Effect Of Mifepristone On The Expression Of Bcl-2 And Bax In Human Luteinizing Granulosa Cells

BackgroundProgesterone (P4) is one of the major steroids synthesized and secreted by the ovary. In addition to its role as a substrate for production of other steroids, progesterone is critical for regulating reproductive events such as ovulation and luteinization. Mifepristone (RU 486) is an 11 β-dimethyl-amino-phenyl derivative of norethindrone with a high affinity to progesterone and glucocorticoid receptors. As an antiprogestin, it has been widely used to terminate early pregnancy. It has other potential uses in fertility control, such as inhibition of ovulation, prevention of implantation, and induction of menstrual bleeding. As a potential contraceptive drug, mifepristone attracts the attention of many researchers. It has been demonstrated that administration of mifepristone during the follicular phase can interfere follicular growth and inhibit ovulation. This effect makes it possible that mifepristone might be an estrogen-free oral contraceptive that suppresses ovulation. However, the underlying mechanism which mifepristone inhibited ovulation is not clear.Granulosa cells, a main cellar component of the ovarian follicle, surrounding and nursing the oocyte, support oocyte maturation. The high level of steroid hormone secretion by these cells ensures a receptive environment for the implantation anddevelopment of the early embryo. Recent observations have suggested that apoptosis in granulosa cells is primarily responsible for follicular atresia and luteal regression. Furthermore, ovarian follicular atresia in all vertebrates is mediated via apoptosis that is initiated in the granulosa cell layer. These studies raise a question about the possible mechanism that inhibiting ovulation by mifepristone may be mediated via granulosa cell apoptosis.Researchers have studied the regulation pathways of apoptosis in granulosa cells induced by mifepristone. Mifepristone has been demonstrated to increase caspase-3 activity in granulose cells after 24-hour incubation. However, to date no work has been reported on the effect of mifepristone on apoptosis and the expression of Bcl-2 gene family in primary human granulosa cells. Therefore, we decided to test in detail the effect of mifepristone on apoptosis and the expression of apoptosis-regulating genes, Bcl-2 and Bax in human granulosa cells.Materials and MethodsHuman granulosa cells were obtained from patients undergoing in vitro fertilization-embryo transfer (IVF-ET) by ultrasound-guided follicular aspiration. Electron microscopy and electrophoresis analysis of DNA fragmentation, were conducted to observe the morphologic and biochemical change of granulosa cells exposed to different concentrations of mifepristone for 24 hours (0, 2.5>5 and 10MM/L mifepristone). Immunocytochemistry was performed to detect the protein expression of Bcl-2 and Bax in granulosa cells exposed to different concentrations of mifepristone for 24 hours. The mRNA levels of Bcl-2 and Bax in granulosa cells were detected by RT-PCR.One-way AN OVA and Student-Newman-Keuls test were used for statistical analysis. Two-tailed test with P<0.05 were considered significant. All data were managed with SPSS 10.0 for windows.Results1. Human granulosa cells cultured within the first 24 h were well spread, showingnumerous stress fibers and numerous cytoplasmic lipid droplets characteristic of steroidogenic cells.2. Electron microscopy demonstrated that granulosa cells treated with differentconcentrations of mifepristone appeared typical morphological characteristics of apoptosis such as condensation of chromatinand cytosolic, and shrink of cells etc.3. No effect on DNA fragmentation was seen in granulosa cells treated with different concentrations of mifepristone, compared with that of control group.4. Granulosa cells treated with mifepristone showed significant decrease of Bcl-2expression, compared with that of control group (P< 0.01), and comparisons among the treated groups had the significant difference. In contrast, the expression of Bax in mifepristone-treated cells showed signicicant increase, compared with that of control group (P< 0.01), and comparisons among the treated groups had the significant difference.5. The mRNA levels of Bcl-2 and Bax in mifepristone-treated granulosa cells did not show difference compared with that of control group (P > 0.05).Conclusions1. Mifepristone was able to induce apoptosis in human luteinizing granulose cells. The finding suggested that, apoptosis induced by mifepristone in granulosa cells might be one of the mechanisms in inhibiting ovulation.2. Regulation the protein expression of Bcl-2 and Bax may be one of the apoptotic pathways of granulosa cells induced by mifepristone.3. Mifepristone might regulate the expression of Bcl-2 and Bax by affecting the post-transcription levels.