Abstract
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.
Highlights
Unlike the adult mammalian species in which the existence of ovarian germline stem cells (OGSCs) is still controversial [1], adult female fish unquestionably have OGSCs within their ovaries, which was clearly confirmed by a study using Japanese medaka (Oryzias latipes) [2]
Fish OGSCs are responsible for the production of a large number of mature eggs throughout the life of fish and have the potential to be used in various fields, including developmental biology, transgenic animal production, reproduction, and species conservation, as if primordial germ cells (PGCs) and spermatogonial stem cells (SSCs) have done [3]
To determine the optimal conditions for OGSC enrichment, the dissociated ovarian cells were separated by Percoll density gradient centrifugation (PDGC), and the cells harvested from the density fractions were compared in terms of morphology and gene expression
Summary
Unlike the adult mammalian species in which the existence of ovarian germline stem cells (OGSCs) is still controversial [1], adult female fish unquestionably have OGSCs within their ovaries, which was clearly confirmed by a study using Japanese medaka (Oryzias latipes) [2]. Several studies have demonstrated the feasibility of the use of fish OGSCs as a biological resource by producing live offspring through the transplantation of crude or enriched ovarian germ cells [4,5,6] or cultured OGSCs [7] into developing larvae. The exact proportion of OGSCs within the ovaries of adult medaka has not been verified yet, it is expected to be small since only 1–5 OGSCs are present in a germinal cradle and the distribution of germinal cradles is restricted to the epithelial cell layer of the ovary [2]. The isolation of such a small number of pure OGSCs from the ovary is a key issue that remains to be addressed for the use of these cells for various applications
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
