Parthenogenetic activation pattern and microtubular organization of the mouse oocyte after exposure to...

We have studied the effect of 1,2-propanediol (PROH) on cumulus-oocyte complexes from the mouse. We determined the morphological survival rate, the pattern of parthenogenetic activation, and the microtubular and chromosomal organization. Cumulus-oocyte complexes were collected at 16 h post hCG from superovulated female hybrid mice. These cumulus-intact oocytes were exposed to 1.5 or 3 M PROH for 6, 12, or 18 min at 0, 22, or 37 degrees C. The cryoprotectant was diluted out in a 1 M sucrose solution at 22 degrees C. After 5-6 h at 37 degrees C, oocytes were denuded and examined under Nomarski optics. The results show that PROH can induce degeneration and parthenogenetic activation in the mouse oocyte in a concentration, temperature, and time-dependent way. As the activation stimulus was strengthened, an increasing proportion of oocytes shifted from parthenogenetic activation with polar body extrusion to parthenogenetic activation with polar body retention and even to immediate cleavage. Nontoxic and nonactivating conditions involved mainly exposure to 1.5 M PROH at 0 degrees C. Spindle integrity and chromosomal organization were analyzed for exposure to 1.5 and 3 M PROH for 12 min at 0 degrees C. The separate effect of cooling and exposure to 1 M sucrose were also evaluated. Microtubules were visualized by monoclonal anti-alpha-tubulin labeling followed by immunogold-silver staining. Cooling and exposure to 1 M sucrose or to 1.5 M PROH did not induce major abnormalities in the microtubular or chromosomal organization. On the other hand, a significant percentage of deformities such as spindle size reduction and loss of bipolarity were observed after exposure to 3 M PROH. The results of the present study demonstrate that the use of PROH as a single cryoprotectant for the freezing of mature unfertilized oocytes cannot be recommended in procedures involving ambient temperature or concentrations exceeding 1.5 M PROH. On the other hand, the potential beneficial effect of low temperatures may outweigh the effect of concentration at subzero temperatures and could be explored further in the tailoring of conditions for slow controlled freezing.