It is widely accepted that spermatogonial stem cells (SSCs) are consistently present in the animal testes and support spermatogenesis. However, whether the mature ovaries of all animals have GSCs or oogonial stem cells (OSCs) remains to be determined. In invertebrate ovaries, the existence of GSCs has been reported by many articles, and Drosophila ovarian GSCs have been a hot topic in scientific research (Cuevas and Spradling, 1998). In vertebrates, GSCs have been identified and labeled by nanos2 in the mature ovaries of medakas by lineage-tracing experiments (Nakamura et al., 2010). Recent studies provide evidence of GSCs or OSCs in zebrafish mature ovaries (Draper et al., 2007; Wong et al., 2011; Beer and Draper, 2013; Cao et al., 2019). For example, vasa-labeled germ cells isolated from adult ovaries and transplanted into sterile host larvae led to the production of zebrafish germline chimeras and restoration of fertility, indicating the presence of mitotically active adult OSCs in zebrafish mature ovaries (Wong et al., 2011). Zebrafish nanos3 can maintain GSCs and expression of the RNA binding gene nanos2, and thus, the female nanos3 homozygous mutants develop into sterile male fish due to loss of GSCs (Draper et al., 2007; Beer and Draper, 2013). Moreover, we knocked out zebrafish nanos2, a marker of GSCs, and found that all of GSCs were absent at 32 dpf, and the female nanos2 mutants developed into sterile male fish at 75 dpf (Figure 1G) (Cao et al., 2019). These results suggest that similar to medaka, GSCs is specifically labeled and regulated by nanos2 in zebrafish.

GSCs stem from primordial germ cells (PGC) and differentiate into oocytes through mitosis and meiosis, which is known as oogenesis. Many genes were reported to be involved in the regulation of oogenesis in zebrafish. vasa express specially in germ cells and is required for germ cell differentiation and GSC maintenance in zebrafish (Hartung et al., 2014). ziwi and zili are also expressed in germ cells and their mutations lead to failure of germ cell maintenance and defect of mature oocytes production in zebrafish (Houwing et al., 2007, 2008). Meiosis defects and loss of germ cells were observed and tdrd12 deficient fish (Dai et al., 2017). ca15b is expressed in PGC and oocytes, and plays an important role in PGC development and oogenesis (Wang et al., 2013). Furthermore, nanos2 and nanos3 are required for maintaining oocytes production and GSCs (Draper et al., 2007; Beer and Draper, 2013; Cao et al., 2019).

Many evidences suggest that the cellular sources underlying germ cell regeneration is the proliferation and differentiation of GSCs, as illustrated in Table 1. In the NTR-MTZ system for germ cell ablation, since zpc only labeled the oocytes, GSCs in the adult Tg(zpc:g4vp16/uas:nfsb-mcherry) transgenic background females were not ablated by MTZ. Therefore, their ovaries were able to recovered completely to regain their reproductive function due to the proliferation of the resident GSCs (Figure 1A) (White et al., 2011). ziwi can labeled all of germ cells but GSCs in the Tg(ziwi:CFP-NTR) transgenic females were not completely ablated by MTZ. Thus, these females reverted into the fertile males (Figure 1B), suggesting that zebrafish ovarian GSCs are bipotential (Dranow et al., 2013, 2016). However, although vasa like ziwi can label all of germ cells, we ablated all of GSCs in the Tg(vasa:dendra2-NTR) by MTZ treatment. These females failed to regain their reproductive functions, indicating that zebrafish ovaries have no regeneration without GSCs (Figure 1C) (Cao et al., 2019). Moreover, our data suggest that GSCs can regenerate the most of removed ovarian tissues in zebrafish after ovariectomy as a result of remaining GSCs proliferation (Figure 1E) (Cao et al., 2019). Other fish ovaries such as Chinese grass carp and Rainbow trout can also regenerate after ovariectomy mainly due to the proliferation of remaining ovarian tissue (Underwood et al., 1986; Kersten et al., 2001). Similarly, newly regenerated germ cells in zebrafish and medaka treated by busulfan originate from the proliferation and differentiation of GSCs (Nakamura et al., 2010; Nóbrega et al., 2010). Furthermore, some reports show that all embryos injected with dead-end MO lose primordial germ cells (PGCs) and develop into sterile males, suggesting that PGCs is essential for fertility in zebrafish (Tzung et al., 2015). Other arguments about the sources of germ cells such as a study reported that regeneration of male germline stem cells occurs in the temperature-sensitive gene stat92E mutants of Drosophila due to dedifferentiation of spermatogonial cells. This indicates that germ cells have dedifferentiation phenomena under some certain microenvironment (Brawley and Matunis, 2004). Moreover, Transplantation of testicular or ovarian germ cell aggregates leads to regeneration of spermatogenesis or oogenesis and production of functional sperm or egg in zebrafish (Kawasaki et al., 2010). Thus, germ cell regeneration in vivo mainly depends on the proliferation and differentiation of GSCs.

Many germ cell-intrinsic factors that regulate the development of GSCs are evolutionarily conserved, especially the functionally conserved nanos family genes which play an important role in maintaining GSCs (Forbes and Lehmann, 1998; Sada et al., 2009). The nanos homologous gene is a RNA-binding protein that can control translation (Kadyrova et al., 2007). In planarians, nanos is expressed specifically in germ cells and essential for germ cell specification and regeneration (Newmark et al., 2008). In Drosophila, nanos is essential for maintaining ovarian GSCs and its fertility (Forbes and Lehmann, 1998). The nanos family genes in vertebrates include three homologous genes, namely, nanos1, nanos2, and nanos3. nanos1 is mainly expressed in the nervous system, while the other two genes nanos2 and nanos3 are both expressed in germ cells (Tsuda et al., 2003; Beer and Draper, 2013). nanos3 is expressed in migrating PGCs and after birth, is found only in the GSCs of adult mouse testis, and its’ targeted disruption leads to the complete loss of germ cells in both sexes (Tsuda et al., 2003). Human NANOS3 mutation results in premature ovarian insufficiency (Wu et al., 2013). However, zebrafish homologous gene nanos3 is expressed in oocytes, but nanos3 mutation leads to loss of ovarian GSCs and sex-reversal, suggesting that nanos3 is essential for the maintenance of ovarian GSCs in zebrafish (Draper et al., 2007; Beer and Draper, 2013). nanos2 is expressed in the spermatogonia at the earliest stage of the mouse testis and is required for maintaining SSCs. nanos2 maintains its long-term stem cell state by inhibiting the specialization of GSC (Sada et al., 2009). However, nanos2 is expressed in early GSCs of zebrafish ovary and testis (Beer and Draper, 2013; Cao et al., 2019). Like nanos3, nanos2 mutations lead to loss of GSCs in zebrafish (Figure 1G), suggesting partially redundant roles of nanos2 and nanos3 in the maintenance of female GSCs (Cao et al., 2019). Furthermore, nanos2 mutation led to the defect of ovarian regeneration (Figure 1H) and continuous overexpression of nanos2 rescued defective ovary regeneration (Cao et al., 2019). Thus, we speculate that ovarian regeneration is also defective in nanos3 mutants, but it needs further verification.

Many reports show that Wnt signaling participates in mammalian gonadal development. Wnt4 homozygous mutant XX mouses have male characteristics, and the loss of oocyte starts to occur, indicating that Wnt4 is necessary for ovary development (Vainio et al., 1999). Moreover, Wnt4 can upregulate the gene nuclear receptor subfamily 0 group B member 1 (NR0B1) and restrict expression of the SRY mammalian male-determining gene (Jordan et al., 2001). Wnt/β-catenin is capable of restricting expression of NR5a1 and Sox9 and the canonical Wnt signaling pathway ligand, Rspo1, is a determinant of mammalian ovary development (Lau and Li, 2009). β-catenin can also regulate expression of gonadal aromatase Cyp19a1a through its interaction with NR5a1 in rat granulosa cells (Parakh et al., 2006). Recent evidences shows that Wnt signaling plays an important role in teleost reproductive development. The specification of zebrafish gonad is regulated by Wnt/β-catenin pathway. Wnt signaling inhibition causes the increase in the proportion of the male and cyp19a1a expressed in granulosa cells may be the target gene of Wnt signaling. These results show that Wnt signaling is very important for ovarian development in zebrafish (Sreenivasan et al., 2014). Additionally, Wnt signaling is also reported to be involved in germ cell regeneration. Our study shows that Wnt signaling is activated after ovarian amputation and inhibition of Wnt signaling impairs ovarian regeneration by reducing proliferation of GSCs (Figure 1E) (Cao et al., 2019).

Many factors were identified to be germ cell-specific genes and involved in germ cell specification and maintainace in zebrafish. These molecular factors are also primarily conserved and may contribute to germ cell regeneration. vasa and dnd are commonly used as germ cell specific markers. vasa is required for germ cell differentiation and GSC maintenance (Hartung et al., 2014). Loss of dnd lead to failure of PGC migration and survival (Slanchev et al., 2005). piwil1 and piwil2, the Piwi homologs in zebrafish, are expressed in germ cells. piwil1, namely ziwi, is essential for germ cell maintenance while piwil2, namely zili, is required for germ cell differentiation and meiosis (Houwing et al., 2007, 2008). azoospermia-like (dazl) is expressed in germ cells and its’ mutation leads to failure of GSC establishment and fertility (Bertho et al., 2021). Based on the fuctions of these molecular factors, we speculate that they may contribute to germ cell regeneration, but it needs further verification.