Effects of freezing and thawing on mammalian oocyte

In an attempt to study the deleterious effects which occur during the freezing and thawing of mammalian oocytes, we developed a cryomicroscope controlled by digital programmable equipment. The program permits any cooling rate between 0.1 and 60 degrees C/min with a precision of 0.6 degrees C. Using a precooled stage, it is possible to obtain rapid cooling (100 degrees C/min). The maximum thawing rate is about 60 degrees C/min. A copper-- constantan microthermocouple allows precise measurement of the specimen temperature. All information (specimen, temperature of the specimen, date, hour, and minutes) is recorded at the same time on photographic film by a camera fitted with a " Recordata Back" and a motor drive which allows three frames per second. Our preliminary results show that: (1) rapid cooling yields a supercooling with simultaneous extra- and intracellular crystallization; (2) slow cooling with seeding at -8 degrees C gives an extracellular crystallization which is achieved by -9 degrees C, followed by an extracellular recrystallization occurring at almost -8 degrees C which alters the morphology of the oocyte and the zona pellucida, without any visible intracellular crystallization; (3) during continued slow cooling the oocytes dehydrate without any intracellular freezing; and (4) during rewarming a partial rehydration of the cell occurs with a swelling of the oocytes to their original volumes after the thawing has been achieved.