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Keywords:

  • adult stem cells;
  • seminiferous tubules;
  • spermatogenesis;
  • spermatogonia;
  • stem cell niche

Abstract

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References

Mammalian testes continually produce a huge number of sperm over a long reproductive period. This constant spermatogenesis is supported by a highly robust stem cell system. Morphological analyses in the 1960s and 70s established the basis of mammalian spermatogenesis and the associated stem cell research. Subsequently, from the 1990s on, functional analyses, which have included post-transplantation colony formation, in vitro spermatogonial culture with persisting stem cell activity, in vivo lineage tracing, and live imaging, and also lines of molecular-genetic analyses, have contributed greatly to our understanding of mammalian spermatogenic stem cells. This review will provide a brief overview of the history of this field and then go on to describe in detail the progress made in recent years.


Anatomy and histology of mammalian spermatogenesis

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References

In the testes of mice and other mammals, spermatogenesis proceeds inside the seminiferous tubules – long, convoluted tubules that have a diameter of approximately 150–200 μm (Fig. 1A) (Russell et al. 1990). Blood vessels run in the interstitial spaces between the tubules but never penetrate the tubules themselves. The blood vessels are surrounded by Leydig cells and other interstitial cells (macrophages and lymphoid epithelial cells) (Fig. 1B) (Russell et al. 1990; Hinton & Turner 1993). Spermatogenesis progresses uniformly over the inner surface of the tubules or the seminiferous epithelium. As shown in Figure 1C–D, two somatic cell types – Sertoli and peritubular myoid cells – together with the basement membrane comprise the main framework of the seminiferous epithelium. Sertoli cells develop a beautiful epithelium inside the tubules that harbors prominent tight junctions between the cells; this structure constitutes the anatomical basis of the blood-testis barrier that separates the basal and adluminal compartments (Fig. 1D). The basal compartment, the space between the tight junction and the basement membrane, is occupied by all the stages of spermatogonia, the mitotic stage of spermatogenic cells (Fig. 1E). On entering the meiotic prophase, the cells translocate to the adluminal compartment across the tight junctions, followed by subsequent movement toward the lumen, and are eventually released as mature spermatozoa. As a result, a beautiful stratification of differentiating germ cells is established among the Sertoli cell epithelium (Fig. 1C–E).

image

Figure 1.  Anatomy of the testis and the process of spermatogenesis in mice. (A) Section of a mature mouse testis in a low magnification. Note that numerous cross-sections of the seminiferous tubules are observed inside tunica albuginea (testicular capsule, shown by arrowheads). Bar, 500 μm. (B) Cross-section of a single seminiferous tubule, which were surrounded by interstitial tissues including blood vessels (yellow arrowheads). Neighboring tubules are shaded. Bar, 100 μm. (C) and (D) represent the actual and schematic organization of the stratified spermatogenesis observed in seminiferous epithelium. The organized cell populations correspond topographically to the stages of spermatogenesis shown in (E). See text for details.

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Spermatogenic cells and the classical stem cell model

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References

What is the nature of the stem cells that facilitates the continuity of spermatogenesis? Before addressing this question, let me explain in more detail the process of spermatogenesis. As shown in Figure 1E, mouse spermatogenesis exhibits a very unique feature: spermatogenenic proliferation/differentiation is accompanied by incomplete cell division that results in the daughter cells remaining interconnected via intercellular bridges (Russell et al. 1990; De Rooij & Russell 2000). The singly isolated spermatogonia (Asingle or As spermatogonia) are considered to be the most primitive cells, and the length of the cell cysts parallels the differentiation status. In mice, after about 10 successive mitotic divisions, theoretically, 1024-cell cysts are believed to enter meiosis in a synchronized manner. This unique feature of interconnected daughter cells is common to germ cells in most (perhaps all) animal species, regardless of the variable numbers of mitotic divisions. It is among the greatest mysteries why differentiating germ cells form cysts.

As regards to the nature of the stem cells, the prevailing model is the “As model,” which was established in 1971 (Huckins 1971; Oakberg 1971; Meistrich & Van Beek 1993). As schematically shown in Figure 2, this model proposes that As cells act as the stem cells, whereas interconnected spermatogonia are committed to differentiation and irreversibly lose the stem cell potential. This model is very comprehensive and has provided the conceptual basis for germ line stem cell biology, regardless of the animal species. However, a logical drawback of this model, which is established on the basis of the morphological observation of fixed specimens, is that it is not from the actual behavior of cells. Accordingly, this model warrants functional evaluation.

image

Figure 2.  A schematic representation of the “As model”. This model suggests that every single As spermatogonium acts as the stem cell. This model also proposes that Apr and subsequent longer cysts are committed to differentiation and do not act as the stem cells.

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Stem cell manipulation: transplantation, purification, and cultivation

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References

Similar to other stem cell systems, the manipulation of mammalian spermatogenic stem cells has provided important breakthroughs. Here, I will present a brief introduction to this research. For a more detailed history and perspective, please refer to the review by Takehashi et al. in this issue.

An outstanding landmark development in this field was the establishment of intra-tubular stem cell transplantation by Brinster and colleagues in 1994 (Brinster & Avarbock 1994; Brinster & Zimmermann 1994; Brinster 2002). After a single cell suspension of the donor testis is transplanted into the recipient’s seminiferous tubules, the injected stem cells will form colonies that exhibit continual spermatogenesis. This experimental system has prompted the purification of stem cells, and it has been demonstrated that stem cell activity is enriched in the so called “undifferentiated spermatogonia,” a collective entity of the primitive As, Apr, and Aal spermatogonia (Fig. 1E) (Shinohara et al. 2000; Ohbo et al. 2003; Tokuda et al. 2007). The establishment of stem cell transplantation has also contributed to the development of long-term spermatogonial cultivation systems with long-lasting colony-forming stem cell activity, namely by Shinohara and colleagues (GS or germline stem cells [Kanatsu-Shinohara et al. 2003]) and Brinster and co-workers (Kubota et al. 2004). These technical improvements will not only extend our biological understanding of stem cells but will also make a significant contribution to the experimental animal and zootechnical sciences (see Takehashi et al.).

Heterogeneity in the stem cell compartment

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References

As mentioned above, so called “undifferentiated spermatogonia,” that is, the As/Apr/Aal population, which comprise <1% of the entire testicular cells, have been shown experimentally to harbor eventually all stem cell activity (Shinohara et al. 2000; Ohbo et al. 2003). Whereas, more advanced, so-called “differentiating spermatogonia” (A1 to B spermatogonia in Figure 1E) has also been suggested to possess a weaker potential of self-renewal (Barroca et al. 2009). This raises the question as to which part of “undifferentiated spermatogonia” contains the stem cells? Furthermore, how is the behavior of these cells in the testis related to the attainment of stem cell functions? Is the stem cell population identical to the As population as the “As model” proposes?

Recently, a number of genes that are expressed in this As/Apr/Aal population have been identified (Meng et al. 2000; Buaas et al. 2004; Costoya et al. 2004; Yoshida et al. 2004; Hofmann et al. 2005; Tokuda et al. 2007; Sada et al. 2009; Suzuki et al. 2009; Zheng et al. 2009). This has led to the finding that, unlike a generally accepted corollary of the “As model,” cysts of the same length are indeed heterogeneous with respect to their gene expression (Fig. 3). Importantly, the fact that As spermatogonia are heterogeneous may suggest that we need to revisit the As model. It will be an intriguing challenge to identify the actual stem cell subset(s) and to reveal their behavior in the context shown in Figure 3.

image

Figure 3.  Heterogeneity of the As/Apr/Aal population revealed by gene expression profile. A schematic representation of the heterogeneity in so-called “undifferentiated spermatogonia” or As/Apr/Aal population. In a classical view, the cyst length (i.e., the morphological entities of As, Apr, and Aal-4 to 16) has been believed as the only heterogeneity among these cells. Recent analyses of gene expression, however, are revealing that cysts with the same number of cells are heterogeneous with regard to their gene expression. Probable combinations of gene expression that may typically represent the cellular heterogeneity are collected from several studies and shown by magenta and green. Genes shown in black are expressed, while those in gray are repressed. Note that relationships among these genes have not been determined precise and there could be a variable combination of expressed genes. This is why the boundaries between the two populations are shown by gradients.

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Intracellular machinery that controls stem cell functions

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References

What mechanism controls the stem cell functions? In this decade, a number of genes have been shown to be involved (Payne & Braun 2006; Seydoux & Braun 2006). Among them, Plzf, a transcriptional repressor, expressed widely in the As/Apr/Aal population, is essential for the maintenance of stem cell activity (Buaas et al. 2004; Costoya et al. 2004) (see also Fig. 3). In mice lacking Plzf, spermatogenesis is initially established but is then gradually lost within months. This phenotype indicates that Plzf plays an essential role in spermatogenic stem cell maintenance, rather than stem cell establishment or the spermatogenesis process per se. It is also suggested that Bcl6b, another BTB/POZ family member, plays essential roles in the stem cell maintenance (Oatley et al. 2006).

In addition, defects in a number of genes resulted in the gradual stem cell dysfunction, including those in the Taf4b (Falender et al. 2005), Utp14b (Bradley et al. 2004; Rohozinski & Bishop 2004), and Etv5 (Chen et al. 2005). It is suggested that Etv5 is required both in the germ cells and the supporting Sertoli cells (Morrow et al. 2007).

Recently, a very important observation was reported, namely that the nanos2 gene product plays central roles in the maintenance of self-renewal in mouse spermatogenic stem cells (Sada et al. 2009; Suzuki et al. 2009). nanos2 is one of the mammalian homologues of Drosophila nanos, which is widely conserved among animal species and plays central roles in several aspects of germ cell development, including the germ line stem cell maintenance (Wang & Lin 2004; Shen & Xie 2009). Sada et al. conducted an elegant set of molecular genetic experiments as if it had been performed in Drosophila, and showed the following: (i) Nanos2 is expressed preferentially in a majority of As and Apr spermatogonia; (ii) constitutive expression of Nanos2 inhibits spermatogenic differentiation and causes the accumulation of stem cell-like immature cells; and (iii) depletion of Nanos2 causes a loss of stem cell maintenance. In addition, fate analysis of the Nanos2-expressing cells using pulse labeling revealed that this population includes many actual stem cells that support normal spermatogenesis (Sada et al. 2009). On the basis of these results, it seems clear that the Nanos2-expressing spermatogonial population is closely related to stem cells and that Nanos2 plays central roles in stem cell function. In medaka fish (Oryzias latipes), a vertebrate species that is distant from mice, Nanos2 expression is similarly restricted to morphologically undifferentiated cell populations in male and female germ cells (Aoki et al. 2009). This raises the possibility that Nanos2 function in stem cells might be evolutionally conserved.

nanos genes encode RNA-binding proteins, and in Drosophlila, nanos acts as a translational repressor of a particular set of genes (Parisi & Lin 2000). Does Nanos2 show similar post-translational control in mammalian spermatogenic stem cells? If it does, which genes are under the control of Nanos2? The controlling mechanisms of stem cells will be revealed through answering these important questions.

Stem cell niche

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References

In the animal body, behavior of stem cells, namely their self-renewal and differentiation, needs to be correctly controlled. Generally speaking, stem cells are under the control of a specialized microenvironment or the stem cell niche. (Spradling et al. 2001; Ohlstein et al. 2004; Morrison & Spradling 2008). However, the cell biological, molecular, and functional nature of this niche is largely unknown in most stem cell systems. Among the few exceptions are the Drosophila germline stem cell niche and the mammalian hematopoietic stem cell niche in bone marrow (Spradling et al. 2001). As mentioned above, seminiferous tubules do not exhibit specific structures, either macroscopically or microscopically, that suggest the location of the stem cell niche. This is in marked contrast to other systems, such as mammalian hair follicles, intestinal crypts, and Drosophila gonads, where the niche region has characteristic structures (Spradling et al. 2001). Moreover, a significant obstacle in the study of mammalian spermatogenesis is that we have yet to identify the actual stem cells unequivocally.

Current knowledge suggests that the As/Apr/Aal population is localized in an area adjacent to the blood vessels and interstitium that surround the seminiferous tubules, and that they migrate out of this region upon differentiation into A1 spermatogonia (Chiarini-Garcia et al. 2001, 2003; Yoshida et al. 2007b). This region could be specialized to provide a microenvironmental niche for As/Apr/Aal spermatogonia, and for stem cells as well (Fig. 4).

image

Figure 4.  The presumptive niche for the As/Apr/Aal population. A model of the microenvironmental niche for so-called “undifferentiated spermatogonia” or the As/Apr/Aal population. Although the seminiferous tubules are uniform around their circumference and along their length, the region adjacent to the surrounding blood vessels and interstitial cells is suggested to be special and to regulate the activity of the As/Apr/Aal population. (Modified from Yoshida 2008[© Kyoritsu Shuppan]).

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Further investigations will hopefully reveal the functions and mediating molecular mechanisms of the local components, that is, Sertoli cells, myoid cells, basement membrane, blood vessels, and/or interstitial cells.

Extracellular controlling factors

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References

Although the cellular and molecular nature of the stem cell niche are yet to be elucidated, there is no doubt that extracellularly secreted factors play essential roles in the stem cell-niche interactions. GDNF (glial cell line-derived neurotrophic factor) signaling is known to be essential for stem cell maintenance. GDNF is secreted by Sertoli cells (Tadokoro et al. 2002), and GFRα1 and Ret, the GDNF receptor components, are expressed in a subset of spermatogonia that is related to the stem cells (Meng et al. 2000; Tokuda et al. 2007; Hofmann 2008; Suzuki et al. 2009). Mutants in Gdnf locus, similar to Plzf and other abovementioned mutants, show gradual loss of spermatogenetic integrity (Meng et al. 2000). Moreover, in vitro, the addition of GDNF to the culture media is essential for the maintenance of spermatogonia with stem cell potential (Kubota et al. 2004; Kanatsu-Shinohara et al. 2003).

Another extracellular factor that may control stem cell function is CSF1 (colony stimulating factor 1, also known as granulocyte-colony stimulating factor, G-CSF) (Kokkinaki et al. 2009; Oatley et al. 2009). Csf1 is expressed in interstitial cells and some peritubular myoid cells, which are among the candidate niche somatic cells (Fig. 4), and increases the stem cell activity in spermatogonia that are maintained in culture in the presence of GDNF (Oatley et al. 2009). Intriguingly, Csf1 enhances the colony-forming activity without influencing the growth rate.

In in vitro cultures, the effects of a number of soluble factors have been investigated. Among these, bFGF (basic fibroblast growth factor, also known as FGF2) action is prominent (Kubota et al. 2004; Kanatsu-Shinohara et al. 2003), although its in vivo role is not clear.

How are these and other unidentified secreted factors involved in the niche function and how do they control stem cell behavior in vivo? What is the basis of the different actions between factors? How do their downstream events affect the intracellular machinery and control the stem cell status? These are important questions that need to be addressed in future investigations.

Stem cell reversibility

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References

Things may not, however, be quite so simple as the foregoing might suggest. When we trace the behavior of the Ngn3-expressing cells, which appear to be relatively “differentiating” within the “undifferentiated” As/Apr/Aal population (Fig. 3), a majority of this population differentiates without self-renewal, whereas only a small proportion function as long-lasting stem cells (Nakagawa et al. 2007; Yoshida et al. 2007a). However, they have the potential to switch back into self-renewing stem cell mode during post-insult regeneration or post-transplantation colony formation. This indicates that, in addition to the actually self-renewing stem cells (“actual stem cells”), the system includes “potential stem cells” that are committed to differentiation but retain stem cell potential (Potten & Loeffler 1990; Nakagawa et al. 2007). Ngn3 expression marks this population, the stem cell potential of which could be captured in response to the tissue conditions. A similar situation has been observed in Drosophila germ line stem cell systems (Brawley & Matunis 2004; Kai & Spradling 2004; Fuller & Spradling 2007). This illustrates the stratified and reversible nature of the stem cell system, which is in contrast to the classical view of stem cells that can be defined uniformly, and that once committed to differentiation irreversibly lose the potential of self-renewal.

Spermatogenesis in other mammals

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References

I have summarized the findings on spermatogenic stem cells that were obtained mainly from mice and other rodents. Please note however, that many of the findings in this field have not been introduced in this review, particularly those in other species. A common feature in mammals and other amniotes (birds and reptiles) is that spermatogenesis occurs in seminiferous tubules. It may be that the basic system is common among these different species, although species-specific modifications could also be involved. For example, in primates, the most primitive population of spermatogonia (type A spermatogonia) are classified into Apale and Adark based on nuclear morphology (Meistrich & Van Beek 1993; Ehmcke et al. 2005). This is a criterion distinct from the cyst length – cysts with the same length may be either Apale or Adark. Does this indicate a heterogeneity that is essentially the same as that visualized in the mouse system by gene expression as shown in Figure 3? Or could this be an evolutionary feature specific to primates? These questions become even more interesting when we ask how these features are related to species-specific longevity (reproduction period) or unique reproductive strategies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References

Thanks to Y. Kitadate, K. Hara, H. Mizugushi-Takase, and R. Sugimoto for comments to this manuscript, and Y. Kuboki for help in preparation of the figures. Studies by our group are partly supported by a Grant-in-Aid for Scientific Research (KAKENHI) on Innovative Areas, “Regulatory Mechanism of Gamete Stem Cells.”

References

  1. Top of page
  2. Abstract
  3. Anatomy and histology of mammalian spermatogenesis
  4. Spermatogenic cells and the classical stem cell model
  5. Stem cell manipulation: transplantation, purification, and cultivation
  6. Heterogeneity in the stem cell compartment
  7. Intracellular machinery that controls stem cell functions
  8. Stem cell niche
  9. Extracellular controlling factors
  10. Stem cell reversibility
  11. Spermatogenesis in other mammals
  12. Acknowledgements
  13. References