For Publishers
DiscoveryMetadataPeer reviewHostingPublishing
For Researchers
JoinPublishReviewCollect
Register
Blog
About
Search
Advanced search
My ScienceOpen
Search
For Publishers
For Researchers
Blog
About
1
views
48
references
 Top references
cited by
1
    
0 reviews
 Review
0
comments
 Comment
0
recommends
+1 Recommend
0 collections
    0
    shares
      224
      similar
       All similar
      • Record: found
      • Abstract: found
      • Article: not found

      Efficiency and cell viability implications using tip type electroporation in zebrafish sperm cells

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Sperm-mediated gene transfer (SMGT) has a potential use for zebrafish transgenesis. However, transfection into fish sperm cells still needs to be improved. The objective was to demonstrate the feasibility of tip type electroporation in zebrafish sperm, showing a protocol that provide high transfection efficiency, with minimal side-effects. Sperm was transfected with a Cy3-labelled DNA using tip type electroporation with voltages ranging from 500 to 1500 V. Sperm kinetics parameters were assessed using Computer Assisted Semen Analysis (CASA) and cell integrity, reactive oxygen species (ROS), mitochondrial functionality and transfection rate were evaluated by flow cytometry. The transfection rates were positively affected by tip type electroporation, reaching 64.9% ± 3.6 in the lowest voltage used (500 V) and 86.6% ± 1.9 in the highest (1500 V). The percentage of overall motile sperm in the electrotransfected samples was found to decrease with increasing field strength ( P < 0.05). Increase in the sperm damaged plasma membrane was observed with increasing field strength ( P < 0.05). ROS and sperm mitochondrial functionality did not present a negative response after the electroporation ( P > 0.05). Overall results indicate that tip type electroporation enhances the internalization of exogenous DNA into zebrafish sperm cells with minimal harmful effects to sperm cells.

          Electronic supplementary material

          The online version of this article (10.1007/s11033-020-05658-2) contains supplementary material, which is available to authorized users.

          Related collections

          Most cited references48

          • Record: found
          • Abstract: found
          • Article: not found

          On the possible origins of DNA damage in human spermatozoa.

          N. De Groot, R. Alan Aitken (2009)
          DNA damage in the male germ line has been linked with a variety of adverse clinical outcomes including impaired fertility, an increased incidence of miscarriage and an enhanced risk of disease in the offspring. The origins of this DNA damage could, in principle, involve: (i) abortive apoptosis initiated post meiotically when the ability to drive this process to completion is in decline (ii) unresolved strand breaks created during spermiogenesis to relieve the torsional stresses associated with chromatin remodelling and (iii) oxidative stress. In this article, we present a two-step hypothesis for the origins of DNA damage in human spermatozoa that highlights the significance of oxidative stress acting on vulnerable, poorly protaminated cells generated as a result of defective spermiogenesis. We further propose that these defective cells are characterized by several hallmarks of 'dysmaturity' including the retention of excess residual cytoplasm, persistent nuclear histones, poor zona binding and disrupted chaperone content. The oxidative stress experienced by these cells may originate from infiltrating leukocytes or, possibly, the entry of spermatozoa into an apoptosis-like cascade characterized by the mitochondrial generation of reactive oxygen species. This oxidative stress may be exacerbated by a decline in local antioxidant protection, particularly during epididymal maturation. Finally, if oxidative stress is a major cause of sperm DNA damage then antioxidants should have an important therapeutic role to play in the clinical management of male infertility. Carefully controlled studies are now needed to critically examine this possibility.
          Article 0 comments Cited 151 times – based on 0 reviews     Review now
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2'-deoxyguanosine, a marker of oxidative stress.

            R. Alan Aitken, Brett Nixon, Adam J Koppers … (2009)
            DNA damage in human spermatozoa has been associated with a range of adverse clinical outcomes, including infertility, abortion, and disease in the offspring. We have advanced a two-step hypothesis to explain this damage involving impaired chromatin remodeling during spermiogenesis followed by a free radical attack to induce DNA strand breakage. The objective of the present study was to test this hypothesis by determining whether impaired chromatin protamination is correlated with oxidative base damage and DNA fragmentation in human spermatozoa. DNA fragmentation, chromatin protamination, mitochondrial membrane potential, and formation of the oxidative base adduct, 8-hydroxy-2'-deoxyguanosine (8OHdG), were monitored by flow cytometry/fluorescence microscopy. Impairment of DNA protamination during late spermatogenesis was highly correlated (P < 0.001) with DNA damage in human spermatozoa. The disruption of chromatin remodeling also was associated with a significant elevation in the levels of 8OHdG (P < 0.001), and the latter was itself highly correlated with DNA fragmentation (P < 0.001). The significance of oxidative stress in 8OHdG formation was demonstrated experimentally using H2O2/Fe2+ and by the correlation observed between this base adduct and superoxide generation (P < 0.001). That 8OHdG formation was inversely associated with mitochondrial membrane potential (P < 0.001) suggested a possible role for these organelles in the creation of oxidative stress. These results clearly highlight the importance of oxidative stress in the induction of sperm DNA damage and carry significant implications for the clinical management of this condition.
            Article 0 comments Cited 105 times – based on 0 reviews     Review now
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Development of a novel CASA system based on open source software for characterization of zebrafish sperm motility parameters.

              M G Leedy, Rolf Ingermann (2007)
              Although computer-assisted sperm analysis (CASA) outperforms manual techniques, many investigators rely on non-automated analysis due to the high cost of commercial options. In this study, we have written and validated a free CASA software primarily for analysis of fish sperm. This software is a plugin for the free National Institutes of Health software ImageJ and is available with documentation at . That it is open source makes possible external validation, should improve quality control and enhance the comparative value of data obtained among laboratories. In addition, we have improved upon the traditional velocity straight line (VSL) algorithm, eliminating inaccurate characterization of highly curved fish sperm paths. Using this system, the motion of zebrafish (Danio rerio) sperm was characterized relative to time post-activation and the impact of acquisition conditions upon data analysis determined. There were decreases in velocity and path straightness (STR), but not linearity (LIN), relative to time. From 30 to 300 frames/s, frame rate significantly affected curvilinear velocity (VCL) and STR measurements. Sperm density in the field of view did not affect any measured parameter. There was significant inter-male variation for VCL, VSL, velocity average path (VAP), percent motility, path character (STR, LIN), and duration of motility. Furthermore, relative sperm output (a measure reflecting both semen volume and concentration) was positively correlated to percent motility. For all motion parameters measured (except duration), the average CV was < or =10%, comparable to values obtained using commercial systems.
              Article 0 comments Cited 99 times – based on 0 reviews     Review now
                Bookmark
                All references

                Author and article information

                Contributors
                Vinicius F. Campos:
                ORCID: http://orcid.org/0000-0003-2119-293X
                fariascampos@gmail.com
                Journal
                Journal ID (nlm-ta): Mol Biol Rep
                Journal ID (iso-abbrev): Mol. Biol. Rep
                Title: Molecular Biology Reports
                Publisher: Springer Netherlands (Dordrecht )
                ISSN (Print): 0301-4851
                ISSN (Electronic): 1573-4978
                Publication date (Electronic): 13 July 2020
                Pages: 1-9
                Affiliations
                [1 ]GRID grid.411221.5, ISNI 0000 0001 2134 6519, Laboratório de Genômica Estrutural, Programa de Pós-Graduação em Biotecnologia, Centro de Desenvolvimento Tecnológico, , Universidade Federal de Pelotas, ; Pelotas, RS Brazil
                [2 ]GRID grid.411221.5, ISNI 0000 0001 2134 6519, ReproPel, Programa de Pós-Graduação em Veterinária, Faculdade de Veterinária, , Universidade Federal de Pelotas, ; Pelotas, RS Brazil
                [3 ]GRID grid.411598.0, ISNI 0000 0000 8540 6536, Laboratório de Reprodução Animal, Programa de Pós-Graduação em Biologia de Ambientes Aquáticos Continentais, , Instituto de Ciências Biológicas - Universidade Federal do Rio Grande, ; Rio Grande, RS Brazil
                [4 ]GRID grid.411221.5, ISNI 0000 0001 2134 6519, Laboratório de Ictiologia, , Faculdade de Zootecnia - Universidade Federal de Pelotas, ; Pelotas, RS Brazil
                Author information
                http://orcid.org/0000-0003-2119-293X
                Article
                Publisher ID: 5658
                DOI: 10.1007/s11033-020-05658-2
                PMC ID: 7356131
                PubMed ID: 32661869
                SO-VID: 09d81a57-8132-4ce5-940a-7b72e8a11cb6
                Copyright © © Springer Nature B.V. 2020
                License:

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                Date received : 7 March 2020
                Date accepted : 8 July 2020
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100004263, Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul;
                Award ID: 19/2551-0002283-2
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award ID: 422292/2016-8
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100002322, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior;
                Award ID: 2537/2018
                Award Recipient :
                Categories
                Subject: Original Article

                ScienceOpen disciplines: Molecular biology
                Keywords: danio rerio,electroporation,spermatozoa,exogenous dna,smgt
                Data availability:
                ScienceOpen disciplines: Molecular biology
                Keywords: danio rerio, electroporation, spermatozoa, exogenous dna, smgt

                Comments

                Comment on this article