Regulation of boar sperm functionality by the nitric oxide synthase/nitric oxide system

Abstract

Purpose Nitric oxide (NO) is a free radical synthesized mainly by nitric oxide synthases (NOSs). NO regulates many aspects in sperm physiology in different species. However, in vitro studies investigating NOS distribution, and how NO influences sperm capacitation and fertilization (IVF) in porcine, have been lacking. Therefore, our study aimed to clarify these aspects. Methods Two main experiments were conducted: (i) boar spermatozoa were capacitated in the presence/absence of S-nitrosoglutathione (GSNO), a NO donor, and two NOS inhibitors, N G -nitro-L-arginine methyl ester hydrochloride (L-NAME) and aminoguanidine hemisulfate salt (AG), and (ii) IVF was performed in the presence or not of these supplements, but neither the oocytes nor the sperm were previously incubated in the supplemented media. Results Our results suggest that NOS distribution could be connected to pathways which lead to capacitation. Treatments showed significant differences after 30 min of incubation, compared to time zero in almost all motility parameters ( P < 0.05). When NOSs were inhibited, three protein kinase A (PKA) substrates (~ 75, ~ 55, and ~50 kDa) showed lower phosphorylation levels between treatments ( P < 0.05). No differences were observed in total tyrosine phosphorylation levels evaluated by Western blotting nor in situ. The percentage of acrosome-reacted sperm and phosphatidylserine translocation was significantly lower with L-NAME. Both inhibitors reduced sperm intracellular calcium concentration and IVF parameters, but L-NAME impaired sperm ability to penetrate denuded oocytes. Conclusions These findings point out to the importance of both sperm and cumulus-oocyte-derived NO in the IVF outcome in porcine. Electronic supplementary material The online version of this article (10.1007/s10815-019-01526-6) contains supplementary material, which is available to authorized users.

Molecular Mechanisms Involved in the Impairment of Boar Sperm Motility by Peroxynitrite-Induced Nitrosative Stress

“…Both signaling pathways, GSK-3α and PKA, are essential in the regulation of boar sperm motility [35][36][37]. Part of our results with ONOO − are in line with a recent study in boar spermatozoa that found that phosphorylation of PKA substrates is significantly lower when using NO synthase inhibitors [27]. The peroxynitrite-induced increase in GSK-3α phosphorylation is greater in TCM (2.4 fold-increase over control), than in TBM (1.7 fold-increase) as stimulating medium TCM alone is already promoting higher levels of phosphorylated GSK-3α than the un-stimulating TBM.…”

“…Specifically, in boar spermatozoa there are discrepancies regarding the involvement of NO pathway in the acrosome reaction. Thus, Staicu et al, (2019) [27] recently found that exogenous NO does not affect acrosome reaction, whereas Hou et al, (2008) [28] described that NO donor increases the number of acrosome-reacted spermatozoa. Our results with ONOO-support the idea that the pathway of NO, including related RNS, is not likely involved in the acrosome reaction of boar spermatozoa.…”

Does Melatonin Exert Its Effect on Ram Sperm Capacitation Through Nitric Oxide Synthase Regulation?

“…Meiser and Schulz [20] localized the nNOS isoform in the apical region of the head and tail of bull spermatozoa, whereas the eNOS was located only in the apical region. In ejaculated boar spermatozoa, eNOS and nNOS were identified in the acrosome and iNOS in the whole head, neck and flagellum [40]. However, no immunotypes or changes in NOS localization after in vitro capacitation were reported for bull or boar spermatozoa.…”

“…This behavior could be explained by the different roles of the NOS isoforms. Staicu et al [40] postulated that the constitutive isoforms (eNOS and nNOS) could be involved in sperm capacitation and that iNOS could be related to inflammation or stress, so the changes observed in iNOS during in vitro capacitation with melatonin could not exactly be related to this process. Taking into account the bimodal role that melatonin has on ram sperm in in vitro capacitation, stimulating this process at low concentrations (100 pM) and preventing it at high doses (1 µM) [27], it is quite surprising that both melatonin concentrations have similar effects on iNOS and nNOS distribution.…”

The Roles of NO and H2S in Sperm Biology: Recent Advances and New Perspectives

“…Furthermore, it is still unclear whether the physiological state (e.g., capacitation) of sperm cells may affect NOS distribution. In a recent study in capacitated boar spermatozoa, Staicu et al [23] found that the eNOS and nNOS are mainly distributed in the sperm head, whereas iNOS is localized in both the sperm head and the flagellum. The study also suggested a link between NOSs distribution and sperm normal function (capacitation, acrosome reaction, tyrosine phosphorylation, and Ca 2+ flux).…”