Advantages, Factors, Obstacles, Potential Solutions, and Recent Advances of Fish Germ Cell Transplantation for Aquaculture-A Practical Review.

Germ cell transplantation technology enables surrogate offspring production in fish. This technology has been expected to mitigate reproductive barriers, such as long generation time, limited fecundity, and complex broodstock management, enhancing seed production and productivity in aquaculture. Many studies of germ cell transplantation in various fish species have been reported over a few decades. So far, surrogate offspring production has been achieved in many commercial species. In addition, the knowledge of fish germ cell biology and the related technologies that can enhance transplantation efficiency and productivity has been developed. Nevertheless, the commercial application of this technology still seems to lag behind, indicating that the established models are neither beneficial nor cost-effective enough to attract potential commercial users of this technology. Furthermore, there are existing bottlenecks in practical aspects such as impractical shortening of generation time, shortage of donor cells with limited resources, low efficiency, and unsuccessful surrogate offspring production in some fish species. These obstacles need to be overcome through further technology developments. Thus, we thoroughly reviewed the studies on fish germ cell transplantation reported to date, focusing on the practicality, and proposed potential solutions and future perspectives.

Enhanced Enrichment of Medaka Ovarian Germline Stem Cells by a Combination of Density Gradient Centrifugation and Differential Plating.

Fish ovarian germline stem cells (OGSCs) have great potential in various biological fields due to their ability to generate large numbers of mature eggs. Therefore, selective enrichment of OGSCs is a prerequisite for successful applications. To determine the optimal conditions for the enrichment of OGSCs from Japanese medaka (Oryzias latipes), we evaluated the effects of Percoll density gradient centrifugation (PDGC), differential plating (DP), and a combination of both methods. Based on cell morphology and gene expression of germ cell-specific Vasa and OGSC-specific Nanos2, we demonstrated that of seven density fractions obtained following PDGC, the 30-35% density fraction contained the highest proportion of OGSCs, and that Matrigel was the most effective biomolecule for the enrichment of Oryzias latipes OGSCs by DP in comparison to laminin, fibronectin, gelatin, and poly-l-lysine. Furthermore, we confirmed that PDGC and DP in combination significantly enhanced the efficiency of OGSC enrichment. The enriched cells were able to localize in the gonadal region at a higher efficiency compared to non-enriched ovarian cells when transplanted into the developing larvae. Our approach provides an efficient way to enrich OGSCs without using OGSC-specific surface markers or transgenic strains expressing OGSC-specific reporter proteins.

Enhanced Adhesion of Fish Ovarian Germline Stem Cells on Solid Surfaces by Mussel-Inspired Polymer Coating.

Development of advanced cell culture methods has gained increasing attention because it allows for efficient genetic engineering and precise regulation of animal reproduction on a cellular basis. Numerous studies have attempted to develop an advanced cell culture method. Previous studies have altered cell culture media and pretreated culture plates with functional molecules. Among them, a mussel-inspired polymer coating has been extensively utilized owing to its wide applicability. For instance, adhesion of human embryonic stem cells and neuronal cells on solid surfaces has been improved. Despite the excellent capability of the mussel-inspired polymer coating, most studies have primarily focused on mammalian cells. However, the efficacy of these coatings on the adhesion of other cell lines is yet unclear. This study aimed to assess the potential of the mussel-inspired polymer coating in the regulation of the adhesion of fish ovarian germline stem cells on solid surfaces. Solid surfaces were coated by polydopamine and poly-L-lysine, and the effect of the coatings on cellular behaviors was investigated.

Non-lethal method for the preparation of metaphase spreads using cultured mantle tissue from live adult abalone.

Metaphase spread preparation in adult abalone has not been successful, which has restricted the applications of karyotyping-based technologies. Here, we present a non-lethal method to enable preparation of metaphase spreads from live adult abalone using a tissue culture method. Mantle tissue fragments from live adult abalone were cultured in vitro and the cultured cells were used for metaphase spread preparation. To retrieve a sufficient number of proliferating cells required for metaphase spread preparation, at least 14 days of culture was required, and culturing the marginal zone of mantle was more optimal than culturing other areas. Additionally, it was shown that simple medium consisting of basal medium, fetal bovine serum and antibiotics could stimulate cellular proliferation followed by metaphase spread preparation.

Identification of embryonic stem cell activities in an embryonic cell line derived from marine medaka (Oryzias dancena).

This study was conducted to identify embryonic stem cell (ESC) activities of a long-term cultured embryonic cell line previously derived from blastula-stage Oryzias dancena embryos. Five sub-cell lines were established from the embryonic cell line via clonal expansion of single cells. ESC activities, including clonogenicity, alkaline phosphatase (AP) activity, and differentiation capacity, were examined in the five sub-cell lines. We observed both clonogenicity and AP activity in all five sub-cell lines, but the proportion of cells that exhibited both properties was significantly different among them. Even though we detected different formation rates and sizes of embryoid body (EB) among these cells, all lines were stably able to form EBs and further induction for differentiation showed their capability to differentiate into other cell types in a spontaneous manner. From this study, we determined that the embryonic cell lines examined possessed heterogeneous ESC activities and can be utilized as a marine model system for fish ESC-based research.