Transcriptomic analysis of ovarian follicles uncovers the crucial genes relevant to follicle selection and preovulatory hierarchy in hens.

Follicle selection and preovulatory hierarchy of hen ovaries were important stages of follicle development and crucially determining egg-laying performance. The selected follicles with a higher expression level of follicle-stimulating hormone receptor (FSHR) mRNA that facilitates response to FSH, and rapidly develops into preovulatory follicles with distinctive characteristics of granulosa cells (GCs) proliferation and differentiation. Identification of the key genes involved in these developmental events is helpful for elucidation of the molecular mechanism underlying egg-laying traits in chicken and other domestic fowl. Herein, the comparative transcriptomic analysis of ovarian prehierarchical follicles before selection (BSF), follicles at selection stage (ASF), and hierarchical follicles (HF) were implemented in the Jilin Black chicken (JB) and Lohmann Brown layer (LB) with the divergences in their egg-laying performance by RNA-sequencing. The results showed that nine deferentially expressed genes (DEGs), including STMN4, FABP3, ROBO2, RSPO4, and DMRT1 were revealed between follicles BSF and ASF; and seventeen DEGs, such as SLC6A15, SLITRK3, PRKG2 and TMC3 were mined between ASF and HF. These two group DEGs being co-expressed between BSF and ASF, and between ASF and HF were compared and substantiated in the JB and LB layers, respectively. Furthermore, 10 signaling pathways, such as cAMP signaling, PPAR signaling pathway, AMPK(Adenosine 5'-monophosphate (AMP)-activated protein kinase) pathway, and estrogen signaling pathway were also identified. Moreover, the roles of two representative candidates ROBO2 and PRKG2 genes presented as downregulated mRNA expression pattern in the transcriptomic profiles were further verified in vitro. The results demonstrated that downregulation of ROBO2 or PRKG2 significantly increased the expression levels of FSHR mRNA and protein with the boosted expression of CCND1, STAR, and BCL-2, whereas remarkably inhibited the expression of Caspase-3, consequently, brought about the decrease of GC apoptosis in the ovarian follicles, but increase of GC proliferation and differentiation serving as the hallmarks for follicle selection. It indicated that ROBO2 and PRKG2 may play indispensable roles in follicle selection and preovulatory hierarchy of hen ovaries separately. Our findings provided a comparative transcriptomic evidence for clarifying the molecular mechanism of the follicle development underlying egg-laying traits in chicken.