ABSTRACT: Background: The initial formation of follicular antrum (iFFA) is a milestone in folliculogenesis, which serves as a boundary between gonadotropin (GTH) -independent folliculogenesis and GTH-dependent folliculogenesis and endows the follicle's ability to acquire response to GTHs and continue to survive. However, iFFA is a barren field in folliculogenesis research, which do not match its physiological significance. Methods:In this study, transcriptome sequencing was used to comprehensively analyze the changes in the life activities of cells in the ovary during iFFA, and then the regulatory mechanism of iFFA was deduced based on the results of omics analysis, and it was systematically verified by experiments. Result: Herein, we conducted in vivo and in vitro experiments to studied iFFA in a mouse model, and revealed that: (1) iFFA is a physiological process of active cell proliferation, increased energy expenditure, enhanced secretory activity and increased moisture absorption, accompanied by complex changes in epigenetic modification, bioclock regulation, and cell junctions; (2) iFFA and blastocoel formation may be conservatively regulated in fluid accumulation, and tight junctions, ion pumps and aquaporins underlie the structural, kinetic and transport foundations for fluid accumulation during iFAA, respectively; (3) mTOR-CNP is the main signal cascade regulating iFFA, it initiated iFFA by targeting tight junction/ion pumps/aquaporins and follicular cell proliferation; (4) FSH controls iFFA by targeting the intra-ovarian mTOR-CNP cascade; (5) mTOR activator could be used as an improver to increase the efficiency of in vitro follicular culture technology. Conclusion: Overall, this study elucidated the changes in follicular cells' life activities in preparation for iFFA, proposed the model of "Dam-Pump-Pipe" to explain the accumulation of follicular fluid, and confirmed that FSH-mTOR-CNP-Tight junction/Ion pumps/Aquaporins is the core cascade controlling iFFA. These findings would help to fill in the gaps in iFFA research, and enrich the theory of folliculogenesis regulation.
