Transcriptional analysis of abdominal fat in chickens divergently selected on bodyweight at two ages reveals novel mechanisms controlling adiposity: validating visceral adipose tissue as a dynamic endocrine and metabolic organ.

BACKGROUND: Decades of intensive genetic selection in the domestic chicken (Gallus gallus domesticus) have enabled the remarkable rapid growth of today's broiler (meat-type) chickens. However, this enhanced growth rate was accompanied by several unfavorable traits (i.e., increased visceral fatness, leg weakness, and disorders of metabolism and reproduction). The present descriptive analysis of the abdominal fat transcriptome aimed to identify functional genes and biological pathways that likely contribute to an extreme difference in visceral fatness of divergently selected broiler chickens. METHODS: We used the Del-Mar 14 K Chicken Integrated Systems microarray to take time-course snapshots of global gene transcription in abdominal fat of juvenile [1-11 weeks of age (wk)] chickens divergently selected on bodyweight at two ages (8 and 36 wk). Further, a RNA sequencing analysis was completed on the same abdominal fat samples taken from high-growth (HG) and low-growth (LG) cockerels at 7 wk, the age with the greatest divergence in body weight (3.2-fold) and visceral fatness (19.6-fold). RESULTS: Time-course microarray analysis revealed 312 differentially expressed genes (FDR ≤ 0.05) as the main effect of genotype (HG versus LG), 718 genes in the interaction of age and genotype, and 2918 genes as the main effect of age. The RNA sequencing analysis identified 2410 differentially expressed genes in abdominal fat of HG versus LG chickens at 7 wk. The HG chickens are fatter and over-express numerous genes that support higher rates of visceral adipogenesis and lipogenesis. In abdominal fat of LG chickens, we found higher expression of many genes involved in hemostasis, energy catabolism and endocrine signaling, which likely contribute to their leaner phenotype and slower growth. Many transcription factors and their direct target genes identified in HG and LG chickens could be involved in their divergence in adiposity and growth rate. CONCLUSIONS: The present analyses of the visceral fat transcriptome in chickens divergently selected for a large difference in growth rate and abdominal fatness clearly demonstrate that abdominal fat is a very dynamic metabolic and endocrine organ in the chicken. The HG chickens overexpress many transcription factors and their direct target genes, which should enhance in situ lipogenesis and ultimately adiposity. Our observation of enhanced expression of hemostasis and endocrine-signaling genes in diminished abdominal fat of LG cockerels provides insight into genetic mechanisms involved in divergence of abdominal fatness and somatic growth in avian and perhaps mammalian species, including humans.

Mapping QTL for growth and shank traits in chickens divergently selected for high or low body weight.

An F(2) population (695 individuals) was established from broiler chickens divergently selected for either high (HG) or low (LG) growth, and used to localize QTL for developmental changes in body weight (BW), shank length (SL9) and shank diameter (SD9) at 9 weeks. QTL mapping revealed three genome-wide QTL on chromosomes (GGA) 2, 4 and 26 and three suggestive QTL on GGA 1, 3 and 5. Most of the BW QTL individually explained 2-5% of the phenotypic variance. The BW QTL on GGA2 explained about 7% of BW from 3 to 7 weeks of age, while that on GGA4 explained 15% of BW from 5 to 9 weeks. The BW QTL on GGA2 and GGA4 could be associated with early and late growth respectively. The GGA4 QTL also had the largest effect on SL9 and SD9 and explained 7% and 10% of their phenotypic variances respectively. However, when SL9 and SD9 were corrected with BW9, a shank length percent QTL was identified on GGA2. We identified novel QTL and also confirmed previously identified loci in other chicken populations. As the foundation population was established from commercial broiler strains, it is possible that QTL identified in this study could still be segregating in commercial strains.

A comprehensive analysis of QTL for abdominal fat and breast muscle weights on chicken chromosome 5 using a multivariate approach.

Quantitative trait loci (QTL) influencing the weight of abdominal fat (AF) and of breast muscle (BM) were detected on chicken chromosome 5 (GGA5) using two successive F(2) crosses between two divergently selected 'Fat' and 'Lean' INRA broiler lines. Based on these results, the aim of the present study was to identify the number, location and effects of these putative QTL by performing multitrait and multi-QTL analyses of the whole available data set. Data concerned 1186 F(2) offspring produced by 10 F(1) sires and 85 F(1) dams. AF and BM traits were measured on F(2) animals at slaughter, at 8 (first cross) or 9 (second cross) weeks of age. The F(0), F(1) and F(2) birds were genotyped for 11 microsatellite markers evenly spaced along GGA5. Before QTL detection, phenotypes were adjusted for the fixed effects of sex, F(2) design, hatching group within the design, and for body weight as a covariable. Univariate analyses confirmed the QTL segregation for AF and BM on GGA5 in male offspring, but not in female offspring. Analyses of male offspring data using multitrait and linked-QTL models led us to conclude the presence of two QTL on the distal part of GGA5, each controlling one trait. Linked QTL models were applied after correction of phenotypic values for the effects of these distal QTL. Several QTL for AF and BM were then discovered in the central region of GGA5, splitting one large QTL region for AF into several distinct QTL. Neither the 'Fat' nor the 'Lean' line appeared to be fixed for any QTL genotype. These results have important implications for prospective fine mapping studies and for the identification of underlying genes and causal mutations.

Functional genomics of the chicken--a model organism.

Since the sequencing of the genome and the development of high-throughput tools for the exploration of functional elements of the genome, the chicken has reached model organism status. Functional genomics focuses on understanding the function and regulation of genes and gene products on a global or genome-wide scale. Systems biology attempts to integrate functional information derived from multiple high-content data sets into a holistic view of all biological processes within a cell or organism. Generation of a large collection ( approximately 600K) of chicken expressed sequence tags, representing most tissues and developmental stages, has enabled the construction of high-density microarrays for transcriptional profiling. Comprehensive analysis of this large expressed sequence tag collection and a set of approximately 20K full-length cDNA sequences indicate that the transcriptome of the chicken represents approximately 20,000 genes. Furthermore, comparative analyses of these sequences have facilitated functional annotation of the genome and the creation of several bioinformatic resources for the chicken. Recently, about 20 papers have been published on transcriptional profiling with DNA microarrays in chicken tissues under various conditions. Proteomics is another powerful high-throughput tool currently used for examining the dynamics of protein expression in chicken tissues and fluids. Computational analyses of the chicken genome are providing new insight into the evolution of gene families in birds and other organisms. Abundant functional genomic resources now support large-scale analyses in the chicken and will facilitate identification of transcriptional mechanisms, gene networks, and metabolic or regulatory pathways that will ultimately determine the phenotype of the bird. New technologies such as marker-assisted selection, transgenics, and RNA interference offer the opportunity to modify the phenotype of the chicken to fit defined production goals. This review focuses on functional genomics in the chicken and provides a road map for large-scale exploration of the chicken genome.

Manipulation of thyroid status and/or GH injection alters hepatic gene expression in the juvenile chicken.

Both thyroid hormone (T3) and growth hormone (GH) are important regulators of somatic growth in birds and mammals. Although T3-mediated gene transcription is well known, the molecular basis of T3 interaction with GH on growth and development of birds remains unknown. In earlier studies, we discovered that exogenous GH alone increased accumulation of visceral fat in young chickens, while the combination of GH injections and dietary T3 worked synergistically to deplete body fat. In the present study, cDNA microarray and quantitative RT-PCR analyses enabled us to examine hepatic gene expression in young chickens after chronic manipulation of thyroid status and GH injection alone or in combination with T3. Thyroid status modulates expression of common and unique sets of genes involved in a wide range of molecular functions (i.e., energy metabolism, storage and transport, signal transduction, protein turnover and drug detoxification). Hepatic expression of 35 genes was altered by hypothyroidism (e.g., ADFP, ANGPTL3, GSTalpha, CAT, PPARG, HMGCL, GHR, IGF1, STAT3, THRSPalpha), whereas hyperthyroidism affected expression of another cluster of 13 genes (e.g., IGFBP1, KHK, LDHB, BAIA2L1, SULT1B, TRIAD3). Several genes were identified which have not been previously ascribed as T3 responsive (e.g., DEFB9, EPS8L2, ARHGAP1, LASS2, INHBC). Exogenous GH altered expression of 17 genes (e.g., CCAR1, CYP2C45, GYS2, ENOB, HK1, FABP1, SQLE, SOCS2, UPG2). The T3+GH treatment depleted the greatest amount of body fat, where 34 differentially expressed genes were unique to this group (e.g., C/EBP, CDC42EP1, SYDE2, PCK2, PIK4CA, TH1L, GPT2, BHMT). The marked reduction in body fat brought about by the T3+GH synergism could involve modulation of hormone signaling via altered activity of the Ras superfamily of molecular switches, which control diverse biological processes. In conclusion, this study provides the first global analysis of endocrine (T3 and GH) regulation of hepatic gene transcription in the chicken.

Functional annotation of genomic data with metabolic inference.

Metabolomics is an appealing new approach in systems biology aimed at enabling an improved understanding of the dynamic biochemical composition of living systems. Biological systems are remarkably complex. Importantly, metabolites are the end products of cellular regulatory processes, and their concentrations reflect the ultimate response of a biological system to genetic or environmental changes. In this article, we describe the components of lipid metabolomics and then use them to investigate the metabolic basis for increased abdominal adiposity in 2 strains of divergently selected chickens. Lipid metabolomics were chosen due to the availability of well-developed analytical platforms and the pervasive physiological importance of lipids in metabolism. The analysis suggests that metabolic shifts that result in increased abdominal adiposity are not universal and vary with genetic background. Metabolomics can be used to reverse engineer selection programs through superior metabolic descriptions that can then be associated with specific gene networks and transcriptional profiles.

Functional genomics in chickens: development of integrated-systems microarrays for transcriptional profiling and discovery of regulatory pathways.

The genetic networks that govern the differentiation and growth of major tissues of economic importance in the chicken are largely unknown. Under a functional genomics project, our consortium has generated 30 609 expressed sequence tags (ESTs) and developed several chicken DNA microarrays, which represent the Chicken Metabolic/Somatic (10 K) and Neuroendocrine/Reproductive (8 K) Systems (http://udgenome.ags.udel.edu/cogburn/). One of the major challenges facing functional genomics is the development of mathematical models to reconstruct functional gene networks and regulatory pathways from vast volumes of microarray data. In initial studies with liver-specific microarrays (3.1 K), we have examined gene expression profiles in liver during the peri-hatch transition and during a strong metabolic perturbation-fasting and re-feeding-in divergently selected broiler chickens (fast vs. slow-growth lines). The expression of many genes controlling metabolic pathways is dramatically altered by these perturbations. Our analysis has revealed a large number of clusters of functionally related genes (mainly metabolic enzymes and transcription factors) that control major metabolic pathways. Currently, we are conducting transcriptional profiling studies of multiple tissues during development of two sets of divergently selected broiler chickens (fast vs. slow growing and fat vs. lean lines). Transcriptional profiling across multiple tissues should permit construction of a detailed genetic blueprint that illustrates the developmental events and hierarchy of genes that govern growth and development of chickens. This review will briefly describe the recent acquisition of chicken genomic resources (ESTs and microarrays) and our consortium's efforts to help launch the new era of functional genomics in the chicken.

Systems-wide chicken DNA microarrays, gene expression profiling, and discovery of functional genes.

The goal of our current consortium project is to launch a new era--functional genomics of poultry--by providing genomic resources [expressed sequence tags (EST) and DNA microarrays] and by examining global gene expression in target tissues of chickens. DNA microarray analysis has been a fruitful strategy for the identification of functional genes in several model organisms (i.e., human, rodents, fruit fly, etc.). We have constructed and normalized five tissue-specific or multiple-tissue chicken cDNA libraries [liver, fat, breast, and leg muscle/epiphyseal growth plate, pituitary/hypothalamus/pineal, and reproductive tract (oviduct/ovary/testes)] for high-throughput DNA sequencing of EST. DNA sequence clustering was used to build contigs of overlapping sequence and to identify unique, non-redundant EST clones (unigenes), which permitted printing of systems-wide chicken DNA microarrays. One of the most promising genetic resources for gene exploration and functional gene mapping is provided by two sets of experimental lines of broiler-type chickens developed at INRA, France, by divergent selection for extremes in growth traits (fast-growing versus slow-growing; fatness versus leanness at a similar growth rate). We are using DNA microarrays for global gene expression profiling to identify candidate genes and to map growth, metabolic, and regulatory pathways that control important production traits. Candidate genes will be used for functional gene mapping and QTL analysis of F2 progeny from intercrosses made between divergent genetic lines (fat x lean lines; fast-growing x slow-growing lines). Using our first chicken liver microarray, we have already identified several interesting differentially expressed genes in commercial broilers and in divergently selected broiler lines. Many of these candidate genes are involved in the lipogenic pathway and are controlled in part by the thyrotropic axis. Thus, genome-wide transcriptional profiling is a powerful tool used to visualize the cascade of genetic circuits that govern complex biological responses. Global gene expression profiling and QTL scans should enable us to functionally map the genetic pathways that control growth, development, and metabolism of chickens. This emerging technology will have broad applications for poultry breeding programs (i.e., use of molecular markers) and for future production systems (i.e., the health and welfare of birds and the quality of poultry products).

Insulin-like growth factors and body growth in chickens divergently selected for high or low growth rate.

Insulin-like growth factors (IGFs) stimulate growth rate in a number of animal species and are likely to contribute to genetic variations of growth potential. The present study was designed to link levels of IGF-I and IGF-II mRNA and peptides with growth rate in divergently selected genotypes of chickens with high (HG) or low (LG) growth rates. Circulating IGF-I and -II and hepatic mRNA levels were measured under ad libitum feeding conditions from 1 to 12 weeks of age, and at 6 weeks of age under three different nutritional conditions (fed, fasted for 16 or 48 h, re-fed for 4 or 24 h after a 48-h fast). IGF binding proteins (IGFBPs) were also measured. Circulating IGFs increased with age and were higher in HG chickens from 1 to 6 weeks. They decreased with fasting and only IGF-II was fully restored after 24 h of re-feeding, while IGF-I remained low. A significant decrease in steady state IGF-I mRNA levels was also observed with fasting. Across the nutritional study, hepatic IGF-I mRNAs were significantly higher in HG chickens. Variations of IGF-II mRNA levels with nutritional state or genotype exhibited a similar trend. IGFBP (28, 34 and 40 kDa) levels increased with age, while only faint differences were observed between genotypes. IGFBP-28 transiently increased with fasting and was inversely related to blood glucose and insulin levels, suggesting that it is equivalent to mammalian IGFBP-1. In HG chickens, IGFBP-28 and IGFBP-34 levels decreased markedly following re-feeding. Therefore, high and low growth rates were respectively associated with high and low IGF-I and -II levels, supporting the hypothesis of a stimulatory role for both IGFs during post-hatching growth of chickens.

Characterization of unique truncated prolactin receptor transcripts, corresponding to the intracellular domain, in the testis of the sexually mature chicken.

We have examined expression of the chicken PRL receptor (cPRLR) gene in different tissues of the chicken by Northern blot analysis. Most tissues examined (ovary, testis, oviduct, kidney, and fat) possess a prominent full-length (4.6-kb) cPRLR transcript. A larger (11.7-kb) transcript is also detected in ovary, oviduct, testis, and kidney after longer exposure. A unique pattern of cPRLR expression was found in the testis of sexually mature chickens, which have an unusually high abundance of three small transcripts (1.2, 1.7, and 2 kb) in addition to the 4.6-kb transcript found in other tissues. Three domain-specific complementary DNA (cDNA) probes were constructed that correspond to the first and second ligand-binding regions in the extracellular domain and the transmembrane-intracellular domain. With these probes, Northern blot analysis of polyadenylated RNA prepared from the testes of a mature (22-week-old) chicken indicates that the highly abundant (1.2- and 1.7-kb) and less abundant (2.0-kb) cPRLR transcripts in testis hybridize only to the intracellular domain probe. Two types of truncated testis-specific cPRLR transcripts were identified using 5'-RACE (rapid amplification of cDNA ends) analysis of polyadenylated RNA from the testis of a 22-week-old chicken. The predominant truncated cDNA sequence contains the highly conserved box 1 motif [(+)box 1 cDNA] and diverges (at nucleotide 1396) from that of the cPRLR cDNA, just downstream of the transmembrane domain. The other truncated cDNA lacks the box 1 motif [(-)box 1 cDNA], which is replaced by 39 bases that could encode a hydrophobic N-terminus with a putative 5'-untranslated region of 131 bases. Young chickens predominately express the full-length cPRLR messenger RNA (4.6 kb) in the testis. At the onset of sexual maturity, there is a dramatic increase in abundance of the testis-specific (+)box 1 transcript, whereas expression of the full-length cPRLR is depressed. The presence of truncated [(+) or (-)box 1] cPRLR transcripts in the sexually mature chicken testis suggests a complex mechanism of PRL action on gonadal function.

Ontogeny of growth hormone receptor gene expression in tissue of growth-selected strains of broiler chickens.

The purpose of this study was to determine the relationship between genetic selection for growth traits and tissue expression of the chicken growth hormone receptor (cGHR) gene. Two different populations of broiler chickens were studied. One population consisted of strain (S) 80, selected for 14 generations for high 9-week body weight (BW), and its progenitor, S90 (a 1950's strain). The second population consisted of S21, selected for 10 generations for high 4-week BW and low abdominal fat, and its progenitor S20 (a 1970's strain). Tissue (liver, fat, breast and leg muscle) and blood samples were collected from six birds/strain at 2-week intervals between 1 and 11 weeks of age. An RNase protection assay was developed to measure mRNA levels of full-length cGHR (3.2 and 4.3 kb) transcripts and chicken glyceraldehyde 3-phosphate dehydrogenase (for normalization) in total RNA prepared from tissue. Analysis of the area-under-curve (AUC) was used for strain comparisons of certain developmental profiles (BW, plasma hormones and tissue cGHR mRNA). The BW AUC showed that the growth rates are different (P < 0.05) among the four strains (S21 > S20 > S80 > S90). Both slow-growing strains (S90 and S80) had a higher (P < 0.05) plasma GH AUC than the two fast-growing strains (S20 and S21). The plasma T3 AUC was highest (P < 0.05) in S90 due to maintenance of higher T3 levels after 3 weeks of age. At 11 weeks of age, hepatic and plasma GH-binding activities were positively related to growth rate (S21 > S20 > S80 > S90). However, the developmental increase in cGHR mRNA in liver and fat was similar among these different populations of growth-selected broiler chickens. Steady-state levels of cGHR mRNA increased in a developmental manner in the liver (5-fold at 9 weeks of age) and abdominal fat (4.5-fold at 11 weeks of age) of all strains. In contrast, there was no developmental increase or strain difference in cGHR mRNA levels in breast and leg muscle. There is a discrepancy between GH-binding activity in liver and plasma, which is different among strains, and steady-state levels of tissue cGHR mRNA which are similar among strains. These observations suggest that the cGHR is under translational or post-translational regulation which would determine the amount of cGHR protein available for GH binding.

Growth hormone down-regulates growth hormone receptor mRNA in chickens but developmental increases in growth hormone receptor mRNA occur independently of growth hormone action.

The purpose of this study was to determine the role of growth hormone (GH) in regulating expression of the chicken GH receptor (cGHR) gene by comparing the levels of cGHR mRNA in livers of normal chickens with that of GHR-deficient dwarf chickens. Since the sex-linked dwarf chicken lacks a functional cGHR, there are no genes activated as a result of GH action. Examination of the early developmental profile of hepatic cGHR mRNA in normal and dwarf chickens should yield information on the relative contribution of developmental and hormonal factors to the regulation of cGHR gene expression. Using a sensitive RNase protection assay, we found that the abundance of the major cGHR transcripts (4.3, 3.2 and 0.8 kb) in normal chickens increases about 2-fold between 1 and 7 weeks of age. Due to a splice site mutation in the dwarf chicken, the two larger transcripts encoding the full-length cGHR are not expressed. However, the expression of the truncated cGHR transcript (0.8 kb) in dwarf chickens increases about 5-fold between 1 and 7 weeks of age which suggests that the cGHR gene is overexpressed when not down-regulated by GH. Furthermore, a single promoter, appears to control expression of cGHR transcripts in liver since primer extension analysis revealed the same 5'-end in both full-length and 0.8 kb transcripts. These observations suggest that even though developmental increases in cGHR gene expression occur independently of GH action, GH, either directly or indirectly, down-regulates expression of the cGHR gene in normal chickens.

Molecular cloning and sequence analysis of chicken type I deiodinase cDNA: expression in normal and dwarf broiler chickens.

A cDNA encoding the chicken type I iodothyronine deiodinase (cDI-1) was isolated and sequenced from a cDNA library prepared from ConA-activated chicken splenic T-lymphocytes. The coding region of cDI-1 cDNA is composed of 738 basepairs (bp) which encodes a 246 amino acid protein. The predicted amino acid sequence of cDI-1 indicates only 60% identity to several mammalian type I deiodinases. The cDI-1 cDNA contains a codon for a highly conserved selenocysteine residue (Cys124). Northern blot analysis of total RNA prepared from different tissues of a 3-week-old broiler chicken shows a single transcript (2 kb) in liver and kidney. The abundance of hepatic cDI-1 transcripts in growth hormone receptor (GHR)-deficient dwarf chicken was similar to normal chickens despite lower levels of plasma T3 (37% lower) and elevated levels of T4 (21% higher) in dwarf chickens. This finding suggests that regulation of hepatic cDI-1 mRNA is GH-independent in the post-hatch chicken.

Cryptic peptides of prepro-TRH antagonize TRH-induced GH secretion in chickens at extrapituitary sites.

Complete processing of the TRH precursor in the rat hypothalamus generates TRH and a number of other "cryptic' peptides that flank the TRH progenitor sequences. Two of these peptides, P4 (Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr; present between amino acids 160 and 169 of rat prepro-TRH) and P5 (Phe-Ile-Asp-Pro-Gly-Leu-Gln-Arg-Ser-Trp- Glu-Glu-Lys-Glu-Gly-Glu-Gly-Val-Leu-Met-Pro-Glu; present between amino acids 178 and 199 of rat prepro-TRH), have recently been shown to modulate TRH-induced GH and thyrotrophin release from rat pituitary glands. The possibility that these peptides might modulate GH secretion in chickens was examined, since TRH is a physiological GH-releasing factor in birds. The administration of P4 and P5 (at doses of 10 and 100 micrograms/kg) consistently lowered basal plasma GH concentrations 30 and 60 min after a bolus i.v. injection. Pretreatment with P4 and P5 similarly suppressed the GH response to systemic TRH challenge. The GH-releasing activity of maximally stimulatory doses of TRH was also reduced by concomitant injections of either P4 (100 micrograms/kg) or P5 (100 micrograms/kg), which blocked the GH-releasing activity of submaximally effective doses of TRH. In marked contrast, neither P4 nor P5 significantly affected basal or TRH-induced GH release from chicken pituitary glands incubated in vitro. These results demonstrate novel actions of P4 and P5 on hypothalamic-pituitary function and, for the first time, indicate extrapituitary sites of action for these cryptic peptides in modulating anterior pituitary function.

Chronic intravenous infusion of chicken growth hormone increases body fat content of young broiler chickens.

The purpose of this study was to determine the effects of programmed intravenous infusion of chicken growth hormone (cGH) on growth and metabolism of young broiler chickens (4-7 weeks of age). Four-week-old broiler cockerels, fitted with indwelling jugular catheters, were randomly assigned to three treatment groups (6 birds/group): pulsatile infusion of buffer (phosphate buffer, pH 7.4)[PB-P] at 3 hr intervals, pulsatile infusion of cGH (15 micrograms/kg at 3 hr intervals)[GH-P], or continuous infusion of cGH (120 micrograms/kg-day)[GH-C]. Birds were bled 5 min before (0-min) and 5 min post-infusion (relative to the pulses of PB and cGH) at 5, 6, and 7 weeks of age. Pulsatile infusion of cGH increased (P < 0.05) feed consumption by 24% and reduced (P < 0.05) feed efficiency by 14% without affecting body weight (BW) gain. The relative weights (%BW) of liver, abdominal fat, and bursa of Fabricius were not affected by the pattern of cGH infusion. However, the body fat content of cGH-infused chickens was increased (P < 0.05) by 13% (GH-C) and 17% (GH-P), while body protein and water contents were slightly reduced. Body ash content was not affected by pattern of cGH infusion. When compared with the PB-P controls, the GH-P treatment depressed (P < 0.05) hepatic GH-binding activity by 52% without affecting plasma insulin-like growth factor-I (IGF-I) levels. Continuous infusion of cGH increased (P < 0.05) plasma IGF-I by 16%, thyroxine (T4) by 31%, and glucagon levels by 55%, although plasma GH levels were only 47% higher than those of the PB-P group. However, the GH-P treatment was only half as effective as the GH-C pattern in elevating plasma levels of T4 and glucagon. This study shows that programmed intravenous infusion of cGH increases deposition of body fat in young rapidly-growing broiler chickens.

Comparison of gene expression in normal and growth hormone receptor-deficient dwarf chickens reveals a novel growth hormone regulated gene.

Because of a dysfunctional growth hormone (GH) receptor there is an absence of GH-dependent gene expression in the sex-linked dwarf chicken. Therefore, a comparison of mRNAs expressed in normal and dwarf chickens should lead to the identification of mRNAs that are regulated by GH action. We have compared gene expression in normal and dwarf chickens using the mRNA differential display technique. A combination of three anchored oligo dT primers and 15 random decamers were used to detect at least 75 differentially expressed mRNAs. One of these, designated GHRG-1, hybridizes to a 0.9 kb transcript found only in liver and in normal chickens shows a pattern of developmental expression which parallels the plasma GH profile. A GHRG-1 cDNA clone was isolated that encodes a 120 amino acid peptide with no homology to any known gene. Sequence of the promoter from a genomic clone shows a region with strong similarity to the GH response element identified in the serine protease inhibitor gene, Spi 2.1. These results suggest that GHRG-1 is a novel GH regulated gene.

Dysfunctional growth hormone receptor in a strain of sex-linked dwarf chicken: evidence for a mutation in the intracellular domain.

The sex-linked dwarf (dwdw) chicken represents a valuable animal model for studying GH insensitivity and the consequence of mutations in the GH receptor (GHR) gene. We have recently reported undetectable hepatic GH-binding activity and an aberrantly sized transcript in a strain of dwdw chickens obtained from Arbor Acre Farms, Inc. (Glastonbury, CT, USA). Southern blot analysis of the chicken GHR (cGHR) gene revealed a restriction-fragment length polymorphism in HindIII and EcoRI digests of genomic DNA in this strain of dwdw chicken. In order to localize the molecular mutation, we analysed the gene structure and determined the complete sequence of the 3' untranslated region (3' UTR) of the normal cGHR. With the use of this information, we located a large deletion in the 3' end of the cGHR gene of the Connecticut (CT) strain of dwdw chicken. This deletion (1773 bp) contained 27 highly conserved amino acids of the 3' end of the coding region, the in-frame stop codon, a less frequently used poly(A) signal that is normally found 445 bp downstream of the stop codon, and a large portion of the 3' UTR. Because of this deletion, 27 novel amino acids were substituted and the open reading frame was extended for an additional 26 amino acids before reaching the transcriptional termination site. The predicted amino acid sequence of the novel carboxyl-terminus of the dwdw cGHR is largely hydrophobic with a polylysine tail, whereas the carboxyl-terminus of the wild-type (DwDw) cGHR is composed of hydrophilic amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine and metabolic responses of intact and hypophysectomized turkey poults given a daily injection of chicken growth hormone.

Female turkey poults were hypophysectomized at 4-5 weeks of age. Beginning at 6 weeks of age, 20 hypophysectomized and 20 intact birds received a daily intramuscular injection of natural chicken growth hormone (cGH, 100 micrograms/kg body weight) or vehicle for 12 days. Blood samples were taken from each bird just before injection and 4 hr post-injection at 6 and 12 days of treatment. Hypophysectomy reduced the growth rate of turkey poults to 75% of that of intact controls, significantly reduced carcass protein and ash percentages, and significantly lower plasma concentrations of GH, insulin-like growth factor-I, triiodothyronine, thyroxine, insulin, glucose, triglycerides, and non-esterified fatty acids. Hypophysectomy was without effect on liver GH receptor binding activity, but increased liver 5'-monodeiodinase activity. Daily cGH injection had no effect on the average daily gain of either hypophysectomized or intact poults when compared to vehicle-injected controls over 12 days of treatment. Daily cGH administration increased plasma insulin-like growth factor-I levels in intact and hypophysectomized turkeys, and increased plasma triiodothyronine, insulin, glucose, and triglyceride concentrations in hypophysectomized birds, but not in intact birds. Responses of young turkeys to hypophysectomy and GH replacement were consistent with the known metabolic role of GH in other species, but the influence of GH on growth appears to be of less importance in poultry than in mammals.

Pulsatile infusion of growth hormone-releasing factor depresses growth of young broiler chickens.

This study was conducted to determine the effects of programmed intravenous infusion of growth hormone-releasing factor (GRF) on the growth and metabolism of young broiler chickens (4-7 weeks of age). Twelve 4-week-old chickens, fitted with jugular catheters, were randomly assigned to three treatment groups (four birds/group): pulsed infusion of saline [SAL-P] at 3 hr intervals, pulsed infusion of GRF1-44 (5 micrograms/kg at 3 hr intervals)[GRF-P], or continuous infusion of GRF (40 micrograms/kg-day)[GRF-C]. The GRF-P treatment depressed (P < 0.05) average daily gain by 32%, average daily feed consumption by 24%, and final body weight by 17% when compared with the SAL-P group. Pulsatile infusion of GRF (GRF-P) reduced (P < 0.05) abdominal fat weight by 39% and body fat content by 28% when compared to the SAL-P group. Plasma GH levels were elevated (P < 0.05) 2.1-fold in the GRF-P group at 15 min-post-infusion, while GH levels in the GRF-C group were maintained about 70% higher than those in the SAL-P group. Plasma levels of insulin-like growth factor-I (IGF-I) were consistently lower in the GRF-P group at all ages. There were no significant differences in plasma levels of triiodothyronine (T3), thyroxine (T4), insulin, or glucose among treatment groups. This study shows that pulsatile infusion of GRF, designed to enhance plasma GH levels, does not improve growth rate, feed efficiency, or body composition of young broiler chickens.

Overexpression of a truncated growth hormone receptor in the sex-linked dwarf chicken: evidence for a splice mutation.

Sex-linked dwarfism in chickens is a form of GH resistance that resembles the Laron syndrome in humans. The dwarfism found in chickens is due to a mutant gene (dw) carried on the sex chromosome. The homozygous dwarf (dwdw) chicken is characterized by reductions in stature and plasma insulin-like growth factor-I (IGF-I) levels. Despite the absence of hepatic GH-binding activity, Southern blot analysis shows that there is no gross structural change in the gene for the GH receptor (GHR) in this strain of dwdw chicken. GH-dependent IGF-I production can be restored in cultured dwdw hepatocytes after transfection and transient expression of a chicken GHR (cGHR) cDNA, indicating that other factors that participate in GH-mediated IGF-I synthesis are intact. Northern blot analysis of liver, muscle, fat, and pituitary RNA from normal (DwDw) chickens shows a major transcript of 4.3 kilobases (kb) and three minor transcripts (0.8, 1.7, and 3.2 kb), which correspond to the cGHR. In contrast, the 0.8-kb transcript is the major cGHR transcript expressed in these tissues from dwdw chickens. Northern blot analysis with domain-specific probes shows that the 0.8-kb transcript in DwDw and dwdw liver contains only a small portion of the extracellular domain of the cGHR. A cDNA clone encoding this transcript has been isolated from a liver library prepared from a normal chicken.(ABSTRACT TRUNCATED AT 250 WORDS)