Yonsei Med J.  2018 Mar;59(2):331-336. 10.3349/ymj.2018.59.2.331.

Improvement in Ovarian Tissue Quality with Supplementation of Antifreeze Protein during Warming of Vitrified Mouse Ovarian Tissue

• Kong HS
• Kim EJ
• Youm HW
• Kim SK
• Lee JR
• Suh CS
• Kim SH

• KMID: 2418799
• DOI: http://doi.org/10.3349/ymj.2018.59.2.331

Abstract

Ice easily recrystallizes during warming after vitrification, and antifreeze protein (AFP) can inhibit the re-crystallization. However, no study has evaluated the effect of AFP treatment only thereon during warming. This study sought to compare AFP treatment protocols: a conventional protocol with AFP treatment during vitrification and first-step warming and a new protocol with AFP treatment during the first-step warming only. According to the protocols, 10 mg/mL of LeIBP (a type of AFP) was used. Five-week-old B6D2F1 mouse ovaries were randomly divided into a vitrified-warmed control and two experimental groups, one treated with the conventional AFP treatment protocol (LeIBP-all) and the other with the new AFP treatment protocol (LeIBP-w). For evaluation, ratios of ovarian follicle integrity, apoptosis, and DNA double-strand (DDS) damage/repairing were analyzed. The LeIBP-treated groups showed significantly higher intact follicle ratios than the control, and the results were similar between the LeIBP-treated groups. Apoptotic follicle ratios were significantly lower in both LeIBP-treated groups than the control, and the results were not significantly different between the LeIBP-treated groups. With regard to DDS damage/repairing follicle ratio, significantly lower ratios were recorded in both LeIBP-treated groups, compared to the control, and the results were similar between the LeIBP-treated groups. This study demonstrated that both protocols with LeIBP had a beneficial effect on maintaining follicle integrity and preventing follicle apoptosis and DDS damage. Moreover, the new protocol showed similar results to the conventional protocol. This new protocol could optimize the mouse ovary vitrification-warming procedure using AFP, while minimizing the treatment steps.

Keyword

Antifreeze protein; cryodamage; ovarian tissue cryopreservation; recrystallization; vitrification

MeSH Terms

Animals
Antifreeze Proteins/*pharmacology
Apoptosis/drug effects
Cryopreservation
Cryoprotective Agents/pharmacology
Female
Mice
Ovarian Follicle/cytology/drug effects
Ovary/cytology/drug effects/*physiology
*Vitrification/drug effects
Antifreeze Proteins
Cryoprotective Agents

Figure

• 

  Fig. 1 Representative images of hematoxylin and eosin stained ovaries (A, B, and C ×100; D, E, and F ×400). Each panel shows the ovaries from different groups: (A and D) vitrified-warmed control, (B and E) LeIBP-all group, and (C and F) LeIBP-w group. Fields indicated by an arrowhead in (A), (B), and (C) are shown in (D), (E), and (F), respectively. Scale bar: 50 µm.

• 

  Fig. 2 Morphologically intact follicle ratio according to developmental stages and groups. *p<0.05 in comparison to control, †p<0.05 in comparison to the other experimental group.

• 

  Fig. 3 Representative images of TUNEL stained ovaries (A, B, and C ×100; D, E, and F ×400). Each panel shows the ovaries from different groups: (A and D) vitrified-warmed control, (B and E) LeIBP-all group, and (C and F) LeIBP-w group. Fields indicated by an arrowhead in (A), (B), and (C) are shown in (D), (E), and (F), respectively. Non-apoptotic cells are stained with blue fluorescence and apoptotic cells with green fluorescence. TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Scale bar: 50 µm.

• 

  Fig. 4 Apoptotic follicle ratios according to the different groups. *p<0.05 in comparison to control.

• 

  Fig. 5 Representative images of immune-stained ovaries (×200). Each panel indicates (A) γH2AX immune-stained and (B) Rad51 immune-stained ovaries. Counterstained cells are stained with blue and immune-stained cells with red. Red arrowheads: immune-stained follicle, white arrowheads: counterstained follicle. Scale bar: 50 µm.

• 

  Fig. 6 Immuno-stained follicle ratios according to the different groups. *p<0.05 in comparison to control.

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