TRANSCRIPTOMIC REGULATION OF EGG FORMATION IN THE OVIDUCT OF HENS

Abstract

Introduction: The oviduct of a hen provides a conducive environment for egg formation. Each part of the oviduct has a unique function: infundibulum (receives egg), magnum (deposits albumen), isthmus (synthesizes eggshell membranes), uterus (forms mineralized eggshell), and vagina (helps in oviposition). Any functional anomaly of the oviduct will lead to small-sized, misshapened, soft-shelled or cracked eggs, and taken together; these defects account for lost revenue in approximately 10% of the total number of eggs produced. At present, the cellular processes and biological pathways involved in the albumen synthesis, and eggshell formation are not clearly understood. Objective: The purposes of this study were to: (1) identify the novel differentially expressed genes (DEGs) and important biological pathways in the oviduct (laying vs. non-laying), and (2) validate the identified novel genes in the laying (3 h and 15-20 h post-ovulation; p.o.), non-laying (no growing ovarian follicles), and molting (reproductive rest) hens. Methods: Hy-line white hens, including laying (n=12) of 35 weeks, non-laying (n=6) of 35-60 weeks, and molting (n=6) hens of 60 weeks were used for this study. Magnum and uterine tissues were collected from laying hens at 3 h p.o. (egg present in the magnum), and 15-20 h p.o. (egg present in the uterus), molting, and non-laying hens for total RNA isolation. Total RNA (n=3/group) from the magnum of laying (3 h p.o.) versus non-laying, and total RNA (n=3/group) from uteri of laying (15-20 h p.o.) versus non-laying hens were subjected to next-generation sequencing (NGS). Raw reads of the sequences were filtered and trimmed (to remove low-quality reads), then aligned with the chicken genome (Galgal 5.0) and analyzed for differential gene expression. The mRNA expression of selected candidate genes in the magnum and uterus of laying, non-laying, and molting hens were also validated using real-time quantitative PCR (qPCR). Results: A total of 152 genes in the magnum, and 229 genes in the uterus of laying hens were up-regulated (FDR_BH <0.05) with the presence of egg in the oviduct. In the magnum, the glycine, serine and threonine metabolism was the most-enriched pathway; whereas, in the uterus, calcium signaling was amongst the most prevalent pathways. Results of qPCR confirmed the higher (P <0.05) mRNA expression of AVD and AvBD11 (antimicrobials); CAPN2, TMPRSS9, MMP1, and MMP9 (ECM remodeling and angiogenesis); REN and RLN3 (albumen secretion and oviposition); and CGN (vascular permeability) in the magnum of layers compared to both molters and non-layers. In the uterus, mRNA expression of MEPE (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 (P <0.05) in laying hens, when compared to either molting or non-laying hens. Conclusions: This study identified several novel genes and biological pathways involved in the albumen synthesis and eggshell formation. These molecules can potentially be used as markers to improve the egg-qualities through nutritional modulation, and genetic improvement.