THE EFFECT OF COOLING AND HYPERTONIC EXPOSURE ON MURINE OOCYTE FUNCTION, FERTILIZATION, AND DEVELOPMENT

Several individual but related steps are involved in the cryopreservat ion process, including the addition of cryoprotectants at various temp eratures, cooling to subzero temperatures, and long-term storage. The process is completed by rewarming and removal of cryoprotectants prior to a return to physiological conditions. In this series of experiment s we have attempted to distinguish the effects of some of these proced ures. Control, untreated ovulated mouse oocytes showed 95% in vitro fe rtilization (190/200) and 92% subsequent development to hatching blast ocyst (184/200). Exposure of oocytes to either isotonic or hypertonic media at 37 degrees C did not significantly change the rate of fertili zation (90%, 108/120; and 89%, 154/174, respectively) or subsequent em bryonic development (85%, 102/120; and 82%, 143/174, respectively). Sl ow cooling in isotonic medium (- 3 degrees C/min) to 0 degrees C had n o effect on the rate of fertilization (83%, 103/124), but rapid coolin g (>1000 degrees C/min) to 0 degrees C resulted in a significant reduc tion in fertilization rate to 75% (151/202). When oocytes suspended in a hypertonic solution were cooled using slow or rapid rates, there we re marked decreases in fertilization to 26% (61/231) and 56% (156/278, respectively. Subsequent embryonic growth was reduced to 15% (34/231) after slow cooling and 26% (72/278) after rapid cooling. Exposure of oocytes to glycerol at 37 degrees C and dimethyl sulfoxide at 0 degree s C reduced the fertilization rate to 57% (67/118) and 73% (103/145), respectively, with a corresponding reduction in embryonic growth to 52 % (61/118) and 65% (94/145), but there were no additional effects of c ooling or hypertonic exposure after addition of cryoprotectants. (C) 1 995 Academic Press, Inc.