亚洲鲈鱼(Lates calcarifer)整个睾丸的冷冻保存及其对凋亡、生殖细胞特异性基因表达、生殖细胞可移植性和 DNA 甲基化的影响

IF 2.4 2区 农林科学 Q3 REPRODUCTIVE BIOLOGY
S. Sreebun , K. Booncherd , S. Thongchaitriwat , K. Ichida , P. Pasomboon , R. Yazawa , S. Boonanuntanasarn
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引用次数: 0

摘要

冷冻保存精原细胞是保存鱼类遗传资源的有用工具,可通过生殖细胞移植进一步恢复遗传资源。本研究优化了冷冻保存亚洲鲈鱼(Lates calcarifer)精原细胞的方案,亚洲鲈鱼是印度-西太平洋地区具有重要经济价值的渔业资源。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相关的几个基因的表达、异体受体的可移植性以及全局 DNA 甲基化的影响。对含有大量精原细胞的未成熟睾丸组织采用了慢冻法进行冷冻保存。解冻后精原细胞回收率最高的最佳条件包括:冷冻培养基中含有莱博维茨(L-15)培养基和10%的二甲亚砜,冷冻前冰平衡60分钟,然后在4 ℃解冻8分钟。此外,在冷冻保存的睾丸中检测到的早期和晚期凋亡细胞数量高于新鲜睾丸,这表明细胞凋亡可能导致存活率降低。冷冻保存的睾丸中 dazl 的表达水平下降;然而,新鲜睾丸和冷冻保存的睾丸中 nanos2 或 nanos3 的表达水平没有显著差异。尽管qRT-PCR显示冷冻保存的睾丸细胞中vasa的表达量较低,但原位杂交显示冷冻保存的睾丸细胞中表达了vasa。解冻后的精原细胞可与异体受体的生殖脊结合,这表明冷冻保存的精原细胞具有可移植性。与新鲜睾丸相比,无冷冻介质的睾丸细胞的DNA甲基化比例发生了显著变化(5-mC和5-caC减少),而冷冻保存的细胞则无显著差异。因此,我们开发的冷冻保存亚洲鲈精原细胞的方法可使解冻后的细胞保留多种干性特征,并保持其表观遗传稳定性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Cryopreservation of the whole testes of Asian sea bass (Lates calcarifer) and its effects on apoptosis, germ cell-specific gene expression, germ cell transplantability, and DNA methylation

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.

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来源期刊
Theriogenology
Theriogenology 农林科学-生殖生物学
CiteScore
5.50
自引率
14.30%
发文量
387
审稿时长
72 days
期刊介绍: Theriogenology provides an international forum for researchers, clinicians, and industry professionals in animal reproductive biology. This acclaimed journal publishes articles on a wide range of topics in reproductive and developmental biology, of domestic mammal, avian, and aquatic species as well as wild species which are the object of veterinary care in research or conservation programs.
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