Anti-Rad51 (L302) Anticorps (A27172)

BACKGROUND:

Ovarian tissue (OT) cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function.

OBJECTIVE:

This study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs) on mouse ovarian tissue cryopreservation and transplantation.

METHODOLOGY:

Ovaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III) and concentration (0.1, 1, 10 mg/mL) used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs) and repair (DDR), respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control). Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared.

PRINCIPAL FINDINGS:

In Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification control. The number of apoptotic (TUNEL-positive) follicles was significantly decreased in the groups treated with 1 and 10 mg/mL LeIBP. The proportion of τH2AX-positive follicles was significantly reduced in all AFP-treated groups, while the proportion of Rad51-positive follicles was significantly decreased in only the FfIBP- and LeIBP-treated groups. In Experiment II, after autotransplantation of OT vitrified with 10 mg/mL of LeIBP, the percentage of total grade 1 and primordial grade 1 follicles, and the extent of the CD31-positive area, were increased significantly. Moreover, the levels of serum FSH and the percentage of TUNEL-positive follicles were significantly lower in the LeIBP-treated than in the control group.

CONCLUSION:

A supplementation with high concentrations of AFPs had protective effects on follicle preservation during OT vitrification-warming procedures. The group treated with LeIBP was protected most effectively. The beneficial effects of LeIBP were also observed after autotransplantation of vitrified-warmed OT. Further studies are necessary to determine the exact mechanism of these protective effects.

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.

© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.