Oxidative stress is the result of a delicate balance between the activity of endogenous or environmental oxidants and the defense mechanisms acting to counteract their effects inside and outside the cell. Mural granulosa cells (GC), living within a confined environment such as the ovarian follicle, are partially preserved from environmental oxidants. However, their high metabolic activity, which is mainly directed to the production of sex hormones, involves the generation of reactive oxygen species (ROS). Moreover, during follicle selection, specific metabolic signals activate apoptosis mechanisms leading to degeneration/death of GC. The aim of this study was to evaluate, in bovine GC, the oxidative stress dynamics related to the size and the quality of the follicle. A total number of 33 ovarian follicles of sexually mature heifers were dissected and morphologically analyzed [1]. The follicle diameter ranged from 9 to 15 mm. GC were collected and the corresponding cumulus-oocyte complexes (COC) were morphologically classified [2]. GC were incubated with specific fluorochromes to evaluate: lipid peroxidation (5 μM C11-BODIPY581,591), the production of peroxides and superoxide radicals by using 10 μM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and 30 μM dihydroethidium (DHE), respectively. A number of 400*103 GC were incubated with C11-BODIPY581,591 and H2DCFDA for 30 min, then, washed and incubated in PBS for additional 30 min before read. Since C11-BODIPY581,591 is an effective tracer of lipid trafficking and can be used to measure antioxidant activity, C11-loaded GC were stressed by exposure to menadione, CuSO4 and H2O2 [3] for 30 min before read. DHE was incubated with a number of 2*106 GC for 20 min. All samples were read with a spectrofluorometer. The excitation wavelengths were set at 490, 350 and 488 nm and the emission spectra were recorded in the range of 500–650, 550-670 and 500-560 nm for C11-BODIPY581,591, DHE and H2DCFDA, respectively. Lipid peroxidation estimation was obtained by comparing the two fluorescence intensity peaks ((Fo~520/ (Fo~520+Fo~594))*100) of C11-BODIPY581,591 whereas the ROS generation was evaluated on DHE and H2DCFDA fluorescence intensity peaks at ~595 and ~520 nm, respectively. The follicle size did not significantly affect the GC ability to counteract lipid peroxidation; however, this ability was higher (P< 0.01) in healthy follicles and decreased in atretic follicles. Both H2DCFDA- and DHE-detected ROS decreased together with increasing follicle atresia. Overall data, COC morphology better discriminated follicle atresia than follicle morphology. In conclusion, bovine GC from healthy follicles generate ROS that are counteracted by a high antioxidant activity. Both ROS generation and antioxidant defense systems are impaired by atresia occurrence and are not affected by follicle size.
Relationship between ovarian follicle characteristics and oxidative stress in bovine granulosa cells
Boni R
;Ostuni A.
2017-01-01
Abstract
Oxidative stress is the result of a delicate balance between the activity of endogenous or environmental oxidants and the defense mechanisms acting to counteract their effects inside and outside the cell. Mural granulosa cells (GC), living within a confined environment such as the ovarian follicle, are partially preserved from environmental oxidants. However, their high metabolic activity, which is mainly directed to the production of sex hormones, involves the generation of reactive oxygen species (ROS). Moreover, during follicle selection, specific metabolic signals activate apoptosis mechanisms leading to degeneration/death of GC. The aim of this study was to evaluate, in bovine GC, the oxidative stress dynamics related to the size and the quality of the follicle. A total number of 33 ovarian follicles of sexually mature heifers were dissected and morphologically analyzed [1]. The follicle diameter ranged from 9 to 15 mm. GC were collected and the corresponding cumulus-oocyte complexes (COC) were morphologically classified [2]. GC were incubated with specific fluorochromes to evaluate: lipid peroxidation (5 μM C11-BODIPY581,591), the production of peroxides and superoxide radicals by using 10 μM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and 30 μM dihydroethidium (DHE), respectively. A number of 400*103 GC were incubated with C11-BODIPY581,591 and H2DCFDA for 30 min, then, washed and incubated in PBS for additional 30 min before read. Since C11-BODIPY581,591 is an effective tracer of lipid trafficking and can be used to measure antioxidant activity, C11-loaded GC were stressed by exposure to menadione, CuSO4 and H2O2 [3] for 30 min before read. DHE was incubated with a number of 2*106 GC for 20 min. All samples were read with a spectrofluorometer. The excitation wavelengths were set at 490, 350 and 488 nm and the emission spectra were recorded in the range of 500–650, 550-670 and 500-560 nm for C11-BODIPY581,591, DHE and H2DCFDA, respectively. Lipid peroxidation estimation was obtained by comparing the two fluorescence intensity peaks ((Fo~520/ (Fo~520+Fo~594))*100) of C11-BODIPY581,591 whereas the ROS generation was evaluated on DHE and H2DCFDA fluorescence intensity peaks at ~595 and ~520 nm, respectively. The follicle size did not significantly affect the GC ability to counteract lipid peroxidation; however, this ability was higher (P< 0.01) in healthy follicles and decreased in atretic follicles. Both H2DCFDA- and DHE-detected ROS decreased together with increasing follicle atresia. Overall data, COC morphology better discriminated follicle atresia than follicle morphology. In conclusion, bovine GC from healthy follicles generate ROS that are counteracted by a high antioxidant activity. Both ROS generation and antioxidant defense systems are impaired by atresia occurrence and are not affected by follicle size.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
