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Proc Natl Acad Sci U S A. Dec 22, 2009; 106(51): 21672–21677.
Published online Dec 14, 2009. doi:  10.1073/pnas.0912432106
PMCID: PMC2799876
Cell Biology

Prepubertal human spermatogonia and mouse gonocytes share conserved gene expression of germline stem cell regulatory molecules

Abstract

In the human testis, beginning at ≈2 months of age, gonocytes are replaced by adult dark (Ad) and pale (Ap) spermatogonia that make up the spermatogonial stem cell (SSC) pool. In mice, the SSC pool arises from gonocytes ≈6 days after birth. During puberty in both species, complete spermatogenesis is established by cells that differentiate from SSCs. Essentially pure populations of prepubertal human spermatogonia and mouse gonocytes were selected from testis biopsies and validated by confirming the presence of specific marker proteins in cells. Stem cell potential of germ cells was demonstrated by transplantation to mouse testes, following which the cells migrated to the basement membrane of the seminiferous tubule and were maintained similar to SSCs. Differential gene expression profiles generated between germ cells and testis somatic cells demonstrated that expression of genes previously identified as SSC and spermatogonial-specific markers (e.g., zinc-finger and BTB-domain containing 16, ZBTB16) was greatly elevated in both human spermatogonia and mouse gonocytes compared to somatic cells. Several genes were expressed at significantly higher levels in germ cells of both species. Most importantly, genes known to be essential for mouse SSC self-renewal (e.g., Ret proto-oncogene, Ret; GDNF-family receptor α1, Gfrα1; and B-cell CLL/lymphoma 6, member B, Bcl6b) were more highly expressed in both prepubertal human spermatogonia and mouse gonocytes than in somatic cells. The results indicate remarkable conservation of gene expression, notably for self-renewal genes, in these prepubertal germline cells between two species that diverged phylogenetically ≈75 million years ago.

Keywords: mouse spermatogonia, spermatogenesis

Spermatogonial stem cells (SSCs) are the foundation for spermatogenesis and are capable of both self-renewal and production of daughter cells that differentiate into spermatozoa. During embryonic development, primordial germ cells (PGCs) migrate to the genital ridge and subsequently differentiate into gonocytes (1, 2). Following birth in the mouse, which has a short (≈3 weeks) prepubertal period, gonocytes undergo a transition to SSCs or develop directly to type A1 spermatogonia, an early differentiation stage, by day six of life (2). However in humans, which have a long (≈12 years) prepubertal period, the gonocytes are gradually replaced in the first 2–3 months by adult dark (Ad) and adult pale (Ap) spermatogonia that are thought to represent the reserve and active SSC pool, respectively (2, 3). Beginning at about age 5 years, these Ad and Ap spermatogonia undergo a modest activation, particularly to type B spermatogonia, that represent ≈10% of total spermatogonia by age 10. During puberty, the SSCs in both human and mouse provide the foundation, through self-renewal and differentiation to daughter cells, for spermatogenesis and fertility.

The study of rodent gonocytes and SSCs was previously hampered by the lack of techniques for purification and long-term in vitro maintenance. However, over the past 15 years, robust methods were developed for rodent germ cell transplantation and SSC culture conditions (1, 48). These techniques led to the characterization of many aspects of SSC biology, including the identification of glial cell line-derived neurotrophic factor (GDNF) as the main regulator of rodent SSC self-renewal (7, 8). GDNF binds to the c-Ret receptor tyrosine kinase (RET) in combination with the cofactor GDNF-family receptor α1 (GFRα1) to initiate intracellular signaling in SSCs (7, 9, 10). Using SSC culture and transplantation in conjunction with combinations of GDNF withdrawal, microarray analysis, small interference RNA (siRNA), and signaling molecule inhibition, several GDNF-regulated genes involved in SSC self-renewal were identified and studied in the mouse, including B-cell CLL/lymphoma 6, member B (Bcl6b), Ets variant gene 5 (Etv5), and LIM homeobox 1 (Lhx1) (10). Three additional GDNF-regulated genes, basic helix–loop–helix family, member e 40 (Bhlhb2), homeobox C4 (Hoxc4), and Tec protein tyrosine kinase (Tec) were validated in rat SSCs (11). Among these six genes, Bcl6b and Etv5 have now been identified as important by several studies and appear to play a central role in rodent SSC self-renewal (1013). GDNF also regulates downstream signaling and ultimately rodent SSC maintenance and self-renewal, which involves phosphatidylinositol 3-kinase/serine-threonine kinase AKT family (PI3K/AKT), and Src family kinase (SFK) signaling mechanisms (1416). In contrast to rodent SSCs, little is known regarding the mechanisms regulating primate SSC function. This lack of knowledge is in part due to the absence of techniques for identification and isolation of essentially pure populations of primate SSCs for in vitro study. Several groups have attempted to characterize gene expression in human testes. However, the lack of purified cell populations made interpretation of results difficult (17, 18).

To develop an understanding of SSCs and their regulation in the human germline, essentially pure populations of prepubertal human spermatogonia were isolated and their gene expression profile determined. Parallel studies were performed on mouse gonocytes for comparison, because extensive data exists regarding mouse SSCs and their regulation. Moreover, a similarity in self-renewal and survival mechanisms between human and mouse SSCs may exist because transplantation of testis cells from nonrodent species, including human, into testes of immunodeficient mice allowed the maintenance and limited replication of spermatogonia in the recipient seminiferous tubules for periods of 6–12 months (1, 19). Thus, comparison of prepubertal human spermatogonia and mouse gonocyte gene expression profiles could confirm the existence of regulatory conservation between human and mouse in male germline cells, provide details regarding species-specific gene expression patterns, potentially allow for the extrapolation of our knowledge about mouse SSCs to the human germline, which is difficult to study, and impact our understanding of human male fertility and infertility.

Results

Human and Mouse Germ Cell Populations Are Predicted by Morphology and Immunostaining.

Before selection of prepubertal human spermatogonia and mouse gonocytes for oligonucleotide microarray analysis, morphology of the germ cells was confirmed using immunostaining. In prepubertal testes from a 9-year-old human (Fig. 1A) and a 3-day-old mouse (Fig. 1B) germ cells were large in diameter and near the basement membrane of the seminiferous tubules. The prepubertal period in human is ≈12 years and in mouse is ≈3 weeks, and the relative immature status of both human and mouse testis is confirmed by the large size of the germ cells and the absence of spermatogenesis. In the mouse, by about day 6, the gonocytes convert to SSCs or type A1 spermatogonia, and both reside on the basement membrane, are smaller than the gonocytes and resemble more differentiated germ cell stages (2, 3). In human, Ad and Ap spermatogonia persist as large cells comprising 80–90% of the germ cells until puberty begins (2, 3). Then, the Ad and Ap stem cells become a small percentage of the total germ cell population and are difficult to distinguish from differentiating germ cells. Thus, unequivocal separation of SSCs from other germ cells once differentiation begins is not possible, and these prepubertal human spermatogonia and mouse gonocytes present an opportunity to potentially isolate essentially pure populations of human SSCs and mouse gonocytes, which include the immediate precursor of mouse SSCs.

Fig. 1.
Germ cells in prepubertal human (age 9 years) and mouse testes (age: 3 days). Histological cross sections of human (A) and mouse (B) testis show large cells (arrows) resting near the basement membrane of the seminiferous tubule. Immunohistochemical staining ...

To confirm that the large cells located in the seminiferous tubules were indeed prepubertal human spermatogonia and mouse gonocytes, histological sections were stained for characteristic germ cell marker proteins. ZBTB16 (previously known as PLZF) (20, 21) is highly expressed in gonocytes and spermatogonia, including SSCs, but not in later differentiation stages of spermatogenesis, and the protein was found to be strongly immunostained in the large cells in the seminiferous tubules of both human and mouse testes (Fig. 1 C and D). Other characteristic spermatogonial proteins also stained in prepubertal human spermatogonia and mouse gonocytes (Fig. S1). In contrast, GATA-binding protein 4 (GATA4), which is found in Sertoli cells but not germ cells (22), was not present in the large cells (Fig. 1 E and F). These data demonstrate that the large cells in the seminiferous tubules in both species are indeed immature germ cells. Following digestion of the testis tissue to a single cell suspension, immunostaining for the presence of germ cell and Sertoli cell-specific markers confirmed germ cell markers were expressed exclusively in large round cells from both human and mouse testes (Fig. S2). GATA4 was not detected in any large cells, and was exclusively found in smaller cells.

Prepubertal Human Spermatogonia and Mouse Gonocytes Can Be Selected and Purified from Testis Cell Suspensions.

Because of the difference in size and morphological characteristics between the immature germ cells and somatic cells, micromanipulation techniques to select these two cell types from single cell suspensions isolated from prepubertal human and mouse testes were developed (SI Materials and Methods). The selected populations of germ cells and somatic cells were distinctly homogenous and essentially pure, as demonstrated by immunofluorescence for marker proteins (Fig. S3). Germ cells were significantly larger than somatic cells for both human (14.8 ± 0.2 and 8.7 ± 0.2 μm, respectively, n = 10, P < 0.05) and mouse (12.7 ± 0.3 and 8.7 ± 0.3 μm, respectively, n = 10, P < 0.05) and prepubertal human spermatogonia were significantly larger than mouse gonocytes (P < 0.05). Selected germ cells from both human and mouse testes were positive for ZBTB16, as well as for three additional spermatogonial and germ cell markers, ubiquitin carboxyl-terminal esterase L1 (UCHL1; previously known as PGP9.5), dead (Asp-Glu-Ala-Asp) box polypeptide 4 (VASA; also known as DDX4), and deleted in azoospermia-like (DAZL) (2325), but were negative for GATA4 (Fig. S3). When 820 selected large cells from human and mouse testes were stained for germ cell markers, 811 were stained (98.9%). In contrast, selected populations of somatic cells were negative for ZBTB16, UCHL1, VASA, and DAZL and some are positive for GATA4 (Fig. S3). When 820 selected somatic cells from human and mouse testes were stained for germ cell markers, none were stained (0%). Collectively, these data indicate that micromanipulator selection of germ cells from prepubertal human testis and neonatal mouse testis cell suspensions is an efficient technique for the enrichment of prepubertal human spermatogonia and mouse gonocytes, which provides essentially pure populations of cells for analysis.

Transplantation of Selected Testis Germ Cells Demonstrates Stem Cell Potential.

Germ cells from neonatal mouse testes, gonocytes, have stem cell potential (4, 26). To prove unequivocally that isolated germ cells are indeed gonocytes, populations of selected putative somatic cells and gonocytes from ROSA26 transgenic mice that express LacZ in all cells, including germ cells, were transplanted into recipient mouse testes. Two months after transplantation, testes were evaluated using 5-bromo-4-chloro-3-indolyl b-d--galactoside (X-gal) staining. No spermatogenic colonies were observed in any testis transplanted with somatic cells (n = 8 testes; Fig. S4). In contrast, colonies were readily observed in testes transplanted with putative gonocytes (n = 8 testes; Fig. S4), thus demonstrating that large cells selected from digested mouse neonatal testes are gonocytes with stem cell potential.

When adult human testis cells are transplanted to immunodeficient mice the somatic cells are lost, as above with mouse testis cell transplantation (19). However, the transplanted human SSCs remain on the recipient mouse seminiferous tubule basement membrane for periods up to 6 months as single cells, with an occasional doublet or slightly higher number of germ cells, representing slow division (19). Differentiation of the human SSCs to later germ cell stages does not occur in mouse seminiferous tubules. When 10 μL of a testis cell suspension from a prepubertal boy (age: 10 years) containing human prepubertal spermatogonia (≈150 cells) plus somatic cells (≈850 cells) was transplanted to each testis of immunodeficient nude mice and the testes (n = 6) of the recipients examined 3 to 6 months later, germ cells were found as singlets or doublets on the seminiferous tubule basement membrane (Fig. 2A). These results indicate that the spermatogonia in the testis cell suspension of the prepubertal boy migrate to the basement membrane of the mouse recipient seminiferous tubule and are maintained as germ cells in a manner similar to transplanted adult human SSCs (19). The appearance of the human cells on the basement membrane after 3–6 months resembles that of mouse gonocytes in the first few days after transplantation (Fig. 2B) suggesting that the prepubertal human spermatogonia home to the basement membrane but are unable to differentiate. UCHL1 immunostaining identifies these cells as spermatogonia(Fig. 2A and Fig. S4). The species origin of the cells was confirmed using an anti-baboon antibody known to stain human germ cells (Fig. S4) (19). Moreover, any residual endogenous mouse SSCs in a recipient mouse would form a dense network of spermatogonia and a colony of spermatogenesis (Fig. S4).

Fig. 2.
Colonization of recipient mouse seminiferous tubules by transplanted donor testis cells at posttransplantation times of approximately 4 months for human, and 2 days or 2 weeks for mouse. Whole-mount staining of transplanted human germ cells with anti-UCHL1 ...

To determine whether the prepubertal human spermatogonia were capable of further differentiation in mouse testes, we made use of a modified Busulfan-treated mouse recipient in which the endogenous Sertoli cells are removed by injecting cadmium into the seminiferous tubules (27), After transplantation of a mouse testis cell suspension into such recipients, donor-derived minitubules and spermatogenesis are established in the recipient tubules (27). When a prepubertal human testis cell suspension from a 10-year-old boy was injected into the tubules of immunodeficient mouse recipients treated with cadmium, after 4 months, approximately seven times as many prepubertal human spermatogonia colonization sites were observed (Table S1). In addition, small colonies of three to six human germ cells could be identified, similar to initial SSC proliferation in the 2 weeks after mouse gonocyte transplantation to recipients (Fig. 2 C and D). No human spermatogonia differentiation or spermatogenesis beyond the small colonies were found (SI Discussion).

Oligonucleotide Microarray Analysis Reveals Genes Expressed in Prepubertal Human Spermatogonia and Mouse Gonocytes.

RNA from selected populations of germ cells and somatic cells from human and mouse testes was subjected to oligonucleotide microarray analysis. This analysis showed that 10,809 and 6,076 gene probes were expressed at significantly different levels between germ cells and somatic cells from prepubertal human and mouse testes, respectively. Of these probes with significantly different expression levels (P < 0.05), 3909 and 2115 were expressed 2-fold or higher in prepubertal human and mouse germ cells, respectively, relative to somatic cells. In addition, 44 and 6 gene probes were expressed 100-fold or higher in human and mouse germ cells, respectively, compared to somatic cells. When the top 100 genes with enriched expression in germ cells were compared between human (Table S2) and mouse (Table S2) samples, seven of these genes including Dazl, were conserved between prepubertal human spermatogonia and mouse gonocytes (Table S3). Expression and enrichment of these seven genes were confirmed using qRT-PCR (Table S3). The specificity of this list to germ cells is validated by the presence of DAZL, which previously has been demonstrated to be characteristically expressed by the germline (25). The two most differentially expressed of the conserved genes, embryonic lethal, abnormal vision, Drosophila-like 2 (Hu antigen B) (Elavl2) and serine/threonine kinase 31(Stk31) were selected for further analysis to confirm their presence in germ cells and validate the oligonucleotide microarrays. Expression of Elavl2 has previously been observed in testis tissue; however, this expression was not characterized (28). Stk31 was recently identified as a novel cancer/testis antigen expressed in adult mouse spermatogonia (29). In human testis, Elavl2 and Stk31 were both expressed in germ cells (Fig. S1). ELAVL2 protein was localized to the nucleus, whereas STK31 was localized to the cytoplasm. ELALVL2 was also expressed in mouse gonocytes; however, no antibodies exist for localizing mouse STK31.

Comparison of Gene Expression between Prepubertal Human Spermatogonia and Mouse Gonocytes Reveals a High Level of Conservation.

Many relevant genes that previously have been reported to be enriched in germ cells were expressed in selected populations of both prepubertal human spermatogonia and mouse gonocytes (Table 1). These genes are important for several aspects of male germ cell biology. Specific extracellular surface marker genes that have been used to enrich for germline stem cells were conserved and expressed significantly higher in germ cells than in somatic cells including, EpCam for human and mouse (8), Gpr125 for human (30), and Itga6 for mouse (31). Several intracellular marker genes associated with germ cell function were also expressed higher in prepubertal human spermatogonia and mouse gonocytes. These included, Dazl, Vasa (24), Zbtb16, TATA box-binding protein (TBP)-associated factor (Taf4b) (32), and Uchl1 (23) but not neurogenin 3 (33). While the oligonucleotide microarray analysis failed to show enrichment of Zbtb16 and Uchl1 gene expression in prepubertal human spermatogonia, protein staining and qRT-PCR indicated the presence of high levels of proteins (Fig. 1 and Figs. S1 and S2) and mRNA (Table 1, legend) for these genes.

Table 1.
Expression of genes in prepubertal human spermatogonia and mouse gonocytes that were previously identified in mice as enriched in spermatogonial stem cells relative to testis somatic cells

Most important, the expression of genes previously demonstrated to be essential for GDNF regulation of mouse and rat SSC self-renewal were also conserved between prepubertal human spermatogonia and mouse gonocytes (Table 1). These genes include the cognate receptors for GDNF, Ret and GFRα1, as well as for the GDNF regulated transcription factors Bcl6b and Etv5 (10, 11), which are all critical for SSC self-renewal. Etv5 is reported to be expressed in mouse Sertoli cells (34), and thus, the signal is high in the microarray data for mouse somatic cells as well as for gonocytes, resulting in an insignificant enrichment calculation for mouse gonocytes. Nonetheless, it has been established that Etv5 is highly expressed in mouse SSCs and critical for self-renewal (10). In contrast, genes (Nanog, Pou5f1, and Sox2) known to be important for self-renewal and pluripotency of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were not expressed at high levels or greatly enriched in prepubertal human spermatogonia and mouse gonocytes (Table S4) (35, 36). An exception appears to be lin-28 homolog B (Caenorhabditis elegans) (Lin28b) in prepubertal human spermatogonia and lin-28 homolog (C. elegans) (Lin28) in mouse gonocytes (Table S4) that are expressed in pluripotent cells and at relatively high levels in the germ cells from prepubertal testes, which may suggest a role for these genes in the biology of these germ cells (SI Text). Thus, prepubertal human spermatogonia and mouse gonocytes share a remarkable similarity in gene expression for surface and intracellular markers, as well as in gene expression for molecules known to be critical to mouse and rat SSC survival and self-renewal, but little similarity in expression of the genes critical for ESC and iPSC pluripotency and self-renewal.

GDNF Is Important for Prepubertal Human Spermatogonia Maintenance.

When mouse gonocytes are placed in serum-free culture medium containing GDNF and GFRα1 on STO (SIM mouse embryo derived thioguanine and ouabain resistant) feeder cells, in conditions that support mouse SSC self-renewal, they quickly form germ cell clumps that contain a high percentage of cells with stem cell potential and the SSCs continue to multiply (7). However, these conditions do not support prepubertal human spermatogonia maintenance, and the human germ cells quickly detach from the feeder cells and are lost. Serum-free medium for culture of rat gonocytes and SSCs is slightly modified from the mouse medium, with several components increased in concentration (8). When this medium is used for prepubertal human spermatogonia culture and the feeder cells are changed to C166 (ATCC), the human germ cells can be maintained for at least 19 days but do not form germ cell clumps (Fig. S5). However, if GDNF and GFRα1 are omitted from the medium, the germ cells quickly detach from the feeder cells and are lost (Fig. S5). By the end of 1 week in culture, 80% of the prepubertal human spermatogonia have detached in medium lacking GDNF and GFRα1 compared to medium with the growth factors (Fig. S5). Clearly, the GDNF signaling pathway is important for prepubertal human spermatogonia maintenance, but other mechanisms are necessary for continual self-renewal and formation of germ cell clumps.

Discussion

Little is known about the biology and regulation of human germline cells, particularly regarding maintenance and regulation of SSCs, which are the foundation of spermatogenesis throughout adult life. The oligonucleotide microarray analyses provided compelling data regarding the similarity of prepubertal human spermatogonia and mouse gonocytes. Remarkably, seven of the 100 most highly differentially expressed genes between germ cells and somatic cells were conserved in human and mice, despite phylogenetic divergence of these species approximately 75 million years ago, attesting to the fundamental importance of germ cells in species evolution (37). While indicative of a high degree of conservation, a specific role for these seven genes in germ cell biology has not been identified. DAZL is present in germ cells, but its function is unclear. More informative is the conservation of expression of genes already known to be present and often of functional importance in germ cells, particularly SSCs. The expression of genes for four surface protein markers found on SSCs was significantly enriched in the germ cell populations. In mice, EPCAM has previously been identified on neonatal male germ cells and in adult testes only on spermatogonia (38). In rat, EPCAM is an excellent, and perhaps the best, surface antigen for enrichment of rat gonocytes and SSCs of pups (8). The expression of Gpr125 is present in prepubertal human spermatogonia but not mouse gonocytes. However, GPR125 has been reported to be present on mouse spermatogonia and useful for selection (30). Expression of Itga6 is enriched in mouse gonocytes, and the antigen is known to be useful in selection (31). The absence of enriched expression in the prepubertal human spermatogonia reflects not so much the absence of expression in the germ cells, but rather its expression in somatic cells as indicated by the high probe signal for human testis somatic cells. KIT is a well-known characteristic surface marker of germ cells, often expressed in the fetal testis and on differentiating spermatogonia (39). While a high signal for mRNA is present in the prepubertal human spermatogonia, the signal is much lower in the mouse gonocyte, and the protein may be absent or low on the cell surface. The relatively high level of the Kit signal in prepubertal human spermatogonia may reflect a signal for some Ad and Ap stem cells to differentiate into type B spermatogonia, a process known to be occurring during the prepubertal period in humans (2, 3). While the match between human and mouse surface antigen gene expression in germ cells is not exact, the similarity is compelling. Moreover, the biological importance of surface antigen similarity is confirmed, in part, by the ability of human SSCs (19) and prepubertal spermatogonia (Fig. 2 and Table S4) after transplantation to migrate to the basement membrane of the mouse seminiferous tubule. This migration of prepubertal human spermatogonia in a direction opposite to normal differentiating germ cell movement, through the mouse Sertoli cell tight junctions, which separate the seminiferous tubule lumen from the basement membrane in the recipient testis, represents a dramatic conservation of the “homing” mechanism in these human germ cells despite an enormous phylogenetic divergence from the recipient.

The enrichment in expression of genes for intracellular markers of SSCs in prepubertal human spermatogonia and mouse gonocytes is just as remarkable and important as found for cell surface proteins. On the basis of oligonucleotide microarray analyses and qRT-PCR, the expression of Dazl, Vasa, Zbtb16, and Uchl1 are all enriched in these prepubertal germ cells of both species as they are in mouse SSCs (20, 21, 2325). Rhesus monkey spermatogonia have also been shown to express Dazl, Vasa, and Zbtb16 (40). While Taf4b expression showed enrichment on the microarray in prepubertal human spermatogonia but not mouse gonocytes, it is known to be important for mouse SSC maintenance (32). In contrast to the previous genes, Ngn3 has been reported to be present in mouse SSCs and early spermatogonia but not in gonocytes (33). Thus, the intracellular markers in these germ cells mirror the similarity between the two species in surface markers and suggest that intracellular regulation in prepubertal human spermatogonia and mouse gonocytes is comparable in important aspects and similar to mouse SSCs.

The most striking and significant pattern of similarity between prepubertal human spermatogonia and mouse gonocytes is regarding expression of genes known to be critical for self-renewal of mouse SSCs. In mouse, rat, and probably hamster, RET and its coreceptor, GFRα1, bind GDNF and signal, in part, through transcription factors ETV5 and BCL6B to regulate self-renewal of the SSC (10, 11). Protein kinases SFK, P13K, and AKT, play a role in the intracellular self-renewal signals from RET in mouse SSCs, and in each of these signaling pathways several molecules are expressed at high levels in prepubertal human spermatogonia and mouse gonocytes (14) (SI Text). Thus, the data in Table 1 suggest that this regulatory pathway is already present in the mouse gonocyte for maintenance and certainly for use immediately when the transition to SSC occurs before puberty. Moreover, these data imply that prepubertal human spermatogonia and mouse gonocytes as well as mouse SSCs share, at least in part, critical aspects of maintenance and self-renewal (Fig. 3). Certainly other regulatory factors are involved in these processes for prepubertal human spermatogonia, because a medium containing GDNF that supports mouse gonocyte transition to SSC is not adequate to support self-renewal and germ cell clump formation for prepubertal human spermatogonia. Nonetheless, the importance of GDNF for prepubertal human spermatogonia is attested to, not only by the microarray data, but also by the maintenance of prepubertal human spermatogonia in medium containing GDNF in contrast to their rapid disappearance when the growth factor is absent (Fig. S5). Moreover, the maintenance of human putative SSCs in mouse testes for long periods indicates important growth factors and gene activity are common to the two species. In a medical context, information regarding maintenance of human SSCs in vitro has particular relevance for prepubertal boys undergoing cancer treatment (SI Discussion).

Fig. 3.
A proposed model of human spermatogonial stem cell (SSC) self-renewal regulation by glial cell line-derived neurotrophic factor (GDNF), which has been demonstrated to have an essential role in regulating rodent SSC self-renewal. The model is similar to ...

The morphology and location of the prepubertal germ cells made identification and isolation possible, and protein markers confirmed the selection of essentially pure populations. Subsequent oligonucleotide microarray analyses demonstrated a remarkable similarity in these cells from human and mouse and demonstrated that they share high expression levels for the receptors for GDNF, the essential self-renewal growth factor for mouse and rat SSCs, as well as for critical intracellular transcription factors controlling SSC self-renewal. These data suggest that prepubertal human spermatogonia and mouse gonocytes share regulatory mechanisms with mouse SSCs. The ability of prepubertal human spermatogonia to migrate to the basement membrane and be maintained as germ cells, and likely stem cells, in mouse seminiferous tubules, as well as the positive influence of GDNF on the human spermatogonia in vitro, lends biological support to the similarity of the two species in their SSCs. This relationship is remarkable because of the large phylogenetic separation between the two species and is important because it opens a window of opportunity to learn about human SSCs through studies on prepubertal human spermatogonia that can be identified and isolated in essentially pure populations and relating observations to the rapidly developing information base about mouse SSCs.

Materials and Methods

Cell Preparation and Selection.

Mouse donor cells were isolated from 3-day-old C57BL/6 or ROSA26 mouse pups. Human donor cells were isolated from testis biopsies from boys aged 2–10 years. An average of 31.5 mg tissue was obtained from human biopsies.

Microarray Processing and Analysis.

Four hundred cells were selected for each group of germ cells or testis somatic cells from three independent testis cell preparations from both human (ages 2, 8, and 10 years) and mouse (age 3 days).

Further details of procedures are described in the SI Text.

While this manuscript was under review, an article appeared describing staining of human adult germ cells for some the same antigenic proteins (41)

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Drs. R. Behringer, S. Goodyear, M. Kotlikoff, Z. Niu, and J. Oatley for critical evaluation of the manuscript; C. Freeman and R. Naroznowski for assistance with animal maintenance; J. Hayden for assistance with photography and art work; and Juliana Burns for histological preparations. Financial support by an Ethel Foerderer Award (J.P.G.), National Institute of Child Health and Human Development (NICHD) Grant HD061217 (to J.P.G.), NICHD Grant HD044445 (to R.L.B.), NICHD Grant HD 052728 (to R.L.B.), and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (R.L.B.).

Footnotes

The authors declare no conflict of interest.

Data deposition: The microarray data reported in this paper has been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE18914).

This article contains supporting information online at www.pnas.org/cgi/content/full/0912432106/DCSupplemental.

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