Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
Oita Marine Biological Technology Center, Nippon Suisan Kaisha, Ltd., 508-8, Ariakrura Turumi, Saiki-shi, Oita 876-1204, Japan.
Gen Comp Endocrinol. 2020 Sep 1;295:113525. doi: 10.1016/j.ygcen.2020.113525. Epub 2020 Jun 2.
We aim to establish a small-bodied surrogate broodstock, such as mackerel, which produces functional bluefin tuna gametes by spermatogonial transplantation. When reproductively fertile fish are used as recipients, endogenous gametogenesis outcompetes donor-derived gametogenesis, and recipient fish predominantly produce their gametes. In this study, we assessed fertility of hybrid mackerel, Scomber australasicus × S. japonicus, and its suitability as a recipient for transplantation of bluefin tuna germ cells. Hybrid mackerel were produced by artificially inseminating S. australasicus eggs with S. japonicus spermatozoa. Cellular DNA content and PCR analyses revealed that F1 offspring were diploid carrying both paternal and maternal genomes. Surprisingly, histological observations found no germ cells in hybrid mackerel gonads at 120 days post-hatch (dph), although they were present in the gonad of 30- and 60-dph hybrid mackerel. The frequency of germ cell-less fish was 100% at 120-dph, 63.1% at 1-year-old, and 81.8% at 2-year-old. We also confirmed a lack of expression of germ cell marker (DEAD-box helicase 4, ddx4) in the germ cell-less gonads of hybrid mackerel. By contrast, expression of Sertoli cell marker (gonadal soma-derived growth factor, gsdf) and of Leydig cell marker (steroid 11-beta-hydroxlase, cyp11b1) were clearly detected in hybrid mackerel gonads. Together these results showed that most of the hybrid gonads were germ cell-less sterile, but still possessed supporting cells and steroidogenic cells, both of which are indispensable for nursing donor-derived germ cells. To determine whether hybrid gonads could attract and incorporate donor bluefin tuna germ cells, testicular cells labeled with PKH26 fluorescent dye were intraperitoneally transplanted. Fluorescence observation of hybrid recipients at 14 days post-transplantation revealed that donor cells had been incorporated into the recipient's gonads. This suggests that hybrid mackerel show significant promise for use as a recipient to produce bluefin tuna gametes.
我们的目标是建立一个小型的代亲群体，例如鲐鱼，通过精原细胞移植来产生功能正常的蓝鳍金枪鱼配子。当使用有生殖能力的鱼作为受体时，内源性配子发生会与供体来源的配子发生竞争，受体鱼主要产生自己的配子。在这项研究中，我们评估了杂交鲐鱼（Scomber australasicus × S. japonicus）的生殖力及其作为蓝鳍金枪鱼生殖细胞移植受体的适宜性。杂交鲐鱼是通过用日本鲐鱼精子人工授精 S. australasicus 卵而产生的。细胞 DNA 含量和 PCR 分析表明，F1 后代是二倍体，携带父本和母本基因组。令人惊讶的是，组织学观察发现，杂交鲐鱼的性腺在孵化后 120 天（dph）时没有生殖细胞，尽管在 30 天和 60 天的杂交鲐鱼的性腺中存在生殖细胞。在 120 天龄时，无生殖细胞鱼的频率为 100%，1 岁时为 63.1%，2 岁时为 81.8%。我们还证实，在杂交鲐鱼无生殖细胞的性腺中，没有检测到生殖细胞标记物（DEAD -box 解旋酶 4，ddx4）的表达。相比之下，在杂交鲐鱼的性腺中，明显检测到了支持细胞标记物（性腺体衍生生长因子，gsdf）和间质细胞标记物（甾体 11-β-羟化酶，cyp11b1）的表达。这些结果表明，大多数杂交鱼的性腺是无生殖细胞的不育的，但仍具有支持细胞和类固醇生成细胞，这两者对于护理供体来源的生殖细胞都是必不可少的。为了确定杂交鱼的性腺是否能够吸引和纳入供体蓝鳍金枪鱼的生殖细胞，将用 PKH26 荧光染料标记的睾丸细胞进行了腹膜内移植。在移植后 14 天对杂交受体的荧光观察表明，供体细胞已被纳入受体的性腺中。这表明，杂交鲐鱼在作为受体产生蓝鳍金枪鱼配子方面具有很大的潜力。
