87 THE EFFECT OF LOW TEMPERATURE ON THE DETECTABILITY/VISUALIZATION OF THE MEIOTIC SPINDLE IN MII MOUSE OOCYTES

Abstract

The polscope uses novel electrooptical hardware and digital processing to image macromolecular structures in cells on the basis of their birefringence (Sato et al. 1975 J. Cell Biol. 76, 501-517). Imaging of the spindle in living oocytes with the polscope is based on birefringence of an inherent physical property of the microtubules. De-polymerization of microtubules arrests meiosis and induces abnormal meiosis generating chromosomally abnormal oocytes which are not capable of being fertilized and developing. In this study we examined whether spindles in living mouse oocytes can be safely imaged/examined by the polscope and investigated the influence of sub-optimal temperature on spindle detection/visualization. For oocyte collection, 6- to 8-week-old CB6F1 female mice were superovulated with PMSG (10 IU, i.p.; Sigma, USA) and 46 to 48 h later they were injected with hCG (10 IU, i.p.; Sigma). Oocytes were collected 20 to 23 h after hCG treatment in MOPS buffered medium (G-MOPS"; Vitrolife, Sweden AB, Kungsbacka, Sweden) supplemented with human serum albumin (HAS"; Vitrolife). Cumulus cells were removed from the zonae pellucidae by exposure to a solution of 40 IU/mL hyaluronidase (HYASE"; Vitrolife). For spindle examination, each oocyte was placed in a 5-µL drop of G-MOPS medium, covered with oil (Ovoil; Vitrolife), and examined in a Delta T.C.O. dish with a specially coated glass bottom (Willco-Dish, Willco Wells, Amsterdam, The Netherlands). Oocytes were imaged by a Nikon Diaphot microscope with a video camera, objective lens, and controller, combined with a computerized imaging analysis system (CRI, Great Britain). For evaluation of the effect of sub-optimal temperature on the visualization/detection of the spindle, mouse oocytes were cultured for a short period of time at sub optimal temperatures (18 to -20°C/25 min and -10 to -15°C/2 min). After that, the oocytes were immediately checked for presence and condition of the meiotic spindle and re-checked after 30-min culture at 37°C with 6% CO₂, 5% O₂, 89% N₂ and maximal humidity in air. There was no difference in the detect ability of the spindle in mouse oocytes with or without polar body (PB+: 21/24, 87%; PB-: 29/32, 90%). We found that the treatment with sub-optimal temperature has an effect to the spindle visualization. Immediately after treatment, the spindle could be detected in 80% of the oocytes (16/20). However, after 30-min culture, the meiotic spindle was detectable in only 25% of the treated oocytes (5/20; P < 0.05). Sub optimal temperature at first increased the birefringence of the meiotic spindle for a short period of time; then the spindle became completely undetectable. The increased sensitivity of the meiotic spindle to low temperature is one of the biggest obstacles to the oocyte cryopreservation. Our results provide further evidence that the spindle in mammalian oocytes is very sensitive to temperature fluctuations. The increase in the birefringence was the first sign that the structure of microtubules and other cytoskeletal factors forming the spindle had changed. Investigations are underway to learn more about the capability of the meiotic spindle to become reorganized.