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-v itro 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 d ays of preincubation. Cultures were assayed for progesterone and oestr adiol production. Fresh cells displayed very high basal progesterone p roduction which could be stimulated with LH but not FSH. After preincu bation, addition of LH and FSH resulted in dose-dependent increases of progesterone and oestradiol. When cultured on human fibronectin-coate d wells, similar basal but higher progesterone concentrations after st imulation were observed. In comparison with serum-free media, addition of Serum-Plus(TM) resulted in higher basal and stimulated progesteron e concentration, possibly due to the presence of serum factors. This s tudy demonstrates firstly that after 7 days preincubation, cultures ga ined responsiveness to FSH but remained responsive to LH during 4 days of stimulation. This suggests a persisting differentiated cell popula tion in vitro. Secondly, the use of human fibronectin extracellular ma trix and serum promotes steroid production, either due to factors prom oting cell growth and function or to availability of steroid precursor s. Therefore one has to be cautious with interpretation of data obtain ed from this widely used culture system, employing highly differentiat ed cells obtained after ovarian stimulation for in-vitro fertilization for study of local regulation of granulosa cell function.