title: Effects of antifreeze proteins on the vitrification of mouse oocytes: comparison of three different antifreeze proteins.
abstract: OBJECTIVE
  Can antifreeze proteins (AFPs) from three different sources improve the efficacy of mouse oocyte vitrification?
  
  
  CONCLUSIONS
  Treatment with AFPs can improve both murine oocyte quality and embryo development, and reduce reactive oxygen species (ROS) production in vitrified-warmed oocytes.
  
  
  BACKGROUND
  A previous study discovered that vitrification of immature oocytes and 2-cell stage embryos of mice augmented with antifreeze glycoproteins at 40 mg/ml dramatically improved the morphological integrity of the samples, suggesting that AFPs have the ability to inhibit ice formation and stabilize the plasma membrane.
  
  
  METHODS
  Metaphase II oocytes were obtained from 4-week-old BD-F1 mice. AFPs from bacteria (Flavobacterium frigoris ice-binding protein (FfIBP)), yeast (Glaciozyma sp. ice-binding protein (LeIBP)) and fish (Type III AFP) were added to the vitrification and warming solutions individually. Survival and development, meiotic spindle organization, intracellular ROS, mitochondrial activity, DNA double-strand breaks (DSBs) and repair of damaged DNA were analyzed.
  
  
  METHODS
  Vitrification of oocytes was performed with the CryoTop (equilibration solution: 7.5% ethylene glycol (EG) and 7.5% 1,2-propandiol (PROH) for 5 min; vitrification solution: 15% EG, 15% PROH and 0.5 M sucrose for 1 min). Warming was performed in three steps with decreasing concentrations of sucrose (1.0, 0.5 and 0.25 M sucrose).
  
  
  RESULTS
  AFP treatment can improve murine oocyte quality and embryo development. Survival rates, cleavage rates and blastocyst rates (blastocyst per cleaved and per survived oocytes) of oocytes in AFP-treated groups were significantly higher than those in the control group [75.0, 89.0, 90.0 and 85.0% for survival rate (P = 0.012); 58.7, 89.0, 87.8 and 81.2% for cleavage rate (P = 0.003); 52.3, 87.7, 78.5 and 76.8% for blastocyst per cleaved oocytes (P < 0.01); 30.7, 78.0, 68.9 and 62.4% for blastocyst per survived oocytes (P < 0.01) in control, FfIBP, LeIBP and Type III AFP-treated groups, respectively]. The mean (±SD) number of apoptotic blastomeres per blastocyst was significantly lower in AFP-treated groups than in the control group (9.1 ± 1.0, 2.0 ± 1.7, 2.3 ± 1.2 and 2.7 ± 2.4 in control, FfIBP, LeIBP and Type III AFP-treated groups, respectively, P = 0.040). FfIBP treatment was the most effective in maintaining normal meiotic spindle organization and chromosome alignment (52.0, 92.0, 80.0 and 83.0% in control, FfIBP, LeIBP and Type III AFP-treated groups, respectively, P < 0.01). Intracellular ROS levels (mean ± SD) significantly decreased in the AFP-treated groups (17.0 ± 11.2, 8.4 ± 8.2, 10.3 ± 6.4 and 11.6 ± 12.3 in control, FfIBP, LeIBP and Type III AFP-treated groups, respectively, P < 0.01), and the FfIBP and LeIBP groups had significantly lower DNA DSBs, compared with controls (65.2, 30.8, 44.4 and 55.8% in control, FfIBP, LeIBP and Type III AFP-treated groups, respectively, P < 0.01).
  
  
  CONCLUSIONS
  The origins of FfIBP and LeIBP were bacteria and yeast, respectively. Therefore, treatment of human oocytes and embryos with these AFPs should be tested before clinical application.
  
  
  CONCLUSIONS
  After further research, AFPs can potentially be applied to human oocyte cryopreservation to improve the efficacy of vitrification.
  
  
  BACKGROUND
  This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0055). The authors have no conflict of interest to declare.
authors.affiliation: Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam 463-707, Korea.
authors.forename: Hyang Heun
authors.initials: HH
authors.lastname: Lee
authors.affiliation: Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam 463-707, Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea Department of Obstetrics and Gynecology, Fertility Center of CHA Gangnam Medical Center, CHA University, Seoul 135-916, Korea.
authors.forename: Hee Jun
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Pukyong National University, Busan 608-737, Korea Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Korea.
authors.forename: Hak Jun
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Korea.
authors.forename: Jun Hyuck
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Biotechnology, Korea University, Seoul 136-713, Korea.
authors.forename: Yong
authors.initials: Y
authors.lastname: Ko
authors.affiliation: Seoul National University Hospital Health Care System Gangnam Center, Seoul 135-984, Korea.
authors.forename: Sun Mie
authors.initials: SM
authors.lastname: Kim
authors.affiliation: Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam 463-707, Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea leejrmd@snu.ac.kr.
authors.forename: Jung Ryeol
authors.initials: JR
authors.lastname: Lee
authors.affiliation: Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam 463-707, Korea Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea.
authors.forename: Chang Suk
authors.initials: CS
authors.lastname: Suh
authors.affiliation: Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea.
authors.forename: Seok Hyun
authors.initials: SH
authors.lastname: Kim
mesh_terms: D000818:Animals; D021301:Antifreeze Proteins; D001426:Bacterial Proteins; D001755:Blastocyst; D015925:Cryopreservation / Q000379:methods*; D047108:Embryonic Development; D005260:Female; D029941:Fish Proteins; D005656:Fungal Proteins; D008677:Metaphase; D051379:Mice; D009865:Oocytes; D058989:Vitrification
chemical_list: D021301:Antifreeze Proteins; D001426:Bacterial Proteins; D029941:Fish Proteins; D005656:Fungal Proteins
keywords: antifreeze protein; cryopreservation; fertility preservation; oocyte; vitrification