Administration of Adrenocorticotropic Hormone during Chicken ...

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Endocrinology 148(8):3914 –3921 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1102

Administration of Adrenocorticotropic Hormone during Chicken Embryonic Development Prematurely Induces Pituitary Growth Hormone Cells S. A. Jenkins, M. Muchow, M. P. Richards, J. P. McMurtry, and T. E. Porter Department of Animal and Avian Sciences (S.A.J., M.M., T.E.P.), and Molecular and Cell Biology Program (M.M., T.E.P.), University of Maryland, College Park, Maryland 20742; and United States Department of Agriculture (M.P.R., J.P.M.), Growth Biology Laboratory, Beltsville, Maryland 20705 Treatment of fetal rats and embryonic chickens with exogenous glucocorticoids induces premature GH cell differentiation. However, it is unknown whether the developing adrenal gland is capable of mounting this response autonomously. The present study determined whether stimulation of the adrenal gland in developing chicken embryos through administration of ACTH could induce a premature increase in GH cells. We found that plasma corticosterone and ACTH levels increased between embryonic day (e) 11 and e17, consistent with GH cell (somatotroph) ontogeny. Injection of ACTH into eggs on e9, e10, or e11 increased somatotrophs on e14. In contrast, thyroid-stimulating hormone, CRH, ␣-MSH, GHRH, and TRH were ineffective. Culture of e11 pituitary cells with ACTH failed to induce somatotrophs, suggesting an indirect action

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UR LABORATORY HAS been using the chicken embryo as a model to study the mechanisms underlying GH cell (somatotroph) differentiation in the anterior pituitary. Chicken embryonic development is a useful model for studies of extracellular or endocrine signals in this process because the embryos can be easily manipulated in the absence of compensatory maternal-fetal interactions. Furthermore, the ability to control the initiation of embryonic development in chickens allows for experiments involving hundreds of embryonic pituitary glands at a single age, which would be difficult in rodent models. In addition, the mechanisms involved in early pituitary differentiation are conserved between mammals and chickens (1), and the pattern of pituitary cell differentiation in chickens is comparable with that in mammals (2). Somatotrophs first become a significant population between embryonic day (e) 14 and e16 during chicken embryonic development (3). GH cells do not differentiate autonomously in cultures of chicken embryonic or fetal rat pituitary cells without an extrapituitary signal (4 –7), and GH cell differentiation can be induced in culture with adrenal glucocorticoids (5–9). Furthermore, treatment of chicken embryos in ovo with corticosterone (CORT) or of fetal rats in utero with dexamethasone increases the number

First Published Online April 26, 2007 Abbreviations: cACTH, Chicken ACTH; CE, capillary electrophoresis; CORT, corticosterone; e, embryonic day; hACTH, human ACTH; ICC, immunocytochemistry; LIF, laser-induced fluorescence. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

of ACTH on GH cells in vivo. Intravenous administration of ACTH dramatically increased plasma levels of corticosterone within 1 h and increased the percentage of pituitary somatotrophs within 24 h. Although ACTH administration increased the relative abundance of pituitary GH cells, there was no effect on plasma levels of GH, IGF-I, or IGF-II, or in hepatic expression of IGF-I or IGF-II mRNA. We conclude that ACTH administration can increase the population of GH cells in the embryonic pituitary. However, this treatment alone does not lead to downstream activation of hepatic IGF production. These findings indicate that the embryonic adrenal gland, and ultimately anterior pituitary corticotrophs, may function to regulate pituitary GH cell differentiation during embryonic development. (Endocrinology 148: 3914 –3921, 2007)

of GH cells prematurely (10 –15). However, the ability of ACTH and endogenous adrenal glucocorticoid production to induce GH cells has not been evaluated in any species. In the present study, experiments were designed to determine whether administration of ACTH to developing chicken embryos could induce premature somatotroph differentiation. We report that CORT production by the embryonic adrenal gland is sufficiently responsive to ACTH to induce GH cells prematurely and that this response is specific to ACTH. Finally, we examine whether the ACTH- or CORT-induced increase in GH cells activates downstream elements of the somatotropic axis by measuring hepatic IGF-I and IGF-II mRNA, and plasma IGF-I and IGF-II concentrations. Our findings implicate embryonic pituitary ACTH and adrenal glucocorticoid production in normal pituitary somatotroph ontogeny. Materials and Methods Animals, egg injections, and pituitary dispersions All animals used were broiler strain chicken embryos purchased from Allen’s Hatchery (Seaford, DE). All procedures with chicken embryos were approved by the Institutional Animal Care and Use Committee of the University of Maryland. All hormones and other chemicals were purchased from Sigma (St. Louis, MO), unless otherwise stated. Embryonic d 0 (e0) was defined as the day when the eggs were placed in a humidified incubator at 37.5 C. The typical length of embryonic development for chickens is 21 d. Treatment solutions (100 ␮l) were injected 8 mm into the egg albumen through a hole punched in the small apical end of the egg on e9, e10, or e11. These ages were chosen because we previously found that injection of CORT on e11 could induce GH cells, and we reasoned that additional time might be required for exogenous ACTH to induce GH through stimulation of endogenous glu-

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Jenkins et al. • ACTH Induces GH Cells

cocorticoid secretion. On e14, the embryos were removed, and their pituitary glands isolated using a dissecting microscope. The pituitaries were dissociated into individual cells by trypsin digestion and mechanical agitation, as described previously (3, 4). The viability of the cells, as assessed by the trypan blue dye-exclusion method, was consistently more than 95%. The dispersed cells were subjected to immunocytochemistry (ICC) to detect cells that contained GH.

ICC Intracellular GH was detected by ICC as described previously (16). In short, dispersed pituitary cells were diluted in DMEM to a concentration of 10,000 cells/ml, and were then attached to poly-l-lysine coated tissue culture plates during a 1-h incubation. The plated cells were fixed with 3.7% formaldehyde in PBS for 10 min, rinsed with PBS, quenched for 10 min with 0.3% H2O2, blocked for 30 min with 1% normal goat serum, and incubated overnight with rabbit antichicken GH (1:4000 in PBS) using antiserum validated previously (3). The cells were further processed using rabbit ABC kits according to the directions supplied by the manufacturer (Vector Laboratories, Burlingame, CA). VIP reagent (Vector Laboratories) was used as substrate for the peroxidase. GHcontaining cells were identified using an inverted light microscope as those exhibiting robust cytoplasmic staining. Cells with no obvious staining were considered GH negative. Results were expressed as the percentage of all pituitary cells present. Controls included cells incubated with normal rabbit serum instead of GH antiserum.

Extended cell cultures Anterior pituitary cells were cultured according to the procedure described previously (3, 4). Cells were plated (2 ⫻ 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 containing a 1:1 mixture of phenol red-free medium 199 and Ham’s F-12 nutrient mixture, supplemented with 0.1% BSA, 5 ␮g/ml human transferrin, 5 ␮g/ml bovine insulin, 100 U/ml penicillin G, and 100 ␮g/ml streptomycin sulfate. Cells were then treated with CORT or ACTH for 3 d in a humidified incubator (37.5 C; 95% air-5% CO2), after which the cells were harvested for detection of GH by ICC.

Plasma and tissue collection and RNA isolation On appropriate days, blood was collected from the chorioallantoic vessels of the embryonic chicks. Blood was allowed to flow into tubes containing 10 ␮l EDTA [0.5 m (pH ⫽ 8)] to prevent clotting and then centrifuged at 14,000 ⫻ g for 10 min to isolate the plasma. Plasma was stored at ⫺20 C until use. Liver samples were obtained and immediately frozen in liquid nitrogen. Tissues were stored at ⫺80 C before RNA isolation. Total RNA was isolated from tissue samples using Trizol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Isolated RNA was quantified using a UV spectrophotometer (Genequant II; Pharmacia Biotech, Piscataway, NJ).

Endocrinology, August 2007, 148(8):3914 –3921

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cycler (PTC-200; MJ Research, Waltham, MA). PCR thermal cycling parameters were as follows: one cycle at 94 C for 2 min, followed by 35 cycles for IGF-I or 30 cycles for IGF-II at 94 C for 30 sec, 60 C for 30 sec, and 72 C for 1 min with a final extension at 72 C for 8 min. The PCR products were then quantified using a procedure previously described and validated (17). Aliquots (2 ␮l) of RT-PCR samples were diluted 1:100 with deionized water before capillary electrophoresis (CE) with laserinduced fluorescence (LIF) detection. A P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Inc., Fullerton, CA) equipped with an argon ion LIF detector was used. Capillaries were 75 ␮m inside diameter ⫻ 32 cm ␮SIL-DNA (J&W Scientific, Folsom, CA). EnhanCE dye (Beckman Coulter, Inc.) was added to the DNA separation buffer (Sigma) to a final concentration of 0.5 ␮g/ml. Samples were loaded by electrokinetic injection at 3.5 kV for 5 sec and run in reverse polarity at 8.1 kV for 5 min. P/ACE MDQ software (Beckman Coulter, Inc.) was used to calculate peak areas for the PCR products separated by CE. The level of gene expression was determined as the ratio of integrated peak area for each individual gene PCR product relative to that of the coamplified 18S internal standard.

RIAs for ACTH, CORT, GH, IGF-I, and IGF-II The RIAs used to measure plasma ACTH and CORT levels during normal development were the ImmuChem Double Antibody ACTH and Corticosterone 125I RIA Kits (ICN Biomedicals, Costa Mesa, CA). All protocols were performed as directed by the supplier, except that in the ACTH kit; 10 ␮l undiluted plasma samples were used instead of a 1/200 dilution of the plasma samples, and half volumes of the steroid diluent, standards, CORT-125I, and anti-CORT were used. These steps were taken to increase the sensitivity of the assays. All samples were assayed in duplicate. The anti-ACTH antibody in the ACTH RIA kit was raised against human ACTH (hACTH) (39 amino acids). The amino acid sequence of chicken ACTH (cACTH) is approximately 77% identical to that of hACTH, so the results should be interpreted as ACTH equivalents, and not the actual plasma concentration of cACTH. The intraassay coefficients of variation were 4.95% and 3.65% for the ACTH and Corticosterone RIA kits, respectively. The GH RIA has been previously described and validated (18). The RIAs for IGF-I/II have been previously validated (19, 20), and the intraassay coefficients of variation for IGF-I and IGF-II assays were 3.39% and 4.22%, respectively.

Synthetic cACTH cACTH 1–24 was synthesized by Genemed Synthesis, Inc. (San Francisco, CA). The sequence used was ’N⬘-SYSMEHFRWGKPVGRKRRPIKVYP-’C’. This predicted cACTH amino acid sequence was derived from the cDNA sequence for chicken proopiomelanocortin (GenBank accession no. AB019555). The synthetic peptide was determined to be 97.6% pure by HPLC.

RT-PCR assay for IGF-I and -II mRNA

Intravenous administration of cACTH and CORT assay

First-strand cDNA synthesis was performed using the SuperScript First-Strand Synthesis System for RT-PCR kit (Invitrogen) on 1 ␮g total RNA in 0.2 ml thin-walled PCR tubes. The primer sequences for amplification of chicken IGF-I were as follows: forward (sense, 5⬘-GCTGAGCTGGTTGATGCTCT); and reverse (antisense, 5⬘-CACGTACAGAGCGTGCAGAT). The primer sequences for chicken IGF-II were as follows: forward (sense, 5⬘-ACACTGCAGTTCGTCTGTGG); and reverse (antisense, 5⬘-GCTGACTGGGCAAGGAGAT). 18S Ribosomal RNA was used as the internal control in the quantitative RT-PCR experiments. These controls were provided with the Universal 18S Internal Standards kit (Ambion, Austin, TX) and were used in a 1:9 18S primer to competimer ratio, as directed by the supplier. PCRs were conducted in a separate 25-␮l reaction volume containing: 2.5 ␮l 10⫻ PCR buffer minus Mg (Invitrogen); 1.0 ␮l 50 mm MgCl2; 0.5 ␮l 10 mm deoxynucleotide triphosphates; 0.2 ␮l PLATINUM Taq DNA Polymerase (Invitrogen); 1 ␮l chicken IGF-I or IGF-II gene-specific primers (10 ␮m); 2 ␮l 1:9 18S primer to competimer mix; 16.8 ␮l autoclaved-distilled water; and 1 ␮l each RT reaction. PCR was performed using a commercial thermal

Chorioallantoic membrane blood vessels were visualized by candling of eggs on e10, and eggshell overlying two vessels for each egg was removed with a Dremel rotary grinder (Dremel, Racine, WI). Synthetic cACTH (100 ␮l at 2 ⫻ 10⫺5 m in saline) was then injected into one of the vessels using a 0.5-ml syringe fitted with a 31-gauge needle. Controls included noninjected embryos and embryos injected with 100 ␮l saline. The eggs were then returned to the incubator. For determination of plasma CORT levels, blood samples were collected from the second chorioallantoic vessel using a 0.5-ml syringe fitted with a 31-gauge needle, 1 and 3 h after injection. Blood samples were immediately placed in tubes containing 5 ␮l of 0.5 m EDTA. The tubes were then centrifuged and resulting plasma samples frozen until assayed. Plasma samples (60 ␮l) were extracted twice with four volumes (240 ␮l) of diethyl ether, and the ether fractions dried and resuspended in 120 ␮l assay buffer (a 1:2 dilution). Concentrations of CORT were then determined using an ELISA kit (no. 500651), according to the manufacturer’s protocol (Cayman Chemical Co., Ann Arbor, MI). Values are expressed as picogram of steroid per milliliter of starting plasma.

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Data analysis All data were analyzed by ANOVA using the GLM or MIXED models procedures of SAS (SAS Institute Inc., Cary, NC). Differences between groups were tested by Duncan’s new multiple range test. Where appropriate, percentage data were transformed by taking the log or arcsine of the percentage to compensate for nonhomogeneity of variance among treatments. Differences were considered significant at P ⬍ 0.05.

Results Circulating levels of ACTH and CORT in embryonic chicks

To define endogenous ACTH and CORT secretion profiles, plasma samples were taken from embryonic chicks on e11, e13, e15, and e17, and were subjected to RIAs for ACTH and CORT. Figure 1 shows the plasma levels of both hormones at the various embryonic ages. Plasma ACTH levels were not significantly elevated, relative to e11, until e17. Plasma CORT levels showed a steady increase from e11– e17. These results confirm that ACTH and CORT levels are low before GH cell differentiation. Pituitary somatotroph abundance exhibits a similar increase between e12 and e16 (3). ACTH induction of somatotrophs

This experiment was conducted to ascertain whether exogenous ACTH administration at an age when endogenous levels are low could induce premature GH-cell differentiation and, if so, to define the ages during embryonic development that this could occur. One hundred microliters of 6 ⫻ 10⫺5 m hACTH (synthetic fragment 1–24) were injected on e9, e10, or e11, and the percentage of GH-containing cells was assessed on e14. This dose of hACTH was selected to achieve an average hACTH concentration in the egg of approximately 1 ⫻ 10⫺7 m, based on a typical egg weight of 60 g. This would be at the high end of the physiological range if all of the ACTH was distributed evenly. GH cells were assessed on e14 based on previous results (10). CORT (500 ng) and deionized water were also injected into eggs on e11 as positive and negative controls, respectively. Results are shown in Fig. 2A. Injection of hACTH into the egg albumen on e9, e10, and e11 significantly increased the number of somatotrophs on e14, FIG. 2. Effect of ACTH and CORT on somatotroph abundance in vivo. A, ACTH (100 ␮l at 6 ⫻ 10⫺5 M) was injected into the albumen of fertile eggs on either e9, e10, or e11. CORT (500 ng) and deionized water were also injected into eggs on e11 as positive and negative controls, respectively. B, ACTH (100 ␮l of 6 ⫻ 10⫺5 M or 6 ⫻ 10⫺6 M) was injected on either e10 or e11. Deionized water was injected into control eggs on e11 as a negative control. C, ACTH, CRH, GHRH, MSH, TRH, or thyroid-stimulating hormone (100 ␮l; 6 ⫻ 10⫺5 M) was injected into eggs on e11. For each series of experiments, the anterior pituitaries of the embryos were isolated on e14 and dispersed into individual cells. The percentage of pituitary cells that contained GH was determined using ICC. Data were analyzed by ANOVA, followed by Duncan’s multiple range test. These results are the means and SEM from four independent experiments. Values denoted by different letters are significantly different (P ⬍ 0.05).

FIG. 1. Embryonic levels of ACTH (top) and CORT (bottom). Plasma samples were collected from the chorioallantoic vessels on e11, e13, e15, or e17, and levels of both hormones were measured by RIA. Values represent the mean ⫾ SEM of four to five plasma samples for each age group. Significantly different values (P ⬍ 0.05) are indicated by different letters.

relative to water-injected and nontreated controls. There were no significant differences in the number of somatotrophs between the ages of hACTH administration tested. In additional experiments, the optimal dose and day of hACTH injection were evaluated further. Two different doses of hACTH 1–24 (100 ␮l of 6 ⫻ 10⫺5 and 6 ⫻ 10⫺6 m)


were injected on e10 or e11, and the population of somatotrophs was assessed on e14 in all groups. Injection of the lower dose of ACTH on e10 and e11 increased the number of GH-containing cells compared with the water-injected controls, but not compared with the nontreated controls (Fig. 2B). Injection of 6 ⫻ 10⫺5 m hACTH on e10 and e11 induced a greater increase in the percentage of somatotrophs on e14, with no significant difference between the two ages tested. These results indicate that treatment with 100 ␮l of 6 ⫻ 10⫺5 m hACTH is an effective dose, and this dose is equally effective when administered on e10 or e11. Specificity of ACTH response

The purpose of this set of experiments was to determine how specific the premature increase in somatotrophs is to ACTH administration, compared with other hypothalamic and pituitary peptide hormones. On e11, along with injection of 100 ␮l 6 ⫻ 10⫺5 m hACTH (full length, 1–39), 100 ␮l 6 ⫻ 10⫺5 m synthetic ovine CRH, human synthetic GHRH, synthetic N-acetylated ␣-MSH, synthetic TRH, or bovine thyroid-stimulating hormone (provided by Dr. John Proudman, U.S. Department of Agriculture, Beltsville, MD) were injected into fertile eggs, and on e14 the percentage of GHcontaining cells was determined. All of the mammalian preparations have been previously shown to be biologically active in chickens. Besides ACTH, only injections of GHRH and TRH were able to affect the percentage of somatotrophs, compared with noninjected and water-injected controls (Fig. 2C). However, these effects were small. Of all the hormones evaluated, ACTH was most effective at increasing the abundance of somatotrophs. Effect of ACTH on somatotroph differentiation in vitro

We next examined whether ACTH could induce GH cell differentiation directly. Anterior pituitary cells from e11 embryos were cultured for 3 d in the presence of deionized water, CORT (1 ⫻ 10⫺9 m), or hACTH (1 ⫻ 10⫺7 m). As Fig. 3 shows, culture of e11 pituitary cells for 3 d in the presence of ACTH failed to increase the number of GH-containing cells compared with the water-treated control group. On the other hand, CORT treatment significantly increased the number of somatotrophs. These results suggest that ACTH induction of GH cells must occur by an indirect mechanism.

FIG. 3. Effect of ACTH and CORT on somatotroph abundance in vitro. Pituitaries from e11 embryos were isolated and dispersed into individual cells. The cells were treated for 3 d with ACTH (1 ⫻ 10⫺7 ⫺9 M), CORT (1 ⫻ 10 M), or deionized water (vehicle control). After 3 d of treatment, the percentage of pituitary cells that contained GH was determined using ICC. Results shown are the means ⫾ SEM of triplicate cultures. Data were analyzed as in Fig. 2, with values denoted by different letters being significantly different (P ⬍ 0.05).


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Hepatic levels of IGF-I and IGF-II mRNA, and plasma levels of IGF-I, IGF-II, and GH

The previous experiments have shown conclusively that exogenous ACTH administration, when endogenous ACTH and CORT levels are low, can prematurely increase GH cells. One question that remains to be resolved is whether an increase in GH cells is associated with activation of other aspects of the somatotropic axis. To address this question, we determined whether injection of ACTH or CORT was associated with increased hepatic levels of IGF-I and IGF-II mRNA or plasma levels of IGF-I and IGF-II. On e11, ACTH and CORT (100 ␮l of 6 ⫻ 10⫺5 m and 500 ng, respectively) were injected into separate eggs. The livers of each treatment group were isolated on e14, e17, or e20, and total RNA was obtained. RT-PCR was performed on the RNA to determine levels of IGF-I and IGF-II mRNA relative to those of 18S ribosomal RNA. Figure 4 shows that neither treatment had any significant effect on levels of IGF-I (Fig. 4A) or IGF-II (Fig. 4B) mRNA. In initial trials there appeared to be a possible effect of ACTH on IGF-I mRNA levels on e17 (Fig. 4A). However, when 12 additional trials were performed on that age, the results were negative (data not shown), indicating that ACTH administration on e11 did not increase hepatic IGF-I gene expression. Plasma samples obtained on e14, e17, or e20 were subjected to RIAs for chicken IGF-I, IGF-II, and GH. Unfortunately, levels of GH were below our limit of detection (2.5 ng/ml) for e14 and e17 embryos (data not shown). ACTH or CORT treatment on e11 had no significant effect (P ⬎ 0.05; n ⫽ 3 samples for each treatment group) on plasma GH levels in e20 embryos (51.0 ⫾ 21.7 and 27.8 ⫾ 2.1 ng/ml, respectively) when compared with the water-injected group (71.7 ⫾ 7.2 ng/ml). Plasma IGF-I and IGF-II levels, shown in Fig. 4, were unaffected by treatment with either ACTH or CORT on any of the days tested (P ⬎ 0.05, n ⫽ 3– 4 samples for each age and treatment group). Effect of cACTH on somatotroph differentiation and adrenal CORT secretion

Up to this point, our experiments evaluated the effects of synthetic hACTH. We next wanted to ensure that the response observed was not due to a pharmacological effect of the hACTH amino acid sequence, relative to the endogenous cACTH sequence. To test this, 100 ␮l 6 ⫻ 10⫺5 m cACTH (synthetic, fragment 1–24) was injected into the albumen of fertile eggs on e11, and the percentage of GH-containing cells was assessed in each group on e13. Dose and timing of administration were selected based on preliminary experiments. Negative controls included noninjected and waterinjected eggs, while eggs injected with CORT (500 ng) served as a positive control. Results are shown in Fig. 5A. We found that cACTH and CORT were equally effective at increasing the abundance of GH cells, relative to noninjected and waterinjected controls. These results confirmed that endogenous cACTH is capable of prematurely inducing somatotrophs in vivo. This finding in combination with the inability of ACTH to induce somatotrophs in vitro implicated involvement of glucocorticoid synthesis by the embryonic adrenal glands in vivo.

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FIG. 4. Effect of CORT and ACTH on hepatic IGF-I and II mRNA levels, and plasma IGF-I and II. One hundred microliters of 6 ⫻ 10⫺5 M ACTH were injected into the albumen of fertile eggs on e11. Five hundred nanograms of CORT and 100 ␮l deionized water were also injected into control eggs on e11 as positive and negative controls, respectively. Embryonic livers were isolated on e14, e17, and e20, and total RNA was extracted. RT-PCR was used to amplify cDNA for IGF-I and IGF-II, and CE with LIF detection was used to quantitate the cDNAs. Vertical bars represent the integrated peak area ratios of the IGF-I/II amplicons to an 18S ribosomal RNA internal standard. The values expressed are the means ⫾ SEM of four independent samples for IGF-I (A) and IGF-II (B). Plasma samples were collected from the chorioallantoic vessels on e14, e17, or e20, and plasma levels of IGF-I (C) and IGF-II (D) were measured by RIA. Values represent the mean ⫾ SEM of three to four plasma samples for each age and treatment group. Significantly different values (P ⬍ 0.05) are indicated by different letters.

Plasma CORT levels after injection of ACTH

Because the main premise of our work is that ACTH elicits a premature increase in somatotroph differentiation by increasing adrenal glucocorticoid secretion, the purpose of this experiment was to verify an elevation in plasma CORT levels


FIG. 5. Effect of cACTH on somatotroph abundance and CORT secretion in vivo. A, cACTH (100 ␮l 6 ⫻ 10⫺5 M) was injected into the albumen of fertile eggs on e11, and the percentage of pituitary cells that contained GH was determined on e13. CORT (500 ng) and deionized water were also injected as positive and negative controls, respectively. These results are the means and SEM from eight independent experiments, with significant differences in the means (P ⬍ 0.05) indicated by the different letters. B, Saline or cACTH (100 ␮l at 2 ⫻ 10⫺5 M) was injected into the chorioallantoic vessels of e10 and e11 embryos, and the abundance of GH cells was determined by ICC on e12. Noninjected embryos (NI) served as controls. Results shown are the means and SEM of three replicate experiments. Values denoted by different letters are significantly different (P ⬍ 0.05). C, Saline or cACTH (100 ␮l at 2 ⫻ 10⫺5 M) was injected into chorioallantoic vessels of e10 embryos, and plasma samples were collected from separate chorioallantoic vessels 1 and 3 h later. Different embryos were used for each time point. Plasma levels of CORT were determined by ELISA. Significant differences (P ⬍ 0.05) between ACTH and saline at each time point are indicated by an asterisk (*).

after the injection of ACTH. To accomplish this aim, chorioallantoic vessels of individual e10 embryos were exposed for iv injection of cACTH. Synthetic cACTH or saline was injected into one vessel, and blood samples were collected 1 and 3 h later from a second vessel. In similar experiments,


cACTH or saline was injected into a chorioallantoic vessel on e10 or e11, and anterior pituitary glands were isolated on e12 for determination of GH cell abundance. Selection of injection and assay ages was based on preliminary experiments to maximize responses. Intravenous administration of cACTH on e10 or e11 increased somatotroph abundance on e12 by approximately 2-fold (Fig. 5B). Furthermore, iv injection of cACTH increased plasma CORT concentrations 26-fold at 1 h and 57-fold at 3 h (Fig. 5C). These results indicate that the embryonic adrenal gland is capable of responding to ACTH sufficiently through secretion of CORT to induce an increase in pituitary somatotrophs. Discussion

The ability of glucocorticoids to induce somatotroph differentiation in vitro and in vivo has been well documented in chickens (8, 10 –12, 16, 21–23) and rats (5–7, 14, 15). Administration of glucocorticoids can advance the appearance of GH cells by 1–2 d. In both species, glucocorticoid induction of GH gene expression requires active protein synthesis (7, 22), suggesting involvement of an intermediate protein and indicating that this mechanism of somatotroph induction during development may be common among vertebrates. However, the ability of endogenous adrenal glucocorticoid production in response to ACTH administration to induce this effect has not been evaluated in any species. Thus, the purpose of the present study was to examine the capacity of exogenous ACTH administration to stimulate adrenal CORT secretion and premature GH cell recruitment. We also measured plasma levels of ACTH and CORT at different embryonic ages, and we studied the plasma concentrations and hepatic mRNA levels of IGF-I and IGF-II, all in relation to elevated somatotroph number. We demonstrated that treatment of chicken embryos with ACTH on e9, e10, or e11 can increase somatotroph abundance on e12 or e14. However, treatment of pituitary cells for 3 d in culture with ACTH could not induce GH cell differentiation. This suggests that ACTH induction of GH cells in vivo must occur by an indirect mechanism, most likely by elevating plasma glucocorticoid levels that subsequently act on the pituitary to increase somatotroph abundance. It has been reported previously that the embryonic adrenal gland can secrete CORT in response to ACTH (24, 25). Our current and previous findings (10, 11) indicate that direct administration of CORT to e11 embryos can induce GH cell differentiation. In the present study we showed that iv administration of ACTH to individual embryos stimulated CORT secretion within 1 h and increased GH cells within 24 h. Therefore, ACTH most likely increases the number of somatotrophs by increasing plasma CORT levels, which in turn induces GH gene expression. This increase in pituitary somatotrophs represents an advancement in the ontogeny of GH cells, rather than a permanent increase in somatotroph abundance (10). Our conclusion that ACTH and CORT induce premature somatotroph differentiation rather than a decrease in other cell types is based on reports from our laboratory using in situ hybridization, whole mount in situ hybridization, Northern blotting, ICC, and reverse hemolytic plaque assays (8 –11, 16, 18, 21, 22, 26, 27). In each case, GH mRNA and mRNA-containing cells, and GH-con-


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taining and GH-secreting cells, increase from near zero after CORT treatment. The results are not due to a decrease in other cell populations because in many cases, little or no GH was detected under basal conditions. The concept of a steroid hormone regulating cellular differentiation in the pituitary gland is not new because it is generally accepted that estrogen stimulates the expansion of the lactotroph population in neonatal rats (28, 29). Development of the adrenal cortex precedes that of pituitary somatotrophs. In chickens, the development and secretory capacity of the adrenal cortex is believed to be independent of pituitary influence until e14 – e16 (30, 31), when its maturation and development become dependent on pituitary ACTH. These reports are consistent with our present findings that CORT levels increased between e11 and e15, even though ACTH levels remained low during that same period. On e17 there was a significant increase in ACTH, sequentially leading to increased secretion of CORT. Conversely, it has been shown in vivo (24) and in vitro (25) that the embryonic adrenal gland can respond to ACTH as early as e5. Thus, adrenal glucocorticoid secretion is largely autonomous, although responsive to ACTH, before the normal increase in ACTH secretion on e17. Somatotroph abundance normally increases at this same age, around e16 (3). Together, our results indicate that normal somatotroph differentiation may result from an increase in endogenous pituitary ACTH and adrenal CORT secretion. It is natural to assume that if CORT can increase GH cell number, then there must also be an increase in GH secretion. In a previous study, it was shown that treatment with CORT can stimulate GH secretion in vitro from e16, e18, and e20 pituitaries (23). However, in another report, it was shown that in ovo administration of CORT on e11 failed to increase serum GH on e14 (11). This lack of an increase in circulating GH levels may be due to the limited number of sampling times in that study or to the existence of pulsatile GH secretion in embryos, as in adult animals. In the present study, we failed to detect GH in the plasma on e14 or e17, or an effect of e11 CORT or ACTH treatment on plasma GH levels on e20. These results suggest that although somatotroph abundance is increased by CORT or ACTH administration in vivo, circulating levels of GH are unaffected. However, small increases on either e14 or e17 that remained below the limit of detection cannot be discounted. The IGFs (IGF-I and -II) play significant roles in growth as mediators of GH. We determined whether plasma IGF concentrations and hepatic IGF mRNA levels increased along with elevated pituitary GH cell numbers. Treatment with ACTH or CORT on e11 had no effect on either plasma concentrations or hepatic mRNA levels of IGF-I or IGF-II. These results are not entirely surprising because hepatic IGF-I production may not depend on GH during early embryonic development (32). In the chicken embryo, circulating GH is not detectable from e12– e15, but GH levels increase sharply near the time of hatching on e21 (33). In contrast, plasma levels of IGF-I reach a peak between e14 and e17, and then decline before hatching (34). Thus, the ontogeny of plasma IGF-I does not run parallel to that of plasma GH. Unlike IGF-I, plasma levels of IGF-II remain elevated throughout embryonic development (20). These divergent profiles in

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hormone secretion suggest that dynamic changes in GH secretion do not affect embryonic IGF-I or IGF-II production. Furthermore, hepatic GH receptor mRNA levels are nondetectable until e15 (35). Our intention in the present study was to assess whether ACTH or CORT-induced increases in pituitary GH cells had any effect at all on downstream elements of the somatotropic axis. Although published reports suggest that embryonic IGF production is independent of GH, we thought it prudent to evaluate this under the current experimental conditions. In addition, rather than reporting a lack of effect on one IGF element, we investigated effects of ACTH and CORT on hepatic mRNA levels, and serum protein levels for both IGF-I and IGF-II. This was done at multiple ages. No effects were found at any age or on any of the four parameters; all were negative. We conclude that ACTH- and CORTinduced increases in pituitary GH cells are not associated with increased plasma levels of IGF-I/II or hepatic levels of IGF-I/II mRNA. In the present study, we also evaluated the effects of other hypothalamic and pituitary hormones on somatotroph abundance. It should be noted that ␣-MSH administration in vivo did not increase the percentage of somatotrophs, even though ␣-MSH and ACTH are both products of the proopiomelanocortin gene, and the first 13 amino acids of ACTH are identical to full-length ␣-MSH. TRH and GHRH were able to increase GH cell numbers on e14 to a small extent. The exact mechanism for this is not known, but TRH and GHRH stimulate GH release (36, 37). Thus, the small increases in GH cells noted may be due to stimulation of GH production in existing but previously undetected somatotrophs. However, it has been shown that GHRH used in combination with CORT can increase somatotroph differentiation in vitro (18). Interestingly, CRH was unable to increase GH cell abundance. One would expect that CRH administration would lead to increased pituitary ACTH release and that ACTH subsequently would incite CORT secretion by the adrenal cortex to induce somatotroph differentiation. Our finding that CRH was unable to increase GH cells when injected into the egg albumen on e11 may indicate that either the pituitary at this age is not responsive to CRH or incapable of secreting sufficient ACTH to induce CORT secretion. In contrast, the normal increases in ACTH and CORT seen by e17 in the present study may be regulated by hypothalamic CRH release. Adrenal growth and development become dependent on ACTH stimulation around e16 (30, 31), and the hypothalamic immunoreactive CRH-neuronal system appears in the chick embryo around e14 (38). The anatomical link between the hypothalamus and the anterior pituitary is established early on in embryonic development. The vascular connection is present by e6 (39), and the hypothalamohypophysial portal vascular plexus is present by e12 (40). On e14, the number of corticotrophs increases significantly (41), signifying the morphological equivalent of a capacity to increase ACTH secretion. We found an increase in plasma ACTH between e15 and e17. So, it would appear that ACTH secretion from the adenohypophysis is regulated by hypothalamic CRH as early as e14, and clearly by e17. If this is true, then the lack of a CRH response in the present study most likely indicates that either the pituitary corticotrophs are unresponsive to CRH at e11 or that the response of the corticotrophs to CRH is not suf-


ficient to elicit an adequate amount of glucocorticoid secretion to subsequently stimulate GH cell differentiation. In conclusion, we found that administration of exogenous ACTH when endogenous levels are low increased adrenal CORT secretion and prematurely induced somatotroph differentiation. These results suggest that normal ontogeny of pituitary somatotrophs may be affected by pituitary corticotroph secretion of ACTH and adrenal production of glucocorticoids. Acknowledgments Received August 11, 2006. Accepted April 18, 2007. Address all correspondence and requests for reprints to: Tom E. Porter, Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland 20742. E-mail: [email protected]. This research was supported by National Research Initiative Competitive Grants 2000-35206-9463, 2003-35206-12836, and 2006-3520616617 from the United States Department of Agriculture Cooperative State Research, Education, and Extension Service. Disclosure Statement: The authors have nothing to declare.

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