NITRIC OXIDE SYNTHASE AND AGE RELATED INFERTILITY: THE ROLE OF ARGINASE II

Abstract

Low L-Arginine (L-Arg) may lead to alteration in nitric oxide synthase (NOS) activity shifting from nitric oxide (NO) synthesis to O2•- generation, a process known as NOS uncoupling. Arginase II, a critical enzyme in the urea cycle, can modulate the availability of L-Arg for NOS, regulating NO release in many types of cells. Based on the relationship between L-Arg and NO modulation, we sought to determine the relationship between differing NO profiles in young and old mouse oocytes and their expression of the arginase gene (ARG1) as well as developmental effects such as fertilization and embryonic development. This is an experimental case-control study. B6D2F1 mice were divided into young (6-10 week) and old (48-60 week) groups. An NO-selective electrode was used for direct NO measurements in ovaries, oviducts, and oocytes. A subset of ocytes were also further exposed to 50 μM Nitro-L-arginine methyl ester (L-NAME), an NOS inhibitor for 20-30 minutes. NO levels in each of those groups were measured using ICSI. ARG1 expression was assessed in ovarian tissue of young versus old mice with real-time PCR. Ornithine, the final product of arginase II, and L-Arg were also measured in oocytes from young (n=100) versus old mouse (n=100) ovaries via high-performance liquid chromatography (HPLC). NO levels in the ovaries of young mice were 20 ± 1.5 nmol. There was a marked reduction, ∼60%, in old mouse NO, with similar results among oocytes, ovaries and oviducts. ARG1 expression was upregulated (threefold) in old versus young mice. Ornithine was significantly increased (fivefold) in old versus young oocytes, while L-Arg levels, in contrast, were markedly decreased (0.25 +/- 0.02 nmol/ml) in old compared to young oocytes (0.50 +/- 0.01 nmol/ml). The upregulation of ARG1, and therefore increased arginase II activity, in older animals was correlated with higher levels of ornithine, decreased L-Arg, and decreased NO. Therefore, our results suggest that increased activity of arginase II may be an important modulator of NOS activity and thus NO bioavailability in aged animals.