Study On The Signaling Pathway Involved In Controlling Oocyte Apoptosis In Neonatal Rat Ovaries

Objectives: To investigate if FOXO3a is involved in the apoptosis of naked oocytes and oocytes of primordial follicles in mammalian ovaries and its signal pathway. Methods: (1) Oocytes from ovarian nests and primordial follicles of postnatal day 2 rat ovaries were cultured. (2) Experiment grouping and medication method: Stem cell factor(SCF)and phosphoinositide 3-kinase (PI3K) inhibitor, LY 294002 were added to the media to final concentrations of 150ng/L and 25μM respectively. The experiment was divided into three groups: Oocytes were cultured without SCF (SCF-), with SCF (SCF+) and with SCF in combination with LY 294002 (SCF+LY). After starvation in serum-free media, oocytes in tissue culture dishes were treated for 5 min or 24 h with SCF. To investigate the effect on AKT and FOXO3a phosphorylation, LY 294002 was added 1h before stimulation with SCF for 5 min. To study the effect of LY 294002 on the expression of Bad, Bax, Bim, Bcl-2, MnSOD and p27kip1, the inhibitor was added together with SCF and incubated for 24 h.(3) Oocyte apoptosis was examined using the TUNEL technique. The percentage of apoptotic oocytes per slide was evaluated by counting the number of TUNEL-positive oocytes and the total number of oocytes in a minimum of three fields (100 cells per slide) under a microscope with a 40X objective. (4) The expression of FOXO3a and its target genes were examined by RT-PCR and Western blot. (5) Immunocytochemistry: The cells were incubated with primary antibody against FOXO3a, BIM, BAD, BAX, BCL-2, MnSOD or p27KIP1 (1:25 or 1:100 diluted in PBS) and corresponding biotinylated secondary antibody. The slide staining was developed with 3,3'-diaminobenzidine(DAB). The percentage of positive oocytes per slide was evaluated by counting the number of positive oocytes and the total number of oocytes in a minimum of three fields (100 cells per slide) under a microscope with a 40X objective. (6) Statistical analysis: Values shown in all the figures are expressed as the means±SEM. The data were analyzed using a one-way ANOVA as appropriate. All experiments were repeated at least three times, and representative data are shown. Results: (1) SCF inhibited oocyte apoptosis and LY 294002 abolished the anti-apoptotic action of SCF. (2)SCF did not affect the level of total FOXO3a protein and total AKT protein, but rapidly elevated the level of their phosphorylation. The phosphorylated AKT indicated functional activation and the phosphorylated FOXO3a indicated functional suppression. (3) SCF down-regulated the expression of Bim, Bax, Bad and p27kip1, and this activity was reversed by LY 294002. (4) SCF up-regulated the expression of MnSOD, and LY 294002 blocked the aboved effects. (5) SCF had no effect on Bcl-2 protein. Conclusions: (1) FOXO3a is involved in oocyte apoptosis in postnatal day 2 rat ovary, and the SCF-PI3K/AKT signaling pathway mediates oocyte apoptosis and primordial follicle formation. (2) The downstream molecules, Bim, Bax, Bad, MnSOD and p27kip1, may be involved in this process.