Nuclear transfer with apoptotic bovine fibroblasts: can programmed cell death be reprogrammed?
Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return." In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 μM of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx+) and Caspase-9-positive (Casp-9+) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx+ (4.6% in control cells; p<0.01) and 24.9% were Casp-9+ (2.4% in control cells; p<0.01). Fusion and cleavage were not affected by the use apoptotic cells (p>0.05). Also, the use of Anx+ cells did not affect blastocyst production compared to control (26.4% vs. 22.9%, respectively; p>0.05). However, blastocyst formation was affected by the use of Casp-9+ cells (12.3%; p<0.05). These findings contribute to the idea of that apoptosis is reversible only at early stages. Additionally, we hypothesize that the "point of no return" for apoptosis may be located around activation of Caspase-9.
Faculdade de Biotecnologia, Universidade Federal do Pará (UFPA), Belém, Pará, Brazil. moyses_m@hotmail.com
Moyses dos Santos
Mdos S
Miranda
Fabiana Fernandes
FF
Bressan
Tiago Henrique Camara
TH
De Bem
Giovana Krempel Fonseca
GK
Merighe
Otávio Mitio
OM
Ohashi
William Alan
WA
King
Flavio Viera
FV
Meirelles
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002417:Cattle / Q000196:embryology; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000502:physiology*; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary; D047108:Embryonic Development / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D005434:Flow Cytometry; D020287:In Situ Nick-End Labeling; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D061251:Primary Cell Culture