ABSTRACT
Background: A unique feature of embryo metabolism is production of reactive oxygen species [ROS]. It is well established that during in vitro culture, ROS levels increase over normal ranges observed for embryos developed in vivo. This study evaluates and compares the stepwise pattern of ROS production during in vitro development of reconstructed goat embryos produced by zona-free method of somatic cell nuclear transfer [SCNT]. Furthermore, the pattern of ROS production of SCNT embryos were compared with zona free embryos derived from in vitro fertilization [IVF]
Materials and Methods: In this experimental study, zona-free oocytes, SCNT and IVF embryos at different stages of in vitro development [2, 4, 8, 16-cells, morula, and blastocyst] were used for assessment of ROS production using 2, 7-dichloro dihydroflourescein diacetate [DCHFDA] probe and the result were presented as fold increase or decrease relative zona free oocytes
Results: The relative level of ROS compared to metaphase-II [MII] oocytes insignificantly decrease during early stages post embryo reconstitution and regained its value by 8-cell and morula stage and, significantly increase compared to MII oocytes by blastocyst stage
Conclusion: The pattern of ROS change in SCNT embryos is similar to zona free IVF derived embryos, except it decrease from two cell stage and regain its value at morula stage. The sudden rise in ROS at blastocyst stage, further emphasizes the special need of IVF and SCNT derived embryos during this stage of development
ABSTRACT
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. Our objective was to determine if treatment of donor cells for 72 hours with 5-aza-2'-deoxycytidine [5-aza-dc; 0-0.3 microM], a DNA methyl transferase inhibitor, improved development and expression of Oct-4. In comparison with untreated cells, 0.01 and 0.08 microM 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 microM 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 microM 5-aza-dc treated cells when compared with 0 and 0.01 microM 5-aza-dc treated cells. Although reconstructed embryos from 0.08 and 0.3 microM 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 microM 5-aza-dc remained unchanged. 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 embryo