Abstract
Follicular fluid serves a crucial role in follicular development and oocyte maturation. Increasing evidence indicates that follicular fluid is rich in proteins and functional cells. In addition to oocyte cells, follicular fluid contains granulosa, thecal and ovarian surface epithelial cells. Granulosa cells (GCs) represent the predominant somatic cell type of the ovarian follicle and are involved in steroidogenesis and folliculogenesis. However, the long-term culture of GCs in vitro remains challenging. The present study aimed to extend the culture of GCs in vitro. Human GCs were collected from the follicular fluid of patients included in an in vitro fertilization program and cultured in the presence of conditioned medium obtained from mouse embryonic fibroblasts. GCs were cultured for over a year and 130 passages, and the population doubling time was ~22 h. Cells presented epithelial-like morphology and a cobblestone-like appearance when they reached confluence. Flow cytometric analysis demonstrated that cells expressed CD29, CD166 and CD49f but not CD31, CD34, CD45, CD90, CD105 or CD13. Immunofluorescence staining revealed that cells expressed follicle stimulating hormone receptor, luteinizing hormone receptor and cytochrome P450 aromatase, which was confirmed by reverse transcription-quantitative polymerase chain reaction. In the presence of androstenedione, cells secreted estradiol. In addition, estradiol level was further stimulated by dibutyryl cAMP treatment. In addition, intracellular cAMP and progesterone expression levels were upregulated by follicle stimulating hormone and/or human chorionic gonadotropin. Furthermore, cells survived in severe combined immunodeficiency mice following intra-ovarian injection. Histological analysis revealed that certain cells formed follicle-like structures. The results from the present study suggested that immortalized GCs may be a useful tool for further research on GC and improve the clinical application of drugs such as follicle-stimulating hormone or human chorionic gonadotropin.