Synthesis of ascorbate and urate in the ovary of water buffalo

Blood flow interruption is associated with oxygen depletion and loss of fac tors for function and survival in downstream tissues or cells. Hypoxia and absence of gonadotropins trigger apoptosis and atresia in the ovary. We stu died the antioxidant response of follicular cells to plasma deprivation in ovaries dissected from water buffalo. Aliquots of follicular fluid were asp irated from each antral follicle, before and during incubation of the ovari es at 39 degreesC. Urate, ascorbate, retinol and alpha -tocopherol in the f luid were, titrated by High Performance Liquid Chromatography (HPLC) with s pectrophotometric or spectrofluorimetric detection. The total antioxidant c apacity of follicular fluid was determined as absorbance decrease, followin g addition of a source of radical chromophores. The more the incubation pro gressed, the higher levels of urate, ascorbate and total antioxidant capaci ty were found. Conversely, changes in concentration of the liposoluble anti oxidants were not observed. Ascorbate synthesizing activity in the follicle was demonstrated by detecting the enzyme L-gulono-gamma -lactone oxidase i n microsomes prepared from granulosa cells. These cells were also analyzed for the expression of the enzyme CPP32. The enzyme level, measured as DEVD- p-nitroanihde cleaving activity, was found related with the immunoreactivit y to anti-CPP32 antibodies. Negative correlation between the enzyme activit y (which is known to be induced by peroxynitrite) and the follicular level of urate (which scavenges peroxynitrite) was also observed. The amount of n itrotyrosine, a product of peroxynitrite attack on proteins, was measured i n follicular fluids by Enzyme Linked ImmunoSorbent Assay (ELISA). This amou nt was found positively correlated with the CPP32 activity, and negatively correlated with the urate level in follicular fluid. Alterations in concent rations of ascorbate or urate may be associated with oxidative stress durin g follicular atresia.