Improved functional oocyte enucleation by actinomycin D for bovine somatic cell nuclear transfer
Marcelo T. Moura
A B C E , Jeferson Badaraco A , Regivaldo V. Sousa A , Carolina M. Lucci B and Rodolfo Rumpf A B D
+ Author Affiliations
- Author Affiliations
A Laboratório de Reprodução Animal, Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, Av. W5 Norte (final), CP 02372, CEP 70770-900, Brasília, DF, Brazil.
B Departamento de Agronomia e Medicina Veterinária, Universidade de Brasília, Instituto Central de Ciências Sul, Campus Universitário Darci Ribeiro, CEP 70297-400, Brasília, DF, Brazil.
C Present address: Laboratório de Biologia Celular, Universidade Federal de São Paulo, Campus Diadema, CEP 09972-270, Diadema, SP, Brazil.
D Present address: Geneal Biotecnologia, Rodovia BR-050, Km 184, CEP 38038-050, Uberaba, MG, Brazil.
E Corresponding author. Email: marcelotmoura@gmail.com
Reproduction, Fertility and Development 31(8) 1321-1329 https://doi.org/10.1071/RD18164
Submitted: 4 May 2018 Accepted: 5 February 2019 Published: 16 April 2019
Abstract
Somatic cell nuclear transfer (SCNT) allows animal cloning but remains technically challenging. This study investigated limitations to functional oocyte enucleation by actinomycin D (AD) as a means of making SCNT easier to perform. Denuding oocytes or inhibiting transcription before AD treatment revealed that the toxicity of this compound during bovine oocyte maturation is mediated by cumulus cells. Exposure of denuded oocytes to higher concentrations of AD (5–20 μg mL−1) and stepwise reductions of the incubation period (from 14.0 to 0.25 h) led to complete inhibition of parthenogenetic development. Bovine SCNT using this improved AD enucleation protocol (NT(AD)) restored cleavage rates compared with rates in the parthenogenetic and SCNT controls (P(CTL) and NT(CTL) respectively). However, NT(AD) was associated with increased caspase-3 activity in cleavage stage embryos and did not recover blastocyst rates. The removal of AD-treated oocyte spindle before reconstruction (NT(AD+SR)) improved embryo development and reduced caspase-3 activity to levels similar to those in the P(CTL) and NT(CTL) groups. Furthermore, mid-term pregnancies were achieved using NT(AD+SR) blastocysts. In conclusion, improvements in AD functional enucleation for bovine SCNT circumvents most cellular roadblocks to early embryonic development and future investigations must focus on restoring blastocyst formation.
Additional keywords: cattle, cytoplast, nuclear transplantation, reprogramming.
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