Circadian regulation of calcium and phosphorus homeostasis during the oviposition cycle in laying hens.

Maintenance of calcium and phosphorus homeostasis in laying hens is crucial for preservation of skeletal integrity and eggshell quality, though physiological regulation of these systems is incompletely defined. To investigate changes in mineral and vitamin D3 homeostasis during the 24-h egg formation cycle, 32-wk-old commercial laying hens were sampled at 1, 3, 4, 6, 7, 8, 12, 15, 18, 21, 23, and 24 h post-oviposition (HPOP; n ≥ 4). Ovum location and egg calcification stage were recorded, and blood chemistry, plasma vitamin D3 metabolites, circulating parathyroid hormone (PTH), and expression of genes mediating uptake and utilization of calcium and phosphorus were evaluated. Elevated levels of renal 25-hydroxylase from 12 to 23 HPOP suggest this tissue might play a role in vitamin D3 25-hydroxylation during eggshell calcification. In shell gland, retinoid-x-receptor gamma upregulation between 6 and 8 HPOP followed by subsequently increased vitamin D receptor indicate that vitamin D3 signaling is important for eggshell calcification. Increased expression of PTH, calcitonin, and fibroblast growth factor 23 (FGF23) receptors in the shell gland between 18 and 24 HPOP suggest elevated sensitivity to these hormones toward the end of eggshell calcification. Shell gland sodium-calcium exchanger 1 was upregulated between 4 and 7 HPOP and plasma membrane calcium ATPase 1 increased throughout eggshell calcification, suggesting the primary calcium transporter may differ according to eggshell calcification stage. Expression in shell gland further indicated that bicarbonate synthesis precedes transport, where genes peaked at 6 to 7 and 12 to 18 HPOP, respectively. Inorganic phosphorus transporter 1 (PiT-1) expression peaked in kidney between 12 and 15 HPOP, likely to excrete excess circulating phosphorus, and in shell gland between 18 and 21 HPOP. Upregulation of FGF23 receptors and PiT-1 during late eggshell calcification suggest shell gland phosphorus uptake is important at this time. Together, these findings identified potentially novel hormonal pathways involved in calcium and phosphorus homeostasis along with associated circadian patterns in gene expression that can be used to devise strategies aimed at improving eggshell and skeletal strength in laying hens.

Physiological regulation of calcium and phosphorus utilization in laying hens.

Commercial laying hens can produce one egg approximately every 24 h. During this process, regulatory systems that control vitamin D3 metabolism, calcium and phosphorus homeostasis, and intestinal uptake of these minerals work in concert to deliver components required for eggshell calcification and bone mineralization. Commercial production cycles have been extended in recent years to last through 100 weeks of age, and older hens often exhibit an increased prevalence of skeletal fractures and poor eggshell quality. Issues such as these arise, in part, through imbalances that occur in calcium and phosphorus utilization as hens age. As a result, an in-depth understanding of the mechanisms that drive calcium and phosphorus uptake and utilization is required to develop solutions to these welfare and economic challenges. This paper reviews factors that influence calcium and phosphorus homeostasis in laying hens, including eggshell formation and development and roles of cortical and medullary bone. Metabolism and actions of vitamin D3 and physiological regulation of calcium and phosphorus homeostasis in key tissues are also discussed. Areas that require further research in avian species, such as the role of fibroblast growth factor 23 in these processes and the metabolism and action of bioactive vitamin D3, are highlighted and the importance of using emerging technologies and establishing in vitro systems to perform functional and mechanistic studies is emphasized.

Delayed access to feed early post-hatch affects the development and maturation of gastrointestinal tract microbiota in broiler chickens.

BACKGROUND: The first two weeks of post-hatch (PH) growth in broilers (meat-type birds) are critical for gut development and microbiota colonization. In the current broiler production system, chicks may not receive feed and water for 24 to 72 h due to variations in hatching time and hatchery management. Post-hatch feed delay affects body weight, feed efficiency, mortality, and gut development. The goal of this study was to investigate changes in the microbiome in broiler chickens early PH and the effect of delayed access to feed on the microbiota. RESULTS: Chicks either received feed and water immediately after hatch or access to feed was delayed for 48 h to mimic commercial hatchery settings (treatment, TRT). Both groups were sampled (n = 6) at -48, 0, 4 h, and 1 (24 h), 2 (48 h), 3 (72 h), 4 (96 h), 6 (144 h), 8 (192 h), 10 (240 h), 12 (288 h) and 14 (336 h) days PH. Ileal (IL) and cecal (CE) epithelial scrapings (mucosal bacteria, M) and digesta (luminal bacteria, L) were collected for microbiota analysis. Microbiota was determined by sequencing the V3-V4 region of bacterial 16S rRNA and analyzed using QIIME2. The microbiota of early ileal and cecal samples were characterized by high abundance of unclassified bacteria. Among four bacterial populations (IL-L, IL-M, CE-L, CE-M), IL-M was the least affected by delayed access to feed early PH. Both alpha and beta diversities were affected by delayed access to feed PH in IL-L, CE-M and CE-L. However, the development effect was more pronounced. In all four bacterial populations, significant changes due to developmental effect (time relative to hatch) was observed in taxonomic composition, with transient changes of bacterial taxa during the first two weeks PH. Delayed access to feed has limited influence on bacterial composition with only a few genera and species affected in all four bacterial populations. Predicted function based on 16S rRNA was also affected by delayed access to feed PH with most changes in metabolic pathway richness observed in IL-L, CE-L and CE-M. CONCLUSIONS: These results show transient changes in chicken microbiota biodiversity during the first two weeks PH and indicate that delayed access to feed affects microbiota development. Proper microbiota development could be an important factor in disease prevention and antibiotic use in broiler chickens. Moreover, significant differences in response to delayed access to feed PH between luminal and mucosal bacterial populations strongly suggests the need for separate analysis of these two populations.

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.

Evaluation of berberine efficacy in reducing the effects of aflatoxin B1 and ochratoxin A added to male broiler rations.

Many types of mycotoxins are found in food sources contaminated with fungi, and if these are ingested in large quantities or over a long period, they can affect the health of humans and domestic animals. Berberine (BBR) is a plant alkaloid with multiple pharmacological functions. This study aimed to investigate the effect of different levels of the plant alkaloid BBR on reducing toxic effects of aflatoxin B1 (AFB) and ochratoxin A (OTA) in broilers by examining performance characteristics, blood biochemistry, antioxidant systems, ileum morphology, and histopathology of the liver. The experiment was performed with 288 Ross 308 broilers reared in floor pens for 42 d in a randomized design with 9 treatments. Each treatment was replicated 4 times, and each replicate contained 8 chicks. Experimental treatments included (1) negative control diet with no additives (NC); (2) NC + 2 ppm AFB (positive control AFB; PCAFB); (3) NC + 2 ppm OTA (positive control OTA; PCOTA); (4) PCAFB + 200 mg/kg BBR; (5) PCAFB + 400 mg/kg BBR; (6) PCAFB + 600 mg/kg BBR; (7) PCOTA + 200 mg/kg BBR; (8) PCOTA + 400 mg/kg BBR; and (9) PCOTA + 600 mg/kg BBR. Compared with NC, feeding PCAFB and PCOTA diets reduced average daily feed intake, weight gain, serum concentrations of superoxide dismutase, glutathione peroxidase, and the length and width of ileum villi (P < 0.05). At the same time, these parameters increased in birds fed PCAFB or PCOTA diets supplemented with 600 mg/kg of BBR (P < 0.05). Feeding PCAFB and PCOTA diets increased feed conversion ratio (FCR), serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and gamma-glutamyl transferase (GGT) activities, serum urea, and liver lesions compared with NC. By contrast, compared with PCAFB and PCOTA, adding 600 mg/kg BBR decreased FCR, AST, LDH, ALT, and GGT activities, urea, and liver lesions (P < 0.05). Overall, supplementation with 600 mg/kg BBR may improve growth performance, liver function, and antioxidant status of broilers fed diets contaminated with AFB and OTA.

Effects of heat stress on performance, blood chemistry, and hypothalamic and pituitary mRNA expression in broiler chickens.

This study was conducted to evaluate potential hormonal mechanisms associated with the stress response, thermoregulation, and metabolic changes of broiler chickens exposed to high environmental temperature. Nine hundred 1-day-old male broiler chicks (Ross 708) were placed in floor pens and raised to 24 d. At 24 d, chicks were randomly assigned to 1 of 2 treatments, heat stress (HS) or no HS, and allocated into battery cages in 8 batteries (10 birds per cage, 2 cages per battery). On day 31, blood was collected prior to HS and analyzed using an iSTAT analyzer. Half of the batteries were then moved into 2 rooms with an elevated ambient temperature (35°C) for 8 h. The remaining batteries stayed in the thermoneutral rooms with an ambient temperature of 22°C. Beginning at 5 h after the initiation of HS, blood was collected and analyzed using an iSTAT analyzer, birds were euthanized, and hypothalamus and pituitary samples were collected (16 birds per treatment), flash frozen, and stored at -80°C until RNA extraction. Reverse transcription-quantitative PCR was used to compare mRNA levels of key corticotropic and thyrotrophic genes in the hypothalamus and pituitary. Levels of mRNA for each target gene were normalized to PGK1 (pituitary) and GAPDH (hypothalamus) mRNA. Differences were determined using mixed model ANOVA. HS decreased (P < 0.05) feed intake, BW, bicarbonate, potassium, CO2, and triiodothyronine, while it increased mortality, glucose, pH, plasma thyroxine, and corticosterone. Expression of pituitary corticotropin-releasing hormone receptor 1 was downregulated (P < 0.001), while corticotropin-releasing hormone receptor 2 mRNA levels were higher (P = 0.001) in HS birds. HS increased expression of thyroid hormone receptor ß (P = 0.01) (2.8-fold) and thyroid stimulating hormone ß (P = 0.009) (1.4-fold). HS did not affect levels of mRNA of genes evaluated in the hypothalamus. Results showed that HS significantly affected both the thyrotropic and corticotropic axes. Understanding the role and regulation of these pathways during HS will allow researchers to better evaluate management strategies to combat HS.

Delayed access to feed alters gene expression associated with hormonal signaling, cellular differentiation, and protein metabolism in muscle of newly hatch chicks.

Birds rely solely on utilization of the yolk sac as a means of nutritional support throughout embryogenesis and early post-hatch, before first feeding occurs. Newly hatched broiler (meat-type) chickens are frequently not given immediate access to feed, and this can result in numerous alterations to developmental processes, including those that occur in muscle. The objective of this study was to characterize the gene expression profile of newly hatched chicks' breast muscle with regards to hormonal regulation of growth and metabolism and development and differentiation of muscle tissue, and determine impacts of delayed access to feed on these profiles. Within 3 h of hatch, birds were placed in battery pens and given immediate access to feed (Fed) or delayed access to feed for 48 h (Delayed Fed). Breast muscle collected from male birds at hatch, or 4 h, 1 day (D), 2D, 4D, and 8D after hatch was used for analysis of mRNA expression by reverse transcription-quantitative PCR. Under fully fed conditions, insulin-like growth factor receptor and leptin receptor mRNA expression decreased as birds aged; however, delayed access to feed resulted in prolonged upregulation of these genes so their mRNA levels were higher in Delayed Fed birds at 2D. These expression profiles suggest that delayed feed access alters sensitivity to hormones that may regulate muscle development. Myogenin, a muscle differentiation factor, showed increasing mRNA expression in Fed birds through 2D, after which expression decreased. A similar expression pattern in Delayed Fed birds was deferred until 4D. Levels of myostatin, a negative regulator of muscle growth, increased in Fed birds starting at 2D, while levels in Delayed Fed birds began to increase at 4D. In Fed birds, levels of transcripts for two genes associated with protein catabolism, F-box protein 32 and forkhead box O3, were lower at 2D, while Delayed Fed mRNA levels did not decrease until 4D. Mechanistic target of rapamycin mRNA levels decreased from 1D through 8D in both treatments, except for a transient increase in the Delayed Fed birds between 1D and 2D. These data suggest that within breast muscle, delayed feeding alters hormonal signaling, interrupts tissue differentiation, postpones onset of growth, and may lead to increased protein catabolism. Together, these processes could ultimately contribute to a reduction in proper growth and development of birds not given feed immediately after hatch, and ultimately hinder the long-term potential of muscle accretion in meat type birds.

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.

Delayed access to feed alters expression of genes associated with carbohydrate and amino acid utilization in newly hatched broiler chicks.

Newly hatched chicks must transition from lipid-rich yolk to carbohydrate-rich feed as their primary nutrient source, and posthatch delays in access to feed can have long-term negative consequences on growth and metabolism. In this study, impacts of delayed access to feed at hatch on expression of genes related to nutrient uptake and utilization in two metabolically important tissues, liver and muscle, were determined in broiler (meat-type) chickens. Hatched chicks were given access to feed within 3 h (fed) or delayed access to feed for 48 h (delayed fed), and liver and breast muscle were collected from males at hatch and 4 h, 1 day, 2 days, 4 days, and 8 days posthatch for analysis of gene expression. Differential expression of carbohydrate response element-binding protein and peroxisome proliferator-activated receptor-γ in muscle and liver was observed, with results indicating a transitional delay from lipid to carbohydrate metabolism when hatched chicks were not given immediate access to feed. Extended upregulation of insulin receptor mRNA was observed in both tissues in delayed fed birds, suggesting increased sensitivity to circulating levels of the hormone. Developmental delays in expression patterns of cationic amino acid transporters 1 and 2 in both tissues and large neutral amino acid transporter 1 in muscle were also apparent when immediate feed access was prevented. These data suggest that delayed transition to carbohydrate use and altered nutrient transport and utilization within liver and breast muscle are key factors negatively affecting growth and metabolism following delayed feed access in broiler chickens.

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.

Mechanisms involved in glucocorticoid induction of pituitary GH expression during embryonic development.

Glucocorticoid hormones are involved in functional differentiation of GH-producing somatotrophs. Glucocorticoid treatment prematurely induces GH expression in mammals and birds in a process requiring protein synthesis and Rat sarcoma (Ras) signaling. The objective of this study was to investigate mechanisms through which glucocorticoids initiate GH expression during embryogenesis, taking advantage of the unique properties of chicken embryos as a developmental model. We determined that stimulation of GH expression occurred through transcriptional activation of GH, rather than enhancement of mRNA stability, and this process requires histone deacetylase activity. Through pharmacological inhibition, we identified the ERK1/2 pathway as a likely downstream Ras effector necessary for glucocorticoid stimulation of GH. However, we also found that chronic activation of ERK1/2 activity with a constitutively active mutant or stimulatory ligand reduced initiation of GH expression by glucocorticoid treatment. Corticosterone treatment of cultured embryonic pituitary cells increased ERK1/2 activity in an apparent cyclical manner, with a rapid increase within 5 minutes, followed by a reduction to near-basal levels at 3 hours, and a subsequent increase again at 6 hours. Therefore, we conclude that ERK1/2 signaling must be strictly controlled for maximal glucocorticoid induction of GH to occur. These results are the first in any species to demonstrate that Ras- and ERK1/2-mediated transcriptional events requiring histone deacetylase activity are involved in glucocorticoid induction of pituitary GH during embryonic development. This report increases our understanding of the molecular mechanisms underlying glucocorticoid recruitment of somatotrophs during embryogenesis and should provide insight into glucocorticoid-induced developmental changes in other tissues and cell types.

Environmental gestagens activate fathead minnow (Pimephales promelas) nuclear progesterone and androgen receptors in vitro.

Gestagen is a collective term for endogenous and synthetic progesterone receptor (PR) ligands. In teleost fishes, 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and 17α,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S) are the predominant progestogens, whereas in other vertebrates the major progestogen is progesterone (P4). Progestins are components of human contraceptives and hormone replacement pharmaceuticals and, with P4, can enter the environment and alter fish and amphibian reproductive health. In this study, our primary objectives were to clone the fathead minnow (FHM) nuclear PR (nPR), to develop an in vitro assay for FHM nPR transactivation, and to screen eight gestagens for their ability to transactivate FHM nPR. We also investigated the ability of these gestagens to transactivate FHM androgen receptor (AR). Fish progestogens activated FHM nPR, with DHP being more potent than 20ß-S. The progestin drospirenone and P4 transactivated the FHM nPR, whereas five progestins and P4 transactivated FHM AR, all at environmentally relevant concentrations. Progestins are designed to activate human PR, but older generation progestins have unwanted androgenic side effects in humans. In FHMs, several progestins proved to be strong agonists of AR. Here, we present the first mechanistic evidence that environmental gestagens can activate FHM nPR and AR, suggesting that gestagens may affect phenotype through nPR- and AR-mediated pathways.

Sources, concentrations, and exposure effects of environmental gestagens on fish and other aquatic wildlife, with an emphasis on reproduction.

Fish and other aquatic wildlife, including frogs, turtles, and alligators, have been used as vertebrate sentinels for the effects of endocrine disrupting and other emerging chemicals of concern found in aquatic ecosystems. Research has focused on the effects of estrogenic, androgenic, and thyroidogenic compounds, but there is a growing body of literature on the reproductive health exposure effects of environmental gestagens on aquatic wildlife. Gestagens include native progestogens, such as progesterone, and synthetic progestins, such as gestodene and levonorgestrel, which bind progesterone receptors and have critically important roles in vertebrate physiology, especially reproduction. Roles for progestogen include regulating gamete maturation and orchestrating reproductive behavior, both as circulating hormones and as secreted pheromones. Gestagens enter the aquatic environment through paper mill effluent, wastewater treatment plant effluent, and agricultural runoff. A number of gestagens have been shown to negatively affect reproduction, development, and behavior of exposed fish and other aquatic wildlife at ng/L concentrations, and these compounds have been measured in the environment at single to 375 ng/L. Given the importance of endogenous progestogens in the regulation of gametogenesis, secondary sex characteristics, and reproductive behavior in vertebrates and the documented exposure effects of pharmaceutical progestins and progesterone, environmental gestagens are an emerging class of contaminants that deserve increased attention from researchers and regulators alike. The potential for environmental gestagens to affect the reproductive health of aquatic vertebrates seems evident, but there are a number of important questions for researchers to address in this nascent field. These include identifying biomarkers of gestagen exposure; testing the effects of environmentally relevant mixtures; and determining what other physiological endpoints and taxa might be affected by exposure to environmental gestagens.

An inter-laboratory study on the variability in measured concentrations of 17ß-estradiol, testosterone, and 11-ketotestosterone in white sucker: implications and recommendations.

Endocrine-disrupting chemicals are exogenous substances that can impact the reproduction of fish, potentially by altering circulating concentrations of 17ß-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT). Common methods to measure steroids in plasma samples include radioimmunoassays (RIAs) and enzyme-linked immunosorbant assays (ELISAs). The present study examines variability in E2, T, and 11-KT across 8 laboratories measuring reference and pulp mill effluent-exposed white sucker (Catostomus commersoni) plasma. We examine the contribution of assay type (RIA vs ELISA), standardized hormone extraction, location of values on the standard curve (upper and lower limits), and other variables on the ability to distinguish hormone levels between reference and exposed fish and the impact of these variables on quantitation of hormones in different laboratories. Of the 8 participating laboratories, 7 of 8 and 7 of 7 identified differences between sites for female E2 and female T, respectively, and 7 of 7 and 4 of 5 identified no differences between male T and male 11-KT. Notably, however, the ng/mL concentration of steroids measured across laboratories varied by factors of 10-, 6-, 14-, and 10-fold, respectively. Within laboratory intra-assay variability was generally acceptable and below 15%. Factors contributing to interlaboratory variability included calculation errors, assay type, and methodology. Based on the interlaboratory variability detected, we provide guidelines and recommendations to improve the accuracy and precision of steroid measurements in fish ecotoxicology studies.

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.

Ras-dva is a novel Pit-1- and glucocorticoid-regulated gene in the embryonic anterior pituitary gland.

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5'-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5'-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland.

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.

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.

Transcriptional and pathway analysis in the hypothalamus of newly hatched chicks during fasting and delayed feeding.

BACKGROUND: The hypothalamus plays a central role in regulating appetite and metabolism. However, the gene networks within the hypothalamus that regulate feed intake and metabolism, and the effects of fasting on those pathways are not completely understood in any species. The present experiment evaluated global hypothalamic gene expression in newly hatched chicks using microarray analysis to elucidate genes and pathways regulated by feeding, fasting, and delayed feeding. Ten groups of chicks were sampled over four days post-hatch, including fed, fasted, and 48 h fasted followed by access to feed for 4 h, 24 h, and 48 h. Hypothalamic samples were collected for microarray analysis (n = 4). Expression patterns of selected genes were confirmed by quantitative real-time PCR. Pathway analysis of the microarray results predicted a network of genes involved in neuropeptide or neurotransmitter signaling. To confirm the functionality of this predicted gene network, hypothalamic neurons from fed and fasted chicks were isolated and cultured in the presence of neuropeptide Y, somatostatin, alpha-melanocyte stimulating hormone, norepinephrine, and L-phospho-serine. Results confirmed functional relationships among members of the predicted gene network. Moreover, the effects observed were dependent upon the nutritional state of the animals (fed vs. fasted). RESULTS: Differences in gene expression (> or = 1.6 fold) were detected in 1,272 genes between treatments, and of those, 119 genes were significantly (P < 0.05) different. Pathway Miner analysis revealed that six genes (SSTR5, NPY5R, POMC, ADRB2, GRM8, and RLN3) were associated within a gene network. In vitro experiments with primary hypothalamic neurons confirmed that receptor agonists involved in this network regulated expression of other genes in the predicted network, and this regulation within the network was influenced by the nutritional status and age of the chick. CONCLUSIONS: Microarray analysis of the hypothalamus during different nutritional states revealed that many genes are differentially regulated. We found that functional interactions exist among six differentially regulated genes associated within a putative gene network from this experiment. Considering that POMC, an important gene in controlling metabolism, was central to this network, this gene network may play an important role in regulation of feeding and metabolism in birds.