Spindle dynamics in living mouse oocytes during meiotic maturation, ageing, cooling and overheating: a study by polarized light microscopy

Abstract

A liquid crystal polarized light microscope (LC PolScope) was used to examine spindle dynamics in living mouse oocytes. Immature oocytes were cultured for 0-48 h and spindles were imaged with the PolScope at various time points of culture. Oocytes at metaphase I (M-I) and metaphase II (M-II) were also exposed to shifts of temperature from 25 to 41 degrees C to examine the effects of fluctuations of temperature on spindle dynamics. After examination with the PolScope, some oocytes were fixed and examined by immunocytochemical staining and confocal microscopy. After culturing for 6 h, 76% and 2% of the oocytes reached M-I and M-II stages and all oocytes had birefringent spindles. When the oocytes were cultured for 14-16 h, 88% and 6% of oocytes were at M-II and M-I stages respectively and all oocytes had birefringent spindles. However, when the oocytes were cultured for 22-48 h, the proportions of oocytes with birefringent spindles decreased as culture time was increased. Exposure of oocytes to 25 degrees C induced spindle disassembly within 10-20 min in both M-I and M-II oocytes. Most (93-100%) oocytes reassembled spindles after warming at 37 degrees C. Furthermore, exposure of oocytes at M-I stage but not at M-II stage, to 30 degrees C also induced significant microtubule disassembly. However, exposure of oocytes to 38-41 degrees C did not obviously change the quantity of microtubules in the spindles, which was measured by retardance. This study indicates that the PolScope can be used to examine spindle dynamics in living oocytes, and it has the advantage over the routine fluorescence microscope in that images can be obtained in the same individual oocyte and the quantity of microtubules can be measured by retardance in living oocytes. These results also indicate that the M-II spindle in mouse oocytes is sensitive to oocyte ageing and cooling, but not heating, and M-I spindle is more sensitive to temperature decline than M-II spindle.