Study on the role of heat shock protein 90 (HSP90) gene in chicken preadipocytes proliferation and differentiation.

In this study, we examined the effects of Heat Shock Protein 90 (HSP90) on adipocyte proliferation and differentiation in chickens. To achieve this, we constructed RNA interference (RNAi) vectors to target HSP90 and transfected the vectors into primary adipocytes. After transfection, oil red O staining was performed to determine the status of triglyceride accumulation in the cells, whereas the CCK-8 cell kit and 5-Ethynyl-2'-Deoxyuridine (EdU) assays were used to determine cell proliferation. Thereafter, the mRNA and protein expression levels of PPARγ, FAS, SREBP-1c, and HSP90 were determined, and the results showed that after the interference of HSP90, the mRNA and protein expression levels of HSP90 in the chicken adipocytes decreased significantly compared to the control and blank groups (p < 0.05). The decreased mRNA and protein expression of PPARγ, FAS, and SREBP-1c was related to adipocyte differentiation (p < 0.05). However, HSP90 interference had no effect on adipocyte proliferation (p > 0.05). Taken together, the results of this study showed that HSP90 influenced the expression of PPARγ and adipose-differentiation-related genes, thereby regulating triglyceride accumulation and adipocyte differentiation in chickens.

Circadian miR-449c-5p regulates uterine Ca2+ transport during eggshell calcification in chickens.

BACKGROUND: miRNAs regulate circadian patterns by modulating the biological clocks of animals. In our previous study, we found that the clock gene exhibited a cosine expression pattern in the fallopian tube of chicken uterus. Clock-controlled miRNAs are present in mammals and Drosophila; however, whether there are clock-controlled miRNAs in the chicken uterus and, if so, how they regulate egg-laying rhythms is unclear. In this study, we selected 18 layer hens with similar ovipositional rhythmicity (each of three birds were sacrificed for study per 4 h throughout 24 h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens. RESULTS: We identified six circadian miRNAs that are mainly associated with several biological processes including ion trans-membrane transportation, response to calcium ion, and enrichment of calcium signaling pathways. Verification of the experimental results revealed that miR-449c-5p exhibited a cosine expression pattern in the chicken uterus. Ca2+-transporting ATPase 4 (ATP2B4) in the plasma membrane is the predicted target gene of circadian miR-449c-5p and is highly enriched in the calcium signaling pathway. We speculated that clock-controlled miR-449c-5p regulated Ca2+ transportation during eggshell calcification in the chicken uterus by targeting ATP2B4. ATP2B4 mRNA and protein were rhythmically expressed in the chicken uterus, and dual-luciferase reporter gene assays confirmed that ATP2B4 was directly targeted by miR-449c-5p. The expression of miR-449c-5p showed an opposite trend to that of ATP2B4 within a 24 h cycle in the chicken uterus; it inhibited mRNA and protein expression of ATP2B4 in the uterine tubular gland cells. In addition, overexpression of ATP2B4 significantly decreased intracellular Ca2+ concentration (P < 0.05), while knockdown of ATP2B4 accelerated intracellular Ca2+ concentrations. We found similar results after ATP2B4 knockdown by miR-449c-5p. Taken together, these results indicate that ATP2B4 promotes uterine Ca2+ trans-epithelial transport. CONCLUSIONS: Clock-controlled miR-449c-5p regulates Ca2+ transport in the chicken uterus by targeting ATP2B4 during eggshell calcification.

Structural determinants for tRNA selective cleavage by RNase 2/EDN.

tRNA-derived fragments (tRFs) have emerged as key players of immunoregulation. Some RNase A superfamily members participate in the shaping of the tRFs population. By comparing wild-type and knockout macrophage cell lines, our previous work revealed that RNase 2 can selectively cleave tRNAs. Here, we confirm the in vitro protein cleavage pattern by screening of synthetic tRNAs, single-mutant variants, and anticodon-loop DNA/RNA hairpins. By sequencing of tRF products, we identified the cleavage selectivity of recombinant RNase 2 with base specificity at B1 (U/C) and B2 (A) sites, consistent with a previous cellular study. Lastly, protein-hairpin complexes were predicted by MD simulations. Results reveal the contribution of the α1, loop 3 and loop 4, and ß6 RNase 2 regions, where residues Arg36/Asn39/Gln40/Asn65/Arg68/Arg132 provide interactions, spanning from P-1 to P2 sites that are essential for anticodon loop recognition. Knowledge of RNase 2-specific tRFs generation might guide new therapeutic approaches for infectious and immune-related diseases.

RNA-Seq Analysis Reveals the Molecular Mechanisms Regulating the Development of Different Adipose Tissues in Broiler Chicks.

In an effort to enhance growth rates, chicken breeders have undertaken intensive genetic selection. In the selection process, the primary aim is to accelerate growth, inadvertently leading to new chicken breeds having an increased capacity for rapid adipose tissue accumulation. However, little is known about the relationship between changes in gene expression and adipose tissue accumulation and deposition in chickens. Therefore, in this study, RNA-seq analysis was utilized, and transcriptome data were obtained from the abdominal fat, thoracic subcutaneous fat, and clavicular fat on day 1 (d1), day 4, day 7, day 11, and day 15 to reveal the molecular mechanisms regulating the development and deposition of different adipose tissues in broiler chicks. The results showed that the key period for adipocyte differentiation and proliferation was between d4 and d7 (abdominal fat development) and between d1 and d4 (chest subcutaneous fat and clavicular fat). In addition, candidate genes such as MYOG, S100A9, CIDEC, THRSP, CXCL13, and NMU related to adipose tissue growth and development were identified. Further, genes (HOXC9, AGT, TMEM182, ANGPTL3, CRP, and DSG2) associated with the distribution of adipose tissue were identified, and genes (MN1, ANK2, and CAP2) related to adipose tissue growth were also identified. Taken together, the results from this study provide the basis for future studies on the mechanisms regulating adipose tissue development in chickens. Further, the candidate genes identified could be used in the selection process.

Integrated Proteomic and Metabolomic Analyses of Chicken Ovary Revealed the Crucial Role of Lipoprotein Lipase on Lipid Metabolism and Steroidogenesis During Sexual Maturity.

During sexual maturation and ovulatory cycle in chickens, ovaries undergo dynamic morphological and functional changes. The aim of this study was to evaluate the integrated proteome and metabolome analyses of chicken ovaries to characterize the changes in protein and metabolite profiles during sexual maturity. The ovary of Rohman layers before (125 days of age) and after (139 days of age) sexual maturation were collected for proteome and metabolome sequencing. The results showed that a total of 680 differentially expressed proteins (DEPs) and 1,046 differential metabolites (DMs) were identified in the chicken ovary during sexual maturity. Among the DEPs, 595 proteins were up-regulated and 85 were down-regulated, whereas 519 metabolites were up-regulated and 527 were down-regulated. KEGG pathway enrichment analysis showed that DEPs were significantly enriched in glycerolipid metabolism, calcium signaling pathway, folate biosynthesis, fat digestion and absorption, NF-kB signaling pathway, and PPAR signaling pathway. However, DMs were significantly enriched in the metabolism pathways, PPAR signalling pathway, glycerolipid metabolism, ferroptosis, biosynthesis of amino acids, and biosynthesis of unsaturated fatty acids. The results of the integrated analyses of DEPs and DMs revealed that the PPAR signaling pathway and glycerolipid metabolism were the most significantly enriched pathways. Among the identified DEPs, lipoprotein lipase (LPL) was upregulated in sexually mature chicken ovaries and was significantly enriched in the glycerolipid metabolism pathway, which may partially explain the possible reasons for steroidogenesis and lipid reserves responsible for oocyte maturation and ovarian follicle development during sexual maturity in chickens. The results further revealed that LPL silencing decreased the content of lipid droplets (LDs), as well as the mRNA expression of lipid metabolism-related genes including; sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase (FASN); and steroidogenesis-related genes such as; cytochrome P450 11A1 (CYP11A1) and steroidogenic acute regulatory (StAR). The present study revealed that upregulation of LPL in the chicken ovary during sexual maturity promotes granulosa cell (GC) lipid metabolism and steroidogenesis. These findings provide a theoretical support for further studies to elucidate the mechanism of lipid metabolism to regulate the function of avian GCs during sexual maturity in chickens.

Dietary supplementation of salidroside alleviates liver lipid metabolism disorder and inflammatory response to promote hepatocyte regeneration via PI3K/AKT/Gsk3-ß pathway.

Fatty liver hemorrhagic syndrome (FLHS) is a chronic hepatic disease which occurs when there is a disorder in lipid metabolism. FLHS is often observed in caged laying hens and characterized by a decrease in egg production and dramatic increase of mortality. Salidroside (SDS) is an herbal drug which has shown numerous pharmacological activities, such as protecting mitochondrial function, attenuating cell apoptosis and inflammation, and promoting antioxidant defense system. We aimed to determine the therapeutic effects of SDS on FLHS in laying hens and investigate the underlying mechanisms through which SDS operates these functions. We constructed oleic acid (OA)-induced fatty liver model in vitro and high-fat diet-induced FLHS of laying hens in vivo. The results indicated that SDS inhibited OA-induced lipid accumulation in chicken primary hepatocytes, increased hepatocyte activity, elevated the mRNA expression of proliferation related genes PCNA, CDK2, and cyclinD1 and increased the protein levels of PCNA and CDK2 (P < 0.05), as well as decreased the cleavage levels of Caspase-9, Caspase-8, and Caspase-3 and apoptosis in hepatocytes (P < 0.05). Moreover, SDS promoted the phosphorylation levels of PDK1, AKT, and Gsk3-ß, while inhibited the PI3K inhibitor (P < 0.05). Additionally, we found that high-fat diet-induced FLHS hens had heavier body weight, liver weight, and abdominal fat weight, and severe steatosis in histology, compared with the control group (Con). However, hens fed with SDS maintained lighter body weight, liver weight, and abdominal fat weight, as well as normal liver without hepatic steatosis. In addition, high-fat diet-induced FLHS hens had high levels of serum total cholesterol (TC), triglyceride (TG), alanine transaminase (ALT), and aspartate aminotransferase (AST) compared to the Con group, however, in the Model+SDS group, the levels of TC, TG, ALT, and AST decreased significantly, whereas the level of superoxide dismutase (SOD) increased significantly (P < 0.05). We also found that SDS significantly decreased the mRNA expression abundance of PPARγ, SCD, and FAS in the liver, as well as increased levels of PPARα and MTTP, and decreased the mRNA expression of TNF-α, IL-1ß, IL-6, and IL-8 in the Model+SDS group (P < 0.05). In summary, this study showed that 0.3 mg/mL SDS attenuated ROS generation, inhibited lipid accumulation and hepatocyte apoptosis, and promoted hepatocyte proliferation by targeting the PI3K/AKT/Gsk3-ß pathway in OA-induced fatty liver model in vitro, and 20 mg/kg SDS alleviated high-fat-diet-induced hepatic steatosis, oxidative stress, and inflammatory response in laying hens in vivo.

Combination of Quercetin and Vitamin E Supplementation Promotes Yolk Precursor Synthesis and Follicle Development in Aging Breeder Hens via Liver-Blood-Ovary Signal Axis.

The fertility of female animals is negatively correlated with increasing chronological age. In aging broiler breeder hens, there is a decline in the functionality of the ovary and liver accompanied by hormonal or endocrine changes, a reduction in antioxidant capacity, and a decrease in folliculogenesis. Therefore, improving the reproductive function in aging breeder hens using dietary strategies is of great concern to the poultry breeder. This study evaluated the capacity of dietary quercetin (Q), vitamin E (VE), and their combination (Q + VE) to promote follicle development and attenuate organ inflammation by improving the antioxidant capacity of aging breeder hens. In this study, 400 broiler breeder hens (Tianfu broilers breeder hens, 435 days old) were allotted into four groups (100 birds each) with four replicates each (25 birds each). They were fed diets containing Q (0.4 g/kg), VE (0.2 g/kg), Q + VE (0.4 g/kg + 0.2 g/kg), and a basal diet for 10 weeks. The results showed that Q + VE improved the organ characteristics (p < 0.05), and also that Q + VE showed protective effects on the liver against injury, as well as increasing the antioxidant capacity of the liver, serum, and ovary (p < 0.05). Furthermore, liver lipid synthesis was increased remarkably, as indicated by the changes in triglyceride levels in hens fed Q + VE (p < 0.05). Levels of E2, FSH, and LH, their receptors, and mRNAs related to yolk precursor synthesis were increased by the Q + VE (p < 0.05). Therefore, the combination of quercetin and vitamin E synergistically promotes and regulates the transportation and exchange of synthetic substances among the liver-blood-ovary alliances to ensure the synchronous development and functional coordination between the liver and ovary in aging breeder hens.

Sexual Maturity Promotes Yolk Precursor Synthesis and Follicle Development in Hens via Liver-Blood-Ovary Signal Axis.

Several reproductive hormones were reported to be involved in regulating egg yolk precursor synthesis in chickens; however, the mechanism that shows how the liver-blood-ovary signal axis works in relation to age changes has not been reported yet. Therefore, in this study, we observe the morphology and histology of the liver and ovary and determine the serum biochemical parameters and the expression abundance of the critical genes from d90 to 153. Results show that the body weight and liver weight were significantly increased from d132, while the ovary weight increased from d139. Aside from the increase in weight, other distinct changes such as the liver color and an increased deposition of large amounts of yolk precursors into the ovarian follicles were observed. On d139, we observed small fatty vacuoles in the hepatocytes. The results of serum biochemical parameters showed a significant increase in the estradiol (E2) level, first on d125, and then it reached its peak on d132. Meanwhile, the levels of follicle-stimulating hormone (FSH) increased initially and then remained at a high level from d146 to d153, while the levels of luteinizing hormone (LH) increased significantly on d132 and reached the top level on d153. Moreover, the levels of lecithin (LEC), vitellogenin (VTG), very low density lipoprotein y (VLDLy), triglyceride (TG), and total cholesterol (TC) were significantly increased at d125 and were close from d146 to d153. The mRNA and protein expression of estrogen receptor-alpha (ER-α) and E2 levels in the liver and serum, respectively, showed similar changes. Moreover, with reference to an increase in serum E2 level, the mRNA expression of genes related to yolk precursor synthesis (very low density apolipoprotein-II, ApoVLDL-II) and vitellogenin-II (VTG-II), lipogenesis (fatty acid synthase, FAS), and lipid transport (microsomal triglyceride transport protein, MTTP) in the liver showed up-regulation. These results suggest that the correlation between liver-blood-ovary alliances regulate the transport and exchange of synthetic substances to ensure synchronous development and functional coordination between the liver and ovary. We also found that E2 is an activator that is regulated by FSH, which induces histological and functional changes in the hepatocytes through the ER-α pathway.