Farlora Rodolfo, Hattori-Ihara Shoko, Takeuchi Yukata, Hayashi Makoto, Octavera Anna, Yoshizaki Goro
Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR), University of Concepcion, Concepción, Chile.
Mar Biotechnol (NY). 2014 Jun;16(3):309-20. doi: 10.1007/s10126-013-9551-y. Epub 2013 Oct 6.
Germ cell transplantation offers promising applications in finfish aquaculture and the preservation of endangered species. Here, we describe an intraperitoneal spermatogonia transplantation procedure in the Nile tilapia Oreochromis niloticus. Through histological analysis of early gonad development, we first determined the best suitable stage at which exogenous germ cells should be transplanted into the recipients. For the transplantation procedure, donor testes from a transgenic Nile tilapia strain carrying the medaka β-actin/enhanced green fluorescent protein (EGFP) gene were subjected to enzymatic dissociation. These testicular cells were then stained with PKH26 and microinjected into the peritoneal cavity of the recipient fish. To confirm colonization of the donor-derived germ cells, the recipient gonads were examined by fluorescent and confocal microscopy. PKH26-labeled cells exhibiting typical spermatogonial morphology were incorporated into the recipient gonads and were not rejected within 22 days posttransplantation. Long-term survival of transgenic donor-derived germ cells was then verified in the gonads of 5-month-old recipients and in the milt and vitelogenic oocytes of 1-year-old recipients, by means of PCR using EGFP-specific primers. EGFP-positive milt from adult male recipients was used to fertilize non-transgenic oocytes and produced transgenic offspring expressing the donor-derived phenotype. These results imply that long-term survival, proliferation, and differentiation of the donor-derived spermatogonia into vitelogenic oocytes and functional spermatozoa are all possible. Upon further improvements in the transplantation efficiency, this intraperitoneal transplantation system could become a valuable tool in the conservation of genetic resources for cichlid species.
生殖细胞移植在硬骨鱼类养殖和濒危物种保护方面有着广阔的应用前景。在此，我们描述了尼罗罗非鱼（Oreochromis niloticus）腹腔精原细胞移植的过程。通过对早期性腺发育的组织学分析，我们首先确定了将外源生殖细胞移植到受体中的最佳合适阶段。在移植过程中，取自携带青鳉β - 肌动蛋白/增强型绿色荧光蛋白（EGFP）基因的转基因尼罗罗非鱼品系的供体睾丸进行酶解。然后将这些睾丸细胞用PKH26染色，并显微注射到受体鱼的腹腔中。为了确认供体来源的生殖细胞的定植情况，通过荧光显微镜和共聚焦显微镜检查受体性腺。表现出典型精原细胞形态的PKH26标记细胞被整合到受体性腺中，并且在移植后22天内未被排斥。然后，通过使用EGFP特异性引物的PCR，在5月龄受体的性腺以及1岁龄受体的精液和卵黄生成卵母细胞中验证了转基因供体来源生殖细胞的长期存活情况。成年雄性受体的EGFP阳性精液用于使非转基因卵母细胞受精，并产生表达供体来源表型的转基因后代。这些结果表明，供体来源的精原细胞长期存活、增殖并分化为卵黄生成卵母细胞和功能性精子都是可能的。随着移植效率的进一步提高，这种腹腔移植系统可能成为丽鱼科物种遗传资源保护的一种有价值的工具。
