The Elusive Spermatogonial Stem Cell Marker?

Abstract

Commentary on Oatley et al., “Inhibitor of DNA binding 4 is expressed selectively by single spermatogonia in the male germline and regulates the self-renewal of spermatogonial stem cells.”

Spermatogonial stem cells (SSCs) are undifferentiated male germ cells that have the potential to self-renew and differentiate into committed progenitors that maintain spermatogenesis throughout adult life. Rodent SSCs can be identified in whole-mount preparations of testicular seminiferous tubules (initially described by Clermont and Bustos-Obregon [1]) as isolated A_single spermatogonia. These A_single SSCs are present on the basement membrane of seminiferous tubules and can be distinguished from committed, transit-amplifying progenitor spermatogonia (some A_paired and A_aligned chains of 4–16 cells) because committed cells are clonally arranged and connected by intercellular cytoplasmic bridges. Here we define progenitors as undifferentiated spermatogonia that are committed to differentiate and can undergo a finite number of self-renewing divisions. SSCs can be definitively identified in a retrospective manner by observing their ability to produce and maintain spermatogenesis in a functional transplantation assay as initially described by Brinster et al. [2, 3]. It has been widely assumed that all A_singles (as the classically-defined SSCs) possess stem cell properties [4–7]. However, it has never been shown whether all or only some A_singles are bona fide SSCs, and cells with a particular morphological/clonal arrangement (e.g., A_single) may or may not comprise the entire SSC pool [8].

Antibody-based studies using intact (whole-mount) seminiferous tubules have provided an avenue for defining proteins with expression patterns that are limited to classically described undifferentiated spermatogonia and, by extension, spermatagonial stem cells (SSCs). Such studies have revealed that CDH1, GFRA1, LIN28, NANOS2, NANOS3, NEUROG3, ZBTB16, and POU5F1 are expressed by undifferentiated stem and progenitor spermatogonia, including A_single, A_paired, and A_{aligned 4–16} [9–16]. However, a unique marker that distinguishes A_single spermatogonia and perhaps spermatogonial stem cells has been elusive.

In the present issue, Oatley et al. identified ID4 as a marker restricted to A_single spermatogonia in the testis and a protein essential for normal spermatogonial stem cell renewal both in vitro and in vivo [17]. The authors found that only a portion of ID4+ spermatogonia also stained for ZBTB16 (i.e., PLZF) in seminiferous tubule cross sections, indicating the presence of both ID4+/PLZF− and ID4+/PLZF+ A_single spermatogonia (Fig. 1) and perhaps distinct subpopulations of SSCs. From the data provided, it is not known what proportion of total A_single spermatogonia are marked by either ID4 or PLZF, so the possible existence of ID4−/PLZF+ or ID4−/PLZF− A_singles cannot be excluded (Fig. 1). These ID4 data support a growing theory in the field that the pool of rodent A_single spermatogonia (and SSCs) is phenotypically heterogeneous, which may define unique subpopulations of these cells with potential functional differences. Heterogeneity among A_singles has been observed for CDH1, LIN28, NANOS2, and NANOS3 [13, 16, 18, 19]. Heterogeneity among transplantable SSCs has been revealed by studies on NEUROG3 and GFRA1 [10, 20]. While the significance of this heterogeneity is not well understood, it seems clear that a one-size-fits-all definition of A_single spermatogonia and SSCs oversimplifies the stem cell system underlying spermatogenesis.

FIG. 1.

Phenotypic and functional heterogeneity among rodent SSCs. Within the pool of A_single spermatogonia, some express only ID4 (red nuclei), while other A_singles also express PLZF (yellow nuclei), suggesting subpopulations of A_singles and SSCs. It is not known whether some A_singles express PLZF only (green nuclei) or neither marker (not shown). It is also not known whether all or some A_single spermatogonia (or cells beyond A_singles) make up the SSC pool. Based on results of in vitro knockdown and in vivo knockout studies that suggest that ID4 participates in SSC self-renewal, Oatley et al. propose that ID4 delineates different functional subpopulations of SSCs (i.e., quiescent and proliferating).

Oatley et al. used two approaches to posit a functional role for ID4 in SSCs. First, ID4 knockdown in cultured SSCs resulted in stunted stem cell renewal without changes in germ cell amplification, suggesting a role for ID4 in maintenance of the SSC pool (i.e., self-renewal). Second, ID4 null mice showed progressive spermatogenic failure characteristic of a SSC self-renewal defect. Based on these data, Oatley's group suggested a model whereby the phenotypically distinct subpopulations of A_single spermatogonia may represent functionally discrete populations. For example, ID4+/PLZF− A_single spermatogonia may represent A_singles that are quiescent (Fig. 1, red nuclei) and/or that have self-renewing capacity and that these cells acquire PLZF expression on entering the cell cycle (green/yellow nuclei). Under this scenario, presence or persistence of ID4 at the subsequent cell division would favor a self-renewal fate decision, while absence or loss of ID4 might favor commitment to differentiation (e.g., production of A_paired and A_aligned spermatogonia).

The concept of a quiescent population of SSCs is similar to the A₀/A₁ model that was originally advanced for mouse and rat spermatogenesis [1, 21–23]. The A₀/A₁ model holds that normal spermatogenesis is maintained by an “active” pool of SSCs (A₁) and that a quiescent “reserve” pool of A₀ is mobilized following an insult to spermatogenesis. This model is also consistent with the A_dark/A_pale “reserve stem cell” model of primate SSCs [24]. Ultimately, the A₀/A₁ model was supplanted by the A_single model [25, 26], in which a single population of stem cells (A_single spermatogonia) divides regularly but infrequently and gives rise to the spermatogenic lineage. Recent pulse-chase experiments lend support for continual, steady-state renewal of the SSC pool in mice [27] but do not rule out the possibility of a phenotypically distinct, quiescent component. A model of rodent SSC contribution to normal spermatogenesis that incorporates a quiescent intermediate (whether truly “reserve” or simply “long cycling” [28]) would help unify our disparate understanding of spermatogenic lineage development in rodents and primates.

ID4 has the most restricted pattern of expression in undifferentiated spermatogonia observed to date and clearly delineates subpopulations of A_single spermatogonia in the mouse testis. It is likely that molecular heterogeneity in the pool of A_single spermatogonia have functional correlates that will be the focus of ongoing investigations. Whether the stem cell pool resides entirely in the population of A_single spermatogonia or might be extended to include some A_paired or possibly larger chains, as suggested by Nakagawa et al. [8], is the subject of ongoing debate.