Published: January 11, 2018

Introduction {#sec1}
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In tissues that require continuous renewal during life, stem cells fuel the generation of differentiated progeny. The ability to identify pathways important for stem cell function and to distinguish between populations with self-renewal capacity or commitment requires a robust method for isolating defined populations as well as a means for testing their stem cell potential via transplantation. In the mammalian testis, spermatogonial stem cells (SSCs) are the mitotic cells that maintain the germline by undergoing both self-renewal and differentiation, eventually yielding haploid sperm ([@bib38]). The exact identity and nature of the SSC pool remains incompletely understood.

SSCs, and all cells with transplantation potential in the testis, reside in a population of "undifferentiated type A" spermatogonia (A-undiff), named originally based on their undifferentiated morphology ([@bib17]). These rare cells on the basement membrane of seminiferous tubules are found as single cells, pairs, and chains of 4 to 16 cells (termed A~single~, A~pair~, and A~aligned4-16~, respectively), as incomplete cell division in this compartment results in elongating cell syncytia. "Undifferentiated" spermatogonia mature to become "differentiated" spermatogonia, which are marked by expression of the cell surface receptor *Kit* ([@bib36]). During each cycle of spermatogenesis, the vast majority of spermatogonia migrate luminally to enter meiosis. Based on histological observations, it was proposed that the SSC pool is comprised only of the A~single~ cells, and that division into A~pair~ represents commitment to a transiently amplifying progenitor ([@bib7], [@bib17], [@bib31]).

Recent studies have identified a number of genes that are expressed on a subset of A~single~ cells, including *Bmi1*, *Pax7*, and *Id4* ([@bib1], [@bib16], [@bib23]). In support of the A~single~ model, transplantation of ID4-GFP^Bright^ spermatogonia from juvenile testis achieved a high transplantation efficiency ([@bib16]). However, whether all ID4+ cells function as SSCs in the adult or whether ID4 marks the entire population of SSCs is unclear.

Short-chain undifferentiated spermatogonia tend to express GFRα1, the cell surface receptor for the key self-renewal factor glial cell line-derived neurotrophic factor (GDNF) ([@bib27]). Lineage tracing using GFRα1-- CreER knockin mice revealed that GFRα1+ cells can give rise to long-term labeling of the germ cell compartment, indicating that SSCs reside within the GFRα1+ population ([@bib12], [@bib29]). Only a subset of undifferentiated spermatogonia express GFRα1. Seventy percent of undifferentiated spermatogonia do not express GFRα1, including 10%--30% of A~single~ and 25%--50% of A~pair~ ([@bib9], [@bib10], [@bib30]), and the functional properties of these cell types are largely unexplored. The behavior of GFRα1-- undifferentiated spermatogonia has been inferred by analyzing Neurogenin3-positive (NGN3+) cells, whose expression imperfectly marks the GFRα1-- state. Analysis of NGN3-CreER knockin mice showed that NGN3+ cells can give rise to long-term labeling in a small subset of tracing events homeostatically, and to a greater degree after injury ([@bib29], [@bib30]). However, approximately 10% of NGN3+ cells are also GFRα1+, so whether self-renewal potential is found outside of the GFRα1+ compartment remains unknown. Alternative approaches are required to understand the properties of GFRα1-- spermatogonia.

Transplantation is a rigorous assay for stem cell potential and has been used extensively to quantify functional SSCs ([@bib4]). Previous work has revealed that the SSC pool may reside within spermatogonia expressing *Thy1*, *Itga6*, *Itgb1*, *Cdh1*, *Id4*, and *Pax7*, among others ([@bib1], [@bib16], [@bib24], [@bib35], [@bib37], [@bib42]). Although GFRα1-- expressing spermatogonia are thought to be among the most primitive cells in the SSC differentiation hierarchy, attempts to transplant this population did not show enrichment for SSC-repopulating activity ([@bib5], [@bib11]).

We previously discovered that PLZF+ undifferentiated spermatogonia are characterized by high levels of telomerase, the enzyme that synthesizes telomere DNA repeats at chromosome ends ([@bib34]). By generating *Tert*^*Tomato/+*^ reporter knockin mice, we identified a gradient of *Tert* transcription in the testis and used it to isolate undifferentiated spermatogonia. We also found that telomere dysfunction in *Tert*^*−/−*^ mice induced depletion of the PLZF+ A-undiff pool over time, providing a cellular mechanism to explain the established infertility phenotype in telomerase knockout mouse strains ([@bib25], [@bib34]). In this study, we develop methods to isolate highly purified populations of GFRα1--positive and GFRα1--negative undifferentiated spermatogonia from the testes of adult *Tert*^*Tomato/+*^ reporter mice and from wild-type mice. We leverage these techniques to define transcriptome-wide features and functional differences between these two cell populations that define the SSC pool.

Results {#sec2}
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Purification of GFRα1+ and GFRα1-- Undifferentiated Spermatogonia from Adult *Tert*^Tomato/+^ Reporter Mice {#sec2.1}
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Using a Tert-Tomato transcriptional reporter of telomerase activity, we previously showed that TERT^High^ KIT-- population represents a pure population of PLZF+ undifferentiated spermatogonia ([@bib34]). To determine the relationship between telomerase expression and GFRα1 expression, we performed whole-mount microscopy on adult seminiferous tubules. Triple-staining for PLZF, GFRα1 and Tomato revealed that effectively all GFRα1+ cells express Tert-Tomato (99.3% ± 0.5%; [Figure 1](#fig1){ref-type="fig"}A). Tert-Tomato expression was similarly homogeneous (99.8% ± 0.1%) within GFRα1-- PLZF+ undifferentiated spermatogonia, which included both long and short chains of cells ([Figure 1](#fig1){ref-type="fig"}B). Thus, both GFRα1+ and GFRα1-- undifferentiated spermatogonia are characterized by high *Tert* promoter activity.Figure 1High Telomerase Expression Enables the Purification and Characterization of GFRα1+ and GFRα1-- Undifferentiated Spermatogonia(A) Whole-mount analysis of adult seminiferous tubules immunostained for GFRα1, PLZF, and anti-RFP in *Tert*^*Tomato/+*^ seminiferous tubules. A total of 99.3% ± 0.5% of GFRα1+ PLZF+ cells were Tert-Tomato+ (N = 370 cells; N = 4 mice); 99.8% ± 0.1% GFRα1-- PLZF+ cells were Tert-Tomato+ (N = 1900 cells; N = 6 mice). Scale bar, 50 μm.(B) Whole-mount analysis of adult seminiferous tubules immunostained for GFRα1, PLZF, and anti-RFP in *Tert*^*Tomato/+*^ seminiferous tubules. White arrows point to TERT^High^ GFRα1− A-paired (left arrow) and TERT^High^ GFRα1− A-single (right arrow) spermatogonia. Scale bar, 50 μm.(C) Flow cytometry measurement of GFRα1 and KIT expression in TERT^High^ cells. Panels are representative of at least six independent FACS runs.(D) *In situ* hybridization for NGN3 mRNA on FACS-sorted cells of the indicated immunophenotypes. Percentage of NGN3+ cells was quantified. Mean and SEM are shown. Scale bar, 25 μm. N = 5--6 mice; at least 2,000 cells counted per condition. ^∗∗^p = 0.012.(E) Interpretation of identities of various sorted cell types, based on whole-mount, cytospin, immunophenotype, and neonatal time course data.

To develop a method for the isolation of GFRα1+ and GFRα1-- undifferentiated spermatogonia using fluorescence-activated cell sorting (FACS), dissociated adult testes were stained with antibodies against GFRα1 and KIT. We previously found that the TERT^High^ population comprised approximately 50% KIT-- cells, representing undifferentiated spermatogonia, and 50% KIT+ cells, representing early differentiating spermatogonia. TERT^High^ KIT-- cells transplanted efficiently, whereas TERT^High^ KIT+ cells failed to transplant ([@bib34]). FACS of whole testes showed a subpopulation of TERT^High^ KIT-- cells were GFRα1+, and that the GFRα1+ cells represented approximately one-third of the TERT^High^ KIT-- population, consistent with whole-mount analysis. GFRα1+ cells were absent in all other populations, including TERT^Low^ cells and TERT^High^ KIT+ ([Figure 1](#fig1){ref-type="fig"}C). To further assess the identity of the purified TERT^High^ KIT-- GFRα1+ and GFRα1-- populations, we isolated them by FACS and employed RNA *in situ* hybridization (ISH) to assay for NGN3 mRNA, whose expression has been used as a surrogate marker for GFRα1-- spermatogonia. RNA ISH on sorted TERT^High^ KIT-- GFRα1+ and GFRα1-- populations showed that 17% ± 1% of GFRα1+ cells express *Ngn3*, whereas 53% ± 7% of GFRα1-- cells express *Ngn3* (p = 0.012, t test) ([Figure 1](#fig1){ref-type="fig"}D). These results indicate that *Ngn3* is differentially expressed between the TERT^High^ KIT-- GFRα1+ and GFRα1-- populations of undifferentiated spermatogonia, but that *Ngn3* expression alone is insufficient to discriminate the GFRα1+ and GFRα1-- populations.

These results provide strong evidence for the successful isolation of GFRα1+ and GFRα1-- undifferentiated spermatogonia, based on intrinsic *Tert* promoter strength and cell surface phenotypes (summary in [Figure 1](#fig1){ref-type="fig"}E). Employing the Tert-Tomato reporter was essential for successful purification of these populations. Sorting based on GFRα1 expression alone did not allow isolation of a pure population of GFRα1+ cells, nor did it allow the discrimination of GFRα1-- undifferentiated spermatogonia due to background staining for GFRα1 in the meiotic cells and spermatids ([Figure S1](#mmc1){ref-type="supplementary-material"}A). Enrichment for A-undiff cells using the *Tert* reporter allowed detection of distinct GFRα1+ and GFRα1-- A-undiff populations, enabling subsequent molecular and functional studies.

Isolation and Transcriptional Profiling of Four Distinct Spermatogonial Populations {#sec2.2}
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To identify the differences between the GFRα1+ and GFRα1-- undifferentiated spermatogonia we performed RNA sequencing (RNA-seq) to identify the transcriptional features of each population. From four independent *Tert*^*Tomato/+*^ mice, we sequenced the transcriptomes of FACS-purified TERT^High^ GFRα1+ KIT-- cells and TERT^High^ GFRα1-- KIT-- cells. To put the transcriptional profiles in the context of spermatogenesis and differentiation, we also sequenced the population of TERT^High^ KIT+ cells and TERT^Low^ KIT+ cells ([@bib34]), which represent early and late differentiating spermatogonia, respectively ([@bib34]) ([Figure S1](#mmc1){ref-type="supplementary-material"}). In addition, we isolated TERT^High^ Oct4-GFP double-positive spermatogonia from postnatal day 6 (P6) juveniles. Spermatogonia isolated from juvenile testes are enriched for stem cell activity and have previously been used to gain insights about adult SSCs ([@bib16], [@bib21]).

By principal-component analysis (PCA) and unsupervised hierarchical clustering, biological replicates from the same populations clustered together, confirming our ability to isolate pure populations with discrete identities ([Figures 2](#fig2){ref-type="fig"}A and [S2](#mmc1){ref-type="supplementary-material"}A). PCA showed three principal components that explain a large proportion of the variance between the populations. The four adult populations lined up along axis PC1 in an order that recapitulated differentiation: the TERT^High^ GFRα1+ KIT-- cells and TERT^High^ GFRα1-- KIT-- populations were the leftmost populations, followed by TERT^High^ KIT+ cells further right, and with TERT^Low^ KIT+ cells as the rightmost population ([Figure 2](#fig2){ref-type="fig"}A). By PCA, the GFRα1+ and GFRα1-- spermatogonia cluster together, and neither population is closer to the more differentiated TERT^High^ KIT+ cells. Our PCA analysis also showed that the P6 spermatogonia were significantly different from adult populations as they were separated from the rest of the samples along the PC2 axis ([Figure 2](#fig2){ref-type="fig"}A). We conclude that the PC1 axis captured the gene expression changes associated with differentiation, while the PC2 axis reflected changes associated with postnatal maturation. These data highlight the relatedness of the TERT^High^ GFRα1+ and TERT^High^ GFRα1-- populations in the undifferentiated spermatogonia compartment.Figure 2RNA-Seq Reveals That GFRα1+ Spermatogonia Are Defined by a Transcriptional Signature of Active GDNF and FGF Signaling(A) Principal-component analysis (PCA) of transcriptomes from five isolated spermatogonial populations from adult and postnatal day 6 (P6) juvenile.(B) Volcano plot of expression profiles comparing TERT^High^ GFRα1+ KIT-- to TERT^High^ GFRα1-- KIT-- cells.(C) MAP/ERK/protein phosphorylation cluster generated by Cytoscape Enrichment Map of gene set enrichment analysis (GSEA) results for TERT^High^ GFRα1+.(D) Cell-cycle/proliferation cluster generated by Cytoscape Enrichment Map of GSEA results for TERT^High^ GFRα1+.(E) Indicated cell types were sorted and cytospun. A 2 hr EdU pulse was visualized using Click chemistry, and the cells were then immunostained for the undifferentiated spermatogonia marker PLZF. Scale bar, 25 μm. Percentage of EdU+ cells was quantified. Mean and SEM are shown. (N = 5 mice; N = 900--10,000 cells per condition). ^∗^p \< 0.05; ^∗∗∗^p \< 0.001.

GFRα1+ Spermatogonia Are Defined by a Transcriptional Signature of Active GDNF and Fibroblast Growth Factor Signaling {#sec2.3}
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By differential expression analysis, we identified 578 significantly upregulated and 430 significantly downregulated genes in TERT^High^ GFRα1+ versus TERT^High^ GFRα1-- cells (5% false discovery rate and 2-fold change cutoff; [Figure 2](#fig2){ref-type="fig"}B; [Table S1](#mmc2){ref-type="supplementary-material"}). The GFRα1 receptor was one of the most differentially expressed genes, and its co-receptor Ret was also highly differentially expressed (16.9-fold change, q = 3.9 × 10^−58^; 2.7-fold, q = 3.2 × 10^−5^, respectively). Id4, a marker of A~single~ cells was also shown to be enriched in the GFRα1+ population (1.4-fold, q = 4.5 × 10^−5^). Furthermore, many of the most significantly upregulated genes in TERT^High^ GFRα1-- cells were factors known to be enriched in long-chained A-undiff cells *Ngn3* (7.3-fold q = 5.3 × 10^−16^), *Nanos3* (2.8-fold, q = 3.9 × 10^−7^), *Sohlh1* (2.1-fold q = 4.3 × 10^−4^), *Sox3* (3.3-fold q = 5.26 × 10^−5^), and *Lin28a* (2.2-fold q = 1.6 × 10^−10^) ([@bib6], [@bib35], [@bib40], [@bib39]) Thus, these transcriptomic studies have captured key markers of both GFRα1+ and GFRα1-- undifferentiated spermatogonia.

GFRα1-- GDNF signaling is required for both the *in vivo* maintenance of SSCs and their *ex vivo* culture ([@bib20], [@bib27]). Many genes originally identified as being GDNF responsive in germline stem cell culture systems ([@bib32]) were highly enriched in TERT^High^ GFRα1+ cells compared with TERT^High^ GFRα1-- spermatogonia: *T* (57.3-fold, q = 2.4 × 10^−10^), *ETV5* (12.3-fold, q = 3.4 × 10^−27^), *EGR2* (19.8-fold, q = 4.4 × 10^−99^), *EGR3* (5.7-fold, q = 4.7 × 10^−5^), *BCL6B* (4.8-fold, q = 4.5 × 10^−12^), *Tspan8* (1.2-fold, q = 7.3 × 10^−3^), and *Lhx1* (15.5-fold, q = 3.0 × 10^−11^) ([Figure 2](#fig2){ref-type="fig"}B). In addition to GDNF, fibroblast growth factor (FGF) signaling is required for maintenance of SSCs *in vivo* and in culture ([@bib14]). Sprouty and Spred families of receptor tyrosine kinase (RTK) inhibitors and the DUSP family of MAPK phosphatases are transcriptionally induced during FGF responses ([@bib3]), and this family of genes was highly upregulated in TERT^High^ GFRα1+ cells ([Figure 2](#fig2){ref-type="fig"}B). These findings provide evidence that TERT^High^ GFRα1+ cells actively receive FGF signals *in vivo*. Taken together, these transcriptomic studies indicate a specific induction of GDNF- and FGF-regulated genes in GFRα1+ cells.

To understand the transcriptomes in these two populations more generally, we analyzed the differentially expressed genes by gene set enrichment analysis and visualized the results using Cytoscape Enrichment Map ([@bib28]). Three major clusters of enriched functional gene sets were found in genes upregulated in GFRα1+ cells (p-value cutoff, 0.001). The first cluster involves RTK-RAS-MAPK signaling and protein phosphorylation ([Figure 2](#fig2){ref-type="fig"}C). The RAS-MAPK pathway is known to be important downstream of GDNF and FGF signaling ([@bib13], [@bib15]). Numerous gene sets related to RTK-RAS-MAPK signaling were associated with genes upregulated in GFRα1+ cells. Many core genes in this enrichment cluster are the GDNF/FGF-responsive genes highlighted in the volcano plot ([Figure 2](#fig2){ref-type="fig"}B).

The second cluster involves cell-cycle progression, E2F transcription factor targets, DNA packaging, and replication ([Figure 2](#fig2){ref-type="fig"}D). These findings suggest a difference in cell-cycle activity between the GFRα1+ and GFRα1-- undifferentiated spermatogonia. To test this idea, we assayed proliferation by *in vivo* 5-ethynyl-2′-deoxyuridine (EdU) labeling, followed by FACS purification, cytospin, and EdU detection. We found a marked difference in cell-cycle status between the two subpopulations of undifferentiated spermatogonia: GFRα1+ cells showed an S-phase fraction of 17.4% ± 1.4%, while GFRα1-- cells exhibited an S-phase fractions of 3.3% ± 0.9% (p = 0.033, t test) ([Figure 2](#fig2){ref-type="fig"}E). These finding are consistent with the role for GDNF in spermatogonial self-renewal and proliferation ([@bib27], [@bib41]). The third, smaller cluster involved genes associated with morphogenesis and epithelium development ([Figure S2](#mmc1){ref-type="supplementary-material"}B). Taken together, transcriptome and cell-cycle data suggest that GFRα1+ undifferentiated spermatogonia receive critical self-renewal and proliferation signals from their environment and that downregulating these pathways is an important characteristic of the transition to the GFRα1-- state.

To understand the relationships between GFRα1+ and GFRα1-- undifferentiated spermatogonia and other selections of undifferentiated spermatogonia, we compared the transcriptomes of GFRα1+ and GFRα1-- cells with those of ID4-Bright cells, which show high transplantation potential, and ID4-Dim cells ([@bib16]). By differential expression analysis, the ID4-Bright and GFRa1+ undifferentiated spermatogonia were similar in expression of genes important for SSC maintenance and self-renewal, including: *GFRa1*, *Taf4b*, *Zbtb16*, *Bcl6b*, *Lhx1*, *T*, and *Pou3f1*, among others ([Figure S3](#mmc1){ref-type="supplementary-material"}A). ID4 was enriched in ID4-Bright cells compared with GFRa1+ cells. Surprisingly, we found a number of genes associated with differentiated spermatogonia enriched in the ID4-Bright cells, including *Stra8*, *Alcam*, *Sycp1*, *Nanos3*, *Dmrt1*, *Sox3*, *Sohlh1*, and *Kit* ([Figure S3](#mmc1){ref-type="supplementary-material"}A). A similar pattern was also seen in a comparison of GFRa1-- cells with ID4-Bright cells ([Figure S3](#mmc1){ref-type="supplementary-material"}B). PCA on the two ID4+ populations and our five populations to assess their relatedness revealed that both the ID4-Bright and the ID4-Dim cells cluster closely with the TERT^High^ cells we isolated from day 6 neonates (P6) ([Figure S3](#mmc1){ref-type="supplementary-material"}C). This relationship was also seen using unsupervised hierarchical clustering ([Figure S3](#mmc1){ref-type="supplementary-material"}D). This similarity may occur because the ID4 populations in Helsel et al. were isolated from day 8 neonatal mice. Thus, the ID4-Bright cells from P8 mice are most similar transcriptionally to TERT^High^ neonatal spermatogonia, and less similar to adult GFRα1+ and GFRα1-- cells. These data highlight potential molecular differences between neonatal and adult SSC populations.

Elevated Melanocyte Cell Adhesion Molecule Is a Cell Surface Marker of the GFRα1+ State {#sec2.4}
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*Tert* reporter mice enabled efficient purification of many spermatogonial subsets, but the lack of good antibodies specific for undifferentiated spermatogonial subpopulations currently makes it difficult to isolate these cells from wild-type mice. To identify new markers, we leveraged our transcriptome datasets from four distinct subpopulations of undifferentiated and differentiated spermatogonia. We used Short Time-series Expression Miner (STEM) ([@bib8]) to compare gene expression simultaneously across all four populations. STEM analysis takes an ordered collection of expression datasets, assigns each gene to a profile bin and computationally identifies statistically enriched profiles. STEM analysis of TERT^High^ GFRα1+ KIT-- cells, TERT^High^ GFRα1-- KIT-- cells, TERT^High^ KIT+, and TERT^Low^ KIT+ cells identified nine enriched profiles ([Figure 3](#fig3){ref-type="fig"}A). We were specifically interested in genes that followed the expression profile of GFRα1 ([Figure 3](#fig3){ref-type="fig"}A, green pattern). STEM identified GFRα1, ETV5, and melanocyte cell adhesion molecules (MCAMs) as having highly similar expression profiles ([Figure 3](#fig3){ref-type="fig"}B and [Table S2](#mmc3){ref-type="supplementary-material"}). MCAM, an immunoglobulin-superfamily surface protein shown to enrich for transplantation activity ([@bib19]), was also one of the top ten most differentially expressed genes between GFRα1+ and GFRα1-- cells ([Figure 2](#fig2){ref-type="fig"}B; 4.6-fold change, q = 4.4 × 10^−44^). Thus, MCAM represents a candidate cell surface marker with an expression pattern similar to GFRα1. To test similarity at the protein level, we investigated MCAM expression using whole-mount immunostaining. MCAM protein was enriched in a subset of PLZF+ undifferentiated spermatogonia ([Figure 3](#fig3){ref-type="fig"}C). Elevated MCAM expression was restricted to short-chain PLZF+ cells. Co-staining revealed that these MCAM^High^ cells were exclusively GFRα1+ PLZF+ spermatogonia ([Figure 3](#fig3){ref-type="fig"}C). Therefore, our analysis enabled the discovery of an independent marker, MCAM, for the GFRα1+ subset of undifferentiated spermatogonia.Figure 3MCAM Is a Cell Surface Marker of the GFRα1+ State(A) Nine patterns of gene expression changes across adult spermatogonial populations identified as statistically significant by Short Time-series Expression Miner (STEM).(B) Details on STEM pattern no. 8, containing genes with peak expression in TERT^High^ GFRα1+ cells, with diminished expression in all other cell types. Genes of interest are highlighted. The entire list of 575 genes is found in [Table S2](#mmc3){ref-type="supplementary-material"}.(C) Whole-mount analysis of tubules triple-stained for MCAM, GFRα1, and PLZF. All 76/76 GFRα1+ cells were MCAM^High^. White arrows point to cells shown in greater magnification in the panels to the right. Scale bar, 50 μm. N = 3 mice.(D) Flow cytometry measurement of GFRα1 and MCAM expression in TERT^High^ KIT-- cells and TERT^Low^ KIT+. Panels are representative of at least six independent FACS runs.

Isolation of Both GFRα1+ and GFRα1-- Undifferentiated Spermatogonia Based on Differential MCAM Expression Using FACS {#sec2.5}
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As MCAM is expressed on the cell surface, we tested its ability to enrich for spermatogonial populations using FACS. Staining dissociated tubules for GFRα1 and MCAM revealed a population of double-positive GFRα1+ MCAM+ cells in the TERT^High^ KIT-- fraction. This double-positive population was not present in the TERT^Low^ KIT+ fraction ([Figure 3](#fig3){ref-type="fig"}D). These GFRα1+ MCAM+ cells isolated by FACS correspond to the same population seen in whole-mount immunostaining ([Figure 3](#fig3){ref-type="fig"}C).

These results suggested that MCAM can be used to isolate GFRα1+ cells without relying on the Tert-Tomato reporter mouse or on GFRα1 staining. To test this hypothesis, we stained dissociated tubules from wild-type mice using antibodies against MCAM and KIT. By flow cytometry, the MCAM signal was sufficiently strong to separate three distinct populations by MCAM expression level: MCAM^High^, MCAM^Med^, and MCAM^Low^ ([Figure 4](#fig4){ref-type="fig"}A). The MCAM^High^ population was highly enriched for KIT-- cells (85%--90% KIT--) ([Figure S4](#mmc1){ref-type="supplementary-material"}A). The MCAM^Med^ and MCAM^Low^ populations were predominantly KIT+ cells ([Figures S4](#mmc1){ref-type="supplementary-material"}B and S4C). To determine if MCAM levels along with staining for KIT allow us to isolate GFRα1+ and GFRα1-- undifferentiated spermatogonia, we sorted MCAM^High^ KIT-- cells, MCAM^Med^ KIT-- cells, MCAM^Med^ KIT+ cells, and MCAM^Low^ KIT+ cells, cytospun them, and stained them for GFRα1 and PLZF ([Figure 4](#fig4){ref-type="fig"}B). The MCAM^High^ KIT-- cells were 84% GFRα1+ PLZF+. The MCAM^Med^ KIT-- population was 77% GFRα1-- PLZF+. The MCAM^Med^ KIT+ population was 90% GFRα1-- PLZF--. And the MCAM^Low^ KIT+ population was 100% GFRα1-- PLZF-- ([Figure 4](#fig4){ref-type="fig"}C). Therefore, this approach allows the isolation of highly enriched populations of A-undiff GFRα1+ spermatogonia as MCAM^High^ KIT-- and A-undiff GFRα1-- spermatogonia as MCAM^Med^ KIT--.Figure 4MCAM Levels Can be Used to Isolate Both GFRα1+ and GFRα1-- Undifferentiated Spermatogonia and Are Responsive to GDNF/FGF(A) Flow cytometry measurement of MCAM levels in whole adult testis from wild-type mice.(B) Indicated populations were sorted from wild-type mice, cytopun, and stained for GFRα1, PLZF, and DAPI.(C) Quantification of (B) showing fraction of GFRα1+ PLZF+, GFRα1-- PLZF+, and GFRα1-- PLZF-- cells in each MCAM population. N = 3 mice pooled; N = 1,524 cells.(D) qRT-PCR for indicated SSC and differentiation markers from cells sorted based on MCAM expression and KIT. Mean and SEM are shown.(E) Experimental outline of cell culture experiments. Indicated cells populations were sorted and cultured in basal GS medium supplemented with or without 50 ng/mL GDNF and 20 ng/mL FGF2. Forty-eight hours later, anti-MCAM immunofluorescence was performed.(F) Effect of GDNF/FGF on MCAM expression. TERT^High^ GFRα1+ cells and TERT^High^ GFRα1-- cells were stained for MCAM and DAPI after 48 hr of culture. Scale bar, 15 μm.(G) Quantification of (E). (N = 4 mice; N = 50 cells). Mean and SEM are shown. ^∗^p \< 0.05; ^∗∗∗^p \< 0.001.

To validate our data, we performed qRT-PCR for a variety of marks of undifferentiated and differentiated spermatogonia. By qPCR, MCAM^High^ KIT+ cells expressed high levels of GFRα1 and PLZF mRNA. MCAM^Med^ KIT-- cells expressed 10-fold less GFRα1 mRNA than MCAM^High^ KIT+ and high levels of PLZF and NGN3. MCAM^Med^ KIT+ cells and MCAM^Low^ KIT+ expressed high levels of KIT and SYCP3 mRNA ([Figure 4](#fig4){ref-type="fig"}D). Our findings provide a robust protocol for isolation of phenotypically defined subtypes of undifferentiated spermatogonia from adult wild-type mice. MCAM provides a marked advantage over GFRα1 staining due to an improved signal-to-noise ratio with this combination of antigen and antibody.

Our RNA-seq analysis suggests that the main difference between GFRα1+ and GFRα1-- undifferentiated spermatogonia is active GDNF/FGF signaling. We wondered if MCAM were a GDNF/FGF-responsive gene. To test this hypothesis, we used FACS to isolate pure populations of GFRα1+ and GFRα1-- cells and then cultured them in germline stem cell medium either with or without GDNF/FGF ([Figure 4](#fig4){ref-type="fig"}E) ([@bib20]). The high level of MCAM expression in freshly isolated SSCs was indeed dependent on exposure to GDNF/FGF: *ex vivo* culture of TERT^High^ GFRα1+ cells in the absence of these cytokines led to a rapid downregulation of surface MCAM levels. Similarly, TERT^High^ GFRα1-- cells exposed to GDNF/FGF had higher MCAM levels than TERT^High^ GFRα1-- cells cultured in the absence of these cytokines ([Figure 4](#fig4){ref-type="fig"}F). Quantifying the changes in MCAM antibody staining showed a significant induction of MCAM expression with GDNF/FGF exposure ([Figure 4](#fig4){ref-type="fig"}G). Thus, both GFRα1+ and GFRα1-- retain the ability to respond in culture to GDNF/FGF, leading to robust induction of MCAM. Taken together, GFRα1+ and GFRα1-- undifferentiated spermatogonia are efficiently isolated on the basis of surface MCAM expression, and elevated MCAM protein in GFRα1+ cells likely reflects active GDNF/FGF signaling in this compartment.

Elevated Stem Cell Repopulating Activity in Both GFRα1+ and GFRα1-- Undifferentiated Spermatogonia {#sec2.6}
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Based on the similarities between the GFRα1+ and GFRα1-- cells, we hypothesized that the self-renewal capacity of GFRα1-- cells may be revealed upon transplantation into an empty niche and we leveraged our ability to isolate both GFRα1+ and GFRα1-- cells to compare their relative transplantation potential side-by-side. FACS-purified cells were assayed in terms of their ability to colonize the tubules of *Kit*^*W/Wv*^ mice, which lack SSCs ([@bib4]). Donor cells were permanently marked by breeding *Tert*^*Tomato/+*^ mice to mouse strains with ubiquitous expression of either EGFP or β-galactosidase. FACS-sorted TERT^High^ GFRα1+ and TERT^High^ GFRα1-- cells from adult donors were injected separately into the seminiferous tubules of *Kit*^*W/Wv*^ recipients via the efferent bundle ([Figure 5](#fig5){ref-type="fig"}A). Stem cell frequency in bulk germ cells was assessed by transplanting FACS-sorted live cells that were not fractionated by antigen expression (unfractionated). Two months later, the total number of colonies was counted ([Figure 5](#fig5){ref-type="fig"}B).Figure 5Elevated Stem Cell Repopulating Activity in GFRα1+ and GFRα1-- Undifferentiated Spermatogonia(A) Experimental outline of transplant experiments. Tert-Tomato cells permanently labeled by ubiquitous GFP or LacZ expression were transplanted into sterile *Kit*^*W/Wv*^ recipients. Colonies were counted 2 months post-injection.(B) Representative EGFP epifluorescence in recipient *Kit*^*W/Wv*^ mice 8 weeks after transplantation of cells shown in (A). White lines represent boundary of the testis. "Unfractionated" represents the transplantation of FACS-sorted live cells not fractionated by Tert-Tomato expression or immunophenotype. Scale bar, 2 μm.(C) Quantification of transplant results shown in (B). Colony counts were normalized to 10^5^ cells. Mean and SEM are shown. p Values are from two-tailed Mann-Whitney test. N = 16--18 recipient testes per condition. ^∗^p = 0.019 ^∗∗∗^p \< 0.0005.

TERT^High^ GFRα1+ cells transplanted at high efficiencies, achieving 65-fold enrichment for stem cell activity over unfractionated germ cells (72.5 ± 37 colonies per 100,000 cells; p \< 0.0001 U test). TERT^High^ GFRα1-- cells also showed robust transplantation, albeit at lower frequencies than TERT^High^ GFRα1+ cells (p \< 0.0005 U test; GFRα1+ versus GFRα1-- ). TERT^High^ GFRα1-- cells showed 25-fold enrichment in stem cell transplantation compared with unfractionated germ cells (28 ± 22 colonies per 100,000 cells; p = 0.0189 U test) ([Figure 5](#fig5){ref-type="fig"}C). Histological analysis confirmed that both types of A-undiff spermatogonia were capable of full reconstitution of spermatogenesis post-transplant ([Figure S5](#mmc1){ref-type="supplementary-material"}B). Importantly, stringent sorting conditions led to very high purity of donor cell preparations, as confirmed by re-analysis of the sorted cells prior to transplant ([Figure S5](#mmc1){ref-type="supplementary-material"}A). Thus, we find that GFRα1+ cells show high transplantation efficiency, but that GFRα1-- cells also retain significant transplantation potential. Given that the pool of GFRα1-- undifferentiated spermatogonia is three times as large as the GFRα1+ pool, the *total* number of GFRα1-- stem cells is comparable with the total number of GFRα1+ stem cells in the testis.

GFRα1-- A-Undiff Cells Regenerate the GFRα1+ State after Transplantation {#sec2.7}
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The considerable stem cell potential in GFRα1-- spermatogonia prompted us to explore the features of GFRα1-- stem cell self-renewal. To address whether spermatogenesis from GFRα1-- cells entailed the regeneration of GFRα1+ cells, whole-mount analysis of GFRα1 expression was performed on recipient testes 2 months post-transplantation of TERT^High^ GFRα1-- cells ([Figure 6](#fig6){ref-type="fig"}A). These GFRα1-- cells robustly gave rise to colonies containing TERT^High^ GFRα1+ cells ([Figure 6](#fig6){ref-type="fig"}B). The colonies also contained TERT^High^ MCAM^High^ cells ([Figure 6](#fig6){ref-type="fig"}C). Our data show the ability, *in vivo*, of GFRα1-- cells to respond to niche signals and convert to the GFRα1+ state. These data highlight the functional similarity between the GFRα1+ and the GFRα1-- undifferentiated spermatogonia as revealed by the transplantation assay.Figure 6*In Vivo* Conversion of GFRα1-- Undifferentiated Spermatogonia to GFRα1+ Undifferentiated Spermatogonia(A) Experimental outline of transplant experiments. GFRα1-- Tert-Tomato cells permanently labeled by ubiquitous GFP or LacZ expression were transplanted into sterile *Kit*^*W/Wv*^ recipients. Tubules were stained for MCAM and GFRα1 2 months post transplant.(B) GFRα1 expression in colonies arising from transplanted TERT^High^ GFRα1-- cells. Tert-Tomato used as a marker for the donor cells. Staining results are compared with regions of testis that were not colonized. Scale bar, 50 μm.(C) MCAM expression in colonies arising from transplanted TERT^High^ GFRα1-- cells. Tert-Tomato used as a marker for the donor cells. Scale bar, 50 μm.(D) Model for a flexible hierarchy of adult spermatogonia. Cell surface features of different spermatogonial subtypes are highlighted. GFRα1-- spermatogonia represent a poised state, competent to either differentiate or convert to GFRα1+ spermatogonia in a context-dependent fashion.

Discussion {#sec3}
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A Heterogeneous Adult Germline Stem Cell Pool: GFRα1+ and GFRα1-- Undifferentiated Spermatogonia Are Closely Related {#sec3.1}
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We leveraged differences in *Tert* promoter strength, together with cell surface marker expression, to isolate pure populations of phenotypically defined spermatogonia subsets from adult testis. Our approach enables the isolation of both GFRα1+ and GFRα1-- undifferentiated spermatogonia for functional and molecular analysis. These two populations of A-undiff cells show overall relatedness with key differences in signaling pathways. Both populations showed elevated frequencies of transplantation compared with unfractionated spermatogonia, with GFRα1+ cells transplanting approximately 2.6-fold more efficiently than GFRα1-- cells. Most data have converged on the idea that SSCs are restricted to the short-chain population of A-undiff cells. Consistent with this idea, short-chain A-undiff cells are present throughout the spermatogenic cycle, whereas longer-chain A-undiff cells are ultimately depleted from the population through differentiation. Our data show that SSCs are highly enriched in the GFRα1+ fraction. These results are consistent with those of other laboratories using reporter mice for GFRα1+ and other markers within this short-chain population, including ID4, BMI1, and PAX7 ([@bib1], [@bib16], [@bib23]). It has been argued that these latter markers of subpopulations of short-chain A-undiff cells represent the true stem cells, but direct molecular and functional comparisons of GFRα1+ cells bearing ID4, BMI1, or PAX7, with their GFRα1+ counterparts without expression of ID4, BMI1, or PAX7, have been lacking. Such direct comparisons will be required to understand functional heterogeneity within the GFRα1+ population. Our studies enabling the isolation of GFRα1+ cells using either GFRα1+ antibodies in conjunction with *Tert*^*Tomato/+*^ mice, or MCAM antibodies in wild-type mice, may allow these ideas to be tested directly. In addition, approximately 20% of short-chain A-undiff cells are GFRα1-- in steady state, and these cells have not been isolated or characterized for stem cell activity.

Our findings indicate that GFRα1-- cells exhibit a surprising capacity for transplantation. SSCs in this fraction may reside in the short-chain GFRα1-- fraction or in the elongating chains of PLZF+ A-undiff cells, or both these populations. If residing within the short-chain GFRα1-- fraction, these cells may be in equilibrium with GFRα1+ cells, or may have unique characteristics that have not yet been revealed. If the SSCs defined here in the GFRα1-- fraction reside in the elongating A-undiff population, the residual stem cell activity may reflect that some or many of these cells have not yet committed to differentiate. Our results showing elevated SSC activity in the TERT^High^ GFRα1-- fraction suggests that many cells in this population have not yet fully committed. The ability of these cells to successfully transplant is also consistent with the likelihood that many GFRα1-- cells are fated to differentiate to A-aligned spermatogonia during the spermatogenic cycle, as transplantation tests the ability of cells to function as stem cells. These distinctions are important to define the cellular and molecular mechanisms of self-renewal in the mammalian testis. We note that although the transplantation activity was 2.6 times lower in GFRα1-- undifferentiated spermatogonia compared with GFRa1+ undifferentiated spermatogonia, GFRα1-- cells are more abundant than their GFRα1+ counterparts, making the total number of potential stem cells comparable in each population. These data provide support for a model in which the GFRα1+ and GFRα1-- cells together comprise a stem cell pool, and that some GFRα1-- cells can convert to the GFRα1+ state based on exposure to niche factors ([Figure 6](#fig6){ref-type="fig"}D).

Transcriptional Similarity, but Distinct Regulation, of GDNF and FGF Signaling in GFRα1+ and GFRα1-- Spermatogonia {#sec3.2}
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Transcriptional analysis of GFRα1+ and GFRα1-- undifferentiated spermatogonia revealed a previously unknown similarity between the two populations, in particular when compared with transcriptomes of neonatal spermatogonia, TERT^High^ KIT+ or TERT^Low^ KIT+ spermatogonia, which each cluster separately based on PCA and unsupervised hierarchical clustering. The differentially expressed genes between GFRα1+ and GFRα1-- undifferentiated spermatogonia were enriched for gene sets including cell-cycle regulation and Ras/MEK/ERK signaling downstream of GFRα1/Ret binding of GDNF. Cyclin D1, D2, and A2 were in the top of differentially expressed genes between GFRα1+ and GFRα1-- cells ([Figures 2](#fig2){ref-type="fig"}B and 2D; [Table S1](#mmc2){ref-type="supplementary-material"}). As cyclin D2 has been shown to be important for GS cell self-renewal and long-term culture ([@bib26]), and is expressed in type A spermatogonia *in vivo* ([@bib2]), we speculate that cell-cycle regulation is key for the SSC population *in vivo*. Differences in the abundance of each population during the seminiferous cycle may also contribute to the differences in S-phase fraction measured here, as NGN3+ cells are more abundant in stages IV-VII, at which time they are not proliferating ([@bib18]).

We found that ID4-Bright cells clustered most closely with our TERT^High^ neonatal spermatogonia, likely reflecting the neonatal origin of the ID4-Bright cells used for RNA-seq studies ([@bib16]). Although the neonatal ID4-Bright cells share expression of many stem cell genes with the adult GFRα1+ population, their overall transcriptomes are sufficiently different that they are most similar to other neonatal populations. These transcriptional differences may relate to expression of both SSC genes and differentiation genes within the ID4+ population. This combination of features reflects the peculiarities of the first, synchronized wave of spermatogenesis, which is faster than the adult cycle and features gonocytes that directly give rise to A~2~ spermatogonia ([@bib22], [@bib43]). The extensive RNA-seq analysis performed here highlights key differences between neonatal and adult populations of spermatogonia.

GFRα1-- Spermatogonia Are Capable of Responding to GDNF/FGF Niche Signals in Culture {#sec3.3}
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We found that, in culture, GFRα1-- cells can respond to GDNF/FGF2 to upregulate MCAM to the levels of GFRα1+ cells. Consistent with this observation, we observed that GFRα1-- cells and GFRα1+ cells share a requirement for GDNF and FGF for even short-term culture. The mechanism by which GFRα1-- cells can sense GDNF/FGF is unclear, but may be due to low level receptor expression. Our molecular profiling showed a gradient of GFRα1 expression, similar to the gradient of MCAM expression ([Figure 3](#fig3){ref-type="fig"}B). Although GFRα1 mRNA is reduced by 16-fold in GFRα1-- A-undiff cells compared with GFRα1+ cells ([Table S1](#mmc2){ref-type="supplementary-material"}), GFRα1 mRNA remains 2.3-fold elevated in GFRα1-- cells compared with TERT^High^ KIT+ early differentiating spermatogonia (q = 1.39 × 10^−17^; [Tables S2](#mmc3){ref-type="supplementary-material"} and [S3](#mmc4){ref-type="supplementary-material"}). Furthermore, FGFR1 and FGFR3 are both expressed on GFRα1+ and GFRα1-- spermatogonia, and FGFR1 significantly decreases in TERT^High^ KIT+ cells compared with GFRα1-- cells ([Table S3](#mmc4){ref-type="supplementary-material"}). Taken together, these results suggest that at least a subpopulation of GFRα1-- cells retains the ability to respond to GDNF and FGF niche factors.

Isolation of Phenotypically Defined Spermatogonial Subpopulations from Adult Wild-Type Mice {#sec3.4}
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Our purification of adult spermatogonia populations, together with RNA-seq, allowed us to identify MCAM as a useful cell surface marker enabling efficient purification of GFRα1+ and GFRα1-- spermatogonial subtypes from adult wild-type mice. MCAM expression on spermatogonia was discovered by [@bib19] in GS cell cultures. *In vivo*, MCAM expression was found on both undifferentiated and differentiating spermatogonia and CD9+ EPCAM^low^ MCAM+ KIT-- cells were enriched for SSC activity by transplantation ([@bib19]). Subsequently, sorting for MCAM+ KIT-- was used to isolate Bmi1+ undifferentiated spermatogonia ([@bib23]). Our results are consistent with MCAM enriching for SSCs; however, we revealed a clear gradient of MCAM surface expression that, when coupled with KIT expression, allows isolation of nearly pure populations of GFRa1+ and GFRa1-- undifferentiated spermatogonia. The ability to isolate these cells from adult wild-type mice will facilitate the study of these populations and allow for future work to define additional molecular and functional features of these cell types in steady-state spermatogenesis.

Experimental Procedures {#sec4}
=======================

Animals {#sec4.1}
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Tert^Tomato/+^ mice were described previously ([@bib34]); *Kit*^*W/Wv*^ mice were purchased (Jackson Laboratory, stock no. 100410). Experiments on adult mice were performed on males between 6 weeks and 3 months of age. All mice were treated in accordance with Association for Assessment and Accreditation of Laboratory Animal Care-approved guidelines at Stanford University.

Antibodies {#sec4.2}
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The following antibodies were used for immunostaining and/or flow cytometry: MCAM-AF488 and MCAM-APC (Biolegend ME-9F1; rat monoclonal), Thy1.2-APC-Cy7 (Biolegend 3OH-12; rat monoclonal), SOHLH1 (gift of A. Rajkovic; rabbit polyclonal). Other antibodies used have been described previously ([@bib34])

Testes Dissociation and FACS Analysis {#sec4.3}
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Testes were dissociated and FACS analyzed as previously described ([@bib34]). For GFRα1 FACS staining, a biotinylated primary antibody was used, together with a secondary streptavidin-APC secondary (Jackson Immunoresearch) for 30 min at 4°C. All FACS experiments were performed on a single BD Aria II machine. Cells were sorted using a 100 μm nozzle in purity mode. Data were analyzed with FlowJo software (Tree Star, San Carlos, CA).

Germ Cell Transplantation {#sec4.4}
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Four independent transplantations were performed. For each transplantation experiment, testes cell suspensions were prepared from two pooled adult mice and sorted as described above. A total of 16--18 recipient testes was analyzed per cell type. Donor cells were introduced into infertile *Kit*^*W/Wv*^ recipients (Jackson Laboratory) via efferent duct injection ([@bib33]). Colonization was determined 8 weeks after injection. Colony numbers were normalized to 100,000 cells transplanted. Statistics were calculated using Prism (GraphPad Software, La Jolla, CA) using the Mann-Whitney non-parametric U test.

RNA-Seq Library Preparation {#sec4.5}
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Dissociated testes cells were prepared and sorted from both testes of adult mice, as described previously ([@bib34]). Four to five biological replicates were sorted per cell population. cDNA was prepared and amplified using the NuGEN Ovation V2 kit, starting from 5 to 10 ng of total RNA. cDNA was sonicated to 200 bp using a Covaris S2 machine, and 25 ng of cDNA was used to make the libraries, following standard Illumina TruSeq v2 protocols. Samples were sequenced on an Illumina HiSeq 2500 machine, with paired-end 101 bp reads.

Author Contributions {#sec5}
====================

A.G. and M.F.P. carried out the majority of the experiments. A.G. performed the bioinformatics analysis. M.F.P. generated the Tert-Tomato mouse and performed whole mounts. K.H. contributed MCAM cytospin and qPCR data. M.S. performed the transplantation experiments with support from K.E.O. R.J.Z. carried out the mouse husbandry. A.G., M.F.P., and S.E.A. conceived the study, designed the experiments, and wrote the manuscript.

Accession Numbers {#app1}
=================

The accession number for the RNA-seq data reported in this paper is GEO: [GSE107694](ncbi-geo:GSE107694){#intref0010}.

Supplemental Information {#app3}
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Document S1. Supplemental Experimental Procedures and Figures S1--S5Table S1. Differentially Expressed Genes between GFRα1+ and GFRα1-- Undifferentiated Spermatogonia, Related to Figure 2Table S2. Short Time-series Expression Miner Table of Genes with Expression Profiles Similar to GFRα1, Related to Figure 3Table S3. Differentially Expressed Genes between GFRα1-- Undifferentiated Spermatogonia and TERTHigh KIT+ Differentiated Spermatogonia, Related to Figure 2Document S2. Article plus Supplemental Information

This work was supported by NIH grants CA197563 and AG056575. We thank members of the Artandi lab for critical comments. A.G. was funded by a National Science Foundation National Research Service Award fellowship. M.P. was supported by a National Science Foundation National Research Service Award fellowship and a T32 training grant (CA09302). We acknowledge the Stanford Neuroscience Microscopy Service (NIH grant NS069375) and the Stanford Shared FACS Facility.

Supplemental Information includes Supplemental Experimental Procedures, five figures, and three tables and can be found with this article online at [https://doi.org/10.1016/j.stemcr.2017.12.009](10.1016/j.stemcr.2017.12.009){#intref0015}.

[^1]: Co-first author
