The effects of vitrification on gene expression in mature mouse oocytes by nested quantitative PCR.
ABSTRACT: this study was conducted on the effects of vitrification cryotop method on gene expression of mature oocytes in Mus musculus.transcript analyses of three mouse genes, namely Mater, Hook1 and Sod1, were performed upon non-vitrified and vitrified oocytes with different concentrations of dimethyl sulfoxide (DMSO) and ethylene glycol (EG),15%: 7.5% DMSO + 7.5% EG, and 30%: 15% DMSO + 15% EG, using cryotop following normalization of transcripts with Hprt1 by nested quantitative PCR.vitrification caused down-regulation of Mater and Hook1 and up-regulation of Sod1 when lower concentrations of cryoprotectants were used as opposed to the control group. The relative expression of Sod1 in vit(2) (30% v/v) was significantly higher than vit(1) (15% v/v)(.) Quantitative transcript analysis of Mater and Hook1 for the vit(2) condition failed to produce any data. Survival rates were the same for both vitrification treatments and significantly lower than control group.although vit(1) treatment had lower survival rate compared to control group, it demonstrated better stability comparing to vit(2) based on the transcript analysis.
Project description:In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new cryoprotective agent (CPA), called as carboxylated ?-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20% (v/v) and COOH-PLL 10% (w/v)] or E15P15 group (EG 15% and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10% and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mouse oocytes.
Project description:This study was designed to compare the efficiency of the Cryotop and Calibrated plastic inoculation loop (CPIL) devices for vitrification of rabbit embryos on in vitro development and implantation rate, offspring rate at birth and embryonic and fetal losses. CPIL is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. In experiment 1, embryos were vitrified using a Cryotop device and a CPIL device. There were no significant differences in hatched/hatching blastocyst stage rates after 48 h of culture among the vitrified groups (62 ± 4.7% and 62 ± 4.9%, respectively); however, the rates were significantly lower (P<0.05) than those of the fresh group (95 ± 3.4%). In experiment 2, vitrified embryos were transferred using laparoscopic technique. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at day 14 of gestation. At birth, total offspring were recorded. Embryonic and fetal losses were calculated as the difference between implanted embryos and embryos transferred and total born at birth and implanted embryos, respectively. The rate of implantation and development to term was similar between both vitrification devices (56 ± 7.2% and 50 ± 6.8% for implantation rate and 40 ± 7.1% and 35 ± 6.5% for offspring rate at birth); but significantly lower than in the fresh group (78 ± 6.6% for implantation rate and 70 ± 7.2% for offspring rate at birth, P<0.05). Likewise, embryonic losses were similar between both vitrification devices (44 ± 7.2% and 50 ± 6.8%), but significantly higher than in the fresh group (23 ± 6.6%, P < 0.05). However, fetal losses were similar between groups (10 ± 4.4%, 15 ± 4.8% and 8 ± 4.2%, for vitrified, Cryotop or CPIL and fresh, respectively). These results indicate that the CPIL device is as effective as the Cryotop device for vitrification of rabbit embryos, but at a cost of €0.05 per device.
Project description:OBJECTIVE:The aim of the study was to compare the effects of two different concentrations of cryoprotectants by cryotopvitrification on survival, developmental capacity and Heat shock protein 72 (Hsp72) expression of two-cell mouse embryos. MATERIALS AND METHODS:In this experimental study, transcript analysis of Hsp72 gene was performed on non-vitrified and vitrified 2-cell mouse embryos via a nested quantitative polymerase chain reaction (nqPCR) subsequent to normalization with Hprt1 as the reference gene. The different cryoprotectant combinations were 15% (vit1:7.5% of each ethylene glycol (EG) and dimethyl sulfoxide (DMSO), 30% (vit2:15% EG + 15% DMSO) and control group with no cryoprotectants. Vitrified and fresh 2-cell embryos were cultured to obtain cleavage and blastocyst formation rates. The results were analyzed via one-way analysis of variance and the mean values were compared with least significant difference (LSD) (p< 0.05). RESULTS:The relative expression of Hsp72 in vit2 (30% v/v) was significantly higher than vit1 (15% v/v). Survival rates were the same for both vitrification treatments and significantly lower than the control group. Cleavage and blastocyst rates in vit1 were significantly higher than vit2 while those in two vitrified groups were significantly lower than the control group. CONCLUSION:Our developmental data demonstrated that vit1 treatment (7.5% EG and 7.5% DMSO) was more efficient than vit2 (15% EG and 15% DMSO) in mouse embryos. The cryotopvitrification with two concentrations of cryoprotectants caused the relative changes of Hsp72 transcript level, but the stability of the gene in vit1 was significantly higher than vit2 and closer to the fresh 2-cell embryos.
Project description:Mesenchymal stem cells (MSCs) are one of the most promising adult stem cells for clinical application in a cell therapy. The development of large-scale cryopreservation techniques, such as vitrification, for MSCs is a prerequisite for clinical therapies. Dimethyl sulfoxide (DMSO) and ethylene glycol (EG) are two types of cryoprotectants widely used for cell vitrification. However, the effects of DMSO and EG on the biological characteristics and transcriptome profiles of MSCs after cryopreservation remain unknown. In the present study, the viability, immunophenotype of cell surface markers, proliferation, differentiation potency, and global gene expression of rhesus macaque bone marrow-derived MSCs vitrified using DMSO and EG were studied. The results showed that vitrification did not affect the morphology, surface markers, and differentiation of the MSCs, and compared to DMSO, EG better protected cell viability and proliferation. Most importantly, vitrification resulted in changes in a large number of transcripts of MSCs either preserved using DMSO or EG. This report is the first to examine the effects of DMSO and EG on global gene expression in stem cells. These results will be beneficial to understanding the biological process involved in MSC vitrification and will contribute to improving cryopreservation protocols that maintain transcriptomic identity with high cryosurvival for preclinical research and clinical long-term storage.
Project description:Changes in gene expression induced by the Cryotop vitrification procedure in bovine blastocysts using Agilent EmbryoGENE microarray slides. Bovine in vitro-produced embryos at the blastocyst stage (144 to 156 hours post insemination) were vitrified using the Cryotop system and compared with non-vitrified (control) embryos. After vitrification, the embryos were warmed and cultured for an additional 4 hours. Embryos that re-expanded or developed to the expanded blastocyst stage were used for microarray analysis. Four pools of vitrified embryos were hybridized against four pools of control embryos in a dye-swap design.
Project description:PURPOSE: To compare closed-system solid surface vitrification with slow freezing. METHODS: Mouse 2-cell embryos (n = 348) were divided into vitrification, slow freezing and non-frozen groups. For vitrification, embryos were exposed to 10% ethylene glycol (EG), 10% dimethylsulfoxide (DMSO) and 10% fetal bovine serum (FBS) in phosphate-buffered saline (PBS) for 10 min, then transferred into 17.5% EG, 17.5% DMSO, 0.25 M trehalose and 10% FBS in PBS. They were placed on hemi-straws and inserted into 0.5 ml straws inside a previously cooled aluminum cylinder. Slow freezing was done in straws by the conventional method. RESULTS: Vitrified embryos had significantly higher survival, further cleavage and blastocyst formation rates than those in the slow freezing group (p < 0.001) and were comparable to controls. Blastocysts in the vitrification and control groups had significantly more cells than those in the slow freezing group (p < 0.05). CONCLUSIONS: Closed-system vitrification was more effective than conventional slow freezing.
Project description:PURPOSE: The objective of this study was to investigate the effects of vitrification on the preimplantation developmental competence of mouse 2-cell, 4-cell and 8-cell stage embryos. METHODS: Mouse 2-cell, 4-cell and 8-cell stage embryos were cryopreserved using the cryotop vitrification method and subsequently warmed on a later date. The embryos were then assessed by their morphology, blastocyst formation and hatching rates. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in hatched blastocysts from the control and experimental groups. RESULTS: Vitrified embryos at the 2-cell, 4-cell and 8-cell stages appeared morphologically normal after warming. The overall survival rate of vitrified embryos at various stages after warming was 96.7% and there were no significant differences among 2-cell stage (96.0%), 4-cell stage (96.8%) and 8-cell stage (97.1%) embryos (P > 0.05). The blastocyst formation rate (69.4%) and hatching rate (52.6%) of vitrified 2-cell embryos were significantly lower than that from the control group and vitrified 8-cell embryos (P < 0.05). In the vitrified 4-cell embryo group, the blastocyst formation rate (90.3%) was similar to the 8-cell group (91.2%), but the hatching rate (60.0%) was significantly lower than that of the non-vitrified control ( 84.1%) and vitrified 8-cell embryo (78.4%) groups (P < 0.05). When further development to the fully hatched blastocyst stage was compared, hatched blastocysts derived from vitrified 2-cell, 4-cell and 8-cell embryos had significantly lower cell counts both in the ICM and TE, as compared to fresh blastocysts (P < 0.05). Among the vitrified 2-cell, 4-cell and 8-cell embryo groups, there were no significant differences in the cell counts of ICM and TE (P > 0.05). CONCLUSIONS: Although cryotop vitrification was suitable for the cryopreservation of mouse embryos from the 2-cell stage, 4-cell stage and 8-cell stage without significant loss of survival, vitrification had an adverse effect on the development of 2-cell embryos. Mouse embryos at the 8-cell stage had the best tolerance for vitrification and would yield the highest level of post-vitrification developmental competence among early cleavage stage embryos. Nevertheless, it is unclear how these findings can be extrapolated to human embryos.
Project description:BACKGROUND:Embryo cryopreservation is the process that water is removed from the cell by cryoprotectant materials, and embryos are stored at temperature below zero. This process may affect the viability and developmental potential of embryos. OBJECTIVE:In this study, the effect of the vitrification cryotop method on the expression level of Oct4 and Mest developmental genes in mouse blastocysts was examined. MATERIALS AND METHODS:The collected 2-cell embryos of superovulated mouse by oviduct flushing were divided into non-vitrified and vitrified groups. These embryos were cultured to the blastocyst stage directly in the non-vitrified group and in the vitrified group, these embryos were cultured to 4-8 cell embryos, vitrified with cryotop in these stages and after 2-6 months, warmed and cultured to blastocyst embryos. Quantitative expression of two developmental genes, namely Oct4 and Mest, were performed in these groups, using RNA purification and Real-time RT-PCR. RESULTS:Quantitative PCR analysis showed that the expression level of both genes, Oct4 and Mest, was reduced significantly in the vitrified-warmed group relative to the control group (p=0.046 and p=0.001). CONCLUSION:This study revealed that morphologically normal embryos show a reduced amount of Oct4 and Mest transcripts which indicate that the vitrification method negatively effects the expression level of these two developmental genes.
Project description:Sugars are commonly supplemented into vitrification solution to dehydrate cells in order to reduce the formation of fatal intracellular ice crystals. Natural honey is a mixture of 25 sugars (mainly fructose and glucose) that have different biological and pharmacological benefits. The present study was designed to determine if honey can be used as a nonpermeating cryoprotectant in vitrification of bovine oocytes. In the first experiment, denuded-MII oocytes were exposed to 0.25, 0.5, 1.0, 1.5 or 2.0 M of honey or sucrose. Natural honey and sucrose caused similar ooplasm dehydration. A significant relationship existed between time and ooplasm volume change (P < 0.05), during dehydration and rehydration phases, in both honey and sucrose solutions. In the second experiment, the immature cumulus-oocyte complexes (COCs) were vitrified in an EG/DMSO-based vitrification solution containing honey (0.5, 1 or 1.5 M) or sucrose (0.5 M) as a gold standard. The vitrified-warmed COCs were matured in vitro and evaluated for nuclear maturation. The maturation (MII) rate was greater in nonvitrified control (81%) than vitrified groups (54%, P < 0.05). In the third experiment, COCs were either remained nonvitrified (control) or vitrified in 1.0 M honey or 0.5 M sucrose, followed by IVM, IVF and IVC (for 9 days). Cleavage rate was greater in control (74%) than in vitrified groups (47%, P < 0.05), without significant difference between sugars. Blastocyst rate was 34, 13 and 3% in control, honey and sucrose groups respectively (P < 0.05). In conclusion, natural honey acted as a nonpermeating cryoprotectant in vitrification solution and improved the embryonic development in vitrified bovine COCs.
Project description:Gorgonian corals are slowly declining due to human interaction and environmental impacts. Cryopreservation of gorgonian corals is an ex-situ method of conservation, ensuring future reproduction. The present study assessed the vitrification properties of cryoprotectant (CPT) mixtures using the cryotop, cryoloop and open pulled straw (OPS) cryopereservation methods prior to experimentation on gorgonian (Junceella juncea) oocytes. Investigations of the equilibration and vitrification solutions' (ES and VS) effect on oocytes throughout different incubation periods were conducted. The cryotop method was found to be the most successful in ensuring vitrification. The most favourable VS was composed of propylene glycol (PG), ethylene glycol (EG) and methanol with concentrations of 3.5 M, 1.5 M and 2 M respectively. Experiments were performed using the cryotop method to cryopreserve Junceella juncea oocytes using VS2, the solution had the least impact on oocytes at 5°C rather than at 26°C. The success of the vitrification procedures was determined by adenosine triphosphate (ATP) levels in cooled-thaw oocytes and the highest viability obtained from the present study was 76.6 ± 6.2%. This study provides information regarding gorgonian corals' tolerance and viability throughout vitrification to further advance the vitrification protocol on whip corals.