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Dose- and time-dependent effects of TNFα and actinomycin D on cell death incidence and embryo growth in mouse blastocysts

Published online by Cambridge University Press:  01 August 2007

D. Fabian ,
S. Juhás ,
G. Il'ková  and
J. Koppel
D. Fabian*
Affiliation:
Institute of Animal Physiology, Slovak Academy of Sciences, Košice, Slovakia
S. Juhás
Affiliation:
Institute of Animal Physiology, Slovak Academy of Sciences, Košice, Slovakia
G. Il'ková
Affiliation:
Institute of Animal Physiology, Slovak Academy of Sciences, Košice, Slovakia
J. Koppel
Affiliation:
Institute of Animal Physiology, Slovak Academy of Sciences, Košice, Slovakia
*
All correspondence to: Dušan Fabian, Institute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4/6, 04001 Košice, Slovakia. Tel: +421 55 728 78 41. Fax: +421 55 728 78 42. e-mail: fabian@saske.sk
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Summary

This study was undertaken to obtain information about characteristics of different types of induced apoptosis in preimplantation embryos. Freshly isolated mouse blastocysts were cultured in vitro with the addition of two apoptotic inductors – TNFα and actinomycin D – at various doses and times. The average number of nuclei and the percentage of dead cells were evaluated in treated embryos. Classification of dead cells was based on morphological assessment of their nuclei evaluated by fluorescence microscopy, the detection of specific DNA degradation (TUNEL assay), the detection of active caspase-3 and cell viability assessed by propidium iodide staining. The addition of both apoptotic inductors into culture media significantly increased cell death incidence in blastocysts. Their effects were dose and time dependent. Lower concentrations of inductors increased cell death incidence, usually without affecting embryo growth after 24 h culture. Higher concentrations of inductors caused wider cell damage and also retarded embryo development. In all experiments, the negative effect of actinomycin D on blastomere survival and blastocyst growth was greater than the effect of TNFα. Furthermore, the addition of actinomycin D into culture media increased cell death incidence even after 6 h culture. Differences resulted probably from diverse specificity of apoptotic inductors. The majority of dead cells in treated blastocysts were of apoptotic origin. Morphological and biochemical features of apoptotic cell death induced by both TNFα and actinomycin D were similar and had homologous profile. In blastomeres, similarly to somatic cells, the biochemical pathways of induced apoptosis included activation of caspase-3 and internucleosomal DNA fragmentation.

Keywords

Actinomycin DCaspase-3Induced apoptosisMouse blastocystsTNFα
Type
Research Article
Information
Zygote , Volume 15 , Issue 3 , August 2007 , pp. 241 - 249
Copyright
Copyright © Cambridge University Press 2007

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