486d448bceeff6c27360038c62ecffaf pmc_34572999.pdf 92e3fe57a1b7fee2820d2821ada6ea31d900c26d pmc_34572999.pdf 27b7601e8cf74d6f5879f754ec4f6b3975dbb7f6419d4a7e2a3b6e503bb81a63 pmc_34572999.pdf Title: Extracellular Reactive Oxygen Species (ROS) Production in Fresh Donkey Sperm Exposed to Reductive Stress, Oxidative Stress and NETosis Subject: Jenny shows a large endometrial reaction after semen influx to the uterus with a large amount of polymorphonuclear neutrophils (PMN) migrating into the uterine lumen. PMN act as a sperm selection mechanism through phagocytosis and NETosis (DNA extrudes and, together with proteins, trap spermatozoa). While a reduced percentage of spermatozoa are phagocytosed by PMN, most are found to be attached to neutrophil extracellular traps (NETs). This selection process together with sperm metabolism produces a large amount of reactive oxygen species (ROS) that influence the reproductive success. The present study aimed to determine the extracellular ROS production in both sperm and PMN. With this purpose, (1) donkey sperm were exposed to reductive and oxidative stresses, through adding different concentrations of reduced glutathione (GSH) and hydrogen peroxide (H2O2), respectively; and (2) PMN were subjected to NETosis in the presence of the whole semen, sperm, seminal plasma (SP) or other activators such as formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular ROS production (measured as H2O2 levels) was determined with the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. Donkey sperm showed more resilience to oxidative stress than to the reductive one, and GSH treatments led to greater H2O2 extracellular production. Moreover, not only did SP appear to be the main inducer of NETosis in PMN, but it was also able to maintain the extracellular H2O2 levels produced by sperm and NETosis. Keywords: reactive oxygen species (ROS); reduced glutathione (GSH); hydrogen peroxide (H2O2); polymorphonuclear neutrophils (PMN); NETosis; seminal plasma (SP); donkey Author: Iván Yánez-Ortiz, Jaime Catalán, Yentel Mateo-Otero, Marta Dordas-Perpinyà, Sabrina Gacem, Natalia Yeste, Anna Bassols, Marc Yeste and Jordi Miró Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Sep 15 03:59:50 2021 CEST ModDate: Wed Sep 15 05:58:36 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2510142 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ZMEFUK+VnURWPalladioL Type 1 Custom yes yes no 10 0 TWOHAU+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 IZSCDZ+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 RJUEYC+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 NEBEKD+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 KUDXZB+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 36 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 58 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 62 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 66 0 PRBWYS+CMSY10 Type 1 Builtin yes yes no 81 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 88 0 FDJELK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 105 0 FDJEML+PalatinoLinotype CID TrueType Identity-H yes yes yes 108 0 FDJEMM+PalatinoLinotype TrueType WinAnsi yes yes no 114 0 FDJEMO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 117 0 EUQOAW+EURM7 Type 1 Builtin yes yes no 125 0 FDJELK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 139 0 FDJEML+PalatinoLinotype CID TrueType Identity-H yes yes yes 142 0 FDJEMM+PalatinoLinotype TrueType WinAnsi yes yes no 148 0 FDJEMO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 151 0 FDJELK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 164 0 FDJEML+PalatinoLinotype CID TrueType Identity-H yes yes yes 167 0 FDJEMM+PalatinoLinotype TrueType WinAnsi yes yes no 173 0 FDJEMO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 176 0 FDJELK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 194 0 FDJEML+PalatinoLinotype CID TrueType Identity-H yes yes yes 197 0 FDJEMM+PalatinoLinotype TrueType WinAnsi yes yes no 203 0 FDJEMO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 206 0 FDJELK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 222 0 FDJEML+PalatinoLinotype CID TrueType Identity-H yes yes yes 225 0 FDJEMM+PalatinoLinotype TrueType WinAnsi yes yes no 231 0 FDJEMO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 234 0 FDJELK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 250 0 FDJEML+PalatinoLinotype CID TrueType Identity-H yes yes yes 253 0 FDJEMM+PalatinoLinotype TrueType WinAnsi yes yes no 259 0 FDJEMO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 262 0 FDJELK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 284 0 FDJEML+PalatinoLinotype CID TrueType Identity-H yes yes yes 287 0 FDJEMM+PalatinoLinotype TrueType WinAnsi yes yes no 293 0 FDJEMO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 296 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-22 02:45:43 CET RepresentationInformation: pmc_34572999.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2021-10-04 03:38:46 CEST Size: 2510142 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 582 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Animals and Statement of Ethics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Experimental Design Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Experiment 1. Sperm Exposure to Reductive Stress and Oxidative Stress Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: Sperm Collection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Seminal Plasma (SP) Collection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Treatments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Evaluation of Sperm Motility Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Evaluation of Sperm Viability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Determination of Extracellular H2O2 Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Experiment 2. Determination of Extracellular H2O2 Production by NETosis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Children: Item: Title: Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: PMN Isolation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Treatments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Evaluation of NETosis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Determination of Extracellular H2O2 Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Children: Item: Title: Experiment 1: Exposure of Sperm to Reductive Stress Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Children: Item: Title: Sperm Motility Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Sperm Viability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Extracellular Hydrogen Peroxide (H2O2) Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Experiment 1: Exposure of Sperm to Oxidative Stress Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Children: Item: Title: Sperm Motility Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Sperm Viability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Extracellular Hydrogen Peroxide (H2O2) Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: Experiment 2. Extracellular H2O2 Production by NETosis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Children: Item: Title: Reacted Polymorphonuclear Neutrophils (PMN) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Item: Title: Extracellular Hydrogen Peroxide (H2O2) Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@74ad1f1f Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6a1aab78 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@462d5aee Info: Title: Extracellular Reactive Oxygen Species (ROS) Production in Fresh Donkey Sperm Exposed to Reductive Stress, Oxidative Stress and NETosis Author: Iván Yánez-Ortiz, Jaime Catalán, Yentel Mateo-Otero, Marta Dordas-Perpinyà, Sabrina Gacem, Natalia Yeste, Anna Bassols, Marc Yeste and Jordi Miró Subject: Jenny shows a large endometrial reaction after semen influx to the uterus with a large amount of polymorphonuclear neutrophils (PMN) migrating into the uterine lumen. PMN act as a sperm selection mechanism through phagocytosis and NETosis (DNA extrudes and, together with proteins, trap spermatozoa). While a reduced percentage of spermatozoa are phagocytosed by PMN, most are found to be attached to neutrophil extracellular traps (NETs). This selection process together with sperm metabolism produces a large amount of reactive oxygen species (ROS) that influence the reproductive success. The present study aimed to determine the extracellular ROS production in both sperm and PMN. With this purpose, (1) donkey sperm were exposed to reductive and oxidative stresses, through adding different concentrations of reduced glutathione (GSH) and hydrogen peroxide (H2O2), respectively; and (2) PMN were subjected to NETosis in the presence of the whole semen, sperm, seminal plasma (SP) or other activators such as formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular ROS production (measured as H2O2 levels) was determined with the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. Donkey sperm showed more resilience to oxidative stress than to the reductive one, and GSH treatments led to greater H2O2 extracellular production. Moreover, not only did SP appear to be the main inducer of NETosis in PMN, but it was also able to maintain the extracellular H2O2 levels produced by sperm and NETosis. 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Annotation: Subtype: Link Rect: 519, 65, 555, 76 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 466, 721, 504, 732 Annotation: Subtype: Link Rect: 510, 721, 546, 732 Annotation: Subtype: Link Rect: 225, 698, 263, 709 Annotation: Subtype: Link Rect: 269, 698, 305, 709 Annotation: Subtype: Link Rect: 224, 675, 262, 686 Annotation: Subtype: Link Rect: 430, 652, 467, 663 Annotation: Subtype: Link Rect: 498, 640, 535, 651 Annotation: Subtype: Link Rect: 357, 617, 395, 628 Annotation: Subtype: Link Rect: 409, 594, 447, 605 Annotation: Subtype: Link Rect: 453, 594, 489, 605 Annotation: Subtype: Link Rect: 173, 572, 210, 582 Annotation: Subtype: Link Rect: 172, 526, 209, 536 Annotation: Subtype: Link Rect: 215, 526, 251, 536 Annotation: Subtype: Link Rect: 510, 502, 547, 513 Annotation: Subtype: Link Rect: 470, 479, 507, 490 Annotation: Subtype: Link Rect: 514, 479, 550, 490 Annotation: Subtype: Link Rect: 133, 457, 170, 467 Annotation: Subtype: Link Rect: 176, 457, 212, 467 Annotation: Subtype: Link Rect: 462, 433, 500, 444 Annotation: Subtype: Link Rect: 419, 410, 456, 421 Annotation: Subtype: Link Rect: 497, 399, 534, 410 Annotation: Subtype: Link Rect: 95, 365, 133, 375 Annotation: Subtype: Link Rect: 312, 341, 350, 352 Annotation: Subtype: Link Rect: 356, 341, 392, 352 Annotation: Subtype: Link Rect: 59, 308, 97, 318 Annotation: Subtype: Link Rect: 103, 308, 139, 318 Annotation: Subtype: Link Rect: 95, 273, 133, 283 Annotation: Subtype: Link Rect: 77, 250, 115, 260 Annotation: Subtype: Link Rect: 319, 226, 357, 237 Annotation: Subtype: Link Rect: 363, 226, 399, 237 Annotation: Subtype: Link Rect: 395, 203, 432, 214 Annotation: Subtype: Link Rect: 439, 203, 475, 214 Annotation: Subtype: Link Rect: 294, 180, 331, 191 Annotation: Subtype: Link Rect: 337, 180, 373, 191 Annotation: Subtype: Link Rect: 104, 158, 141, 168 Checksum: e3ce1f46 Type: CRC32 Checksum: 486d448bceeff6c27360038c62ecffaf Type: MD5 Checksum: 92e3fe57a1b7fee2820d2821ada6ea31d900c26d Type: SHA-1