High survival of mouse oocytes using an optimized vitrification protocol.
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ABSTRACT:
SUBMITTER: Zhou CJ
PROVIDER: S-EPMC4726034 | biostudies-other | 2016 Jan
REPOSITORIES: biostudies-other
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Project description:The open carrier system (OC) is used for vitrification due to its high efficiency in preserving female fertility, but concerns remain that it bears possible risks of cross-contamination. Closed carrier systems (CC) could be an alternative to the OC to increase safety. However, the viability and developmental competence of vitrified/warmed (VW) oocytes using the CC were significantly lower than with OC. We aimed to improve the efficiency of the CC. Metaphase II oocytes were collected from mice after superovulation and subjected to in vitro fertilization after vitrification/warming. Increasing the cooling/warming rate and exposure time to cryoprotectants as key parameters for the CC effectively improved the survival rate and developmental competence of VW oocytes. When all the conditions that improved the outcomes were applied to the conventional CC, hereafter named the modified vitrification/warming procedure using CC (mVW-CC), the viability and developmental competence of VW oocytes were significantly improved as compared to those of VW oocytes in the CC. Furthermore, mVW-CC increased the spindle normality of VW oocytes, as well as the cell number of blastocysts developed from VW oocytes. Collectively, our mVW-CC optimized for mouse oocytes can be utilized for humans without concerns regarding possible cross-contamination during vitrification in the future.
| S-EPMC8304188 | biostudies-literature
Project description:Vitrification is now the main route to the cryopreservation of human and animal oocytes and preimplantation embryos. A central belief is that for success, the cells must be placed in very high concentrations of cryoprotective solutes and must be cooled extremely rapidly. We have shown recently that these beliefs are incorrect. Over 90% of mouse oocytes and embryos survive being cooled relatively slowly even in solutions containing only 1/3(rd) the normal solute concentrations, provided that they are warmed ultra-rapidly at 10(7)°C/min by a laser pulse. Nearly all vitrification solutions contain both permeating and non-permeating solutes, and an important question is whether the former protect because they permeate the cells and promote intracellular vitrification (as is almost universally believed), or because they osmotically withdraw a large fraction of intracellular water prior to cooling. The answer for the mouse system is clearly the latter. When oocytes or embryos are placed in 1 molal concentrations of the impermeable solute sucrose, they osmotically lose ~85% of their cellular water in less than 2 minutes. If the cells are then cooled rapidly to -196°C, nearly 90% remain viable after warming, again provided that the warming is ultra rapid.
| S-EPMC4365397 | biostudies-other
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.
| S-EPMC6099150 | biostudies-literature
Project description:Oocyte freezing confers thermal and chemical stress upon the oolemma and various other intracellular structures due to the formation of ice crystals. The lipid profiles of oocytes and embryos are closely associated with both, the degrees of their membrane fluidity, as well as the degree of chilling and freezing injuries that may occur during cryopreservation. In spite of the importance of lipids in the process of cryopreservation, the phospholipid status in oocytes and embryos before and after freezing has not been investigated. In this study, we employed mass spectrometric analysis to examine if vitrification has an effect on the phospholipid profiles of mouse oocytes. Freshly prepared metaphase II mouse oocytes were vitrified using copper grids and stored in liquid nitrogen for 2 weeks. Fresh and vitrified-warmed oocytes were subjected to phospholipid extraction procedure. Mass spectrometric analyses revealed that multiple species of phospholipids are reduced in vitrified-warmed oocytes. LIFT analyses identified 31 underexpressed and 5 overexpressed phospholipids in vitrified mouse oocytes. The intensities of phosphatidylinositol (PI) {18∶2/16∶0} [M-H]- and phosphatidylglycerol (PG) {14∶0/18∶2} [M-H]- were decreased the most with fold changes of 30.5 and 19.1 in negative ion mode, respectively. Several sphingomyelins (SM) including SM {d38∶3} [M+H]+ and SM {d34∶0} [M+K]+ were decreased significantly in positive ion mode. Overall, the declining trend of multiple phospholipids demonstrates that vitrification has a marked effect on phospholipid profiles of oocytes. These results show that the identified phospholipids can be used as potential biomarkers of oocyte undergoing vitrification and will allow for the development of strategies to preserve phospholipids during oocyte cryopreservation.
| S-EPMC4102530 | biostudies-literature
Project description:Necrostatin 1 (Nec1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase 1 in cell death. The biological actions of Nec1 are blocking necrotic cell death. The purpose of this study was to investigate whether adding Nec1 into in vitro maturation (IVM) media, followed by vitrification procedures, could enhance the survival and developmental competency of oocytes. Germinal vesicle oocytes were matured in IVM medium containing 2 different doses of Nec1 (0.5 and 1 μmol/L). After IVM, the oocytes were vitrified using a 2-step exposure to equilibrium and vitrification solutions. After warming, the rates of survival, fertilization, embryonic development up to blastocyst in vitro, morphology of spindle and chromosome, membrane integrity, mitochondria integrity, and several gene expressions were evaluated. The survival and developmental competency of oocytes were higher in the 1 μmol/L Nec1-treated group than control. The proportion with intact spindles/chromosomes and stable membranes was similar in all the groups. The mitochondrial integrity of all Nec1-treated groups showed a higher score with strong staining. The 1 μmol/L Nec1 showed significantly increased expressions of Mad2, Gdf9, and Bcl2. The Cirp level had a tendency to be downregulated in the 0.5 µmol/L Nec1 but upregulated in the 1 μmol/L Nec1, compared with the control. The Mtgenome expressions were significantly decreased in both Nec1 groups. The supplementation of 1 μmol/L Nec1 into the IVM medium could be beneficial for the survival and development of immature oocytes after vitrification.
| S-EPMC4519766 | biostudies-literature
Project description: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.
| S-EPMC3359366 | biostudies-literature
Project description:BackgroundOocyte cryopreservation is an essential part of the assisted reproductive technology (ART), which was recently introduced into clinical practice. This study aimed to evaluate the effects of two vitrification systems-Cryotop and Open Pulled Straw (OPS)-on mature oocytes gene expressions.Materials and methodsIn this experimental study, the survival rate of metaphase II (MII) mouse oocytes were assessed after cryopreservation by vitrification via i. OPS or ii. Cryotop. Then we compared the fertilization rate of oocytes produced via these two methods. In the second experiment, we determined the effects of the two vitrification methods on the expression of Hspa1a, mn-Sod, and ß-actin genes in vitrified-warmed oocytes. Denuded MII oocytes were vitrified in two concentrations of vitrification solution (VS1 and VS2) by Cryotop and straw. We then compared the results using the two vitrification methods with fresh control oocytes.Resultsmn-Sod expression increased in the vitrified-warmed group both in OPS and Cryotop compared with the controls. We only detected Hspa1a in VS1 and control groups using Cryotop. The survival rate of the oocytes was 91.2% (VS1) and 89.2% (VS2) in the Cryotop groups (P=0.902) and 85.5% (VS1) and 83.6% (VS2) in the OPS groups (P=0.905). There were no significant differences between the Cryotop and the OPS groups (P=0.927). The survival rate in the Cryotop or the OPS groups was, nevertheless, significantly lower than the control group (P<0.001). The fertilization rates of the oocytes were 39% (VS1) and 34% (VS2) in the Cryotop groups (P=0.902) and 29 %( VS1) and 19.7% (VS2) in the OPS groups (P=0.413). The fertilization rates were achieved without significant differences among the Cryotop and OPS groups (P=0.755).ConclusionOur results indicated that Cryotop vitrification increases both cooling and warming rates, but both Cryotop and OPS techniques have the same effect on the mouse oocytes after vitrification.
| S-EPMC5767935 | biostudies-literature
Project description:The ability to cryopreserve human oocytes has significant potential for fertility preservation. Current cryopreservation methods still suffer from the use of conventional cryoprotectants, such as dimethyl sulphoxide (DMSO), causing loss of viability and function. Such injuries result from the toxicity and high concentration of cryoprotectants, as well as mechanical damage of cells due to ice crystal formation during the cooling and rewarming processes. Here we report the preservation of human oocytes following vitrification using an innovative bio-inspired cryoprotectant integrated with a minimum volume vitrification approach. The results demonstrate that the recovered human oocytes maintained viability following vitrification and rewarming. Moreover, when this approach was used to vitrify mouse oocytes, the recovered oocytes preserved their viability and function following vitrification and rewarming. This bio-inspired approach substitutes DMSO, a well-known toxic cryoprotectant, with ectoine, a non-toxic naturally occurring solute. The bio-inspired vitrification approach has the potential to improve fertility preservation for women undergoing cancer treatment and endangered mammal species.
| S-EPMC5766438 | biostudies-literature
Project description:In vitro maturation (IVM) and vitrification have been widely used to prepare oocytes before fertilization; however, potential effects of these procedures, such as expression profile changes, are poorly understood. In this study, mouse oocytes were divided into four groups and subjected to combinations of in vitro maturation and/or vitrification treatments. RNA-seq and in silico pathway analysis were used to identify differentially expressed genes (DEGs) that may be involved in oocyte viability after in vitro maturation and/or vitrification. Our results showed that 1) 69 genes were differentially expressed after IVM, 66 of which were up-regulated. Atp5e and Atp5o were enriched in the most significant gene ontology term "mitochondrial membrane part"; thus, these genes may be promising candidate biomarkers for oocyte viability after IVM. 2) The influence of vitrification on the transcriptome of oocytes was negligible, as no DEGs were found between vitrified and fresh oocytes. 3) The MII stage is more suitable for oocyte vitrification with respect to the transcriptome. This study provides a valuable new theoretical basis to further improve the efficiency of in vitro maturation and/or oocyte vitrification.
| S-EPMC5643491 | biostudies-literature
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.
| S-EPMC7828578 | biostudies-literature