Abstract
Eggshell is the outermost calcified covering of an egg that protects it from microbial invasion and physical damage, and is critical for egg quality. However, understanding of the genes/proteins and the biological pathways regulating the eggshell formation is still obscure. We hypothesized that the transcriptomic analysis of the chicken uteri using RNA-sequencing may reveal novel genes and biological pathways involved in the eggshell biomineralization. RNA-sequence analysis using uteri of laying hens at 15–20 h post-ovulation (layers, n = 3) and non-laying (non-layers, n = 3) hens was carried out. About 229 differentially expressed genes (DEGs) were up-regulated in the layers compared to the non-layers. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Ingenuity Pathway Analysis (IPA) revealed more than ten novel genes and biological pathways related to calcium transport and mineralization in the uterus. Based on the enriched pathways and molecular function analysis, 12 DEGs related to eggshell mineralization were further analyzed in the uteri of layers (3 h and 15–20 h post-ovulation), non-layers and molters using qPCR. Expressions of OC-116 (regulator of mineralization), OTOP2 (modulator of cellular calcium influx), CALCB (intracellular release of Ca-ions), STC2 (increases alkaline phosphatase activity), and ATP2C2 (cellular import of Ca-ions) were significantly higher in the uteri of laying hen at 15–20 h post-ovulation. This study identified the involvement of novel genes and their proposed biological pathways in the regulation of eggshell formation.
Highlights
The oviduct of a laying hen provides a conducive biological environment for the formation and potential fertilization of the egg
We hypothesized that RNA sequencing (RNA-seq) of a laying versus non-laying hen’s oviduct can reveal novel genes and biological pathways having a significant role in eggshell formation
Based on the enriched pathways and molecular functions, and their expression levels, twelve genes were identified as potential regulator of calcium-homeostasis during eggshell mineralization
Summary
The oviduct of a laying hen provides a conducive biological environment for the formation and potential fertilization of the egg. With the use of cutting-edge techniques and more precise time of sampling, each study reported some unpredicted genes and proposed their potential function in the eggshell mineralization. The eggshell mineralization in chicken is the most efficient biological process of calcium mobilization and biomineralization. This premise leads to the belief that several calcium-transporting genes and proteins must be involved, through independent or integrated biological pathways, for eggshell biomineralization. The genes responsible for eggshell calcification are suppressed in hens before laying period and are activated once the hens reach sexual maturity for egg production[8]. We hypothesized that RNA sequencing (RNA-seq) of a laying versus non-laying hen’s oviduct can reveal novel genes and biological pathways having a significant role in eggshell formation. The objectives of this study were to analyze the transcriptome of the uteri in layers and non-layers for identifying the differentially expressed genes (DEGs) for eggshell formation, and to further confirm their expression in laying hens at the early egg formation period comparing them with molter and non-laying hens
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