biomolecules Review Spermatogonial Stem Cells in Fish: Characterization, Isolation, Enrichment, and Recent Advances of In Vitro Culture Systems Xuan Xie 1,* , Rafael Nóbrega 2 and Martin Pšeniˇcka 1 1 Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, University of South Bohemia in Ceske Budejovice, Zátiší 728/II, 389 25 Vodˇnany, Czech Republic; [email protected] 2 Reproductive and Molecular Biology Group, Department of Morphology, Institute of Biosciences, São Paulo State University, Botucatu, SP 18618-970, Brazil; [email protected] * Correspondence: [email protected]; Tel.: +420-606-286-138 Received: 9 March 2020; Accepted: 14 April 2020; Published: 22 April 2020 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. Keywords: spermatogonial stem cell (SSC); fish; spermatogenesis; florescence-activated cell sorting (FACS); magnetic-activated cell sorting (MACS); germ cell culture 1. Overview of Fish Germ Cell Biology Spermatogenesis is a complex and orderly developmental process in which a single diploid spermatogonial stem cell (SSC) proliferates and differentiates to form mature spermatozoa. Spermatogenesis depends on the activity of the SSC, which can both self-renew to produce more stem cells or differentiate into daughter cells committed to spermatogenesis [1–5]. The proper balance between SSC self-renewal and differentiation is essential to assure the continuous homeostasis of spermatogenesis. The decision as to a SSC’s self-renewal or differentiation is mediated by cell–cell communication, and in vitro germ cell culture provides a novel platform with which to investigate the regulatory network that determines cell fate. Furthermore, germ cell culture can be combined with gene editing techniques such as clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated(Cas) for germ-line transmission, cell transplantation, nuclear transfer, and in vitro spermatogenesis [6–9]. In the following section, we will review fish spermatogonial cell morphology, distribution, identification, and niche, and the endocrine and paracrine regulation of Biomolecules 2020, 10, 644; doi:10.3390/biom10040644 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, x 2 of 31 Biomolecules 2020, 10, 644 2 of 31 and in vitro spermatogenesis [6–9]. In the following section, we will review fish spermatogonial cell morphology, distribution, identification, and niche, and the endocrine and paracrine regulation of spermatogenesis. In t thehe subsequent section, we will summarize the available protocols and advances in enrichingenriching undi undifferentiatedfferentiated spermatogonia spermatogonia according according to their to unique their physiochemical unique physiochemical and biochemical and properties.biochemical Finally, properties. we will Finally, review we developments will review developments of traditional ofin traditional vitro germ in cell vitro culture germ conditions cell culture to maintainconditions proliferation to maintain andproliferation survival of and spermatogonia survival of spermatogonia in selected fish in species, selected as fish well species, as organ as culturewell as andorgan three-dimensional culture and three models-dimensional for in vitromodels investigation for in vitro of investigation fish spermatogenesis. of fish spermatogenesis. 1.1. Spermatogenesis—an Overview 1.1. Spermatogenesis—an Overview Spermatogenesis is a continuous and dynamic developmental process which can be divided into Spermatogenesis is a continuous and dynamic developmental process which can be divided into three phases: the mitotic, or spermatogonial, phase with the generation of spermatogonia; the meiotic three phases: the mitotic, or spermatogonial, phase with the generation of spermatogonia; the meiotic phase with primary and secondary spermatocytes; and the spermiogenic phase with the haploid phase with primary and secondary spermatocytes; and the spermiogenic phase with the haploid spermatids emerging from meiosis and differentiating into motile, flagellated, haploid spermatozoa. spermatids emerging from meiosis and differentiating into motile, flagellated, haploid spermatozoa. In the spermatogonial phase, the primary increase in germ cell numbers occurs during successive In the spermatogonial phase, the primary increase in germ cell numbers occurs during roundssuccessive of mitoticrounds duplicationof mitotic duplication of the spermatogonia. of the spermatogonia The number. T ofhe spermatogonialnumber of spermatogonial generations, andgenerations hence the, and number hence of the mitotic number divisions of mitotic before divisions differentiation before differentiation into spermatocytes, into spermatocytes varies among,, butvaries not among within,, but species. not within There, species. can be as The fewre ascan two be as (humans) few as two and (humans) as many and as 14 as (guppy) many as generations, 14 (guppy) but,generations most commonly,, but, most five commonly to eight generations, five to eight are generations reported [10 ].are In reported this phase, [10] in. allIn invertebratesthis phase, in and all vertebrates,invertebrates at and the vertebrates end of mitosis,, at the incomplete end of mitosis cytokinesis, incomplete occurs, cytokinesis and the two occurs, newly-generated and the two spermatogonianewly-generated remain spermatogonia connected remain by a cytoplasmicconnected by bridge, a cytop insteadlasmic ofbridge, forming instead individual of forming cells (Figureindividual1A,B). cells However, (Figure 1A,B). the cytoplasmic However, the bridge cytoplasmic is not present bridge inis not the present descendants in the of descendants an SSC that of entersan SSC a that self-renewal enters a self pathway-renewal in pathway which two in single,which two isolated single, daughter isolated cells daughter are generated. cells are generate Therefore,d. cytoplasmicTherefore, cytoplas bridgesmic are consideredbridges are aconsidered sign of SSC a di signfferentiation of SSC differentiation and are present and during are present all subsequent during germall subsequent cell divisions germ (Figure cell divisions1B). All (Figure di fferentiated 1B). All descendantsdifferentiated of descendants an SSC form of clones an SSC connected form clone bys cytoplasmicconnected by bridges cytoplasmic through bridges which through their developmental which their developmental steps are synchronized steps are synchronized (Figure1A). (Figure These bridges1A). These are bridges cleaved are when cleaved spermatogenesis when spermatogenesis is complete, is and complete germ cells, and leave germ the cells germinal leave the epithelium germinal asepithelium spermatozoa as spermatozoa [11]. [11]. Figure 1.1. MitoticMitotic/spermatogonial/spermatogonial phase. ( A) Mitosis produces spermatogonium clones. In rodents, type A A single single spermatogonia spermatogonia (As) (As) harbor harbor the the spermatogonial spermatogonial stem stem cell population, cell population, which which can either can eitherself-renew self-renew or generate or generate two interconnected two interconnected cells named cells namedtype A- typepaired A-paired spermatogonia spermatogonia (Apr). The (Apr). A- Thepaired A-paired spermatogonia spermatogonia are interconnected are interconnected by cytoplasmic by cytoplasmic bridge bridge (CB) (CB) as as a aconsequence consequence of of incomplete incomplete cytokinesis duringduring cellcell division.division. Amplifying Amplifying divisions divisions beyond beyond the A A-paired-paired also do not complete cytokinesis and continue to generate longer syncytial syncytial chains, termed termed A A-aligned-aligned spermatogonia spermatogonia (Aal). (Aal). ((B).). AnAn electron electron micrograph micrograph showing showing a cytoplasmic a cytoplasmic bridge bridge (arrow) (arrow) connecting connecting daughter daughter cells resulting cells fromresulting spermatogonium from spermatogonium division. The division. mitochondria The m (M),itochondria nucleus (N), (M), smooth nucleus endoplasmic (N), smooth reticulum endoplasmic (SER), Biomolecules