用冻干精子生产ICSI小鼠

前期研究中，我们采用小鼠卵母细胞胞浆内精子注射（ICSI）技术，成功地用新鲜精子获得了生理健康的ICSI小鼠。在此基础上，本文结合细胞冻干技术，进行了冻干精子ICSI生产试管小鼠的尝试。B6D2F1成年公鼠的精子在Tris-HCl-EDTA（pH8.2）溶液中冻干4 h后解冻，将其精子头注入到KM小鼠成熟卵母细胞的胞质中。6 h后，83.0%的卵子受精。用CZB溶液体外培养发现，冻干精子生产的胚胎，体外发育至2-C (92.0% vs 99.5%)、4-C（52.7% vs 97.2%）、桑椹胚（36.6% vs 86.3%）和囊胚的比例（21.4% vs 68.7%）极显著地（p<0.01）低于新鲜精子生产的ICSI胚胎。移植2-C期冻干精子ICSI胚胎检测体内发育，结果发现，D10的着床率为63.0%（17/27）；胎鼠的出生比例为21.7%（13/60），这些ICSI小鼠成年后的生理和生殖能力正常。试验结果说明，精子冻干后，仍能生产ICSI动物，冻干技术可以用于哺乳动物精子的保存。

Normal Mice Produced from Oocytes Injected with Freeze-dried Sperm

In our previous study, we had successfully obtained normal mice derived from oocytes following intracytoplasmic fresh sperm injection (ICSI). In the current study, we tested the developmental potency of ICSI embryos produced using freeze-dried sperm. The sperm of B6D2F1 mice were firstly freeze-dried in Tris-HCl-EDTA（pH8.2）solution for 4 h, then were individually injected into mature KM oocytes. Six hours later, 83.0% of fertilized eggs shown two pronuclei and extruded the 2nd polar body. The development rates of these fertilized embryos at the 2-cell (92.0% vs 99.5%), 4-cell (52.7% vs 97.2%), morulae (36.6% vs 86.3%) and blastocyst stages (21.4% vs 68.7%), were significantly (p<0.01) lower than that of the embryos fertilized by fresh sperm injection. After transferred to pseudo-pregnant KM female mice, 63.0% (17/27) of the 2-cell embryos were implanted on D10 and 21.7% (13/60) of them developed to term. Eleven pups developed into normal and fertile adult. Here we reported that the first birth of mouse offspring following ICSI using freeze-dried sperm in China. It was valid to store mammalian sperm using freeze-dried procedure.