Introduction: Cloning is an asexual reproductive method that results in genetically identical offsprigs. The principle of cloning is transfer of a donated uncleus (from a somatic or stem cell) to an enucleated oocyte. Nuclear transfer procedure includes two parts: 1) Enucleation: removal of nucleus from an. 2) Nuclear transfer: transfer of the donated nucleus to the enucleated oocyte. The role of oocyte for initiation, and direction the developmental procedure of reconstructed zygote is so important, because it is not only, as a nutritional support? but also has so many important and special factors for reprogramming the donated nucleus and fetal development. Therefore, finding a method that able to enucleate the oocyte without damaging it’s cytoplasm and mitochondrial DNA, is and important prognostic factor of cloning.
Purpose: The goal of this project is the nucleation of the matured mouse oocytes, arrested at MII (metaphase II).
Method and Materials: matured oocytes, arrested at MII, were collected after superovulation by using 7.5IU PMSG, followed by 7.5IU HCG, 48h later. UP to the method using for identifying the location of chromosomes, we arranged 3 groups of oocyte:
1) oocytes stained by Hoechst- 33342, followed by UV irradiation (Hoechst group).
2) oocytes placed in the media contains 3% sucrose that biuldges the chromatin (sucrose group).
3) oocytes that their chromatin identified by the ability of researcher and with no staining, substance or irradiation. After identification of chromatin, enucleation was done by microinjection tool.
Result: The ratio of intact and degenerated oocytes in sucrose group was lower than to all of other groups (46.8%), and has shown a significant difference (p<0.001). On the other hand the best result was achieved from control group with no staining, substance or irradiation.
Conclusion and Discussion: It is no important to find a method that not only able to successfully enucleate the oocyte, but also it would be able to guaranty the oocyte’s integrity.
Most of staining or other substances and methods, used for identifying the location of chromosome, have adverse effects on mitochondrial DNA and cytoplasm is oocytes.
Although in the experienced hands, with no exposure to any substance, enucleation can be done successfully (success rate: 80%). As mentioned previously, in this project, the best result has been achieved in control group? success rate: 82%, compatible with the results of other studies.