Related Experiment Video
Updated: Jul 8, 2026
Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes
Published on: May 11, 2017
Analysis of actinomycin D treated cattle oocytes and their use for somatic cell nuclear transfer
Marcelo Tigre Moura1, Regivaldo Vieira de Sousa, Ligiane de Oliveira Leme
1Laboratório 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.
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.
Insights
Actinomycin D effectively inhibits cattle oocyte development and embryonic progression. However, reconstructed embryos using treated oocytes show normal apoptotic cell indices, suggesting potential for further research in assisted reproduction.
Area of Science:
- Reproductive Biology
- Developmental Biology
- Cell Biology
Background:
- Chemical enucleation is a critical step in assisted reproductive technologies.
- Actinomycin D is a known inhibitor of transcription and replication.
- Evaluating novel agents for oocyte manipulation is essential for improving reproductive efficiency.
Purpose of the Study:
- To assess the efficacy of actinomycin D as a chemical enucleation agent for cattle oocytes.
- To investigate the impact of actinomycin D on oocyte maturation, cleavage, and blastocyst development.
- To evaluate the apoptotic status of embryos reconstructed using actinomycin D-treated oocytes.
Main Methods:
- Cattle oocytes were matured in vitro and treated with varying concentrations and durations of actinomycin D.
- Oocyte maturation, chromosome morphology, and embryonic development (cleavage and blastocyst rates) were assessed.
- Somatic cell nuclear transfer (SCNT) and parthenogenetic activation were performed using treated oocytes.
- Apoptosis was quantified using the TUNEL assay in both parthenogenetic and SCNT embryos.
Main Results:
- Actinomycin D significantly reduced oocyte maturation, cleavage, and blastocyst rates in a dose-dependent manner.
- Higher concentrations of actinomycin D induced chromosome uncoiling.
- SCNT embryos derived from treated oocytes had reduced cleavage rates but improved blastocyst rates compared to the treated control.
- Parthenogenetic embryos from treated oocytes showed increased apoptosis, while SCNT embryos had comparable apoptotic indices to controls.
Conclusions:
- Actinomycin D demonstrates efficiency in blocking embryonic development in cattle oocytes.
- While impacting early development, actinomycin D-treated oocytes can be used for SCNT, yielding reconstructed embryos with normal apoptotic cell profiles.
- Further research is warranted to optimize actinomycin D application for potential use in oocyte enucleation techniques.
Related Concept Videos
01:08Cloning of Dolly the Sheep
01:24Methods of Nuclear Reprogramming