In Vitro Ovarian Culture System

A safe and effective in vitro ovarian culture system has been established in our lab (10 (link), 11 (link)). Neonatal mice were sacrificed by cervical dislocation at the designated times. The ovaries were separated in cold phosphate-buffered saline (PBS) under the microscope. The isolated ovaries were incubated in 6-well culture dishes (NEST, China), and an insert (PICM0RG50, Millipore, USA) was placed in every well with 3 mL Dulbecco Modified Eagle Medium/Ham F12 nutrient mixture (DMEM/F12) (Gibco, Life Technologies, CA) supplemented with insulin-transferrin-sodium selenite (1:100, Sigma, USA) and penicillin-streptomycin solution at 37 °C, 5% CO₂ and saturated humidity. Ovaries were randomly assigned, and cultured for 1–7 days in basal medium alone or basal medium supplemented with either dbcAMP (10 μM, D0627, Sigma, USA), MDL-12,330 (5 μM, M182, Sigma, USA), Milrinone (10 μM, 78415-72-2, J&K, China) or H 89 2HCL (5 μM, S1582, Selleck, China), respectively.