| Embryos cryopreservation plays a key role for the development of embryology and embryo toxicology; it has become a useful tool in the term of the animal resource preservation and the human artificial assist technology (ART). In this study, we hope to select a suitable of cryoprotectant to cryopreserve the two cell and four cell embryos in mice, and to investigate the appearance of cell membrane, to observe the development of embryos after thawed and to analysis the mechanism of embryos damage. In addition, we propose to establish a suitable system of cryopreservation and developmental by optimal cryopreservation proceduce and culture condition in vitro of two and four cell mouse embryo, to supply sufficient sample of experiment for the study of space biology, to provide the evidence and reference for the study of cryobiology at the same time.In order to select an optimal cryoprotectant for used in two cell embryos frozen, four cryoprotectants (1, 2-propanediol, PROH; ethylene glycol, ETG; glycerol; dimethyl sulfoxide, DMSO) were compared for their permeability and toxicity on two cell mouse embryo cryopreservation. Results indicated that PROH and in ETG shows higher permeable than DMSO, glycerol shows the lowest permeation for two cell embryo. However, the toxicity result indicate that 1.5 mol/L DMSO has most toxic than other cryoprotectants. So, our results demonstrate that PROH and ETG are suitable cryoprotectants using in slow freezing procedure for two cell mouse embryo according to the index of permeability and toxicity.To further compared the effective of PROH and ETG for cryopreservation of two cell embryo and for their subsequent development in mice. Embryos were freezed with PROH and ETG using a fast freeze-fast thaw method. The rates of blastomeres integrity of two cell embryos after thawed were assessed and the rate of cleavage, blastocyst, hatched blastocyst and offspring were compared with nonfrozen embryos. In addition, to document the integrity of frozen embryos between PROH and ETG, the level of cytoskeleton disruption of mouse embryos was detected. The rate of developed to four cell and the expanded blastocyst rate of embryos frozen with PROH was significantly better than with ETG (82.7% vs. 64.6% and 61.2% vs. 29.1%, respectively, P<0.01), however, there were no statistics difference in hatched blastocyst rate and birth rate between PROH and ETG (32.4% vs. 32.2% and 26.9% vs. 23.5%, P>0.05). Moreover, to further document the integrity of frozen embryos between PROH and ETG, the level of cytoskeleton disruption of mouse embryos was detected. Result shows that the higher cytoskeleton disruption of embryo in ETG group than in PROH group is the main reasons for their difference in development rate. Therefore, PROH does appear to be a good alternative to the ETG for two cell embryo cryopreservation in mice.Freezing and warming can dramatic effect on embryo survival and future development inevitable. To further analyze the mechanism of cryoinjury of embryos and investigate the reasons of development block for two cell mouse embryos after thawed, we detected the mechanism of development block that detect in term of integrity of cell membrane, cytoskeleton distribution, distribution and metabolism of mitochondria and cell apoptosis for frozen embryos. Here, our results shows that there are some embryos can derived to develop block neither integrity of cell membrane and cytoskeleton or not, the distribution of mitochondria was disrupted supplying the key reasons for development block of two cell embryo, moreover, it can be led to cell apoptosis.Cryothaw produces often result in cell damage. It is likely that such damage may also result in alterations in autocrine secretion of growth factor and effect on the subsequent development of embryo in vitro. Therefore, heighten the impact of the post-thaw culture environment and improved the development rate of blastocyst is very important. In order to investigated the effects of epidermal growth factor on the development of 4-cell mouse cryopreservation embryos. 4-cell embryos were frozen by slow cryopreservation and embryos post thawed were culture in the CZB medium supplemented with 10 ng/mL EGF or without EGF. In addition, embryos were cultured in CZB medium containing 0.1μg/mL monoclonal antibody EGFR. The rate of blastocyst and the cell number of blastocyst in each group were examined. Results indicated that presence of EGF in medium can significantly enhanced the development rate of expanded blastocyst(82.4% vs. 68.9%, P<0.01)and hatched blastocyst (61.3% vs. 40.3%, P<0.01), increased the number of cells in a blastocyst (P<0.01). However, the effect was neutralized by an antibody against the extracelluar domain of EGFR (P<0.01). Furthermore, we are found that the stage of embryonic development primarily affected by the treatment of EGF was between from 8-cell/morula and blastocyst for four cell cryopreservation embryos according to the ratio of each developmental stage. The study presents evident that treatment with EGF could condition the cultured mouse four cell cryopreservation embryos for optimal progression into blastocysts.In conclusion, in the study of two cell embryo cryopreservation, we have determined the 1,2-propanediol as an optimal cryoprotectant suitable for two cell mouse embryo cryopreservation. The main reasons of damage for two cell embryos post thawed that may lead to cell apoptosis and death is the structure of mitochondrial disruption. In the study of four cell embryo cryoreservation, we found that supplementation with 10 ng/mL EGF could improvement the cultured condition for mouse four cell cryopreservation embryos from four cell into blastocysts,however, this role was observed initial at the 8-cell/morula stage of embryo.
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