Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling
Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling
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DOI：
10.3791/56118
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发表时间：
2018-03-01
影响因子：
1.2
通讯作者：
Horvath, Akos
中科院分区：
文献类型：
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作者：
Marinovic, Zoran;Lujic, Jelena;Horvath, Akos
目前的科学和生物技术趋势导致在模式生物中创造出数以千计的新品系，从而导致有必要在保持繁殖群体的常见做法之外，采用新的方法来安全储存遗传资源。本研究的主要目的是适应针浸玻璃化冷冻（NIV）程序冷冻保存整个斑马鱼睾丸。通过全睾丸NIV冷冻保存早期生殖细胞为储存斑马鱼遗传资源提供了可能性，特别是因为移植后它们可以成熟为雄性和雌性配子。将睾丸切除，固定在针灸针上，在两种冷冻保护介质（含有1.5 M甲醇和1.5 M丙二醇的平衡溶液;和含有3 M二甲基亚砜和3 M丙二醇的玻璃化溶液）中平衡，并投入液氮中。将样品在一系列的三种随后的加温溶液中加温。该技术的主要优点是（1）在消化温热的睾丸后没有精子，因此便于下游操作;（2）超快速冷却使得组织能够最佳地暴露于液氮，因此最大化冷却并降低冷冻保护剂的所需浓度，从而降低其毒性;（3）几个睾丸同步暴露于冷冻保护剂和液氮;和（4）通过在五种不同的斑马鱼品系中获得超过50%的存活率证明的可重复性。
Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.