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Comparison between electroporation and polyfection in pig sperm: efficiency and cell viability implications

Published online by Cambridge University Press:  13 September 2018

Zigomar da Silva Open the ORCID record for Zigomar da Silva [Opens in a new window] ,
Andressa Pereira de Souza ,
José Rodrigo Claudio Pandolfi ,
Francisco Noé da Fonseca ,
Carlos André da Veiga Lima-Rosa  and
Mariana Groke Marques
Zigomar da Silva
Affiliation:
Santa Catarina State University. College of Agriculture and Veterinary – CAV, Av. Luiz de Camões, 2090, Conta Dinheiro, CEP: 88.520–000, Lages City, SC, Brazil
Andressa Pereira de Souza
Affiliation:
Santa Catarina State University. College of Agriculture and Veterinary – CAV, Av. Luiz de Camões, 2090, Conta Dinheiro, CEP: 88.520–000, Lages City, SC, Brazil Concordia College. Anita Garibaldi Street, 3185, Primavera 89701-130, Concórdia, SC, Brazil
José Rodrigo Claudio Pandolfi
Affiliation:
Brazilian Agricultural Research Corporation, Embrapa Swine and Poultry, 13 BR-153, Km 110, Tamandua District, Post Box: 321 CEP: 89715-899, Concórdia, SC. Brazil
Francisco Noé da Fonseca
Affiliation:
Brazilian Agricultural Research Corporation, Embrapa Swine and Poultry, 13 BR-153, Km 110, Tamandua District, Post Box: 321 CEP: 89715-899, Concórdia, SC. Brazil
Carlos André da Veiga Lima-Rosa
Affiliation:
Santa Catarina State University, College of Southern Region – Ceres, Street Cel. Fernandes Martins, 270, Progresso, CEP: 88.790-000, Laguna City, SC, Brazil
Mariana Groke Marques*
Affiliation:
Brazilian Agricultural Research Corporation, Embrapa Swine and Poultry, 13 BR-153, Km 110, Tamandua District, Post Box: 321 CEP: 89715-899, Concórdia, SC. Brazil
*
Author for correspondence: Mariana Grokes Marques. Brazilian Agricultural Research Corporation. Embrapa Swine and Poultry, BR-153, Km 110, Tamandua District, Postal Box: 321 CEP: 89715–899, Concórdia, SC. Brazil. Tel: +55 493441 0400. Fax: +55 493441 0497. E-mail: mariana.marques@embrapa.br
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Summary

The aim of this study was to optimize protocols for electroporation (EP) and polyfection (PLF) using polyethyleneimine (PEI) for pig sperm transfection and to determine which method was the most efficient. For EP standardization, different voltages, amounts and times of electric pulses were tested using propidium iodide (PI) as reporter. For PLF standardization, different concentrations of fluorescein isothiocyanate (FITC)-labelled PEI (PEI/FITC) were incubated with sperm for different periods of time. Flow cytometry was performed to evaluate the best protocol in terms of cell viability, including cytoplasmic membrane, acrosome, chromatin integrities and mitochondrial potential using the FITC probe, PI, acridine orange (AO) and JC1. Transfections with the plasmid pmhyGENIE-5 were carried out under optimum conditions for each procedure (EP: 500 volts, 500 μs and two pulses; PLF: PEI 0.5 mg/ml and incubation time 10 min). Transfection efficacy was assessed by fluorescence in situ hybridization (FISH). A lower transfection rate was observed for sperm in the control group (17.8%) compared with EP (36.7%), with PLF (76.8%) being the most efficient. These results suggest that the EP and PEI could be an efficient and low cost transfection method for swine sperm. Notably, treated cells showed higher plasmatic the membrane damage (PMD) and/or acrosome damage (AD) indexes, therefore the combination of this procedure with biotechniques that facilitate fecundation (i.e. in vitro fertilization or intracytoplasmic sperm injection) or even inclusion of antioxidant or anti-apoptotic drugs to improve spermatozoa viability would be important.

Keywords

ElectroporationFlow cytometryPigPolyethyleneimineSperm viability
Type
Research Article
Information
Zygote , Volume 26 , Issue 4 , August 2018 , pp. 286 - 293
Copyright
© Cambridge University Press 2018 

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References

Bieber, T, Meissner, W, Kostin, S, Niemann, A Elsasser, H-P (2002) Intracellular route and transcriptional competence of polyethyleneimine–DNA complexes. J Control Release 82, 441–454.Google Scholar
Boe-Hansen, GB, Morris, ID, Ersboll, AK, Greve, T Christensen, P (2005) DNA integrity in sexed bull sperm assessed by neutral comet assay and sperm chromatin structure assay. Theriogenology 63, 1789–1802.Google Scholar
Bou, G, Sun, M, Lv, M, Zhu, J, Li, H, Wang, J, Li, L, Liu, Z, Zheng, Z, He, W, Kong, Q Liu, Z (2014) A pre-breeding screening program for transgenic boars based on fluorescence in situ hybridization assay. Transgenic Res 23, 679–689.Google Scholar
Boussif, O, Lezoualc’h, F, Zanta, MA, Mergny, MD, Scherman, D, Demeneix, B Behr, JP (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethyleneimine. Proc Natl Acad Sci USA 92, 7297–7301.Google Scholar
Breunig, M, Lungwitz, U, Liebl, R Goepferich, A (2007) Breaking up the correlation between efficacy and toxicity for nonviral gene delivery. Proc Natl Acad Sci USA 104, 14454–14459.Google Scholar
CBRA (1998) Manual para exame andrológico e avaliação de sêmen animal, Colégio Brasileiro de Reprodução Animal: Belo Horizonte.Google Scholar
da Silva, Z, de Souza, AP, Superti, BFV, Lima-Rosa, CAV Marques, MG (2017) Medium and temperature optimization for the recovery of membrane integrity of swine spermatozoa submitted to electroporation. In Proceedings of the 30th Annual Meeting of the Brazilian Embryo Technology Society (SBTE). Cabo de Santo Agostinho, PE, Vol. 14 Animal Reproduction, 877 pp.Google Scholar
Dang, SP, Wang, RX, Qin, MD, Zhang, Y, Gu, YZ, Wang, MY, Yang, QL, Li, XR Zhang, XG (2011) A novel transfection method for eukaryotic cells using polyethyleneimine coated albumin microbubbles. Plasmid 66, 19–25.Google Scholar
Fang, G-F, Chen, W, Wang, S-D, Wang, Y-D, Li, C-H, Zhu, H-L, Wang, H Zeng, Y-Q (2017) Generation of transgenic pigs overexpressing PID1 gene mediated by magnetic nanoparticles and sperm. Asian J Anim Vet Adv 12, 161–168.Google Scholar
Feitosa, WB, Milazzotto, MP, Simoes, R, Rovegno, M, Nicacio, AC, Nascimento, AB, Goncalves, JS, Visintin, JA Assumpcao, ME (2009) Bovine sperm cells viability during incubation with or without exogenous DNA. Zygote 17, 315–320.Google Scholar
Fischer, D, Bieber, T, Li, Y, Elsässer, H-P Kissel, T (1999) A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethyleneimine: effect of molecular weight on transfection efficiency and cytotoxicity. Pharm Res 16, 1273–1279.Google Scholar
Francolini, M, Lavitrano, M, Lamia, CL, French, D, Frati, L, Cotelli, F Spadafora, C (1993) Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells. Mol Reprod Dev 34, 133–139.Google Scholar
Gagne, MB, Pothier, F Sirard, MA (1991) Electroporation of bovine spermatozoa to carry foreign DNA in oocytes. Mol Reprod Dev 29, 6–15.Google Scholar
Godbey, WT Mikos, AG (2001) Recent progress in gene delivery using non-viral transfer complexes. J Control Res 72, 115–125.Google Scholar
Horan, R, Powell, R, Bird, JM, Gannon, F Houghton, JA (1992) Effects of electropermeabilization on the association of foreign DNA with pig sperm. Arch Androl 28, 105–114.Google Scholar
Hsu, CY Uludag, H (2012) A simple and rapid nonviral approach to efficiently transfect primary tissue-derived cells using polyethyleneimine. Nat Protoc 7, 935–945.Google Scholar
Hunter, AC Moghimi, SM (2010) Cationic carriers of genetic material and cell death: a mitochondrial tale. Biochim Biophys Acta 1797, 1203–1209.Google Scholar
Kafil, V Omidi, Y (2011) Cytotoxic impacts of linear and branched polyethyleneimine nanostructures in A431 cells. BioImpacts: BI 1, 23–30.Google Scholar
Kurz, A, Viertel, D, Herrmann, A Muller, K (2005) Localization of phosphatidylserine in boar sperm cell membranes during capacitation and acrosome reaction. Reproduction 130, 615–626.Google Scholar
Lavitrano, M, Forni, M, Bacci, ML, Di Stefano, C, Varzi, V, Wang, H Seren, E (2003) Sperm mediated gene transfer in pig: selection of donor boars and optimization of DNA uptake. Mol Reprod Dev 64, 284–291.Google Scholar
Lavitrano, M, Maione, B, Forte, E, Francolini, M, Sperandio, S, Testi, R Spadafora, C (1997) The interaction of sperm cells with exogenous DNA: a role of CD4 and major histocompatibility complex class II molecules. Exp Cell Res 233, 56–62.Google Scholar
Pramod, RK, Kumar, R Mitra, A (2016) Transgenic expression of green fluorescent protein in caprine embryos produced through electroporation-aided sperm-mediated gene transfer. Gene 576, 505–511.Google Scholar
Rieth, A, Pothier, F Sirard, MA (2000) Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequences into oocytes and detection of homologous recombination events. Mol Reprod Dev 57, 338–345.Google Scholar
Saito, M Saitoh, H (2012) Labeling of polyethyleneimine with fluorescent dye to image nucleus, nucleolus, and chromosomes in digitonin-permeabilized HeLa cells. Biosci Biotechnol Biochem 76, 1777–1780.Google Scholar
Schackmann, RW, Christen, R Shapiro, BM (1981) Membrane potential depolarization and increased intracellular pH accompany the acrosome reaction of sea urchin sperm. Proc Natl Acad Sci USA 78, 6066–6070.Google Scholar
Simões, R, Feitosa, WB, Milazzotto, MP, Nicacio, AC, Barros, FRO d, Gonçalves, JSDA, Marques, MG, Visintin, JA Assumpção, MEODÁ (2015) Comparison of different methods for exogenous DNA uptake by bovine spermatozoa. Braz. J Vet Res Anim Sci 52, 78.Google Scholar
Tros de Ilarduya, C, Sun, Y Duzgunes, N (2010) Gene delivery by lipoplexes and polyplexes. Eur J Pharm Sci 40, 159–170.Google Scholar
Tsai, H-J (2000) Electroporated sperm mediation of a gene transfer system for finfish and shellfish. Mol Reprod Dev 56, 4.Google Scholar
Zoraqi, G Spadafora, C (1997) Integration of foreign DNA sequences into mouse sperm genome. DNA Cell Biol 16, 291–300.Google Scholar