CYTOSKELETAL ORGANIZATION AND ZONA SENSITIVITY TO DIGESTION BY CHYMOTRYPSIN OF FROZEN - THAWED MOUSE OOCYTES

Mouse oocyte-cumulus masses were added to 1.5 dimethyl sulphoxide (DMSO) + 20% fetal bovine serum (FBS) that had been precooled at + 4-degrees-C, were frozen by slow cooling to an intermediate temperature of -60-degrees-C before being plunged into liquid nitrogen at -196-degrees-C, subjected to a controlled thaw, expelled into 1.5 M DMSO + FBS at 4-degrees-C, and then washed in medium + FBS at 37-degrees-C. Of 7733 oocytes treated, 78.4% were viable (controls; no treatment: 94.2% of 2764 oocytes; cryoprotectant only: 92.2% of 2991 oocytes). The oocyte losses were not due to complete loss of all oocytes from some straws or mice, since analysis of individual straws containing oocytes from a single mouse revealed considerable inter-straw/mouse variation. Amongst surviving oocytes, no significant differences between frozen and control oocytes in spindle, chromosomal or microfilament organization were recorded. Two significant differences were observed: (i) fewer frozen - thawed oocytes had zonae resistant to chymotrypsin digestion, and (ii) spindle organization in control oocytes, but not frozen - thawed oocytes, was improved by 3 h incubation at 37-degrees-C. More of the abnormal than the normal frozen-thawed and control oocytes were surrounded by zonae which were resistant to digestion by chymotrypsin.