| Intracytoplasmic sperm injection(ICSI) is an new assisted reproductive technique developed in the 1980s. Its essence is that a single sperm inject ooplasm directly by inject pipette to fertilize a oocyte.The establishment of ICSI bring the infertile couple gospel and become an regular therapeutic tool for male sterility. However, the lack of animal model hinder the research of ICSI of human. Though mouse oocyte is ideal animal model, the ICSI research made low progess because the mouse oocyte have a small volume and poor capacity of preventing damage. It got higher mouse oocyte survival rate, fertilization rate and blastocyst rate until Kimura et al use the instrument of piezo on mouse ICSI in 1995. Nevertheless, owing to the unstability and potential mercury contamination, the application of piezo-ICSI in clinic and scientific reseach is limit.This acticle applied and improved the new ICSI technology (New-ICSI, N-ICSI). The method of N-ICSI is as follows:The holding pipette was modified to a trumpet shape and part of zona pellucida and cytoplasm were sucked in by the modified holding pipette. Then, the conventional sharp injection pipette inserted the cytoplasm of oocyte, aspirated and ruptured of membrane, and injected the head of mouse spermatozoa at last. Now, the experimental result are listed as follows:①Compare the oocytes developmental condition in vitro which treated by N-ICSI or C-ICSI. The result of experiment suggested that a significantly higher survival rate (89%) was achieved by N-ICSI than the conventional one (C-ICSI,22%; P< 0.01) and the normal fertilization rates, cleavage rates and morula rates were respective 85%,71%and 61%, which were not obvious different with C-ICSI (88%,84%,76%, P>0.05). The blastocyst rates between N-ICSI and C-ICSI were also not obvious different (65% vs 62%, P>0.05) but both significantly lower than the activation group (97%, P<0.05). optimizing and improving the experimental condition of N-ICSI may can improve the blastocyst rate.②Compare the blastocyst rate effected by different combinations of two main parameter (inner diameter of holding pipette opening and the sucked length of cytoplasm by holding pipette). We found that a low survival rate of oocyte(64%) was gained when the inner diameter of holding pipette opening was 45μm, which significantly lower than the one of 50μm or 55μm. When the sucked length of cytoplasm by holding pipette was 75μm, either the blastocyst rate (0% and 33%) was significantly lower than most other combinations or the normal fertilization rates were extra-low (only 36%), and the triploid and polyploidy were common found. However, superior survival rate, normal fertilization rate, cleavage rate and morula rate and balstocyst rate was achieved when the inner diameter of holding pipette opening was 50 u m and the sucked length of cytoplasm by holding pipette was 40μm. So, this combination of parameter is the best condition of N-ICSI.③Use the best condition of N-ICSI in interspecies ICSI that human sperm injected mouse oocyte and compare the different mouse oocyte activate capacity effected by different immobilization time of human sperm. Our experiment results suggested that high survival rate(over 80%), extra-high normal fertilization rate(over 90%) and accepted cleavage rate(70%) can acquired by human sperm injected mouse oocytc via N-ICSI. And we also found that the capacity that human sperm activated mouse oocyte does not change very much during the 30min after sperm immobilization.In conclusion, our reseach establish a high efficiency, easy and minor damage for mouse oocyte new ICSI technique. Better result has been achieved when human sperm injected mouse oocyte. However, whether the part of cytoplasm distortion sucked by holding pipette effect later development or not is deserved study. Nevertheless, owing the simpleness and practicability of this technique, it is worthy applying in clinic and scientific reseach.(?)#[(?)#]... |
PDF Full Text Request