Nitric oxide-targeted protein phosphorylation during human sperm capacitation

Florentin-Daniel Staicu,Carmen Matás,Juan Carlos Martínez-Soto,Sebastian Canovas

doi:10.1038/s41598-021-00494-1

Abstract

Among many other molecules, nitric oxide insures the correct progress of sperm capacitation by mediating phosphorylation events. For a more comprehensive understanding of how this happens, we capacitated human spermatozoa from healthy men in the presence/absence of S-Nitrosoglutathione, a nitric oxide donor, two nitric oxide synthase inhibitors, NG-Nitro-l-arginine Methyl Ester Hydrochloride and Aminoguanidine Hemisulfate salt and, finally, with/without l-Arginine, the substrate for nitric oxide synthesis, and/or human follicular fluid. When analyzing the phosphorylation of protein kinase A substrates and tyrosine residues, we particularly observed how the inhibition of nitric oxide synthesis affects certain protein bands (~ 110, ~ 87, ~ 75 and ~ 62 kD) by lowering their phosphorylation degree, even when spermatozoa were incubated with l-Arginine and/or follicular fluid. Mass spectrometry analysis identified 29 proteins in these species, related to: spermatogenesis, binding to the zona pellucida, energy and metabolism, stress response, motility and structural organization, signaling and protein turnover. Significant changes in the phosphorylation degree of specific proteins could impair their biological activity and result in severe fertility-related phenotypes. These findings provide a deeper understanding of nitric oxide’s role in the capacitation process, and consequently, future studies in infertile patients should determine how nitric oxide mediates phosphorylation events in the species here described.

Highlights

Among many other molecules, nitric oxide insures the correct progress of sperm capacitation by mediating phosphorylation events
Another pathway through which Nitric oxide (NO) can modulate protein phosphorylation is by activating the soluble isoform of guanylate cyclase and, increasing the intracellular concentration of cyclic guanosine monophosphate17. cGMP activates the cGMP-dependent protein kinase (PKG)[18,19] which leads to the serine/threonine phosphorylation of proteins that promote sperm capacitation and acrosome reaction[20,21]
When adding l-Arginine, the relative amount of signal for the ~ 87 kD species was lower with l-NAME than in the GSNO group (P < 0.05; Fig. 2a,e; Supplementary Fig. S2)

Summary

Introduction

Nitric oxide insures the correct progress of sperm capacitation by mediating phosphorylation events. Significant changes in the phosphorylation degree of specific proteins could impair their biological activity and result in severe fertility-related phenotypes These findings provide a deeper understanding of nitric oxide’s role in the capacitation process, and future studies in infertile patients should determine how nitric oxide mediates phosphorylation events in the species here described. NO can modulate the capacitation via protein S-nitrosylation[12] and activation of the cyclic adenosine monophosphate/ protein kinase A (cAMP/PKA) pathway[13] The latter leads to an increase in the phosphorylation levels of sperm proteins, in serine, threonine and tyrosine r esidues[14,15]. Since cGMP and cAMP compete for the catalytic sites of p hosphodiesterases[22,23], an increase in the Physiology, Nursery Faculty, University of Murcia, International Excellence Campus for Higher Education and Scientific Reports | (2021) 11:20979

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Conclusion