Transcriptome analysis of the hypothalamus and pituitary of turkey hens with low and high egg production.

BACKGROUND: High egg producing hens (HEPH) show increased hypothalamic and pituitary gene expression related to hypothalamo-pituitary-gonadal (HPG) axis stimulation as well as increased in vitro responsiveness to gonadotropin releasing hormone (GnRH) stimulation in the pituitary when compared to low egg producing hens (LEPH). Transcriptome analysis was performed on hypothalamus and pituitary samples from LEPH and HEPH to identify novel regulators of HPG axis function. RESULTS: In the hypothalamus and pituitary, 4644 differentially expressed genes (DEGs) were identified between LEPH and HEPH, with 2021 genes up-regulated in LEPH and 2623 genes up-regulated in HEPH. In LEPH, up-regulated genes showed enrichment of the hypothalamo-pituitary-thyroid (HPT) axis. Beta-estradiol was identified as an upstream regulator regardless of tissue. When LEPH and HEPH samples were compared, beta-estradiol was activated in HEPH in 3 of the 4 comparisons, which correlated to the number of beta-estradiol target genes up-regulated in HEPH. In in vitro pituitary cell cultures from LEPH and HEPH, thyroid hormone pretreatment negatively impacted gonadotropin subunit mRNA levels in cells from both LEPH and HEPH, with the effect being more prominent in HEPH cells. Additionally, the effect of estradiol pretreatment on gonadotropin subunit mRNA levels in HEPH cells was negative, whereas estradiol pretreatment increased gonadotropin subunit mRNA levels in LEPH cells. CONCLUSIONS: Up-regulation of the HPT axis in LEPH and upstream beta-estradiol activation in HEPH may play a role in regulating HPG axis function, and ultimately ovulation rates. Thyroid hormone and estradiol pretreatment impacted gonadotropin mRNA levels following GnRH stimulation, with the inhibitory effects of thyroid hormone more detrimental in HEPH and estradiol stimulatory effects more prominent in LEPH. Responsiveness to thyroid hormone and estradiol may be due to desensitization to thyroid hormone and estradiol in LEPH and HEPH, respectively, due to up-regulation of the HPT axis in LEPH and of the HPG axis in HEPH. Further studies will be necessary to identify possible target gene desensitization mechanisms and elicit the regulatory role of the HPT axis and beta-estradiol on ovulation rates in turkey hens.

Transcriptome analyses of liver in newly-hatched chicks during the metabolic perturbation of fasting and re-feeding reveals THRSPA as the key lipogenic transcription factor.

BACKGROUND: The fasting-refeeding perturbation has been used extensively to reveal specific genes and metabolic pathways that control energy metabolism in the chicken. Most global transcriptional scans of the fasting-refeeding response in liver have focused on juvenile chickens that were 1, 2 or 4 weeks old. The present study was aimed at the immediate post-hatch period, in which newly-hatched chicks were subjected to fasting for 4, 24 or 48 h, then refed for 4, 24 or 48 h, and compared with a fully-fed control group at each age (D1-D4). RESULTS: Visual analysis of hepatic gene expression profiles using hierarchical and K-means clustering showed two distinct patterns, genes with higher expression during fasting and depressed expression upon refeeding and those with an opposing pattern of expression, which exhibit very low expression during fasting and more abundant expression with refeeding. Differentially-expressed genes (DEGs), identified from five prominent pair-wise contrasts of fed, fasted and refed conditions, were subjected to Ingenuity Pathway Analysis. This enabled mapping of analysis-ready (AR)-DEGs to canonical and metabolic pathways controlled by distinct gene interaction networks. The largest number of hepatic DEGs was identified by two contrasts: D2FED48h/D2FAST48h (968 genes) and D2FAST48h/D3REFED24h (1198 genes). The major genes acutely depressed by fasting and elevated upon refeeding included ANGTPL, ATPCL, DIO2, FASN, ME1, SCD, PPARG, SREBP2 and THRSPA-a primary lipogenic transcription factor. In contrast, major lipolytic genes were up-regulated by fasting or down-regulated after refeeding, including ALDOB, IL-15, LDHB, LPIN2, NFE2L2, NR3C1, NR0B1, PANK1, PPARA, SERTAD2 and UPP2. CONCLUSIONS: Transcriptional profiling of liver during fasting/re-feeding of newly-hatched chicks revealed several highly-expressed upstream regulators, which enable the metabolic switch from fasted (lipolytic/gluconeogenic) to fed or refed (lipogenic/thermogenic) states. This rapid homeorhetic shift of whole-body metabolism from a catabolic-fasting state to an anabolic-fed state appears precisely orchestrated by a small number of ligand-activated transcription factors that provide either a fasting-lipolytic state (PPARA, NR3C1, NFE2L2, SERTAD2, FOX01, NR0B1, RXR) or a fully-fed and refed lipogenic/thermogenic state (THRSPA, SREBF2, PPARG, PPARD, JUN, ATF3, CTNNB1). THRSPA has emerged as the key transcriptional regulator that drives lipogenesis and thermogenesis in hatchling chicks, as shown here in fed and re-fed states.

Dynamics of transcriptome changes during subcutaneous preadipocyte differentiation in ducks.

BACKGROUND: Pekin duck is an important animal model for its ability for fat synthesis and deposition. However, transcriptional dynamic regulation of adipose differentiation driven by complex signal cascades remains largely unexplored in this model. This study aimed to explore adipogenic transcriptional dynamics before (proliferation) and after (differentiation) initial preadipocyte differentiation in ducks. RESULTS: Exogenous oleic acid alone successfully induced duck subcutaneous preadipocyte differentiation. We explored 36 mRNA-seq libraries in order to study transcriptome dynamics during proliferation and differentiation processes at 6 time points. Using robust statistical analysis, we identified 845, 652, 359, 2401 and 1933 genes differentially expressed between -48 h and 0 h, 0 h and 12 h, 12 h and 24 h, 24 h and 48 h, 48 h and 72 h, respectively (FDR < 0.05, FC > 1.5). At the proliferation stage, proliferation related pathways and basic cellular and metabolic processes were inhibited, while regulatory factors that initiate differentiation enter the ready-to-activate state, which provides a precondition for initiating adipose differentiation. According to weighted gene co-expression network analysis, pathways positively related to adipogenic differentiation are significantly activated at the differentiation stage, while WNT, FOXO and other pathways that inhibit preadipocyte differentiation are negatively regulated. Moreover, we identified and classified more than 100 transcription factors that showed significant changes during differentiation, and found novel transcription factors that were not reported to be related to preadipoctye differentiation. Finally, we manually assembled a proposed regulation network model of subcutaneous preadipocyte differentiation base on the expression data, and suggested that E2F1 may serve as an important link between the processes of duck subcutaneous preadipocyte proliferation and differentiation. CONCLUSIONS: For the first time we comprehensively analyzed the transcriptome dynamics of duck subcutaneous preadipocyte proliferation and differentiation. The current study provides a solid basis for understanding the synthesis and deposition of subcutaneous fat in ducks. Furthermore, the information generated will allow future investigations of specific genes involved in particular stages of duck adipogenesis.

Transcriptional profiling and pathway analysis reveal differences in pituitary gland function, morphology, and vascularization in chickens genetically selected for high or low body weight.

BACKGROUND: Though intensive genetic selection has led to extraordinary advances in growth rate and feed efficiency in production of meat-type chickens, endocrine processes controlling these traits are still poorly understood. The anterior pituitary gland is a central component of the neuroendocrine system and plays a key role in regulating important physiological processes that directly impact broiler production efficiency, though how differences in pituitary gland function contribute to various growth and body composition phenotypes is not fully understood. RESULTS: Global anterior pituitary gene expression was evaluated on post-hatch weeks 1, 3, 5, and 7 in male broiler chickens selected for high (HG) or low (LG) growth. Differentially expressed genes (DEGs) were analyzed with gene ontology categorization, self-organizing maps, gene interaction network determination, and upstream regulator identification to uncover novel pituitary genes and pathways contributing to differences in growth and body composition. A total of 263 genes were differentially expressed between HG and LG anterior pituitary glands (P ≤ 0.05 for genetic line-by-age interaction or main effect of line; ≥1.6-fold difference between lines), including genes encoding four anterior pituitary hormones. Genes involved in signal transduction, transcriptional regulation, and vesicle-mediated transport were differentially expressed and are predicted to influence expression and secretion of pituitary hormones. DEGs involved in immune regulation provide evidence that inflammation and response to cellular stressors may compromise pituitary function in LG birds, affecting their ability to adequately produce pituitary hormones. Many DEGs were also predicted to function in processes that regulate organ morphology and angiogenesis, suggesting pituitary gland structure differs between the divergently selected lines. CONCLUSIONS: The large number of DEGs within the anterior pituitary gland of birds selected for high or low body weight highlights the importance of this gland in regulating economically important traits such as growth and body composition in broiler chickens. Intracellular signaling, transcriptional regulation, and membrane trafficking are important cellular processes contributing to proper hormone production and secretion. The data also suggest that pituitary function is intimately tied to structure, and organization of the gland could influence hypothalamic and systemic metabolic inputs and delivery of hormones regulating growth and metabolism into peripheral circulation.

The effects of replacing eggs with chicks on mesotocin, dopamine, and prolactin in the native Thai hen.

The mesotocinergic (MTergic) and dopaminergic (DAergic) systems have been documented to play pivotal roles in maternal behaviors in native Thai chickens. In native Thai chickens, plasma prolactin (PRL) concentrations are associated with maternal behaviors, which are also controlled by the DAergic system. However, the role of MT in conjunction with the roles of DA and PRL on the neuroendocrine regulation of the transition from incubating to rearing behavior has never been studied. Therefore, the aim of this study was to investigate the association of MT, DA, and PRL during the transition from incubating to rearing behavior in native Thai hens. Using an immunohistochemistry technique, the numbers of MT-immunoreactive (-ir) and tyrosine hydroxylase-ir (TH-ir, a DA marker) neurons were compared between incubating hens (INC; n = 6) and hens for which the incubated eggs were replaced with 3 newly hatched chicks for 3 days after 6, 10, and 14 days of incubation (REC; n = 6). Plasma PRL concentrations were determined by enzyme-linked immunosorbent assay. The results revealed that the numbers of MT-ir neurons within the nucleus supraopticus, pars ventralis (SOv), nucleus preopticus medialis (POM), and nucleus paraventricularis magnocellularis (PVN) increased in the REC hens when compared with those of the INC hens at 3 different time points (at days 9, 13, and 17). On the other hand, the number of TH-ir neurons in the nucleus intramedialis (nI) decreased in the REC13 and REC17 hens when compared with those of the INC hens. However, the number of TH-ir neurons in the nucleus mamillaris lateralis (ML) only decreased in the REC13 hens when compared with the INC13 hens. The decrease in the numbers of TH-ir neurons within the nI and ML is associated with the decrease in the levels of plasma PRL. This study suggests that the presence of either eggs or chicks is the key factor regulating the MTergic system within the SOv, POM, and PVN and the DAergic system within the nI and ML during the transition from incubating to rearing behavior in native Thai chickens. The results further indicate that these two systems play pivotal roles in the transition from incubating to rearing behavior in this equatorial species.

Differential expression of hepatic genes with embryonic exposure to an environmentally relevant PCB mixture in Japanese quail (Coturnix japonica).

The upper Hudson River was contaminated with polychlorinated biphenyls (PCB) Aroclor mixtures from the 1940s until the late 1970s. Several well-established biomarkers, such as induction of hepatic cytochrome P450 monooxygenases, were used to measure exposure to PCBs and similar contaminants in birds. In the present study, Japanese quail eggs were injected with a PCB mixture based upon a congener profile found in spotted sandpiper eggs at the upper Hudson River and subsequently, RNA was extracted from hatchling liver tissue for hybridization to a customized chicken cDNA microarray. Nominal concentrations of the mixture used for microarray hybridization were 0, 6, 12, or 49 µg/g egg. Hepatic gene expression profiles were analyzed using cluster and pathway analyses. Results showed potentially useful biomarkers of both exposure and effect attributed to PCB mixture. Biorag and Ingenuity Pathway Analysis® analyses revealed differentially expressed genes including those involved in glycolysis, xenobiotic metabolism, replication, protein degradation, and tumor regulation. These genes included cytochrome P450 1A5 (CYP1A5), cytochrome b5 (CYB5), NADH-cytochrome b5 reductase, glutathione S-transferase (GSTA), fructose bisphosphate aldolase (ALDOB), glycogen phosphorylase, carbonic anhydrase, and DNA topoisomerase II. CYP1A5, CYB5, GSTA, and ALDOB were chosen for quantitative real-time polymerase chain reaction confirmation, as these genes exhibited a clear dose response on the array. Data demonstrated that an initial transcriptional profile associated with an environmentally relevant PCB mixture in Japanese quail occurred.

Identification of microRNAs controlling hepatic mRNA levels for metabolic genes during the metabolic transition from embryonic to posthatch development in the chicken.

BACKGROUND: The transition from embryonic to posthatch development in the chicken represents a massive metabolic switch from primarily lipolytic to primarily lipogenic metabolism. This metabolic switch is essential for the chick to successfully transition from the metabolism of stored egg yolk to the utilization of carbohydrate-based feed. However, regulation of this metabolic switch is not well understood. We hypothesized that microRNAs (miRNAs) play an important role in the metabolic switch that is essential to efficient growth of chickens. We used high-throughput RNA sequencing to characterize expression profiles of mRNA and miRNA in liver during late embryonic and early posthatch development of the chicken. This extensive data set was used to define the contributions of microRNAs to the metabolic switch during development that is critical to growth and nutrient utilization in chickens. RESULTS: We found that expression of over 800 mRNAs and 30 miRNAs was altered in the embryonic liver between embryonic day 18 and posthatch day 3, and many of these differentially expressed mRNAs and miRNAs are associated with metabolic processes. We confirmed the regulation of some of these mRNAs by miRNAs expressed in a reciprocal pattern using luciferase reporter assays. Finally, through the use of yeast one-hybrid screens, we identified several proteins that likely regulate expression of one of these important miRNAs. CONCLUSIONS: Integration of the upstream regulatory mechanisms governing miRNA expression along with monitoring the downstream effects of this expression will ultimately allow for the construction of complete miRNA regulatory networks associated with the hepatic metabolic switch in chickens. Our findings support a key role for miRNAs in controlling the metabolic switch that occurs between embryonic and posthatch development in the chicken.

Progestin priming before gonadotrophin stimulation and AI improves embryo development and normalises luteal function in the cat.

Exogenous gonadotrophins administered before AI can adversely alter endocrine dynamics and inhibit embryo development in felids. In the present study, we tested the hypothesis that priming the domestic cat ovary with progestin mitigates the negative influence of gonadotrophin therapy by normalising early embryogenesis and luteal function. Queens were given either: (1) progestin pretreatment plus chorionic gonadotrophins (n=8; primed); or (2) gonadotrophins only (n=8; unprimed). Ovulatory response was assessed laparoscopically, and cats with fresh corpora lutea (CL) were inseminated in utero. Ovariohysterectomy was performed 3 days later to recover intra-oviductal embryos for in vitro culture; one ovary was prepared for histology, and CL from the remaining ovary were excised and assessed for progesterone content and targeted gene expression. Of the six primed and seven unprimed queens inseminated, embryo(s) were recovered from five individuals per group. Embryos from progestin-primed donors more closely simulated normal stage in vivo development (P<0.05). No 2- or 4-cell embryos from either group developed beyond 16-cells in vitro; however, 50% of unprimed and 66.7% of primed (P>0.05) 5-16-cell embryos progressed to morulae or blastocysts by Day 4 of culture. Although histological characteristics were unaffected by progestin priming (P>0.05), luteal progesterone was unusually high (P<0.05) in unprimed compared with primed cats (72.4±5.8 vs. 52.2±5.5 ng mg(-1), respectively). Two genes associated with progesterone biosynthesis (luteinising hormone receptor and 3ß-hydroxysteroid dehydrogenase) were upregulated in unprimed versus primed individuals (P=0.05 and P<0.05, respectively), indicating potential mechanistic pathways for the protective influence of pre-emptive progestin treatment. Building on earlier findings that progestin priming prevents spontaneous ovulation, increases ovarian sensitivity to gonadotrophins and ensures a normative endocrine environment, the present study demonstrates that pretreatment with this steroid also benefits embryo development and normalisation of early luteal function.

Glucocorticoid-induced changes in gene expression in embryonic anterior pituitary cells.

Within the anterior pituitary gland, glucocorticoids such as corticosterone (CORT) provide negative feedback to inhibit adrenocorticotropic hormone secretion and act to regulate production of other hormones including growth hormone (GH). The ontogeny of GH production during chicken embryonic and rat fetal development is controlled by glucocorticoids. The present study was conducted to characterize effects of glucocorticoids on gene expression within embryonic pituitary cells and to identify genes that are rapidly and directly regulated by glucocorticoids. Chicken embryonic pituitary cells were cultured with CORT for 1.5, 3, 6, 12, and 24 h in the absence and presence of cycloheximide (CHX) to inhibit protein synthesis. RNA was analyzed with custom microarrays containing 14,053 chicken cDNAs, and results for selected genes were confirmed by quantitative reverse transcription real-time PCR (qRT-PCR). Levels of GH mRNA were maximally induced by 6 h of CORT treatment, and this response was blocked by CHX. Expression of 396 genes was affected by CORT, and of these, mRNA levels for 46 genes were induced or repressed within 6 h. Pathway analysis of genes regulated by CORT in the absence of CHX revealed networks of genes associated with endocrine system development and cellular development. Eleven genes that were induced within 6 h in the absence and presence of CHX were identified, and eight were confirmed by qRT-PCR. The expression profiles and canonical pathways defined in this study will be useful for future analyses of glucocorticoid action and regulation of pituitary function.

Transcriptional analysis of abdominal fat in genetically fat and lean chickens reveals adipokines, lipogenic genes and a link between hemostasis and leanness.

BACKGROUND: This descriptive study of the abdominal fat transcriptome takes advantage of two experimental lines of meat-type chickens (Gallus domesticus), which were selected over seven generations for a large difference in abdominal (visceral) fatness. At the age of selection (9 wk), the fat line (FL) and lean line (LL) chickens exhibit a 2.5-fold difference in abdominal fat weight, while their feed intake and body weight are similar. These unique avian models were originally created to unravel genetic and endocrine regulation of adiposity and lipogenesis in meat-type chickens. The Del-Mar 14K Chicken Integrated Systems microarray was used for a time-course analysis of gene expression in abdominal fat of FL and LL chickens during juvenile development (1-11 weeks of age). RESULTS: Microarray analysis of abdominal fat in FL and LL chickens revealed 131 differentially expressed (DE) genes (FDR≤0.05) as the main effect of genotype, 254 DE genes as an interaction of age and genotype and 3,195 DE genes (FDR≤0.01) as the main effect of age. The most notable discoveries in the abdominal fat transcriptome were higher expression of many genes involved in blood coagulation in the LL and up-regulation of numerous adipogenic and lipogenic genes in FL chickens. Many of these DE genes belong to pathways controlling the synthesis, metabolism and transport of lipids or endocrine signaling pathways activated by adipokines, retinoid and thyroid hormones. CONCLUSIONS: The present study provides a dynamic view of differential gene transcription in abdominal fat of chickens genetically selected for fatness (FL) or leanness (LL). Remarkably, the LL chickens over-express a large number of hemostatic genes that could be involved in proteolytic processing of adipokines and endocrine factors, which contribute to their higher lipolysis and export of stored lipids. Some of these changes are already present at 1 week of age before the divergence in fatness. In contrast, the FL chickens have enhanced expression of numerous lipogenic genes mainly after onset of divergence, presumably directed by multiple transcription factors. This transcriptional analysis shows that abdominal fat of the chicken serves a dual function as both an endocrine organ and an active metabolic tissue, which could play a more significant role in lipogenesis than previously thought.

Global gene expression analysis of the turkey hen hypothalamo-pituitary-gonadal axis during the preovulatory hormonal surge.

The preovulatory hormonal surge (PS) consists of elevated circulating luteinizing hormone (LH) and progesterone levels and serves as the primary trigger for ovarian follicle ovulation. Increased LH and progesterone, produced by the pituitary and the granulosa layer of the largest ovarian follicle (F1), respectively, result from hypothalamic stimulation and steroid hormone feedback on the hypothalamo-pituitary-gonadal (HPG) axis. The hypothalamus, pituitary, F1 granulosa, and granulosa layer of the fifth largest follicle (F5) were isolated from converter turkey hens outside and during the PS and subjected to RNA sequencing (n = 6 per tissue). Differentially expressed genes were subjected to functional annotation using DAVID and IPA. A total of 12, 250, 1235, and 1938 DEGs were identified in the hypothalamus, pituitary, F1 granulosa, and F5 granulosa respectively (q<0.05, |fold change|>1.5, FPKM>1). Gene Ontology (GO) analysis revealed key roles for metabolic processes, steroid hormone feedback, and hypoxia induced gene expression changes. Upstream analysis identified a total of 4, 42, 126, and 393 potential regulators of downstream gene expression in the hypothalamus, pituitary, F1G, and F5G respectively, with a total of 63 potential regulators exhibiting differential expression between samples collected outside and during the PS (|z-score|>2). The results from this study serve to increase the current knowledge base surrounding the regulation of the PS in turkey hens. Through GO analysis, downstream processes and functions associated with the PS were linked to identified DEGs, and through upstream analysis, potential regulators of DEGs were identified for further analysis. Linking upstream regulators to the downstream PS and ovulation events could allow for genetic selection or manipulation of ovulation frequencies in turkey hens.

Insulin immuno-neutralization in fed chickens: effects on liver and muscle transcriptome.

Chickens mimic an insulin-resistance state by exhibiting several peculiarities with regard to plasma glucose level and its control by insulin. To gain insight into the role of insulin in the control of chicken transcriptome, liver and leg muscle transcriptomes were compared in fed controls and "diabetic" chickens, at 5 h after insulin immuno-neutralization, using 20.7K-chicken oligo-microarrays. At a level of false discovery rate <0.01, 1,573 and 1,225 signals were significantly modified by insulin privation in liver and muscle, respectively. Microarray data agreed reasonably well with qRT-PCR and some protein level measurements. Differentially expressed mRNAs with human ID were classified using Biorag analysis and Ingenuity Pathway Analysis. Multiple metabolic pathways, structural proteins, transporters and proteins of intracellular trafficking, major signaling pathways, and elements of the transcriptional control machinery were largely represented in both tissues. At least 42 mRNAs have already been associated with diabetes, insulin resistance, obesity, energy expenditure, or identified as sensors of metabolism in mice or humans. The contribution of the pathways presently identified to chicken physiology (particularly those not yet related to insulin) needs to be evaluated in future studies. Other challenges include the characterization of "unknown" mRNAs and the identification of the steps or networks, which disturbed tissue transcriptome so extensively, quickly after the turning off of the insulin signal. In conclusion, pleiotropic effects of insulin in chickens are further evidenced; major pathways controlled by insulin in mammals have been conserved despite the presence of unique features of insulin signaling in chicken muscle.

Identification of cis elements necessary for glucocorticoid induction of growth hormone gene expression in chicken embryonic pituitary cells.

Glucocorticoid (GC) treatment of rat or chicken embryonic pituitary (CEP) cells induces premature production of growth hormone (GH). GC induction of the GH gene requires ongoing protein synthesis, and the GH genes lack a canonical GC response element (GRE). To characterize cis-acting elements and identify trans-acting proteins involved in this process, we characterized the regulation of a luciferase reporter containing a fragment of the chicken GH gene (-1727/+48) in embryonic day 11 CEP cells. Corticosterone (Cort) increased luciferase activity and mRNA expression, and mRNA induction was blocked by protein synthesis inhibition. Through deletion analysis, we identified a GC-responsive region (GCRR) at -1045 to -954. The GCRR includes an ETS-1 binding site and a degenerate GRE (dGRE) half site. Nuclear proteins, including ETS-1, bound to a GCRR probe in electrophoretic mobility shift assays, and Cort regulated protein binding. Using chromatin immunoprecipitation, we found that ETS-1 and GC receptor (GR) were associated with the GCRR in CEP cells, and Cort increased GR recruitment to the GCRR. Mutation of the ETS-1 site or dGRE site in the -1045/+48 GH reporter abolished Cort responsiveness. We conclude that GC regulation of the GH gene during development requires cis-acting elements in the GCRR and involves ETS-1 and GR binding to these elements. Similar ETS-1 elements/dGREs are located in the 5'-flanking regions of GH genes in mammals, including rodents and humans. This is the first study to demonstrate involvement of ETS-1 in GC regulation of the GH gene during embryonic development in any species, enhancing our understanding of GH regulation in vertebrates.

The Effect of Commercial Genetic Selection on Somatotropic Gene Expression in Broilers: A Potential Role for Insulin-Like Growth Factor Binding Proteins in Regulating Broiler Growth and Body Composition.

The somatotropic axis influences growth and metabolism, and many of its effects are a result of insulin-like growth factor (IGF) signaling modulated by IGF-binding proteins (IGFBPs). Modern commercial meat-type (broiler) chickens exhibit rapid and efficient growth and muscle accretion resulting from decades of commercial genetic selection, and it is not known how alterations in the IGF system has contributed to these improvements. To determine the effect of commercial genetic selection on somatotropic axis activity, two experiments were conducted comparing legacy Athens Canadian Random Bred and modern Ross 308 male broiler lines, one between embryonic days 10 and 18 and the second between post-hatch days 10 and 40. Gene expression was evaluated in liver and breast muscle (pectoralis major) and circulating hormone concentrations were measured post-hatch. During embryogenesis, no differences in IGF expression were found that corresponded with difference in body weight between the lines beginning on embryonic day 14. While hepatic IGF expression and circulating IGF did not differ between the lines post-hatch, expression of both IGF1 and IGF2 mRNA was greater in breast muscle of modern broilers. Differential expression of select IGFBPs suggests their action is dependent on developmental stage and site of production. Hepatic IGFBP1 appears to promote embryonic growth but inhibit post-hatch growth at select ages. Results suggest that local IGFBP4 may prevent breast muscle growth during embryogenesis but promote it after hatch. Post-hatch, IGFBP2 produced in liver appears to inhibit body growth, but IGFBP2 produced locally in breast muscle facilitates development of this tissue. The opposite appears true for IGFBP3, which seems to promote overall body growth when produced in liver and restrict breast muscle growth when produced locally. Results presented here suggest that paracrine IGF signaling in breast muscle may contribute to overall growth and muscle accretion in chickens, and that this activity is regulated in developmentally distinct and tissue-specific contexts through combinatorial action of IGFBPs.

Remodeling of Hepatocyte Mitochondrial Metabolism and De Novo Lipogenesis During the Embryonic-to-Neonatal Transition in Chickens.

Embryonic-to-neonatal development in chicken is characterized by high rates of lipid oxidation in the late-term embryonic liver and high rates of de novo lipogenesis in the neonatal liver. This rapid remodeling of hepatic mitochondrial and cytoplasmic networks occurs without symptoms of hepatocellular stress. Our objective was to characterize the metabolic phenotype of the embryonic and neonatal liver and explore whether these metabolic signatures are preserved in primary cultured hepatocytes. Plasma and liver metabolites were profiled using mass spectrometry based metabolomics on embryonic day 18 (ed18) and neonatal day 3 (nd3). Hepatocytes from ed18 and nd3 were isolated and cultured, and treated with insulin, glucagon, growth hormone and corticosterone to define hormonal responsiveness and determine their impacts on mitochondrial metabolism and lipogenesis. Metabolic profiling illustrated the clear transition from the embryonic liver relying on lipid oxidation to the neonatal liver upregulating de novo lipogenesis. This metabolic phenotype was conserved in the isolated hepatocytes from the embryos and the neonates. Cultured hepatocytes from the neonatal liver also maintained a robust response to insulin and glucagon, as evidenced by their contradictory effects on lipid oxidation and lipogenesis. In summary, primary hepatocytes from the embryonic and neonatal chicken could be a valuable tool to investigate mechanisms regulating hepatic mitochondrial metabolism and de novo lipogenesis.

Ontogenic characterization of gene expression in the developing neuroendocrine system of the chick.

The neuroendocrine system consists of five major hypothalamic-pituitary hormone axes that regulate several important metabolic processes, and it develops in all vertebrates during embryogenesis. In order to define initiation and establishment of these five axes, mRNA expression profiles of hypothalamic releasing and release-inhibiting factors, their pituitary receptors, and pituitary hormones were characterized during the second half of embryogenesis and first week post-hatch in the chick. Axis initiation was defined as the age when pituitary hormone mRNA levels began to increase substantially, and establishment was defined as the age when mRNA for all components had reached maximum expression levels. The adrenocorticotropic axis appears established by e12, as there were no major increases in gene expression after that age. Hypothalamic thyrotropin-releasing hormone and pituitary thyroid-stimulating hormone ß-subunit increased between e10 and e18, indicating establishment of the thyrotropic axis during this period. Pituitary growth hormone substantially increased on e16, and hypothalamic growth hormone-releasing hormone did not increase until e20, indicating that somatotropic axis activity is established late in embryonic development. Lactotropic axis initiation is evident just prior to hatch, as pituitary prolactin and vasoactive intestinal peptide receptor 1 did not increase until e18 and e20, respectively. Hypothalamic gonadotropin-releasing hormone 1 increased after hatch, and pituitary luteinizing hormone ß-subunit expression remained low until d3, indicating the gonadotropic axis is not fully functional until after hatching. This study is the first to characterize major hypothalamic and pituitary components of all five neuroendocrine axes simultaneously and considerably increases our understanding of neuroendocrine system establishment during development.

Changes in vasoactive intestinal peptide and tyrosine hydroxylase immunoreactivity in the brain of nest-deprived native Thai hen.

Hyperprolactinemia is associated with incubation behavior and ovarian regression in birds. To investigate the association of prolactin (PRL), vasoactive intestinal peptide (VIP), and dopamine (DA) with the neuroendocrine regulation of incubation behavior, changes in the number of visible VIP-immunoreactive (VIP-ir) neurons in the nucleus inferioris hypothalami (IH) and nucleus infundibuli hypothalami (IN) and tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the nucleus intramedialis (nI) and nucleus mamillaris lateralis (ML) of incubating native Thai hens were compared with those of nest-deprived hens. TH was used as a marker for dopaminergic (DAergic) neurons. Blood samples were collected to determine PRL levels. The localization and the number of visible VIP-ir and TH-ir neurons were determined by immunohistochemistry. Disruption of incubation behavior was accompanied by a precipitous decline in plasma PRL levels. The number of visible VIP-ir neurons in the IH-IN and TH-ir neurons in the nI and ML were high during incubation and decreased when hens were deprived of their nests. This study indicated an association between VIP neurons in the IH-IN and DA neurons in the nI and ML with the degree of hyperprolactinemia, suggesting that the expression of incubation behavior in birds might be, in part, regulated by the DAergic input from the nI and ML to VIP neurons in the IH-IN and subsequent PRL release.

Characterization of hypothalamo-pituitary-thyroid axis gene expression in the hypothalamus, pituitary gland, and ovarian follicles of turkey hens during the preovulatory surge and in hens with low and high egg production.

Dysregulation of the preovulatory surge (PS) leads to lowered egg production. The hypothalamo-pituitary-thyroid (HPT) axis has been shown to influence plasma progesterone levels and follicle ovulation. The presence of thyroid hormone receptors (THR) in the reproductive axis suggests possible effects of thyroid hormone. To further understand the potential role of thyroid hormone on the PS, HPT axis plasma hormone concentrations and gene expression were characterized surrounding the PS in average egg producing hens (AEPH), low egg producing hens (LEPH), and high egg producing hens (HEPH) (n = 3 hens/group). Data were analyzed using the mixed models procedure of SAS, with significance indicated at P < 0.05. Average egg producing hens and HEPH displayed lower levels of triiodothyronine (T3) and higher levels of thyroxine (T4) inside of the PS, whereas LEPH showed inverse T3 and T4 levels relative to the PS. Expression of mRNA for hypothalamic thyrotropin-releasing hormone (TRH), pituitary thyrotropin (TSHB), and the main thyroid hormone metabolism enzyme (DIO2) were downregulated during the PS in AEPH and HEPH. Low egg producing hens displayed higher expression of mRNA for hypothalamic TRH as well as pituitary TSHB and DIO2 compared with HEPH. Average egg producing hens expression of THR mRNAs was upregulated during the PS in the hypothalamus but downregulated in the pituitary. High egg producing hens showed decreased expression of THR mRNAs in both the hypothalamus and pituitary when compared with LEPH. In ovarian follicles, THR mRNAs were more prevalent in the thecal layer of the follicle wall compared with the granulosa layer, and expression tended to decrease with follicle maturity. Minimal differences in follicular THR expression were seen between LEPH and HEPH, indicating that THR expression is unlikely to be responsible for steroid hormone production differences occurring between LEPH and HEPH. Generally, downregulation of the HPT axis was seen during the PS in AEPH and HEPH, whereas upregulation of the HPT axis was seen in LEPH. Further studies will be required to clarify the role of the HPT axis in the regulation of ovulation and egg production rates in turkey hens.

Transcriptome Analysis During Follicle Development in Turkey Hens With Low and High Egg Production.

Low and high egg producing hens exhibit gene expression differences related to ovarian steroidogenesis. High egg producing hens display increased expression of genes involved in progesterone and estradiol production, in the granulosa layer of the largest follicle (F1G) and small white follicles (SWF), respectively, whereas low egg producing hens display increased expression of genes related to progesterone and androgen production in the granulosa (F5G) and theca interna layer (F5I) of the fifth largest follicle, respectively. Transcriptome analysis was performed on F1G, F5G, F5I, and SWF samples from low and high egg producing hens to identify novel regulators of ovarian steroidogenesis. In total, 12,221 differentially expressed genes (DEGs) were identified between low and high egg producing hens across the four cell types examined. Pathway analysis implied differential regulation of the hypothalamo-pituitary-thyroid (HPT) axis, particularly thyroid hormone transporters and thyroid hormone receptors, and of estradiol signaling in low and high egg producing hens. The HPT axis showed up-regulation in high egg producing hens in less mature follicles but up-regulation in low egg producing hens in more mature follicles. Estradiol signaling exclusively exhibited up-regulation in high egg producing hens. Treatment of SWF cells from low and high egg producing hens with thyroid hormone in vitro decreased estradiol production in cells from high egg producing hens to the levels seen in cells from low egg producing hens, whereas thyroid hormone treatment did not impact estradiol production in cells from low egg producing hens. Transcriptome analysis of the major cell types involved in steroidogenesis inferred the involvement of the HPT axis and estradiol signaling in the regulation of differential steroid hormone production seen among hens with different egg production levels.

Transcriptional profiling of hypothalamus during development of adiposity in genetically selected fat and lean chickens.

The hypothalamus integrates peripheral signals to regulate food intake, energy metabolism, and ultimately growth rate and body composition in vertebrates. Deviations in hypothalamic regulatory controls can lead to accumulation of excess body fat. Many regulatory genes involved in this process remain unidentified, and comparative studies may be helpful to unravel evolutionarily conserved mechanisms controlling body weight and food intake. In the present study, divergently selected fat (FL) and lean (LL) lines of chickens were used to characterize differences in hypothalamic gene expression in these unique genetic lines that develop differences in adiposity without differences in food intake or body weight. Hypothalamic transcriptional profiles were defined with cDNA microarrays before and during divergence of adiposity between the two lines. Six differentially expressed genes identified in chickens are related to genes associated with control of body fat in transgenic or knockout mice, supporting the importance of these genes across species. We identified differences in expression of nine genes involved in glucose metabolism, suggesting that alterations in hypothalamic glycolysis might contribute to differences in levels of body fat between genotypes. Expression of the sweet taste receptor (TAS1R1), which in mammals is involved in glucose sensing and energy uptake, was also higher in FL chickens, suggesting that early differences in glucose sensing might alter the set point for subsequent body composition. Differences in expression of genes associated with tumor necrosis factor (TNF) signaling were also noted. In summary, we identified alterations in transcriptional and metabolic processes within the hypothalamus that could contribute to excessive accumulation of body fat in FL chickens in the absence of differences in food intake, thereby contributing to the genetic basis for obesity in this avian model.