PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of transbThe Royal Society PublishingPhilosophical Transactions BAboutBrowse By SubjectAlertsFree Trial
 
Philos Trans R Soc Lond B Biol Sci. 2010 May 27; 365(1546): 1663–1678.
PMCID: PMC2871929

Spermatogonial stem cell regulation and spermatogenesis

Abstract

This article will provide an updated review of spermatogonial stem cells and their role in maintaining the spermatogenic lineage. Experimental tools used to study spermatogonial stem cells (SSCs) will be described, along with research using these tools to enhance our understanding of stem cell biology and spermatogenesis. Increased knowledge about the biology of SSCs improves our capacity to manipulate these cells for practical application. The chapter concludes with a discussion of future directions for fundamental investigation and practical applications of SSCs.

Keywords: spermatogonial stem cells, spermatogenesis, fertility

1. Introduction

Spermatogonial stem cells (SSCs) are at the foundation of spermatogenesis and male fertility. Similar to other tissue-specific stem cells, SSCs are rare, representing only 0.03 per cent of all germ cells in rodent testes (Tegelenbosch & de Rooij 1993). This is because SSCs are heavily outnumbered by the differentiating spermatogonia, spermatocytes, spermatids and sperm that they produce (detailed below). SSCs are defined like all other stem cells, by their ability to balance self-renewing divisions and differentiating divisions. This balance maintains the stem cell pool and meets the proliferative demand of the testis to produce millions of sperm each day. Studies of SSCs are complicated because these cells are few in number and no unique identifying characteristics have been reported to date. We will review experimental tools used to study SSCs and summarize current knowledge about the characteristics and regulation of these adult tissue stem cells. We will focus primarily on rodent models, which have generated the majority of data about SSCs and the spermatogenic lineage.

2. Origin of the spermatogonial stem cell pool

SSCs arise from gonocytes in the postnatal testis, which arise from primordial germ cells (PGCs) during foetal development. PGCs are a transient cell population that is first observed as a small cluster of alkaline phosphatase-positive cells in the epiblast stage embryo at about 7–7.25 days post coitum (dpc). PGC specification is dependent on the expression of BMP4 and BMP8b from the extraembryonic ectoderm (Ginsburg et al. 1990; Lawson et al. 1999; Ying et al. 2001). During the formation of the allantois, the PGCs are passively swept out of the embryo before they start migrating via the hindgut to arrive at the indifferent gonad between 8.5 and 12.5 dpc in mice. PGCs replicate during the migratory phase and approximately 3000 PGCs colonize the genital ridges (Bendel-Stenzel et al. 1998). In the male gonad at about 13.5 dpc, PGCs give rise to gonocytes, which become enclosed in testicular cords formed by Sertoli precursor cells and peritubular myoid cells. Gonocyte is a general term that can be subcategorized into mitotic (M)-prospermatogonia, T1-prospermatogonia and T2-propsermatogonia (McCarrey 1993). M-prospermatogonia are located in the centre of the testicular cords, away from the basal membrane and continue proliferating until about 16.5 dpc of mouse development when they become T1-prospermatogonia and enter G0 mitotic arrest (McLaren 2003; Tohonen et al. 2003). Gonocytes resume proliferation during the first week after birth (marking their transition to T2-prospermatogonia), concomitant with migration to the seminiferous tubules basement membrane (Clermont & Perey 1957). T2-prospermatogonia that colonize the basement membrane give rise to the first round of spermatogenesis as well as establish the initial pool of SSCs that maintain spermatogenesis throughout postpubertal life (Kluin & de Rooij 1981; McCarrey 1993; Yoshida et al. 2006).

3. The spermatogenic cycle

Spermatogenic lineage development is a complex process, but occurs in an orderly manner, referred to as the spermatogenic cycle (Clermont 1972), which is divided in a species-specific number of stages or cell associations (i.e. 12 stages in the mouse (Oakberg 1956a,b) and 14 stages in the rat (Leblond & Clermont 1952a,b)). This synchronized spermatogenic development may be facilitated by incomplete cytokinesis during mitotic divisions that lead to maintenance of cytoplasmic bridges among germ cells. Proteins and messenger RNAs are exchanged via the cytoplasmic bridges and may help in coordinating the synchronized development of germ cell clones (Braun et al. 1989). Each stage is characterized by a combination of the types of spermatogonia, spermatocytes and spermatids that synchronously proceed through the spermatogenic process (figure 1). For example, the basement membrane of a stage V seminiferous tubule depicted in figure 1 is mostly filled with preleptotene primary spermatocytes (blue cells of a large clone). By stage VI, these spermatocytes migrate off the basement membrane and will be replaced by spermatogonia. Thus, stage V can be distinguished from stage VI by the presence or absence of spermatocytes on the basement membrane. The duration of each stage is precisely timed, and the complete spermatogenic cycle was determined to be around 8.6 days in the mouse (Oakberg 1956b), and 12.8 days in the rat (Hilscher et al. 1969). One complete cycle (12 stages) of the mouse seminiferous epithelium is depicted in figure 1.

Figure 1.

Mouse spermatogenic clone development by stage. The mouse spermatogenic cycle contains twelve stages (I–XII). Each stage is temporally unique, and the stages in the diagram represent the relative time each stage lasts in the mouse. Each stage ...

4. Spermatogenic lineage development

In order to understand the regulation of spermatogonial stem cells, it is important to understand them in the context of the spermatogenic lineage that they produce. Spermatogonia are primitive diploid germ cells, located on the basement membrane of the seminiferous tubules. Three types of spermatogonia were initially described based on their nuclear morphology (Roosen-Runge & Giesel 1950; Clermont & Leblond 1953; Monesi 1962). Type A spermatogonia were considered the most primitive because heterochromatin is absent from the nucleus, a general characteristic of undifferentiated cells. The nuclei of intermediate type spermatogonia contain a small amount of heterochromatin and type B spermatogonia contain a large amount of heterochromatin, indicating a more differentiated state.

Histological staining of whole-mount preparations of seminiferous tubules provided additional level of detail about spermatogonial morphometry compared with tissue sections alone and broadened the knowledge of the spermatogonial cell types in the testis. To facilitate the following discussion, figure 1 depicts one compete cycle of the mouse seminiferous epithelium and represents whole-mount perspective as well as corresponding cross-section and longitudinal section perspectives. Figure 1 traces the development of three putative clones (green, red and blue) through one cycle of the seminiferous epithelium. Based on whole-mount examination of seminiferous tubules, Huckins & Oakberg (Huckins 1971c; Oakberg 1971) reported that undifferentiated type A spermatogonia can be subdivided into Asingle (As), Apaired (Apr) and Aaligned (Aal) spermatogonia, which differ only in their topographical arrangement on the seminiferous tubule basement membrane. When an As (see green clone in figure 1, stage VII) spermatogonium divides, it produces an Apr that either (i) completes cytokinesis to produce two new As spermatogonia (self-renewing division, see green clone in figure 1, stages IX, X and XI) or (ii) remains connected by an intercellular cytoplasmic bridge and produces a chain of four Aal spermatogonia at the next division (differentiating division, see red clone in figure 1, stages IX and X). Further cell divisions lead to the formation of chains of 8, 16 and sometimes 32 Aal spermatogonia (see red clone in stages XII and I and blue clone in stage VII, figure 1). Chains of 4–16 Aal are generally considered committed to the differentiation process. Thus, the stem cell pool includes As and at least some Apr spermatogonia. Some have argued that stem cell potential may extend to larger clones (e.g. Aal4 or beyond; (Yoshida et al. 2007a; Morimoto et al. 2009)), but this is difficult to confirm experimentally. Note that while each clone can be observed in histological sections as well as in whole-mount preparations of seminiferous tubules, clone size can only be observed in the whole-mount preparations (figure 1).

As, Apr and smaller chains of four Aal spermatogonia are evenly distributed along the seminiferous epithelium (Huckins 1971a,b; Tegelenbosch & de Rooij 1993). Larger chains of Aal (8, 16 and 32) become differentiating A1 spermatogonia between stages IV and VIII of the seminiferous epithelium (there is no cell division at this transition, see blue clone in figure 1, stages VII and VIII) and these give rise to A2 spermatogonia at stage IX (see blue clone in figure 1, stage IX). Thus, in contrast to undifferentiated spermatogonia, differentiating spermatogonia (A1, A2, A3, A4, intermediate and B) divide in a synchronized manner and are found at specific stages of the seminiferous epithelium (for detailed description see Oakberg 1971). B spermatogonia give rise to primary spermatocytes that progress into meiosis. Two meiotic divisions lead to the formation of secondary spermatocytes and haploid spermatids respectively, which undergo 16 steps of morphological changes to finally become spermatozoa ready to be released from the seminiferous epithelium (Oakberg 1956a).

An alternative to the As model of SSC self-renewal described above is the A0/A1 model (Clermont & Bustos-Obregon 1968; Dym & Clermont 1970; Clermont & Hermo 1975). This model is very similar to the Adark and Apale model that has been used to describe stem cell activity in non-human primates (Clermont & Leblond 1959; Clermont & Antar 1973). Briefly, A0 spermatogonia were observed as singles or pairs of cells that were present at all stage of the seminiferous epithelium. Mitotic figures were rarely observed in these cells, so they were considered ‘reserve stem cells’ not contributing to steady-state spermatogenesis. These reserve stem cells are only activated when spermatogenesis is destroyed by toxic insult (i.e. radiation). The ‘active stem cell’ pool is comprised of A1–A4 spermatogonia. When A4 spermatogonia divide, they give rise either to new A1 spermatogonia (self-renewal) or to intermediate spermatogonia (differentiation). While there continues to be vigorous debate about the merits of the As versus the A0/A1 models, the As model is currently favoured by most investigators in the field and will be the basis for further discussion of spermatogonial self-renewal in this review.

5. Experimental tools for studying spermatogonial stem cells

As discussed above, experimental investigation of SSCs is complicated because these cells are rare and are difficult to distinguish from the differentiating progeny that they produce. Whole-mount analyses of seminiferous tubules help in distinguishing As from Apr and Aal spermatogonia, but there is continuing debate about whether the stem cell pool is restricted to As or might be expanded to include Apr and some Aal (Nakagawa et al. 2007; Yoshida et al. 2007a). Thus, the only way to definitively identify an SSC is by observing its biological capacity to produce and maintain spermatogenesis in a transplant paradigm.

6. Spermatogonial stem cell transplantation

A technique for transplanting SSCs was first described by Brinster and colleagues in 1994 (Brinster & Avarbock 1994; Brinster & Zimmermann 1994). Briefly, germ cells are isolated from the testes of donor animals and transplanted into the testicular seminiferous tubules of infertile recipients, where they produce normal colonies of spermatogenesis and functional sperm (figure 2). Infertility of recipients is because of genetic mutation (i.e. W mutant mice, (Ogawa et al. 2000)) or induced experimentally (e.g. busulphan treatment (Brinster & Zimmermann 1994)). In mice, these studies are facilitated by the availability of transgenic donors (e.g. lacZ and GFP) with germ cells that can be readily identified in the testes of non-transgenic recipients. By definition, only a stem cell can produce and maintain a colony of spermatogenesis and each colony arises from the clonogenic proliferation and differentiation of a single SSC (Dobrinski et al. 1999; Zhang et al. 2003; Kanatsu-Shinohara et al. 2006). Therefore, the SSC transplantation technique provides a quantitative functional assay to characterize stem cell activity in any donor cell population. SSC transplantation remains the gold standard method for identifying SSCs, but this approach can be technically challenging. In addition, SSC transplantation is a retrospective assay with an inherent two to three months timeframe between transplant and analysis. To accelerate investigations of SSCs, Nagano and co-workers recently suggested that the SSC culture system (described below) may provide a shorter term, in vitro assay for SSCs (Yeh et al. 2007). However, culture does not assess regenerative activity.

Figure 2.

Spermatogonial stem cell (SSC) transplant assay. The functional analysis of SSCs is a retrospective assay of spermatogenic function. In this example, cells are isolated from a lacZ donor mouse testis and digested to produce a single cell suspension. Cells ...

7. Dissecting the molecular phenotype of spermatogonial stem cells

Fluorescence-activated cell sorting (FACS), combined with SSC transplantation is a powerful tool that has enabled investigators to systematically characterize cell surface molecules of SSCs. This experimental approach is patterned after similar studies to characterize and enrich haematopoietic stem cells (Spangrude 1989; Smith et al. 1991; Osawa et al. 1996). Briefly, a heterogeneous testis cell suspension is stained with a fluorescent-conjugated antibody that recognizes a cell surface antigen. Marker+ and marker cells are fractionated by FACS and each fraction is transplanted into the seminiferous tubules of infertile recipient mice to determine the relative stem cell activity. The first application of this approach for characterizing SSCs was reported by Shinohara and co-workers, who demonstrated that SSCs specifically bind to laminin-coated plates. The laminin-binding cells were enriched for β1-integrin, making this surface molecule a candidate for enriching SSCs (Shinohara et al. 1999). Subsequent transplantation of magnetic-activated cell-sorted (MACS) and FACS-sorted testis fractions indicated that SSCs express β1-integrin and α6-integrin, but are negative for αv-integrin and the c-KIT receptor tyrosine kinase (Shinohara et al. 1999, 2000). Based on several similar studies, mouse SSCs can now be described by the cell surface phenotype, α6-Integrin (CD49f)+, β1-Integrin (CD29)+, THY-1 (CD90)+, CD9+, GFRα1+, CDH1+, αv-Integrin (CD51), c-KIT (CD117), major histocompatibility complex class I (MHC-I), CD45 (Shinohara et al. 1999, 2000; Kubota et al. 2003; Kanatsu-Shinohara et al. 2004b; Buageaw et al. 2005; Fujita et al. 2005; Hofmann et al. 2005b; Lo et al. 2005; Tokuda et al. 2007, table 1). Using combinations of positive and negative markers, it is now possible to achieve significant enrichment (100- to 200-fold) of mouse SSCs (Shinohara et al. 2000; Kubota et al. 2003). However, it should be noted that none of these markers are exclusive to SSCs and no marker or combination of markers has produced a pure population of SSCs. Also, while FACS and MACS sorting followed by transplantation are powerful tools for characterizing the cell surface phenotype of SSCs, this approach has limited utility for characterizing cytoplasmic or nuclear markers.

Table 1.
Germ cell markers in the rodent testis.

Genetic mouse models in which GFP expression is driven by a promoter from a putative SSC gene provide an alternative approach for characterizing SSCs. For example, Schöler and co-workers reported that the OCT-4 transcription factor is expressed by gonocytes and type A spermatogonia of newborn, pup and adult mouse testes (Pesce et al. 1998). This group subsequently characterized an 18 kb promoter/enhancer fragment of the Oct-4 gene that directed faithful expression of lacZ and GFP transgenes (Yeom et al. 1996; Yoshimizu et al. 1999). The Oct-4–GFP mouse is a valuable tool that enabled FACS-based isolation and transplantation of Oct4 expressing germ cells from a heterogeneous testis cell suspension (Ohbo et al. 2003; Ohmura et al. 2004). Stem cell activity was significantly enriched in the Oct4 expressing (GFP+) population compared with the Oct4 negative (GFP−) population of mouse testis cells (Ohmura et al. 2004). Interestingly, gonocytes and pre-spermatogonia from neonatal mice with an Oct4–EGFP+/c-Kit phenotype had a greater repopulation capacity than Oct4–EGFP+/c-Kit+ cell fractions (Ohbo et al. 2003). These data suggest that there is molecular heterogeneity among pre-spermatogonia. This observation is consistent with reports suggesting that some gonocytes/pre-spermatogonia establish the initial pool of SSCs, while other gonocytes/pre-spermatogonia differentiate to produce the first round of spermatogenesis (Kluin & de Rooij 1981; Yoshida et al. 2006).

Transgenic and conditional knock-in approaches were recently used to demonstrate that neurogenin 3 (Ngn3) is expressed by the earliest spermatogonia (Yoshida et al. 2004), including at least 11 per cent of transplantable SSCs (Nakagawa et al. 2007). The fact that Ngn3 was not expressed by all transplantable stem cells in that study provides additional evidence that there may be heterogeneity among SSCs. A conditional knock-in approach was also used to demonstrate that Nanos2 is expressed by SSCs (Sada et al. 2009). Finally, transgenic models suggest that Stra-8 (stimulated by retinoic acid-8) is expressed by undifferentiated spermatogonia, including SSCs (Giuili et al. 2002; Guan et al. 2006; Sadate-Ngatchou et al. 2008), although the transplant data in the Stra-8 studies were limited.

Knock-out models have also been used to demonstrate that specific genes/proteins are required for SSC function. Male mice carrying the luxoid (lu) mutation are subfertile and show abnormal sperm development. Progression of infertility is caused by gradual loss of SSCs (Buaas et al. 2004). The mutation was shown to affect the Zfp145 locus, which encodes the transcriptional repressor PLZF (promyelocytic leukaemia zinc-finger). PLZF is expressed during embryogenesis and plays a crucial role during limb and axial skeletal patterning. Targeted disruption of Zfp145 resulted in a testicular phenotype similar to that of luxoid mutant mice (Costoya et al. 2004). In the testis, PLZF expression is restricted to As, Apr and Aal undifferentiated spermatogonia, including SSCs as demonstrated by transplantation experiments of testicular cells from luxoid or PLZF−/−mice that failed to initiate donor-derived spermatogenesis in recipient mice (Buaas et al. 2004; Costoya et al. 2004). A possible role for PLZF in spermatogonia could be the maintenance of an undifferentiated state (Filipponi et al. 2007), similar to the role suggested for Plzf in haematopoietic precursor cells (Reid et al. 1995).

Similar knock-out and over-expression studies implicate glial cell line-derived neurotrophic factor (GDNF) and its receptor GFRα1 in stem cell self-renewal (Meng et al. 2000). GDNF signalling has since been shown to be required for in vitro expansion of SSCs, and it has been demonstrated that a combination of GDNF and soluble GFRα1 is most favourable for the self-renewal of SSCs in vitro (Kubota et al. 2004a,b; see below). Finally, knock-out studies implicate Sox3 in the differentiation of the earliest germ cells (Raverot et al. 2005). The latter study indicated that Ngn3 expression is dependent on SOX3 and suggested that SOX3 may act directly or indirectly through Ngn3 to regulate spermatogonial differentiation (Raverot et al. 2005).

In addition to data derived from flow cytometry, genetic models and transplantation, immunohistochemistry in tissue sections or intact seminiferous tubules (whole mount) has been widely used to investigate the expression patterns of various proteins in the male germ lineage. In this context, a candidate SSC marker would be expressed by cells located on the basement membrane of seminiferous tubules and be co-expressed with confirmed markers of SSCs. This histochemical approach is most convincing in whole-mount preparations of seminiferous tubules in which it is possible to correlate marker expression with clone size (i.e. As, Apr, Aal). Several established markers of stem, progenitor and differentiating spermatogonia are listed in figure 3. Here we define progenitors as undifferentiated spermatogonia that are committed to differentiate. An example of this approach is shown in figure 4 for the putative SSC marker, Spalt-like 4 (SALL4). SALL4 is a zinc finger transcription factor that is expressed in the inner cell mass of the late blastocyst in a pattern similar to OCT4 and SOX2 (Elling et al. 2006). In vitro, SALL4 stimulates embryonic stem (ES) cell proliferation (Sakaki-Yumoto et al. 2006) and maintains pluripotency by repressing trophectoderm differentiation (Yuri et al. 2009), possibly by binding the Oct-4 proximal promoter (Zhang et al. 2006) and by interacting with NANOG (Wu et al. 2006). Thus, SALL4 is an important stemness factor and together with OCT-4, SOX2 and NANOG constitutes a tightly regulated transcription circuit important for stem cell pluripotency (Lim et al. 2008; Yang et al. 2008). Postnatally, Sall4 expression is restricted to the gonads and is expressed by isolated spermatogonia (Wang et al. 2001; Sall4 was identified as testis-expressed gene 20 (Tex20) in that paper). Co-stained whole-mount seminiferous tubules (figure 4) indicated that SALL4 is expressed by single, paired and aligned cells on the seminiferous tubule basement membrane and overlaps with consensus SSC markers, PLZF (figure 4ac) and GFRα1 (figure 4df). However, these whole-mount immunohistochemistry results highlight the heterogeneity among undifferentiated spermatogonia, including As spermatogonia (see figure 4f with examples of SALL4+/GFRα1 and SALL4+/GFRα1+ As spermatogonia). GFRα1 appears to have the most restricted expression (limited to singles, pairs and chains of four), while PLZF and SALL4 are also expressed by larger chains of 8 and 16 Aal spermatogonia.

Figure 3.

Genes expressed by stem, progenitor and differentiating spermatogonia. The As, seen at the top of the diagram, is responsible for self renewal and differentiation. Self-renewal is represented here by the Apair dividing to form two As. Differentiation ...

Figure 4.

Immunofluorescent co-staining of adult mouse whole-mount seminiferous tubules. (a) SALL4 labels undifferentiated As, Apr and Aal spermatogonia. (b) PLZF labels undifferentiated As, Apr and Aal spermatogonia. (c) Merged picture from (a,b). SALL4 and PLZF ...

Similar observations of molecular heterogeneity among undifferentiated As, Apr and Aal spermatogonia have been reported in several recent studies (Tokuda et al. 2007; Grisanti et al. 2009; Sada et al. 2009; Suzuki et al. 2009; Zheng et al. 2009). The functional significance of this heterogeneity remains to be determined. Through the combination of FACS and MACS analyses, transplantation, genetic models and histochemical approaches, the phenotype of rodent SSCs is beginning to emerge. A list of putative SSC and undifferentiated spermatogonia markers is provided in table 1 along with the experimental evidence used to characterize each marker.

8. The spermatogonial stem cell niche

SSCs reside within a specialized microenvironment called ‘niche’ that regulates testicular homeostasis by balancing SSC self-renewal and differentiation. A stem cell niche is comprised of cells, extracellular matrix components, and local soluble factors present in the vicinity of the stem cell that regulates cell fate. The structural basis for the SSC niche in the mammalian testis is the basal compartment of the seminiferous tubules that is composed of Sertoli cells and peritubular myoid cells (Dadoune 2007) (figure 5). Together, Sertoli and peritubular myoid cells secrete the basement membrane components to which the SSCs are connected via adhesion molecules (Tung et al. 1984). Sertoli cells are polarized columnar epithelial cells that support SSCs and differentiating germ cells by providing nutrients and mediating external signals in order to support spermatogenesis (Griswold 1998). The importance of Sertoli cells for germ cell differentiation is demonstrated by the transplantation of normal Sertoli cells into the testis of infertile mutant recipients with a Sertoli cell defect and successful initiation of spermatogenesis by recipient-derived spermatogonia (Kanatsu-Shinohara et al. 2003b, 2005b). Tight junctions between adjacent Sertoli cells constitute a protective blood–testis barrier (BTB) that divides the seminiferous epithelium into basal and adluminal compartments (figure 5a) and plays an important role in the regulation of germ cell differentiation (Cheng & Mruk 2002). The BTB maintains a selective substance flow between luminal fluid, blood plasma and interstitial fluid, thereby creating an immune-privileged environment for haploid germ cells in the adluminal compartment of the seminiferous tubules.

Figure 5.

SSC niche. The SSC (dark blue) is diagrammed in its physical niche (a) surrounded by Sertoli cells (orange) and differentiating germ cells (light blue) within the seminiferous tubule. Niche components outside the tubule itself include myoid cells (green), ...

Along the length of the tubule, SSCs are thought to be localized in areas adjacent to the interstitial space (Chiarini-Garcia et al. 2003). Undifferentiated spermatogonia are observed predominantly in tubule areas adjacent to vasculature (Yoshida et al. 2007b; figure 5a).

The SSC niche mediates endocrine and paracrine signals that regulate self-renewal and differentiation (figure 5b). A key regulator of the SSC niche is GDNF which is secreted by Sertoli cells and acts through Ret receptor tyrosine kinase and GFRα1 co-receptor, which form a receptor complex on the surface of As, APr and Aal (Meng et al. 2000). Downstream signalling pathways that are activated by GDNF in undifferentiated spermatogonia are the PI3K/Akt pathway, members of the Src kinase family and the Ras/Erk1/2 pathway (Braydich-Stolle et al. 2007; Oatley et al. 2007; He et al. 2008). GDNF is thought to act through these pathways to regulate SSC self-renewal.

Targeted disruption of the Ets variant gene 5 (Etv5) results in defective maintenance of the SSC pool, whereas spermatogonial differentiation appears to be unaffected by this mutation (Chen et al. 2005). The transcription factor Etv5 is expressed in Sertoli cells and loss of Etv5 appears to impair the ability of Sertoli cells to support spermatogonia, possibly by disrupted BTB function as indicated by decreased Claudin-5 (CLDN5) levels in mutant mice (Morrow et al. 2009). In Sertoli cells, Etv5 is upregulated by FGF2 in vitro, which is important for SSC renewal in culture (Kanatsu-Shinohara et al. 2003a; Kubota et al. 2004a; Yoon et al. 2009). Therefore, in addition to a direct effect of FGF2 on SSCs, an indirect paracrine effect of FGF2 on Sertoli cells appears possible.

The importance of peritubular myoid cells for spermatogonia maintenance has long been discussed. New data now suggest a role for the peritubular cell product colony-stimulating factor 1 (CSF1) on SSC maintenance (Oatley et al. 2009). Csf1 was found to be expressed in interstitial Leydig and peritubular myoid cells, whereas the Csf1 receptor (Csf1r) was highly enriched in THY1+ cell fractions from pre-pubertal and adult mouse testis.

9. SSC culture

SSC culture provides a new approach for investigating the molecular mechanisms and cell-signalling pathways that regulate SSC function. While methods for maintaining and amplifying pluripotent ES and embryonic germ cells in culture are routine, methods for culturing adult tissue stem cells (including SSCs) had been more difficult to establish. However, tremendous progress culturing mouse and rat SSCs has been reported during the past 5–6 years (Kanatsu-Shinohara et al. 2003a; Kubota et al. 2004a,b; Hamra et al. 2005; Ryu et al. 2005). Rodent SSCs can now be maintained for a very long time (perhaps indefinitely) with a significant amplification in numbers. Stem cell activity in these cultures was confirmed by SSC transplantation, as diagrammatically represented in figure 2. The doubling time for mouse SSCs was determined to be 5.6 days (Kubota et al. 2004b), while the doubling time for rat SSCs is 3–4 days (Hamra et al. 2005) or 11 days (Ryu et al. 2005).

Several factors were critical to the establishment of long-term SSC cultures. First, methods to fractionate testis cell populations (FACS or MACS sorting and/or differential attachment and replating) resulted in the enrichment of SSCs and the removal of somatic cells that promote germ cell differentiation. Second, development of a serum-free, defined medium facilitated the discovery of essential growth factors. Specifically, GDNF is necessary to maintain and expand rodent SSCs in culture (Kanatsu-Shinohara et al. 2003a; Kubota et al. 2004b). The trophic effects of GDNF in both mice and rats is enhanced by the addition of soluble GFRα1 (the receptor for GDNF) and FGF2 (Kubota et al. 2004a; Ryu et al. 2005). Unlike mouse ES cells, the additions of leukaemia inhibitory factor (LIF) and foetal bovine serum (FBS) to cultures are superfluous and detrimental, respectively, in SSC cultures (Kubota et al. 2004b). Third, STO or mouse embryonic fibroblast (MEF) feeder cells are usually required. Whereas Shinohara's group has demonstrated that mouse SSCs can also be maintained in feeder-free conditions (Kanatsu-Shinohara et al. 2005a). SSC cultures are usually established from mouse pup testes (5–12 days postpartum) because SSCs are enriched at this stage of development. However, SSC cultures can be established from neonate (Kanatsu-Shinohara et al. 2003a) and adult mouse testis cells (Kubota et al. 2004a). Immortalized SSC lines have been established by the introduction of a retroviral telomerase gene (Feng et al. 2002) or treatment with the SV40 large T-antigen (Hofmann et al. 2005a). Evidence that each of these immortalized cell lines is spermatogonial-like is based primarily on genetic or immunocytochemical data, but transplantation data are lacking.

Stable SSC culture provides a valuable tool for dissecting mechanisms that regulate SSC renewal and differentiation. GDNF is required for SSC renewal in vitro (Kubota et al. 2004a) and in vivo (Meng et al. 2000). Through withdrawal and/or addition of GDNF to SSC cultures, two groups have now demonstrated that GDNF action is mediated by Src family kinases acting through PI3 kinase/Akt-dependent pathways (Braydich-Stolle et al. 2007; Oatley et al. 2007). In addition, microarray analysis identified genes that are regulated by GDNF withdrawal in SSC cultures. The importance of three of these genes (Bcl6b, Erm and Lhx1) was confirmed by transfecting SSC cultures with siRNAs specific for each gene. siRNA treatment caused decreased clump formation in vitro and decreased colonization of recipient testes after transplantation (Oatley et al. 2006, 2007).

Transfection of SSC cultures with siRNA, as described above, enables temporary knockdown of the target gene. To achieve stable knockdown of a target gene, short hairpin RNAs (shRNAs) can be coupled with lentiviral vectors. Dann et al. (2008) recently treated cultured SSCs with a lentiviral vector containing on Oct-4-targeted shRNA. The treatment caused a significant reduction in OCT-4 expression and reduced colonizing activity in the transplant assay by sixfold. Thus, through genetic manipulation and transplantation of SSC cultures, studies will continue to unravel regulatory pathways required for SSC self-renewal and differentiation.

10. Future directions

We have attempted to review the current state of knowledge and research in the biology of SSCs, focused primarily on the rodent model. Many areas of research are only beginning to be thoroughly investigated in SSCs, such as the molecular regulation of stem cell fate decisions and SSC heterogeneity. Recent progress characterizing, manipulating and culturing SSCs has opened the door to new experimental approaches for fundamental investigation and possible practical applications discussed below.

In vitro derivation of haploid gametes (elongated spermatids or sperm) may help to overcome spermatogenic barriers in infertile men. Feng et al. (2002) reported the production of spermatocytes and spermatids from a stable mouse SSC line, though the fertilization potential of these cells was not tested. Haploid male germ cells have also been generated by differentiation of ES cells in mice (Toyooka et al. 2003; Geijsen et al. 2004; Nayernia et al. 2006) and humans (Kee et al. 2009). Two studies demonstrated that in vitro, ESC-derived spermatids were competent to fertilize mouse eggs, generating blastocysts (Geijsen 2004) and live progeny (Nayernia et al. 2006), respectively. However, because of the epigenetic reprogramming that occurs during in vivo germ cell development, the epigenetic regulation of in vitro gametogenesis must be carefully assessed before clinical applications ensue (Georgiou et al. 2007). Generation of haploid germ cells from primary SSC cultures has not yet been reported, but this approach may have epigenetic advantages over ESC-derived gametes. Furthermore, progress establishing human SSC cultures will be an important experimental tool in a species where transplantation is not an option for characterizing SSCs.

In addition to trying to drive SSCs towards their typical biological end, there is evidence that SSCs are a source of pluripotent stem cells (Kanatsu-Shinohara et al. 2004a; Guan et al. 2006; Seandel et al. 2007). The ability to derive pluripotent stem cells from adult tissues, with the consent of the donor, may have some advantages over other approaches to pluripotency. However, more details are needed to understanding the genetic, epigenetic constitution of these cells, as well as their developmental potential.

Testicular tissues or testicular cell suspensions (containing SSCs) can be cryopreserved and may provide an avenue for preservation of valuable strains or species. Honaramooz et al. (2002) recently demonstrated that testicular tissues from newborn mice, pigs or goats can be grafted under the skin of immune-deficient mice and generate complete spermatogenesis. This approach has now been reported for several species (Oatley et al. 2004, 2005; Snedaker et al. 2004; Zeng et al. 2006; Kim et al. 2007; Arregui et al. 2008; Rodriguez-Sosa et al. 2010) and may allow germline preservation for endangered species or valuable domestic strains. Alternatively, valuable germlines can be preserved by freezing testis cell suspensions (containing SSCs) for future SSC transplantation. The proof in principle for this approach is already established for mice, rats, goats and dogs (Brinster & Avarbock 1994; Brinster et al. 2003; Honaramooz et al. 2003; Ryu et al. 2003; Kim et al. 2008).

SSC transplantation may have application for treating some cases of male infertility. For example, high-dose chemotherapy and total body radiation treatment of cancer can cause permanent infertility. While adult men can cryopreserve a semen sample prior to their oncologic treatment, this is not an option for pre-adolescent boys who are not yet making sperm. Using methods similar to those already established for other species, it may be possible for these young cancer patients to cryopreserve testis cells or tissue prior to cancer treatment and use those tissues to achieve fertility after they are cured (Orwig & Schlatt 2005; Goossens et al. 2008; Hermann et al. 2009). We have recently established a non-human primate model of cancer survivorship to test the safety and feasibility of SSC transplantation in a species that is relevant to human physiology (Hermann et al. 2007). Although SSC transplantation is not yet ready for the human fertility clinic, it may be reasonable for young cancer patients, with no other options to preserve their fertility, to cryopreserve testicular cells (Schlatt et al. 2009). Ginsberg and co-workers have been cryopreserving testicular tissue for young cancer patients since 2008 and report that this intervention is acceptable to parents and that testicular biopsies caused no acute adverse effects (Ginsberg et al. 2010). A human SSC culture system would be particularly useful in this setting because a few SSCs could be obtained in a small biopsy and expanded to a number sufficient for transplant therapy.

Progress studying SSC origins, regulation and activity over the past half century, has laid the foundation to pursue the clinical and veterinary options described in the preceding paragraphs. The field of SSC biology has grown substantially in the past two decades, fuelled in part by development of the SSC transplantation technique (Brinster & Avarbock 1994; Brinster & Zimmermann 1994), which impacted fundamental investigations as well as clinical application. Growth was also fuelled by the explosive development of the pluripotent stem cell and regenerative medicine fields. The next half century should bring many new discoveries about the biology and regenerative potential of SSCs that parallels the development of the haematopoietic stem cell field in the 1980s and 1990s.

Acknowledgements

The authors would like to thank Dr Brian Hermann for critically reviewing the chapter. B.T.P. produced the artwork in figures 1, ,2,2, ,33 and and5.5. K.E.O. is supported by NIH grants HD055475 and HD008610 and the Magee-Womens Research Institute and Foundation.

Footnotes

One contribution of 17 to a Theme Issue ‘The biology and regulation of spermatogenesis’.

References

  • Anderson R., Schaible K., Heasman J., Wylie C. 1999. Expression of the homophilic adhesion molecule, Ep-CAM, in the mammalian germ line. J. Reprod. Fertil. 116, 379–384 [PubMed]
  • Antonangeli F., Giampietri C., Petrungaro S., Filippini A., Ziparo E. 2009. Expression profile of a 400-bp Stra8 promoter region during spermatogenesis. Microsc. Res. Tech. 72, 816–822 [PubMed]
  • Arregui L., Rathi R., Megee S. O., Honaramooz A., Gomendio M., Roldan E. R., Dobrinski I. 2008. Xenografting of sheep testis tissue and isolated cells as a model for preservation of genetic material from endangered ungulates. Reproduction 136, 85–93 (doi:10.1530/REP-07-0433) [PubMed]
  • Ballow D., Meistrich M. L., Matzuk M., Rajkovic A. 2006. aSohlh1 is essential for spermatogonial differentiation. Dev. Biol. 294, 161–167 (doi:10.1016/j.ydbio.2006.02.027) [PubMed]
  • Ballow D. J., Xin Y., Choi Y., Pangas S. A., Rajkovic A. 2006. bSohlh2 is a germ cell-specific bHLH transcription factor. Gene Expr. Patterns 6, 1014–1018 (doi:10.1016/j.modgep.2006.04.007) [PubMed]
  • Bendel-Stenzel M., Anderson R., Heasman J., Wylie C. 1998. The origin and migration of primordial germ cells in the mouse. Semin. Cell Dev. Biol. 9, 393–400 [PubMed]
  • Braun R. E., Behringer R. R., Peschon J. J., Brinster R. L., Palmiter R. D. 1989. Genetically haploid spermatids are phenotypically diploid. Nature 337, 373–376 (doi:10.1038/337373a0) [PubMed]
  • Braydich-Stolle L., Kostereva N., Dym M., Hofmann M. C. 2007. Role of Src family kinases and N-Myc in spermatogonial stem cell proliferation. Dev. Biol. 304, 34–45 [PMC free article] [PubMed]
  • Brinster R. L., Avarbock M. R. 1994. Germline transmission of donor haplotype following spermatogonial transplantation. Proc. Natl Acad. Sci. USA 91, 11 303–11 307 (doi:10.1073/pnas.91.24.11303) [PubMed]
  • Brinster R. L., Zimmermann J. W. 1994. Spermatogenesis following male germ-cell transplantation. Proc. Natl Acad. Sci. USA 91, 11 298–11 302 (doi:10.1073/pnas.91.24.11298) [PubMed]
  • Brinster C. J., Ryu B. Y., Avarbock M. R., Karagenc L., Brinster R. L., Orwig K. E. 2003. Restoration of fertility by germ cell transplantation requires effective recipient preparation. Biol. Reprod. 69, 412–420 (doi:10.1095/biolreprod.103.016519) [PubMed]
  • Buaas F. W., Kirsh A. L., Sharma M., McLean D. J., Morris J. L., Griswold M. D., de Rooij D. G., Braun R. E. 2004. Plzf is required in adult male germ cells for stem cell self-renewal. Nat. Genet. 36, 647–652 (doi:10.1038/ng1366) [PubMed]
  • Buageaw A., Sukhwani M., Ben-Yehudah A., Ehmcke J., Rawe V. Y., Pholpramool C., Orwig K. E., Schlatt S. 2005. GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes. Biol. Reprod. 73, 1011–1016 (doi:10.1095/biolreprod.105.043810) [PubMed]
  • Chen C., et al. 2005. ERM is required for transcriptional control of the spermatogonial stem cell niche. Nature 436, 1030–1034 (doi:10.1038/nature03894) [PMC free article] [PubMed]
  • Cheng C. Y., Mruk D. D. 2002. Cell junction dynamics in the testis: sertoli–germ cell interactions and male contraceptive development. Physiol. Rev. 82, 825–874 [PubMed]
  • Chiarini-Garcia H., Raymer A. M., Russell L. D. 2003. Non-random distribution of spermatogonia in rats: evidence of niches in the seminiferous tubules. Reproduction 126, 669–680 (doi:10.1530/rep.0.1260669) [PubMed]
  • Clermont Y. 1972. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol. Rev. 52, 198–236 [PubMed]
  • Clermont Y., Antar M. 1973. Duration of the cycle of the seminiferous epithelium and the spermatogonial renewal in the monkey, Macaca arctoides. Am. J. Anat. 136, 153–165 (doi:10.1002/aja.1001360204) [PubMed]
  • Clermont Y., Bustos-Obregon E. 1968. Re-examination of spermatogonial renewal in the rat by means of seminiferous tubules mounted ‘in toto’. Am. J. Anat. 122, 237–247 (doi:10.1002/aja.1001220205) [PubMed]
  • Clermont Y., Hermo L. 1975. Spermatogonial stem cells in the albino rat. Am. J. Anat. 142, 159–175 (doi:10.1002/aja.1001420203) [PubMed]
  • Clermont Y., Leblond C. P. 1953. Renewal of spermatogonia in the rat. Am. J. Anat. 93, 475–501 (doi:10.1002/aja.1000930308) [PubMed]
  • Clermont Y., Leblond C. P. 1959. Differentiation and renewal of spermatogonia in the monkey, Macacus rhesus. Am. J. Anat. 104, 237–273 (doi:10.1002/aja.1001040204) [PubMed]
  • Clermont Y., Perey B. 1957. Quantitative study of the cell population of the seminiferous tubules in immature rats. Am. J. Anat. 100, 241–267 (doi:10.1002/aja.1001000205) [PubMed]
  • Cooke H. J., Lee M., Kerr S., Ruggiu M. 1996. A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads. Hum. Mol. Genet. 5, 513–516 (doi:10.1093/hmg/5.4.513) [PubMed]
  • Corallini S., Fera S., Grisanti L., Falciatori I., Muciaccia B., Stefanini M., Vicini E. 2006. Expression of the adaptor protein m-Numb in mouse male germ cells. Reproduction 132, 887–897 (doi:10.1530/REP-06-0062) [PubMed]
  • Costoya J. A., Hobbs R. M., Barna M., Cattoretti G., Manova K., Sukhwani M., Orwig K. E., Wolgemuth D. J., Pandolfi P. P. 2004. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat. Genet. 36, 653–659 (doi:10.1038/ng1367) [PubMed]
  • Dadoune J. P. 2007. New insights into male gametogenesis: what about the spermatogonial stem cell niche? Folia Histochem. Cytobiol. 45, 141–147 [PubMed]
  • Dann C. T., Alvarado A. L., Molyneux L. A., Denard B. S., Garbers D. L., Porteus M. H. 2008. Spermatogonial stem cell self-renewal requires OCT4, a factor downregulated during retinoic acid-induced differentiation. Stem Cells 26, 2928–2937 (doi:10.1634/stemcells.2008-0134) [PubMed]
  • Dettin L., Ravindranath N., Hofmann M. C., Dym M. 2003. Morphological characterization of the spermatogonial subtypes in the neonatal mouse testis. Biol. Reprod. 69, 1565–1571 (doi:10.1095/biolreprod.103.016394) [PubMed]
  • Dobrinski I., Ogawa T., Avarbock M. R., Brinster R. L. 1999. Computer assisted image analysis to assess colonization of recipient seminiferous tubules by spermatogonial stem cells from transgenic donor mice. Mol. Reprod. Dev. 53, 142–148 (doi:10.1002/(SICI)1098-2795(199906)53:2<142::AID-MRD3>3.0.CO;2-O) [PubMed]
  • Dym M., Clermont Y. 1970. Role of spermatogonia in the repair of the seminiferous epithelium following x-irradiation of the rat testis. Am. J. Anat. 128, 265–282 (doi:10.1002/aja.1001280302) [PubMed]
  • Ebata K. T., Zhang X., Nagano M. C. 2005. Expression patterns of cell-surface molecules on male germ line stem cells during postnatal mouse development. Mol. Reprod. Dev. 72, 171–181 (doi:10.1002/mrd.20324) [PubMed]
  • Elling U., Klasen C., Eisenberger T., Anlag K., Treier M. 2006. Murine inner cell mass-derived lineages depend on Sall4 function. Proc. Natl Acad. Sci. USA 103, 16 319–16 324 (doi:10.1073/pnas.0607884103) [PubMed]
  • Enders G. C., May J. J., 2nd 1994. Developmentally regulated expression of a mouse germ cell nuclear antigen examined from embryonic day 11 to adult in male and female mice. Dev. Biol. 163, 331–340 (doi:10.1006/dbio.1994.1152) [PubMed]
  • Falender A. E., Freiman R. N., Geles K. G., Lo K. C., Hwang K., Lamb D. J., Morris P. L., Tjian R., Richards J. S. 2005. Maintenance of spermatogenesis requires TAF4b, a gonad-specific subunit of TFIID. Genes Dev. 19, 794–803 (doi:10.1101/gad.1290105) [PubMed]
  • Feng L. X., Chen Y., Dettin L., Pera R. A., Herr J. C., Goldberg E., Dym M. 2002. Generation and in vitro differentiation of a spermatogonial cell line. Science 297, 392–395 (doi:10.1126/science.1073162) [PubMed]
  • Filipponi D., Hobbs R. M., Ottolenghi S., Rossi P., Jannini E. A., Pandolfi P. P., Dolci S. 2007. Repression of kit expression by Plzf in germ cells. Mol. Cell. Biol. 27, 6770–6781 (doi:10.1128/MCB.00479-07) [PMC free article] [PubMed]
  • Fujita K., Ohta H., Tsujimura A., Takao T., Miyagawa Y., Takada S., Matsumiya K., Wakayama T., Okuyama A. 2005. Transplantation of spermatogonial stem cells isolated from leukemic mice restores fertility without inducing leukemia. J. Clin. Invest. 115, 1855–1861 (doi:10.1172/JCI24189) [PMC free article] [PubMed]
  • Fujiwara Y., Komiya T., Kawabata H., Sato M., Fujimoto H., Furusawa M., Noce T. 1994. Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage. Proc. Natl Acad. Sci. USA 91, 12 258–12 262 (doi:10.1073/pnas.91.25.12258) [PubMed]
  • Geijsen N., Horoschak M., Kim K., Gribnau J., Eggan K., Daley G. Q. 2004. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427, 148–154 (doi:10.1038/nature02247) [PubMed]
  • Georgiou I., et al. 2007. In vitro spermatogenesis as a method to bypass pre-meiotic or post-meiotic barriers blocking the spermatogenetic process: genetic and epigenetic implications in assisted reproductive technology. Andrologia 39, 159–176 (doi:10.1111/j.1439-0272.2007.00778.x) [PubMed]
  • Ginsberg J. P., Carlson C. A., Lin K., Hobbie W. L., Wigo E., Wu X., Brinster R. L., Kolon T. F. 2010. An experimental protocol for fertility preservation in prepubertal boys recently diagnosed with cancer: a report of acceptability and safety. Hum. Reprod. 25, 37–41 (doi:10.1093/humrep/dep371) [PMC free article] [PubMed]
  • Ginsburg M., Snow M. H., McLaren A. 1990. Primordial germ cells in the mouse embryo during gastrulation. Development 110, 521–528 [PubMed]
  • Giuili G., Tomljenovic A., Labrecque N., Oulad-Abdelghani M., Rassoulzadegan M., Cuzin F. 2002. Murine spermatogonial stem cells: targeted transgene expression and purification in an active state. EMBO Rep. 3, 753–759 (doi:10.1093/embo-reports/kvf149) [PubMed]
  • Goossens E., Geens M., De Block G., Tournaye H. 2008. Spermatogonial survival in long-term human prepubertal xenografts. Fertil. Steril. 90, 2019–2022 (doi:10.1016/j.fertnstert.2007.09.044) [PubMed]
  • Grisanti L., et al. 2009. Identification of spermatogonial stem cell subsets by morphological analysis and prospective isolation. Stem Cells 27, 3043–3052 [PubMed]
  • Griswold M. D. 1998. The central role of Sertoli cells in spermatogenesis. Semin. Cell Dev. Biol. 9, 411–416 (doi:10.1006/scdb.1998.0203) [PubMed]
  • Guan K., et al. 2006. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 440, 1199–1203 (doi:10.1038/nature04697) [PubMed]
  • Hamra F. K., Schultz N., Chapman K. M., Grellhesl D. M., Cronkhite J. T., Hammer R. E., Garbers D. L. 2004. Defining the spermatogonial stem cell. Dev. Biol. 269, 393–410 [PubMed]
  • Hamra F. K., Chapman K. M., Nguyen D. M., Williams-Stephens A. A., Hammer R. E., Garbers D. L. 2005. Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture. Proc. Natl Acad. Sci. USA 102, 17 430–17 435 (doi:10.1073/pnas.0508780102) [PubMed]
  • He Z., Jiang J., Kokkinaki M., Golestaneh N., Hofmann M. C., Dym M. 2008. Gdnf upregulates c-Fos transcription via the Ras/Erk1/2 pathway to promote mouse spermatogonial stem cell proliferation. Stem Cells 26, 266–278 (doi:10.1634/stemcells.2007-0436) [PMC free article] [PubMed]
  • Hermann B. P., et al. 2007. Characterization, cryopreservation and ablation of spermatogonial stem cells in adult rhesus macaques. Stem Cells 25, 2330–2338 (doi:10.1634/stemcells.2007-0143) [PMC free article] [PubMed]
  • Hermann B. M., Sukhwani M., Hansel M., Orwig K. 2009. Spermatogonial stem cells in higher primates: are there differences to those in rodents? Reproduction 139, 479–493 (doi:10.1530/REP-09-0255) [PMC free article] [PubMed]
  • Hilscher B., Hilscher W., Maurer W. 1969. Autoradiographic studies on the modus of proliferation and regeneration of the seminiferous epithelium of Wistar rats. Z. Zellforsch. Mikrosk. Anat. 94, 593–604 (doi:10.1007/BF00936064) [PubMed]
  • Hofmann M. C., Braydich-Stolle L., Dettin L., Johnson E., Dym M. 2005. aImmortalization of mouse germ line stem cells. Stem Cells 23, 200–210 (doi:10.1634/stemcells.2003-0036) [PMC free article] [PubMed]
  • Hofmann M. C., Braydich-Stolle L., Dym M. 2005. bIsolation of male germ-line stem cells; influence of GDNF. Dev. Biol. 279, 114–124 (doi:10.1016/j.ydbio.2004.12.006) [PMC free article] [PubMed]
  • Honaramooz A., Snedaker A., Boiani M., Scholer H., Dobrinski I., Schlatt S. 2002. Sperm from neonatal mammalian testes grafted in mice. Nature 418, 778–781 (doi:10.1038/nature00918) [PubMed]
  • Honaramooz A., Behboodi E., Megee S. O., Overton S. A., Galantino-Homer H., Echelard Y., Dobrinski I. 2003. Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats. Biol. Reprod. 69, 1260–1264 (doi:10.1095/biolreprod.103.018788) [PubMed]
  • Huckins C. 1971. aThe spermatogonial stem cell population in adult rats. II. A radioautographic analysis of their cell cycle properties. Cell Tissue Kinet. 4, 313–334 [PubMed]
  • Huckins C. 1971. bCell cycle properties of differentiating spermatogonia in adult Sprague–Dawley rats. Cell Tissue Kinet. 4, 139–154 [PubMed]
  • Huckins C. 1971. cThe spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. Anat. Rec. 169, 533–557 (doi:10.1002/ar.1091690306) [PubMed]
  • Jarvis S., Elliott D. J., Morgan D., Winston R., Readhead C. 2005. Molecular markers for the assessment of postnatal male germ cell development in the mouse. Hum. Reprod. 20, 108–116 (doi:10.1093/humrep/deh565) [PubMed]
  • Kanatsu-Shinohara M., Ogonuki N., Inoue K., Miki H., Ogura A., Toyokuni S., Shinohara T. 2003. aLong-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol. Reprod. 69, 612–616 (doi:10.1095/biolreprod.103.017012) [PubMed]
  • Kanatsu-Shinohara M., Ogonuki N., Inoue K., Ogura A., Toyokuni S., Shinohara T. 2003. bRestoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells. Hum. Reprod. 18, 2660–2667 (doi:10.1093/humrep/deg483) [PubMed]
  • Kanatsu-Shinohara M., et al. 2004. aGeneration of pluripotent stem cells from neonatal mouse testis. Cell 119, 1001–1012 (doi:10.1016/j.cell.2004.11.011) [PubMed]
  • Kanatsu-Shinohara M., Toyokuni S., Shinohara T. 2004. bCD9 is a surface marker on mouse and rat male germline stem cells. Biol. Reprod. 70, 70–75 (doi:10.1095/biolreprod.103.020867) [PubMed]
  • Kanatsu-Shinohara M., Miki H., Inoue K., Ogonuki N., Toyokuni S., Ogura A., Shinohara T. 2005. aLong-term culture of mouse male germline stem cells under serum- or feeder-free conditions. Biol. Reprod. 72, 985–991 [PubMed]
  • Kanatsu-Shinohara M., Miki H., Inoue K., Ogonuki N., Toyokuni S., Ogura A., Shinohara T. 2005. bGermline niche transplantation restores fertility in infertile mice. Hum. Reprod. 20, 2376–2382 [PubMed]
  • Kanatsu-Shinohara M., Inoue K., Miki H., Ogonuki N., Takehashi M., Morimoto T., Ogura A., Shinohara T. 2006. Clonal origin of germ cell colonies after spermatogonial transplantation in mice. Biol. Reprod. 75, 68–74 (doi:10.1095/biolreprod.106.051193) [PubMed]
  • Kanatsu-Shinohara M., et al. 2008. Homing of mouse spermatogonial stem cells to germline niche depends on beta1-integrin. Cell Stem Cell 3, 533–542 [PubMed]
  • Kee K., Angeles V. T., Flores M., Nguyen H. N., Reijo Pera R. A. 2009. Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature 462, 222–225 (doi:10.1038/nature08562) [PMC free article] [PubMed]
  • Kim Y., Selvaraj V., Pukazhenthi B., Travis A. J. 2007. Effect of donor age on success of spermatogenesis in feline testis xenografts. Reprod. Fertil. Dev. 19, 869–876 (doi:10.1071/RD07056) [PubMed]
  • Kim Y., Turner D., Nelson J., Dobrinski I., McEntee M., Travis A. J. 2008. Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog. Reproduction 136, 823–831 (doi:10.1530/REP-08-0226) [PMC free article] [PubMed]
  • Kluin P. M., de Rooij D. G. 1981. A comparison between the morphology and cell kinetics of gonocytes and adult type undifferentiated spermatogonia in the mouse. Int. J. Androl. 4, 475–493 (doi:10.1111/j.1365-2605.1981.tb00732.x) [PubMed]
  • Koshimizu U., Nishioka H., Watanabe D., Dohmae K., Nishimune Y. 1995. Characterization of a novel spermatogenic cell antigen specific for early stages of germ cells in mouse testis. Mol. Reprod. Dev. 40, 221–227 (doi:10.1002/mrd.1080400211) [PubMed]
  • Kubota H., Avarbock M. R., Brinster R. L. 2003. Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proc. Natl Acad. Sci. USA 100, 6487–6492 (doi:10.1073/pnas.0631767100) [PubMed]
  • Kubota H., Avarbock M. R., Brinster R. L. 2004. aGrowth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc. Natl Acad. Sci. USA 101, 16 489–16 494 (doi:10.1073/pnas.0407063101) [PubMed]
  • Kubota H., Avarbock M. R., Brinster R. L. 2004. bCulture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol. Reprod. 71, 722–731 (doi:10.1095/biolreprod.104.029207) [PubMed]
  • Lawson K. A., Dunn N. R., Roelen B. A., Zeinstra L. M., Davis A. M., Wright C. V., Korving J. P., Hogan B. L. 1999. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 13, 424–436 (doi:10.1101/gad.13.4.424) [PubMed]
  • Leblond C. P., Clermont Y. 1952. aSpermiogenesis of rat, mouse, hamster and guinea pig as revealed by the periodic acid-fuchsin sulfurous acid technique. Am. J. Anat. 90, 167–215 (doi:10.1002/aja.1000900202) [PubMed]
  • Leblond C. P., Clermont Y. 1952. bDefinition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N.Y. Acad. Sci. 55, 548–573 (doi:10.1111/j.1749-6632.1952.tb26576.x) [PubMed]
  • Lim C. Y., et al. 2008. Sall4 regulates distinct transcription circuitries in different blastocyst-derived stem cell lineages. Cell Stem Cell 3, 543–554 (doi:10.1016/j.stem.2008.08.004) [PubMed]
  • Lo K. C., Brugh V. M., 3rd, Parker M., Lamb D. J. 2005. Isolation and enrichment of murine spermatogonial stem cells using rhodamine 123 mitochondrial dye. Biol. Reprod. 72, 767–771 (doi:10.1095/biolreprod.104.033464) [PubMed]
  • Lolicato F., Marino R., Paronetto M. P., Pellegrini M., Dolci S., Geremia R., Grimaldi P. 2008. Potential role of Nanos3 in maintaining the undifferentiated spermatogonia population. Dev. Biol. 313, 725–738 (doi:10.1016/j.ydbio.2007.11.011) [PubMed]
  • Manova K., Nocka K., Besmer P., Bachvarova R. F. 1990. Gonadal expression of c-kit encoded at the W locus of the mouse. Development 110, 1057–1069 [PubMed]
  • McCarrey J. 1993. Development of the germ cell. In Cell and molecular biology of the testis (eds Desjardins C., Ewing L., editors. ), pp. 58–89 New York, NY: Oxford University Press
  • McLaren A. 2003. Primordial germ cells in the mouse. Dev. Biol. 262, 1–15 (doi:10.1016/S0012-1606(03)00214-8) [PubMed]
  • Meng X., et al. 2000. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287, 1489–1493 (doi:10.1126/science.287.5457.1489) [PubMed]
  • Monesi V. 1962. Autoradiographic study of DNA synthesis and the cell cycle in spermatogonia and spermatocytes of mouse testis using tritiated thymidine. J. Cell Biol. 14, 1–18 (doi:10.1083/jcb.14.1.1) [PMC free article] [PubMed]
  • Morimoto H., Kanatsu-Shinohara M., Takashima S., Chuma S., Nakatsuji N., Takehashi M., Shinohara T. 2009. Phenotypic plasticity of mouse spermatogonial stem cells. PLoS One 4, e7909 (doi:10.1371/journal.pone.0007909) [PMC free article] [PubMed]
  • Morrow C. M., Tyagi G., Simon L., Carnes K., Murphy K. M., Cooke P. S., Hofmann M. C., Hess R. A. 2009. Claudin 5 expression in mouse seminiferous epithelium is dependent upon the transcription factor Ets-variant 5 and contributes to blood–testis barrier function. Biol. Reprod. 81, 871–879 [PMC free article] [PubMed]
  • Nakagawa T., Nabeshima Y., Yoshida S. 2007. Functional identification of the actual and potential stem cell compartments in mouse spermatogenesis. Dev. Cell 12, 195–206 (doi:10.1016/j.devcel.2007.01.002) [PubMed]
  • Naughton C. K., Jain S., Strickland A. M., Gupta A., Milbrandt J. 2006. Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate. Biol. Reprod. 74, 314–321 (doi:10.1095/biolreprod.105.047365) [PubMed]
  • Nayernia K., et al. 2006. In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Dev. Cell 11, 125–132 (doi:10.1016/j.devcel.2006.05.010) [PubMed]
  • Niederberger C., Agulnik A. I., Cho Y., Lamb D., Bishop C. E. 1997. In situ hybridization shows that Dazla expression in mouse testis is restricted to premeiotic stages IV–VI of spermatogenesis. Mamm. Genome 8, 277–278 (doi:10.1007/s003359900409) [PubMed]
  • Oakberg E. F. 1956. aA description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am. J. Anat. 99, 391–413 (doi:10.1002/aja.1000990303) [PubMed]
  • Oakberg E. F. 1956. bDuration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium. Am. J. Anat. 99, 507–516 (doi:10.1002/aja.1000990307) [PubMed]
  • Oakberg E. F. 1971. Spermatogonial stem-cell renewal in the mouse. Anat. Rec. 169, 515–531 (doi:10.1002/ar.1091690305) [PubMed]
  • Oatley J. M., de Avila D. M., Reeves J. J., McLean D. J. 2004. Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue. Biol. Reprod. 71, 494–501 (doi:10.1095/biolreprod.104.027953) [PubMed]
  • Oatley J. M., Reeves J. J., McLean D. J. 2005. Establishment of spermatogenesis in neonatal bovine testicular tissue following ectopic xenografting varies with donor age. Biol. Reprod. 72, 358–364 (doi:10.1095/biolreprod.104.030783) [PubMed]
  • Oatley J. M., Avarbock M. R., Telaranta A. I., Fearon D. T., Brinster R. L. 2006. Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc. Natl Acad. Sci. USA 103, 9524–9529 (doi:10.1073/pnas.0603332103) [PubMed]
  • Oatley J. M., Avarbock M. R., Brinster R. L. 2007. Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on SRC family kinase signaling. J. Biol. Chem. 282, 25 842–25 851 [PubMed]
  • Oatley J. M., Oatley M. J., Avarbock M. R., Tobias J. W., Brinster R. L. 2009. Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal. Development 136, 1191–1199 (doi:10.1242/dev.032243) [PubMed]
  • Ogawa T., Dobrinski I., Avarbock M. R., Brinster R. L. 2000. Transplantation of male germ line stem cells restores fertility in infertile mice. Nat. Med. 6, 29–34 [PubMed]
  • Ohbo K., et al. 2003. Identification and characterization of stem cells in prepubertal spermatogenesis in mice. Dev. Biol. 258, 209–225 (doi:10.1016/S0012-1606(03)00111-8) [PubMed]
  • Ohmura M., Yoshida S., Ide Y., Nagamatsu G., Suda T., Ohbo K. 2004. Spatial analysis of germ stem cell development in Oct-4/EGFP transgenic mice. Arch. Histol. Cytol. 67, 285–296 (doi:10.1679/aohc.67.285) [PubMed]
  • Ohmura M., et al. 2008. Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity. Stem Cells 26, 3237–3246 [PubMed]
  • Orwig K. E., Schlatt S. 2005. Cryopreservation and transplantation of spermatogonia and testicular tissue for preservation of male fertility. J. Natl Cancer Inst. Monogr. 2005, 51–56 (doi:10.1093/jncimonographs/lgi029) [PubMed]
  • Osawa M., Hanada K., Hamada H., Nakauchi H. 1996. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273, 242–245 (doi:10.1126/science.273.5272.242) [PubMed]
  • Oulad-Abdelghani M., Bouillet P., Decimo D., Gansmuller A., Heyberger S., Dolle P., Bronner S., Lutz Y., Chambon P. 1996. Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene. J. Cell Biol. 135, 469–477 (doi:10.1083/jcb.135.2.469) [PMC free article] [PubMed]
  • Pesce M., Wang X., Wolgemuth D. J., Scholer H. 1998. Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation. Mech. Dev. 71, 89–98 (doi:10.1016/S0925-4773(98)00002-1) [PubMed]
  • Raverot G., Weiss J., Park S. Y., Hurley L., Jameson J. L. 2005. Sox3 expression in undifferentiated spermatogonia is required for the progression of spermatogenesis. Dev. Biol. 283, 215–225 (doi:10.1016/j.ydbio.2005.04.013) [PubMed]
  • Reid A., et al. 1995. Leukemia translocation gene, PLZF, is expressed with a speckled nuclear pattern in early hematopoietic progenitors. Blood 86, 4544–4552 [PubMed]
  • Rodriguez-Sosa J. R., Foster R. A., Hahnel A. 2010. Development of strips of ovine testes after xenografting under the skin of mice and co-transplantation of exogenous spermatogonia with grafts. Reproduction 139, 227–235 (doi:10.1530/REP-09-0176) [PubMed]
  • Roosen-Runge E. C., Giesel L. O., Jr 1950. Quantitative studies on spermatogenesis in the albino rat. Am. J. Anat. 87, 1–30 (doi:10.1002/aja.1000870102) [PubMed]
  • Ruggiu M., Speed R., Taggart M., McKay S. J., Kilanowski F., Saunders P., Dorin J., Cooke H. J. 1997. The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 389, 73–77 [PubMed]
  • Ryu B. Y., Orwig K. E., Avarbock M. R., Brinster R. L. 2003. Stem cell and niche development in the postnatal rat testis. Dev. Biol. 263, 253–263 (doi:10.1016/j.ydbio.2003.07.010) [PubMed]
  • Ryu B. Y., Kubota H., Avarbock M. R., Brinster R. L. 2005. Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. Proc. Natl Acad. Sci. USA 102, 14 302–14 307 (doi:10.1073/pnas.0506970102) [PubMed]
  • Sada A., Suzuki A., Suzuki H., Saga Y. 2009. The RNA-binding protein NANOS2 is required to maintain murine spermatogonial stem cells. Science 325, 1394–1398 (doi:10.1126/science.1172645) [PubMed]
  • Sadate-Ngatchou P. I., Payne C. J., Dearth A. T., Braun R. E. 2008. Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice. Genesis 46, 738–742 (doi:10.1002/dvg.20437) [PMC free article] [PubMed]
  • Sakaki-Yumoto M., et al. 2006. The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development. Development 133, 3005–3013 (doi:10.1242/dev.02457) [PubMed]
  • Schlatt S., Ehmcke J., Jahnukainen K. 2009. Testicular stem cells for fertility preservation: preclinical studies on male germ cell transplantation and testicular grafting. Pediatr. Blood Cancer 53, 274–280 (doi:10.1002/pbc.22002) [PubMed]
  • Schrans-Stassen B. H., van de Kant H. J., de Rooij D. G., van Pelt A. M. 1999. Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia. Endocrinology 140, 5894–5900 (doi:10.1210/en.140.12.5894) [PubMed]
  • Seandel M., et al. 2007. Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature 449, 346–350 (doi:10.1038/nature06129) [PMC free article] [PubMed]
  • Shinohara T., Avarbock M. R., Brinster R. L. 1999. beta1- and alpha6-Integrin are surface markers on mouse spermatogonial stem cells. Proc. Natl Acad. Sci. USA 96, 5504–5509 (doi:10.1073/pnas.96.10.5504) [PubMed]
  • Shinohara T., Orwig K. E., Avarbock M. R., Brinster R. L. 2000. Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells. Proc. Natl Acad. Sci. USA 97, 8346–8351 (doi:10.1073/pnas.97.15.8346) [PubMed]
  • Smith L. G., Weissman I. L., Heimfeld S. 1991. Clonal analysis of hematopoietic stem-cell differentiation in vivo. Proc. Natl Acad. Sci. USA 88, 2788–2792 (doi:10.1073/pnas.88.7.2788) [PubMed]
  • Snedaker A. K., Honaramooz A., Dobrinski I. 2004. A game of cat and mouse: xenografting of testis tissue from domestic kittens results in complete cat spermatogenesis in a mouse host. J. Androl. 25, 926–930 [PubMed]
  • Spangrude G. J. 1989. Enrichment of murine haemopoietic stem cells: diverging roads. Immunol. Today 10, 344–350 (doi:10.1016/0167-5699(89)90192-8) [PubMed]
  • Suzuki A., Tsuda M., Saga Y. 2007. Functional redundancy among Nanos proteins and a distinct role of Nanos2 during male germ cell development. Development 134, 77–83 (doi:10.1242/dev.02697) [PubMed]
  • Suzuki H., Sada A., Yoshida S., Saga Y. 2009. The heterogeneity of spermatogonia is revealed by their topology and expression of marker proteins including the germ cell-specific proteins Nanos2 and Nanos3. Dev. Biol. 336, 222–231 [PubMed]
  • Tanaka S. S., Toyooka Y., Akasu R., Katoh-Fukui Y., Nakahara Y., Suzuki R., Yokoyama M., Noce T. 2000. The mouse homolog of Drosophila vasa is required for the development of male germ cells. Genes Dev. 14, 841–853 [PubMed]
  • Tegelenbosch R. A., de Rooij D. G. 1993. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat. Res. 290, 193–200 [PubMed]
  • Tohonen V., Ritzen E. M., Nordqvist K., Wedell A. 2003. Male sex determination and prenatal differentiation of the testis. In The developing testis, vol. 5 (ed. Soder O., editor. ), pp. 1–23 Basel, Switzerland: Karger
  • Tokuda M., Kadokawa Y., Kurahashi H., Marunouchi T. 2007. CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes. Biol. Reprod. 76, 130–141 (doi:10.1095/biolreprod.106.053181) [PubMed]
  • Toyooka Y., Tsunekawa N., Takahashi Y., Matsui Y., Satoh M., Noce T. 2000. Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development. Mech. Dev. 93, 139–149 (doi:10.1016/S0925-4773(00)00283-5) [PubMed]
  • Toyooka J., Tsunekawa N., Akasu R., Noce T. 2003. Embryonic stem cells can form germ cells in vitro. PNAS 100, 11 457–11 462 (doi:10.1073/pnas.1932826100) [PubMed]
  • Tsuda M., Sasaoka Y., Kiso M., Abe K., Haraguchi S., Kobayashi S., Saga Y. 2003. Conserved role of nanos proteins in germ cell development. Science 301, 1239–1241 (doi:10.1126/science.1085222) [PubMed]
  • Tung P. S., Skinner M. K., Fritz I. B. 1984. Cooperativity between Sertoli cells and peritubular myoid cells in the formation of the basal lamina in the seminiferous tubule. Ann. N.Y. Acad. Sci. 438, 435–446 (doi:10.1111/j.1749-6632.1984.tb38304.x) [PubMed]
  • van Bragt M. P., Roepers-Gajadien H. L., Korver C. M., Bogerd J., Okuda A., Eggen B. J., de Rooij D. G., van Pelt A. M. 2008. Expression of the pluripotency marker UTF1 is restricted to a subpopulation of early A spermatogonia in rat testis. Reproduction 136, 33–40 (doi:10.1530/REP-07-0536) [PubMed]
  • van der Wee K. S., Johnson E. W., Dirami G., Dym T. M., Hofmann M. C. 2001. Immunomagnetic isolation and long-term culture of mouse type A spermatogonia. J. Androl. 22, 696–704 [PubMed]
  • Wang P. J., McCarrey J. R., Yang F., Page D. C. 2001. An abundance of X-linked genes expressed in spermatogonia. Nat. Genet. 27, 422–426 (doi:10.1038/86927) [PubMed]
  • Wu Q., Chen X., Zhang J., Loh Y. H., Low T. Y., Zhang W., Sze S. K., Lim B., Ng H. H. 2006. Sall4 interacts with Nanog and co-occupies Nanog genomic sites in embryonic stem cells. J. Biol. Chem. 281, 24 090–24 094 (doi:10.1074/jbc.C600122200) [PubMed]
  • Yamaguchi Y. L., Tanaka S. S., Kasa M., Yasuda K., Tam P. P., Matsui Y. 2006. Expression of low density lipoprotein receptor-related protein 4 (Lrp4) gene in the mouse germ cells. Gene Expr. Patterns 6, 607–612 (doi:10.1016/j.modgep.2005.11.013) [PubMed]
  • Yang J., et al. 2008. SALL4 is a key regulator of survival and apoptosis in human leukemic cells. Blood 112, 805–813 (doi:10.1182/blood-2007-11-126326) [PubMed]
  • Yeh J. R., Zhang X., Nagano M. C. 2007. Establishment of a short-term in vitro assay for mouse spermatogonial stem cells. Biol. Reprod. 77, 897–904 [PubMed]
  • Yeom Y. I., Fuhrmann G., Ovitt C. E., Brehm A., Ohbo K., Gross M., Hubner K., Scholer H. R. 1996. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894 [PubMed]
  • Ying Y., Qi X., Zhao G. Q. 2001. Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways. Proc. Natl Acad. Sci. USA 98, 7858–7862 (doi:10.1073/pnas.151242798) [PubMed]
  • Yoon K. A., Chae Y. M., Cho J. Y. 2009. FGF2 stimulates SDF-1 expression through the Erm transcription factor in Sertoli cells. J. Cell Physiol. 220, 245–256 (doi:10.1002/jcp.21759) [PubMed]
  • Yoshida S., Takakura A., Ohbo K., Abe K., Wakabayashi J., Yamamoto M., Suda T., Nabeshima Y. 2004. Neurogenin3 delineates the earliest stages of spermatogenesis in the mouse testis. Dev. Biol. 269, 447–458 (doi:10.1016/j.ydbio.2004.01.036) [PubMed]
  • Yoshida S., Sukeno M., Nakagawa T., Ohbo K., Nagamatsu G., Suda T., Nabeshima Y. 2006. The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage. Development 133, 1495–1505 (doi:10.1242/dev.02316) [PubMed]
  • Yoshida S., Nabeshima Y., Nakagawa T. 2007. aStem cell heterogeneity: actual and potential stem cell compartments in mouse spermatogenesis. Ann. N.Y. Acad. Sci. 1120, 47–58 (doi:10.1196/annals.1411.003) [PubMed]
  • Yoshida S., Sukeno M., Nabeshima Y. 2007. bA vasculature-associated niche for undifferentiated spermatogonia in the mouse testis. Science 317, 1722–1726 (doi:10.1126/science.1144885) [PubMed]
  • Yoshimizu T., et al. 1999. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev. Growth Differ. 41, 675–684 (doi:10.1046/j.1440-169x.1999.00474.x) [PubMed]
  • Yoshinaga K., Nishikawa S., Ogawa M., Hayashi S., Kunisada T., Fujimoto T. 1991. Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development 113, 689–699 [PubMed]
  • Yuri S., et al. 2009. Sall4 is essential for stabilization, but not for pluripotency, of embryonic stem cells by repressing aberrant trophectoderm gene expression. Stem Cells 27, 796–805 (doi:10.1002/stem.14) [PubMed]
  • Zeng W., Avelar G. F., Rathi R., Franca L. R., Dobrinski I. 2006. The length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts. J. Androl. 27, 527–533 [PubMed]
  • Zhang X., Ebata K. T., Nagano M. C. 2003. Genetic analysis of the clonal origin of regenerating mouse spermatogenesis following transplantation. Biol. Reprod. 69, 1872–1878 (doi:10.1095/biolreprod.103.019273) [PubMed]
  • Zhang J., et al. 2006. Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nat. Cell Biol. 8, 1114–1123 (doi:10.1038/ncb1481) [PubMed]
  • Zheng K., Wu X., Kaestner K. H., Wang P. J. 2009. The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse. BMC Dev. Biol. 9, 38 (doi:10.1186/1471-213X-9-38) [PMC free article] [PubMed]

Articles from Philosophical Transactions of the Royal Society B: Biological Sciences are provided here courtesy of The Royal Society