REGULATION OF THE STEROIDOGENIC RESPONSE OF CULTURED HUMAN GRANULOSA-CELLS - EFFECTS OF SERUM AND 25-HYDROXYCHOLESTEROL

Objective: To examine the effect of serum and 25-hydroxycholesterol on steroidogenesis in cultured human granulosa cells from women undergoi ng assisted fertilization. Design: Retrospective. Setting: Private Fer tility Clinic and National Research Institute. Patients: Women undergo ing IVF-ET or GIFT programs. Results: In serum-free medium P productio n decreased significantly with culture time (2, 4, 6, and 8 days: 566 +/- 128, 161 +/- 50, 71 +/- 16, and 36 +/- 7 ng/mL P, respectively; co nversion factor to SI unit, 3.180; mean +/- SEM). The addition of 25-h ydroxycholesterol (10 mu g/mL), a substrate for steroidogenesis, did n ot prevent the decrease in P levels. However, P production was greater in the presence of this substrate at all times. The presence of fetal bovine serum (10% FBS) in the cultures allowed the maintenance of 75% of P production with respect to the initial time considered (at which maximal P values are detected). Cultured granulosa cells treated with 10 ng/mL LH in the presence of FBS showed an increase in the percenta ge of stimulation with culture time (2, 4, and 7 days: 2.4%; 54.8%, an d 55.1%, respectively). This effect was not observed when 25-hydroxych olesterol was added to the cultures. Similar results to that obtained by LH were attained when steroidogenesis was stimulated with 0.1 mM di butyryl cyclic adenosine 3':5' monophosphate (cAMP). In addition, cAMP production in response to 100 ng/mL LH in the presence of 0.1 mM meth yl-isobutyl-xanthine decreased with culture time, showing a time depen dency similar to that observed for P. Conclusion: Our results demonstr ate that the decrease in granulosa cell steroidogenic activity with cu lture time is inhibited by serum but not by 25-hydroxyeholesterol, sug gesting that other factors despite LH and cholesterol are necessary to support the luteal function.