Neonatal Mouse Ovary Culture and Whole-mount Follicle Quantification: An Efficient <em>In Vitro</em> Approach for Studying Primordial Follicle Regulation

In female mammals, primordial follicles form during fetal ovarian development and serve as the sole source for sustaining adult ovarian function. Mechanisms underlying how primordial follicles assemble, maintain dormancy, activate for follicular development, and undergo cell death are important for understanding ovarian physiology and pathological conditions. This study presents a protocol for culturing postnatal mouse ovaries on membrane inserts, an approach that enables the culture and pharmaceutical treatment of intact ovaries for up to 10 days, depending on the developmental stage of the ovary. Changes in the culture conditions can be achieved by transferring inserts containing cultured ovaries between wells on a plate, thereby avoiding physical interference with the tissues during culture. P5 ovaries were isolated and cultured on a 12 mm insert in a 24-well plate as an example. Each ovary was separated within a droplet of DMEM/F12 medium supplemented with 10% FBS, 3 mg/mL BSA, 10 mIU/mL FSH, and Antibiotic-Antimycotic, and gently stabilized on the membrane insert. The medium was changed every two days, and the culture was maintained for a total of five days. Following the culture, ovaries were fixed in 4% paraformaldehyde for 2 h and processed for whole-mount antibody staining of the oocyte marker DDX4. Follicles were staged and quantified based on oocyte size and the nuclear morphology of somatic follicle cells. The results showed that the number of primordial follicles in each ovary was significantly affected by the proper placement of tissues on the membrane insert. In addition, differences in the number of ovaries on each insert may introduce non-biological variations and should be avoided.