نبذة مختصرة : Abstract Background There are many gaps in our understanding of the mechanisms involved in ovarian follicular development in cattle, particularly regarding follicular deviation, acquisition of ovulatory capacity, and preovulatory changes. Molecular evaluations of ovarian follicular cells during follicular development in cattle, especially serial transcriptomic analyses across key growth phases, have not been reported. This study aims to address this gap by analyzing gene expression using RNA-seq in granulosa and antral cells recovered from ovarian follicular fluid during critical phases of ovarian follicular development in Holstein cows. Results Integrated analysis of gene ontology (GO), gene set enrichment (GSEA), protein–protein interaction (PPI), and gene topology identified that differentially expressed genes (DEGs) in the largest ovarian follicles at deviation (Dev) were primarily involved in FSH-negative feedback, steroidogenesis, cell proliferation, apoptosis, and the prevention of early follicle rupture. In contrast, DEGs in the second largest follicles (DevF2) were mainly related to loss of cell viability, apoptosis, and immune cell invasion. In the dominant (PostDev) and preovulatory (PreOv) follicles, DEGs were associated with vascular changes and inflammatory responses. Conclusions The transcriptome of ovarian follicular fluid cells had a predominance of granulosa cells in the dominant follicle at deviation, with upregulation of genes involved in cell viability, steroidogenesis, and apoptosis prevention, whereas in the non-selected follicle there was upregulation of cell death-related transcripts. Immune cell transcripts increased significantly after deviation, particularly in preovulatory follicles, indicating strong intrafollicular chemotactic activity. We inferred that immune cell invasion occurred despite an intact basal lamina, contributing to follicular maturation. Graphical Abstract
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