Freezing mouse blastocysts

Summary

A good survival rate in culturing mouse blastocysts can be obtained in Ovum Culture Medium, enriched with 20 per cent inactivated Foetal Bovine Serum or Sheep Serum under air. The transfer of fresh blastocysts gives the best results if the recipients are on day 3 of the pseudo‐pregnancy, but with 20 hours’ cultured blastocysts it is better to use recipients on day 4.

Exposure to 1.5 M DMSO has no harmful effect, provided that the DMSO is added at 5° C in 6 steps and is removed, again in 6 steps, at 35° C. The crystallization of the medium containing the embryos at ‐5° C to ‐6° C doet not appear te have a harmful influence on culture results of the blastocysts.     

小鼠卵母细胞孤雌激活试验

应用两种激活方法(乙醇和离子霉素 6-DMAP)激活和三种培养液(CZB，KSOM，M199)对90只小鼠卵母细胞进行了孤雌激活研究。结果显示，离子霉素结合6-DMAP的激活效果高于乙醇；三种培养液中，以CZB激活效果较为理想。在随后的卵母细胞发育率方面，CZB、KSOM和M199三者之间的差异极为明显，以CZB最高，KSOM次之，M199的发育率最低。     

Ovarian choriocarcinoma in the mouse

Choriocarcinoma is one of the rarest ovarian tumors in any animal species. This paper describes the gross and microscopic appearance of seven such neoplasms in B6C3F1 mice. Mean age at death was 47 weeks. Tumors were described at necropsy as dark or hemorrhagic cystic lesions. On microscopic examination tumors were composed of hematocysts, intercellular hemorrhage, and cytotrophoblasts, syncytiotrophoblasts, and/or trophoblastic giant cells. Cytotrophoblasts, syncytiotrophoblasts, and occasional giant cells were present in three cases while the other four tumors contained only trophoblastic giant cells.     

Vitrification of biopsied mouse embryos

Solid surface vitrification (SSV) was compared with in-straw vitrification for cryopreservation of biopsied mouse embryos. Eight-cell stage embryos were zona drilled and one blastomere was removed. Developed morulae or blastocysts were vitrified in microdrop (35% EG + 5% PVP + 0.4 M trehalose) or in straw (7.0 M EG + 0.5 M sucrose). Following recovery, embryos were cultivated in vitro or transferred into recipients. Cryopreservation had an effect not only on the survival of biopsied embryos but also on their subsequent development in vitro. Cryosurvival of biopsied morulae vitrified in straw was significantly inferior to SSV. The post-warm development of biopsied and non-biopsied morulae was delayed on Day 3.5 and 4.5 in both vitrification groups. A delay in development was observed on Day 5.5 among vitrified non-biopsied blastocysts. The percentage of pups born from biopsied morulae or blastocysts following cryopreservation did not differ from that of the control. No significant differences could be detected between methods within and between embryonic stages in terms of birth rate. The birth rate of biopsied embryos vitrified in straw was significantly lower compared to the non-biopsied embryos. The novel cryopreservation protocol of SSV proved to be effective for cryopreservation of morula- and blastocyst-stage biopsied embryos.     

Glycidol degrades scrapie mouse prion protein

Agents of transmissible spongiform encephalopathy (prion) are known to be extremely resistant to physicochemical inactivation procedures such as heat, radiation, chemical disinfectants such as detergents, alcohols, glutaraldehyde, formalin, and so on. Because of its remarkable resistance, it is difficult to inactivate prion. Chemical inactivation seems to be a practical method because it is applicable to large or fixed surfaces and complicated equipment. Here, three epoxides: beta-propiolactone, propylene oxide, and glycidol (GLD) were examined of their inactivation ability against scrapie-mouse prion protein (PrP(Sc)) under various conditions of chemical concentration, incubation time, and temperature. Among these chemicals, GLD worked most effectively and degraded PrP into small fragments. As a result of the bioassay, treatment with 3% GLD for 5 hr and 5% GLD for 2, 5 hr or 12 hr at room temperature prolonged the mean incubation time by 44, 30, 110 and 73 days, respectively. From dose-incubation time standard curve, the decrease in infectivity titers were estimated as 10(3) or more. Therefore, degradation of PrP(Sc) by GLD decreased the scrapie infectivity. It is also suggested that pH and salt concentrations influence the effect of GLD. Although further study is necessary to determine the optimal condition, GLD may be a potential prion disinfectant.     

甲醛对小鼠的病理性损伤试验

作为合成树脂的原料,甲醛已被广泛应用于各种建筑和装饰材料而进入千家万户,室内挥发性甲醛超标已经成为备受关注的公共卫生问题;在高校特别是农业院校的生物学实验室内,甲醛也常被用来制备和保存标本,期间甲醛的缓慢挥发会令工作人员眼、鼻、咽喉不适或疼痛,肺功能异常;甚至记忆力减退,神经行为和情绪、精神状态改变等非特异性症状.     

Pulmonary pathology of the motheaten mouse

Mice, homozygous for the motheaten gene, developed an unusual pneumonia that was the cause of natural death of mice by 7 weeks of age. Initial lesions consisted of focal accumulations of alveolar macrophages in alveoli, especially adjacent ot bronchioles. Needle-like crystals formed in lysosomes of macrophages and numerous macrophages with crystals filled most alveoli in 5- to 7-week-old mice. Although motheaten mice had lesions in other tissues and were shown by other investigators to have immunological defects, the unusual pneumonia was the only lesion severe enough to cause death.     

Cryopreservation of isolated mouse germinal vesicles

The storage of unfertilized oocytes, either immature, maturing or mature, is still unsatisfactory. Here we describe an approach in which germinal vesicles isolated as karyoplasts from immature oocytes are vitrified by open the pulled straws (OPS) method in evacuated porcine zonae pellucidae. After thawing, their survival was almost absolute. Moreover, when thawed GV-karyoplasts were fused to immature oocyte cytoplasts the maturation of reconstructed cells resulted in the production of secondary oocytes--metaphase II.     

小鼠胚胎冷冻后线粒体损伤的研究

为了研究玻璃化冷冻后小鼠胚胎线粒体的损伤,试验将V1冷冻的囊胚和V2冷冻的扩张囊胚解冻后在体外进行培养,分别在培养0,4,8,12,18,24 h后观察线粒体的损伤情况.结果表明:无论是用V1还是用V2对胚胎进行玻璃化冷冻,线粒体的正常聚集率随着培养时间的延长均呈现出下降的趋势;V1冷冻的囊胚在培养0,4 h后的线粒体正常聚集率极显著高于培养8,12,18,24 h后的正常聚集率(P<0.01),V2冷冻的扩张囊胚在培养0 h后线粒体的正常聚集率极显著高于培养4,8,12,18,24 h后的正常聚集率(P<0.01).     

Transgenic mouse offspring generated by ROSI

The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes.     

小鼠卵母细胞冷冻技术研究

采用PBS作为冷冻基础液,分别用甘油和二甲基亚砜(DMSO)作为冷冻保护液,在程序化冷冻保存和玻璃化冷冻保存条件下,研究小鼠生发泡期(GV期)卵母细胞的抗冻能力。结果表明,2种冷冻方法对小鼠GV期卵母细胞解冻后形态正常率和存活率无显著影响(P>0.05)。冷冻保护剂种类对小鼠GV期卵母细胞解冻后形态正常率无显著影响(P>0.05);但对存活率有显著影响,玻璃化冷冻采用二甲基亚砜作为冷冻保护液效果极显著优于甘油(P<0.01)。以冷冻效果较好的二甲基亚砜作为冷冻保护液,采用玻璃化冷冻不同发育阶段(GV期和MⅡ期)的小鼠卵母细胞,解冻后形态正常率无显著差异(P>0.05),但存活率GV期要显著优于MⅡ期卵母细胞(P<0.05)。