PARENT SESSION
SLIDE SESSION 21: FOLLICLE DEVELOPMENT: INPUTS TO FUNCTIONAL MATURATION
Chairs: Jeff May, Jerry Menon, Kari Doyle (Trainee)
Univ Ottawa-Lamoureaux 122
1:30 PM-3:30 PM

ESTRADIOL INHIBITS APOPTOSIS OF GRANULOSA CELLS INDUCED BY FAS LIGAND.

Quirk, Susan¹, Cowan, Robert¹, Harman, Rebecca¹, ¹

ABSTRACT- A hallmark of healthy preovulatory follicles is the capacity of their granulosa cells (GC) to secrete large amounts of estradiol (E2). This study examined the ability of E2 to inhibit apoptosis of cultured GC induced by Fas ligand (FasL). FasL induces apoptosis through its receptor, Fas antigen, and may regulate follicular atresia. GC were cultured for 2 days in DMEM-F12 + FBS with or without 0.5 M E2. On day 2, media was changed to DMEM-F12 + transferrin, selenium, and 100 ng/ml insulin (ITS) ± FasL or with ITS+E2 ± FasL. On day 3 FasL-induced killing was determined based on cell counts using trypan blue exclusion. FasL-induced killing was 39±5% in GC cultured in ITS and was reduced to 21±5% in ITS+E2 (P<0.01). The proportion of cells expressing Ki-67, a protein expressed only by cells actively progressing through the cell cycle, was determined by immunocytochemistry. In GC cultured in ITS, 13±2% were positive for Ki67, while in those cultured in ITS+E2, 19±3% expressed Ki67 (P<0.01). The proportion of cells expressing cyclin D2, a G1 cyclin which promotes entry into the cell cycle, was increased from 10±5% of cells cultured in ITS to 28±7% of cells cultured in ITS+E2 (p<0.05). The data indicate that E2 treatment increases proliferation of GC and increases resistance to FasL-induced apoptosis. Recent data from our laboratory suggests that cells which are detained in the S phase of the cell cycle are susceptible to apoptosis, while those which rapidly traverse the S phase are resistant. Accordingly, we examined the effect of hydroxyurea (HU), which blocks cells in early S, on the ability of E2 to protect cells from FasL-induced apoptosis. When GC were cultured in ITS, 42±8% died in response to FasL, and this was not affected by HU (40±1% killing). In ITS+E2, protection from apoptosis was observed (26±2% killing, p<0.05 ), but this protective effect was abolished when the cells were also treated with HU (39±5% killing, p<0.05 vs E2 treatment, p>0.05 vs ITS). Flow cytometric analysis of GC DNA content confirmed that HU blocked cells in S phase. This suggests that the protective effect of E2 may be due to timely progression through the cell cycle. We conclude that follicles expressing higher levels of E2 may be selected to develop further while those expressing less E2 may succumb to apoptotic stimuli and undergo atresia.

KEY WORDS: granulosa cells, Fas, apoptosis, cell cycle