The effects of cooling mouse oocytes

Abstract

The effects of cooling and warming on meiotic spindles of mouse oocytes have been assessed by transmission electron microscopy. Intact cumulus—oocyte complexes were immediately cooled from 37 to 15, 4, 0, and −7°C (seeding temperature) for 15 min in a programmed biological freezer and fixed at these temperatures. Other complexes, cooled to these temperatures, were rapidly warmed to 37°C and incubated for 2 hr before fixation at 37°C. Of 334 oocytes assessed at various temperatures, at least 100 were examined for metaphase II spindles. Spindle microtubules completely disappear at 0 and −7°C, while complete or partial depolymerization of microtubules was observed at 4°C. Cooling to 15°C did not cause major disruptions of spindle structure in most oocytes. Chromosomes tended to rotate or clump at lower temperatures but chromosome scatter outside the spindle zone was rarely observed. Centrosomal material was fragmented at 4°C and occasionally at 15°C and was not evident at the spindle poles at 0 and −7°C. Kinetochores were seen at all temperatures. Spindle structure was evidently restored in the majority of oocytes on rewarming at 37°C. Changes in the ooplasm induced by cooling were elongation and disruption of vesicular smooth endoplasmic reticulum, especially between lipid globules and disappearance of fibrillar inclusions. Cortical granule exocytosis was not observed on cooling, while microfilaments were intact. Swelling of membranous organelles was also observed in cumulus cells. Most of the cytoplasmic changes were also reversed on rewarming. The response of mouse oocytes to cooling is compared to that of human oocytes, reported previously.