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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cell Cycle. Author manuscript; available in PMC 2010 July 26.
Published in final edited form as:
PMCID: PMC2909758
NIHMSID: NIHMS217538

Mechanistic Insights into the Regulation of the Spermatogonial Stem Cell Niche

Abstract

Potential therapeutic use of stem cells in the treatment of human diseases depends on our ability to control the balance of their differentiation and self-renewal in vitro and in vivo. The stem cell “niche,” or specialized microenvironment, is now recognized as one of the major contributors to this regulation in many species. Our recent study, which was reported in Nature, was the first to demonstrate that expression of a vertebrate animal transcription factor is essential for the maintenance of a stem cell niche. In that letter, targeted disruption of ERM (ets-related molecule), which was localized only in the somatic support cell of the testis, the Sertoli cell, resulted in failure of self-renewal by spermatogonial stem cells, following the first wave of spermatogenesis. One of the more important conclusions drawn was the realization that regulation of the stem cell niche during the perinatal period, a phase characterized by rapid mitosis of both spermatogonial stem cells and Sertoli cells, differed from that in the adult. It appears that the ERM-regulated pathways are coincident with the termination of Sertoli cell proliferation and commencement of the cycle of spermatogenesis, which is sustained by the same cell that regulates the stem cell niche. Several likely targets for ERM regulation are discussed, as well as their potential implications for increasing our understanding of spermatogonial stem cell activity and the uniqueness of the Sertoli cell's immune privilege and possible utility for the protection of transplanted adult stem cells.

Keywords: stem cell, niche, Sertoli, Ets-related molecule, chemokine, differentiation, GDNF, PLZF

Introduction

In a recent study of spermatogonial stem cells, we demonstrated that Ets-related molecule (ERM) is required for their self-renewal and maintenance of spermatogenesis in the adult mouse.1 This was the first evidence that a transcription factor regulates a stem cell niche in a vertebrate animal. The study found that in testis ERM was localized in the Sertoli cell, the only somatic cell of the seminiferous epithelium, and firmly established that in adult testes Sertoli cells maintain the spermatogonial stem cell niche. However, the molecular mechanisms involved and signaling pathways responsible for this activity have only begun to be answered, despite the critical nature of this Sertoli-germ cell interaction. Spermatogonia are the consummate stem cells because they not only self-renew throughout life, but they have the capacity for immortality by serving as genomic vectors capable of perpetuating the genome from one generation to another. Thus, the spermatogonial stem cell niche provides an excellent model for the study of mechanisms that may be common for the regulation of adult stem cell proliferation and differentiation. In this brief review, we will highlight the significance of data obtained from targeted disruption of the ERM gene and discuss potential implications for future research.

The stem cell niche is a specialized microenvironment provided by supporting cells, which promotes self-renewal and retention of stem cells in their undifferentiated state. The current scientific attention that is given to this biological compartment came about in part because the adult stem cell, although often being pluripotential in its ability to differentiate into multiple cell types, has unique requirements for its maintenance within specific organs and tissues. These requirements include both intrinsic as well as extrinsic signals. The Sertoli cell is recognized as one of those unique support cells, as it provides extrinsic signals for establishment and coordination of the complicated steps associated with spermatogenesis. As early as 1865, this tree-like cell was identified as being “sustentacular”, or the “mother” cell,2 because it formed an intimate physical relationship with germ cells (Fig. 1). However, until recently this label was not completely understood, especially regarding maintenance of the spermatogonial stem cell niche.

Figure 1
The spermatogonial stem cell niche. This electron micrograph shows the physical attachment of a Sertoli cell to a type A spermatogonium in situ, with both cell types lying on a basement membrane that is common with the surrounding peritubular cell. Plasmalemma ...

ERM is Essential for Maintenance of the Adult Spermatogonial Stem Cell

In mice with a targeted deletion of ERM (ERM-/-), spermatogonial stem cells eventually disappear, but this only occurs after completion of the first wave of spermatogenesis.1 By six weeks of age, the germinal epithelium is lost one layer at a time, beginning with spermatogonia and ending with elongated spermatids (Fig. 2). Thus, with maturation of the epithelium more advanced germ cells appear normal, but the underlying new generation of spermatogonia, spermatocytes and eventually spermatids fail to appear, which leads to seminiferous tubules surrounded only by Sertoli cells (Fig. 3). In the normal testis, Sertoli cells maintain stem cell self-renewal and differentiation, but in ERM-/- mice spermatogonial stem cells fail to renew, and these cells are lost through differentiation (Fig. 4). In this first study of ERM inactivation, considerable effort was made to determine in which cell types it was expressed, and the conclusion, based upon at least six different types of data, was that ERM expression is exclusive to Sertoli cells in testis.

Figure 2
ERM -/- testis at 6 weeks of age. One tubule lacks spermatogonia but otherwise appears normal (A); Sertoli cells line the basement membrane (arrows) and other germ cell types fill the epithelium, including pachytene spermatocytes (P), round spermatids ...
Figure 3
A timeline for the spermatogonial stem cell niche. In the wild-type (WT), GDNF is expressed from birth through adult, while ERM is expressed approximately during the period when Sertoli cells cease to divide. In the ERM-/- testis, GDNF continues to be ...
Figure 4
Sertoli cell expression of ERM is essential for maintenance of the spermatogonial stem cell (SSC) niche. In the ERM-/- mice, Sertoli cells do not maintain SSC self-renewal, but rather show a continuation of stem cell differentiation.

ERM, along with PEA3 and ER81, comprise the PEA3 group of transcription factors that are members of the large Ets protein family. Although the mouse testis expresses high levels of ERM mRNA,3,4 and this organ, along with brain, colon and lung, has the highest mRNA levels, its function is not known for any organ. Thus, its role in the Sertoli cell remains unanswered. However, by comparing isolated Sertoli cells from wild-type (ERM+/+) and ERM-/- testes,1 we were able to demonstrate that this transcription factor is unique, not only in its regulation of Sertoli cell factors, but also of spermatogonial cell markers, which coincided with the sequential depletion of germ cell layers (Tables 1 and and22).

Table 1
Effect of targeted disruption of ERM on the expression of germ cell genes
Table 2
Effect of targeted disruption of ERM on the expression of Sertoli cell genes

What is regulated by ERM?

ERM is a Sertoli cell gene, but it has an impact on germ cells as well. Using selective microarray and RT-PCR analyses,1 it was demonstrated that spermatogonia-specific genes had the greatest reduction (3.5- to 14-fold) in expression (Table 1), while genes associated with mature or more differentiated germ cells were unchanged at four weeks of age. However, over time all germ cells are lost in the ERM-/- mice, but the first wave of spermatogenesis appears normal until the spermatogonia begin to disappear. Several genes were also found to be regulated by ERM in Sertoli cells (see appendix Table 41). One of the more significant findings detected by microarray analysis of ERM-/- Sertoli cells was a 9- to 25-fold reduction in several chemokines, including SDF-1 or CXCL-12 (Stromal cell-derived factor), and a tenfold reduction in matrix metalloproteinase-12 (MMP-12). Chemokines are well known for their involvement in cell migration and in particular, hematopoietic stem cell attraction and self-renewal.5-11 Furthermore, new data suggest that chemokines and MMPs may be working together in the regulation of stem cell recruitment and migration.12,13 Thus, it appears that in Sertoli cells, ERM is responsible for maintaining the production of factors common to other organs and tissues, which may help to retain stem cells inside their niches and balance their self-renewal and differentiation pathways. With ERM inactivation, spermatogonial stem cells are not maintained and differentiation is facilitated (Fig. 5), until the germinal epithelium is depleted (Fig. 2).

Figure 5
The stem cell population requires a balance between differentiation, proliferation and self-renewal. During the perinatal period of development, the balance is higher for self-renewal, as the testis size becomes established through Sertoli cell proliferation. ...

The Spermatogonial Stem Cell Niche Changes with Age

The niche has been shown to be essential for maintenance of stem cell populations in the gut, skin, bone marrow and other organs.14-16 Regulation of stem cell development typically depends on supporting cells that not only physically establish the niche but also communicate with stem cells to regulate their proliferation and differentiation. For example, in bone marrow, the stromal fibroblast and osteoblast are the essential supporting cells that regulate development of hematopoietic stem cells.17 Much of our understanding of the stem cell niche came from the study of Drosophila, whose maintenance of germline stem cells is also dependent on signals from a somatic cell, which ensures asymmetric division of stem cells, producing one cell for self-renewal and one for differentiation.15,18,19 In mammals, the somatic Sertoli cell is responsible for maintaining the germinal stem cell.

Until recently, most data suggested that a single Sertoli cell factor, GDNF, was most likely responsible for maintaining the spermatogonial stem cells20-28 and that this activity extended throughout the life of the animal. GDNF is protein member of the TGF-β superfamily that is secreted by Sertoli cells (Table 2). The GDNF knockout is neonatally lethal. Therefore, evidence for its essential role in stem cell maintenance was unconvincing until fetal testes from GDNF knockout mice were transplanted under the back skin of the nude mouse host, thus circumventing neonatal lethality.20 In the transplanted testes, spermatogonial stem cells quickly become depleted, as the germ cells differentiated but also experienced reduced proliferation. Others have shown that mice heterozygous for the GDNF knockout are viable but also have a progressive depletion of testicular stem cells.28 Conversely, mice that over express GDNF have an increased number of undifferentiated spermatogonia.29 Extensive in vitro data also indicate that GDNF stimulates spermatogonial stem cell proliferation without causing differentiation22,23,26,30 and its coreceptor GFRα1 is now used as a marker for their isolation and identification.22,23,31,32

Based on the study of GDNF, one might assume that regulation of spermatogonial stem cells is straightforward and dependent only on the secretion of this Sertoli cell protein. However, two sets of data now suggest that spermatogonia stem cell regulation changes as the testis develops from perinatal to the pubertal age:

  1. The perinatal period is regulated by GDNF. The recent study by Naughton's laboratory20 demonstrated that transplanted fetal GDNF-/- testes do not maintain spermatogonial stem cells nor support proliferation, but rather allow differentiation. The transplanted seminiferous tubules do not enter meiosis and thus never establish spermatogenesis. Sertoli cells do not express GDNF receptor; thus, ERM must be regulated by another pathway.
  2. The pubertal period is dependent on ERM. In ERM-/- mice, spermatogenesis is normal during the perinatal period, with completion of the first wave of spermatogenesis and formation of spermatozoa.1 However, GDNF expression is normal in ERM-/-testes, even during the period when germ cells become depleted. Thus, GDNF is not regulated by ERM and is sufficient to maintain spermatogonial stem cells in the perinatal period, but ERM is essential for stem cell renewal in pubertal and adult testes.

Important Questions Raised by these New Findings

When are ERM-regulated factors expressed?

It now appears that multiple factors are involved in the regulation of spermatogonial stem cells, but the critical windows of time during which these factors are expressed must be determined. From birth to puberty, the seminiferous epithelium shows tremendous change and the testis grows over 80-fold in the mouse. Rapid growth occurs during the perinatal period, as Sertoli cells proliferate quickly after birth and continue to divide until about day 12–16, which establishes the seminiferous epithelial foundation. The number of Sertoli cells determines the ultimate adult testis size and sperm production, because the number of germ cells supported by the Sertoli cell is finite.33 During this period of Sertoli cell mitosis, gonocytes migrate to the basement membrane, become spermatogonial stem cells and also begin rapid proliferation and differentiation. Thus, it is possible that this early time period, during which there is simultaneous proliferation of both Sertoli cells and spermatogonia, may have regulatory requirements that are unique. With each Sertoli cell division a new stem cell niche is created during this period of rapid growth, which would be filled by the continued proliferation of the new spermatogonial stem cells. Stem cells during this period of growth are likely to favor self-renewal (Fig. 4), similar to spermatogonia after irradiation of the testis.34-37 Thus, GDNF is sufficient for stem cell maintenance during the perinatal period because it enhances self-renewal,20-22,32 as would be required in order to fill expanding niches provided by new Sertoli cells. However, ERM is required for the continuation of stem cell maintenance beyond the first wave of spermatogenesis. The reason for this change in niche regulation remains to be determined.

Is ERM expression dependent upon Sertoli cell maturation and cessation of its proliferation?

Sertoli cells cease to divide as they mature. They establish Sertoli-Sertoli junctional complexes and begin physiological support of germ cell differentiation (Fig. 3), which is vital for spermatogonial entrance into meiosis and subsequent spermiogenesis.38,39 This sudden change in both Sertoli cell structure and function occurs around the time that ERM begins to be expressed and may signal the requirement for establishing a balance between spermatogonial stem cell differentiation (an adult need) and self-renewal (required developmentally and in the adult) (Fig. 5). Mature Sertoli cells regulate the simultaneous maintenance of several different types of developing germ cells within a carefully timed cycle of spermatogenesis.40,41 The cycle has been divided into numerous epithelial stages or cellular associations,42,43 with each stage exhibiting specific molecular and hormonal responsive patterns that are unique to the Sertoli and germ cells.44-46 Thus, stage specificity of Sertoli cell function may be a major reason that a factor/s other than GDNF is required for regulation of the stem cell niche in adult testes, compared to the perinatal period.

What are the potential targets for ERM regulation?

As a transcription factor in Sertoli cells, ERM could act through several different pathways, not exclusive to but including the following: (a) enhancing spermatogonia stem cell proliferation through production by the Sertoli cells of growth factors or other molecules that alter the cell cycle in spermatogonial stem cells; (b) balancing asymmetric and symmetric division of the stem cells; (c) modulating the Sertoli-spermatogonial cell junction; (d) altering basement membrane factors or stem cell membrane receptors that interact with basement membrane; and (e) recruiting or attracting stem cells to remain in the niche by increasing Sertoli cell secretion of chemokines. In the next few paragraphs, these ideas will be examined for their potential to explain downstream actions of ERM.

The phenotypic similarities between ERM and GDNF knockouts and the fact that GDNF continues to be expressed in the ERM-/-testis suggests that an overlapping pathway/s may involve these two factors. However, current data suggest that neither factor would be capable of directly regulating the other, but there is the potential for ERM to indirectly alter GDNF activity, especially if such regulation is required in the adult testis to reach equilibrium between stem cell self-renewal and differentiation (Fig. 5). Spermatogonia stem cells express both GFRα1 and RET, which are coreceptors for GDNF. GFRα1 (GDNF Family Receptor α1) contains the ligand-binding region and is bound extracellularly to the germ cell plasmalemma. Since GFRα1 lacks a transmembrane or intracellular region, signaling transduction requires the intracellular tyrosine kinase receptor, RET. Like the GDNF knockout, both GFRα1 and RET knockouts are neonatally lethal, but involvement of GFRα1 and RET in stem cell maintenance was demonstrated by grafting neonatal testes from these mice into nude mice hosts. These studies showed that both GFRα1 and RET are necessary for maintaining undifferentiated spermatogonia stem cells, and knockout of either results in loss of the stem cells and failure of spermatogenesis.20 These data from knockout mice confirm earlier studies with the RET hypomorphic mice, which showed abnormalities in germ cell maturation.47 In addition, these findings are consistent with in vitro studies indicating that treatment of spermatogonial stem cell cultures with soluble GFRα1 promoted cell proliferation.26 Thus, ERM expression with the onset of puberty may be involved in an indirect regulation of the GDNF pathway, which could occur by altering expression of either receptor or other factors that influence their expression.

Taf4b and PLZF (Promyelocytic leukemia zinc-finger) are also essential for spermatogonia stem cell maintenance, but these factors are only found in germ cells. TAF4b is a germ cell specific component of the RNA polymerase complex and plays an important role in transcriptional regulation. Testicular phenotypes of TAF4b and ERM-/- knockouts are quite similar, as they both experience completion of the first wave of spermatogenesis, but undergo progressive losses of subsequent germ cell progenitor cells, leading to Sertoli cell only seminiferous tubules.1,48 In both cases, the loss of stem cells is hormone independent. The difference is found in their exclusive cell type expression; ERM is in Sertoli cells, while TAF4b is only found in germ cells. Impairment produced in the TAF4b knockout testis reflects germ cell deficits exclusively, as the Sertoli cells support spermatogenesis of transplanted WT spermatogonia.48 Based on the depletion of germ cells in the TAF4b knockout mouse, it is not surprising to find that the expression of RET, GDNF's coreceptor, is decreased significantly in the TAF4b-/- germ cells,48 which provides a potential link between ERM, TAF4b and the GDNF pathway.

PLZF is a transcriptional repressor protein that inhibits stem cell differentiation and helps to maintain their presence in the testis and other organs. In hematopoietic cells there is high expression of PLZF in undifferentiated multipotential precursor cells, and low expression in differentiated cells49 PLZF also exerts growth suppressive activities and induces accumulation of cells in G0/G1 of the cell cycle,50,51 possibly through its repression of the protooncogene c-myc.52 Data reporting that PLZF is important for stem cell differentiation are consistent with two recent papers suggesting that PLZF is vital in the regulation of spermatogonia stem cells.53,54 Both the naturally occurring mutant lacking PLZF (Luxoid) and the PLZF knockout mice53,54 have progressive losses of spermatogonia with age. The phenotypic similarities between PLZF and ERM knockouts suggest that PLZF could be downstream of ERM or at the very least that PLZF and ERM deficiencies act through common pathways, which produces in both knockout mice a delayed loss of stem cells.

Finally, ERM-/- mice survive into adulthood without obvious phenotypic abnormalities,1 suggesting there are no major problems with hematopoietic, skin or intestinal stem cells, and these animals have no other obvious structural/functional deficits. Thus, ERM is obligatory for stem cell maintenance only in the testis, as far as we presently know. However, it is now recognized that many of the pathways involved in stem cell maintenance are conserved across species. Therefore, the challenge will be to determine how and for what reasons ERM provides a unique regulation of adult spermatogonial stem cell maintenance. Two important areas of future study that are common to other stem cell niches are the cell-to-cell adhesion junctions that are formed55-57 and the role of basement membrane interactions with both spermatogonia and Sertoli cells.58-61 It is possible that ERM will participate in the regulation of both these components. For example, the formation of adherens junctions62 and cell cycle regulation63 have been associated with Jagged-1 and its receptors Notch 1, 2 and 3. ERM could regulate the Sertoli cell expression of Jagged-1, as its receptors Notch-1, 2 and 3 are found on the spermatogonial membrane.

In conclusion, the Sertoli cell transcription factor ERM is unique in that its expression is required after the first wave of spermatogenesis has been established. A known secretory protein of Sertoli cells, GDNF, is sufficient for maintaining spermatogonial stem cell self-renewal during the perinatal period, but other factors are needed in the adult testis, possibly associated with the cessation of Sertoli cell proliferation and the onset of stage-specific activities that are required for continual output of sperm. Disruption of the ERM pathways may contribute to several unresolved testicular problems. For example, recent work by the Brinster laboratory indicates that the decrease in spermatogenesis with aging reflects a degradation of the stem cell niche, rather than stem cell abnormalities.64 Thus, experimental modulation of ERM expression may help to uncover new methods for the treatment of male infertility or possibly lead to the development of a novel male contraceptive. The discovery of ERM expression in Sertoli cells is also important for a less obvious reason-this cell type exhibits immune privilege and is being cotransplanted with other cell types, such as pancreatic islet beta cells.65-69 Thus, it may be possible to use the Sertoli cell also for the protection of transplanted adult stem cells in specific organs or tissues.

Acknowledgments

We wish to acknowledge the outstanding work by Dr. Chen Chen (now at Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1×5, Canada) on ERM expression in the mouse. The original work was supported by the Howard Hughes Medical Institute (KMM). Our collaborative effort has been supported in part by the following: Subproject CIG-05-111 (RAH) provided by CICCR, a program of CONRAD, Eastern Virginia Medical School; NIH grants ES11590 (PSC), P01AG024387 (PSC), and HD44543 (M-CH). The views expressed by the authors do not necessarily reflect the views of CONRAD or CICCR.

References

1. Chen C, Ouyang W, Grigura V, Zhou Q, Carnes K, Lim H, Zhao GQ, Arber S, Kurpios N, Murphy TL, Cheng AM, Hassell JA, Chandrashekar V, Hofmann MC, Hess RA, Murphy KM. ERM is required for transcriptional control of the spermatogonial stem cell niche. Nature. 2005;436:1030–4. [PMC free article] [PubMed]
2. Hess R, França LR. History of the sertoli cell discovery. In: Griswold M, Skinner M, editors. Sertoli Cell Biology. New York: Academic Press; 2005.
3. Chotteau-Lelievre A, Desbiens X, Pelczar H, Defossez PA, de Launoit Y. Differential expression patterns of the PEA3 group transcription factors through murine embryonic development. Oncogene. 1997;15:937–52. [PubMed]
4. Monte D, Baert JL, Defossez PA, de Launoit Y, Stehelin D. Molecular cloning and characterization of human ERM, a new member of the Ets family closely related to mouse PEA3 and ER81 transcription factors. Oncogene. 1994;9:1397–406. [PubMed]
5. Luttichaux IV, Notohamiprodjo M, Wechselberger A, Peters C, Henger A, Seliger C, Djafarzadeh R, Huss R, Nelson PJ. Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4. Stem Cells Dev. 2005;14:329–36. [PubMed]
6. Kouroumalis A, Nibbs RJ, Aptel H, Wright KL, Kolios G, Ward SG. The chemokines CXCL9, CXCL10, and CXCL11 differentially stimulate G alpha i-independent signaling and actin responses in human intestinal myofibroblasts. J Immunol. 2005;175:5403–11. [PubMed]
7. Sun CX, Downey GP, Zhu F, Koh AL, Thang H, Glogauer M. Rac1 is the small GTPase responsible for regulating the neutrophil chemotaxis compass. Blood. 2004;104:3758–65. [PubMed]
8. Ni HT, Hu S, Sheng WS, Olson JM, Cheeran MC, Chan AS, Lokensgard JR, Peterson PK. High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells. Brain Res Dev Brain Res. 2004;152:159–69. [PubMed]
9. De Falco E, Porcelli D, Torella AR, Straino S, Iachininoto MG, Orlandi A, Truffa S, Biglioli P, Napolitano M, Capogrossi MC, Pesce M. SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells. Blood. 2004;104:3472–82. [PubMed]
10. Hattori K, Heissig B, Rafii S. The regulation of hematopoietic stem cell and progenitor mobilization by chemokine SDF-1. Leuk Lymphoma. 2003;44:575–82. [PubMed]
11. Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR, Crystal RG, Besmer P, Lyden D, Moore MA, Werb Z, Rafii S. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell. 2002;109:625–37. [PMC free article] [PubMed]
12. Son BR, Marquez-Curtis LA, Kucia M, Wysoczynski M, Turner AR, Ratajczak J, Ratajczak MZ, Janowska-Wieczorek A. Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by SDF-1-CXCR4 and HGF-c-met axes and involves matrix metalloproteinases. Stem Cells. 2006 In press. [PubMed]
13. El Ramy R, Verot A, Mazaud S, Odet F, Magre S, Le Magueresse-Battistoni B. Fibroblast growth factor (FGF) 2 and FGF9 mediate mesenchymal-epithelial interactions of peritubular and Sertoli cells in the rat testis. J Endocrinol. 2005;187:135–47. [PubMed]
14. Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: Stem cells and their niche. Cell. 2004;116:769–78. [PubMed]
15. Li L, Xie T. Stem cell niche: Structure and function. Annu Rev Cell Dev Biol. 2005;21:605–31. [PubMed]
16. Yamashita YM, Fuller MT, Jones DL. Signaling in stem cell niches: Lessons from the Drosophila germline. J Cell Sci. 2005;118:665–72. [PubMed]
17. Heissig B, Ohki Y, Sato Y, Rafii S, Werb Z, Hattori K. A role for niches in hematopoietic cell development. Hematology. 2005;10:247–53. [PubMed]
18. Tulina N, Matunis E. Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling. Science. 2001;294:2546–9. [PubMed]
19. Kiger AA, White-Cooper H, Fuller MT. Somatic support cells restrict germline stem cell self-renewal and promote differentiation. Nature. 2000;407:750–4. [PubMed]
20. Naughton CK, Jain S, Strickland AM, Gupta A, Milbrandt J. Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate. Biol Reprod. 2006;74:314–21. [PubMed]
21. Kanatsu-Shinohara M, Ogonuki N, Iwano T, Lee J, Kazuki Y, Inoue K, Miki H, Takehashi M, Toyokuni S, Shinkai Y, Oshimura M, Ishino F, Ogura A, Shinohara T. Genetic and epi-genetic properties of mouse male germline stem cells during long-term culture. Development. 2005;132:4155–63. [PubMed]
22. Hofmann MC, Braydich-Stolle L, Dym M. Isolation of male germ-line stem cells: Influence of GDNF. Dev Biol. 2005;279:114–24. [PMC free article] [PubMed]
23. Buageaw A, Sukhwani M, Ben-Yehudah A, Ehmcke J, Rawe VY, Pholpramool C, Orwig KE, Schlatt S. GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes. Biol Reprod. 2005;73:1011–6. [PubMed]
24. Aponte PM, Soda T, van de Kant HJ, de Rooij DG. Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor. Theriogenology. 2005 [PubMed]
25. Oatley JM, Reeves JJ, McLean DJ. Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture. Biol Reprod. 2004;71:942–7. [PubMed]
26. Kubota H, Avarbock MR, Brinster RL. Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA. 2004;101:16489–94. [PubMed]
27. Tadokoro Y, Yomogida K, Ohta H, Tohda A, Nishimune Y. Homeostatic regulation of germinal stem cell proliferation by the GDNF/FSH pathway. Mech Dev. 2002;113:29–39. [PubMed]
28. Meng X, Lindahl M, Hyvonen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, Saarma M, Sariola H. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science. 2000;287:1489–93. [PubMed]
29. Meng X, de Rooij DG, Westerdahl K, Saarma M, Sariola H. Promotion of seminomatous tumors by targeted overexpression of glial cell line-derived neurotrophic factor in mouse testis. Cancer Res. 2001;61:3267–71. [PubMed]
30. Kanatsu-Shinohara M, Miki H, Inoue K, Ogonuki N, Toyokuni S, Ogura A, Shinohara T. Long-term culture of mouse male germline stem cells under serum-or feeder-free conditions. Biol Reprod. 2005;72:985–91. [PubMed]
31. Ebata KT, Zhang X, Nagano MC. Expression patterns of cell-surface molecules on male germ line stem cells during postnatal mouse development. Mol Reprod Dev. 2005;72:171–81. [PubMed]
32. Hofmann MC, Braydich-Stolle L, Dettin L, Johnson E, Dym M. Immortalization of mouse germ line stem cells. Stem Cells. 2005;23:200–10. [PMC free article] [PubMed]
33. Russell LD, Peterson RN. Determination of the elongate spermatid-Sertoli cell ratio in various mammals. J Reprod Fertil. 1984;70:635–41. [PubMed]
34. van den Aardweg GJ, de Ruiter-Bootsma AL, Kramer MF, Davids JA. Growth and differentiation of spermatogenetic colonies in the mouse testis after irradiation with fission neutrons. Radiat Res. 1983;94:447–63. [PubMed]
35. van Beek ME, Meistrich ML, de Rooij DG. Probability of self-renewing divisions of spermatogonial stem cells in colonies, formed after fission neutron irradiation. Cell Tissue Kinet. 1990;23:1–16. [PubMed]
36. De Rooij DG, Van Dissel-Emiliani FM, Van Pelt AM. Regulation of spermatogonial proliferation. Ann NY Acad Sci. 1989;564:140–53. [PubMed]
37. Meistrich ML, Hunter NR, Suzuki N, Trostle PK, Withers HR. Gradual regeneration of mouse testicular stem cells after exposure to ionizing radiation. Radiat Res. 1978;74:349–62. [PubMed]
38. Russell LD. Form, dimensions, and cytology of mammalian Sertoli cells. In: Russell LD, Griswold MD, editors. The Sertoli Cell. Clearwater: Cache River Press; 1993. pp. 1–37.
39. Russell LD. The blood-testis barrier and its formation relative to spermatocyte maturation in the adult rat: A lanthanum tracer study. Anat Rec. 1978;190:99–111. [PubMed]
40. Joyce KL, Porcelli J, Cooke PS. Neonatal goitrogen treatment increases adult testis size and sperm production in the mouse. J Androl. 1993;14:448–55. [PubMed]
41. Van Haaster LH, De Jong FH, Docter R, De Rooij DG. The effect of hypothyroidism on Sertoli cell proliferation and differentiation and hormone levels during testicular development in the rat. Endocrinology. 1992;131:1574–6. [PubMed]
42. Hess RA. Quantitative and qualitative characteristics of the stages and transitions in the cycle of the rat seminiferous epithelium: Light microscopic observations of perfusion-fixed and plastic-embedded testes. Biol Reprod. 1990;43:525–42. [PubMed]
43. Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann NY Acad Sci. 1952;55:548–73. [PubMed]
44. Parvinen M. Regulation of the seminiferous epithelium. Endocr Rev. 1982;3:404–17. [PubMed]
45. Parvinen M. Cyclic function of Sertoli cells. In: Russell LD, Griswold MD, editors. The Sertoli Cell. Clearwater: Cache River Press; 1993. pp. 39–86.
46. Griswold MD, McLean D. Sertoli cell gene expression and protein secretion. In: Griswold M, Skinner M, editors. Sertoli Cell Biology. New York: Academic Press; 2005. pp. 95–106.
47. Jain S, Naughton CK, Yang M, Strickland A, Vij K, Encinas M, Golden J, Gupta A, Heuckeroth R, Johnson EM, Jr, Milbrandt J. Mice expressing a dominant-negative Ret mutation phenocopy human Hirschsprung disease and delineate a direct role of Ret in spermatogenesis. Development. 2004;131:5503–13. [PubMed]
48. Falender AE, Freiman RN, Geles KG, Lo KC, Hwang K, Lamb DJ, Morris PL, Tjian R, Richards JS. Maintenance of spermatogenesis requires TAF4b, a gonad-specific subunit of TFIID. Genes Dev. 2005;19:794–803. [PubMed]
49. Reid A, Gould A, Brand N, Cook M, Strutt P, Li J, Licht J, Waxman S, Krumlauf R, Zelent A. Leukemia translocation gene, PLZF, is expressed with a speckled nuclear pattern in early hematopoietic progenitors. Blood. 1995;86:4544–52. [PubMed]
50. Shaknovich R, Yeyati PL, Ivins S, Melnick A, Lempert C, Waxman S, Zelent A, Licht JD. The promyelocytic leukemia zinc finger protein affects myeloid cell growth, differentiation, and apoptosis. Mol Cell Biol. 1998;18:5533–45. [PMC free article] [PubMed]
51. Barna M, Hawe N, Niswander L, Pandolfi PP. Plzf regulates limb and axial skeletal patterning. Nat Genet. 2000;25:166–72. [PubMed]
52. McConnell MJ, Chevallier N, Berkofsky-Fessler W, Giltnane JM, Malani RB, Staudt LM, Licht JD. Growth suppression by acute promyelocytic leukemia-associated protein PLZF is mediated by repression of c-myc expression. Mol Cell Biol. 2003;23:9375–88. [PMC free article] [PubMed]
53. Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, Orwig KE, Wolgemuth DJ, Pandolfi PP. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet. 2004;36:653–9. [PubMed]
54. Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet. 2004;36:647–52. [PubMed]
55. Lui WY, Mruk DD, Cheng CY. Interactions among IQGAP1, Cdc42, and the cadherin/ catenin protein complex regulate Sertoli-germ cell adherens junction dynamics in the testis. J Cell Physiol. 2005;202:49–66. [PubMed]
56. Lee NP, Mruk DD, Wong CH, Cheng CY. Regulation of sertoli-germ cell adherens junction dynamics in the testis via the nitric oxide synthase (NOS)/cGMP/protein kinase G (PRKG)/{beta}-catenin (CATNB) signaling pathway: An in vitro and in vivo study. Biol Reprod. 2005;73:458–71. [PubMed]
57. Zhang J, Wong CH, Xia W, Mruk DD, Lee NP, Lee WM, Cheng CY. Regulation of Sertoli-germ cell adherens junction dynamics via changes in protein-protein interactions of the N-cadherin-beta-catenin protein complex which are possibly mediated by c-Src and myotubularin-related protein 2: An in vivo study using an androgen suppression model. Endocrinology. 2005;146:1268–84. [PubMed]
58. Siu MK, Cheng CY. Dynamic cross-talk between cells and the extracellular matrix in the testis. Bioessays. 2004;26:978–92. [PubMed]
59. Dym M. Basement membrane regulation of Sertoli cells. Endocr Rev. 1994;15:102–15. [PubMed]
60. Richardson LL, Kleinman HK, Dym M. Basement membrane gene expression by Sertoli and peritubular myoid cells in vitro in the rat. Biol Reprod. 1995;52:320–30. [PubMed]
61. van der Wee K, Hofmann MC. An in vitro tubule assay identifies HGF as a morphogen for the formation of seminiferous tubules in the postnatal mouse testis. Exp Cell Res. 1999;252:175–85. [PubMed]
62. Mizuhara E, Nakatani T, Minaki Y, Sakamoto Y, Ono Y, Takai Y. MAGI1 recruits Dll1 to cadherin-based adherens junctions and stabilizes it on the cell surface. J Biol Chem. 2005;280:26499–507. [PubMed]
63. Duncan AW, Rattis FM, DiMascio LN, Congdon KL, Pazianos G, Zhao C, Yoon K, Cook JM, Willert K, Gaiano N, Reya T. Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance. Nat Immunol. 2005;6:314–22. [PubMed]
64. Ryu BY, Orwig KE, Oatley JM, Avarbock MR, Brinster RL. Effects of aging and niche microenvironment on spermatogonial stem cell self-renewal. Stem Cells. 2006 In press. [PubMed]
65. Teng Y, Xue WJ, Ding XM, Feng XS, Xiang HL, Jiang YZ, Tian PX. Isolation and culture of adult Sertoli cells and their effects on the function of cocultured allogeneic islets in vitro. Chin Med J (Engl) 2005;118:1857–62. [PubMed]
66. Wang DZ, Skinner S, Elliot R, Escobar L, Salto-Tellez M, Garkavenko O, Khoo A, Lee KO, Calne R, Isaac JR. Xenotransplantation of neonatal porcine islets and Sertoli cells into nonimmunosuppressed streptozotocin-induced diabetic rats. Transplant Proc. 2005;37:470–1. [PubMed]
67. Luca G, Calafiore R, Basta G, Ricci M, Calvitti M, Neri L, Nastruzzi C, Becchetti E, Capitani S, Brunetti P, Rossi C. Improved function of rat islets upon comicroencapsulation with Sertoli's cells in alginate/poly-L-ornithine. AAPS Pharm Sci Tech. 2001;2:E15. [PMC free article] [PubMed]
68. Dufour JM, Rajotte RV, Korbutt GS, Emerich DF. Harnessing the immunomodulatory properties of Sertoli cells to enable xenotransplantation in type I diabetes. Immunol Invest. 2003;32:275–97. [PubMed]
69. Cameron DF, Hushen JJ, Nazian SJ. Formation of insulin-secreting, Sertoli-enriched tissue constructs by microgravity coculture of isolated pig islets and rat Sertoli cells. In Vitro Cell Dev Biol Anim. 2001;37:490–8. [PubMed]