Oxidative stress induces many serious reproductive diseases in female mammals and thus poses a serious threat to reproductive health. However, the relationship between reactive oxygen species (ROS)—induced oxidative stress and follicular development, oocyte and embryo quality is not clear. The aim of this study was to investigate the effect of ovarian oxidative stress on the health of follicle and oocyte development. Female ICR mice were dosed with 3-nitropropionic acid (3-NPA) at three different concentrations (6.25, 12.5 and 25 mg/kg) and saline (control) via continuous intraperitoneal injection for 7 days. The treatment with 12.5 mg/kg reduced the weight of mouse ovaries, and significantly increased ROS levels and the activities of antioxidant enzymes—total superoxide dismutase (T-SOD), glutathione peroxidase (GPx) and catalase (CAT) — in granulosa cells and ovarian tissues, but not in other tissues (brain, liver, kidney and spleen). The same treatment significantly increased the percentage of atretic large follicles, and reduced the number of large follicles, the number of ovulated oocytes, and the capacity for early embryonic development compared with controls. It also significantly decreased the ratio of Bcl-2 to Bax, while causing an increase in the mRNA expression of (SOD2, CAT and GP X) and ROS levels in granulosa cells. Collectively, these data indicate that 3-NPA induces granulosa cell apoptosis, large follicle atresia, and an increase of ROS levels in the ovary. Therefore, we have established an in vivo model of ovarian oxidative stress for studying the mechanism of resulting damage induced by free radicals and for the screening of novel antioxidants.
ABSTRACT. Vascular endothelial growth factor (VEGF) has been found responsible for the induction of proliferation and differentiation in granulosa cells. We constructed four short hairpin RNA (shRNA) expression plasmids targeting the mouse VEGFA gene, and examined their effect on VEGF expression in mouse granulosa cells (MGC) in vitro. Four different shRNA oligonucleotides targeting the coding sequence of mouse VEGFA mRNA and one negative control (shNC) were designed and cloned into a pGPU6/GFP/Neo siRNA expression vector, and transiently transfected into MGC. At 48 h post-transfection, total RNA was extracted from the cells and subjected to qRT-PCR analysis. The most effective interference vector, shVEGF1487 was chosen for lentiviral construction. The recombinant plasmid was then transfected into 293FT cells via Lipofectamine TM 2000-mediated gene transfer, for the production of lentivirus, and then concentrated via ultracentrifugation. This lentiviral vector was then used for the transduction of MGC. VEGFA gene expression, apoptosis genes and VEGFA receptor genes were detected by qRT-PCR, the VEGFA protein level in culture media by ELISA assay and protein levels in MGC by Western blot analysis. The four VEGFA expression plasmids were successfully constructed and the most effective interference vector, shVEGF1487, was chosen for lentiviral production and MGC transduction. There was significant knockdown of the VEGFA gene, receptor genes and apoptosis genes for all the shVEGF constructs, compared with the shNC and Mock controls. The lentiviral vector also gave significant knockdown of the VEGFA gene. Protein levels were lower for most of the shVEGFs based on Western blot analysis with exception of VEGF1359; in this case, it was higher than shNC but lower than for the Mock group. Lentivector-transduced MGC also gave lower levels of protein. We conclude that shVEGF expression plasmids and lentivector carrying RNAi are promising tools for the inhibition of VEGF, the corresponding receptor genes, and apoptosis gene expression in MGC.
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