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The beneficial effects of antifreeze proteins in the vitrification of immature mouse oocytes.

ABSTRACT: Antifreeze proteins (AFPs) are a class of polypeptides that permit organismal survival in sub-freezing environments. The purpose of this study was to investigate the effect of AFP supplementation on immature mouse oocyte vitrification. Germinal vesicle-stage oocytes were vitrified using a two-step exposure to equilibrium and vitrification solution in the presence or absence of 500 ng/mL of AFP III. After warming, oocyte survival, in vitro maturation, fertilization, and embryonic development up to the blastocyst stage were assessed. Spindle and chromosome morphology, membrane integrity, and the expression levels of several genes were assessed in in vitro matured oocytes. The rate of blastocyst formation was significantly higher and the number of caspase-positive blastomeres was significantly lower in the AFP-treated group compared with the untreated group. The proportion of oocytes with intact spindles/chromosomes and stable membranes was also significantly higher in the AFP group. The AFP group showed increased Mad2, Hook-1, Zar1, Zp1, and Bcl2 expression and lower Eg5, Zp2, Caspase6, and Rbm3 expression compared with the untreated group. Supplementation of the vitrification medium with AFP has a protective effect on immature mouse oocytes, promoting their resistance to chilling injury. AFPs may preserve spindle forming ability and membrane integrity at GV stage. The fertilization and subsequent developmental competence of oocytes may be associated with the modulation of Zar1, Zp1/Zp2, Bcl2, Caspase6, and Rbm3.

SUBMITTER: Jo JW

PROVIDER: S-EPMC3359366 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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The beneficial effects of antifreeze proteins in the vitrification of immature mouse oocytes.

Jo Jun Woo JW Jee Byung Chul BC Suh Chang Suk CS Kim Seok Hyun SH

PloS one 20120523 5

Antifreeze proteins (AFPs) are a class of polypeptides that permit organismal survival in sub-freezing environments. The purpose of this study was to investigate the effect of AFP supplementation on immature mouse oocyte vitrification. Germinal vesicle-stage oocytes were vitrified using a two-step exposure to equilibrium and vitrification solution in the presence or absence of 500 ng/mL of AFP III. After warming, oocyte survival, in vitro maturation, fertilization, and embryonic development up t ...[more]

PMID: 22649508

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Project description:Objective:Over the last years, vitrification has been widely used for oocyte cryopreservation, in animals and humans; however, it frequently causes minor and major epigenetic modifications. The effect of oocyte vitrification on levels of acetylation of histone H4 at lysine 12 (AcH4K12), and histone acetyltransferase (Hat) expression, was previously assessed; however, little is known about the inhibition of Hat expression during oocyte vitrification. This study evaluated the effect of anacardic acid (AA) as a Hat inhibitor on vitrified mouse oocytes. Materials and Methods:In this experimental study, 248 mouse oocytes at metaphase II (MII) stage were divided in three experimental groups namely, fresh control oocytes (which were not affected by vitrification), frozen/thawed oocytes (vitrified) and frozen/thawed oocytes pre-treated with AA (treatment). Out of 248 oocytes, 173 oocytes were selected and from them, 84 oocytes were vitrified without AA (vitrified group) and 89 oocytes were pretreated with AA, and then vitrified (treatment group). Fresh MII mouse oocytes were used as control group. Hat expression and AcH4K12 levels were assessed by using real-time quantitative polymerase chain reaction (PCR) and immunofluoresce staining, respectively. In addition, survival rate was determined in vitrified and treatment oocytes. Results:Hat expression and AcH4K12 modification significantly increased [4.17 ± 1.27 (P≤0.001) and 97.57 ± 6.30 (P<0.001), respectively] in oocytes that were vitrified, compared to the fresh oocytes. After treatment with AA, the Hat mRNA expression and subsequently H4K12 acetylation levels were significantly reduced [0.12 ± 0.03 (P≤0.001) and 89.79 ± 3.20 (P≤0.05), respectively] in comparison to the vitrified group. However, the survival rate was not significantly different between the vitrified (90.47%) and treatment (91.01%) groups (P>0.05). Conclusion:The present study suggests that AA reduces vitrification risks caused by epigenetic modifications, but does not affect the quality of vitrification. In fact, AA as a Hat inhibitor was effective in reducing the acetylation levels of H4K12.

Project description:BackgroundOvarian 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.ObjectiveThis study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs) on mouse ovarian tissue cryopreservation and transplantation.MethodologyOvaries 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 findingsIn 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.ConclusionA 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.

Project description:As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental competence of vitrified oocytes has remained low. The underlying mechanisms responsible for reduced oocyte quality post-vitrification are largely unknown. Mouse cumulus-oocyte complexes were vitrified using a cryoloop technique and a mixture of dimethylsulphoxide, ethylene glycol and trehalose as cryoprotectants. Fresh and vitrified/thawed oocytes were compared for chromosome alignment, spindle morphology, kinetochore-microtubule attachments, spindle assembly checkpoint (SAC) and aneuploidy. Although the majority of vitrified oocytes extruded the first polar body (PB), they had a significant increase of chromosome misalignment, abnormal spindle formation and aneuploidy at metaphase II. In contrast to controls, vitrified oocytes extruded the first PB in the presence of nocodazole and etoposide, which should induce metaphase I arrest in a SAC-dependent manner. The fluorescence intensity of mitotic arrest deficient 2 (MAD2), an essential SAC protein, at kinetochores was reduced in vitrified oocytes, indicating that the SAC is weakened after vitrification/thawing. Furthermore, we found that vitrification-associated stress disrupted lysosomal function and stimulated cathepsin B activity, with a subsequent activation of caspase 3. MAD2 localization and SAC function in vitrified oocytes were restored upon treatment with a cathepsin B or a caspase 3 inhibitor. This study was conducted using mouse oocytes, therefore confirming these results in human oocytes is a prerequisite before applying these findings in IVF clinics. Here, we uncovered underlying molecular pathways that contribute to an understanding of how vitrification compromises oocyte quality. Regulating these pathways will be a step toward improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.

Dataset Information

The beneficial effects of antifreeze proteins in the vitrification of immature mouse oocytes.

Publications

The beneficial effects of antifreeze proteins in the vitrification of immature mouse oocytes.

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