Gonadotropin-releasing hormones I and II induce apoptosis in human granulosa cells.
BACKGROUND
The direct effects of GnRH-I or GnRH-II on apoptosis in human granulosa cells are unknown and, if present, can be influenced by FSH. Apoptosis involves activation of the intracellular proteolytic cascade of caspases. We therefore evaluated the roles of GnRH-I and -II, and the effects of FSH, on apoptosis in human granulosa cells and on caspases.


METHODS
Human immortalized granulosa cells treated with GnRH-I or GnRH-II or nothing were cultured with and without antide (a GnRH-I antagonist), a broad-spectrum caspase inhibitor or selective caspase-8, -3, or -7 inhibitor, or FSH in replicates for 72 h. Apoptotic changes were evaluated by terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick-end labeling (TUNEL) assays, immunoblotting, and expression levels of caspases and compared by ANOVA.


RESULTS
GnRH-I and -II induced TUNEL-positive apoptotic cells and increased cleavage activities of caspase-8, -3, and -7 by 48 h and peaked at 72 h, changes that were blocked by FSH cotreatment. Antide also effectively blocked these TUNEL-positive changes and expression levels of caspase-3 induced by GnRH-I or -II. Activation of caspase-8, -3, and -7 was inhibited by the corresponding caspase inhibitor. Caspase-8 inhibitor also abolished cleavages of caspase-3 and -7 induced by GnRH-I and -II.


CONCLUSIONS
GnRH-I and -II induce apoptosis in human granulosa cells through GnRH-I receptors, which mediate the proteolytic caspase cascade involving caspase-8 (the initiator) and caspase-3 and -7 (the effectors). FSH protects human granulosa cells from apoptosis induced by GnRH-I or -II. This raises potentially important roles of GnRH-I and GnRH-II in regulating follicle development and atresia together with FSH.
Hong
In-Sun
IS
Department of Obstetrics and Gynecology, University of British Columbia, 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5.
Cheung
Anthony P
AP
Leung
Peter C K
PC
D017209:Apoptosis; D061945:Caspase Inhibitors; D020169:Caspases / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism; D006107:Granulosa Cells / Q000166:cytology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D009842:Oligopeptides / Q000494:pharmacology; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism*
D061945:Caspase Inhibitors; D009842:Oligopeptides; D011498:Protein Precursors; C050866:progonadoliberin I; D007987:Gonadotropin-Releasing Hormone; D005640:Follicle Stimulating Hormone; C047717:LHRH, His(5)-Trp(7)-Tyr(8)-; C057022:iturelix; D020169:Caspases