Expression of Neuronal Nitric Oxide Synthase in the Extratesticular Pathway and Its Role in Murine Sperm Maturation

Abstract

Neuronal nitric oxide synthase (nNOS) is one of two constitutive enzyme variants responsible for the production of nitric oxide (NO) from L‐arginine in mammalian cells. In somatic cells, this membrane‐associated protein has been shown to be activated by Ca2+ and to interact with Plasma Membrane Calcium ATPase 4 (PMCA4), the major calcium efflux pump in murine sperm (Wennemuth et. al. 2003), which negatively regulates it. NO is an important second messenger, and is required for a variety of sperm functions, including motility and fertilizing ability (Ramya et. al. 2011). Recently, the DeLeon lab has shown the expression of PMCA4 in the murine epididymis. However, no work has been done on the expression of nNOS in the extratesticular pathway of any mammalian species. Therefore, we set out to investigate the expression pattern of nNOS in the post‐testicular pathway and its role in sperm maturation. Here, we show the presence of nNOS in all three regions of the murine epididymis (caput, corpus, and cauda), including the basal and apical regions of the cells, via immunofluorescence for the first time. Western blotting results confirmed the expression of nNOS throughout the epididymis, and showed differential expression of nNOS, with highest expression in the corpus. Furthermore, preliminary immunofluorescence results suggest that the process of capacitation, in which the sperm experience a major influx of calcium, may influence the localization of nNOS. Experiments to isolate epididymosomes and determine if they are able to deliver nNOS to sperm are in progress.