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A transcriptome analysis for 24-hour continuous sampled uterus reveals circadian regulation of the key pathways involved in eggshell formation of chicken.

ABSTRACT: Circadian timing system controlled the rhythmic events, for example, ovulation and oviposition in chickens. However, how biological clock mediates eggshell formation remains obscure. Here, A 24-h mRNA transcriptome analysis was carried out in the uterus of 18 chickens with similar oviposition time points to identify the rhythmic genes and to reveal critical genes and biological pathways involved in the eggshell biomineralization. JTK_CYCLE analysis and real-time PCR revealed a total of 1,793 genes from the sequencing database with 23,513 genes (FPKM>1) were rhythmic genes regulating the rhythmic system and the expression of typical clock genes Per2, Cry1, Bmal1, Clock, Per3, and Rev-erbβ were rhythmically expressed, which suggested that endogenous clock in uterus might control the eggshell mineralization. Time of peak expression of the rhythmic genes was analyzed based on their acrophase. The main phases clustered at the periods from Zeitgeber time 0 (ZT0) to ZT4 (6:00-10:00) and from ZT10 to ZT14 (16:00-20:00). The rhythmic genes were annotated to the following Gene Ontology terms rhythmic process, lyase, ATP binding, cell membrane component. KEGG pathway enrichment analysis revealed the top 15 rhythmic genes were involved in vital biological pathways, including syndecan (1, 2, 3)-mediated signaling, post-translational regulation of adheres junction stability and disassembly, FoxO family signaling, TGF-β receptor and transport of small molecular pathways. 166 of total 1,235 genes (13.4%) were defined as rhythmic transfer factors (TFs) and they were investigated expression time distribution of cis-elements of circadian clock system D-box, E-box, B-site, and Y-Box within 24 h. Results indicated that rhythmic TFs at each phase are potential drivers of their circadian transcription activities. Compared with the control, the expression abundances of ion transport elements SCNN1G, CA2, SPP1, and ATP1B1 were significantly decreased after the interference of Bmal1 gene in synchronized uterine tubular gland cells. Clock genes changed their expression along with the eggshell formation, indicating that there is circadian clock in the uterus of chicken and it regulates the expression of eggshell formation genes.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC8628016 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Calcium (Ca) and phosphorus (P) transporters are responsible for their absorption and transport in small intestine and kidney, contributing to eggshell formation. The light-dark cycle is a primary cue in the reproduction of laying hen. In this study, we investigated the effect of different light-dark programs on eggshell quality and the expression of genes related to Ca and P transportation in laying hens. Seventy-two 56-week-old laying hens were randomly divided into two groups and reared at 16-h light and 8-h dark (control) or 9-h light and 15-h dark regime (long dark phase, LDP). The expressions of calcium transporter calbindin-D28k (CaBP-D28k), plasma membrane Ca ATPase 1b (PMCA1b), and phosphorus transporter NaPi-IIb (NPt2b) and NaPi-IIa (NPt2a) were measured in the small intestine, kidney, and eggshell gland. The results showed that feed intake (P < 0.001) and egg weight (P = 0.05) were decreased by LDP treatment. Compared with control, the eggshell hardness was increased (P = 0.011) by LDP treatment, but the eggshell thickness and the percentage of eggshell were not changed. The Ca and P contents in eggshell were increased by LDP treatment. During the scotophase, LDP-hens showed higher serum Ca (P = 0.0056) and P levels (P = 0.079) but lower alkaline phosphatase (ALP) activity than that of control hens. In the duodenum, the relative higher expression of CaBP-D28k and PMCA1b in scotophase compared to photophase was masked by LDP treatment. The expression of CaBP-D28k and osteopontin (OPN) in the eggshell gland were increased by LDP treatment, compared to control hens. In the jejunum, the protein expression levels of CaBP-D28k and PMCA1b decreased during photophase in LDP-hens. The result indicates that the increased blood Ca and P concentration during scotophase by LPD treatment is beneficial to the deposition of Ca and P in the eggshell. The result offers an alternative strategy for managing laying hens with poor eggshell quality.

Project description:BACKGROUND: As uricoletic animals, chickens produce cleidoic eggs, which are self-contained bacteria-resistant biological packages for extra-uterine development of the chick embryo. The eggshell constitutes a natural physical barrier against bacterial penetration if it forms correctly and remains intact. The eggshell's remarkable mechanical properties are due to interactions among mineral components and the organic matrix proteins. The purpose of our study was to identify novel eggshell proteins by examining the transcriptome of the uterus during calcification of the eggshell. An extensive bioinformatic analysis on genes over-expressed in the uterus allowed us to identify novel eggshell proteins that contribute to the egg's natural defenses. RESULTS: Our 14 K Del-Mar Chicken Integrated Systems microarray was used for transcriptional profiling in the hen's uterus during eggshell deposition. A total of 605 transcripts were over-expressed in the uterus compared with the magnum or white isthmus across a wide range of abundance (1.1- to 79.4-fold difference). The 605 highly-expressed uterine transcripts correspond to 469 unique genes, which encode 437 different proteins. Gene Ontology (GO) analysis was used for interpretation of protein function. The most over-represented GO terms are related to genes encoding ion transport proteins, which provide eggshell mineral precursors. Signal peptide sequence was found for 54 putative proteins secreted by the uterus during eggshell formation. Many functional proteins are involved in calcium binding or biomineralization--prerequisites for interacting with the mineral phase during eggshell fabrication. While another large group of proteins could be involved in proper folding of the eggshell matrix. Many secreted uterine proteins possess antibacterial properties, which would protect the egg against microbial invasion. A final group includes proteases and protease inhibitors that regulate protein activity in the acellular uterine fluid where eggshell formation takes place. CONCLUSIONS: Our original study provides the first detailed description of the chicken uterus transcriptome during formation of the eggshell. We have discovered a cache of about 600 functional genes and identified a large number of encoded proteins secreted into uterine fluid for fabrication of the eggshell and chemical protection of the egg. Some of these uterine genes could prove useful as biological markers for genetic improvement of phenotypic traits (i.e., egg and eggshell quality).

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A transcriptome analysis for 24-hour continuous sampled uterus reveals circadian regulation of the key pathways involved in eggshell formation of chicken.

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