Abstract：
Primordial germ cells (PGCs) constitute an important cell lineage that directly impacts genetic dissemination and species conservation through the creation of cryobanks. In order to advance the field of animal genetic cryopreservation, this work aimed to recover intact PGCs cryopreserved in embryonic tissues during the segmentation phase for subsequent in vitro maintenance, using the yellow-tailed tetra (Astyanax altiparanae) as a model organism. For this, a total of 202 embryos were distributed in two experiments. In the first experiment, embryos in the segmentation phase were dissociated, and isolated PGCs were maintained in vitro. They were visualized using gfp-Pm-ddx4 3\'UTR labeling. The second experiment aimed to vitrify PGCs using 3 cryoprotective agents or CPAs (dimethyl sulfoxide, ethylene glycol, and 1,2 propanediol) at 3 molarities (2, 3, and 4 M). The toxicity, somatic cell viability, and recovery of intact PGCs were evaluated. After cryopreservation and thawing, 2 M ethylene glycol produced intact PGCs and somatic cells (44 ± 11.52 % and 42.35 ± 0.33 %, respectively) post-thaw. The recovery of PGCs from frozen embryonic tissues was not possible without the use of CPAs. Thus, the vitrification of PGCs from an important developmental model and Neotropical species such as A. altiparanae was achieved, and the process of isolating and maintaining PGCs in a culture medium was successful. Therefore, to ensure the maintenance of genetic diversity, PGCs obtained during embryonic development in the segmentation phase between 25 and 28 somites were stored through vitrification for future applications in the reconstitution of species through germinal chimerism.
摘要：
原始生殖细胞（PGCs）构成了重要的细胞谱系，通过创建冷冻库直接影响遗传传播和物种保护。为了推进动物基因冷冻保存领域,这项工作的目的是恢复完整的PGCs冷冻保存在胚胎组织在分割阶段的后续体外维持，使用黄尾草（Astyanaxaltiparanae）作为模型生物。为此，共有202个胚胎分布在两个实验中。在第一个实验中,分裂阶段的胚胎被分离，并在体外维持分离的PGCs。使用gfp-Pm-ddx43'UTR标记对它们进行可视化。第二个实验旨在使用3种冷冻保护剂或CPAs（二甲基亚砜，乙二醇,和1,2丙二醇），3摩尔（2、3和4M）。毒性,体细胞活力，并对完整PGCs的回收率进行评价。冷冻保存和解冻后，2M乙二醇产生完整的PGCs和体细胞(44±11.52%和42.35±0.33%，分别)解冻后。在不使用CPA的情况下,从冷冻胚胎组织中回收PGCs是不可能的。因此，从一个重要的发育模型和新热带物种如Altiparanae中玻璃化PGCs，并且在培养基中分离和维持PGCs的过程是成功的。因此,为了确保遗传多样性的维持，在25-28个体节之间的分割阶段的胚胎发育过程中获得的PGCs通过玻璃化进行存储，以便将来通过生发嵌合在物种重建中应用。
