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BOR - Papers in Press, published online ahead of print March 19, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.012112
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Submitted October 10, 2002
Returned for revision November 8, 2002
Accepted February 28, 2003

Reproductive Technology


Induction of a Senescent-Like Phenotype Does Not Confer the Ability of Bovine Immortal Cells to Support the Development of Nuclear-Transfer Embryos

Wei Shi , Andreas Hoeflich , Heinrich Flaswinkel , Miodrag Stojkovic , Eckhard Wolf , and Valeri Zakhartchenko *

* To whom correspondence should be addressed. E-mail: v.zakhartchenko{at}gen.vetmed.uni-muenchen.de.

Abstract

Previously, we reported that cloned embryos derived from an immortalized bovine mammary epithelial cell line (MECL) failed to develop beyond 12-16 cell stage. To analyze whether induction of a senescent-like phenotype in MECL can improve their ability to support the development after transferred into enucleated oocytes, we treated MECL with DNA methylation inhibitor 5-aza-2- deoxycytidine (Aza-C), histone deacetylase inhibitors trichostatin A (TSA) or sodium butyrate (NaBu), 5- bromodeoxyuridine (BrdU) 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 (SA-{beta} gal) 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 to 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.



Key words: Embryo • Apoptosis • Early development • Mammary glands • Oocyte development


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