Single-cell transcriptome profiling of primordial germ cells reveals differences between right and left gonads in chickens.

In female birds, the left ovary develops while the right gonad regresses. Whether and how PGCs differ between the two sides during early gonadal differentiation remains unclear. We performed single-cell RNA-seq on right and left gonads of female chicken embryos at HH29 and HH34. After quality control, cells were clustered, PGCs transcriptional programs compared, GO/KEGG enrichment and cell communication analyzed, and genes validated by qPCR. We found that at HH29, right-left PGCs differences were enriched for DNA damage response, p53-mediated cell-cycle arrest, ribosome, and translation-related terms. KEGG pathways included ribosome, ubiquitin-proteolysis, and oxidative phosphorylation. At HH34, divergence shifted to cellular senescence, cAMP signaling, and necroptosis. Genes like JUN, ID2, GADD45A, and GTSE1 were differentially expressed. Across developmental stages, right-side PGCs additionally showed enrichment in steroid hormone biosynthesis, estrogen signaling and ovarian steroidogenesis. Cell-cell communication analysis indicated that somatic-PGCs interactions within the NOTCH pathway were present only in the right gonad at both stages. Single‑cell profiling reveals a stage‑dependent shift of right-left PGCs differences from DNA damage/cell‑cycle programs at HH29 to senescence‑associated programs at HH34, together with right-specific NOTCH interactions. These data offer insights into microenvironmental regulation of PGCs fate during avian ovarian asymmetry.