Crotamine is capable of penetrating cells and embryos and transfecting cells with exogenous DNA. However, no studies are available regarding its uptake by parthenogenetic (PA) embryos or its use for transfection in in vitro fertilized (IVF) embryos. This study aimed to determine the translocation kinetics of crotamine into PA and IVF bovine embryos and assess its effect over in vitro development of PA embryos. Moreover, crotamine-DNA complexes were used to test the transfection ability of crotamine in bovine IVF zygotes.
PA and IVF embryos were exposed to labeled crotamine for four interval times. Embryo toxicity was assayed over PA embryos after 24 h of exposure to crotamine. Additionally, IVF embryos were exposed to or injected with a complex formed by crotamine and pCX-EGFP plasmid.
Confocal images revealed that crotamine was uptaken by PA and IVF embryos as soon as 1 h after exposure. Crotamine exposure did not affect two to eight cells and blastocyst rates or blastocyst cell number (p > 0.05) of PA embryos. Regarding transfection, exposure or injection into the perivitelline space with crotamine-DNA complex did not result in transgene-expressing embryos. Nevertheless, intracytoplasmic injection of plasmid alone showed higher expression rates than did injection with crotamine-DNA complex at days 4 and 7 (p < 0.05).
Crotamine is able to translocate through zona pellucida (ZP) of PA and IVF embryos within 1 h of exposure without impairing in vitro development. However, the use of crotamine does not improve exogenous DNA expression in cattle embryos, probably due to the tight complexation of DNA with crotamine.
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The authors are grateful to CIALE for donating the semen and Frigorífico I.F.F.S.A for providing the biological material. The authors are also thankful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) for providing the scholarship support.
Conflict of Interest
The authors declare that they have no conflict of interest.
Crotamine is able to translocate through zona pellucida of PA and IVF embryos, but did not improve the efficiency of transgene expression in cattle embryo.
Electronic supplementary material
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Confocal imaging projections of PA embryos exposed to RhoB-crotamine for 17 h time interval (MP4 1087 kb)
Confocal imaging projections of IVF embryos exposed to RhoB-crotamine for 17 h time interval (MP4 980 kb)
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Campelo, I.S., Canel, N.G., Bevacqua, R.J. et al. Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression. J Assist Reprod Genet 33, 1405–1413 (2016) doi:10.1007/s10815-016-0772-7
- Peptide uptake
- Gene delivery
- Preimplantation embryo
- Transgene expression