Arginase II and nitric oxide synthase uncoupling – a culprit for oocyte quality deterioration in older patients?

Abstract

Utilizing nitric oxide (NO) selective electrode, recently we have shown that the intraoocyte NO concentration of oocytes obtained from young are significantly higher than those from old animals. NO supplementation of old and young oocytes not only resulted in sustained the ability of oocytes to undergo normal fertilization and development, but also prevented the onset of apoptosis in the embryos created from aged oocytes. NO is generated enzymatically by three distinct isoforms of NO synthase (NOS): neuronal, inducible, and endothelial. All three isoforms typically require molecular oxygen, NADPH, tetrahydrobiopterin (H4B), and oxygen to convert L-arginine (L-Arg) to NO and citrulline. Deficiency of L-Arg or H4B, leads NOS to generate O2•- instead of NO, in a process called NOS uncoupling. Arginase II is a critical enzyme in the urea cycle that converts arginine to ornithine and urea and, by modulating the availability of L-Arg for NOS, influences NO release in a variety of cells. Based on the above we hypothesized that enhancement in arginase II levels/activity in old oocytes mediates the decrease in NO and L-Arg levels and the increase in superoxide generation due NOS uncoupling, being a major cause of oocyte deterioration and infertility. To test our hypothesis, we first measured NO levels in young (8-14 weeks) vs. old mice (40-45 weeks) in normal uninjured ovaries. NO measurements were performed by monitoring NO concentration with an NO-electrode that was placed gently over the surface of the ovary, as far as possible from any visible blood vessels and in the oviducts of the mice. In order to verify the role of arginase II in regulating NOS activity, we next measured the expression of arginase II in both young and old ovaries. Gene expression levels in the ovaries of young vs old mice were determined for the ARG1 gene, which regulates expression of arginase I and II. Real-time qPCR was run in triplicate. To see the impact of this on the actual oocytes, we next measured the concentration of ornithine, final product of arginase II, in freshly harvested oocytes obtained from young (n=100; 6-10 week) as compared to old (n=100; 48-60 week) mice utilizing high-performance liquid chromatography (HPLC). Normal tissue NO levels of the uninjured ovary in young animals were in the 20 ± 1.5 nmol ranges, as opposed to, in old animals, the NO levels were markedly reduced by ∼60%. Comparable results were obtained when the NO-selective electrode was inserted in the oviducts of young and old mice. The levels of ornithine were dramatically increased in old compared to young oocytes. In contrast, arginine levels decreased by 50% in oocytes obtained from old mice as compared to young. There was also an enhancement of arginase II expression in old vs young mice as a ratio. These results suggest that arginase II activity is higher in old compared to young oocytes. Thus, the catalytic activity of NOS in old oocytes may be mediated by increased levels and activity of arginase II, thereby influencing NO bioavailability.