Multiparameter Flow Cytometry Assay for Analysis of Nitrosative Stress Status in Human Spermatozoa. Issue 12 (1st July 2020)

• Record Type: Journal Article
• Authors: Uribe, Pamela; Meriño, Juan; Manquemilla, Emilio; Villagrán, Camila; Vega, Etelinda; Zambrano, Fabiola; Schulz, Mabel; Pezo, Felipe; Villegas, Juana V.; Boguen, Rodrigo; Sánchez, Raúl
• Is Part Of: Cytometry. Volume 97:Issue 12(2020)
• Page Start: 1238
• Page End: 1247
• Publication Date: 2020-07-01
• Subjects: human sperm -- sperm function -- mitochondria -- reactive nitrogen species -- multiparametric flow cytometry; Flow cytometry -- Periodicals; Imaging systems in biology -- Periodicals; Imaging systems in medicine -- Periodicals; Diagnostic imaging -- Periodicals; 571.605
• DOI: 10.1002/cyto.a.24170
• Languages: English
• ISSNs: 1552-4922

Abstract

Abstract: 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 Abstract : Presentation of data obtained from multiparameter assay of different quality spermatozoa as density plots. Representative examples of two different combinations of fluorochromes, viability versus mitochondrial membrane potential (MMP) and thiol oxidation versus peroxynitrite levels and the distribution of sperm populations from swim up spermatozoa (A, D ), swim up spermatozoa exposed to SIN‐1 (B, E ), and spermatozoa from a native semen sample (C, F ) are shown. Three populations are clearly seen in the PI versus TMRM plot: live‐high MMP, live‐low MMP, and dead‐low MMP. These three subpopulations were gated from the native semen sample (C) and analyzed for thiol oxidation and peroxynitrite production in G, H, and I respectively. SIN‐1: 3‐morpholinosydnonimine; PI: propidium iodide; TMRM: tetramethylrhodamine methyl ester perchlorate; Fl‐B: fluorescein‐boronate; mBBr: monobromobimane.