AbstractObjectiveEfficiency of preantral follicle culture in vitro is low and is dependent on species, development stage, and follicle‐stimulating hormone (FSH) concentration. Here, we optimized the preantral follicle in vitro culture system in mice.MethodsThe primary follicles (PM follicles, 80‐100 μm diameter ) and early secondary follicles (ES follicles, 110‐130 μm diameter) isolated from 14‐day female mice were cultured in mediums containing 10 mIU/mL or 100 mIU/mL r‐FSH. The follicle growth and oocyte maturation were observed. Estradiol (E2) was detected by ELISA. FSH receptor (FSHR), Ki‐67, 3β‐HSD, CYP17, and CYP19 levels were detected by immunofluorescence and Western blot.ResultsThe antrum formation and oocyte maturation rates of ES follicles were significantly higher than those of PM follicles (P < .05). They were also significantly higher in ES follicles with 100 mIU/mL r‐FSH than with 10 mIU/mL r‐FSH (P < .05). A higher FSHR level was found in ES follicles. Meanwhile, with 10 mIU/mL r‐FSH, the ES follicles exhibited a pattern of flat growth, whereas a pattern of stereoscopic spatial growth was observed with 100 mIU/mL r‐FSH. The 100 mIU/mL r‐FSH stimulated granulosa cell proliferation more significantly than 10 mIU/mL r‐FSH. Moreover, FSH significantly promoted ES follicle granulosa cell proliferation compared to PM follicular granulosa cells. The secretion of E2 and the expressions of 3β‐HSD, CYP 17, and CYP 19 in ES follicles with 100 mIU/mL r‐FSH were significantly higher than those with 10 mIU/mL r‐FSH.ConclusionsThe 100 mIU/mL r‐FSH ideally promotes the development of ES follicles, whose growth pattern can more reasonably simulate the growth of follicles in vivo.