Multiparameter Flow Cytometry Assay for Analysis of Nitrosative Stress Status in Human Spermatozoa.
human sperm
mitochondria
multiparametric flow cytometry
reactive nitrogen species
sperm function
Journal
Cytometry. Part A : the journal of the International Society for Analytical Cytology
ISSN: 1552-4930
Titre abrégé: Cytometry A
Pays: United States
ID NLM: 101235694
Informations de publication
Date de publication:
12 2020
12 2020
Historique:
received:
08
01
2020
revised:
20
05
2020
accepted:
08
06
2020
pubmed:
13
6
2020
medline:
19
8
2021
entrez:
13
6
2020
Statut:
ppublish
Résumé
Male infertility is an increasing health problem, and oxidative/nitrosative stress plays an important role in the etiology of this condition. Nitrosative stress due to excessive levels of reactive nitrogen species (RNS) is associated with impaired male fertility. Flow cytometry may be a useful tool for semen evaluation, but the availability of multiparameter assays for analysis of sperm quality is limited. The present study standardized a multiparameter flow cytometry analysis for nitrosative stress status in human spermatozoa in a single assay. A suitable multicolor fluorochrome panel was designed and consisted of fluorescein-boronate to detect peroxynitrite, a highly RNS, propidium iodide to analyze viability, tetramethylrhodamine methyl ester perchlorate to detect mitochondrial membrane potential (MMP) and monobromobimane to analyze thiol oxidation. Proper positive and negative controls for each fluorochrome were used to establish the technique, and sperm cells of different qualities and spermatozoa subjected to cryopreservation were analyzed. The results showed that the controls clearly discriminated between the high and low fluorescence intensities for each fluorochrome. The analysis of sperm cells of different quality demonstrated that the assay properly detected differences in all parameters analyzed according to sperm quality. The results may be reported as the mean fluorescence intensity of each fluorochrome and the percentage of cells exhibiting different characteristics. In conclusion, a protocol was standardized to analyze nitrosative stress status, including peroxynitrite production, viability, MMP, and thiol oxidation, in a single analysis using flow cytometry. This protocol may be applied to research approaches and clinical andrology to improve the evaluation of sperm quality and provide a promising tool to increase the use of clinical flow cytometry. © 2020 International Society for Advancement of Cytometry.
Identifiants
pubmed: 32530108
doi: 10.1002/cyto.a.24170
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1238-1247Informations de copyright
© 2020 International Society for Advancement of Cytometry.
Références
Szkodziak P, Wozniak S, Czuczwar P, Wozniakowska E, Milart P, Mroczkowski A, Paszkowski T. Infertility in the light of new scientific reports - Focus on male factor. Ann Agric Environ Med 2016;23:227-230.
Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol 2015;13:37.
Aitken RJ, Smith TB, Jobling MS, Baker MA, De Iuliis GN. Oxidative stress and male reproductive health. Asian J Androl 2014;16:31-38.
Agarwal A, Parekh N, Panner Selvam MK, Henkel R, Shah R, Homa ST, Ramasamy R, Ko E, Tremellen K, Esteves S, et al. Male oxidative stress infertility (MOSI): Proposed terminology and clinical practice guidelines for management of idiopathic male infertility. World J Mens Health 2019;37:296-312.
Szabo C, Ischiropoulos H, Radi R. Peroxynitrite: Biochemistry, pathophysiology and development of therapeutics. Nat Rev Drug Discov 2007;6:662-680.
Salvolini E, Buldreghini E, Lucarini G, Vignini A, Di Primio R, Balercia G. Nitric oxide synthase and tyrosine nitration in idiopathic asthenozoospermia: An immunohistochemical study. Fertil Steril 2012;97:554-560.
Vignini A, Nanetti L, Buldreghini E, Moroni C, Ricciardo-Lamonica G, Mantero F, Boscaro M, Mazzanti L, Balercia G. The production of peroxynitrite by human spermatozoa may affect sperm motility through the formation of protein nitrotyrosine. Fertil Steril 2006;85:947-953.
Vignini A, Buldreghini E, Nanetti L, Amoroso S, Boscaro M, Ricciardo-Lamonica G, Mazzanti L, Balercia G. Free thiols in human spermatozoa: Are Na+/K+-ATPase, Ca2+-ATPase activities involved in sperm motility through peroxynitrite formation? Reprod Biomed Online 2009;18:132-140.
Khosravi F, Valojerdi MR, Amanlou M, Karimian L, Abolhassani F. Relationship of seminal reactive nitrogen and oxygen species and total antioxidant capacity with sperm DNA fragmentation in infertile couples with normal and abnormal sperm parameters. Andrologia 2014;46:17-23.
Cassina A, Silveira P, Cantu L, Montes JM, Radi R, Sapiro R. Defective human sperm cells are associated with mitochondrial dysfunction and oxidant production. Biol Reprod 2015;93:119.
Oztezcan S, Turkoglu UM, Kervancioglu E, Kocak T, Kocak-Toker N, Aykac-Toker G. In vitro effects of peroxynitrite on human spermatozoa. Andrologia 1999;31:195-198.
Uribe P, Boguen R, Treulen F, Sanchez R, Villegas JV. Peroxynitrite-mediated nitrosative stress decreases motility and mitochondrial membrane potential in human spermatozoa. Mol Hum Reprod 2015;21:237-243.
Uribe P, Treulen F, Boguen R, Sanchez R, Villegas JV. Nitrosative stress by peroxynitrite impairs ATP production in human spermatozoa. Andrologia 2017;49:e12615.
Cabrillana ME, Uribe P, Villegas JV, Alvarez J, Sanchez R, Fornes MW. Thiol oxidation by nitrosative stress: Cellular localization in human spermatozoa. Syst Biol Reprod Med 2016;62:325-334.
Uribe P, Cabrillana ME, Fornes MW, Treulen F, Boguen R, Isachenko V, Isachenko E, Sanchez R, Villegas JV. Nitrosative stress in human spermatozoa causes cell death characterized by induction of mitochondrial permeability transition-driven necrosis. Asian J Androl 2018;20:600-607.
World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. Geneva, Switzerland: WHO Press, 2010;p. 271.
Martinez-Pastor F, Mata-Campuzano M, Alvarez-Rodriguez M, Alvarez M, Anel L, de Paz P. Probes and techniques for sperm evaluation by flow cytometry. Reprod Domest Anim 2010;45(Suppl 2):67-78.
Ortega-Ferrusola C, Gil MC, Rodriguez-Martinez H, Anel L, Pena FJ, Martin-Munoz P. Flow cytometry in spermatology: A bright future ahead. Reprod Domest Anim 2017;52:921-931.
Pena FJ, Ortiz Rodriguez JM, Gil MC, Ortega Ferrusola C. Flow cytometry analysis of spermatozoa: Is it time for flow spermetry? Reprod Domest Anim 2018;53(Suppl 2):37-45.
Pena FJ, Ball BA, Squires EL. A new method for evaluating stallion sperm viability and mitochondrial membrane potential in fixed semen samples. Cytometry B Clin Cytom 2018;94:302-311.
Ortega-Ferrusola C, Anel-Lopez L, Martin-Munoz P, Ortiz-Rodriguez JM, Gil MC, Alvarez M, de Paz P, Ezquerra LJ, Masot AJ, Redondo E, et al. Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes. Reproduction 2017;153:293-304.
Gil MC, Ferrusola CO, Anel-Lopez L, Ortiz-Rodriguez JM, Alvarez M, de Paz P, Anel L, Pena FJ. A simple flow cytometry protocol to determine simultaneously live, dead and apoptotic stallion spermatozoa in fresh and frozen thawed samples. Anim Reprod Sci 2018;189:69-76.
Boguen R, Treulen F, Uribe P, Villegas JV. Ability of Escherichia coli to produce hemolysis leads to a greater pathogenic effect on human sperm. Fertil Steril 2015;103:1155-1161.
Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985;44:493-498.
Rios N, Piacenza L, Trujillo M, Martinez A, Demicheli V, Prolo C, Alvarez MN, Lopez GV, Radi R. Sensitive detection and estimation of cell-derived peroxynitrite fluxes using fluorescein-boronate. Free Radic Biol Med 2016;101:284-295.
Uribe P, Villegas JV, Boguen R, Treulen F, Sanchez R, Mallmann P, Isachenko V, Rahimi G, Isachenko E. Use of the fluorescent dye tetramethylrhodamine methyl ester perchlorate for mitochondrial membrane potential assessment in human spermatozoa. Andrologia 2017;49:e12753.
Garner DL, Johnson LA. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod 1995;53:276-284.
Kosower NS, Kosower EM. Thiol labeling with bromobimanes. Methods Enzymol 1987;143:76-84.
Baldi E, Tamburrino L, Muratori M, Degl'Innocenti S, Marchiani S. Adverse effects of in vitro manipulation of spermatozoa. Anim Reprod Sci 2020. https://doi.org/10.1016/j.anireprosci.2020.106314.
Hezavehei M, Sharafi M, Kouchesfahani HM, Henkel R, Agarwal A, Esmaeili V, Shahverdi A. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reprod Biomed Online 2018;37:327-339.
Kumar A, Prasad JK, Srivastava N, Ghosh SK. Strategies to minimize various stress-related freeze-thaw damages during conventional cryopreservation of mammalian spermatozoa. Biopreserv Biobank 2019;17:603-612.
Ortega Ferrusola C, Gonzalez Fernandez L, Macias Garcia B, Salazar-Sandoval C, Morillo Rodriguez A, Rodriguez Martinez H, Tapia JA, Pena FJ. Effect of cryopreservation on nitric oxide production by stallion spermatozoa. Biol Reprod 2009;81:1106-1111.
Chatterjee S, Gagnon C. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Mol Reprod Dev 2001;59:451-458.
Uribe P, Rojas C, Merino J, Zambrano F, Villegas JV, Treulen F, Boguen R, Isachenko V, Isachenko E, Sanchez R. Effect of incubation temperature after devitrification on quality parameters in human sperm cells. Cryobiology 2017;79:78-81.
De Jonge C, Barratt CLR. The present crisis in male reproductive health: An urgent need for a political, social, and research roadmap. Andrology 2019;7:762-768.
Doshi SB, Khullar K, Sharma RK, Agarwal A. Role of reactive nitrogen species in male infertility. Reprod Biol Endocrinol 2012;10:109.
Yaniz JL, Soler C, Alquezar-Baeta C, Santolaria P. Toward an integrative and predictive sperm quality analysis in Bos taurus. Anim Reprod Sci 2017;181:108-114.
Agarwal A, Parekh N, Panner Selvam MK, Henkel R, Shah R, Homa ST, Ramasamy R, Ko E, Tremellen K, Esteves S, et al. Male oxidative stress infertility (MOSI): Proposed terminology and clinical practice guidelines for management of idiopathic male infertility. World J Mens Health 2019;37:296-312.
Amaral A, Ramalho-Santos J. Assessment of mitochondrial potential: Implications for the correct monitoring of human sperm function. Int J Androl 2010;33:e180-e186.
Espinoza JA, Schulz MA, Sanchez R, Villegas JV. Integrity of mitochondrial membrane potential reflects human sperm quality. Andrologia 2009;41:51-54.
Gallon F, Marchetti C, Jouy N, Marchetti P. The functionality of mitochondria differentiates human spermatozoa with high and low fertilizing capability. Fertil Steril 2006;86:1526-1530.
Marchetti P, Ballot C, Jouy N, Thomas P, Marchetti C. Influence of mitochondrial membrane potential of spermatozoa on in vitro fertilisation outcome. Andrologia 2012;44:136-141.
Galluzzi L, Bravo-San Pedro JM, Vitale I, Aaronson SA, Abrams JM, Adam D, Alnemri ES, Altucci L, Andrews D, Annicchiarico-Petruzzelli M, et al. Essential versus accessory aspects of cell death: Recommendations of the NCCD 2015. Cell Death Differ 2015;22:58-73.
Kosaka S, Takeda M, Ochi T, Miyahara K, Nakamura E, Tada N, Lane GJ, Yamataka A. Compromised vitality of spermatozoa after contact with colonic mucosa in mice: Implications for fertility in colon vaginoplasty patients. Pediatr Surg Int 2019;35:71-75.
Sancho S, Briz M, Yeste M, Bonet S, Bussalleu E. Effects of the antimicrobial peptide protegrine 1 on sperm viability and bacterial load of boar seminal doses. Reprod Domest Anim 2017;52(Suppl 4):69-71.
de Vasconcelos Franco JS, Faheem M, Chaveiro A, Moreira da Silva F. Effects of alpha-tocopherol and freezing rates on the quality and heterologous in vitro fertilization capacity of stallion sperm after cryopreservation. Theriogenology 2016;86:957-962.
Treulen F, Arias ME, Aguila L, Uribe P, Felmer R. Cryopreservation induces mitochondrial permeability transition in a bovine sperm model. Cryobiology 2018;83:65-74.
Mizrahi R, Breitbart H. Mitochondrial PKA mediates sperm motility. Biochim Biophys Acta 1840;2014:3404-3412.
Arias ME, Andara K, Briones E, Felmer R. Bovine sperm separation by swim-up and density gradients (Percoll and BoviPure): Effect on sperm quality, function and gene expression. Reprod Biol 2017;17:126-132.
Arroyo-Salvo C, Sanhueza F, Fuentes F, Treulen F, Arias ME, Cabrera P, Silva M, Felmer R. Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation. Reprod Domest Anim 2019;54:184-194.
Cabrillana ME, Monclus MA, Saez Lancellotti TE, Boarelli PV, Clementi MA, Vincenti AE, Yunes RF, Fornes MW. Characterization of flagellar cysteine-rich sperm proteins involved in motility, by the combination of cellular fractionation, fluorescence detection, and mass spectrometry analysis. Cytoskeleton (Hoboken) 2011;68:491-500.
Dias GM, Lopez ML, Ferreira AT, Chapeaurouge DA, Rodrigues A, Perales J, Retamal CA. Thiol-disulfide proteins of stallion epididymal spermatozoa. Anim Reprod Sci 2014;145:29-39.