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Expression of Mitochondria-Dependent Apoptosis Genes (p53, Bax, and Bcl-2) in Rat Granulosa Cells During Follicular Development

SeongSoo Hwang, PhD

EunYoung Lee, MSc

Sarah Yoon, MSc

Byung-Koo Yoon, MD,PhD

Department of Obstetrics and Gynecology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul; Center for Clinical Research, Samsung Biomedical Research Institute, Seoul, Korea

DukSoo Bae, MD,PhD

Department of Obstetrics and Gynecology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul; Center for Clinical Research, Samsung Biomedical Research Institute, Seoul, Korea; dschoi{at}smc.samsung.co.kr

Objective: We examined rat ovarian granulosa cells at different follicular stages and evaluated the apoptosis pattern of the mitochondria-dependent genes during folliculogenesis.

Methods: After down-regulating ovarian function with gonadotropin-releasing hormone agonist (Gn-RHa), granulosa cells were collected from the rat ovary at different stages of the following different hormonal treatment paradigms: stage E (after estrogen treatment), EF (after E + follicle-stimulating hormone [FSH] treatment), and EF hCG (after E + FSH + human chorionic gonadotropin treatment). To evaluate the in vitro susceptibility of granulosa cells at different developmental stages to apoptosis, the collected cells were cultured in a serum-free medium with or without E2 for 24 hours. The regulation of apoptosis in the granulosa cells was analyzed using fluorescein-activated cell sorting, quantitative competitive polymerase chain reaction, and western blot methods.

Results: The apoptosis rate of the freshly isolated granulosa cells tended to increase according to the hormonal treatment paradigm. In addition, during the hormone treatment, mitochondria-dependent apoptosis genes showed the following changes: although the Bax mRNA level did not change, the Bcl-2 mRNA level decreased significantly (P < .05). The p53 mRNA level increased significantly (P < .05) and closely matched the apoptosis rate (R = 0.7, P < .05). The expression of the active form of the caspase-3 protein (the final executioner of cell death) tended to increase and showed a good correlation with the apoptosis rate (R = 0.96, P < .01). After an in vitro culture of the granulosa cells, the apoptosis rate tended to increase at all stages, particularly stage EF hCG (P < .05). Bax and p53 mRNA tended to increase and showed a good correlation with the apoptosis rate (R = 0.64, P < .05 and 0.86, P < .01). The Bcl-2 mRNA level tended to decrease at all stages showing no correlation with the apoptosis rate. The expression level of the active caspase-3 protein tended to increase at all stages and showed a good correlation with the apoptosis rate (R = 0.93, P < .01).

Conclusion: Apoptosis of rat ovarian granulosa cells tends to increase according to the stage of follicular development. Among the mitochondria-dependent genes, p53 closely correrlates with granulosa cell apoptosis during follicular development.

Key Words: Apoptosis • mitochondria-dependent genes • follicular development • rat granulosa cells

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