INTERLEUKIN-6 STIMULATES APOPTOSIS IN FSH-STIMULATED RAT GRANULOSA-CELLS IN-VITRO - DEVELOPMENT AND UTILIZATION OF AN IN-VITRO MODEL

A temporal relationship between apoptotic death of granulosa cells and the onset of ovarian follicular atresia has recently been demonstrate d in vivo. On the basis of this relationship, we hypothesized that ini tiation of apoptosis in the granulosa cell layer is the triggering eve nt which culminates in follicular atresia. To investigate this hypothe sis we have developed an in vitro model to study apoptosis in the gran ulosa cell. Moreover, we have utilized this system to examine a potent ial apopotic-regulatory activity for the pleiotropic cytokine, interle ukin-6 (IL-6). Immature female rats were injected with 15 IU PMSG and killed 24 h later, a time at which apoptosis is not occurring in granu losa cells. Granulosa cells were harvested by needle puncture, seeded at 10(6) cells/ml in 35 x 10 mm cultures dishes containing 1 ml Dulbec co's Modified Eagle medium/5% fetal bovine serum and cultured for 24 h (37 degrees C, 95% air/5% CO2). Cells were then washed (3 x) and cult ured an additional 24 h in serum-free medium containing FSH (20 ng/ml) and/or androstenedione (10(-7) M). After this incubation, media were removed and progesterone levels quantified by RIA. Genomic DNA was iso lated from cells, electrophoretically separated on a 2% agarose gel an d visualized by ethidium bromide staining and u.v. transillumination. The results demonstrate that FSH stimulated progesterone production to levels sevenfold over non-stimulated cultures while androstenedione e xerted no significant effect. Analysis of DNA banding patterns reveale d faint apoptotic-like fragments in samples from FSH-stimulated cultur es, however, the intensity of these bands was enhanced in cultures mai ntained without FSH, demonstrating that apoptosis in PMSG-primed granu losa cells is suppressed by FSH and stimulated by its withdrawal. Agai n androstenedione exerted no detectable effect. Utilization of this in vitro model will allow for the investigation of various substances wh ich may either directly stimulate or suppress apoptosis in granulosa c ells. Indeed, when granulosa cells cultured with FSH were treated for 24 h with increasing concentrations of IL-6 (125, 250 and 500 U/ml), a poptotic DNA fragmentation was stimulated in a concentration-dependent manner. FSH-stimulated progesterone production was inhibited by appro ximately 80% at the highest dose of IL-6 examined. These data implicat e IL-6 (for which granulosa cells are both a source and target) in the regulation of granulosa cell apoptosis and suggest a role for this cy tokine in the atretic process.