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

Abstract

Oocytes enucleated at metaphase II stage can support reprogramming of transferred nucleus and further developing to term. However, the first polar body 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 position of the first polar body. Here we use sucrose treatment to visualize metaphase spindle fibers and chromosomes with standard light microscopy. In the manipulation medium containing 3% sucrose, oocytes of poor quality become shrunken, deformed or fragmented, while oocytes of good quality in the same medium would show a swelling around the metaphase chromosomes and a transparent spindle area, shaped like "infinity" and "0". So it is easy to remove the well-distinguished spindle and chromosomes in oocytes of good quality. Re-examined by Hoechst 33342 stain under the UV light, the enucleation rate was 100%. There was no significant difference in IVF and cleavage rates between the sucrose treatment and the control group. In conclusion, this study demonstrated that 3% sucrose pretreatment can give a method for evaluating 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.

Hypertonicity-Induced Projections Reflect Cell Polarity in Mouse Metaphase II Oocytes: Involvement of Microtubules, Microfilaments, and Chromosomes1

“…It is important to point out that the sucrose-induced shrinkage of the oocytes does not significantly reduce the viability of the oocytes or the subsequent development of the embryos, as shown by our studies [10,11,24,25] and by those of others [18-23, 26, 27]. The electrolyte NaCl, on the other hand, has a deleterious effect on the viability of oocytes and is not suitable for the induction of projection for purposes of micromanipulation [31,32].…”

“…We surmised that such treatment of oocytes could induce or enhance the possibility of the spindle undergoing changes distinct from those of other cytoplasmic components. However, systematic analyses to test this hypothesis remained to be performed [10]. Here, we examined the factors responsible for inducing changes in the spindle area of mouse MII oocytes, and we found that hypertonic sucrose, not serum or standard media, is important for the swelling of the spindle area and the formation of projections.…”

“…Our previous study [10] showed that after incubation in M2 Ϫ medium (i.e., M2 medium without glucose and phosphate) containing 90 mM sucrose and 20% (v/v) FBS, the cytoplasm surrounding the meiotic apparatus of the MII oocyte swelled and the vicinity of the spindle or chromosomes became transparent and easily discerned under standard light microscopy. We surmised that such treatment of oocytes could induce or enhance the possibility of the spindle undergoing changes distinct from those of other cytoplasmic components.…”

“…In our previous study [10], we found that after a 3% (w/ v) (90 mM) sucrose treatment, the region including the MII spindle and chromosomes in mouse oocytes could be visualized as a projection under standard light microscopy. A similar phenomenon has also been found in bovine oocytes [11].…”

Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

“…This somatic cell nuclear transfer manipulation was a combination of the Roslin technique and the Honolulu technique [14,15]; after culture under serum-starvation culture, a 3 h post-electrofusion interval and strontium activation were adopted. The nuclear transfer manipulation solution is CZB medium supplemented with 3% sucrose, 10% FCS, and 5 Ag/ml cytochalasin B [16]. The CZB culture medium free of glucose was used for early embryo development [15,17].…”