Protocol for ex vivo culture of neonate mouse ovaries to study early follicle activation

Establishing an ex vivo culture of neonatal ovaries enables controlled investigation of early follicle dynamics. Here, we present a protocol for isolating ovaries from neonatal female mice for maintaining intact ovarian architecture by ex vivo culture on a membrane allowing access to defined medium. We describe procedures for media preparation, ovary dissection, culture setup, and follicle development by stereomicroscopy and histological assessment. This protocol provides a platform for examining the regulation of the primordial-to-primary transition and growth. For complete details on the use and execution of this protocol, please refer to Amoushahi et al.¹.