Sucrose pretreatment for enucleation: An efficient and non-damage method for removing the spindle of the mouse MII oocyte

Oocytes enucleated at metaphase II stage can support reprogramming of trans ferred nucleus and further developing to term. However, the first polar bod y in mice sometimes migrates away from the original place of expulsion, so the chromosomes of the oocyte will displace from the first polar body. Thus , it is not always possible to successfully enucleate according to the posi tion of the first polar body. Here we use sucrose treatment to visualize me taphase spindle fibers and chromosomes with standard light microscopy. In t he manipulation medium containing 3% sucrose, oocytes of poor quality becom e shrunken, deformed or fragmented, while oocytes of good quality in the sa me medium would show a swelling around the metaphase chromosomes and a tran sparent spindle area, shaped like "infinity" and "0". So it is easy to remo ve the well-distinguished spindle and chromosomes in oocytes of good qualit y. Re-examined by Hoechst 33342 stain under the UV light, the enucleation r ate was 100%. There was no significant difference in IVF and cleavage rates between the sucrose treatment and the control group. In conclusion, this s tudy demonstrated that 3% sucrose pretreatment can give a method for evalua ting embryo quality and more importantly, it can, under a common microscope , allow the visualization of the spindle and chromosomes in oocytes of good quality and hence efficiently improve enucleation rate without any harm. ( C) 2001 Wiley-Liss, Inc.