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Effect of crotamine, a cell-penetrating peptide, on blastocyst production and gene expression of in vitro fertilized bovine embryos

Published online by Cambridge University Press:  23 December 2014

Iana S. Campelo
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil.
Alexsandra F. Pereira
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil.
Agostinho S. Alcântara-Neto
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil.
Natalia G. Canel
Affiliation:
Laboratory of Animal Biotechnology, University of Buenos Aires, Buenos Aires, Argentina.
Joanna M.G. Souza-Fabjan
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil.
Dárcio I.A. Teixeira
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil.
Luiz S.A. Camargo
Affiliation:
Embrapa Dairy Cattle, Juiz de Fora-MG, Brazil.
Luciana M. Melo
Affiliation:
Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará, Fortaleza-CE, Brazil.
Gandhi Rádis-Baptista
Affiliation:
Laboratory of Biochemistry and Biotechnology, Institute of Marine Science, Federal University of Ceará, Fortaleza-CE, Brazil.
Daniel F. Salamone
Affiliation:
Laboratory of Animal Biotechnology, University of Buenos Aires, Buenos Aires, Argentina.
Vicente J.F. Freitas*
Affiliation:
Universidade Estadual do Ceará/Faculdade de Veterinária; Av. Dr. Silas Munguba, 1700–Fortaleza-CE, 60714–903Brazil.
*
All correspondence to: Vicente José de Figueirêdo Freitas. Universidade Estadual do Ceará/Faculdade de Veterinária; Av. Dr. Silas Munguba, 1700–Fortaleza-CE, 60714–903Brazil. Tel: +55 85 31019861. Fax: +55 85 31019840. e-mail: vicente.freitas@uece.br

Summary

The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 μM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 μM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 μM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 μM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 μM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1–10 μM for 6–24 h.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2014 

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References

Augustin, R., Pocar, P., Navarrete-Santos, A., Wrenzycki, C., Gandolfi, F., Niemann, H. & Fischer, B. (2001). Glucose transporter expression is developmentally regulated in in vitro derived bovine preimplantation embryos. Mol. Reprod. Dev. 60, 370–6.CrossRefGoogle ScholarPubMed
Barcroft, L.C., Offenberg, H., Thomsen, P. & Watson, A.J. (2003). Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation. Dev. Biol. 256, 342–54.CrossRefGoogle ScholarPubMed
Camargo, L.S., Boite, M.C., Wohlres-Viana, S., Mota, G.B., Serapião, R.V., Sa, W.F., Viana, J.H. & Nogueira, L.A. (2011). Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro . Cryobiology 63, 256–62.Google Scholar
Dussault, A.A. & Pouliot, M. (2006). Rapid and simple comparison of messenger RNA levels using real-time PCR. Biol. Proc. Online 8, 110.CrossRefGoogle ScholarPubMed
Gordon, J.W., Scangos, G.A., Plotkin, D.J., Barbosa, J.A. & Ruddle, F.H. (1980). Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA 77, 7380–4.Google Scholar
Gupta, B., Levchenko, T.S. & Torchilin, V.P. (2005). Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv. Drug Deliv. Rev. 57, 637–51.CrossRefGoogle ScholarPubMed
Gupta, S.K., Bhandari, B., Shrestha, A., Biswal, B.K., Palaniappan, C., Malhotra, S.S. & Gupta, N. (2012). Mammalian zona pellucida glycoproteins: structure and function during fertilization. Cell Tissue Res. 349, 665–78.CrossRefGoogle ScholarPubMed
Hammer, R.E., Pursel, V.G., Rexroad, Jr C.E., Wall, R.J., Bolt, D.J., Ebert, K.M., Palmiter, R.D. & Brinster, R.L. (1985). Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315, 680–3.Google Scholar
Hayashi, M.A., Nascimento, F.D., Kerkis, A., Oliveira, V., Oliveira, E.B., Pereira, A., Rádis-Baptista, G., Nader, H.B., Yamane, T., Kerkis, I. & Tersariol, I.L. (2008). Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization. Toxicon 52, 508–17.Google Scholar
Holm, P., Booth, P.J., Schmidt, M.H., Greve, T. & Callesen, H. (1999). High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology 52, 683700.Google Scholar
Houdebine, L.M. (2007). Transgenic animal models in biomedical research. Methods Mol. Biol. 360, 163202.Google Scholar
Kerkis, A., Kerkis, I., Rádis-Baptista, G., Oliveira, E.B., Vianna-Morgante, A.M., Pereira, L.V. & Yamane, T. (2004). Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus . FASEB J. 18, 1407–9.Google Scholar
Kues, W.A. & Niemann, H. (2011). Advances in farm animal transgenesis. Prev. Vet. Med. 102, 146–56.Google Scholar
Kuzmany, A., Havlicek, V., Wrenzycki, C., Wilkening, S., Brem, G. & Besenfelder, U. (2011). Expression of mRNA, before and after freezing, in bovine blastocysts cultured under different conditions. Theriogenology 75, 482–94.Google Scholar
Livak, K.J. & Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC T method. Methods 25, 402–8.Google Scholar
Nascimento, F.D., Hayashi, M.A., Kerkis, A., Oliveira, V., Oliveira, E.B., Rádis-Baptista, G., Nader, H.B., Yamane, T., Tersariol, I.L.S. & Kerkis, I. (2007). Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans. J. Biol. Chem. 282, 21349–60.Google Scholar
Nascimento, F.D., Sancey, L., Pereira, A., Rome, C., Oliveira, V., Oliveira, E.B., Nader, H.B., Yamane, T., Kerkis, I., Tersariol, I.L.S., Coll, J. & Hayashi, M.A.F. (2012). The natural cell-penetrating peptide crotamine targets tumor tissue in vivo and triggers a lethal calcium-dependent pathway in cultured cells. Mol. Pharmaceutics 9, 211–21.CrossRefGoogle ScholarPubMed
Palmiter, R.D., Brinster, R.L., Hammer, R.E., Trumbauer, M.E., Rosenfeld, M.G., Birnberg, N.C. & Evans, R.M. (1982). Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300, 611–5.Google Scholar
Purcell, S.H. & Moley, K.H. (2009). Glucose transporters in gametes and preimplantation embryos. Trends Endocrinol. Metab. 20, 483–9.Google Scholar
Pursel, V.G., Wall, R.J., Rexroad, C.E. Jr, Hammer, R.E. & Brinster, R.L. (1985). A rapid whole-mounted staining products for nuclei of mammalian eggs. Theriogenology 24, 687–91.CrossRefGoogle Scholar
Rádis-Baptista, G. & Kerkis, I. (2011). Crotamine, a small basic polypeptide myotoxin from rattlesnake venom with cell-penetrating properties. Curr. Pharm. Des. 17, 4351–61.Google Scholar
Rádis-Baptista, G., de la Torre, B.G. & Andreu, D. (2008). A novel cell-penetrating peptide sequence derived by structural minimization of a snake toxin exhibits preferential nucleolar localization. J. Med. Chem. 51, 7041–4.CrossRefGoogle ScholarPubMed
Richard, J.P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M.J., Chernomordik, L.V. & Lebleu, B. (2003). Cell-penetrating peptides: a reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 278, 585–90.CrossRefGoogle ScholarPubMed
Rideout, W.M., Eggan, K. & Jaenisch, R. (2001). Nuclear cloning and epigenetic reprogramming of the genome. Science 293, 1093–8.CrossRefGoogle ScholarPubMed
Rizos, D., Clemente, M., Bermejo-Alvarez, P., Fuente, J., Lonergan, P. & Gutiérrez-Adán, A. (2008). Consequences of in vitro culture conditions on embryo development and quality. Reprod. Dom. Anim. 43, 4450.Google Scholar
Rodrigues, M., Santos, A., Torre, B.G., Rádis-Baptista, G., Andreu, D. & Santos, N.C. (2012). Molecular characterization of the interaction of crotamine-derived nucleolar targeting peptides with lipid membranes. Biochim. Biophys. Acta 1818, 2707–17.Google Scholar
Schwarze, S.R. & Dowdy, S.F. (2000). In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol. Sci. 21, 45–8.Google Scholar
Tervit, H., Whittingham, D. & Rowson, L. (1972). Successful culture in vitro of sheep and cattle ova. J. Reprod. Fertil. 30, 493–7.Google Scholar
Vandesompele, J., Preter, D.K., Pattynm, F., Poppe, B., Roy, N.V., Paepe, A.D. & Speleman, F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Gen. Biol. 3, 112.CrossRefGoogle ScholarPubMed
Watson, A.J., Westhusin, M.E., De Sousa, P.A., Betts, D.H. & Barcroft, L.C. (1999). Gene expression regulating blastocyst formation. Theriogenology 51, 117–33.CrossRefGoogle ScholarPubMed