Liver transcriptome of sexually mature hens in relation to the formation of the egg yolk
Ontology highlight
ABSTRACT: We used a transcriptomic approach based on the comparison of the expression between the liver of sexually mature hens versus pre-laying pullets to better appreciate which hepatic proteases and antiproteases are specifically expressed in relation to vitellogenesis. Using a 20K chicken oligoarray corresponding to 12 595 different chicken genes, a total of 582 genes were shown to be over-expressed in the liver at sexual maturity of hens (1.2 to 67 fold- difference). Most of the top ten over-expressed genes are known components of egg yolk or of the perivitelline membrane. The combination of different bioinformatic tools reveals 12 proteases and 3 antiproteases amongst the over-expressed genes, including many predicted proteins with yet unknown functions.
Project description:We used a transcriptomic approach based on the comparison of the expression between the liver of sexually mature hens versus pre-laying pullets to better appreciate which hepatic proteases and antiproteases are specifically expressed in relation to vitellogenesis. Using a 20K chicken oligoarray corresponding to 12 595 different chicken genes, a total of 582 genes were shown to be over-expressed in the liver at sexual maturity of hens (1.2 to 67 fold- difference). Most of the top ten over-expressed genes are known components of egg yolk or of the perivitelline membrane. The combination of different bioinformatic tools reveals 12 proteases and 3 antiproteases amongst the over-expressed genes, including many predicted proteins with yet unknown functions. 8 samples of each condition( liver of 38 weeks old mature hens and of 10 weeks old immature pullets) were analysed, with an experimental design in dye switch (half of the slides labeled with fluorophore Alexa 555 and half with Alexa 647 for each condition)
Project description:Purpose: With the advent of Next-generation sequencing (NGS), several novel genes/proteins and cellular pathways in wide varitey of tissues has discovered. The aim of this study are to perform transcriptome profiling (RNA-seq) of magnum to determine differently expressed genes in laying and non-laying hens and to further validate the expression of candidate genes using real-time quantitative reverse transcription polymerase chain reaction (qRT–PCR) in laying, non-laying and molting hens. Methods: Magnum mRNA profiles of 35-60 weeks-old laying and non-laying hens, three each, were generated with NextSeq 500 sequencer in single-end mode with a read length of 1x76 bp. Raw sequencing reads were cleaned and trimmmed with Prinseq tool and good reads were aligned against the chicken reference gemone (Galgal 5.0) in Array Studio. Differential gene expression analysis was performed by the DESeq2 algorithm as implemented in Array Studio. The genes with at least three-fold change (FC) and Benjamini and Hochberg q-value < 0.05 were called differentially expressed. Results: Using an optimized data analysis workflow, we mapped about 30.5 million reads from layers and 33.4 million reads from non-layers to the chicken genome. A total of 19,152 gene transcripts were annotated from Ensembl alignment which represents 50.24% of the chicken genome assembly. Differential gene expression analysis showed 540 were differentially expressed between layer and non-layer hens. 152 DEGs were significantly up-regulated and 388 were significantly down-regulated in the laying hens when compared to the non-laying hens. Conclusions: Our study reports the expression of several pre-discovered and many novel genes that may be involved in the transport of precurosor molecules for biosynthesis and secretion of the egg-white proteins in the magnum. These genes can be used as quantitative basis of selecting hens with an improved egg quality.
Project description:In this study, RNA-Seq technology was adopted to investigate the differences in expression profiles of the hepatic lipid metabolism-related genes and the associated pathways between juvenile and laying hens. RNA-Seq analysis was carried out to estimate total RNA harvested from the liver of juvenile hens (n = 3) and laying hens (n = 3). Compared with juvenile hens, 2574 differentially expressed (DE) genes (1487 down and 1087 up) with P ⤠0.05 were obtained, and 955 of these genes were significantly DE (SDE) at a false discovery rate (FDR) of 0.05 and fold-change ⥠2 in laying hens. There were 198 SDE novel genes (107 down-regulated and 91 up-regulated) (FDR ⤠0.05) that were obtained from the transcriptome, and most of them were highly expressed. Moreover, 332 SDE isoforms were identified. Gene Ontology (GO) enrichment and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed that SDE genes were significantly associated with steroid biosynthesis, PPAR signaling pathway, biosynthesis of unsaturated fatty acids, glycerophospholipid metabolism, three amino acid pathways, and pyruvate metabolism (P ⤠0.05). The top significantly enriched GO terms included lipid biosynthesis, cholesterol and sterol metabolic, and oxidation reduction suggesting the principal lipogenesis in the liver of laying hens. This study suggests that the major changes at the level of transcriptome in laying hen liver are closely related to fat metabolism. Some highly differentially expressed uncharacterized novel genes and alternative splicing isoforms detected might also take part in lipid metabolism, though it needs investigation. Therefore, this study provides valuable information of mRNA of chicken liver, and deeper functional investigations on the mRNAs could help explore or provide new insights into molecular networks of lipid metabolism in chicken liver. The liver expression profile of juvenile hens and laying hens were generated by RNA-seq.
Project description:Purpose: With the advent of Next-generation sequencing (NGS), several novel genes/proteins and cellular pathways in wide variety of tissues has been discovered. The aim of this study are to perform uterine transcriptome profiling (RNA-seq) to determine differently expressed genes in laying and non-laying hens and to further validate the expression of candidate genes using real-time quantitative reverse transcription polymerase chain reaction (qRT–PCR) in laying, non-laying and molting hens. Methods: Uterine mRNA profiles of 35-60 weeks-old laying and non-laying hens, three each, were generated with NextSeq 500 sequencer in single-end mode with a read length of 1x76 bp. Raw sequencing reads were cleaned and trimmmed with Prinseq tool and good reads were aligned against the chicken reference gemone (Galgal 5.0) in Array Studio. Differential gene expression analysis was performed by the DESeq2 algorithm as implemented in Array Studio. The genes with at least two-fold change (FC) and Benjamini and Hochberg q-value < 0.05 were called differentially expressed. Results: Using an optimized data analysis workflow, we mapped about 32 million reads from layers and 28 million reads from non-layers to the chicken genome. A total of 19,152 gene transcripts were annotated from Ensembl alignment which represents 50.24% of the chicken genome assembly. Differential gene expression analysis showed 616 were differentially expressed between layer and non-layer hens. 229 DEGs were significantly up-regulated and 286 were significantly down-regulated in the laying hens when compared to the non-laying hens. Twelve candiate genes, linked to calcium remodeling, were identified by gene function analysis and validated using qPCR. MEPE, CALCB, OTOP2, STC2 and ATP2C2 were confirmed to be highly expressed in laying hens as compared to molting and non-laying hens. RNA-seq and qPCR data for relative gene expression were highly correlated (R2 =0.99). Conclusions: Our study reports the expression of four novel genes that are speculated to transport calcium ions across the uterine epithellium for eggshell mineralization. These genes can be used as quantitative basis of selecting hens with an improved eggshell quality.
Project description:In this study, RNA-Seq technology was adopted to investigate the differences in expression profiles of the hepatic lipid metabolism-related genes and the associated pathways between juvenile and laying hens. RNA-Seq analysis was carried out to estimate total RNA harvested from the liver of juvenile hens (n = 3) and laying hens (n = 3). Compared with juvenile hens, 2574 differentially expressed (DE) genes (1487 down and 1087 up) with P ≤ 0.05 were obtained, and 955 of these genes were significantly DE (SDE) at a false discovery rate (FDR) of 0.05 and fold-change ≥ 2 in laying hens. There were 198 SDE novel genes (107 down-regulated and 91 up-regulated) (FDR ≤ 0.05) that were obtained from the transcriptome, and most of them were highly expressed. Moreover, 332 SDE isoforms were identified. Gene Ontology (GO) enrichment and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed that SDE genes were significantly associated with steroid biosynthesis, PPAR signaling pathway, biosynthesis of unsaturated fatty acids, glycerophospholipid metabolism, three amino acid pathways, and pyruvate metabolism (P ≤ 0.05). The top significantly enriched GO terms included lipid biosynthesis, cholesterol and sterol metabolic, and oxidation reduction suggesting the principal lipogenesis in the liver of laying hens. This study suggests that the major changes at the level of transcriptome in laying hen liver are closely related to fat metabolism. Some highly differentially expressed uncharacterized novel genes and alternative splicing isoforms detected might also take part in lipid metabolism, though it needs investigation. Therefore, this study provides valuable information of mRNA of chicken liver, and deeper functional investigations on the mRNAs could help explore or provide new insights into molecular networks of lipid metabolism in chicken liver.
Project description:Heat stress (HS) can damage the integrity of the intestinal mucosal barrier, leading to decreased poultry productivity. This study aimed to identify candidate genes related to acute HS in breeder hens and provide insight into the molecular mechanisms underlying acute HS in gut health. Fifty 28-week-old breeder hens were divided into two groups (25 hens each) raised under thermoneutral zone (23 °C) and acute HS (36 °C, 6 hours) conditions. The heart rate and cloacal temperature were measured in all hens, and jejunal mucosa tissues were randomly collected from 12 hens per treatment for RNA-sequencing analysis. The results indicated a significant increase in the heart rate and cloacal temperature in hens exposed to acute HS (P < 0.05). Transcriptome analysis identified 138 differentially expressed genes (DEGs) in heat-stressed breeder hens, including 75 upregulated DEGs containing heat shock protein (HSP), energy homeostasis metabolism-related gene (PDK4), and fat metabolism-related genes (PPARA and CD36), and 63 downregulated DEGs containing the bile acid transporter gene (SLC10A2). Gene ontology analysis revealed significant enrichment in biological processes related to heat response and cholesterol biosynthesis. Kyoto Encyclopedia of Genes and Genomes analysis highlighted several significant pathways, including steroid biosynthesis, steroid hormone biosynthesis, protein processing in endoplasmic reticulum, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, and the adipocytokine signaling pathway. Protein-protein interaction network analysis involving two large networks: one containing several upregulated HSPs and genes related to energy homeostasis and fat metabolism (PDK4, PPARA, and CD36) and glucose transporter (SLC2A5), and the other containing downregulated DEGs related to cholesterol biosynthesis. Overall, acute HS might affect energy metabolism, fat metabolism, and glucose transport in the jejunal mucosa of breeder hens. Heat-stressed hens could restore the nutritional function of the jejunal mucosa by increasing the expression of HSPs. These findings provide a theoretical framework for further investigation into the molecular regulatory mechanisms responsible for HS-induced changes in the gut health of poultry.
Project description:Ovarian dysfunction is observed in broiler breeder hens fed ad libitum that is most apparent by a reduction in fertility success. Restricting caloric intake in these hens is currently the only effective treatment to improve reproductive efficiency. This analysis sought to determine the transcriptional changes in the ovarian cortex of prepubertal hens given full access to feed (FF) or were placed on a restricted feed (RF) diet. Microarrays were employed to detail the differences in global gene expression that underlie the ovarian dysfunction observed in ad libitum fed hens.
Project description:Ovarian follicle selection plays an important role in the reproduction of sexually mature hens, and this process can directly affect the growth and development of follicles until the final ovulation, thus affecting laying performance and fecundity of hens. In the laying hen ovary, one follicle from a cohort of 8-13 follicles of 6-8 mm in diameter is selected daily to enter the preovulatory hierarchy. In this study, we globally compared the proteomes of chicken ovarian follicles before and after follicle selection. A total of 5883 proteins were identified in the proteomes of chicken 6-8 mm prehierarchical follicles and 12-15 mm hierarchical follicles. 259 proteins are differentially expressed in 12-15 mm hierarchical follicle compared with prehierarchical follicle, of which 175 proteins are up-regulated and 84 proteins are down-regulated. The Gene Ontology enrichment of differentially expressed proteins revealed enriched GO terms for peptidase activity, acrosin binding for their molecular function and in the process of negative regulation of peptidase activity, and regulation of fertilization. The KEGG pathway analysis indicated that differentially expressed proteins were enriched for ribosome, lysine degradation, and endocytosis pathways. Nine differentially expressed proteins including vitellogenin-1 were validated with Parallel Reaction Monitoring (PRM) analysis, and their functions were discussed. This study provided a global proteomic view of the development of chicken ovarian follicles, which will serve as a foundation for understanding the molecular signatures and pathways of follicle selection in hens.
Project description:Intense selective breeding of broiler breeds of chickens has resulted in suboptimal egg production in broiler breeder hens. Ad libitum feeding which leads to excessive and disorganized follicular growth exacerbates this reproductive phenotype. One strategy used to improve broiler breeder hen reproductive efficiency is restricted feeding. In this study, we sought to identify transcriptional changes which translate level of dietary intake to increased follicle selection. Broiler breeder hens were raised according to commercial guidelines until 28 weeks of age and then randomly assigned to an ad libitum diet (FF) or continued on a restricted diet (RF) for 6 weeks. Following dietary treatment, granulosa cells from growing 6-8 mm follicles from FF hens (n=3) and RF hens (n=3) were collected, RNA was extracted, and samples were processed for RNA-sequencing on Illumina NextSeq 500. Transcriptomes of granulosa cells from 6-8 mm follicles were sequenced to identify transcriptional differences in the population from which follicles are selected into the preovulatory stage. FastQ files were first processed through trim-galore and reads were aligned to the Galgal6 genome using the RNA-seq aligner, STAR. A cluster analysis using hclust in R identified a FF sample as an outlier and this sample was removed from the analysis. Differential expression analysis was conducted using DeSEQ2 and resulted in 350 differentially expressed genes. Several genes involved in follicle selection were upregulated in prehierarchal follicles of FF hens, suggesting an effect of dietary treatment at early stages in follicle development.