Hyang Heun Lee1, Hee Jun Lee2, Hak Jun Kim3, Jun Hyuck Lee4, Yong Ko5, Sun Mie Kim6, Jung Ryeol Lee7, Chang Suk Suh8, Seok Hyun Kim9. 1. Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam 463-707, Korea. 2. 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. 3. Department of Chemistry, Pukyong National University, Busan 608-737, Korea Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Korea. 4. Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Korea. 5. Department of Biotechnology, Korea University, Seoul 136-713, Korea. 6. Seoul National University Hospital Health Care System Gangnam Center, Seoul 135-984, Korea. 7. 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. 8. 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. 9. Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea.
Abstract
STUDY QUESTION: Can antifreeze proteins (AFPs) from three different sources improve the efficacy of mouse oocyte vitrification? SUMMARY ANSWER: Treatment with AFPs can improve both murine oocyte quality and embryo development, and reduce reactive oxygen species (ROS) production in vitrified-warmed oocytes. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, 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). MAIN RESULTS AND THE ROLE OF CHANCE: 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). LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: After further research, AFPs can potentially be applied to human oocyte cryopreservation to improve the efficacy of vitrification. STUDY FUNDING/COMPETING INTERESTS: 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.
STUDY QUESTION: Can antifreeze proteins (AFPs) from three different sources improve the efficacy of mouse oocyte vitrification? SUMMARY ANSWER: Treatment with AFPs can improve both murine oocyte quality and embryo development, and reduce reactive oxygen species (ROS) production in vitrified-warmed oocytes. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, 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). MAIN RESULTS AND THE ROLE OF CHANCE: 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). LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: After further research, AFPs can potentially be applied to human oocyte cryopreservation to improve the efficacy of vitrification. STUDY FUNDING/COMPETING INTERESTS: 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: Predrag Jevtić; K Wade Elliott; Shelby E Watkins; Jonathan A Sreter; Katarina Jovic; Ian B Lehner; Paul W Baures; John G Tsavalas; Daniel L Levy; Krisztina Varga Journal: J Exp Biol Date: 2022-02-15 Impact factor: 3.312