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.

Circadian miR-218-5p targets gene CA2 to regulate uterine carbonic anhydrase activity during egg shell calcification

“…Five hundred 30-wk-old laying hens (BH-01, a dual-purpose line with black-shank and yellow-dotted feather cultivated by the Poultry Breeding Group of Sichuan Agricultural University) were caged individually and had access to feed and water ad libitum. The feeding procedures were consistent with those described in our previous study ( Zhang et al, 2022 ).…”

“…We found functional clocks in the chicken infundibulum and uterus ( Zhang et al, 2016 ; Zhang et al, 2017 ). Moreover, clock and ion transport related genes showed cosine expression patterns in the oviduct ( Zhang et al, 2022 ). However, there are few studies on the role of clock-controlled miRNAs in eggshell formation in chicken eggshell gland ( ESG ).…”

“…Our previous study found that circadian clocks are also involved in the function of the poultry reproductive system, especially in the chicken uterus ( Zhang et al, 2016 ; Cui et al, 2021 ; Zhang et al, 2022 ). Some rhythmic miRNAs have been identified by 24 h high-throughput sequencing of the chicken uterus and we found that circadian miR-449c-5p regulates uterine Ca 2+ transport during eggshell calcification in chickens ( Cui et al, 2021 ).…”

High albumen height by expression of GALNT9 and thin eggshell by decreased Ca2+ transportation caused high hatchability in Huainan partridge chicken

“…Studies have shown that there is rhythmic expression of clock genes in the uterus part of chicken fallopian tube. The rhythmic expression of downstream eggshell formation related genes, such as eggshell formation-related ions, ion channel proteins, ion-binding proteins, and eggshell-specific matrix proteins, was regulated to ensure the sequential synthesis of eggshells and the continuity of egg production of laying hens ( Zhang et al, 2022 ). The CaCO 3 precursors are composed of Ca 2+ and HCO 3 − , neither of which are stored in the uterus ( Stapane et al, 2020 ).…”

Characterization of clutch traits and egg production in six chicken breeds

“…Under the 24 h based light-dark period, the pronounced cyclic process of chickens’ egg formation requires an average a bit over 24 h [ 3 – 5 ]. In a sequence of consecutive days, the first oviposition normally occurs in the morning, and the following oviposition is postponed day by day until a laying cessation for one or more days when the oviposition is postponed to the afternoon.…”