title: Analysis of actinomycin D treated cattle oocytes and their use for somatic cell nuclear transfer.
abstract: The present work aimed to evaluate the transcription and replication inhibitor, actinomycin D, for oocyte chemical enucleation. Cattle oocytes matured in vitro were treated with actinomycin D according to the following treatments: T1, control; T2=1.0 microg/ml for 16 h; T3=1.0 microg/ml for 14 h; T4=2.5 microg/ml for 14 h; T5=5.0 microg/ml for 14 h. The oocytes were denuded and activated during 24-26 h of maturation. Oocytes were fixed to determine the maturation status and for chromosome morphology evaluation. Furthermore, oocytes treated with actinomycin D were used for somatic cell nuclear transfer (SCNT). Parthenogenetic and SCNT embryos were fixed to evaluate the percentage of apoptotic nuclei by the TUNEL assay. The maturation (T1=90.4%; T2=82.3%; T3=79.1%; T4=83.4%; T5=74.7%), cleavage (T1=68.9%; T2=46.0%; T3=49.7%; T4=33.4%; T5=29.3%) and blastocyst rate at D8 (T1=41.1%; T2=1.8%; T3=1.3%; T4=0.9%; T5=0.0%) after actinomycin D treatment were significantly different. There was a significant chromosome uncoiling when treated with greater concentrations (2.5 and 5.0 microg/ml). After SCNT, the cleavage rate (61.3%) was similar to the actinomycin D-treated control group (61.3%) and less than the non-treated control (70.2%), although the blastocyst rate was greater in the SCNT group (11.8%) comparing with the treated control (3.6%) and less than the untreated control (38.0%). Treated parthenogenetic embryos had more apoptotic cells than the parthenogenetic controls (24.2% compared with 4.8%). However, the SCNT group using treated cytoplasts was similar from the SCNT control (9.3 compared with 13.0%). Actinomycin D treatment was efficient in blocking embryonic development. Moreover, it was possible to obtain reconstructed embryos that possess an apoptotic cell index indistinguishable from controls.
authors.affiliation: Laboratório de Reprodução Animal, Embrapa Recursos Genéticos e Biotecnologia, C.P. 02372 Brasília, DF, CEP 70770-900, Brazil; Departamento de Agronomia e Medicina Veterinária, Universidade de Brasília, CEP 70910-900 Brasília, DF, Brazil.
authors.forename: Marcelo Tigre
authors.initials: MT
authors.lastname: Moura
authors.forename: Regivaldo Vieira
authors.initials: RV
authors.lastname: de Sousa
authors.forename: Ligiane
authors.initials: L
authors.lastname: de Oliveira Leme
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Rumpf
mesh_terms: D000003:Abattoirs; D000818:Animals; D002417:Cattle; D003609:Dactinomycin / Q000494:pharmacology*; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology; D005260:Female; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D006080:Ovarian Follicle / Q000502:physiology; D010053:Ovary / Q000502:physiology; D010312:Parthenogenesis; D010641:Phenotype; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D015053:Zygote / Q000502:physiology
chemical_list: D019384:Nucleic Acid Synthesis Inhibitors; D011500:Protein Synthesis Inhibitors; D003609:Dactinomycin