title: The effect of cooling and hypertonic exposure on murine oocyte function, fertilization, and development.
abstract: Several individual but related steps are involved in the cryopreservation process, including the addition of cryoprotectants at various temperatures, 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 experiments we have attempted to distinguish the effects of some of these procedures. Control, untreated ovulated mouse oocytes showed 95% in vitro fertilization (190/200) and 92% subsequent development to hatching blastocyst (184/200). Exposure of oocytes to either isotonic or hypertonic media at 37 degrees C did not significantly change the rate of fertilization (90%, 108/120; and 89%, 154/174, respectively) or subsequent embryonic development (85%, 102/120; and 82%, 143/174, respectively). Slow cooling in isotonic medium (-3 degrees C/min) to 0 degree C had no effect on the rate of fertilization (83%, 103/124), but rapid cooling (> 1000 degrees C/min) to 0 degree C resulted in a significant reduction in fertilization rate to 75% (151/202). When oocytes suspended in a hypertonic solution were cooled using slow or rapid rates, there were 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 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 cooling or hypertonic exposure after addition of cryoprotectants.
authors.affiliation: Department of Obstetrics & Gynaecology, Royal Free Hospital School of Medicine, London, United Kingdom.
authors.forename: J E
authors.initials: JE
authors.lastname: Hunter
authors.forename: B J
authors.initials: BJ
authors.lastname: Fuller
authors.forename: A
authors.initials: A
authors.lastname: Bernard
authors.forename: R W
authors.initials: RW
authors.lastname: Shaw
mesh_terms: D000818:Animals; D003080:Cold Temperature; D015925:Cryopreservation / Q000379:methods*; D004121:Dimethyl Sulfoxide; D005314:Embryonic and Fetal Development; D005069:Evaluation Studies as Topic; D005260:Female; D005307:Fertilization in Vitro; D005990:Glycerol; D006982:Hypertonic Solutions; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D009865:Oocytes / Q000254:growth & development / Q000502:physiology
chemical_list: D006982:Hypertonic Solutions; D005990:Glycerol; D004121:Dimethyl Sulfoxide