Effects of antifreeze proteins on the vitrification of mouse oocytes

Comparison of three different antifreeze proteins

Hyang Heun Lee, Hee Jun Lee, Hak Jun Kim, Jun Hyuck Lee, Yong Ko, Sun Mie Kim, Jung Ryeol Lee, Chang Suk Suh, Seok Hyun Kim

Research output: Contribution to journalArticle

24 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2110-2119
Number of pages10
JournalHuman Reproduction
Volume30
Issue number9
DOIs
Publication statusPublished - 2015 Apr 9

Fingerprint

Antifreeze Proteins
Vitrification
Flavobacterium
Ice
Oocytes
Type III Antifreeze Proteins
Carrier Proteins
Blastocyst
Sucrose
Reactive Oxygen Species
Spindle Apparatus
Ethylene Glycol
Embryonic Development
Embryonic Structures
Yeasts
Bacteria
Blastomeres
Control Groups
Double-Stranded DNA Breaks
Cryopreservation

Keywords

  • antifreeze protein
  • cryopreservation
  • fertility preservation
  • oocyte
  • vitrification

ASJC Scopus subject areas

  • Rehabilitation
  • Obstetrics and Gynaecology
  • Reproductive Medicine

Cite this

Effects of antifreeze proteins on the vitrification of mouse oocytes : Comparison of three different antifreeze proteins. / Lee, Hyang Heun; Lee, Hee Jun; Kim, Hak Jun; Lee, Jun Hyuck; Ko, Yong; Kim, Sun Mie; Lee, Jung Ryeol; Suh, Chang Suk; Kim, Seok Hyun.

In: Human Reproduction, Vol. 30, No. 9, 09.04.2015, p. 2110-2119.

Research output: Contribution to journalArticle

Lee, Hyang Heun ; Lee, Hee Jun ; Kim, Hak Jun ; Lee, Jun Hyuck ; Ko, Yong ; Kim, Sun Mie ; Lee, Jung Ryeol ; Suh, Chang Suk ; Kim, Seok Hyun. / Effects of antifreeze proteins on the vitrification of mouse oocytes : Comparison of three different antifreeze proteins. In: Human Reproduction. 2015 ; Vol. 30, No. 9. pp. 2110-2119.
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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.",
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T1 - Effects of antifreeze proteins on the vitrification of mouse oocytes

T2 - Comparison of three different antifreeze proteins

AU - Lee, Hyang Heun

AU - Lee, Hee Jun

AU - Kim, Hak Jun

AU - Lee, Jun Hyuck

AU - Ko, Yong

AU - Kim, Sun Mie

AU - Lee, Jung Ryeol

AU - Suh, Chang Suk

AU - Kim, Seok Hyun

PY - 2015/4/9

Y1 - 2015/4/9

N2 - 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.

AB - 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.

KW - antifreeze protein

KW - cryopreservation

KW - fertility preservation

KW - oocyte

KW - vitrification

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