Development of a human granulosa cell culture model with follicle stimulating hormone responsiveness

In order to study the effects of follicle-stimulating hormone (FSH) on differentiation of granulosa cells, a well-defined and validated in-vitro culture system is indispensable. In this study, pooled follicular aspirates were stimulated in vitro with FSH and luteinizing hormone (LH) for 2, 4 and 6 days, either immediately after plating or after 7 days of preincubation. Cultures were assayed for progesterone and oestradiol production. Fresh cells displayed very high basal progesterone production which could be stimulated with LH but not FSH. After preincubation, addition of LH and FSH resulted in dose-dependent increases of progesterone and oestradiol. When cultured on human fibronectin-coated wells, similar basal but higher progesterone concentrations after stimulation were observed. In comparison with serum-free media, addition of Serum-Plus resulted in higher basal and stimulated progesterone concentration, possibly due to the presence of serum factors. This study demonstrates firstly that after 7 days preincubation, cultures gained responsiveness to FSH but remained responsive to LH during 4 days of stimulation. This suggests a persisting differentiated cell population in vitro. Secondly, the use of human fibronectin extracellular matrix and serum promotes steroid production, either due to factors promoting cell growth and function or to availability of steroid precursors. Therefore one has to be cautious with interpretation of data obtained from this widely used culture system, employing highly differentiated cells obtained after ovarian stimulation for in-vitro fertilization for study of local regulation of granulosa cell function.