Nitric oxide in frozen-thawed equine sperm: Effects on motility, membrane integrity and sperm capacitation.
Abstract: Nitric oxide (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. NO can affect the functionality of spermatozoa through free radical scavenging, deactivating and inhibiting the production of superoxide anions (O). However, the role of NO in mammalian spermatozoa physiology seems paradoxical. The aim of this study was to investigate the effects of 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 - Ih); 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-BODIPY)] 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. 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. 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 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.
Copyright © 2018 Elsevier B.V. All rights reserved.
Publication Date: 2018-05-22 PubMed ID: 29861341DOI: 10.1016/j.anireprosci.2018.05.022Google Scholar: Lookup
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Summary
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This research explores the effect of nitric oxide on the motility, membrane integrity and potentiation (capacitation) of frozen-thawed equine sperm. The study finds that nitric oxide plays a crucial role in these processes, contributing significantly towards the fertility potential of horse sperm.
Objective of the Study
- The study was designed to investigate the influence of nitric oxide (NO) on several key parameters of equine sperm functionality: motility, hyperactivation, membrane integrity, oxidative stress, and potentiation (capacitation).
Methodology
- Equine sperm were frozen, thawed, and then suspended in an ‘in vitro’ fertilization medium.
- These samples were exposed to various treatments involving different combinations of l-arginine (a precursor to NO), L-NAME (an NO synthesis inhibitor), and methylene blue (a reductant).
- Following these treatments, the sperm samples went through evaluation for spermatic kinetics and various other analyses.
- The latter involved checking the integrity of the plasma and acrosomal membranes, tracking the phosphorylation of tyrosine residues, assessing NO production and lipid peroxidation.
Findings
- The study found that treatment with l-arginine initially reduced several motility characteristics of the sperm, although this effect was short-lived, with motility recovery observed after an incubation period.
- Interestingly, when NO was removed by methylene blue, sperm motility was almost completely inhibited. However, these samples showed the highest percentages of intact membranes.
- The l-arginine treatments were observed to improve acrosome reactions (possibly pointing towards successful capacitation) compared to other treatments.
- NO production, tyrosine phosphorylation, and lipid peroxidation levels did not present significant differences between treatments, with the exception of the treatments with methylene blue where these variables were reduced.
Conclusion
- Ultimately, the study concludes that nitric oxide plays a critical role in preserving and regulating motility, hyperactivation, and potentially capacitation in equine sperm – processes crucial for sperm fertility.
- Furthermore, these processes, including capacitation and acrosome reaction appear to be part of an oxidative process involving reactive oxygen species (ROS).
Implications
- The findings of this study could have significant implications for improving the success rates of ‘in vitro’ fertilization procedures and overall equine breeding practices.
- Further, they also contribute to the broader understanding of the complex relationships between NO and other reactive species in regulating sperm function.
Cite This Article
APA
de Andrade AFC, Arruda RP, Torres MA, Pieri NCG, Leite TG, Celeghini ECC, Oliveira LZ, Gardés TP, Bussiere MCC, Silva DF.
(2018).
Nitric oxide in frozen-thawed equine sperm: Effects on motility, membrane integrity and sperm capacitation.
Anim Reprod Sci, 195, 176-184.
https://doi.org/10.1016/j.anireprosci.2018.05.022 Publication
Researcher Affiliations
- Laboratory of Andrology and Technology of Swine Embryos, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Pirassununga, SP, Brazil. Electronic address: andrefc@usp.br.
- Laboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction,School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Pirassununga, SP, Brazil.
- Laboratory of Andrology and Technology of Swine Embryos, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Pirassununga, SP, Brazil.
- Laboratory of Andrology and Technology of Swine Embryos, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Pirassununga, SP, Brazil.
- Laboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction,School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Pirassununga, SP, Brazil.
- Laboratory of Teaching and Research in Pathology of Reproduction, Department of Animal Reproduction,School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Pirassununga, SP, Brazil.
- Department of Animal Pathology and Clinic, School of Veterinary Medicine, Federal Fluminense University, Niterói, RJ, Brazil.
- Laboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction,School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Pirassununga, SP, Brazil.
- Laboratory of Animal Reproduction and Genetics, Norte Fluminense University, Campus dos Goytacazes, RJ, Brazil.
- Laboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction,School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Pirassununga, SP, Brazil.
MeSH Terms
- Animals
- Cell Membrane / drug effects
- Cryopreservation / veterinary
- Horses
- Image Processing, Computer-Assisted
- Male
- Nitric Oxide / pharmacology
- Semen Preservation / veterinary
- Sperm Capacitation / drug effects
- Sperm Motility / drug effects
Citations
This article has been cited 6 times.- Staicu FD, Martínez-Soto JC, Canovas S, Matás C. Nitric oxide-targeted protein phosphorylation during human sperm capacitation.. Sci Rep 2021 Oct 25;11(1):20979.
- Ciani F, Maruccio L, Cocchia N, d'Angelo D, Carotenuto D, Avallone L, Namagerdi AA, Tafuri S. Antioxidants in assisted reproductive technologies: An overview on dog, cat, and horse.. J Adv Vet Anim Res 2021 Mar;8(1):173-184.
- Kadlec M, Ros-Santaella JL, Pintus E. The Roles of NO and H(2)S in Sperm Biology: Recent Advances and New Perspectives.. Int J Mol Sci 2020 Mar 21;21(6).
- Miguel-Jiménez S, Carvajal-Serna M, Calvo S, Casao A, Cebrián-Pérez JÁ, Muiño-Blanco T, Pérez-Pe R. Does Melatonin Exert Its Effect on Ram Sperm Capacitation Through Nitric Oxide Synthase Regulation?. Int J Mol Sci 2020 Mar 18;21(6).
- Alsalman ARS, Almashhedy LA, Alta'ee AH, Hadwan MH. Effect of Zinc Supplementation on Urate Pathway Enzymes in Spermatozoa and Seminal Plasma of Iraqi Asthenozoospermic Patients: A Randomized Controlled Trial.. Int J Fertil Steril 2020 Jan;13(4):315-323.
- Staicu FD, Lopez-Úbeda R, Romero-Aguirregomezcorta J, Martínez-Soto JC, Matás Parra C. Regulation of boar sperm functionality by the nitric oxide synthase/nitric oxide system.. J Assist Reprod Genet 2019 Aug;36(8):1721-1736.
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