(-)-Hydroxycitric Acid Influenced Fat Metabolism via Modulating of Glucose-6-phosphate Isomerase Expression in Chicken Embryos.

The current research aimed to explore the impact of (-)-hydroxycitric acid (HCA) on fat metabolism and investigate whether this action of (-)-HCA was associated with modulation of glucose-6-phosphote isomerase (GPI) expression in chicken embryos. We constructed a recombinant plasmid (sh2-GPI) to inhibit GPI expression, and then embryos were treated with (-)-HCA. Results showed that (-)-HCA reduced lipid droplet accumulation, triglyceride content, and lipogenesis factors mRNA level and increased lipolysis factors mRNA expression, while this effect caused by (-)-HCA was markedly reversed when the chicken embryos were pretreated with sh2-GPI. (-)-HCA increased phospho (p)-acetyl-CoA carboxylase, enoyl-CoA hydratase short chain-1, carnitine palmitoyl transferase 1A, p-AMP-activated protein kinase, and peroxisome proliferators-activated receptor α protein expression, and this action of (-)-HCA also dispelled when the chicken embryos were pretreated with sh2-GPI. These data demonstrated that (-)-HCA decreased fat deposition via activation of the AMPK pathway, and the fat-reduction action of (-)-HCA was due to the increasing of GPI expression in chicken embryos.

Detailed expression profile of the six Glypicans and their modifying enzyme, Notum during chick limb and feather development.

The development of vertebrate appendages, especially the limb and feather buds are orchestrated by numerous secreted signalling molecules including Sonic Hedgehog, Bone Morphogenetic Proteins, Fibroblast Growth Factors and Wnts. These proteins coordinate the growth and patterning of ectodermal and mesenchymal cells. The influence of signalling molecules is affected over large distances by their concentration (morphogen activity) but also at local levels by the presence of proteins that either attenuate or promote their activity. Glypicans are cell surface molecules that regulate the activity of the major secreted signalling molecules expressed in the limb and feather bud. Here we investigated the expression of all Glypicans during chick limb and feather development. In addition we profiled the expression of Notum, an enzyme that regulates Glypican activity. We show that five of the six Glypicans and Notum are expressed in a dynamic manner during the development of limbs and feathers. We also investigated the expression of key Glypicans and show that they are controlled by signalling molecules highlighting the presence of feedback loops. Lastly we show that Glypicans and Notum are expressed in a tissue specific manner in adult chicken tissues. Our results strongly suggest that the Glypicans and Notum have many as yet undiscovered roles to play during the development of vertebrate appendages.

Detailed expression profile of all six Glypicans and their modifying enzyme Notum during chick embryogenesis and their role in dorsal-ventral patterning of the neural tube.

Vertebrate development is orchestrated by secreted signalling molecules that regulate cell behaviour and cell fate decisions during early embryogenesis. The activity of key signalling molecules including members of Hedgehog, Bone Morphogenetic Proteins and Wnt families are regulated by Glypicans, a family of GPI linked polypeptides. Glypicans either promote or inhibit the action of signalling molecules and add a layer of complexity that needs to be understood in order to fully decipher the processes that regulate early vertebrate development. Here we present a detailed expression profile of all six Glypicans and their modifying enzyme Notum during chick embryogenesis. Our results strongly suggest that these proteins have many as yet undiscovered roles to play during early embryogenesis. Finally, we have taken an experimental approach to investigate their role during the patterning of a key embryonic structure - the neural tube. In particular, we show that over-expression of Notum leads to the dorsalisation of this structure.

Similar developmental fluctuations of hepato-renal xanthine oxidoreductase gene expression and xanthine oxidase activity in layer and broiler chicken embryos.

1. Xanthine oxidase (XO) has many physiological functions associated with the synthesis of both antioxidant (uric acid: UA) and numerous oxidants (e.g. H2O2), which makes it an important regulator of the cellular redox potential involving organogenesis. The ontogenetic study of hepatic and renal XO makes a better understanding of the putative role of this enzyme in the development of these tissues. 2. Developmental changes of gene expression of xanthine oxidoreductase (XOR), XO activity and UA content of liver and kidney tissues in both broiler and layer chicken embryos were examined during incubation d 14-21. 3. In both strains, hepatic XOR gene expression peaked on d 21 while renal XOR gene expression did not change. 4. The XO activity was higher in kidney than liver in both strains. Hepatic XO activity of both strains peaked on d 18 and thereafter was decreased on d 21. Renal XO activity peaked on d 18 and from then on did not show any significant changes until d 21 in both strains. 5. The UA content was higher in kidney vs. liver in both strains. The hepatic and renal UA values of the both strains increased significantly from d 14 to d 21. 6. The present results showed dissimilar behaviour of XOR gene expression, XO activity and UA content of liver and kidney tissues in both broiler and layer chicken embryos.

Effect of egg composition and oxidoreductase on adaptation of Tibetan chicken to high altitude.

Tibetan chickens have good adaptation to hypoxic conditions, which can be reflected by higher hatchability than lowland breeds when incubated at high altitude. The objective of this trial was to study changes in egg composition and metabolism with regards the adaptation of Tibetan chickens to high altitude. We measured the dry weight of chicken embryos, egg yolk, and egg albumen, and the activity of lactate dehydrogenase (LDH) and succinic dehydrogenase (SDH) in breast muscle, heart, and liver from embryos of Tibetan chicken and Dwarf chicken (lowland breed) incubated at high (2,900 m) and low (100 m) altitude. We found that growth of chicken embryos was restricted at high altitude, especially for Dwarf chicken embryos. In Tibetan chicken, the egg weight was lighter, but the dry weight of egg yolk was heavier than that of Dwarf chicken. The LDH activities of the three tissues from the high altitude groups were respectively higher than those of the lowland groups from d 15 to hatching, except for breast muscle of Tibetan chicken embryos on d 15. In addition, under the high altitude environment, the heart tissue from Tibetan chicken had lower LDH activity than that from Dwarf chicken at d 15 and 18. The lactic acid content of blood from Tibetan chicken embryos was lower than that of Dwarf chicken at d 12 and 15 of incubation at high altitude. There was no difference in SDH activity in the three tissues between the high altitude groups and the lowland groups except in three tissues of hatchlings and at d 15 of incubation in breast muscle, nor between the two breeds at high altitude except in the heart of hatchlings. Consequently, the adaptation of Tibetan chicken to high altitude may be associated with higher quantities of yolk in the egg and a low metabolic oxygen demand in tissue, which illuminate the reasons that the Tibetan chicken have higher hatchability with lower oxygen transport ability.

DNA methylation and histone modification patterns during the late embryonic and early postnatal development of chickens.

Early mammalian embryonic cells have been proven to be essential for embryonic development and the health of neonates. A series of epigenetic reprogramming events, including DNA methylation and histone modifications, occur during early embryonic development. However, epigenetic marks in late embryos and neonates are not well understood, especially in avian species. To investigate the epigenetic patterns of developing embryos and posthatched chicks, embryos at embryonic day 5 (E5), E8, E11, E14, E17, and E20 and newly hatched chicks on day of life 1 (D1), D7, D14, D21 were collected. The levels of global DNA methylation and histone H3 at lysine 9 residue (H3K9) modifications were measured in samples of liver, jejunum, and breast skeletal muscles by Western blotting and immunofluorescence staining. According to our data, decreased levels of proliferating cell nuclear antigen expression were found in the liver and a V-shaped pattern of proliferating cell nuclear antigen expression was found in the jejunum. The level of proliferating cell nuclear antigen in muscle was relatively stable. Caspase 3 expression gradually decreased over time in liver, was stable in the jejunum, and increased in muscle. Levels of DNA methylation and H3K9 acetylation decreased in liver over time, while the pattern was N-shaped in jejunal tissue and W-shaped in pectoral muscles, and these changes were accompanied by dynamic changes of DNA methyltransferases, histone acetyltransferases 1, and histone deacetylase 2. Moreover, dimethylation, trimethylation, and acetylation of H3K9 were expressed in a time- and tissue-dependent manner. After birth, epigenetic marks were relatively stable and found at lower levels. These results indicate that spatiotemporal specific epigenetic alterations could be critical for the late development of chick embryos and neonates.

Inhibition of lysozyme by taurine dibromamine.

Hypobromous acid (HOBr) is a powerful oxidant produced by stimulated neutrophils and eosinophils. Taurine, a non-protein amino acid present in high amounts in the leukocytes, reacts instantaneously with HOBr leading to their haloamine derivative taurine dibromamine (Tau-NBr2). Lysozyme is a bactericidal enzyme also present in leukocytes and in secretory fluids. The inhibition of lysozyme is a pathway for bacterial proliferation in inflammatory sites. Here, we investigated the inhibition of the enzymatic activity of lysozyme when it was submitted to oxidation by Tau-NBr2. We found that the oxidation of lysozyme by Tau-NBr2 decreased its enzymatic activity in 80%, which was significant higher compared to the effect of its precursor HOBr (30%). The study and comparison of Tau-NBr2 and HOBr regarding the alterations provoked in the intrinsic fluorescence, synchronous fluorescence, resonance light scattering and near and far-UV circular dichroism spectra of lysozyme and oxidized lysozyme revealed that tryptophan residues in the active site of the protein were the main target for Tau-NBr2 and could explain its efficacy as inhibitor of lysozyme enzymatic activity. This property of Tau-NBr2 may have pathological significance, since it can be easily produced in the inflammatory sites.

RALDH2, the enzyme for retinoic acid synthesis, mediates meiosis initiation in germ cells of the female embryonic chickens.

Meiosis is a process unique to the differentiation of germ cells and exhibits sex-specific in timing. Previous studies showed that retinoic acid (RA) as the vitamin A metabolite is crucial for controlling Stra8 (Stimulated by retinoic acid gene 8) expression in the gonad and to initiate meiosis; however, the mechanism by which retinoid-signaling acts has remained unclear. In the present study, we investigated the role of the enzyme retinaldehyde dehydrogenase 2 (RALDH2) which catalyzes RA synthesizes by initiating meiosis in chicken ovarian germ cells. Meiotic germ cells were first detected at day 15.5 in chicken embryo ovary when the expression of synaptonemal complex protein 3 (Scp3) and disrupted meiotic cDNA 1 homologue (Dmc1) became elevated, while Stra8 expression was specifically up-regulated at day 12.5 before meiosis onset. It was observed from the increase in Raldh2 mRNA expression levels and decreases in Cyp26b1 (the enzyme for RA catabolism) expression levels during meiosis that requirement for RA accumulation is essential to sustain meiosis. This was also revealed by RA stimulation of the cultured ovaries with the initiation of meiosis response, and the knocking down of the Raldh2 expression during meiosis, leading to abolishment of RA-dependent action. Altogether, these studies indicate that RA synthesis by the enzyme RALDH2 and signaling through its receptor is crucial for meiosis initiation in chicken embryonic ovary.

Effect of manipulation of incubation temperature on fatty acid profiles and antioxidant enzyme activities in meat-type chicken embryos.

Eggs (n = 1,800) obtained from Ross broiler breeders at 32 and 48 wk of age were incubated at either a constant temperature of 37.6°C throughout (T1), or the temperature was reduced for 6 h to 36.6°C each day during embryonic age (EA) 10 to 18 (T2). Yolk sac, liver, and brain fatty acid profiles and oxidant and antioxidant status of liver and brain were measured at EA 14, 19, and day of hatch (DOH). Fatty acid profiles of yolk sac, liver, and brain were influenced by age of breeder with significant breeder hen age × incubation temperature interactions. At EA 14, higher levels of 20:4n-6 and 22:6n-3 had been transferred from the yolk sac to T2 embryos from younger than older breeders, whereas for T1 and T2 embryos, yolk sac 20:4n-6 and 22.6n-3 values were similar for older breeders. Accumulation of 20:4n-6 and 22:6n-3 fatty acids in the liver of T1 and T2 embryos from younger breeders was similar; however, T2 embryos from older breeders had higher liver levels of 20:4n-6 and 22:6n-3 than T1 embryos. At EA 19, liver nitric oxide levels were higher for T2 embryos from younger breeders than those from breeders incubated at T1. Brain catalase levels of T2 embryos from younger breeders were higher than those from older breeders at DOH. Thus, changes in fatty acid profiles and catalase and nitric oxide production of brain and liver tissues resulting from 1°C lower incubation temperature from EA 10 to 18 reflect adaptive changes.

Cadmium effects on early development of chick embryos.

The toxic potential of cadmium (Cd) is well-documented for young and adult vertebrates, but it is still poorly understood in the early stages of development. In this study, cadmium effects were investigated on Gallus gallus embryos after injection of CdCl(2) (5µM and 50µM) within the egg air chamber, and incubation for 48 and 72h. After exposure, morphological and enzymatic analyses for glucose-6-phosphate dehydrogenase (G6PDH) and glutathione S-transferase (GST) were performed. Critical morphological abnormalities occurred after exposure to the highest concentration of cadmium, mainly in the cephalic region, indicating the powerful teratogenic effect of Cd to chick embryos. Cd exposure did not alter enzymatic activities when compared to the control group, but the levels of G6PDH activity were highest in older embryos at stage 19, indicating that antioxidant defenses are not so robust in the earliest embryo stages.

Effects of estrogen on estrogen receptor expression in the bursal cells of chick embryos and steroidogenic enzymes gene expression in the bursa: relevance of estrogen receptor and estrogen synthesis in the bursa of chick embryos.

Estrogen has been reported to act on B cell genesis in the bursa of Fabricius of chick embryos. In this study, we attempted to demonstrate the hypothesis that B cell genesis is controlled by estrogen receptor (ER) in the bursal cells and steroidogenic enzymes synthesized in the bursa. We previously reported the presence of estrogen receptor α (ERα) in the bursa during the late stage of embryogenesis and an increase in the expression of ERα messenger RNA (mRNA) between the 13th day and 16th day. The number of ER-positive cells was maximal on the 16th day. In the present study, ER-positive cells in the bursa during the late stage of embryogenesis increased 4 h after ß-estradiol treatment on the 14th to 18th day. The concentration of ß-estradiol in the embryonic bursa increased. These results suggest that this stage of embryogenesis is critical in B cell development in the bursa in connection with the effect of estrogen treatment. Our findings also showed that the mRNA expression of five steroidogenic enzymes occurred in the bursa of chick embryos. These results suggest that estrogen is synthesized in the embryonic bursa and estrogen acts on the bursal cells in a paracrine fashion.

Expression pattern of iodotyrosine dehalogenase 1 (DEHAL1) during chick ontogeny.

The iodotyrosine dehalogenase1 (DEHAL1) enzyme is a transmembrane protein that belongs to the nitroreductase family and shows a highly conserved N-terminal domain. DEHAL1 is present in the liver, kidney and thyroid of mammals. DEHAL1 is known to act on diiodotyrosine (DIT) and monoiodotyrosine (MIT), and is involved in iodine recycling in relation to thyroglobulin. Here, we show the distribution of DEHAL1 during gastrulation to neurulation in developing chick. Immunocytochemistry using an anti-serum directed against the N-terminal domain (met(27)-trp(180) fragment) of human DEHAL1 revealed labelled cells in the embryonic ectoderm, embryonic endoderm, neural plate and in the yolk platelets of the chick embryo at gastrulation stage. Distinct DEHAL1 positive cells were located in the presumptive head ectoderm, presumptive neural crest, head mesenchymal cells and in the dorsal, lateral and ventral parts of neural tube during neurulation. Some cells located at the margin of the developing notochord and somites were also DEHAL1-positive. While the functional significance of this observation is not known, it is likely that DEHAL1 might serve as an agent that regulates cell specific deiodination of MIT and DIT before the onset of thyroidal secretion. The presence of DEHAL1 in different components of the chick embryo suggests its involvement in iodine turnover prior to the formation of functional thyroid.

Spots and stripes: pleomorphic patterning of stem cells via p-ERK-dependent cell chemotaxis shown by feather morphogenesis and mathematical simulation.

A key issue in stem cell biology is the differentiation of homogeneous stem cells towards different fates which are also organized into desired configurations. Little is known about the mechanisms underlying the process of periodic patterning. Feather explants offer a fundamental and testable model in which multi-potential cells are organized into hexagonally arranged primordia and the spacing between primordia. Previous work explored roles of a Turing reaction-diffusion mechanism in establishing chemical patterns. Here we show that a continuum of feather patterns, ranging from stripes to spots, can be obtained when the level of p-ERK activity is adjusted with chemical inhibitors. The patterns are dose-dependent, tissue stage-dependent, and irreversible. Analyses show that ERK activity-dependent mesenchymal cell chemotaxis is essential for converting micro-signaling centers into stable feather primordia. A mathematical model based on short-range activation, long-range inhibition, and cell chemotaxis is developed and shown to simulate observed experimental results. This generic cell behavior model can be applied to model stem cell patterning behavior at large.

Identification and characterization of the precursor of chicken matrix metalloprotease 2 (pro-MMP-2) in hen egg.

Using zymography and mass spectrometry, we identified for the first time the precursor of chicken matrix metalloprotease 2 (pro-MMP-2) as a complex with TIMP-2 (tissue inhibitor of metalloproteinases) in egg white and yolk. Real-time polymerase chain reaction confirmed that MMP-2 and its inhibitors TIMP-2 and TIMP-3 were expressed all along the oviduct and in the liver of laying hens. We also demonstrated that the processing of pro-MMP-2 into mature MMP-2 by serine proteases does not occur in vivo, although purified pro-MMP-2 undergoes proteolytic maturation by these proteases in vitro. Moreover, the relative pro-MMP-2 activity assessed by gelatin zymography was shown to decrease in egg white during the storage of unfertilized or fertilized eggs. However, the mature form of 62 kDa MMP-2 could not be detected. The fact that MMP-2 is found as a proform in fresh eggs suggests that the activity of this metalloprotease is regulated under specific conditions during embryonic development.

[Efficacy of various methods of a reporter gene transfer to chicken embryonic cells].

The methods of transfection ofa plasmid with a reporter gene involving DNA injection into chicken embryonic cells were studied. The parameters of the efficient transfection of chicken blastodermal cells with a foreign gene have been determined (20-24 and up to 40% in culture and embryos, respectively). A high efficiency of transfection of primordial germ cells isolated from the gonads has been obtained after DNA injection into the dorsal aorta of 2.5-day-old chicken embryos.

Hepatic lipogenesis in broiler chickens with different fat deposition during embryonic development.

In order to identify the genes involved in the fatness variability, we studied the expression of several genes implicated in the hepatic lipid metabolism of broiler chickens with different fat deposition patterns during embryonic development. The mRNA expression of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME) and apolipoprotein B100 (apoB100) genes were determined using reverse transcriptase-polymerase chain reaction (RT-PCR). Samples of livers were collected from Arbor Acres (AA) and Sanhuang (SH) chickens on day 9, 14 and 19 of embryonic development as well as at hatching. This study showed that hepatic triglyceride (TG) level was found to increase suddenly during day 14 of embryonic development, to gradually increase thereafter, and to remain relatively constant at hatching. FAS gene expression in AA and SH broilers occurred prior to hatching and at hatching. The gene was expressed more in the former breed. ACC gene expression was observed beginning at the earlier development stage of days 9. No breed difference was observed in ME and apoB gene expression. This study indicated that the expression of lipogenic enzyme genes of the liver in broiler chickens exhibited scheduling during embryogenesis. The ACC gene started to express earlier than the FAS gene during embryonic development. This suggested that embryonic liver synthesized fatty acid, and breed difference was noticed prior to hatching.

Cyp26 genes a1, b1 and c1 are down-regulated in Tbx1 null mice and inhibition of Cyp26 enzyme function produces a phenocopy of DiGeorge Syndrome in the chick.

Cyp26a1, a gene required for retinoic acid (RA) inactivation during embryogenesis, was previously identified as a potential Tbx1 target from a microarray screen comparing wild-type and null Tbx1 mouse embryo pharyngeal arches (pa) at E9.5. Using real-time PCR and in situ hybridization analysis of Cyp26a1 and its two functionally related family members Cyp26b1 and c1, we demonstrate reduced and/or altered expression for all three genes in pharyngeal tissues of Tbx1 null embryos. Blockade of Cyp26 function in the chick embryo using R115866, a specific inhibitor of Cyp26 enzyme function, resulted in a dose-dependent phenocopy of the Tbx1 null mouse including loss of caudal pa and pharyngeal arch arteries (paa), small otic vesicles, loss of head mesenchyme and, at later stages, DiGeorge Syndrome-like heart defects, including common arterial trunk and perimembranous ventricular septal defects. Molecular markers revealed a serious disruption of pharyngeal pouch endoderm (ppe) morphogenesis and reduced staining for smooth muscle cells in paa. Expression of the RA synthesizing enzyme Raldh2 was also up-regulated and altered Hoxb1 expression indicated that RA levels are raised in R115866-treated embryos as reported for Tbx1 null mice. Down-regulation of Tbx1 itself was observed, in accordance with previous observations that RA represses Tbx1 expression. Thus, by specifically blocking the action of the Cyp26 enzymes we can recapitulate many elements of the Tbx1 mutant mouse, supporting the hypothesis that the dysregulation of RA-controlled morphogenesis contributes to the Tbx1 loss of function phenotype.

Transition from ectothermy to endothermy: the development of metabolic capacity in a bird (Gallus gallus).

The evolution of endothermy is one of the most significant events in vertebrate evolution. Adult mammals and birds are delineated from their early ontogenetic stages, as well as from other vertebrates, by high resting metabolic rates and consequent internal heat production. We used the embryonic development of a bird (Gallus gallus) as a model to investigate the metabolic transition between ectothermy and endothermy. Increases in aerobic capacity occur at two functional levels that are regulated independently from each other: (i) upregulation of gene expression; and (ii) significant increases in the catalytic activity of the main oxidative control enzymes. Anaerobic capacity, measured as lactate dehydrogenase activity, is extremely high during early development, but diminishes at the same time as aerobic capacity increases. Changes in lactate dehydrogenase activity are independent from its gene expression. The regulatory mechanisms that lead to endothermic metabolic capacity are similar to those of ectotherms in their response to environmental change. We suggest that the phylogenetic occurrence of endothermy is restricted by its limited selective advantages rather than by evolutionary innovation.

Prokaryotic and eukaryotic monothiol glutaredoxins are able to perform the functions of Grx5 in the biogenesis of Fe/S clusters in yeast mitochondria.

The Saccharomyces cerevisiae monothiol glutaredoxin Grx5 participates in the mitochondrial biogenesis of iron-sulfur clusters. Grx5 homologues exist in organisms from bacteria to humans. Chicken (cGRX5) and human (hGRX5) homologues contain a mitochondrial targeting sequence, suggesting a mitochondrial localization for these two proteins. We have compartmentalized the Escherichia coli and Synechocystis sp. homologues, and also cGRX5 and hGRX5, in the mitochondrial matrix of a yeast grx5 mutant. All four heterologous proteins rescue the defects of the mutant. The chicken cGRX5 gene was significantly expressed throughout the embryo stages in different tissues. These results underline the functional conservation of Grx5 homologues throughout evolution.

Regulation of thyroid hormone availability by iodothyronine deiodinases at the blood-brain barrier in birds.

It is accepted that type II iodothyronine deiodinase (D2) is predominantly found in brain, where it maintains homeostasis of thyroid hormone (TH) levels. The current study describes the production of a polyclonal D2 antiserum and its use in the comparison of D2 protein distribution with that of type I (D1) and type III (D3) deiodinase protein in the chicken choroid plexus (CP). Immunocytochemistry showed high D2 protein expression in the epithelial cells of the CP, whereas the D1 and D3 proteins were absent. Furthermore, dexamethasone treatment led to an upregulation of the D2 protein in these cells.