Intraperitoneal Germ Cell Transplantation in the Nile Tilapia Oreochromis niloticus

Abstract

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.

Identifying the germ cells during embryogenesis and gametogenesis by germ‐line gene vasa in an anadromous fish, American shad Alosa sapidissima

“…In D. rerio, the PGCs were migrating before 7 days post fertilization (dpf), starting proliferation at c. 12 dpf and their meiosis are initiated at c. 14 dpf (Tzung et al, 2015). In tilapia Oreochromis niloticus (L. 1758), PGCs had migrated to settle in the genital ridge at 6-8 dpf and gonadal somatic cells were found covering the PGCs at 11 dpf (Farlora et al, 2014). In yellow fin tuna Thunnus albacares (Bonnaterre 1788), the PGCs at 3 dpf came into contacting with stromal cells (Kobayashi et al, 2015).…”

Application of dead end‐knockout zebrafish as recipients of germ cell transplantation

“…For example, when spermatogonial stem cells from rainbow trout ( Oncorhynchus mykiss ) were transplanted into embryonic male and female recipient masu salmon ( Oncorhynchus masou ), the recipients generated donor‐derived, functional sperm and eggs, respectively (Okutsu et al, , ). The spermatogonial transplantation technique has also been expanded to other species, such as Nile tilapia (Farlora et al, ), medaka (Seki et al, ), and several marine fish species, including members of the Sciaenidae (Higuchi et al, ; Takeuchi, Higuchi, Yatabe, Miwa, & Yoshizaki, ; Yoshikawa et al, ), Scombridae (Yazawa et al, ), as well as Carangidae (Bar et al, ; Morita et al, , ).…”

Successful production of functional Y eggs derived from spermatogonia transplanted into female recipients and subsequent production of YY supermales in rainbow trout, Oncorhynchus mykiss

“…Theoretically, the testicular germ-cell transplantation-mediated production of YY supermales could be applied to many other teleosts. In fact, the technique has been demonstrated to be applicable to other species, including Japanese charr, Salvelinus leucomaenis (Salmonidae; Okutsu et al, 2008), tilapia (Cichlidae; Farlora et al, 2014), Nibe croaker, Nibea mitsukurii (Sciaenidae; Takeuchi et al, 2009;Higuchi et al, 2011), yellowtail, Seriola quinqueradiata (Carangidae; Morita et al, 2012), and grass puffer fish, Takifugu niphobles (Tetraodontidae; Hamasaki, unpublished). Thus, the technology developed in the present study can be applied to a wide range of teleosts as long as they possess an XY sexdetermining system.…”