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RAPID COMMUNICATIONS Expression Profiles of Growth Hormone-Releasing Hormone and Growth Hormone-Releasing Hormone Receptor During Chicken Embryonic Pituitary Development C. Y. Wang, Y. Wang, J. Li, and F. C. Leung1 Department of Zoology, The University of Hong Kong, Hong Kong, China GHRHR during embryonic pituitary development. The expression of GHRHR on embryonic d 8 was much lower, but abundant expression was noticed as early as embryonic d 12. In contrast, the level of pituitary GHRH mRNA peaked on d 8 and declined sharply afterwards. Interestingly, unlike those of pituitary GHRH and GHRHR, the higher expression levels of GH appeared much later (from d 16 to 20). The differential expressions of GHRH, GHRHR, and GH in the developing embryonic pituitaries not only imply that pituitary-derived GHRH (or pituitary adenylate cyclase-activating polypeptide) and GHRHR may have a paracrine/autocrine role in the expansion of undifferentiated somatotroph precursor cells, but also suggest that GHRHR is likely to be involved in the somatotroph differentiation occurring at the later developmental stages.

ABSTRACT Growth hormone-releasing hormone (GHRH) and its receptor (GHRHR) have long been regarded as the critical molecules for the stimulation of growth hormone (GH) synthesis and release, as well as the regulation of pituitary somatotroph expansion in vertebrates. However, little is known about their expression in the embryonic pituitaries of birds. In this study, the full-length cDNA for chicken GHRHR was cloned from the chicken pituitary. It encodes 419 amino acids and shares high homology with that of the human, rat, and mouse. As in those in mammals, chicken GHRHR is predominantly expressed in the pituitary and weakly expressed in several extra-pituitary tissues including brain, pancreas, testis, and kidney, among 12 tissues examined. Using semiquantitative reverse transcription-PCR, we further examined the expression of GH, GHRH, and

Key words: chicken, embryonic pituitary, growth hormone, growth hormone-releasing hormone, growth hormone-releasing hormone receptor 2006 Poultry Science 85:569–576

which activates protein kinase A to stimulate the transcription of GH gene (Muller et al., 1999). The role of GHRHR in normal pituitary development is underscored by increasing evidence that mutations in the mouse and human GHRHR genes lead to growth hormone deficiency, stunted growth, and somatotroph hypoplasia (Lin et al., 1992, 1993; Godfrey et al., 1993; Wajnrajch et al., 1996; Netchine et al., 1998). These genetic disorders strongly suggest that GHRHR plays a key role in the hypothalamo-pituitary GH axis. In mammals, GHRHR mRNA is predominantly expressed in the pituitary (Mayo, 1992), and its expression in the pituitary varies with developmental stage (Korytko et al., 1996; Nogami et al., 1999). However, information on the expression profiles of both GHRH and GHRHR in the embryonic pituitaries is still limited in vertebrates including birds. In the present study, using chicken as the experimental model, we cloned GHRHR from chicken pituitary and characterized the expression profiles of GHRH, GHRHR, and GH during embryonic pituitary development. The aim of this study was to provide insights into the physiological roles of pituitary GHRH and GHRHR in the soma-

INTRODUCTION The synthesis and release of growth hormone (GH) are regulated by the hypothalamic polypeptides including growth hormone-releasing hormone (GHRH) and somatostatin. Growth hormone-releasing hormone stimulates the production and release of growth hormone, whereas somatostatin suppresses the release of growth hormone (Petersenn and Schulte, 2000; Frohman and Kineman, 2002; Lin-Su and Wajnrajch, 2002; Porter, 2005). The biological actions of GHRH are mediated by GHRH receptor (GHRHR), a G-protein-coupled receptor specifically localized in the somatotroph of the pituitary (Godfrey et al., 1993; Morel et al., 1999). Activation of GHRHR by GHRH stimulates G-protein and then increases the intracellular calcium, which causes release of premade GH, and causes an increase in the level of the intracellular cyclic adenosine monophosphate level,

2006 Poultry Science Association, Inc. Received December 14, 2005. Accepted December 19, 2005. 1 Corresponding author: [email protected]

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WANG ET AL. Table 1. Primers used for cloning growth hormone-releasing hormone (GHRHR), growth hormone-releasing hormone (GHRH), and growth hormone (GH) Primer sequence1

Size, bp

GenBank accession no.

5′-CAGAGGAGCATGAGAATGCAA-3′ 5′-CTCTACCAGCAGCCATATGAA-3′ 5′-GACATTGACCATTGCAGCTT-3′ 5′-CGTCTGTACTGAGATGAGTTATTGA-3′ 5′-CAGTACAGACGCCTCTCAAGGTCA-3′ 5′-GAGGTGACTGCTCAGGATTTGGTGA-3′ 5′-CAACTGGACAAGCAATGTGATAGGA-3′ 5′-CTATCCCAGCTGATGTTGACTTCTCA-3′ 5′-GAGGTGTACGCGCTGTACTA-3′ 5′-GCTCATCGCTACAAATACGCTA-3′ 5′-CAGAAGTCAGACATGGAGCT-3′ 5′-CTCAGATGGTGCAGTTGCT-3′ 5′-CCCAGACATCAGGGTGTGATG-3′ 5′-GTTGGTGACAATACCGTGTTCAAT-3′

539

DQ230840

Gene GHRHR

GHRH GH β-Actin

Sense2 Antisense2 Sense3 Antisense3 Sense4 Sense4 Antisense4 Antisense4 Sense2,3 Antisense2,3 Sense3 Antisense3 Sense2,3 Antisense2,3

407

326

AY956323

373

NM_204359

123

L08165

1

All primers were synthesized by Invitrogen (Invitrogen, Hong Kong). Primers used for reverse transcription-PCR. 3 Primers used for semiquantitative reverse transcription-PCR. 4 Primers used for rapid amplification of 5′ and 3′ cDNA ends (5′ and 3′ RACE) PCR. 2

totroph proliferation and differentiation during embryonic development.

MATERIALS AND METHODS Animal Tissues Adult chickens and chicken embryos were provided by Kardoorie Agricultural Research Center (University of Hong Kong). All embryos were incubated at 37.5°C in a humidified incubator. Chicken pituitaries from embryonic d 8 (E8), 12 (E12), 16 (E16), and 20 (E20) were isolated, and the attached brain tissue was removed carefully under a dissection microscope. Embryos from d 2, 3, 4, and 5 were rapidly frozen in liquid nitrogen. Adult chickens were decapitated and 12 tissues (including brain, pituitary, lung, heart, liver, kidney, intestine, pancreas, breast muscle, spleen, ovary, and testis) were collected for total RNA extraction.

Cloning the Full-Length cDNA for Chicken GHRHR Gene-specific primers were designed based on the predicted sequence for chicken GHRHR (GenBank accession no.: XM_418490; Table 1). A 1,020-bp cDNA fragment was amplified from the adult chicken pituitary by reverse transcription-PCR (RT-PCR). The fragment was then cloned into pBluescript II KS (+) through T/A cloning (Stratagene, La Jolla, CA). Based on the sequence of the cloned cDNA fragment, gene-specific primers were designed to amplify the 5′ and 3′ cDNA ends using the SMART-RACE cDNA Amplification Kit (Clontech, Palo Alto, CA; Table 1). The amplified 5′ and 3′ cDNA ends were cloned into pBluescript II KS (+) and sequenced. New primers located in the 5′ and 3′ untranslated regions were designed to amplify the full-length cDNA by using the high fidelity Taq DNA polymerase (Roche, Switzer-

land; Table 1). The full-length cDNA for GHRHR was determined by sequencing 3 independent clones.

RNA Extraction, RT-PCR, and Semiquantitative RT-PCR Total RNA was extracted from pituitaries and 11 other adult tissues using Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. Reverse transcription was performed at 42°C for 2 h in a total volume of 10 ␮L consisting of 2 ␮g of RNA, 1× PCR buffer, 10 mM dithiothreitol, 0.5 ␮M of each dNTP, 0.5 ␮g of oligo(dT), and 100 U of Superscript II (Invitrogen, Carlsbad, CA). One microliter of the firststrand cDNA was used as the template for each PCR reaction. According to our previously established methods (Wang et al., 2003; Wang and Ge, 2003, 2004), a semiquantitative RT-PCR assay was also performed to examine the expression of GHRH, GHRHR, and GH in the embryonic pituitaries from d 8, 12, 16, and 20. The PCR was performed under following conditions: 2 min at 94°C for denaturation, followed by 23 cycles (for β-actin: 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C), 29 cycles (for GHRHR and GH: 30 s at 94°C, 30 s at 54°C, and 60 s at 72°C), or 33 cycles (for GHRH: 30 s at 94°C, 30 s at 54°C, and 60 s at 72°C) of reactions, ending with a 7-min extension at 72°C. The primers used are listed in Table 1. The PCR products were electrophoresed in 1.5% agarose gels, stained with ethidium bromide, and visualized under UV illumination. The intensity of each band was quantified using Quantity One software (version 4.5.2, Bio-Rad, Hercules, CA). Moreover, the PCR products were sequenced directly using an ABI 3100 Genetic Analyzer (PE Biosystems, Foster City, CA) to confirm the specificity of amplification.

Data Analysis The mRNA level of each gene was first calculated relative to that of β-actin (which was amplified as the internal

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Figure 1. Comparison of the deduced amino acid sequence of chicken growth hormone-releasing hormone receptor (GHRHR) with that of human, rat, and mouse. The 7 predicted transmembrane domains (TM1 to 7) are underlined. A deletion detected in chicken GHRHR (exon 3, 36 amino acids) is shaded.

control) and expressed as a ratio, and then expressed as a percentage of the level at d 8 (E8). The data were analyzed by one-way ANOVA followed by Dunnett’s test. We performed all the experiments at least twice to confirm the results using different batches of embryos and chickens.

RESULTS Cloning of Full-Length cDNA for Chicken GHRHR The full-length cDNA for chicken GHRHR is 1,672 bp (GenBank accession no.: DQ230840) and encodes a precursor of 419 amino acids, which shares high sequence identity with that of human (61%; Gaylinn et al., 1993), rat (57%; Mayo, 1992), and mouse (56%; Lin et al., 1992), as shown in Figure 1. Comparison of the GHRHR cDNA sequence with the chicken genome database (http:// www.ensembl.org/Gallus_gallus) revealed that the ghrhr gene is located on chromosome 2, and consists of 13 exons (data not shown).

Tissue Distribution of GHRH and GHRHR Expression To examine the mRNA expression of GHRHR in adult chicken tissues, RT-PCR was used. As shown in Figure 2, GHRHR was predominantly expressed in the pituitary. However, a low level of GHRHR mRNA was also found in extra-pituitary tissues including brain, kidney, pan-

creas, and testis (Figure 2). The possibility of amplifying genomic DNA was ruled out by using intron-spanning primers (Table 1). Interestingly, a minor band was consistently observed in the pituitary when a high PCR cycle number was used (Figure 2). Sequencing analysis revealed that the minor band was due to deletion of exon 3 (108 bp, 36 amino acids; Figure 1). To determine the paracrine/autocrine roles of GHRH in pituitary and extra-pituitary tissues, the expression of GHRH was also examined in 12 tissues from adult chickens. Interestingly, the highest expression of GHRH was detected in brain and heart. Only moderate expression was noticed in the testis and intestine. Similar to the situation in lower vertebrates (Sherwood et al., 2000; Wang et al., 2003), the mRNA for chicken GHRH and pituitary adenylate cyclase-activating polypeptide (PACAP) are transcribed from the same gene (Figure 2; GenBank accession no. AY956323). Surprisingly, however, alternative splicing of the mRNA, which may control the differential expression of 2 important bioactive polypeptides in various tissues of lower vertebrates, was not observed in chicken adult tissues even though the higher cycle number was used (Figure 2).

Expression Profiles of GHRH, GHRHR, and GH During Embryonic Pituitary Development To elucidate the roles of GHRH and GHRHR in pituitary development, we investigated the expression of GHRH, GHRHR, and GH in embryonic pituitaries. The

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Figure 2. A) DNA and deduced amino acid sequence of chicken growth hormone-releasing hormone-pituitary adenylate cyclase-activating polypeptide (GHRH-PACAP) gene (GenBank Accession no. AY956323). The shaded amino acid sequences are the putative mature GHRH and PACAP38 sequences. The shaded DNA sequences indicate the locations of sense and antisense primers used for reverse transcription-PCR analyses. B) Tissue distributions of growth hormone-releasing hormone receptor (GHRHR) and GHRH mRNA. C) Detection of GHRHR and GHRH mRNA in whole embryos on d 2, 3, 4, and 5 (embryonic days E2, E3, E4, and E5). Numbers in parentheses show the PCR cycle numbers used.

level of GHRHR mRNA was lowest on embryonic d 8; however, its abundant expression was noticed on d 12 and later (Figure 3). In contrast, pituitary GHRH did not follow the same expression pattern. Its expression was highest on d 8 (E8) and decreased dramatically on d 12, 16, and 20. The expression of GH mRNA had changes similar to that of GHRHR; however, its higher expression significantly lagged behind (from E16 to E20; Figure 3). In addition, we noted that the level of GHRHR mRNA in the whole embryos was the lowest on d 2 and increased slightly on d 4 and 5. In contrast, GHRH mRNA was highly expressed at all stages investigated (Figure 2). The limited expression of GHRHR suggests that GHRH may play a limited role in earlier embryogenesis.

DISCUSSION In the present study, we cloned the full-length cDNA for chicken GHRHR and investigated the expression profiles of GH, GHRH, and GHRHR in chicken embryonic pituitaries. The results from our experiments suggest that pituitary GHRH and GHRHR are likely to be involved in somatotroph development at the embryonic stage. Chicken GHRHR (419 amino acids) is slightly shorter than that of human, rat, and mouse (423 amino acids); however, high sequence identity (∼56%) could be observed between mammals and chickens (Lin et al., 1992; Mayo, 1992; Gaylinn et al., 1993). The amino acid residue (Asp60) critical for ligand binding is conserved in the

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Figure 3. Temporal expression profiles of A) growth hormone-releasing hormone (GHRH), B) growth hormone-releasing hormone receptor (GHRHR), and C) growth hormone (GH) during embryonic pituitary development in the chicken. The expression levels were normalized to that of β-actin and expressed as a percentage of the level on embryonic d 8 (E8). The electrophoretic images from one experiment are shown in panel D for embryonic days E8, E12, E16, and E20. Each data point represents the mean ± SEM of 3 individual chicken embryos. *P < 0.05 vs. d 8 (E8); **P < 0.001 vs. d 8 (E8).

chicken, rat, mouse, and human (Godfrey et al., 1993; Lin et al., 1993). As in rat, mouse, and human, GHRHR in adult chickens is predominantly expressed in the pituitaries. Although the alternatively spliced transcripts of mammalian GHRHR have been noted (Zeitler et al., 1998; Miller et al., 1999), the isoforms identified in mammals were not found in the present study. Interestingly, a minor band was consistently observed in adult pituitaries (Figure 2) and embryonic pituitaries (data not shown). Sequencing analyses indicated that it resulted from a deletion of exon 3, which causes a deletion of 36 amino acid residues (including Asp60) in the extracellular ligandbinding domain. The full-length cDNA for this novel isoform has not yet been obtained, but the structural change in ligand-binding domain may modify GHRHR signaling in the pituitary.

Despite the abundant expression of GHRHR in the pituitary, GHRH displays a distinct spatial expression pattern in chickens. In agreement with previous findings (McRory et al., 1997), little or no expression of GHRH could be detected in the adult chicken pituitary, supporting the hypothesis that hypothalamus-derived GHRH, but not pituitary-produced GHRH, plays a major role in the pituitary of adult chickens. It is well documented that both GHRH and PACAP are encoded on one gene and the mRNA alternative splicing (deletion of exon 4) occurs widely in nonmammalian species (Parker et al., 1997; Hu et al., 2000; Sherwood et al., 2000; Wang et al., 2003; Sherwood and Wu, 2005). However, we could not find spliced transcripts in brain, heart, intestine, or testis from adult chickens. This finding differs slightly from a previous report (McRory et al., 1997). The reason for the dis-

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crepancies in the 2 studies is unknown. However, the deduced amino acid sequence of GHRH/PACAP (Figure 2) differs from the previously identified one in the Nterminal region (McRory et al., 1997). It is generally believed that GHRH and GHRHR play critical roles in somatotroph development in mammals (Petersenn and Schulte, 2000; Frohman and Kineman, 2002). In this study, the expression of pituitary GHRHR was detected at all embryonic stages examined and its expression showed a significant stage-dependent variation. The mRNA level of GHRHR on d 8 is the lowest among all of the stages examined. In contrast to that of GHRHR, GHRH reaches its highest expression at the same stage. The co-expression of GHRH and GHRHR suggests that pituitary-produced GHRH may have an autocrine/paracrine role in the earlier development of embryonic pituitary. The higher expression level of GHRH in the embryonic pituitary has not been reported in any other species. However, it should be noted that both GHRH and PACAP are encoded on the same gene and translated from a common mRNA transcript. Thus, the high level of GHRH mRNA suggests that PACAP may be also involved in this developmental process. Interestingly, the expression of GHRHR dramatically increases on d 12, when rare somatotroph cells are detected by immunostaining analyses and reverse hemolytic plaque assay (Porter et al., 1995; Porter, 2005). The GHRHR is localized specifically in the somatotrophs of vertebrates, and GHRH has strong effects on GH synthesis and release (Leung and Taylor, 1983; Morel et al., 1999; Muller et al., 1999; Frohman and Kineman, 2002). Hence, the high expression of GHRHR occurring before somatotroph differentiation not only indicates that a significant population of somatotroph precursor cells may already exist on embryonic d 12 but also suggests that GHRHR may be actively involved in the rapid expansion of undifferentiated somatotrophs. Our data also support the findings in chickens that there is a preexisting pool of somatotroph precursors on d 12, which could undergo premature differentiation with glucocorticoid (corticosterone) treatment (Bossis and Porter, 2000; Bossis et al., 2004). Because pituitary GHRH decreases dramatically on embryonic d 12 when the hypothalamus is less mature or nonfunctional, the high level of GHRH produced remains unknown. The abundant expression of GHRH in the chicken embryonic brain (data not shown) suggests that the embryonic brain is one of the potential sources. It has been reported that a transcriptional factor, pit-1, can enhance the promoter activity of the human and rat ghrhr gene in vitro (Petersenn et al., 1998; Miller et al., 1999). However, a high level of pituitary pit-1 mRNA appears as early as d 5 (Van As et al., 2000; Nakamura et al., 2004). Thus, it is likely that other pituitary-derived factors including GHRH (or extra-pituitary signals) other than pit-1 are responsible for initiating a dramatic increase in GHRHR expression or GHRHR-producing cells on d 12 (E12). Substantial evidence shows that the differentiation of somatotrophs occurs between embryonic d 12 and 16 (Porter, 2005). In agreement with this concept, a dramatic increase

in the mRNA level of GH was also noticed within this 4-d window. Interestingly, a slight but not significant increase in GHRHR mRNA also appeared. The slight increase in the GHRHR expression, to some extent, supports the findings in chickens that GHRH has a synergistic, but not decisive, role in glucocorticoid-induced differentiation of the somatotroph because glucocorticoid alone can effectively induce the differentiation of somatotroph in vitro in the absence of GHRH (Dean and Porter, 1999; Bossis et al., 2004). It is proposed that the synergistic role of GHRH may be attributed to the glucocorticoidinduced GHRHR expression as demonstrated in mammals (Dean et al., 1999; Nogami et al., 1999). However, considering the significant increase in pituitary size during embryonic development, the possibility that the synergistic role may be in part due to mitogenic effect of GHRH on somatotrophs (Billestrup et al., 1986) cannot be ruled out. After embryonic d 16, the abundant expression of GHRHR mRNA could still be detected before hatching (d 20). This finding is consistent with the ability of cultured E16 somatotrophs (or later stage) to release GH in response to exogenous GHRH (Porter et al., 1995; Dean et al., 1997). Interestingly, somatotroph cells at a stage earlier than d 16 could not release GH in response to GHRH (Porter et al., 1995; Dean et al., 1997). The little or no responsiveness is likely due to the lower maturity of somatotrophs, rather than the delayed functioning of GHRHR. The study on the timing of GHRH expression in developing hypothalamus, as well as the mitogenic action of GHRH on undifferentiated somatotroph cells, would provide clues to this issue. Similar to that in chickens, clear stage-dependent expression of GHRHR was noted in the later embryonic stage of rats by using an RNase protection assay (Nogami et al., 1999). The GHRHR mRNA was first detected at d 19 and increased linearly until E21 (Nogami et al., 1999). This stage-dependent expression is synchronous to the differentiation of GH cells in vivo (Nogami and Tachibana, 1993; Nogami et al., 1999). In contrast to that in rats, the abundant expression of GHRHR in chickens appears before GH cell differentiation (E12). The discrepancy may be due to species difference in pituitary organogenesis. However, similar to rats (Nogami et al., 1999), high levels of GHRHR and GH mRNA were simultaneously detected on d 16, suggesting that GHRHR may have a conserved role in GH cell differentiation in the later embryonic stages across vertebrates. It has been hypothesized that glucocorticoid may be one of the factors to trigger the simultaneous expressions of GHRHR and GH in rats (Nogami and Tachibana, 1993; Nogami et al., 1997, 1999). Similar to that in rats, glucocorticoid is also capable of inducing differentiation of somatotrophs in chickens (Dean et al., 1999; Porter, 2005). Whether glucocorticoid and its receptors (type I and type II glucocorticoid receptors) are also involved in initiating the expression of GHRHR, or whether the differential expression of GHRHR and GH is attributed to the different responsiveness of 2 genes to serum glucocorticoid levels remains to be determined in future studies.

RAPID COMMUNICATION: EXPRESSION OF GHRH AND GHRHR

In summary, a chicken GHRHR has been cloned in the present study. Reverse transcription-PCR demonstrated that chicken GHRHR is predominantly expressed in the pituitary and its expression is stage-dependent. The abundant expression of GHRHR mRNA appears before (E12) and continues during and after somatotroph differentiation. In contrast, pituitary-derived GHRH is only highly expressed on embryonic d 8, at which time GHRHR has its lowest expression. The expressions of pituitary GHRH and GHRHR suggest that GHRH and GHRHR may have an important role in the expansion and differentiation of GH cells in the embryonic pituitary of birds.

ACKNOWLEDGMENT This work was supported by Research Grant Council of the Hong Kong Government, HKU7345/03M.

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