Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil; Laboratory of Fish Biotechnology, National Center for Research and Conservation of Continental Aquatic Biodiversity, Chico Mendes Institute of Biodiversity Conservation, Pirassununga, São Paulo, Brazil.
Institute of Bioscience, São Paulo State University, Botucatu, São Paulo, Brazil; Laboratory of Fish Biotechnology, National Center for Research and Conservation of Continental Aquatic Biodiversity, Chico Mendes Institute of Biodiversity Conservation, Pirassununga, São Paulo, Brazil.
Cryobiology. 2024 Sep;116:104929. doi: 10.1016/j.cryobiol.2024.104929. Epub 2024 Jun 20.
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.
原始生殖细胞（PGCs）是一个重要的细胞谱系，通过创建冷冻库直接影响遗传传播和物种保护。为了推进动物遗传冷冻保存领域的发展，本研究旨在从分割阶段胚胎组织中回收完整的 PGC 进行冷冻保存，以便随后进行体外培养，以黄尾副鲱脂鲤（Astyanax altiparanae）为模型生物。为此，共将 202 个胚胎分配到两个实验中。在第一个实验中，将分割阶段的胚胎进行解离，并在体外维持分离的 PGC，并用 GFP-Pm-ddx4 3'UTR 标记进行可视化。第二个实验旨在使用 3 种冷冻保护剂（二甲亚砜、乙二醇和 1,2-丙二醇）在 3 个摩尔浓度（2、3 和 4 M）下对 PGC 进行玻璃化冷冻。评估了冷冻保护剂的毒性、体细胞活力和完整 PGC 的回收率。冷冻保存和解冻后，2 M 乙二醇产生完整的 PGC 和体细胞（分别为 44±11.52%和 42.35±0.33%）。如果不使用冷冻保护剂，从冷冻胚胎组织中回收 PGC 是不可能的。因此，成功实现了从重要发育模型和新热带物种（如 A. altiparanae）的 PGC 玻璃化冷冻，并且成功地在培养基中分离和维持 PGC。因此，为了确保遗传多样性的维持，在 25-28 个体节之间的胚胎发育的分割阶段获得的 PGC 通过玻璃化冷冻保存，以备将来通过生殖嵌合体重建物种时使用。
