Isolation, Identification, Culture, Cryopreservation, Genetic Transformation and Transplantation of Catfish Germline Stem Cells
Type of Degreedissertation
Fisheries and Allied Aquacultures
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The hybrid of channel catfish (Ictalurus punctatus) ♀ × blue catfish (Ictalurus furcatus) ♂ (C × B) is the best catfish for pond culture. The current technology, artificial fertilization and handstripping is labor intensive, time consuming, and requires sacrifice of mature 5 ~ 6 year old blue catfish males. Xenogenic catfish produced by transplanting blue catfish stem cell into the gonads of triploid channel catfish could reduce the generation interval to produce blue catfish and might result in channel catfish males that are able to produce blue catfish sperm, and increase the efficiency of C × B hybrid catfish embryo production. Gonadal cell size and type of 90 ~ 100 day - old fry (TL: 5 ~ 6 cm), two - year old juvenile (TL: 25 ~ 30cm) and mature adults (TL: 65 ~ 70 cm) blue catfish was examined. Gonads of all ages of fish contained oogonia (12 ~ 15 μm diameter, distinct nucleus 7 ~ 8 μm) and spermatogonia (12 ~ 15 μm, distinct nucleus 6 ~ 7.5 μm). Male juvenile blue catfish, which had suitable size and higher percentage of germline stem cells, were used for stem cell isolation for further study. After testes were dissected from the peritoneal cavity, germline stem cells were isolated with discontinuous density gradient centrifugation to enrich the percentage of spermatogonia. Four distinct cell bands were generated after centrifugation. It was estimated that 55% of the total cells in top - first bands, cell band I, were type A spermatogonia (diameter 12 ~ 15 µm) and type B spermatogonia (diameter 10 ~ 11 µm), 35% were spermatocytes (diameter 5 ~ 9 µm), 10% were spermatids (diameter < 1 µm). In the second band - cell bands II, most of the cells were spermatocytes (60%, diameter 5 ~ 9 µm), some were type B spermatogonia (30%), a few were spermatids (10%) and cell clusters could also be observed. The third band ~ cell band III contained most of the spermatids (95%) and very few secondary spermatocytes (5%, diameter 5 ~ 8 µm). Red blood cells (95%) and a few spermatids (5%) were the predominant cells in the fourth cell band - cell band IV. Different bands generated from Percoll density gradient centrifugation were used for germ cell marker identification studies. Expression of seventeen genes (pfkfb4, urod, Oct4, Plzf, sycp3, SOX2, Integrin6, neurogenin3, ID - 4, integrinV, Thy1, GFRα, CDH1, Smad, Pum2 and Prdm14 and Kit) from cells of different bands were analyzed by qRT - PCR in both channel and blue catfish. pfkfb4, urod, Plzf, Integrin6, ID - 4, integrinV, Thy1 and CDH1 genes, which showed the same expression change pattern in different types of testicular germ cells of both channel and blue catfish, were identified as spermatogonia marker. SOX2 gene, which was up - regulated in spermatocytes and even higher up - regulated in spermatids, was identified as spermatids marker. Blue catfish testicular and ovarian germ cell in vitro cultures were studied. When testicular tissues were used as the initial culture materials, testicular germ cells migrated outward the edges of tissues and formed monolayers during the first two - week culture. Cultured testicular germ cells were fibroblast - like, growing fast after the third passage. For ovaries, ovarian germ cell isolated after trypsin digestion was used as the initial culture material, and the cells attached to the plate during the first week of culture. Cells from ovaries were also fibroblast - like in culture. Electroporation was used for both channel and blue catfish germ cell genetic transformation. Both blue catfish ovarian germ cells and channel catfish testicular germ cells expressed green florescence protein (GFP) for a long time period after cells were transformed by the expression construct FRMwg driven by the carp β - actin promoter. Two transplantation techniques, surgery and catheterization, were studied for transplanting spermatogonian stem cell enrichment and testicular germ cells into triploid channel catfish gonads. Ten months after the surgery transplantation, eight fish were recaptured; all three recipient fish which were transplanted by surgery were proved containing blue catfish cells; four out of five recipient fish which were transplanted by catheterization contained blue catfish cells. Both surgery and catheterization were efficiency transplantation method and had a very high transplantation ratio.