Nanopore long-read RNA sequencing reveals key genes regulating pre-hierarchical follicle development in the chicken ovary.

During the egg laying period of a hen, one pre-hierarchical follicle, approximately 6-8 mm in size, is selected into the preovulatory hierarchy for ovulatory stage development. These follicles develop from white follicles that are 2-5 mm in size. Although numerous studies have investigated follicle selection, the molecular mechanisms underlying pre-hierarchical follicle development and recruitment remain unclear. In this study, we explored the genes regulating chicken ovarian pre-hierarchical follicular development by sequencing RNA from ≤ 2 mm follicles in ovarian stroma (OS), 3-4 mm small white follicles (SWFs), and 6-8 mm small yellow follicles (SYFs) using Oxford Nanopore long-read transcriptome sequencing technology. Herein, we obtained 2,164 differentially expressed transcripts (DETs) from the three phases. The steroidogenesis and angiogenesis signaling pathways were enriched in SWFs compared with in OS. In contrast, the endocytosis, p53, FoxO, and oocyte meiotic signaling pathways were significantly enriched during SYF development compared with during SWF development. The focal adhesion, apelin, and TGF-β signaling pathways were significantly enriched in all three phases. The time-course analysis of DETs and cluster Kyoto Encyclopedia of Genes and Genomes enrichment revealed dynamic changes in gene expression during pre-hierarchical follicle development. Key examples include the genes associated with follicle initial recruitment and development (AMH, GDF9, INHBB, BMPR1B, StAR, and FST), angiogenesis-related genes (ANGPT2, VEGFA, VEGFC, VEGFD, and VEGFRs), extracellular matrix remodeling-related genes (MMP2 and MMP10), and Wnt signaling pathway genes (WNT4, WNT9A, CTNNB1, RSPO3, and WIF1). Moreover, RSPO3 upregulated the expression of FSHR and StAR in cultured chicken Pre-hierarchical follicle granulosa cells and activated the Wnt signaling pathway. The results obtained in this study demonstrate the transcriptional dynamics during small folliculogenesis; the identified signaling pathways and DETs can be used as candidate targets for investigating pre-hierarchical follicle development in chickens.