hr and stimulated with biotinylated GnRH (Bio-GnRH). The populations were double-labeled for Bio-GnRH and/or luteinizing hormone-beta (LH-beta), FSH-beta, or GH antigens. In both diestrous and proestrous rats, activin stimulated more LH and FSH cells and increased the percentages of GnRH target cells. In diestrous rats, activin stimulated increases in the average area and density of Bio-GnRH label on target cells. In addition, more FSH, LH, and GH cells bound Bio-GnRH. The increment in binding by gonadotropes was not as great as that normally seen from diestrus to proestrus, suggesting that additional factors (such as estradiol) may be needed. These data suggest that activin plays an important role in the augmentation of Bio-GnRH target cells normally seen before ovulation. Its actions on GH cells may reflect a role in the transitory change from a somatotrope to a somatogonadotrope that is seen from diestrus to proestrus.