Vitrification of mature mouse oocytes: improved results following addition of polyethylene glycol to a dimethyl sulfoxide solution.
Oocytes have been successfully cryopreserved using rapid and slow freezing procedures; however, variability in the success of replicates has limited its practical application. We have evaluated the potentially beneficial effects of adding 1 mg/ml of the polymer polyethylene glycol (PEG) (M(r) 8000) to a 6 M dimethyl sulfoxide (Me2SO) vitrification solution. Stepwise addition of cryoprotectant, either with or without PEG, was performed at room temperature (19-21 degrees C). Oocytes were then loaded in plastic insemination straws and held in liquid nitrogen vapour at -140 degrees C for 3 min prior to storage in liquid nitrogen. Oocytes were warmed rapidly to room temperature and removal of cryoprotective agent was effected in the presence of 1 M sucrose solution. Viability was assessed by vitro fertilization. Oocytes cryopreserved after exposure to 6 M Me2SO in the absence of PEG showed 60% normality, 80% fertilization, and 55% development to blastocyst, median of 11 replicate experiments (191 oocytes). Individual replicates yield highly variable survival which ranged from 0 to 100%. The addition of PEG significantly improved oocyte normality to 95% (range 76-100%; median of 9 replicate experiments, 301 oocytes). Rates of fertilization (91%; 60-100%) and development of blastocyst (73%; 67-92) were also improved. The addition of 1 mg/ml PEG to a 6 M Me2SO solution resulted in greatly improved viability of oocytes following cryopreservation and vastly reduced the variability seen with Me2SO solution alone.
O'Neil
L
L
Department of Obstetrics and Gynaecology, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom.
Paynter
S J
SJ
Fuller
B J
BJ
D000818:Animals; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide; D005314:Embryonic and Fetal Development; D005069:Evaluation Studies as Topic; D005260:Female; D005307:Fertilization in Vitro; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D009865:Oocytes / Q000166:cytology; D011092:Polyethylene Glycols; D012996:Solutions
D003451:Cryoprotective Agents; D012996:Solutions; D011092:Polyethylene Glycols; D004121:Dimethyl Sulfoxide