An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm
Abstract Background In vitro incubation of epididymal and vas deferens sperm with Mn2+ induces Sperm Chromatin Fragmentation (SCF), a mechanism that causes double-stranded breaks in toroid-linker regions (TLRs). Whether this mechanism, thought to require the participation of topoisomerases and/or DN...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-10-01
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| Series: | Biological Research |
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| Online Access: | https://doi.org/10.1186/s40659-023-00467-w |
| _version_ | 1827635567520645120 |
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| author | Estel Viñolas-Vergés Marc Yeste Ferran Garriga Sergi Bonet Yentel Mateo-Otero Jordi Ribas-Maynou |
| author_facet | Estel Viñolas-Vergés Marc Yeste Ferran Garriga Sergi Bonet Yentel Mateo-Otero Jordi Ribas-Maynou |
| author_sort | Estel Viñolas-Vergés |
| collection | DOAJ |
| description | Abstract Background In vitro incubation of epididymal and vas deferens sperm with Mn2+ induces Sperm Chromatin Fragmentation (SCF), a mechanism that causes double-stranded breaks in toroid-linker regions (TLRs). Whether this mechanism, thought to require the participation of topoisomerases and/or DNAses and thus far only described in epididymal mouse sperm, can be triggered in ejaculated sperm is yet to be elucidated. The current study aimed to determine if exposure of pig ejaculated sperm to divalent ions (Mn2+ and Mg2+) activates SCF, and whether this has any impact on sperm function and survival. For this purpose, sperm DNA integrity was evaluated through the Comet assay and Pulsed Field Gel Electrophoresis (PFGE); sperm motility and agglutination were assessed with computer assisted sperm analysis (CASA); and sperm viability and levels of total reactive oxygen species (ROS) and superoxides were determined through flow cytometry. Results Incubation with Mn2+/Ca2+ activated SCF in a dose-dependent (P < 0.05) albeit not time-dependent manner (P > 0.05); in contrast, Mg2+/Ca2+ only triggered SCF at high concentrations (50 mM). The PFGE revealed that, when activated by Mn2+/Ca2+ or Mg2+/Ca2+, SCF generated DNA fragments of 33–194 Kb, compatible with the size of one or multiple toroids. Besides, Mn2+/Ca2+ affected sperm motility in a dose-dependent manner (P < 0.05), whereas Mg2+/Ca2+ only impaired this variable at high concentrations (P < 0.05). While this effect on motility was concomitant with an increase of agglutination, neither viability nor ROS levels were affected by Mn2+/Ca2+ or Mg2+/Ca2+ treatments. Conclusion Mn2+/Ca2+ and Mn2+/Ca2+ were observed to induce SCF in ejaculated sperm, resulting in DNA cleavage at TLRs. The activation of this mechanism by an intracellular, non-oxidative factor sheds light on the events taking place during sperm cell death. |
| first_indexed | 2024-03-09T15:28:25Z |
| format | Article |
| id | doaj.art-9669be472a604397b9e249e996a6e939 |
| institution | Directory Open Access Journal |
| issn | 0717-6287 |
| language | English |
| last_indexed | 2024-03-09T15:28:25Z |
| publishDate | 2023-10-01 |
| publisher | BMC |
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| series | Biological Research |
| spelling | doaj.art-9669be472a604397b9e249e996a6e9392023-11-26T12:23:52ZengBMCBiological Research0717-62872023-10-0156111610.1186/s40659-023-00467-wAn intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig spermEstel Viñolas-Vergés0Marc Yeste1Ferran Garriga2Sergi Bonet3Yentel Mateo-Otero4Jordi Ribas-Maynou5Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of GironaBiotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of GironaBiotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of GironaBiotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of GironaBiotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of GironaBiotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of GironaAbstract Background In vitro incubation of epididymal and vas deferens sperm with Mn2+ induces Sperm Chromatin Fragmentation (SCF), a mechanism that causes double-stranded breaks in toroid-linker regions (TLRs). Whether this mechanism, thought to require the participation of topoisomerases and/or DNAses and thus far only described in epididymal mouse sperm, can be triggered in ejaculated sperm is yet to be elucidated. The current study aimed to determine if exposure of pig ejaculated sperm to divalent ions (Mn2+ and Mg2+) activates SCF, and whether this has any impact on sperm function and survival. For this purpose, sperm DNA integrity was evaluated through the Comet assay and Pulsed Field Gel Electrophoresis (PFGE); sperm motility and agglutination were assessed with computer assisted sperm analysis (CASA); and sperm viability and levels of total reactive oxygen species (ROS) and superoxides were determined through flow cytometry. Results Incubation with Mn2+/Ca2+ activated SCF in a dose-dependent (P < 0.05) albeit not time-dependent manner (P > 0.05); in contrast, Mg2+/Ca2+ only triggered SCF at high concentrations (50 mM). The PFGE revealed that, when activated by Mn2+/Ca2+ or Mg2+/Ca2+, SCF generated DNA fragments of 33–194 Kb, compatible with the size of one or multiple toroids. Besides, Mn2+/Ca2+ affected sperm motility in a dose-dependent manner (P < 0.05), whereas Mg2+/Ca2+ only impaired this variable at high concentrations (P < 0.05). While this effect on motility was concomitant with an increase of agglutination, neither viability nor ROS levels were affected by Mn2+/Ca2+ or Mg2+/Ca2+ treatments. Conclusion Mn2+/Ca2+ and Mn2+/Ca2+ were observed to induce SCF in ejaculated sperm, resulting in DNA cleavage at TLRs. The activation of this mechanism by an intracellular, non-oxidative factor sheds light on the events taking place during sperm cell death.https://doi.org/10.1186/s40659-023-00467-wDNAEjaculated spermFragmentationPigToroid-linker regions |
| spellingShingle | Estel Viñolas-Vergés Marc Yeste Ferran Garriga Sergi Bonet Yentel Mateo-Otero Jordi Ribas-Maynou An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm Biological Research DNA Ejaculated sperm Fragmentation Pig Toroid-linker regions |
| title | An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm |
| title_full | An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm |
| title_fullStr | An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm |
| title_full_unstemmed | An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm |
| title_short | An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm |
| title_sort | intracellular non oxidative factor activates in vitro chromatin fragmentation in pig sperm |
| topic | DNA Ejaculated sperm Fragmentation Pig Toroid-linker regions |
| url | https://doi.org/10.1186/s40659-023-00467-w |
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