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RNA sequencing-based analysis of the magnum tissues revealed the novel genes and biological pathways involved in the egg-white formation in the laying hen.

ABSTRACT:

Background

The mechanism of egg formation in the oviduct of laying hens is tightly controlled; each segment of the oviduct contributes a unique component of the egg. Several genes/proteins are involved in the synthesis of a completely healthy egg. This implies a time- and tissue-specific expression of genes and proteins in the different oviductal segments. We used hens at different physiological stages and time points to understand the transcriptional regulation of egg-white (albumen) synthesis and secretion onto the eggs in the magnum of laying hens. This study used Next-Generation Sequencing and quantitative real-time PCR (qPCR) to detect the novel genes and the cognate biological pathways that regulate the major events during the albumen formation.

Results

Magnum tissues collected from laying (n = 5 each at 3 h post-ovulation, p.o. and 15-20 h p.o.), non-laying (n = 4), and molting (n = 5) hens were used for differential gene expression analyses. A total of 540 genes (152 upregulated and 388 down-regulated) were differentially expressed at 3 h p.o. in the magnum of laying hens. Kyoto Encyclopedia of Genes and Genomes pathways analysis of the 152 upregulated genes revealed that glycine, serine, and threonine metabolism was the most-enriched biological pathway. Furthermore, the top two most enriched keywords for the upregulated genes were amino-acid biosynthesis and proteases. Nine candidate genes associated with albumen formation were validated with qPCR to have differential expression in laying, non-laying, and molting hens. Proteases such as TMPRSS9, CAPN2, MMP1, and MMP9 (protein maturation, ECM degradation, and angiogenesis); enzymes such as PSPH, PHGDH, and PSAT1 (amino-acid biosynthesis); RLN3, ACE, and REN (albumen synthesis, secretion and egg transport); and AVD, AvBD11, and GPX3 (antimicrobial and antioxidants) were recognized as essential molecules linked to albumen deposition in the magnum.

Conclusions

This study revealed some novel genes that participate in the signaling pathways for egg-white synthesis and secretion along with some well-known functional genes. These findings help to understand the mechanisms involved in albumen biosynthesis.

SUBMITTER: Sah N

PROVIDER: S-EPMC8088581 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:1. Homogenates of the magnum of the hen oviduct have been fractionated by differential centrifuging. 2. Centrifuging at 600g for 10min. gave a pellet containing most of the particulate material of the cell, but on washing this fraction some particles were removed from it. The washed 600g pellet contained most of the DNA of the homogenate. 3. Centrifuging the 600g supernatants at 10000g for 10min. gave particulate fractions (I particles) richer in RNA than other fractions which were active in incorporating amino acids into protein in isolation. When minced tissue was incubated with radioactive amino acids before homogenizing these particles were the most radioactive of the cell fractions. 4. The pellet obtained by centrifuging the 10000g supernatant at 105000g for 60min. was very small; its RNA/protein ratio was raised compared with that of the homogenate. It only incorporated amino acids in isolation to a small extent or not at all. 5. The 105000g supernatant contained a large proportion of the protein of the homogenate. 6. The I particles could be subfractionated by layering over denser sucrose to give a pellet with lower RNA content and incorporating activity, and also suspended material richer in both these properties. 7. Treatment of the I particles with deoxycholate gave rise to particles sedimenting at 105000g for 60min. containing most of the RNA of the original particles, but only about 34% of the protein, and with a high activity in incorporating amino acids. 8. The I particles, or particles derived from them by deoxycholate treatment, required GTP and phosphoenolpyruvate for the incorporation of free amino acids. The omission of ATP reduced the incorporation to varying extents. Chicken-liver cell sap stimulated the activity. 9. Radioactive amino acids linked to transfer RNA were transferred to protein by I particles. GTP and phosphoenolpyruvate were required for this transfer. The phosphoenolpyruvate requirement could not be replaced by increasing the GTP concentration. ATP partially inhibited the transfer. 10. After incorporation by the cell-free system, the hot-trichloroacetic acid extract, but not the lipid extract, was radioactive. Ribonuclease and puromycin inhibited at low concentrations. Lecithinase-C was much less inhibitory. Transfer of amino acid, from a radioactive lipid-amino acid complex prepared from hen oviduct, was not detected. 11. After short periods of incubation of minced tissue with [(14)C]lysine some of the radioactive protein of the isolated I particles behaved as ovalbumin. The distribution of radioactivity in the protein resembled that in ovalbumin in soluble extracts of the tissue obtained after longer periods of incubation.