Janik M, Kleinhans F W, Hagedorn M
National Zoological Park and Conservation and Research Center, Smithsonian Institution, Washington, DC 20008, USA.
Cryobiology. 2000 Aug;41(1):25-34. doi: 10.1006/cryo.2000.2261.
The goal of this research was to examine the developmental effects on zebrafish embryos (Brachydanio rerio) when cryoprotectants were directly microinjected into the yolk. Our objectives were to: (i) determine the final concentration of propylene glycol (PG) and dimethyl sulfoxide (Me(2)SO) that the embryos could tolerate without causing teratogenic effects; (ii) determine if the toxicity of Me(2)SO could be reduced by the simultaneous presence of various proportions of amides; and (iii) examine whether this intracellular cryoprotectant incorporation could reduce the cryodamage to the yolk syncytial layer (YSL) after vitrification trials. The rationale for conducting these microinjection experiments was to overcome the permeability barrier of the YSL. Intracellular PG produced better survival than Me(2)SO (P < 0.05). Embryos tolerated both 10- and 30-nl microinjections of PG, yielding final concentrations of 2.3 and 5.0 M within the yolk, resulting in 70 +/- 3 and 35 +/- 4% survival at day 5, respectively. In similar experiments with Me(2)SO, survival was lower than PG at 60 +/- 4 and 14 +/- 4% at 2.4 and 5.2 M. Unlike other cellular systems, the presence of amides, specifically acetamide or formamide, did not reduce the toxicity of Me(2)SO in zebrafish embryos (P > 0.05). During vitrification trials, we estimated a 25% dehydration of the yolk, yielding an effective PG concentration of 5.9 M. However, the incorporation of this vitrifiable concentration of PG was not sufficient to improve the postthaw morphology of the YSL (P > 0.05). Clearly, other factors need to be examined in establishing a successful vitrification protocol for zebrafish embryos.
本研究的目的是检查当将冷冻保护剂直接显微注射到斑马鱼胚胎（短担尼鱼）的卵黄中时对其发育的影响。我们的目标是：（i）确定胚胎能够耐受而不产生致畸作用的丙二醇（PG）和二甲基亚砜（Me₂SO）的最终浓度；（ii）确定同时存在不同比例的酰胺是否可以降低Me₂SO的毒性；以及（iii）检查这种细胞内冷冻保护剂的掺入是否可以减少玻璃化试验后对卵黄合胞体层（YSL）的冷冻损伤。进行这些显微注射实验的基本原理是克服YSL的渗透屏障。细胞内PG产生的存活率优于Me₂SO（P < 0.05）。胚胎耐受10 nl和30 nl的PG显微注射，卵黄内的最终浓度分别为2.3 M和5.0 M，在第5天的存活率分别为70±3%和35±4%。在使用Me₂SO的类似实验中，在2.4 M和5.2 M时存活率低于PG，分别为60±4%和14±4%。与其他细胞系统不同，酰胺（特别是乙酰胺或甲酰胺）的存在并未降低斑马鱼胚胎中Me₂SO的毒性（P > 0.05）。在玻璃化试验期间，我们估计卵黄脱水25%，产生的有效PG浓度为5.9 M。然而，这种可玻璃化浓度的PG掺入不足以改善YSL的解冻后形态（P > 0.05）。显然，在建立成功的斑马鱼胚胎玻璃化方案时需要检查其他因素。
