Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

Abstract

Purpose 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. Methods 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. Results 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 transgeneexpressing 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). Conclusions 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.

Disulphide-less crotamine is effective for formation of DNA–peptide complex but is unable to improve bovine embryo transfection

“…When comparing embryos microinjected with plasmid alone or with the DNA-dLCr complex, the rate of DNA expression of exogenous DNA in bovine embryos was not comparatively improved. The high affinity for DNA and binding capacity of dLCr might again restrain the release of the GFP plasmid gene in the complex for protein expression, similar to found previously when using native crotamine to induce exogenous DNA expression in bovine embryos (Campelo et al, 2016b). Therefore, it was hypothesized that dLCr remains complexed to exogenous DNA even inside the cytoplasm of embryo cells, and this possibly led to very low GFP expression and detectable fluorescence.…”

“…The rational to use disulphide-less crotamine as the delivery tool for embryo transfection instead of the native disulphide-bond folded crotamine relied on the fact that CPPs should make a complexation between the peptide and DNA complex that was strong enough to permit stable uptake through the zona pellucida and/or cell membrane translocation, but should be sufficiently weak to permit the release of DNA though dissociation into the cell cytoplasm to enable it to traffic to the nucleus. In our previous studies (Campelo et al, 2016a(Campelo et al, , 2016b, bovine embryos were used as a model for gene transfer with DNA-NCr complexes. However, the use of the DNA-NCr complex did not improve the transgene expression rate, supposedly because of strong DNA-peptide binding within embryo cells (Campelo et al, 2016b).…”

“…In our previous studies (Campelo et al, 2016a(Campelo et al, , 2016b, bovine embryos were used as a model for gene transfer with DNA-NCr complexes. However, the use of the DNA-NCr complex did not improve the transgene expression rate, supposedly because of strong DNA-peptide binding within embryo cells (Campelo et al, 2016b). Therefore, it was desirable to evaluate whether modified crotamine peptides, such as dLCr could reduce the binding strength of DNA-peptide and circumvent such physicochemical behaviour.…”

“…It was also demonstrated that this venom-derived peptide is able to successfully transfect cells in murine model (Nascimento et al, 2007). However, when using bovine as a model for gene delivery, crotamine did not improve gene transfer possibly due to its high affinity for DNA molecules (Campelo et al, 2016b).…”

“…A few years ago, our group started studies with crotamine, one of these CPPs, for potential use in animal transgenesis (Campelo et al, 2016a(Campelo et al, , 2016b, exploring its ability to stable binding to nucleic acids by electrostatic interaction and forming DNA-peptide complex. Crotamine was first reported back in 1947 as one of the predominant components in the venom of South American rattlesnake Crotalus durissus terrificus (Gonçalves and Polson, 1947).…”