DNA methylation profiling of the granulosa layer reveals epigenetic features of excessive hierarchical follicle development in chickens.

The excessive hierarchical follicles (EHF) trait is a form of ovarian dysfunction in poultry, characterized by an accumulation of hierarchical follicles; however, the developmental dynamics and epigenetic mechanisms underlying this trait remain incompletely understood. In this study, ultrasonography was applied to monitor follicle development dynamics in chickens. Furthermore, whole-genome bisulfite sequencing was performed to identify DNA methylation alterations in the granulosa layer relevant to the EHF trait. During the early laying period, the EHF trait exhibited a fluctuating downward trend and was associated with impaired egg production. DNA methylation occurred predominantly in the CG context and displayed a pattern of promoter hypomethylation and genebody hypermethylation. In EHF chickens, more pronounced hypomethylation was detected in CpG islands, promoters, 5'UTRs, and exons in the small yellow follicle granulosa layer (SYG). Several genes overlapping promoter-related differentially methylated regions were significantly enriched in pathways such as Wnt signaling, Calcium signaling, and Focal adhesion. Among these genes, AKT1, PIK3CA, FZD2, CAMK2A, and RYR2 exhibited significant promoter hypomethylation together with increased mRNA expression. In summary, this study suggests that region-specific promoter hypomethylation in SYG may contribute to EHF development. These findings enhance understanding of the epigenetic regulation of the EHF trait and may support efforts in laying management.