Abstract: BACKGROUND AND AIM: To obtain and identify cultured granulosa cells from the ovary of rats so as to establish a convenient and stable experimental model. MATERIALS AND METHODS: Female SD rats were subcutaneously treated with pregnant mare serum gonadotropin (PMSG). Forty-eight hours after dosing with PMSG, the animals were decapitated and the ovaries were aseptically removed. Granulosa cells were then released by mechanical method, trypsin digestion and low-speed centrifugation separation were used for granulosa cells isolation. Granulosa cells were diluted and incubated in fresh DMEM-Ham's F-12 medium (1∶1) containing 15％ of fetal bovine serum at 37 ℃ in water-saturated environment of 5％ CO2. Hematoxylin ＆ Eosin (HE) and immunohistochemical staining of FSHR were used for ovarian granulosa cell identification. Additionally, the growth curves of granulosa cells and hormone levels at different incubation times were evaluated as well. RESULTS: Over 95％ of the cultured cells were ovarian granulosa cells．The exponential phase of growth was between 48 and 96 hours of incubation. CONCLUSION: More than 95％ of highly purified granulosa cells could be obtained by mechanical method combined with trypsin digestion and low-speed centrifugation. Moreover, identifications of granulosa cells by HE and FSHR staining were quick and convenient approaches.

Key words: rat, ovary, granulosa cell, culture, identification