Identification of Key Differentially Expressed Genes During Early Sex Determination in Chicken Embryos

In the chicken industry, sex determination significantly affects production efficiency and raises ethical concerns in poultry farming. As a key economic species, maximizing the advantages of each sex is vital in modern intensive breeding. Therefore, understanding the mechanisms of sex determination and regulation is critical to advancing the poultry industry. Transcriptome analysis of 3.5-day-old White Leghorn chicken embryonic genital ridges (n = 30, 15 males and 15 females) was performed using sex-pooled samples (five embryos/replicate, three replicates/sex). Sequencing generated 39.6 GB of high-quality reads for inter-sex comparative analysis, revealing 283 significantly differentially expressed genes (DEGs). The DEGs were primarily enriched in pathways such as ribosome biogenesis, glycan biosynthesis and metabolism, and TGF-β signaling, which are potential candidate pathways for the differentiation of chicken embryonic gonads. Key DEGs (including SMAD2Z, FREM1, NR2F1, SEMA6A, NFIB, RNF165, SMAD7B, SMAD2W, SPIN1W, and HINTW) were validated by RT-qPCR, confirming the transcriptome sequencing results. Among the DEGs, we predict binding sites for NR2F1 and NFIB within the DMRT1 gene promoter and suggest that these factors may serve as potential upstream activators for the expression of DMRT1, and they may initiate high DMRT1 expression in the subsequent stages of male embryos and regulate testicular development. In conclusion, this study investigated DEGs in the gonads of male and female chicken embryos after 3.5 days of incubation and found that NR2F1 and NFIB may serve as potential upstream activators for the expression of DMRT1, which is involved in the early determination of chicken sex.