Developmental Competence and Pluripotency Gene Expression of Cattle Cloned Embryos Derived from Donor Cells Treated with 5-aza-2'-deoxycytidine.
BACKGROUND
Reconstructed embryos from terminally differentiated somatic cells have revealed high levels of genomic methylation which results in inappropriate expression patterns of imprinted and non-imprinted genes. These aberrant expressions are probably responsible for different abnormalities during the development of clones. Improvement in cloning competency may be achieved through modification of epigenetic markers in donor cells.


METHODS
Our objective was to determine if treatment of donor cells for 72 hours with 5-aza-2'-deoxycytidine (5-aza-dc; 0-0.3 μM), a DNA methyl transferase inhibitor, improved development and expression of Oct-4.


RESULTS
In comparison with untreated cells, 0.01 and 0.08 μM 5-aza-dc treated cells insignificantly decreased the blastocyst rate (32.1% vs. 28.6% and 27.2%, respectively) while it was significant for 0.3 μM treated cells (6.5%). Embryo quality as measured by the total cell number (TCN) decreased in a dose-related fashion, which was significant at 0.08 and 0.3 μM 5-aza-dc treated cells when compared with 0 and 0.01 μM 5-aza-dc treated cells. Although reconstructed embryos from 0.08 and 0.3 μM 5-aza-dc treated cells showed lower levels of DNA methylation and histone H3 acetylation, development to blastocyst stage was decreased. The epigenetic markers of embryos cloned from 0.01 μM 5-aza-dc remained unchanged.


CONCLUSIONS
These results show that 5-aza-dc is not a suitable choice for modifying nuclear reprogramming. Finally, it was concluded that the wide genomic hypomethylation induced by 5-aza-dc deleteriously impacts the developmental competency of cloned embryos.
Department of Anatomy and Embryology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.
Farnoosh
F
Jafarpour
Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran.
Sayed Morteza
SM
Hosseini
Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran.
Mahdi
M
Hajian
School of Sciences, Islamic Azad University, Marvdasht Branch, Marvdasht, Iran.
Mohsen
M
Forouzanfar
Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran.
Parvaneh
P
Abedi
Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran.
Laleh
L
Hosseini
Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran.
Somaye
S
Ostadhosseini
Department of Anatomy and Embryology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.
Soghra
S
Gholami
Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran.
Mohammad Hossein
MH
Nasr Esfahani
Epigenesis; Nuclear Reprogramming; Nuclear Transfer