Human spermatozoa contain multiple targets for protein S-nitrosylation: An alternative mechanism of the modulation of sperm function by nitric oxide? (original) (raw)
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Development, 2008
Generation of NO by nitric oxide synthase (NOS) is implicated in gamete interaction and fertilisation. Exposure of human spermatozoa to NO donors caused mobilisation of stored Ca 2+ by a mechanism that did not require activation of guanylate cyclase but was mimicked by S-nitrosoglutathione (GSNO; an S-nitrosylating agent). Application of dithiothreitol, to reduce protein-SNO groups, rapidly reversed the actions of NO and GSNO on [Ca 2+ ] i. The effects of NO, GSNO and dithiothreitol on sperm protein S-nitrosylation, assessed using the biotin switch method, closely paralleled their actions on [Ca 2+ ] i. Immunofluorescent staining revealed constitutive and inducible NOS in human oviduct and cumulus (the cellular layer investing the oocyte). 4,5diaminofluorescein (DAF) staining demonstrated production of NO by these tissues. Incubation of human sperm with oviduct explants induced sperm protein S-nitrosylation resembling that induced by NO donors and GSNO. Progesterone (a product of cumulus cells) also mobilises stored Ca 2+ in human sperm. Pre-treatment of sperm with NO greatly enhanced the effect of progesterone on [Ca 2+ ] i , resulting in a prolonged increase in flagellar excursion. We conclude that NO regulates mobilisation of stored Ca 2+ in human sperm by protein S-nitrosylation, that this action is synergistic with progesterone and that this synergism is potentially highly significant in gamete interactions leading to fertilisation.
Nitric Oxide: Key Features in Spermatozoa
Nitric Oxide Synthase - Simple Enzyme-Complex Roles, 2017
Several in vitro studies have pointed to the importance of nitric oxide (NO) in the female and male reproductive system in mammals. Its functions vary from preventing oocyte aging, improving the integrity of the microtubular spindle apparatus in aged oocytes, modulating the contraction of the oviduct, to regulating sperm physiology by affecting the motility, inducing chemotaxis in spermatozoa, regulating tyrosine phosphorylation, enhancing the sperm-zona pellucida binding ability, and modulating the acrosomal reaction. In spermatozoa, NO exerts its functions in different ways, which involve key elements such as the soluble isoform of guanylate cyclase, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), adenylate cyclase, and the extracellular signal-regulated kinase (ERK) pathway. Furthermore, NO leads to the S-nitrosylation of several sperm proteins, among them a substantial group associated with energy generation and cell movement, but also with signal transduction, suggesting a role for S-nitrosylation in sperm motility and in modulating the sperm function, respectively. In this chapter, an overview of how NO modulates the sperm physiology is presented, based on the knowledge acquired to this day.
The Roles of NO and H2S in Sperm Biology: Recent Advances and New Perspectives
International Journal of Molecular Sciences, 2020
After being historically considered as noxious agents, nitric oxide (NO) and hydrogen sulfide (H2S) are now listed as gasotransmitters, gaseous molecules that play a key role in a variety of cellular functions. Both NO and H2S are endogenously produced, enzymatically or non-enzymatically, and interact with each other in a range of cells and tissues. In spite of the great advances achieved in recent decades in other biological systems, knowledge about H2S function and interactions with NO in sperm biology is in its infancy. Here, we aim to provide an update on the importance of these molecules in the physiology of the male gamete. Special emphasis is given to the most recent advances in the metabolism, mechanisms of action, and effects (both physiological and pathophysiological) of these gasotransmitters. This manuscript also illustrates the physiological implications of NO and H2S observed in other cell types, which might be important for sperm function. The relevance of these gasot...
Fertility and Sterility, 2007
Objective: To investigate whether nitric oxide (NO) may attract human spermatozoa via activation of the soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway. Design: Prospective study. Setting: Academic research institution. Patient(s): Seven normozoospermic patients belonging to couples presenting for infertility evaluation. Intervention(s): Sperm samples were processed by the swim-up technique. Main Outcome Measure(s): Sperm chemotaxis detected by a choice device (specially designed three-well plexiglass chamber), intracellular level of cGMP (by radioimmunoassay), and sperm motility parameters (by computer-assisted sperm analysis).
Biology of Reproduction, 2003
Reactive oxygen species (superoxide anion, hydrogen peroxide, and nitric oxide) are involved in human sperm capacitation and associated tyrosine (Tyr) phosphorylation through a cAMPand protein kinase A-mediated pathway. Recently, we evidenced the double phosphorylation of the threonine-glutamine-Tyr motif (P-Thr-Glu-Tyr-P) in human sperm proteins of 80 and 105 kDa during capacitation. The objective of the present study was to investigate the role of reactive oxygen species in the regulation of this process and to immunolocalize the P-Thr-Glu-Tyr-P motif in human spermatozoa. Superoxide dismutase and catalase did not prevent, and exogenous addition of superoxide anion or hydrogen peroxide did not trigger, the increase in P-Thr-Glu-Tyr-P related to sperm capacitation. However, L-NAME (a competitive inhibitor of L-arginine for nitric oxide synthase) prevented, and a nitric oxide donor promoted, the increase in P-Thr-Glu-Tyr-P related to sperm capacitation. In addition, L-arginine reversed the inhibitory effect of L-NAME on capacitation and the associated increase of P-Thr-Glu-Tyr-P. Therefore, the regulation of P-Thr-Glu-Tyr-P is specific to nitric oxide and not to superoxide anion or hydrogen peroxide. The nitric oxide-mediated increase of P-Thr-Glu-Tyr-P involved protein Tyr kinase, MEK or MEK-like kinase, and protein kinase C but not protein kinase A. The P-Thr-Glu-Tyr-P motif was immunolocalized to the principal piece region of spermatozoa. In conclusion, nitric oxide regulates the level of P-Thr-Glu-Tyr-P in sperm proteins of 80 and 105 kDa during capacitation. These data evidence, to our knowledge for the first time, a specific role for nitric oxide in signal transduction events leading to sperm capacitation.
Signaling Pathway of Nitric Oxide-Induced Acrosome Reaction in Human Spermatozoa1
Biology of Reproduction, 2001
Nitric oxide (NO) has been recently shown to modulate in vitro motility, viability, the acrosome reaction (AR), and metabolism of spermatozoa in various mammalian species, but the mechanism or mechanisms through which it influences sperm functions has not been clarified. In human capacitated spermatozoa, both the intracellular cGMP level and the percentage of AR-positive cells were significantly increased after 4 h of incubation with the NO donor, sodium nitroprusside (SNP). SNPinduced AR was significantly reduced in the presence of the soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ; this block was bypassed by adding 8-bromo-cGMP, a cell-permeating cGMP analogue, to the incubation medium. Finally, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, two inhibitors of the cGMP-dependent protein kinases (PKGs), inhibited the SNP-induced AR. Furthermore, SNP-induced AR did not occur in Ca 2؉free medium or in the presence of the protein kinase C (PKC) inhibitor, calphostin C. This study suggests that the AR-inducing effect of exogenous NO on capacitated human spermatozoa is accomplished via stimulation of an NO-sensitive sGC, cGMP synthesis, and PKG activation. In this effect the activation of PKC is also involved, and the presence of extracellular Ca 2؉ is required.
Effects of Nitric Oxide Exposure on Human Sperm Function and Apoptosis Markers
The Open Reproductive Science Journal, 2014
Nitric oxide (NO) is a signaling molecule produced by intracellular nitric oxide synthase (NOS) enzymes. Studies have shown that this free radical affect sperm capacitation, a maturation step preceding acrosome reaction. This study was aimed at investigating the effects of exogenously administered NO through its donor, sodium nitroprusside (SNP) has on human sperm motility, viability and apoptosis markers. Increased concentrations of SNP (10, 30, 50, 100 M) were administered to human spermatozoa in the presence or absence of NO synthase inhibitor, N-nitro-Larginine methyl ester. Spermatozoa motility and viability were assessed at 60 and 90 minutes of incubation. The caspase activity was assessed after 90 minutes of incubation. SNP significantly decreased spermatozoa motility and viability in a dose and time dependent manner (p < 0.05). The caspase activity was significantly increased with increasing concentration of SNP (p < 0.05). This study therefore conclude that high concentrations of NO result in the decrease of sperm function and increase of germ cell apoptosis rate that may contribute to male infertility.
Sperm nitric oxide and motility: the effects of nitric oxide synthase stimulation and inhibition
Molecular Human Reproduction, 1997
Nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as the nitric oxide synthases (NOS). We have recently shown a NOS similar to constitutive brain NOS (bNOS) and endothelial NOS (ecNOS) to be present in spermatozoa. The aim of this study is to investigate NO production by human spermatozoa and the effects of stimulation and inhibition of NOS. This was carried out using the Iso-NO, an isolated NO meter and sensor, which provides rapid, accurate and direct measurements of NO. Semen samples with normozoospermic and asthenozoospermic profiles were prepared using a direct swim-up technique. Basal concentrations of NO and stimulated NO production were measured after exposure to the calcium ionophore (A23187; 0.01-10 µM) a potent activator of constitutive NOS. NO production in human spermatozoa was significantly increased by the addition of A23187 30 seconds after stimulation. Furthermore, this response was greatly diminished by pre-incubating the samples with competitive inhibitors of L-arginine, the substrate for NOS, before treatment with calcium ionophore. In the presence of N G-nitro-L-arginine methyl ester (L-NAME), N G-nitro-L-arginine (L-NA) or N G-methyl-L-arginine (L-NMMA; all at 10 µM), NO production was inhibited with a rank order of potency L-NAME Ͼ L-NMMA Ͼ L-NA which is in accordance with the inhibition of an endothelial type of constitutive NOS.
Nitric Oxide Regulates Human Sperm Capacitation and Protein-Tyrosine Phosphorylation In Vitro
Biology of Reproduction, 1999
The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.
Role of Nitric Oxide Concentrations on Human Sperm Motility
2004
Nitric oxide (NO) is a free radical generated from the oxidation of L-arginine to L-citrulline by 3 isoforms of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent NO synthases. Several data suggest a relevant role in sperm cell pathophysiology, but any conclusive data on its role in spermatozoa motility are still lacking. In the present study, we have correlated NO concentration in semen and kinetic features of sperm cells from normozoospermic fertile donors and infertile patients affected by idiopathic asthenozoospermia. Normozoospermic fertile men exhibited NO concentrations that were significantly lower than those of asthenozoospermic infertile men. A significant linear negative correlation was evident between NO concentration and percentage of total sperm motility. A further significant linear negative correlation was found between NO concentration and spermatozoa kinetic characteristics determined by a computerized analysis (curvilinear and straight progressive velocity). These data suggest that the overproduction of this free radical and the consequent excessive exposure to oxidative conditions have a potential pathogenetic implication in the reduction of sperm motility. The positive role played by NO in spermatozoa capacitation leads us to speculate that such paradoxical involvement in both pathologic and physiologic processes depends on the alternative redox state and relative level of NO.