哺乳动物胚胎冷冻技术研究进展

胚胎冷冻保存已广泛用于胚胎移植，动物克隆以及动物资源保护。此技术的应用需保证胚胎在冷冻一解冻后具有较高的成活率。自1972年小鼠胚胎冷冻获得成功以来，许多学者在简化冷冻程序，缩短冷冻时间等方面进行了深入研究，试图寻找一种可用于生产的冷冻方法。本文全面地比较了不同的冷冻方法及其结果，籍此为该领域工作者提供一定帮助。     

玻璃化冷冻对小鼠GV期卵母细胞发育能力的影响

试验首次采用OPS法玻璃化冷冻小鼠GV期卵母细胞(不带卵丘细胞,下同),同时尝试用EDFS30对小鼠卵巢进行细管法玻璃化冷冻,以研究GV期卵母细胞冷冻后的发育潜力。首先,利用MEM培养和MEM-腔前卵泡培养新鲜GV期卵母细胞,并把较好的培养方式用于冷冻后培养试验。2种培养方式培养24h后新鲜GV期卵母细胞成熟率无显著性差异;OPS法冷冻的GV期卵母细胞解冻后成熟率及体外受精后卵裂率与对照组差异不显著(P>0.05)。细管法冷冻卵巢组织的GV期卵母细胞成熟率极显著低于对照组(P<0.01),其受精后未获得受精卵。结果表明:OPS法可有效地冷冻保存小鼠GV期卵母细胞,而细管法冷冻小鼠卵巢对GV期卵母细胞损伤较大。     

Droplet-vitrification of apical shoot tips of Rubus fruticosus L. and Prunus cerasifera Ehrh.

Apical shoot tips excised from in vitro plantlets of blackberry (Rubus fruticosus L. ‘?a?anska Bestrna’) and cherry plum (Prunus cerasifera Ehrh.) were tested for recovery after cryopreservation using the droplet-vitrification technique. Following treatment for 30 min with a loading solution comprising 1.9 M glycerol and 0.5 M sucrose, explants were dehydrated with a highly concentrated cryoprotectant solution, so called vitrification solution. Shoot tips were dehydrated for 10, 20 and 30 min at room temperature with a solution derived from the original PVS2 solution (containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose) and for 60, 90 and 120 min using the PVS3 solution (containing 50% (w/v) glycerol and 50% (w/v) sucrose). Explants were cooled by direct immersion in LN in 10 μl droplets of vitrification solution placed on aluminium foil strips. Rewarming was done by direct plunging of foil strips in a preheated (37 °C) unloading solution (0.8 M sucrose) for 30 s, after which an equal volume of unloading solution (at room temperature) was added for further incubation for 30 min. As for regrowth of blackberry, PVS3 proved more effective than the modified PVS2, but the difference was significant (P < 0.05) only for the shortest treatment duration. The duration of PVS3 treatment had no significant effect on regrowth of cryopreserved shoot tips (45.8–70%). By contrast, a 30-min treatment with modified PVS2 solution resulted in a significant increase in regeneration percentage (30%), as compared with a 10-min treatment with the same solution (5%). Cherry plum shoot tips were very sensitive to both vitrification solutions and growth recovery of cryopreserved samples was generally lower (5–20%) than that of blackberry explants. No significant influence of PVS treatment (both type of solution and treatment duration) on regrowth of cryopreserved shoot tips was observed with cherry plum shoot tips. Experiments performed in France and in Serbia produced similar results, thereby showing the robustness and reproducibility of the protocols developed.     

Microtubule assembly crucial to bovine embryonic development in assisted reproductive technologies

Centrosome integrity and microtubule network are crucial to the events around fertilization, including pronuclear development, migration and fusion, and the first mitotic division. The present review highlights the importance of bull spermatozoal centrosomes to function as a microtubule‐organizing center for successful fertilization and the subsequent embryonic development. Spermatozoal centrosomes need to be blended with ooplasmic pericentriolar materials accurately to nucleate and organize the sperm aster. Dysfunction of the spermatozoal centrosomes is associated with fertilization failure, which has been overcome with supplemental stimuli for oocyte activation following intracytoplasmic sperm injection in humans. Even though the spermatozoal centrosomes are functionally intact, abnormal sperm aster formation was frequently observed in vitrified‐warmed bovine oocytes, with delayed pronuclear development and migration. Treatment of the post‐warm oocytes with Rho‐associated coiled‐coil kinase inhibitor or α‐tocopherol inhibited the incidence of the abnormal aster formation, resulting in higher blastocyst yields following in vitro fertilization and culture. Thus, understanding of centrosomal function made it possible to improve the performance of advanced reproductive technologies.     

酸樱桃组织培养中试管苗玻璃化的发生与防止

通过在MS培养基中加入不同浓度的蔗糖、琼脂、细胞分裂素(BA和KT)及不同温度下对酸樱桃进行组培研究，发现酸樱桃玻璃苗的数量随着糖分及琼脂浓度增加而减少．BA会提高玻璃化率，KT对玻璃化现象无影响，适当降低温度能有效降低试管苗玻璃化现象发生的频率。     

741杨和苹果试管苗玻璃化现象及其转化研究

以７４１杨和苹果矮化砧Ｍ２６和Ｍ７为材料，研究了影响７４１杨试管苗玻璃化现象的因素。除培养基中ＢＡ和ＩＢＡ的浓度、琼脂浓度外，光强和温度组合是影响试管苗玻璃化现象的重要因素。７４１杨试管苗玻璃化与其内源ＡＢＡ水平有关。７４１杨无根试管苗的玻璃化率和器官分化能力存在季节变化，春季和夏季较强，冬季较弱；７４１杨无根试管苗的内源ＡＢＡ含量则是春季和夏季低，冬季高。无根正常试管苗ＡＢＡ含量为无根玻璃苗的５．５９倍。苹果无根玻璃化试管苗如在其培养基中加入低浓度（Ｏ．１～０．５ｍｇ／Ｌ）ＡＢＡ，或在较强的光照条件下培养，可以转化为无根正常试管苗，并能正常进行继代和生根。     

生菜遗传转化过程中克服玻璃苗的研究

为了克服生菜遗传转化过程中玻璃化现象,对生菜选择培养基中不同浓度的琼脂、蔗糖、细胞激动素（ＢＡ、ＫＴ）、ＡｇＮＯ３及抗生素浓度进行了研究。结果表明：下班苗的数量随琼脂、蔗糖浓度的增加而减少;高浓度的细胞激动素和抗生素会增加下班苗的数量;加入一定量的ＡｇＮＯ３可抑制下班苗的产生,其最佳浓度为２．０ｍｇ／Ｌ。     

玻璃化冻存对羊软骨细胞的影响

对玻璃化冻存后羊Ovis aries软骨细胞的存活率和琥珀酸脱氢酶（SDH）活性进行了初步研究。结果表明：细胞存活率随着冻存时间的延长而逐渐下降,但下降速度不明显;SDH活性在玻璃化冻存1 ~ 20 d变化幅度较大,20 ~ 30 d下降趋势减弱,保持相对稳定。从各项指标来看,认为玻璃化溶液VSb组的冻存效果最好,在经过5,10, 15,20和30 d的冻存后,其存活率分别为85.47% ± 1.78%,80.73% ± 1.81%,78.62% ± 2.06%, 76.35% ± 2.58%和73.83% ± 1.49%,细胞SDH活性吸光度值分别为0.49 ± 0.064,0.444 ± 0.073,0.394 ± 0.039,0.354 ± 0.082和0.339 ± 0.053。该玻璃化冻存方法对长期冻存羊软骨细胞具有一定的实际应用意义。图4表2参17     

红叶石楠组培苗玻璃化影响因子及其克服技术研究

通过改良MS+6-BA无菌培养红叶石楠,结果表明:培养基离子浓度或细胞分裂素浓度偏高,组培苗玻璃化的比例均增加,降低培养温度或提高培养基硬度玻璃化苗均减少,相近凝固效果的卡拉胶比琼脂易导致玻璃化苗;繁殖代数增加,玻璃化比例降低.培养温度由恒温改为变温、6-BA浓度由1.5 mg/L降为0.5 mg/L、卡拉胶由7.0 g/L增加到8.0 g/L,培养60 d后分别能将61.67%、57.92%和47.08%的仅部分叶片出现玻璃化的红叶石楠组培苗转为正常,部分叶片玻璃化如培养条件不变,或茎、叶完全玻璃化,即使改变培养条件,均不能转变.     

中华绒螯蟹胚胎的玻璃化冷冻保存

研究了中华绒螯蟹(Eriocheir sinensis)4个不同发育时期胚胎对A号玻璃化液的耐受性和玻璃化冷冻保存。结果表明,不同时期的胚胎对玻璃化液的耐受能力不同,其中卵裂期胚胎对玻璃化液的耐受能力较差(20~30 min),前无节幼体期和原溞状幼体期胚胎在玻璃化液中的适应时间较长(40~60 min);随着平衡时间的延长,中华绒螯蟹各个时期的胚胎成活率逐渐下降。中华绒螯蟹前无节幼体期胚胎在A号玻璃化液中平衡40 min,0.25mol/L的蔗糖分别洗脱5、10、15、20 min后,胚胎成活率无显著性差异(P>0.05)。前无节幼体期胚胎在A号玻璃化液中平衡40 min,在–196℃冷冻40 min,快速解冻后用0.25 mol/L的蔗糖洗脱10 min,有8个胚胎成活,成活率为(9.3±2.5)%,胚胎培养至第4天死亡;原溞状幼体期胚胎在A号玻璃化液中平衡40 min,在–196℃冷冻35 min,经相同浓度的蔗糖洗脱相同的时间,有7个胚胎成活,成活率(11.3±3.6)%,培养至第6天时,1个胚胎孵化出膜,出膜胚胎成活1d后死亡。