Ward Monika A, Kaneko Takehito, Kusakabe Hirokazu, Biggers John D, Whittingham David G, Yanagimachi Ryuzo
Institute for Biogenesis Research, University of Hawaii Medical School, Honolulu, Hawaii 96822, USA.
Biol Reprod. 2003 Dec;69(6):2100-8. doi: 10.1095/biolreprod.103.020529. Epub 2003 Aug 20.
The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at 4 degrees C. Samples frozen without cryoprotection were maintained at -196 degrees C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P > 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P > 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P < 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P < 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P > 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then be stored at 4 degrees C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.
转基因、基因敲除和基因突变小鼠的广泛培育，使得维持这些小鼠品系活体种群的可用资源变得紧张，因此需要可靠的方法来保存这些品系的配子和胚胎。在此，我们报告了对储存长达1.5年的冻干或无冷冻保护剂冷冻精子进行胞浆内精子注射（ICSI）的结果。冻干样本储存在4℃。无冷冻保护剂冷冻的样本保存在-196℃。储存后，通过ICSI将精子注射到卵母细胞中。在精子储存0、1、3、6、9和12个月后，检查合子染色体和妊娠第15天的胎儿发育情况。当使用新鲜精子进行ICSI时，96%的所得合子含有正常染色体，移植的二细胞胚胎中有58%发育为正常活胎儿。当精子无冷冻保护剂冷冻后用于ICSI时，也得到了类似的结果（分别为87%和45%；P>0.05），以及在精子储存12个月后（六个检测终点的平均值：分别为87%和52%；P>0.05）。冻干降低了具有正常核板的合子比例（75%对96%；P<0.001）和发育为胎儿的胚胎比例（35%对58%；P<0.001），但与冷冻相似，在12个月的储存期间没有进一步恶化（六个检测终点的平均值：分别为68%和34%；P>0.05）。储存1.5年后，从冻干和冷冻精子中均获得了活后代。结果表明：1）冻干过程本身会导致精子出现一些异常，但无冷冻保护剂冷冻则不会；2）冷冻和冻干精子的长期储存对其遗传完整性无害。无冷冻保护剂冷冻非常成功、简单且高效，但与所有常规精子储存方法一样，需要液氮。冻干也需要液氮，但精子随后可储存在4℃并在室温下运输。两种保存方法都很成功，但无冷冻保护剂的快速冷冻是保存携带独特基因和突变的小鼠品系精子的首选方法。
