title: Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression.
abstract: OBJECTIVE
  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 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).
  
  
  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.
authors: Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará, Fortaleza, CE, Brazil.
authors: Iana S
authors: IS
authors: Campelo
authors: Laboratory of Animal Biotechnology, Faculty of Agronomy, University of Buenos Aires, Av. San Martín 4453, Buenos Aires, 1417, Argentina.
authors: Natalia G
authors: NG
authors: Canel
authors: Laboratory of Animal Biotechnology, Faculty of Agronomy, University of Buenos Aires, Av. San Martín 4453, Buenos Aires, 1417, Argentina.
authors: Romina J
authors: RJ
authors: Bevacqua
authors: Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará, Fortaleza, CE, Brazil.
authors: Luciana M
authors: LM
authors: Melo
authors: Laboratory of Biochemistry and Biotechnology, Institute of Marine Science, Federal University of Ceará, Fortaleza, CE, Brazil.
authors: Gandhi
authors: G
authors: Rádis-Baptista
authors: Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará, Fortaleza, CE, Brazil.
authors: Vicente J F
authors: VJ
authors: Freitas
authors: Laboratory of Animal Biotechnology, Faculty of Agronomy, University of Buenos Aires, Av. San Martín 4453, Buenos Aires, 1417, Argentina. salamone@agro.uba.ar.
authors: Daniel F
authors: DF
authors: Salamone
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects; D002417:Cattle; D057846:Cell-Penetrating Peptides / Q000008:administration & dosage*; D003435:Crotalid Venoms / Q000008:administration & dosage*; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian; D005260:Female; D005307:Fertilization in Vitro; D010312:Parthenogenesis / Q000187:drug effects / Q000235:genetics; D015053:Zygote
chemical_list: D057846:Cell-Penetrating Peptides; D003435:Crotalid Venoms; C010563:crotamine
keywords: Cattle; Gene delivery; Peptide uptake; Preimplantation embryo; Transgene expression