Yonsei Med J.  2015 May;56(3):778-784. 10.3349/ymj.2015.56.3.778.

Effect of Antifreeze Protein on Mouse Ovarian Tissue Cryopreservation and Transplantation

• Lee JR
• Youm HW
• Lee HJ
• Jee BC
• Suh CS
• Kim SH

• KMID: 2450353
• DOI: http://doi.org/10.3349/ymj.2015.56.3.778

Abstract

PURPOSE
To investigate the effect of antifreeze protein (AFP) supplementation on ovarian vitrification and transplantation.
MATERIALS AND METHODS
In this experimental study, we researched a total of 182 ovaries from 4-week-old ICR mice. The equilibration solution included 20% ethylene glycol (EG), and the vitrification solution included 40% EG, 18% Ficoll, and 0.3 M sucrose. Intact ovaries were first suspended in 1 mL of equilibration solution for 10 min, and then mixed with 0.5 mL of vitrification solution for 5 min. Ovaries were randomly assigned to 3 groups and 0, 5, or 20 mg/mL of type III AFP was added into the vitrification solution (control, AFP5, and AFP20 groups, respectively). The vitrified ovaries were evaluated after warming and 2 weeks after autotransplantation. The main outcome measurements are follicular morphology and apoptosis assessed by histology and the TUNEL assay.
RESULTS
A significantly higher intact follicle ratio was shown in the AFP treated groups (control, 28.9%; AFP5, 42.3%; and AFP20, 44.7%). The rate of apoptotic follicles was significantly lower in the AFP treated groups (control, 26.6%; AFP5, 18.7%; and AFP20, 12.6%). After transplantation of the vitrified-warmed ovaries, a significantly higher intact follicle ratio was shown in the AFP20 group. The rate of apoptotic follicles was similar among the groups.
CONCLUSION
The results of the present study suggest that supplementing AFP in the vitrification solution has beneficial effects on the survival of ovarian tissue during cryopreservation and transplantation.

Keyword

Antifreeze protein; ovarian tissue; vitrification; transplantation; fertility preservation

MeSH Terms

Animals
Antifreeze Proteins/*pharmacology
Apoptosis/drug effects
Cryopreservation/*methods
Cryoprotective Agents/*pharmacology
Female
Fertility Preservation
Humans
Mice
Mice, Inbred ICR
Ovarian Follicle/drug effects
Ovary/*drug effects/*transplantation
*Vitrification
Antifreeze Proteins
Cryoprotective Agents

Figure

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  Fig. 1 Schematic representation of the experimental design. n, number of ovaries in each step; AFP5, antifreeze protein, 5 mg/mL treated group; AFP20, antifreeze protein 20 mg/mL treated group.

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  Fig. 2 Histology of ovaries in control, antifreeze protein (AFP) 5 mg/mL treated group, and AFP 20 mg/mL treated group after vitrification-warming and autotransplantation (×200).

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  Fig. 3 Fluorescent TUNEL staining for vitrified-warmed ovaries of control group, antifreeze protein (AFP) 5 mg/mL treated group, AFP 20 mg/mL treated group, positive and negative control (×100, DAPI-counterstained). DAPI, 4',6-Diamidino-2-phenylindole.

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  Fig. 4 Ratios of morphologically intact (grade I) follicle and apoptotic follicle according to supplementation of antifreeze protein (AFP) in vitrification solution after warming.

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  Fig. 5 Ratios of morphologically intact (grade I) follicle and apoptotic follicle according to supplementation of antifreeze protein (AFP) in vitrification solution after autotransplantation of vitrified-warmed ovary.

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