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Review
. 2012 Apr;92(2):577-95.
doi: 10.1152/physrev.00025.2011.

The Germline Stem Cell Niche Unit in Mammalian Testes

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Free PMC article
Review

The Germline Stem Cell Niche Unit in Mammalian Testes

Jon M Oatley et al. Physiol Rev. .
Free PMC article

Abstract

This review addresses current understanding of the germline stem cell niche unit in mammalian testes. Spermatogenesis is a classic model of tissue-specific stem cell function relying on self-renewal and differentiation of spermatogonial stem cells (SSCs). These fate decisions are influenced by a niche microenvironment composed of a growth factor milieu that is provided by several testis somatic support cell populations. Investigations over the last two decades have identified key determinants of the SSC niche including cytokines that regulate SSC functions and support cells providing these factors, adhesion molecules that influence SSC homing, and developmental heterogeneity of the niche during postnatal aging. Emerging evidence suggests that Sertoli cells are a key support cell population influencing the formation and function of niches by secreting soluble factors and possibly orchestrating contributions of other support cells. Investigations with mice have shown that niche influence on SSC proliferation differs during early postnatal development and adulthood. Moreover, there is mounting evidence of an age-related decline in niche function, which is likely influenced by systemic factors. Defining the attributes of stem cell niches is key to developing methods to utilize these cells for regenerative medicine. The SSC population and associated niche comprise a valuable model system for study that provides fundamental knowledge about the biology of tissue-specific stem cells and their capacity to sustain homeostasis of regenerating tissue lineages. While the stem cell is essential for maintenance of all self-renewing tissues and has received considerable attention, the role of niche cells is at least as important and may prove to be more receptive to modification in regenerative medicine.

Conflict of interest statement

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

FIGURE 1
FIGURE 1
Spermatogenesis in mammalian testes. Testicular parenchyma consists of seminiferous tubules and interstitial tissue. A: cross-section of testicular parenchyma from an adult mouse stained with hematoxylin and eosin. B: schematic recreation of the seminiferous epithelium and interstitial tissue. Germ cell maturation from undifferentiated spermatogonia to elongate spermatids occurs within seminiferous tubules that are bound by a basement membrane. Within seminiferous tubules, the developing germ cells associate with somatic Sertoli cells to make up the seminiferous epithelium. Tight junctions between adjacent Sertoli cells separate the seminiferous epithelium into a basal compartment that houses the spermatogonia and an adluminal compartment, which contains maturing meiotic spermatocytes, haploid spermatids, and spermatozoa. Rigidity of the seminiferous tubules is provided by myoepithelial cells or myoid cells, which line the outside of the basement membrane. Interstitial tissue is located between seminiferous tubules and consists primarily of clusters of Leydig cells that secrete testosterone, the vascular network, and various immune cell populations. C: potential outcomes of SSC division in mammalian testes. During neonatal development of the germline and regeneration of steady-state spermatogenesis after cytotoxic insult, symmetrical self-renewal may predominate to establish a stem cell pool. During steady-state spermatogenesis, a balance of symmetrical self-renewal and differentiation may occur at defined frequencies to maintain a stem cell pool and provide the next cohort of progenitor spermatogonia that are committed to further differentiation. Recent evidence suggests that during steady-state conditions in rodent testes true SSCs and transient amplifying progenitor spermatogonia that have limited proliferative potential before giving rise to committed progenitor are present. This model suggests that asymmetric division of an SSC produces one new SSC and one transient amplifying progenitor.
FIGURE 2
FIGURE 2
Spermatogonial hierarchy in rodents (A) and primates (B). In rodents, the undifferentiated spermatogonial population consists of Asingle (As), Apaired (Apr), and Aaligned (Aal) spermatogonia. Most Aal undifferentiated spermatogonia are capable of giving rise to differentiating spermatogonia at the A1 spermatogonial stage, which develop further into A2–4 spermatogonia followed by intermediate (IN) and B spermatogonia, which initiate meiotic prophase to become preleptotene spermatocytes (PL). The As spermatogonia are widely considered to represent the true SSC population that self-renew and differentiate to produce Apr spermatogonia, which marks the beginning of spermatogenesis. In primates, the undifferentiated spermatogonial population consists of Adark (Ad) and Apale (Ap) spermatogonia. These germ cells have been considered the resting (Ad) and active (Ap) SSC populations. After a set number of divisions that is currently undefined, the Ap spermatogonia give rise to differentiating spermatogonia beginning with B1 spermatogonia followed by B2–4 spermatogonia from which preleoptotene spermatocytes (PL) are derived.
FIGURE 3
FIGURE 3
The functional SSC transplantation assay in mice. Stem cells are defined by the functional ability to maintain and reestablish a tissue lineage. For spermatogenesis, SSCs are defined by an ability to reform spermatogenesis following colonization in a recipient’s testis. For this methodology, a testis cell population can be collected fresh from a donor testis or following a culture period and microinjected into the testes of recipient males that have been depleted of germ cells by treatment with chemotoxic compounds or by using sterile mutant males that lack germ cells (e.g., W/Wv mice). Colonies of donor-derived spermatogenesis can be detected several months later in the recipient testes if SSCs were present in the injected cell suspension. Colonies of donor-derived spermatogenesis can easily be detected and quantified if transgenic donors that express a marker gene (e.g., LacZ or GFP) are used. In particular is the use of Rosa mice as donors which contain the LacZ gene within the Rosa26 locus, thereby providing ubiquitous expression in all germ cell types. Spermatogenesis arising from transplanted Rosa SSCs is easily identifiable in testes of non-Rosa recipient mice following incubation with the X-Gal substrate, which is converted to a blue product by the β-galactosidase enzyme produced from the LacZ gene. Because each reformed colony is clonally derived from an individual SSC, this system provides a quantifiable measure of SSC number in the injected cell suspension. Also, the number of reformed colonies reflects (a measure of) the number of accessible niches within recipient testes that can be colonized by injected donor SSCs.
FIGURE 4
FIGURE 4
Impact of niche developmental stage on support of SSC activity in mice. The functional transplantation assay was used to measure niche activity in testes of adult and prepubertal pup recipient male mice. The number of colonies of reestablished spermatogenesis (red bars) is a reflection of the number of accessible niches for colonization. The length of reestablished colonies of spermatogenesis (blue bars) is a reflection of niche support of SSC expansion upon colonization. Regardless of donor SSC age, a greater number of colonies are formed (9.4×), and each colony is longer (4×) in recipient testes of young prepubertal pups than adults. These findings indicate a greater number of accessible niches and greater niche support of SSC expansion in seminiferous tubules of pups during development of the germline compared with adults in which steady-state spermatogenesis has occurred (131, 132).
FIGURE 5
FIGURE 5
Impact of aging on niche support of SSC activity in testes of mice. A: assessment of SSC number in testes of mice at different time periods of postpubertal aging. These findings suggest an age-related decline in SSC number, which could be a result of impaired SSC function or niche support. B: effect of age on the ability of SSCs to expand in number and regenerate spermatogenesis when serially transplanted into testes of young mice. These findings indicate that aging does not impair SSC activity when the SSCs are continually exposed to niches in testes of young males. Collectively, these findings indicate a decline in niche support of SSC functions in aged males leading to impaired SSC maintenance. For A and B, data were obtained using the functional germ cell transplantation assay to measure SSC numbers in donor testes (A) and biological activity of aged colonizing SSCs within niches of testes from young recipient mice. Colony length is longer in the first transplant because the cells are taken from cryptorchid mice in the original transplantation of the series. These stem cells are likely in a different status than those taken for subsequent transplantations, which came from full spermatogenesis (119).
FIGURE 6
FIGURE 6
Influence of colony stimulating factor-1 (CSF-1) on self-renewal of mouse SSCs. Cultures of undifferentiated spermatogonia were maintained in serum-free medium with supplementation of GDNF and FGF2, which supports self-renewal of SSCs and generation of Apr/Aal-like spermatogonia for long periods of time. Exposure to CSF-1 in addition to GDNF and FGF2 for longer than 2 mo increased SSC number specifically (orange area) without affecting the expansion of total spermatogonial number (green area). Data are fold-difference of SSC and spermatogonial number in CSF-1-treated cultures compared with control cultures exposed to GDNF and FGF2 only, and SSC numbers were determined by transplantation analyses. These findings indicate that exposure to CSF-1 selectively promoted a greater frequency of SSC self-renewal to generate more SSCs at the expense of differentiation to produce Apr/Aal spermatogonia (102).
FIGURE 7
FIGURE 7
Impact of Sertoli cell number on SSC niches in testes of adult mice. Transplantation analyses were used to investigate whether an alteration in Sertoli cell number influences the number of niches accessible for colonization by transplanted SSCs in testes of mice. These experiments utilized Rosa mice as SSC donors which express the LacZ marker transgene in all germ cell types. Thus colonies of spermatogenesis arising from transplanted SSCs within accessible niches of testes from non-LacZ expressing recipient mice could be easily quantified. An increase of ~50% in Sertoli cell number resulted in greater than a 3-fold increase in accessible niches for colonization by transplanted SSCs from LacZ-expressing Rosa donor mice. These findings indicate that Sertoli cells influence SSC niche function in seminiferous tubules of mice (104).
FIGURE 8
FIGURE 8
Schematic depicting the current understanding of determinants of the spermatogonial stem cell (SSC) niche in mammalian testes. Sertoli cells are known to dictate the formation of niche microenvironments and have been shown to produce the growth factors GDNF and FGF2 which regulate SSC proliferation and survival. Leydig cells are a source of CSF-1 which specifically regulates self-renewal of SSCs. The differentiation of SSCs is influenced by BMP4 and Neuregulin 1; however, the source of these factors is currently unknown. It is believed that upon differentiation from SSCs the resulting progenitor spermatogonia (i.e., Apr/Aal) migrate away from the niche and continue to develop as a cohort of maturing germ cells.

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