Studies On The Intracytoplasmic Sperm Injection In The Mouse

In the present study, intracytoplasmic sperm injection (ICS1) was successful in the mouse when the piezo-driven micropipette was used and the possibility of making transgenic mouse by the ICSI method was tested. The experimental results demonstrated were: a) the piezo-driven micropipette caused intensive damage to mouse oocyte plasma, 55.5% and 23.3% of sperm-injected oocytes from the B6D2F1 and KM strain survived respectively. The survival rates in B6D2F1 and KM groups (88.5% and 84.5%) could be significantly increased (p<0.01) when 3% sucrose solution was used. HEPES-CZB media containing 3 % sucrose was used in the following experiments. Fresh sperm heads separated from tails by piezo, as well as thawed sperm heads mixed with pEGFP-Nl plasmid, were individually injected into oocytes for normal fertilization. 83.3% of oocytes survived after injection procedure, the fertilization rates of the fresh and the frozen groups were 92.3% and 83.0%, respectively. Fertilized oocytes were culture in vitro in CZB medium containing 5 mM glucose. Developmental rates in vitro at 2-cell, morula and blastocyst stage were (97.8% and 94.7%), (64 % and 64.5%),(5% and 24.7%), respectively, the percentage of the ICSI embryoes developed to morula, blastocyst shown great significantly (p<0.01) difference from the control group (fertilized in vivo and cultured in vitro)(88%,70%). Furthermore, after 96 h culture in vitro, 5 of 16 blastocysts from frozen groups expressed GFP protein, c) When 337 ICSI embryos at pronuclear or 2-cell stages from fertilized with fresh or frozen sperm heads were transferred into recipients, respectively. 23.3% (28/120), 13.3 % (18/135), 4 % (3/82) of embryos at pronuclear stages in the groups of fresh and frozen sperm heads and 2-cell embryos from fresh group reached the full term. 31 of them grew into normal adults and shown no physiological and behavior abnormality. All of them could produce offspring normally. These studies demonstrates that not only we could make transgenic mouse embryo with high efficiency by the method of ICSI, but also the fresh sperm from cauda epididymis or freeze-thawed dead sperm pre-mixed with exogenous DNA had the full development potential to develop into term.