Expression of mitochondria-dependent apoptosis genes (p53, Bax, and Bcl-2) in rat granulosa cells during follicular development.
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 (GnRHa), 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).


CONCLUSIONS
Apoptosis of rat ovarian granulosa cells tends to increase according to the stage of follicular development. Among the mitochondria-dependent genes, p53 closely correlates with granulosa cell apoptosis during follicular development.
Choi
DooSeok
D
Department of Obstetrics and Gynecology, Sungkyunkwan University School of Medicine, Samsung Medical Center, 50 Irwon-dong Gangnam-gu, Seoul 135-710, South Korea. dschoi@smc.samsung.co.kr
Hwang
SeongSoo
S
Lee
EunYoung
E
Yoon
Sarah
S
Yoon
Byung-Koo
BK
Bae
DukSoo
D
D000818:Animals; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D004967:Estrogens / Q000494:pharmacology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D019254:Genes, bcl-2 / Q000502:physiology*; D016158:Genes, p53 / Q000502:physiology*; D006107:Granulosa Cells / Q000187:drug effects / Q000502:physiology*; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006080:Ovarian Follicle / Q000187:drug effects / Q000502:physiology*; D010053:Ovary / Q000187:drug effects / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D051381:Rats; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D006063:Chorionic Gonadotropin; D004967:Estrogens; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D005640:Follicle Stimulating Hormone