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Regulation of Somatotroph Differentiation and Growth Hormone (GH) Secretion by Corticosterone and GH-Releasing Hormone during Embryonic Development¹

Department of Poultry Science, Texas A&M University (C.E.D.), College Station, Texas 77843; and the Department of Animal and Avian Sciences, University of Maryland (T.E.P.), College Park, Maryland 20742

Address all correspondence and requests for reprints to: Dr. Tom E. Porter, Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland 20742. E-mail: tp44{at}umail.umd.edu

	Abstract

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The role of extracellular factors in the regulation of anterior pituitary cell differentiation and GH secretion during embryonic development was investigated. Previously, we reported that somatotrophs become a significant population by embryonic day (e-) 16 of the chick and that corticosterone is the active compound responsible for the observed GH cell-differentiating activity of e-16 serum. More recently, the influence of hormone interactions on somatotroph differentiation and GH secretion during mid- to late embryogenesis was evaluated. Anterior pituitary cells from e-12, -14, and -17 chicks were cultured for 2, 3, and 6 days with corticosterone (10^-9 M) and GH-releasing hormone (GHRH; 10^-10-10^-7 M) alone and in combination. Medium samples were analyzed for GH concentrations, and recovered cells were subjected to GH reverse hemolytic plaque assay for determination of somatotroph percentages and the relative amount of GH secretion from individual somatotrophs. GHRH significantly (P < 0.05) increased GH secretion from e-17, but not e-12 and e-14, pituitary cells during 2 and 3 days of culture. Corticosterone alone failed to increase GH secretion from e-12, -14, and -17 pituitary cells; however, corticosterone in combination with GHRH increased GH secretion from cells of all three ages. Culture with GHRH decreased percentages of e-17 GH-secreting cells in a concentration-dependent manner (from basal levels of 12.3 ± 2.4% to 3.2 ± 0.7% by 2 days), but did not affect percentages of e-12 and e-14 somatotrophs. Conversely, corticosterone increased percentages of e-12 and e-14 GH-secreting cells (by as much as 14- and 3-fold above basal levels, respectively), but did not alter the proportions of e-17 GH cells. Corticosterone in combination with GHRH was more effective than either hormone alone for increasing percentages of e-12 GH-secreting cells (from 9.6 ± 0.8% with corticosterone to 15.9 ± 1.5% with corticosterone plus GHRH), but this synergistic effect was not apparent until after 3 days of culture. Exposure to corticosterone in culture for 2, 3, and 6 days increased subsequent GH release from e-12 and e-14 pituitary cells during reverse hemolytic plaque assay. Combined treatment with corticosterone and GHRH further increased subsequent GH release from e-12 and e-14 cells. We conclude that glucocorticoids induce GH cell differentiation and that corticosterone and GHRH can interact at specific stages of embryonic development to regulate somatotroph differentiation and GH secretion.

	Introduction

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DURING embryonic development, corticotrophs are the first functional cell type to appear in the anterior pituitary, followed by gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. Corticotrophs differentiate autonomously in unstimulated cultures, whereas the other anterior pituitary cell types apparently do not differentiate without extracellular signals (1). Studies with rats suggest that several hormones, acting alone or in combination, may function as differentiation factors during specific stages of pituitary gland development (2, 3). Glucocorticoids can induce differentiation of fetal rat somatotrophs in vitro (4, 5, 6) and in vivo (7, 8), and thyroid hormones appear to act synergistically with glucocorticoids to increase somatotroph differentiation (7, 8). Hypothalamic factors are apparently not required for GH cell differentiation; somatotrophs are present in anencephalic human fetuses (9) and encephalectomized rat embryos (10), and GH cells can differentiate in the absence of hypothalamic factors in vitro (4, 5, 11). However, previous reports indicate that GH-releasing hormone (GHRH) is involved in the clonal expansion of differentiated somatotrophs (12, 13, 14).

The purpose of the present study was to investigate the role of extracellular factors in regulation of somatotroph differentiation and GH secretion during embryonic development, using the chick embryo as a model system. The chicken embryo is a useful model for studying the influence of extracellular factors on pituitary cell differentiation due to its isolation from maternal influences and the relative ease with which the endocrine environment of individual embryos can be manipulated. Somatotrophs first appear between embryonic day (e-) 12 and e-14 and become a significant population by e-16 of chick development (15, 16, 17). This ontogenic profile of somatotroph differentiation correlates with the capacity of embryonic serum to induce GH cell differentiation in vitro (18). Full differentiation of functional chicken somatotrophs continues gradually from e-16 through at least e-20 of the 21-day incubation period (19). The mean proportion of GH cells in pituitaries from a mixed sex population of e-20 chicks was determined to be 19.5% (19). Typical somatotroph percentages in male and female chickens between 4 and 5 weeks of age are about 20% and 13% of the total pituitary cells, respectively (20). Thus, practically all somatotrophs present in the posthatch chicken are differentiated during the embryonic period. Chicken GH cells do not differentiate in culture without an extrapituitary signal (18); however, somatotroph differentiation in the chicken embryonic pituitary can be induced in vitro by glucocorticoids, and corticosterone is responsible for the GH cell-differentiating activity of e-16 chicken serum (21). A role for induction of somatotroph differentiation by corticosterone in vivo is supported by reports that an increase in circulating corticosterone levels occurs before GH cell differentiation on e-16 (22). Pituitary GH release in chickens, as in other vertebrates, is controlled by stimulatory and inhibitory factors from the hypothalamus. GHRH and TRH are thought to be the primary GH secretagogues, whereas somatostatin decreases GH secretion, possibly by antagonizing the effects of GHRH and TRH. Synthetic human GHRH (hGHRH) has been shown to release GH in vivo in both young (23, 24) and adult chickens (24). hGHRH also stimulated GH release from chicken pituitaries in vitro (24, 25). Although TRH is an effective GH secretagogue in immature and anesthetized birds, it has little effect on GH secretion in conscious adult chickens (26). The effects of hGHRH and TRH on in vitro GH release during the embryonic period depend on developmental stage. Between 50–70% of initial somatotrophs present on e-16 released GH in response to hGHRH-(1–40) (15, 19), whereas only 30% of GH cells responded to TRH (19). By e-20, the proportions of somatotrophs that responded to hGHRH1–40 and TRH were approximately equal at about 40% (19). hGHRH and TRH were shown to increase plasma GH concentrations approximately equally on e-18 (27).

To date, the effect of corticosterone on GH cell differentiation in the chicken has been evaluated using only e-12 pituitary cells, an age when somatotrophs are rare. Moreover, potential interactions between glucocorticoids and other extracellular factors that could affect somatotroph differentiation and GH secretion have not been investigated. Therefore, the objective of the present study was to evaluate the effects of corticosterone and GHRH on GH secretion and somatotroph differentiation at different stages of embryonic development, using pituitary cells from e-12, -14, and -17 chicks.

	Materials and Methods

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Animals and pituitary dispersion
Unless stated otherwise, all cell culture reagents were obtained from Life Technologies (Grand Island, NY), and hormones and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). All media were supplemented with 0.1% BSA, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate. All animals used in this study were Single Comb White Leghorn chicken embryos. Fertile eggs were placed in a humidified incubator (G.Q.F. Manufacturing, Savannah, GA) at 37.5 C. The normal duration of incubation for chickens is 21 days. For the in vitro studies, embryos were removed on days 12, 14, and 17 of incubation, and their anterior pituitary glands were isolated with the aid of a dissecting microscope. Isolated pituitaries were placed in Spinner’s Minimum Essential Medium until all glands were removed. Then, the anterior pituitary glands were dissociated into individual cells by trypsin digestion and mechanical agitation as described previously (15). Briefly, anterior pituitaries from 20–30 embryos were placed in 10 ml Spinner’s Minimum Essential Medium with trypsin (1 mg/ml; Difco, Detroit, MI) and incubated at 37.0 C for 45 min under 95% O₂-5% CO₂ in a Spinner flask (Bellco, Vineland, NJ). Tissue dissociation was aided during the incubation with gentle trituration using a siliconized flame-polished Pasteur pipette at 15-min intervals. The resulting monodispersed cells were washed twice with 10 ml DMEM followed by centrifugation. The viability of the cells was assessed by the trypan blue dye exclusion method and was consistently greater than 95%.

Extended cell cultures
Anterior pituitary cells were cultured according to the procedure described previously (18). Cells were plated (2.0 x 10⁵ cells/well) in poly-L-lysine-coated 12-well tissue culture plates and allowed to attach for 45 min. Wells were then filled (2 ml) with serum-free medium alone or medium containing corticosterone (10^-9 M) or synthetic hGHRH-(1–40) (10^-10-10^-7 M; Sigma Chemical Co., G8770) alone or in combination. The medium consisted of a 1:1 mixture of phenol red-free medium 199 and Ham’s F-12 nutrient mixture supplemented with 0.1% BSA, 5 µg/ml human transferrin, 5 µg/ml bovine insulin, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate. Culture plates were prepared in triplicate, and after culture intervals of 2, 3, and 6 days in a humidified incubator (37.5 C; 95% air-5% CO₂), cells were harvested for detection of GH-secreting cells by reverse hemolytic plaque assay (RHPA). Culture medium was aspirated from the wells and replaced with fresh medium and treatments after 3 days of incubation. The GH content of medium samples collected at the end of each incubation interval was assessed by RIA (28). All samples were assayed in a single RIA, and the intraassay coefficient of variation was 5.6%. The sensitivity of this assay was 2.5 ng/ml.

RHPA
The RHPA procedure allows for detection of hormone secretion from individual cells (29). The assays were performed according to the protocol described in detail previously (30), using rabbit antiserum against chicken GH and modifications described previously (15). Briefly, recovered anterior pituitary cells were mixed with an equal volume of an 18% suspension of protein A-coated ovine erythrocytes and infused by capillary action into previously constructed Cunningham chambers. After cells were allowed to attach for 45 min (37.5 C; 95% air-5% CO₂), chambers were rinsed with DMEM to remove unattached cells. DMEM containing GH antiserum (1:40) and hGHRH-(1–40) (10^-7 M) was then added to the resulting monolayer of cells, and replicate chambers were incubated for 8 or 20 h (three chambers per treatment per time point). Plaque formation was subsequently induced by a 45-min incubation with guinea pig complement (1:40, in DMEM). The cells were then fixed with 2% glutaraldehyde in 0.9% saline and stained with methyl green. Chambers were analyzed in two ways using a light microscope. First, the percentage of all pituitary cells that formed plaques was determined, with at least 200 pituitary cells counted/assay chamber. Second, the area of each plaque formed was measured with the aid of an ocular reticle.

Statistical analysis
The data reported are the mean ± SEM from at least 3 completely separate experiments, with the number of replicate experiments provided in the legend to each figure. For each replicate experiment, percentages of GH plaque-forming cells were determined for each combination of treatment and time point using the three replicate chambers for that combination (for a total of at least 600 pituitary cells analyzed for each combination). All data were analyzed using the general linear models procedure of SAS (31). Differences between treatments were tested using Tukey’s Studentized range test and were considered significant at P < 0.05.

	Results

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Effect of corticosterone and GHRH on total GH secretion during culture
The GH concentration in medium samples collected from all culture wells after each incubation interval was measured to determine the effect of treatments on total GH secretion during culture. GH concentrations after short (2 days) and long (6 days) term culture are presented in Figs. 1 and 2 for e-12 and e-17 pituitary cells, respectively. As fresh medium and treatments were added after 3 days of incubation, the 6-day data represent accumulation of GH during the final 3 days of culture only. Neither corticosterone nor GHRH alone significantly changed the amount of GH released by e-12 pituitary cells after 2 and 6 days of incubation relative to basal control levels (Fig. 1). In contrast, the combination of corticosterone and GHRH increased GH release by e-12 cells in a concentration-dependent manner to levels significantly higher than control values or either hormone alone after 2 and 6 days. The results were similar for e-12 cells exposed to corticosterone and GHRH alone and in combination for 3 days (data not shown). Data for e-14 cultures (not shown) were essentially the same as those for e-12 cells. Corticosterone alone did not affect GH release from e-17 cells (Fig. 2); however, GHRH alone significantly increased medium GH content relative to basal controls after 2 and 3 days. Corticosterone plus GHRH also increased GH release from e-17 cells in a concentration-dependent manner after 2 days, but this treatment was no more effective than GHRH alone.

View larger version (53K): [in this window] [in a new window]   Figure 1. The effect of corticosterone and GHRH on GH secretion during extended culture (upper panels), somatotroph differentiation (middle panels), and GH secretion during RHPA (lower panels) on embryonic day 12. Embryonic day 12 pituitary cells were treated with corticosterone or increasing concentrations of GHRH alone and in combination for 2 and 6 days. After each incubation interval, medium GH content was determined by RIA. Cells were harvested and subjected to RHPA for GH. Slides were incubated for 8 h in the presence of GHRH (10-7 M). A minimum of 200 pituitary cells were counted on each of 3 replicate chambers for each treatment to determine the percentage of GH plaque-forming cells. To determine the relative amounts of GH secreted from individual plaque-forming cells, the area of each plaque in all chambers was measured using an ocular reticle. These results are the means and SEM from 6, 5, and 4 independent experiments for the RIA, plaque percentage, and plaque area data, respectively. Within each graph, values with no letters in common are significantly different (P < 0.05). Nearly identical results were found in RHPAs performed for 20 h.

View larger version (60K): [in this window] [in a new window]   Figure 2. The effects of corticosterone and GHRH on GH secretion during extended culture (upper panels), somatotroph abundance (middle panels), and GH secretion during RHPA (lower panels) on embryonic day 17. These results are the means and SEM from three independent experiments. Within each graph, values with no letters in common are significantly different (P < 0.05). See Fig. 1 for additional details.

The effects of corticosterone and GHRH on proportions of e-17 GH cells were markedly different from those on e-12 and e-14 cells (Fig. 2). First, treatment with corticosterone alone did not change percentages of e-17 somatotrophs relative to basal levels of 12.3 ± 2.4%, 11.2 ± 1.6%, and 8.5 ± 2.3% after 2, 3, and 6 days, respectively. In contrast, GHRH decreased the percentages of GH cells detected in a concentration-dependent manner after all three incubation intervals. Treatment with corticosterone plus GHRH tended to counteract the reduction of somatotroph proportions induced by GHRH alone. The percentages of e-17 GH cells detected after combined treatment with corticosterone and GHRH for 2 and 6 days did not differ from those after treatment with corticosterone alone.

Effects of corticosterone and GHRH on GH secretion in RHPA
To determine the influence of exposure to treatments during extended culture on subsequent GH secretion, we measured plaque area as an index of the relative amount of GH secreted from individual somatotrophs. These results are shown for e-12 and e-17 cells in Figs. 1 and 2, respectively. Cells from e-12 that were exposed to GHRH alone in culture for 2, 3, and 6 days subsequently released more GH in the RHPA than cells that were cultured under basal conditions. These increases were apparent for cells cultured with each concentration of GHRH for 2 days and with at least one concentration of GHRH tested for 3 and 6 days. Cells cultured with corticosterone alone and corticosterone in combination with GHRH for 2, 3, and 6 days subsequently released more GH in RHPA than cells cultured under basal conditions or with GHRH alone. Finally, cells exposed to corticosterone in combination with GHRH for 2 and 3 days, but not 6 days, released more GH in RHPA than cells cultured with corticosterone alone (Fig. 1). Similarly, e-14 pituitary cells cultured with corticosterone alone and in combination with GHRH for 2, 3, and 6 days subsequently released more GH in RHPA than cells cultured under basal conditions, and exposure to both corticosterone and GHRH was significantly more effective than that to either hormone alone for increasing subsequent GH secretion (data not shown).

Culture of e-17 pituitary cells with GHRH alone tended to decrease subsequent GH secretion, with exposure to the highest concentration for 2 days resulting in a significant decrease relative to control values (Fig. 2). Both short and long term exposures to corticosterone alone failed to increased subsequent GH secretion by e-17 cells relative to controls. Finally, e-17 cells exposed to corticosterone plus GHRH for 6 days, but not for 2 and 3 days, subsequently released significantly more GH than cells exposed to corticosterone alone.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study was designed to determine the influence of corticosterone and GHRH at different stages of chicken embryonic development on somatotroph proportions and GH secretion in vitro. Synthetic hGHRH-(1–40) was used in these experiments as an alternative to purified chicken GHRH (cGHRH), which is not available. In fact, a cGHRH had never been reported at the time this work was initiated. Recently, the cGHRH gene was isolated and sequenced (32). Alternative splicing results in three different messenger RNAs, isolated from brain and gonads, encoding GHRH-(1–46), GHRH-(1–43), and GHRH-(33–46). The deduced amino acid sequence of the cGHRH peptide has only 42% identity to hGHRH. Thus, it should be emphasized that although hGHRH is an effective secretagogue for cGH in vitro and in vivo, it may not have the same biological activity as cGHRH.

Pituitary cells were subjected to GH RHPA after hormone exposure for 2–6 days to obtain two types of information. First, percentages of GH-secreting cells were determined as a measure of how corticosterone and GHRH affected the extent of somatotroph differentiation. Second, the mean plaque area was measured as an index of the relative amount of GH secreted by the somatotrophs after acute (8-h) GHRH stimulation. The results indicate that both corticosterone and GHRH affected e-12 and e-17 pituitary cells differently. Furthermore, the responses of cells from the two developmental stages were affected by the duration of exposure to the hormones. Short and long term exposures to corticosterone dramatically increased somatotroph percentages among pituitary cells from e-12, an age when somatotrophs are normally rare. Glucocorticoids have also been shown to induce premature differentiation of fetal rat somatotrophs in vitro (4, 5, 6). Corticosterone also increased GH cell proportions on e-14, but not e-17, in the present study, suggesting that the induction of somatotroph differentiation by glucocorticoids is not possible at this later stage of development in the chicken. Treatment of e-12 pituitary cells with corticosterone alone raised somatotroph proportions to as high as 9.6%, which was less than the maximum percentage of e-17 GH cells (12.3%) detected under basal conditions; however, corticosterone increased the proportion of e-14 somatotrophs to as much as 15.7%. Thus, corticosterone was capable of increasing somatotroph proportions on e-14 to levels greater than those observed under basal conditions on e-17 and to levels comparable with those observed in posthatch chickens. Although treatment of e-12 pituitary cells with GHRH alone did not affect somatotroph proportions, combined treatment with corticosterone and GHRH for 6 days increased e-12 GH cell percentages to levels exceeding those observed after treatment with corticosterone alone. The effects of corticosterone and GHRH were truly synergistic, because GHRH alone was ineffective, and the combination increased somatotroph proportions to as much as 15.9% compared to 9.6% with corticosterone alone. One possible explanation for this effect is that GHRH stimulated mitosis of somatotrophs that had been induced to differentiate by corticosterone. Indeed, GHRH has been shown to stimulate the proliferation of differentiated rat somatotrophs in vitro (12). Interestingly, the synergistic effect of corticosterone and GHRH required exposure to the hormones for more than 3 days. Perhaps cells were first induced to express GHRH receptors by corticosterone and then stimulated to release GH by GHRH. The mechanism of this effect cannot be determined from this study, because no attempt was made to evaluate somatotroph proliferation. The synergistic effect of corticosterone and GHRH on GH cell proportions was essentially lost by e-14, about the time when somatotroph differentiation normally begins in vivo.

The effects of corticosterone and GHRH on e-17 pituitary cells were markedly different from those on e-12 and e-14 cells. Although corticosterone did not increase the percentages of e-17 GH cells, treatment with GHRH decreased the percentages of e-17 somatotrophs detected in a concentration-dependent manner; however, this apparent reduction in somatotroph proportions probably reflects decreased sensitivity of the cells to GHRH rechallenge in the RHPA. This phenomenon, referred to as homologous desensitization, was observed when rat anterior pituitary cells were pretreated for 24 h with GHRH (33). When those cells were rechallenged with GHRH, maximal GH secretion was significantly lower than the control value, and releasable GH pools were reduced by 5-fold. Recently, GHRH was shown to inhibit the production of its own receptor in as few as 4 h by reduction of GHRH receptor messenger RNA accumulation in cultures of rat anterior pituitary cells (34). In the present study, treatments were removed from cells for approximately 3 h during the transition from cell culture to RHPA. During this time, cells were recovered from the culture wells, rinsed, and allowed to attach to the RHPA chambers. As all RHPAs were performed under stimulatory conditions in this study (i.e. in the presence of GHRH), cells that were initially cultured with GHRH were, in effect, rechallenged with GHRH during the RHPA. Because detection of somatotrophs by RHPA depends on GH secretion, desensitization to GHRH rechallenge might explain the apparent dose-related reduction in GH cell percentages determined by RHPA. In other words, exposure to GHRH in culture may have decreased the percentage of somatotrophs that released GH when rechallenged with GHRH during RHPA. Treatment of e-17 pituitary cells with corticosterone and GHRH in combination appeared to moderate or eliminate the loss of responsiveness caused by prolonged exposure to GHRH. This finding could reflect corticosterone-induced sensitization of somatotrophs to GHRH. Glucocorticoids have been shown to enhance GHRH-induced GH secretion in primary cultures of rat anterior pituitary cells (35, 36) and in freely moving rats (37). Furthermore, glucocorticoids have been shown to increase GHRH receptor gene expression (38) and the number of GHRH-binding sites on rat anterior pituitary cells (39). Another possibility is that corticosterone increased GH protein synthesis in e-17 cells, an effect that has also been documented in rat anterior pituitary cells (4, 5, 6, 40). Thus, corticosterone could be important for sustaining releasable GH pools and regulating sensitivity to secretagogues during late embryonic development when chicken somatotrophs become responsive to GHRH and TRH (19).

That treatment of e-12 and e-14 pituitary cells with GHRH alone failed to significantly increase the medium GH content was not surprising, as only small percentages of somatotrophs were detected at these ages. Others have failed to observe GHRH-stimulated GH release from chicken pituitaries until after e-14 (41), when the adenohypophyseal vascular sinusoids attain their adult structure (42). That e-17 cells did release GH in response to GHRH was also not surprising, as 50–70% of somatotrophs become responsive to GHRH by e-16 (15, 19). Unexpectedly, the medium GH content was not increased by treatment of e-12 and e-14 cells with corticosterone alone, even though corticosterone substantially increased somatotroph percentages at these ages. This result was surprising in view of previous findings that somatotrophs induced to differentiate prematurely by corticosterone synthesized and released GH in the absence of GHRH (18, 21). However, others have reported that dexamethasone had little effect on basal GH release (38, 43). Although corticosterone alone failed to increase GH secretion in the present study, combined treatment with corticosterone and GHRH increased GH secretion from pituitary cells of all ages in a concentration-dependent manner. This finding confirms that somatotrophs, whether differentiated normally in vivo or induced to differentiate prematurely with corticosterone in vitro, are responsive to GHRH.

The effects of treatments in culture on subsequent GH release in RHPA were determined by measuring plaque area. Interestingly, the responses of e-12 and e-17 cells were different under most conditions. Treatment with GHRH may have stimulated the few somatotrophs present on e-12 to synthesize more GH, thus increasing the pool available for release during RHPA. Indeed, GHRH increased GH gene transcription (44) and GH synthesis (33) in rat anterior pituitary cells in vitro. The increased mean plaque area of e-12 cells cultured with corticosterone alone might also reflect increased GH synthesis as well as greater responsiveness to GHRH. Glucocorticoids have been shown to induce GH synthesis (4, 5, 6, 40), to increase GHRH binding capacity (39), and to enhance GHRH-induced GH secretion in cultures of rat anterior pituitary cells (35, 36). In contrast, the reduced plaque area of e-17 cells after treatment with GHRH could reflect decreased sensitivity due to down-regulation of GHRH receptors. The increased capacity of e-12 cells to secrete GH after exposure to corticosterone and GHRH in combination for 2 days suggests an additive effect of the treatments; however, 6 days of combined treatment failed to increase GH secretion more than corticosterone alone, suggesting that the latter treatment exerted a maximal effect by 6 days.

In conclusion, corticosterone induced GH cell differentiation in vitro on e-12 and e-14, and the somatotroph-differentiating activity of corticosterone was magnified by concomitant exposure to GHRH. Corticosterone did not induce GH cell differentiation in vitro on e-17, but sustained the capacity of e-17 somatotrophs to release GH in response to GHRH. Somatotrophs normally appear during chicken embryonic development after a rise in circulating glucocorticoid levels (22), and corticosterone is responsible for the GH cell-differentiating activity of e-16 chicken serum (21). Taken together, these findings provide strong evidence that glucocorticoids are involved in GH cell differentiation during embryonic development, and that GHRH may act in concert with corticosterone to stimulate differentiation and expansion of the somatotroph population.

	Footnotes

 
¹ This work was supported by USDA Grants 94–3206-1097 (to T.E.P.) and 96–35206-3493 to (C.E.D.) and by the Texas Agricultural Experiment Station.

Received July 20, 1998.

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