Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits

Abstract

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.

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].…”

“…Sucrose has been widely used in embryo and oocyte cryopreservation [18][19][20][21][22][23] as well as micromanipulations, such as intracytoplasmic sperm injection and nuclear transfer [24][25][26][27]. Unable to permeate through the plasma membrane, sucrose induces water molecules to move out of the oocytes and, thus, effectively cause them to shrink.…”

Effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos in vitro

“…After 96 h of the culture the intrazonal blastocyst diameters were quickly measured using a microscope fitted with a calibrated eyepiece micrometer, as described by Hallden and colleagues (1992). Immediately after this measurement, the blastocysts were stained with Hoechst 33342 dye (Sigma), as described previously (Pursel et al 1985, as modified by Yang et al 1990), and the number of total cells per blastocyst were counted using a Nikon inverted microscope equipped with an ultraviolet light source and a ×40 fluorescence objective (Nikon Inc., Melville, NY, USA).…”

Rabbit blastocyst: Allocation of cells to the inner cell mass and trophectoderm

“…The mucin coat and zona pellucida were removed (Yang et al, 1990). Briefly, embryos were incubated in acidic PBS (pH 2.4) for 20-60 sec at room temperature until the mucin coat turned light brown.…”