Regulation of the steroidogenic response of cultured human granulosa cells: effects of serum and 25-hydroxycholesterol

To examine the effect of serum and 25-hydroxycholesterol on steroidogenesis in cultured human granulosa cells from women undergoing assisted fertilization. Retrospective. Private Fertility Clinic and National Research Institute. Women undergoing IVF-ET or GIFT programs. In serum-free medium P production decreased significantly with culture time (2, 4, 6, and 8 days: 566 ± 128, 161 ± 50, 71 ± 16, and 36 ± 7 ng/mL P, respectively; conversion factor to SI unit, 3.180; mean ± SEM). The addition of 25-hydroxycholesterol (10 μg/mL), a substrate for steroidogenesis, did not 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 percentage of stimulation with culture time (2, 4, and 7 days: 2.4%; 54.8%, and 55.1%, respectively). This effect was not observed when 25-hydroxycholesterol was added to the cultures. Similar results to that obtained by LH were attained when steroidogenesis was stimulated with 0.1 mM dibutyryl cyclic adenosine 3′:5′ monophosphate (cAMP). In addition, cAMP production in response to 100 ng/mL LH in the presence of 0.1 mM methyl-isobutyl-xanthine decreased with culture time, showing a time dependency similar to that observed for P. Our results demonstrate that the decrease in granulosa cell steroidogenic activity with culture time is inhibited by serum but not by 25-hydroxycholesterol, suggesting that other factors despite LH and cholesterol are necessary to support the luteal function.