Induction of a senescent-like phenotype does not confer the ability of bovine immortal cells to support the development of nuclear transfer embryos.
Previously, we reported that cloned embryos derived from an immortalized bovine mammary epithelial cell line (MECL) failed to develop beyond 12- to 16-cell stage. To analyze whether induction of a senescent-like phenotype in MECL can improve their ability to support the development after transfer into enucleated oocytes, we treated MECL with DNA methylation inhibitor 5-aza-2-deoxycytidine (Aza-C), histone deacetylase inhibitors trichostatin A (TSA), sodium butyrate (NaBu), or 5-bromodeoxyuridine and used those cells for nuclear transfer. Primary bovine fetal fibroblasts (BFF) were used as control. All agents were capable to induce features of senescence including reduced cell proliferation, enlarged cell size with a considerable proportion of cells stained positive for acidic senescence-associated beta-galactosidase and G1/S cell cycle boundary arrest in MECL. Aza-C treatment induced genome demethylation. Acetylation of H3 and H4 was increased after TSA treatment in both MECL and BFF, whereas no obvious changes in global H3 or H4 acetylation were detected after NaBu treatment. Nuclear transfer experiments following diverse treatments demonstrated that the induced senescent-like phenotype of MECL did not confer their ability to support embryonic development, although 7.3% of reconstructed embryos derived from NaBu-treated cells developed to morula stage. Intriguingly, a much higher proportion of cloned embryos developed to blastocysts when using NaBu-treated BFF, compared with using untreated BFF (59% versus 26%). Our results suggest that the developmental failure of donor nuclei from bovine immortal cells could not be reversed by induction of senescent-like phenotype. The beneficial effect of NaBu on the developmental potential of cloned embryos reconstructed from BFF merits further studies.
Shi
Wei
W
Department of Molecular Animal Breeding and Biotechnology, University of Munich, D-85764 Munich, Germany.
Hoeflich
Andreas
A
Flaswinkel
Heinrich
H
Stojkovic
Miodrag
M
Wolf
Eckhard
E
Zakhartchenko
Valeri
V
D000818:Animals; D017209:Apoptosis; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002087:Butyrates / Q000494:pharmacology; D002417:Cattle; D002454:Cell Differentiation; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D016922:Cellular Senescence; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D005314:Embryonic and Fetal Development; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D005260:Female; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids / Q000494:pharmacology; D066298:In Vitro Techniques; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000254:growth & development*; D010641:Phenotype; D011003:Ploidies
D002087:Butyrates; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C012589:trichostatin A; D000077209:Decitabine; D001973:Bromodeoxyuridine; D001374:Azacitidine