Screening and identification of genes for cell autonomous sex identity in chicken somatic cells

Chicken somatic cells exhibit cell autonomous sex identity (CASI), where sexual features are determined by sex chromosomes rather than hormones. CASI arises from sex‑biased gene expression due to lack of dosage compensation on sex chromosomes. To identify CASI‑determining genes (CASIDGs), we analyzed transcriptomes of wattles (phenotypically hormone‑sensitive) and pectoral muscles (phenotypically hormone‑unresponsive) from 6‑week‑old roosters and hens, with or without sex hormone treatment. Inter‑sex differentially expressed genes (DEGs) mainly located on the Z chromosome and autosomes 1‑2, while hormone‑affected DEGs predominantly on autosomes 1‑3. After removing hormone‑affected DEGs from the inter‑sex DEGs, 285 DEGs (51.23% on sex chromosome) remained for pectoral muscle, and 255 DEGs (63.93% on sex chromosome) for wattle. Out of the 89 DEGs shared by these two tissues, 88 were located on sex chromosomes and were considered candidate CASIDGs. These candidate CASIDGs were mainly enriched in mitochondrial protein synthesis and isomerase activity-related pathways. Among the CASIDGs, HINT1 and MED18 were validated according to the screening criteria. Overexpressing these two genes in primary muscle cells could affect immune/inflammatory responses, cell proliferation and migration, cytoskeleton, and extracellular matrix interactions. Additionally, 139 autosomal DEGs for pectoral muscle and 92 for wattle were identified as candidate sex marker genes. Among these, IRF9 and CCL4 were co‑regulated by HINT1 and MED18. In conclusion, the sex characteristics of chicken somatic cells reflect the combined effects of CASIDGs and sex hormones. HINT1 and MED18 are confirmed as candidate CASIDGs. These findings lay a solid foundation for elucidating the CASI mechanism in avian somatic cells.