School of Animal Technology and Innovation, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Suranaree, Muang, Nakhon Ratchasima, 30000, Thailand.
Institute for Aquaculture Biotechnology (IAB), Tokyo University of Marine Science and Technology, 4-5-7 Konan Minato-ku, Tokyo, 108-8477, Japan.
Theriogenology. 2024 Nov;229:178-190. doi: 10.1016/j.theriogenology.2024.08.025. Epub 2024 Aug 22.
Cryopreservation of spermatogonia could be a useful tool to preserve the genetic resources of fish, which could be further restored via germ cell transplantation. In this study, the protocol for the cryopreservation of the spermatogonia of Asian sea bass (Lates calcarifer), an economically important fishery resource in the Indo-West Pacific, was optimised. The impact of the cryopreservation technique on cell viability and apoptosis, expression of several genes related to immature germ cell markers, transplantability in allogeneic recipients, and global DNA methylation was evaluated. The slow-freezing method was performed for the cryopreservation of immature testis tissue, which contains a high proportion of spermatogonia. The optimal condition that yielded the highest recovery rate of post-thawed spermatogonia included a cryomedium containing Leibovitz's (L-15) medium and 10 % dimethyl sulfoxide, ice equilibration for 60 min before freezing, and subsequent thawing at 4 °C for 8 min. Moreover, a higher number of early and late apoptotic cells was detected in the cryopreserved than in the fresh testes, suggesting that apoptosis could result in reduced viability. The expression levels of dazl decreased in the cryopreserved testes; however, there were no significant differences in the expression levels of nanos2 or nanos3 between the fresh and cryopreserved testes. Although qRT-PCR showed lower vasa expression in cryopreserved testicular cells, in situ hybridisation showed expressed vasa in the cryopreserved testicular cells. Post-thawed spermatogonia could be incorporated into the genital ridge of allogeneic recipients, suggesting that cryopreserved spermatogonia exhibit transplantability characteristics. Compared with fresh testes, significant changes in the proportion of DNA methylation (decreased 5-mC and 5-caC) were observed in cryomedium-free testicular cells, whereas those of the cryopreserved cells were not significantly different. Therefore, the method we developed for the cryopreservation of the spermatogonia of Asian sea bass enabled post-thaw cells to retain several stemness characteristics and maintain their epigenetic stability.
精原细胞的冷冻保存可以成为保存鱼类遗传资源的有用工具，这些资源可以通过生殖细胞移植进一步恢复。在这项研究中，优化了印度-西太平洋地区经济重要渔业资源亚洲海鲈（Lates calcarifer）精原细胞的冷冻保存方案。评估了冷冻保存技术对细胞活力和凋亡、与未成熟生殖细胞标记物相关的几个基因的表达、同种异体受体中的移植能力以及全基因组 DNA 甲基化的影响。采用缓慢冷冻法对含有高比例精原细胞的未成熟睾丸组织进行冷冻保存。在冷冻前进行 60 分钟的冰平衡，然后在 4°C 下解冻 8 分钟，这是获得解冻后精原细胞回收率最高的最佳条件。此外，冷冻保存的睾丸中检测到比新鲜睾丸中更多的早期和晚期凋亡细胞，表明凋亡可能导致活力降低。冷冻保存的睾丸中 dazl 的表达水平降低；然而，新鲜和冷冻保存的睾丸之间的 nanos2 或 nanos3 的表达水平没有显著差异。虽然 qRT-PCR 显示冷冻保存睾丸细胞中 vasa 的表达水平较低，但原位杂交显示冷冻保存睾丸细胞中表达了 vasa。解冻后的精原细胞可以整合到同种异体受体的生殖嵴中，表明冷冻保存的精原细胞具有移植特性。与新鲜睾丸相比，在无冷冻保存液的睾丸细胞中观察到 DNA 甲基化比例（5-mC 和 5-caC 减少）发生显著变化，而冷冻保存细胞的变化则不显著。因此，我们为亚洲海鲈精原细胞开发的冷冻保存方法使解冻后的细胞保留了一些干性特征，并保持了其表观遗传稳定性。
