title: Melatonin protects the integrity of granulosa cells by reducing oxidative stress in nuclei, mitochondria, and plasma membranes in mice.
abstract: Melatonin protects luteinized granulosa cells (GCs) from oxidative stress in the follicle during ovulation. However, it is unclear in which cellular components (e.g., nuclei, mitochondria, or plasma membranes) melatonin works as an antioxidant. GCs from immature (3 wks) ICR mice were incubated with hydrogen peroxide (H2O2; 0.01, 0.1, 1, 10 mM) in the presence or absence of melatonin (100 μg/ml) for 2 h. DNA damage was assessed by fluorescence-based immunocytochemistry using specific antibodies for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative guanine base damage in DNA, and for histone H2AX phosphorylation (γH2AX), a marker of double-strand breaks of DNA. Mitochondrial function was assessed by the fluorescence intensity of MitoTracker Red probes, which diffuse across the membrane and accumulate in mitochondria with active membrane potentials. Lipid peroxidation of plasma membranes was analyzed by measuring hexanoyl-lysine (HEL), a oxidative stress marker for lipid peroxidation. Apoptosis of GCs was assessed by nuclear fragmentation using DAPI staining, and apoptotic activities were evaluated by caspase-3/7 activities. H2O2 treatment significantly increased the fluorescence intensities of 8-OHdG and γH2AX, reduced the intensity of MitoTracker Red in the mitochondria, increased HEL concentrations in GCs, and enhanced the number of apoptotic cells and caspase-3/7 activities. All these changes were significantly decreased by melatonin treatment. Melatonin reduced oxidative stress-induced DNA damage, mitochondrial dysfunction, lipid peroxidation, and apoptosis in GCs, suggesting that melatonin protects GCs by reducing oxidative stress of cellular components including nuclei, mitochondria, and plasma membranes. Melatonin helps to maintain the integrity of GCs as an antioxidant in the preovulatory follicle.
authors.affiliation: Department of Obstetrics and Gynecology, Yamaguchi University Graduate School of Medicine, Ube 755-8505, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Tanabe
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tamura
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Taketani
authors.forename: Maki
authors.initials: M
authors.lastname: Okada
authors.forename: Lifa
authors.initials: L
authors.lastname: Lee
authors.forename: Isao
authors.initials: I
authors.lastname: Tamura
authors.forename: Ryo
authors.initials: R
authors.lastname: Maekawa
authors.forename: Hiromi
authors.initials: H
authors.lastname: Asada
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Yamagata
authors.forename: Norihiro
authors.initials: N
authors.lastname: Sugino
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D004249:DNA Damage; D005260:Female; D006107:Granulosa Cells / Q000166:cytology*; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D015227:Lipid Peroxidation; D008550:Melatonin / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D008928:Mitochondria / Q000378:metabolism*; D018384:Oxidative Stress; D017382:Reactive Oxygen Species
chemical_list: D000975:Antioxidants; D017382:Reactive Oxygen Species; D006151:Guanosine; C046215:8-hydroxyguanosine; D006861:Hydrogen Peroxide; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D008550:Melatonin