美洲鲥雄性生殖细胞冷冻保存及移植

采用分离细胞冻存和组织块直接冻存2种方法, 进行美洲鲥(American shad, Alosa sapidissima)精巢细胞的长时间冷冻保存(>250d), 并比较分析2种不同冻存方法对美洲鲥雄性生殖细胞的冻存效果。解冻复苏后用Hochest33342和PI共染细胞核, 分析统计各期雄性生殖细胞的存活率, 结果显示组织块冻存方法所得精原干细胞和精母细胞的存活率明显高于分离细胞冻存的; 而精细胞及其他细胞存活率在2种方法间无显著差异; 特别是, 镜检发现组织块冻存方法所得精子存活率高达93.83%, 说明此冻存方法能同时高效地冻存美洲鲥各期生殖细胞, 包括成熟的精子。同时, 将组织块冻存的美洲鲥生殖细胞用PKH26染色标记后移植到出苗第1天的斑马鱼仔鱼中, 在细胞植入后5d仍能在受体中检测到供体细胞, 且有部分供体细胞能与内源生殖细胞共定位, 表明经过长时间冷冻保存的美洲鲥生殖细胞仍具有生殖细胞特性, 且能整合到斑马鱼受体性腺原基。研究结果为进一步开展美洲鲥, 或其他洄游性鱼类的生殖细胞发育、培养及种质资源保存等研究工作奠定了技术理论基础。

STUDIES ON CRYOPRESERVATION AND TRANSPLANTATION OF THE MALE GERM CELL IN AMERICAN SHAD (ALOSA SAPIDISSIMA)

Germ cells, different from somatic cells, carry the hereditary information and can transmit it to next generation. The germ cells cryopreservation and culture are the key techniques for cell engineer breeding and genetic resource preservation. This study investigated the effects of cryopreservation of the testicular blocks or dissociated cells on the viability of male germ cells of American shad after 250 days using Hochest33342/Propidium Iodide (PI) staining. The results showed that the overall viability of germ cells from the testis blocks cryopreservation was higher than that of dissociated cells cryopreservation, suggesting that the tissue frozen could efficiently cryopreserve the germ cells at different stages in American shad. Moreover, the thawed germ cells of American shad were stained with PKH26 before transplantation into newly-hatched zebrafish fries (1 days post hatching). At the 5th day post transplantation, the donor cells were still detected in some recipients and some donor cells were co-localized with the endogenous PGCs of recipient, implying that the germ cells of American shad after a long-term cryopreservation still possess the properties of germ cells and could integrate to the genital ridge of zebrafish host. The findings of this study provide technological and theoretical basis for further investigations on germ cell development, culture and genetic resource preservation in American shad or other anadromous fish in wild field.