Vitrification Of Mouse Early Cleavage Embryo And Study Of Quantities Crypreservation Technology

With the progress of vitrification technology, the efficient of vitrification mammalian embryos is increasingly. Existing vitrification technology in mammalian early embryos still save a lot of problems:inefficiency, chilling injury. In this study, we ues nylon mesh vitrification method to cryopreserve the mouse 2-cell embryos, and optimized the method to vitrifiy the mouse early cleavage embryos. Now,100 mouse 2-cell embryos were loaded on the nylon mesh, and vitrified successsfuly and to meet the needs of the embryo development experiments on space. After the vitrification of mouse 2-cell embryos, and we also cultured a lot of vitrified mouse 2-cell embryos in sealed culture system which had been developed for many years, and this system condition is close to the space. Furthermore, compared the results from different culture system, and assessed the development competence of vitrified mouse 2-cell embryos. To get more information information about the quality of mouse 2-cell embryos post-thawed, cell membrane integrity, cytoskeletal, mitochondrial distribution, as well as the distribution and the expression of aquaporins also had been determined to investigate factors which affected the development of mouse 2-cell embryos post-thawed. Research content and results:1. Using nylon mesh vitrification method, EFS40 as vitrification solution containing 40% EG (V/V),0.5 M sucrose and 25% Ficoll 70 (W/V), and mouse 2-cell embryos were treated in this solution, and loaded on a nylon mesh immidiatly, and then immersed in vial containing liquid nitrogen, and stored in liquid nitrogen. There are about 100 mouse 2-cell embryos on nylon mesh. Subusequently using 0.5 M,0.25 M,0.125 M,0M of sucrose-basal medium treated, and at each concentration equilibrium 5min, removing the cryoprotectant rapidly. The survival rate of vitrified 2-cell embryos post-thawed was 96.22±2.74% (1791/1857), in which the IEs (Intact Embryos) accounted for 89.88±4.32(1669/1857), PEs (Partial Embryos) accounted for 6.95±3.44 (122/1857). So we can vitrify quantities of mouse 2-cell stage embryos using nylon mesh vitrificaiton method and get more survival frozen mice embryos.2. The recovered embryos were cultured in microdrop condition an sealed cluture condition for 72 h and 96 h, and the results indicated that the blastocyst formation rates, hatching rates in vitrified groups were lower than fresh groups. The blastocyst rate in Vitrified-Sealed group (90.86 ± 1.43%) is lower than Fresh-Sealed group (94.10±0.89%) after cultured for 72 h, but the difference was not significant. After cultured for 96 h, Vitrified-Sealed group blastocyst rate and hatching blastocyst rate in vitrified-sealed group (91.38±0.29%,32.02±2.30%) was significantly lower than fresh-sealed group (93.72± 0.95%,39.02±2.99%) (P< 0.05). Subusequently, the total blastomere number in blastocyst showed that there was no difference between the vitrified groups and fresh groups after cultured for 72 h and 96 h in different culture system. From those results, we can find that vitrified mouse 2-cell embryos can be cultured in sealed cuture system and develop to blastocyst, and vitrified embryos also can meet requirements of embryon development experiments in space.3. Those vitrified embryos development results is less than fresh embryos, and this may be due to the vitrified mouse 2-cell embryos having damage potentially during vitrification. Therefore, from the cell membrane integrity, cytoskeleton, mitochondria three aspects, we investigate the potential damage during nylon mesh vitrification, and the results showed vitrified mouse 2-cell embryos cell membrane was integrity, microfilament cytoskeleton have some changes, and those changes does not recover in a short time. With the development of the vitrified 2-cell embryos, we found that microfilament cytoskeleton was recovery and the distribution of microfilament in vitrified embryos was consistent with fresh embryos. There is no difference in the a-tubulin which is mainly composed to spindles in embryos, and vitrified embryos is similar to fresh embryos. Moreover, the distribution of mitochondria in vitrified 2-cell embryos also changed in the cytoplasm, with randomly distributed and irregular aggregation, while in fresh embryos, mitochondria evenly distributed in the cytoplasm, the gathered around the nucleus. From these results, we suppose that the distribution of mitochondria may be the main factor decreasing the embryo development rate. In addition, we guess vitrification may cause mitochondrial dysfunction.4. To further investigate the other possible chilling injuries which also have an effect on the development of embryo post-thawing during nylon mesh vitrification, we have determined aquaporin3 (AQP3) and aquaporin7 (AQP7) distribution and expression in those vitrified mouse 2-cell embryos by nylon mesh. Reverse transcription-polymerase chain reaction (RT-PCR) showed gene Aqp3 and gene Aqp7 express in mouse 2-cell embryos post-thawing. Quantitative real time polymerase chain reaction (qRT-PCR) results showed that the relative expression of AQP3 mRNA and AQP7 mRNA does not exist significant differences between vitrified group and fresh group. Immunofluorescence results showed that AQP3 protein in vitrified embryos was mainly distributed in the cytoplasm, nuclear membrane and the cell membrane, which is consistent with the fresh mouse 2-cell embryo. AQP7 protein was mainly distributed in cytoplasm and cell membrane in the two groups is similar, and the AQP7 also distributed in cytoplasm, cell membrane, and there is a part of AQP7 in the nuclear membrane. Western blot results also confirm the above results, and the nylon mesh vitrification does not affect the AQP3 and AQP7 in mouse 2-cell embryos.