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Identification of the Blood-Borne Somatotroph-Differentiating Factor during Chicken Embryonic Development¹

Department of Poultry Science and Center for Animal Biotechnology (T.E.P.), Institute of Biosciences and Technology, Texas A&M University, College Station, Texas 77843-2472

Address all correspondence and requests for reprints to: Dr. T. E. Porter, Department of Animal and Avian Sciences, 4111 Animal Sciences Center, University of Maryland, College Park, Maryland 20742-2311.

	Abstract

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Somatotrophs become a significant population by day 16 of chicken embryonic development. We have previously demonstrated that an earlier induction of GH cell differentiation is possible with the addition of day 16 embryonic serum to cultures of day 12 pituitary cells, an age when somatotrophs are rare. The present study was designed to identify the blood-borne signal(s) responsible for the serum activity, using reverse hemolytic plaque assays to identify individual GH-secreting cells. The activity was found to be a heat-stable, ether-soluble compound(s) that is bound or inhibited by a trypsin-sensitive protein. The extent of GH cell differentiation was greater (P < 0.05; n = 3) in response to the ether phases of heated day 16 (14.1 ± 0.4% of all cells) and day 12 sera (9.3 ± 0.4%) than with untreated serum from days 16 and 12 (6.1 ± 0.4% and 0.82 ± 0.4%, respectively). Furthermore, ether-extracted day 16 serum was more effective than ether-extracted day 12 serum, which was also different from basal (0.85 ± 0.4%; P < 0.05). Based on this biochemical profile, the abilities of various steroids to stimulate differentiation were tested. Three steroids were found to stimulate somatotroph differentiation in vitro: 17ß-estradiol, corticosterone, and progesterone. However, the estradiol receptor antagonist, tamoxifen, while abolishing the effect of estradiol, had no effect on the induction of differentiation by day 16 serum. In contrast, RU486, a specific glucocorticoid receptor antagonist in chickens, blocked the stimulatory effects of corticosterone, progesterone, and day 16 serum on somatotroph differentiation. We next tested whether the active compound in day 16 embryonic serum was corticosterone, the predominant glucocorticoid in chickens. Incubation of day 16 serum with corticosterone antiserum, but not control antiserum, suppressed day 16 serum-induced GH cell differentiation. Therefore, we conclude that corticosterone is the blood-borne signal capable of stimulating somatotroph differentiation in vitro. The present findings together with previous reports indicate that somatotroph differentiation during embryonic development may result from an increase in circulating glucocorticoid concentrations.

	Introduction

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THE ANTERIOR pituitary differentiates into five cell types during embryonic development. The first cell type to appear, corticotrophs, is the only type to differentiate in culture without extracellular signals. The other cell types will not differentiate autonomously in unstimulated cultures (1). Studies with rats indicate that glucocorticoids can stimulate premature expression of GH (2, 3) and GH messenger RNA (4, 5), suggesting that these steroids may be involved in somatotroph differentiation. Our laboratory is studying the mechanisms regulating differentiation of GH-secreting cells in the chicken anterior pituitary. During chicken embryonic development, which lasts 21 days, somatotrophs first appear between embryonic days (e-) 12 and 16 (6). We have demonstrated previously that somatotroph differentiation can be stimulated in cultures of e-12 pituitary cells by serum obtained from e-16 embryos, and differentiation, which appears to be a postmitotic event, does not occur in vitro without an extrapituitary signal (7). The objective of the present study was to identify the blood-borne somatotroph-differentiating signal during chicken embryonic development, using biochemical and immunological techniques.

	Materials and Methods

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Animals and pituitary dispersion
Unless stated otherwise, all chemicals used in this study were obtained from Sigma Chemical Co. (St. Louis, MO), and cell culture reagents were purchased from Life Technologies (Grand Island, NY). All animals used were Single Comb White Leghorn chicken embryos. Fertile eggs were maintained in a humidified incubator (G.Q.F. Manufacturing, Savannah, GA) at 37.5 C. The duration of incubation in chickens is normally 21 days. On day 12 of incubation, the embryos were removed, and their anterior pituitary glands were isolated with the aid of a dissecting microscope. Isolated pituitaries were placed in Spinner’s MEM until all glands were isolated. Then, anterior pituitaries were dissociated into individual cells by a combination of trypsin digestion and mechanical agitation as described previously (6). In short, pituitaries were placed in 10 ml Spinner’s MEM with trypsin (1 mg/ml; Difco, Detroit, MI), and then incubated at 37.0 C for 45 min under 95% O₂ and 5% CO₂ in a Spinner flask (Bellco, Vineland, NJ). Dissociation was aided with gentle trituration using a flame-polished siliconized Pasteur pipette at 15-min intervals. The resulting dispersed cells were washed twice with 10 ml DMEM at ambient temperature, followed by centrifugation. The viability of the monodispersed cells was assessed by the trypan blue dye exclusion method and was consistently greater than 95%.

Cell cultures
Cells were cultured according to the procedure described previously (7). Recovered anterior pituitary cells were plated (2.0 x 10⁵ cells/well) in poly-L-lysine-coated 12-well tissue culture plates. Cells were allowed to attach for 45 min, and wells were then filled (2 ml) with serum-free medium. 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. Cells were cultured for 2 days in a humidified tissue culture incubator (37.5 C; 5% CO₂) and then harvested for detection of GH-secreting cells by reverse hemolytic plaque assays. Approximately 80% of all cells plated were harvested after culture, and no differences were observed between treatments in the numbers of cells recovered throughout this study. The GH content of the culture medium was assessed by RIA (8). All samples were assayed in a single RIA, and the intraassay coefficient of variance was 8.3%. The sensitivity of this assay was 2.5 ng/ml.

Reverse hemolytic plaque assay (RHPA)
The RHPA procedure, which allows detection of hormonal secretion by individual cells, was originally described by Neill and Frawley (9). The assays were performed according to the protocol described in detail previously (10), using rabbit antiserum against chicken GH and modifications described earlier (6). Briefly, pituitary cells (1.0 x 10⁵/ml) 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. Cells were allowed to attach for 45 min (37.5 C; 95% air and 5% CO₂), then chambers were rinsed with DMEM to remove unattached cells. DMEM containing GH antiserum (1:40) with or without human GH-releasing hormone (GHRH; 1.0 x 10^-7 M), was then added to the resulting monolayer of cells, and replicate chambers were incubated for 8 or 20 h (three chambers each). Plaque formation was induced by a 45-min incubation with guinea pig serum as a source of complement (1:80). The cells were then fixed with 2% glutaraldehyde in 0.9% saline and stained with methyl green. The proportions of plaque formers were determined by evaluation under a light microscope, with a minimum of 100 pituitary cells counted per chamber.

Biochemical characterization
Column chromatography.
Three samples of lyophilized e-16 serum (150 µl each) were dissolved in 1 M acetic acid for 30 min at room temperature. Samples were then centrifuged to remove undissolved material, and the supernates were applied to a Sephadex G-50 column, (32 x 1.6 cm; equilibrated with 1 M acetic acid). Twenty-five fractions (3 ml each) were collected and lyophilized. Each fraction was dissolved in 3 ml water and lyophilized again to remove any residual acetic acid. The fractions were then dissolved in 3 ml PBS (pH 7.4, 0.04 M) and tested for their ability to stimulate somatotroph differentiation in cultures of e-12 pituitary cells (bioassay). Relative molecular mass was determined by elution of protein standards. First, fractions were combined into three pools: void volume (>25 kDa; pool I), fractions between void volume and salt fraction (25–1.5 kDa; pool II), and salt fraction (<1.5 kDa; pool III). Each pool was tested for bioactivity. The fractions of the pool that showed bioactivity were then tested individually. Volumes of individual fractions were selected to yield an effective concentration of 1% serum in culture based on the assumption that all of the bioactivity was contained in a single fraction or a pool of fractions.

Proteolytic hydrolysis.
Serum samples from e-12 and e-16 (three of each age) were subjected to enzymatic digestion with trypsin conjugated to agarose beads (Sigma) for 1 h in PBS (1.8 U; 37.0 C; pH 7.4). After digestion, the trypsin-coated beads were removed by centrifugation, and the supernates were applied to day 12 pituitary cells in culture for bioassay. As controls, PBS alone and after incubation with the enzyme was bioassayed as well.

Ether extraction.
Serum samples from e-12 and e-16 (three of each age) were first heated to 70 C for 1 h and then extracted twice with 5 vol ethyl ether. Samples that were not treated and samples that were heated but not extracted were included as controls. All samples were lyophilized and resuspended in PBS, then samples (1% by volume) were applied to day 12 pituitary cells in culture for bioassay.

Steroids and steroid receptor antagonists
All steroids and steroid receptor antagonists were dissolved in 100% ethanol initially (1 x 10^-3 M). Further dilutions were made in PBS. Each steroid was tested for bioactivity at different concentrations (10^-12–10^-7 M). The final concentration of ethanol was consistent across all wells (0.1%), including control wells.

Immunoprecipitation
Rabbit anticorticosterone and rabbit anti-GH were tested for their ability to block serum-induced somatotroph differentiation in vitro. Treatments consisted of heated e-16 serum and corticosterone. Rabbit anti-GH serum was included as a control for the endogenous effects of serum. Each of the treatments and antisera were also bioassayed alone as controls. The GH antiserum control was that used for the RHPA (6). The corticosterone antiserum was obtained from Fitzgerald (Concord, MA). Its cross-reactivity is less than 0.3% with cortisol and 0.01% with progesterone and other steroids. The antisera (10 µl diluted 1:5 with PBS/treatment) were first incubated (1 h, 37.0 C) with protein A-bound to acrylic beads (Sigma) to eliminate steroids and other components in the antisera and to test the IgG fraction alone. After incubation, the tubes were centrifuged, supernatants were removed, and the IgG-protein A-bead complexes were rinsed with equal volumes of PBS. Rinsing was repeated five times. Steroid or embryonic serum treatments were then added to the IgG-protein A beads and incubated at 37.0 C for 2 h. Finally, tubes were centrifuged, and resulting supernatants were diluted in 2 ml culture medium (to achieve estimated concentrations of 0.5% e-16 serum by volume or 1 x 10^-10 M corticosterone). These 2-ml treatments were placed on day 12 pituitary cells for 48 h to test bioactivity.

Statistical analysis
Data were analyzed by ANOVA using the general linear models procedure of SAS (11). The main effects tested were trial and treatment. Differences between treatments were then tested by orthogonal contrasts. Differences were considered significant at P < 0.05. All data presented are the least squares means and SEs from the ANOVA of at least three independent replicate experiments.

	Results

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Gel filtration chromatography revealed that all of the somatotroph-differentiating activity eluted from the Sephadex column in pool II, between the void volume and the salt fraction. Individual fractions within pool II were then assayed separately. In addition, pools I and III as well as the first fraction of pool III were tested again. The results are presented in Fig. 1. Unfractionated serum from e-16 embryos significantly stimulated somatotroph differentiation, with 7.8 ± 0.9% of the population secreting GH (mean ± SE; n = 3). The bioactivity eluted as a single peak with a relative molecular mass less than that of the smallest standard (bacitracin; 1.45 kDa), and with maximal induction of somatotroph differentiation of 11.1 ± 0.9% of all pituitary cells. Next, we tested whether the bioactivity was sensitive to proteolytic digestion. The results are presented in Fig. 2. Untreated serum from e-16 embryos, but not from e-12 embryos, significantly stimulated somatotroph differentiation (Fig. 2A). Trypsin digestion increased the effect of e-16 serum and exposed somatotroph-differentiating activity in serum from e-12 embryos. The effect of trypsin digestion on GH accumulation in culture medium is shown in Fig. 2B. Untreated serum from e-16, but not e-12, embryos stimulated GH secretion (10.1 ± 0.3 ng/ml with e-16 compared with 5.6 ± 0.3 ng/ml with e-12 serum and 3.6 ± 0.3 ng/ml under basal conditions). Digestion with trypsin increased the effectiveness of both e-12 and e-16 serum on medium GH content. However, e-16 serum remained significantly more effective than e-12 serum.

View larger version (21K): [in this window] [in a new window]   Figure 1. The effect of Sephadex G-50 chromatography on the bioactivity of day 16 chicken embryonic serum. Bioassays were performed on separate fractions between the void volume and the salt fraction and on the pooled void volume and salt fractions of day 16 serum samples. Wells were also treated with unfractionated serum (serum) and with medium alone (basal) for 48 h, and then subjected to reverse hemolytic plaque assays for GH. Slides were incubated for 8 h in the presence of GHRH (10-7 M). A minimum of 100 cells were counted on each of 3 replicate chambers for each treatment. These results are the least squares mean and SE from 3 independent experiments. Significant differences from medium alone (P < 0.05) are indicated by an asterisk.

View larger version (27K): [in this window] [in a new window]   Figure 2. The effect of trypsin digestion on the ability of day 12 and day 16 chicken embryonic serum to induce differentiation of GH-secreting cells. Results are expressed as a percentage of total pituitary cells (A) and as the concentration of GH in culture medium (B). Serum samples were treated with trypsin (37 C, 1 h) and then applied to day 12 pituitary cells. Wells were also exposed to untreated serum, medium alone (medium), and trypsin-treated PBS for 48 h, and then subjected to reverse hemolytic plaque assays for GH. Slides were incubated for 8 h in the presence of GHRH (10-7 M). A minimum of 100 cells were counted on each of 3 replicate chambers for each treatment. These results are the least squares mean and SE from 3 independent experiments. Significant differences (P < 0.05) are indicated by different letters.

View larger version (28K): [in this window] [in a new window]   Figure 3. The effect of heating and ether extraction on bioactivity of serum from day 12 and day 16 chicken embryos for stimulating differentiation of GH-secreting cells. Results are expressed as a percentage of the total pituitary cells (A) and GH accumulation in culture medium (B). Serum samples were heated (1 h, 70 C) and then extracted with ether. Heated serum and the ether and aqueous phases of heated serum were applied to e-12 pituitary cells. Significant differences (P < 0.05) between serum of e-12 and e-16 embryos are indicated by different letters. Significant differences (P < 0.05) between untreated and ether-extracted serum are denoted by asterisks. See Fig. 2 for further details.

View larger version (27K): [in this window] [in a new window]   Figure 4. The effects of steroids on somatotroph differentiation. Embryonic day 12 pituitary cells were treated with 10-11 and 10-9 M progesterone (Prog.), corticosterone (Cort.), DEX, testosterone (Test.), DHT, or 17ß-estradiol (E2). These results are the least squares means and SE from three independent experiments. Significant differences (P < 0.05) from medium alone are indicated by asterisks. See Fig. 2 for additional details.

View larger version (27K): [in this window] [in a new window]   Figure 5. The effect of tamoxifen on day 16 serum and estradiol activation of somatotroph differentiation. Day 12 pituitary cells were treated with estradiol (10-11 or 10-9 M) and 1% day 16 serum with (10-9 or 10-7 M) and without tamoxifen. Final ethanol concentrations were equal among treatments (0.1%). These results are the least squares mean and SE from three independent experiments. Significant differences (P < 0.05) are indicated by different letters. See Fig. 2 for details.

View larger version (27K): [in this window] [in a new window]   Figure 6. The effect of RU486 on day 16 serum, corticosterone, or progesterone activation of somatotroph differentiation. Day 12 pituitary cells were treated with corticosterone, progesterone (10-9 M), and 1% day 16 serum with (10-9 or 10-6 M) and without RU486. Final ethanol concentrations were equal among treatments (0.1%). See Fig. 2 for details. These results are the least squares mean and SE from three independent experiments. Significant differences (P < 0.05) are indicated by different letters.

View larger version (21K): [in this window] [in a new window]   Figure 7. The effects of corticosterone and GH antibodies on day 16 serum (serum) and corticosterone (Cort.) induction of somatotroph differentiation. Day 12 pituitary cells were treated for 48 h with corticosterone (10-10 M) and 0.5% heated day 16 serum that were first preincubated with the IgG fraction of corticosterone antiserum. Rabbit anti-GH serum served as a control for potential purified antibody effects. After this culture interval, the cells were subjected to RHPA as described for Fig. 2. These results are the least squares mean and SE from five independent experiments. Significant differences (P < 0.05) from cells treated with medium alone (basal) without prior incubation with antisera are indicated by asterisks. Significant reduction in the day 16 serum response by prior treatment with the corticosterone antibody (P < 0.05) is indicated (#). Preabsorption of the corticosterone treatment with the corticosterone antibody yielded an intermediate response, one not different from the basal level or the response to corticosterone alone.

View larger version (26K): [in this window] [in a new window]   Figure 8. The dose-related effect of corticosterone on somatotroph differentiation. Day 12 pituitary cells were treated with increasing concentrations of corticosterone. These results are the least squares mean and SE from three independent experiments. Significant differences (P < 0.05) from medium alone are indicated by asterisks. The letter a indicates the most effective concentration of the hormone. See Fig. 2 for details about the RHPA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We found that the somatotroph-differentiating activity in chicken embryonic serum was a small, ether-soluble compound, bound or inhibited by a trypsin-sensitive protein. This biochemical profile is typical of a steroid hormone. Previous reports using rats have indicated that glucocorticoids can induce premature expression of GH protein in vitro (2, 3) and messenger RNA in vivo (4) and in vitro (5). In the present study, we showed with anterior pituitary cells from chicken embryos that somatotroph differentiation can be stimulated by several steroids, including corticosterone, 17ß-estradiol, and progesterone. The effect of estradiol was successfully blocked with the estradiol receptor antagonist tamoxifen. However, tamoxifen did not affect the response to day 16 serum, indicating that the blood-borne, somatotroph-differentiating signal did not function through the estrogen receptor. The role of the glucocorticoid receptor in the serum response was evaluated using RU486. Progesterone and glucocorticoids have been reported to activate each other’s receptors (14), and the receptor antagonist RU486 blocks both receptors in mammals. However, RU486 interacts only with the glucocorticoid receptor in chickens (14, 15). As RU486 blocked both corticosterone and the progesterone induction of somatotroph differentiation in the present study, both compounds were shown to act via the glucocorticoid receptor. More importantly, RU486 abolished the stimulatory effect of day 16 serum, indicating that the active steroid in embryonic serum functioned through the glucocorticoid receptor. After demonstrating this requirement for the glucocorticoid receptor, we tested whether the blood-borne compound was corticosterone. Incubation of day 16 serum and corticosterone with antiserum against corticosterone suppressed the effect of corticosterone and, more importantly, the stimulatory effect of day 16 serum. In contrast, the GH antiserum, used as a control for serum effects, had no effect on the embryonic serum response. Therefore, the blood-borne, somatotroph-differentiating compound in chicken embryonic serum is corticosterone.

The present study along with our previous results (7) indicate that levels of somatotroph-differentiating activity in the serum increase between days 12 and 16 of embryonic development, the period of normal somatotroph differentiation (6). This conclusion is supported by bioactivity detected in untreated serum and serum after heating, ether extraction, or trypsin digestion. Interestingly, levels of adrenal steroids increase around day 15 of chicken embryonic development (16, 17) and day 17 of rat embryonic development (18), just before somatotroph differentiation in both species. This correlation between an increase in serum concentrations of corticosterone, shown to be the blood-borne somatotroph-differentiating compound in the present study, and the ontogeny of GH-secreting cells during normal development suggests that increased adrenal glucocorticoid secretion stimulates the differentiation of pituitary somatotrophs. However, we observed in the present study that levels of a corticosterone-binding/inhibiting protein decreased between embryonic days 12 and 16. Thus, somatotroph differentiation during normal embryonic development could occur in response to an increase in adrenal corticosterone production, a decrease in serum corticosterone-binding/inhibiting activity, or a combination of both events. Additional research is necessary to distinguish between these possibilities.

The mechanism underlying glucocorticoid induction of somatotroph differentiation has not been defined. The effects of glucocorticoids on GH gene expression vary between in vitro and in vivo preparations. In most vertebrate species studied, glucocorticoids stimulate GH gene expression in intact animals (17, 19). In contrast, the effects of glucocorticoids on GH gene expression seem to be culture and cell line dependent (20). The lack of stimulatory effects of glucocorticoids on GH gene expression in culture might be due to the absence of synergism with thyroid hormones. Treatment with DEX in the absence of thyroid hormone stimulated GH expression less than simultaneous DEX and thyroid hormone treatment. However, premature expression of the GH gene cannot be stimulated by thyroid hormones without the presence of endogenous or exogenous glucocorticoids (4). In the present study, we demonstrated that glucocorticoids can induce GH cell differentiation in the absence of thyroid hormones. In our previous report (7), we noted that day 16 embryonic serum induced GH cell differentiation, as detected by immunocytochemistry and RHPAs without GHRH. Therefore, the newly recruited somatotrophs, induced to differentiate by corticosterone, synthesize and release GH in the absence of GHRH. This was also shown in the present study by RIA, in that day 16 serum and corticosterone increased medium GH content. Thus, three different assays (RHPA, RIA, and immunocytochemistry) indicate that somatotroph differentiation occurred in response to corticosterone and in the absence of GHRH. Our laboratory is currently exploring the possibility that corticosterone can interact with other factors to alter the extent of somatotroph differentiation.

Therefore, we conclude that somatotroph differentiation in the chicken embryonic pituitary can by induced in vitro by glucocorticoids and that corticosterone is the active compound responsible for the observed GH cell-differentiating activity of day 16 embryonic serum. The fact that normal GH cell differentiation during embryonic development occurs after an increase in circulating corticosterone levels in chickens (17) and rats (18) supports the involvement of glucocorticoids in GH cell differentiation during normal development in vivo.

	Footnotes

 
¹ This work was supported by USDA Grant 94–3206-1097 (to T.E.P.) and the Texas Agricultural Experiment Station.

Received June 5, 1997.

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