Obstet Gynecol Sci.  2013 Nov;56(6):382-388. 10.5468/ogs.2013.56.6.382.

Influence of the vitrification solution on the angiogenic factors in vitrificated mouse ovarian tissue

• Choi WJ
• Lee JH
• Park MH
• Choi IY
• Park JK
• Shin JK
• Lee SA
• Paik WY
• Lee JH

• KMID: 1500372
• DOI: http://doi.org/10.5468/ogs.2013.56.6.382

Abstract

OBJECTIVE
To investigate the effect of the dimethyl sulfoxide (DMSO) and EFS-40 during vitrification on the expression of angiogenic factors in vitrified mouse ovarian tissue.
METHODS
The ovarian tissues were obtained from 5 or 6 weeks aged ICR mouse. Ovarian tissues were divided into four groups: ovarian tissue without cryopreservation (control, group I), ovarian tissue vitrified with 15% DMSO (group II), ovarian tissue vitrified with EFS-40 (group III), and ovarian tissue slowly frozen with 10% DMSO (group IV). Thawing was carried out at room temperature. Levels of messenger RNA (mRNA) and protein for vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Angpt-2) were checked in ovarian tissues of four groups recovered on day 7 after cryopreservation. Reverse transcription-polymerase chain reaction and Western blot analysis were used to identify the levels of angiogenic factors in mouse ovarian tissues.
RESULTS
Levels of mRNA and protein for VEGF-A and Angpt-2 were significantly decreased in cryopreserved group (group II, III and IV) than control group (group I) (P< 0.05). The significant differences of levels of mRNA and protein for VEGF-A and Angpt-2 between cryopreservation methods were observed (P< 0.05). Group III showed highest expression of mRNA and protein for VEFG-A and Angpt-2 than other cryopreservation groups (P< 0.05).
CONCLUSION
These findings suggest that EFS-40 is more efficient vitrification solution for preservation of angiogenic factors than 15% DMSO during vitrification of mouse ovarian tissue. Future studies should investigate to improve the vitrification solution for ovarian tissue vitrification.

Keyword

Angiopoietin-2; Ovary; Vascular endothelial growth factor A; Vitrification

MeSH Terms

Angiopoietin-2
Animals
Blotting, Western
Cryopreservation
Dimethyl Sulfoxide
Female
Methods
Mice*
Mice, Inbred ICR
Ovary
Reverse Transcriptase Polymerase Chain Reaction
RNA, Messenger
Vascular Endothelial Growth Factor A
Vitrification*
Angiopoietin-2
Dimethyl Sulfoxide
RNA, Messenger
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Reverse transcription-polymerase chain reaction analysis of mRNA levels for (A) VEGF-A and (B) Angpt-2 after variable cryopreservation methods and quantitative analysis of each group. GAPDH was used as internal standard. The data represents mean±standard error of three independent experiments. DMSO, dimethyl sulfoxide; EFS, 40% EG, 18% ficoll, 0.5 mol/L sucrose, and 20% FBS in modified Dulbecco's phosphate buffered saline; Angpt-2, angiopoietin-2; VEGF-A, vascular endothelial growth factor-A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. a)P<0.05 vs. cryopreservation groups (vitrification using DMSO, EFS, and slow freezing; b)P<0.05 vs. other cryopreservation groups (vitrification using DMSO and slow freezing).

• 

  Fig. 2 Western blot analysis of (A) VEGF-A and (B) Angpt-2 after variable cryopreservation methods and quantitative analysis of each group. β-Actin was used as internal standard. The data represents mean±standard error of three independent experiments. DMSO, dimethyl sulfoxide; EFS, 40% EG, 18% ficoll, 0.5 mol/L sucrose, and 20% FBS in modified Dulbecco's phosphate buffered saline; Angpt-2, angiopoietin-2; VEGF-A, vascular endothelial growth factor-A. a)P<0.05 vs. cryopreservation groups (vitrification using DMSO, EFS, and slow freezing); b)P<0.05 vs. other cryopreservation groups (vitrification using DMSO and slow freezing).

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