title: Sperm genome cloning used in biparental bovine embryo reconstruction.
abstract: The generation of androgenetic haploid embryos enables several haploid blastomeres to be obtained as identical copies of a single spermatozoon genome. In the present study, we compared the developmental ability of bovine androgenetic haploid embryos constructed by different methods, namely IVF and intracytoplasmic sperm injection (ICSI) before and after oocyte enucleation. Once obtained, the blastomeres of these androgenetic haploid embryos were used as male genome donors to reconstruct biparental embryos by fusion with matured oocytes. To verify the cytoplasmic contribution of androgenetic haploid blastomeres, we used spermatozoa incubated previously with exogenous DNA that coded for a green fluorescent protein gene (pCX-EGFP) and the enhanced green fluorescent protein (EGFP)-positive androgenetic haploid blastomeres generated were fused with mature oocytes. Of the reconstructed embryos reaching the cleavage and blastocyst stages, 85.1% and 9.0%, respectively, expressed EGFP (P>0.05). EGFP expression was observed in 100% of reconstructed embryos, with 91.2% exhibiting homogenic expression. To confirm sperm genome incorporation, androgenetic haploid blastomeres generated by ICSI prior to enucleation and using Y chromosome sexed spermatozoa were used for biparental embryo reconstruction. Incorporation of the Y chromosome was confirmed by polymerase chain reaction and fluorescence in situ hybridisation analysis. In conclusion, the results of the present study prove that it is possible to use sperm genome replicates to reconstruct biparental bovine embryos and that it is a highly efficient technique to generate homogeneous transgene-expressing embryos.
authors.affiliation: Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, C1417 Buenos Aires, Argentina.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Vichera
authors.forename: Ramiro
authors.initials: R
authors.lastname: Olivera
authors.forename: Pablo
authors.initials: P
authors.lastname: Sipowicz
authors.forename: Martín
authors.initials: M
authors.lastname: Radrizzani
authors.forename: Daniel
authors.initials: D
authors.lastname: Salamone
mesh_terms: D000818:Animals; D001757:Blastomeres / Q000166:cytology / Q000378:metabolism; D001947:Breeding / Q000379:methods*; D002417:Cattle; D003001:Cloning, Molecular; D046149:Embryo Culture Techniques / Q000662:veterinary*; D004622:Embryo, Mammalian / Q000502:physiology*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D016678:Genome / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006238:Haploidy; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary; D013094:Spermatozoa / Q000737:chemistry*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins