The effects of cooling human oocytes.

Preovulatory human oocytes were cooled to 0 degrees C at 1 degree C/min, with or without the cryoprotectant dimethyl sulphoxide (DMSO), to assess the effects of cooling on the meiotic spindles and on oocyte structure. Batches of oocytes, cultured for 3-9 h, were held at 0 degrees C for 20 or 60 min and then fixed for transmission electron microscopy (TEM) either at 0 or 8 degrees C. Control oocytes were not cooled and were fixed at 22 or 37 degrees C for comparison. TEM revealed that 80% of the oocytes were at metaphase II, while 20% were at metaphase I and most had resumed meiosis recently. Control oocytes had more or less barrel-shaped meiotic spindles composed of microtubules (MT), some associated with chromosomes at kinetochores. Both metaphase I and II spindles were disassembled when cooled and fixed at 0 degrees C, with or without DMSO, due to extensive depolymerization of MT. The few MT that survived were found at the poles or were bundled together or were associated with chromosomes. Kinetochores were not prominent. Some oocytes cooled with DMSO and fixed at 0 or 8 degrees C showed evidence of MT, but the spindles were still disorganized and were abnormal in structure. Chromosomes tended to clump together or were dislocated in the cortical ooplasm in cooled oocytes, but widespread scattering was not observed. This was particularly evident in the absence of DMSO. Elements of the endoplasmic reticulum, Golgi, mitochondria and the cytosol were also adversely affected in some of the cooled oocytes and their surrounding cumulus cells. The results show that meiotic spindles are very sensitive to simple cooling and that DMSO does not provide substantial stabilization of the meiotic spindle even at 0 degrees C. The findings are discussed with reference to recent work on frozen human and mouse oocytes.