Nitric oxide in frozen-thawed equine sperm: Effects on motility, membrane integrity and sperm capacitation

Nitric oxide (center dot NO) is a reactive nitrogen species (RSN) that, over the years, has been shown to be integrated with biological and physiological events, including reproductive processes. center dot NO can affect the functionality of spermatozoa through free radical scavenging, deactivating and inhibiting the production of superoxide anions (O-2(center dot-)). However, the role of center dot NO in mammalian spermatozoa physiology seems paradoxical. The aim of this study was to investigate the effects of center dot NO on motility, hyperactivation, membrane integrity, peroxidation, and capacitation in cryopreserved equine sperm. Ejaculates were collected and cryopreserved. After thawing, samples were centrifuged, suspended in an in vitro fertilization (IVF) medium and incubated with the following treatments: 1) C = control (IVF); 2) A = L-arginine (10 mM - In); 3) L = L-NAME (1 mM 1h); 4) M = methylene blue (100 mM- Re); 5) AL = L-arginine + L-NAME (In + Ih); 6) AM = L-arginine + methylene blue (In + Re). The samples were evaluated for spermatic kinetics by CASA and other analyses [plasma and acrosomal membranes used the propidium iodide (PI) and Pisum sativum agglutinin (PSA), detection of tyrosine residues phosphorylation in the membrane (F0426), nitric oxide (DAF-2/DA), lipid peroxidation (C11-BODIPY581/591)] by flow cytometry. The L-arginine treatments reduced MOT, PROG, RAP and LIN only at time 0 min compared to the control and L-NAME. These treatments (MT and MP, VAP, VSL, LIN, RAP) also reduced the sperm movement characteristics but only at the beginning of the incubation period. After this period of incubation, motility recovered. center dot NO removal by methylene blue almost completely inhibited sperm motility, but these treatments had the highest percentages of intact membranes. L-arginine treatments improved acrosome reactions and differed from M and AM. center dot NO production, tyrosine phosphorylation and lipid peroxidation did not differ among treatments, except for M and AM, where a reduction in these variables was detected. Therefore, equine sperm capacitation and the acrosome reaction are part of an oxidative process that involves the participation of ROS, and center dot NO plays an important role in the maintenance and regulation of motility, hyperactivation, induction of acrosome reaction and possibly in capacitation, which are indispensable processes for the fertility of equine sperm.