Spermatogonial Stem Cells in Fish: Characterization, Isolation, Enrichment, and Recent Advances of In Vitro Culture Systems

Abstract

Spermatogenesis is a continuous and dynamic developmental process, in which a single diploid spermatogonial stem cell (SSC) proliferates and differentiates to form a mature spermatozoon. Herein, we summarize the accumulated knowledge of SSCs and their distribution in the testes of teleosts. We also reviewed the primary endocrine and paracrine influence on spermatogonium self-renewal vs. differentiation in fish. To provide insight into techniques and research related to SSCs, we review available protocols and advances in enriching undifferentiated spermatogonia based on their unique physiochemical and biochemical properties, such as size, density, and differential expression of specific surface markers. We summarize in vitro germ cell culture conditions developed to maintain proliferation and survival of spermatogonia in selected fish species. In traditional culture systems, sera and feeder cells were considered to be essential for SSC self-renewal, in contrast to recently developed systems with well-defined media and growth factors to induce either SSC self-renewal or differentiation in long-term cultures. The establishment of a germ cell culture contributes to efficient SSC propagation in rare, endangered, or commercially cultured fish species for use in biotechnological manipulation, such as cryopreservation and transplantation. Finally, we discuss organ culture and three-dimensional models for in vitro investigation of fish spermatogenesis.

Surrogate broodstock to enhance biotechnology research and applications in aquaculture

“…Although PGCs can also be cryopreserved, they require a lengthy procedure and the low number of cells is a limitation ( Robles et al, 2017 ). Although labelling of gonial cells would be useful for isolation and monitoring of the transplanted germ cells, gonial cells can be isolated without labelling procedure from testes or ovaries using their physiochemical and biochemical properties such as size, density and specific receptors ( Xie et al, 2020 ). The simplest way to enrich for gonial cells is to enzymatically dissociate gonads and filtrate the suspension using a filter with a pore size bigger than somatic cells but smaller than gonial cells.…”

“…Alternative methods to enrich gonial cells include cell sorting based on their light-scattering characteristics ( Ichida et al, 2017 ; Kise et al, 2012 ), or the use of gonial cell specific antibodies with flow cytometry, FACS or magnetic-activated cell sorting (MACS) ( Hayashi et al, 2019 ; Ichida et al, 2020 , Ichida et al, 2019 , Ichida et al, 2019 ). However, it should be noted that these processes may damage gonial cells or alter their physiological features ( Xie et al, 2020 ).…”

“…Spermatogonial cells are favoured due to their higher abundance, and they can result in either eggs or sperm according to the phenotypic sex of the surrogate recipients. Gonial cell culture requires appropriate sera, feeder cells and growth factors to suppress growth of gonadal somatic cells, promote proliferation of GSCs, and maintain stemness and transplantability of GSCs ( Xie et al, 2020 ). Further studies in other target aquaculture species are needed to determine optimal culture conditions.…”

Isolation and Characterization of Highly Pure Type A Spermatogonia From Sterlet (Acipenser ruthenus) Using Flow-Cytometric Cell Sorting

“…A und retain the ability of self-renewal, differentiation, and transformation into different germ cell types (biopotency, De Rooij and Russell, 2000;Schulz et al, 2010). A und are single cells and the largest germ cells in the fish testis, with a large nucleus (Xie et al, 2020). To obtain efficient germ cell in vitro culture, cryopreservation and transplantation, a highly purified population of A und cells is essential.…”

“…Mammalian SSCs have been sorted by FACS based on SSCs surface markers, such as α6-integrin, CD9, SSEA-4, GFRA1, THY1, and MCAM (Kokkinaki et al, 2011;Kanatsu-Shinohara et al, 2012;Valli et al, 2014). However, to date, fish SSCs surface markers, sgsa-1, as well as cytoplasm markers, such as plzf, gfrα1, and nanos2, have been identified in only a few species (Xie et al, 2020). Some commercially important or endangered species lack transgenic strains or specific molecular markers to label SSCs, presenting limitations in detecting and isolating SSCs.…”