Page 33 - 《水产学报》2026年第3期

3 期 水 产 学 报 50 卷

Efficient establishment and application studies of the gonadal cell
lines from Japanese eel (Anguilla japonica)

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ZHONG Zhaowei , XI Ning , YANG Zhen , DING Zhen , SHI Shengkai ,
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LUO Dingyuan , LU Yizhen , JIANG Yonghua 1,3*
(1. State Key Laboratory of Mariculture Breeding, Jimei University, Xiamen 361021, China;
2. College of Ocean and Earth Sciences, Xiamen University, Xiamen 361005, China;
3. Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture and Rural Affairs,
Jimei University, Xiamen 361021, China)
Abstract: Fish cell lines hold significant value in various fields such as resource conservation, genetic breeding, disease con-
trol, and environmental toxicological assessment. The establishment of gonadal cell lines provides critical in vitro models for
elucidating sex determination mechanisms and gonad-related genes function regulations. In this study, we successfully estab-
lished Japanese eel (Anguilla japonica) ovarian tissue cell line (AJOTCL) and testicular tissue cell line (AJTTCL) via optimiz-
ing L-15 medium supplemented with 15% fetal bovine serum (FBS), growth factors, A. japonica serum, and embryonic
extracts, combined with tissue explant culture. Both cell lines were stably passaged to 120 and 100 generations, respectively,
with cell survival rates exceeding 90% after cryopreservation and recovery. Morphologically, the cells predominantly exhibited
fibroblast-like characteristics (spindle, prism-shaped, polygonal, or irregular shapes) with whorl-like arrangements, while
AJOTCL displayed oval morphology at high density. Growth characteristic analysis revealed that medium containing 15%-20%
FBS, culture temperature of 26 ℃, and a 1∶2 subculturing ratio significantly promoted proliferation capacity of AJOTCL and
AJTTCL. Gene expression analysis showed that AJOTCL highly expressed ovarian-specific genes foxl2 and sox3 (similar to
ovarian tissue), while AJTTCL highly expressed testicular-specific genes dmrt1 and amh (similar to testicular tissue), with both
cell lines showing weak expression of the germ cell marker gene nanog or dnd, validating that both cell lines were predomin-
antly composed of somatic cells. Therefore, they were designated as A. japonica ovarian tissue cell line and A. japonica testicu-
lar tissue cell line, respectively. Electroporation successfully mediated the transfection of the exogenous pEGFP-N1 plasmid
into both cell lines with high efficiency (≥60%), validating their applicability for genetic manipulation. Gene function valida-
tion indicated that overexpression of nanog, oct4, and lin28a/b activated downstream pathways thereby promoting cell prolifer-
ation, whereas nanog knockdown resulted in significant functional inhibition leading to cell senescence. These results demon-
strate that both cell lines are suitable for gene function research. In conclusion, the gonadal somatic cell lines, AJOTCL and
AJTTCL, established in this study possess stable growth characteristics and efficient gene manipulation capabilities, providing
reliable in vitro models for in-depth investigation of somatic-germ cell interactions, sex determination mechanisms, and repro-
ductive regulation.
Key words: Anguilla japonica; cell line; primary culture; ovary; testis; gene transfection
Corresponding author: JIANG Yonghua. E-mail: yhjiang1974@jmu.edu.cn

Funding projects: National Key R & D Program of China (2024YFD2401002); National Natural Science Foundation of China
(42476103, 41306174); Natural Science Foundation of Fujian Province (2024J01704); Natural Science Foundation of Xiamen
Municipality (3502Z202473056)

https://www.china-fishery.cn 中国水产学会主办 sponsored by China Society of Fisheries
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