Role of apoptosis controlled by cytochrome c released from mitochondria for luteal function in human granulosa cells.
OBJECTIVE
The aim of this study was to investigate the relationship between apoptosis by the mitochondrial pathway and luteal function in human granulosa cells.


METHODS
Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization and embryo transfer. After the addition of RU486, cells were stained with a mitochondria-specific fluorescent dye, MitoTracker Red CM x Ros. Using flow cytometry and National Institute of Health image, the mitochondrial fluorescent area was measured. After staining with Hoechst 33258 dye, the number of apoptotic bodies per 1000 cells were counted at random on photomicrographs. Homogenates were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against cytochrome c or caspase-3.


RESULTS
The incidence of apoptotic bodies increased and the mitochondrial membrane potential decreased time dependently. The opposite effect was observed dose dependently with RU486 treatment. Western blot analysis showed increased cytochrome c expression, after treatment with 1-2 microg/mL of RU486 which then decreased with 5-10 microg/mL of RU486. Caspase-3 expression increased dose dependently with RU486.


CONCLUSIONS
These results suggest that the activation of caspase-3 caused by cytochrome c released from mitochondria plays an important role in apoptosis-related luteal function in human granulosa cells.
Makino
Aiko
A
Department of Obstetrics and Gynecology, Nagoya City University Medical School, Mizuho-ku Nagoya, Japan. aikomaki@med.nagoya-cu.ac.jp
Ozaki
Yasuhiko
Y
Matsubara
Hirokazu
H
Sato
Takeshi
T
Ikuta
Katsuo
K
Nishizawa
Yuji
Y
Suzumori
Kaoru
K
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000502:physiology*; D005260:Female; D005434:Flow Cytometry; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D008184:Luteal Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008186:Luteolytic Agents / Q000494:pharmacology; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D015735:Mifepristone / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011374:Progesterone / Q000378:metabolism
D008186:Luteolytic Agents; D015735:Mifepristone; D011374:Progesterone; D045304:Cytochromes c; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases