bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Single-cell RNA sequencing of , macaque, and mouse testes uncovers conserved and divergent features of mammalian

Adrienne Niederriter Shami1,7, Xianing Zheng1,7, Sarah K. Munyoki2,7, Qianyi Ma1, Gabriel L. Manske3, Christopher D. Green1, Meena Sukhwani2, , Kyle E. Orwig2*, Jun Z. Li1,4*, Saher Sue Hammoud1,3,5,6,8*

1Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA 2 Department of Obstetrics, Gynecology and Reproductive Sciences, Integrative Systems Biology Graduate Program, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA. 3 Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI, USA 4 Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA 5 Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA 6Department of Urology, University of Michigan, Ann Arbor, MI, USA 7 These authors contributed equally 8 Lead Contact

* Correspondence: [email protected] (K.E.O.), [email protected] (J.Z.L.), [email protected] (S.S.H.)

bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Summary

Spermatogenesis is a highly regulated process that produces to transmit genetic information to the next generation. Although extensively studied in mice, our current understanding of spermatogenesis is limited to populations defined by state-specific markers defined from rodent data. As between-species differences have been reported in the process duration and hierarchy, it remains unclear how molecular markers and cell states are conserved or have diverged from mice to man. To address this challenge, we employ single-cell RNA-sequencing to identify transcriptional signatures of major germ and somatic cell-types of the testes in human, macaque and mice. This approach reveals differences in expression throughout spermatogenesis, including the stem/progenitor pool of spermatogonia, classical markers of differentiation, potential regulators of meiosis, the kinetics of RNA turnover during spermatid differentiation, and germ cell-soma communication. These datasets provide a rich foundation for future targeted mechanistic studies of primate germ cell development and in vitro gametogenesis. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Introduction

Sperm are highly specialized terminally differentiated cells that carry the genetic information from father to offspring, thus providing a continuous link between the past and future of a species. In all male , the foundational unit of fertility is the spermatogonial stem cell, which balances self-renewal with differentiation to sustain continuous sperm production throughout a male’s adult life. In all mammals, isolated spermatogonial stem cells divide to form networks of connected cells which progress through a series of mitotic and meiotic divisions followed by post-meiotic morphological changes of spermiogenesis to produce mature sperm. This differentiation process is reliant on intrinsic (germ-cell mediated) and extrinsic (soma mediated) signals. Despite these global similarities, it is difficult to translate knowledge generated in mice to higher , especially as it relates to spermatogonial development, because the classical vocabulary that is used to describe those cells in rodents [Asingle (As), 1-3 Apaired (Apr), Aaligned (Aal), A1-4, Int, B] is different than monkeys (Adark, Apale, B1-4) and 4-7 (Adark, Apale, B) For example, based on the existing terminology, it may not be obvious that the undifferentiated to differentiated transition that is marked by expression of cKIT corresponds to the Aal to A1 transition in mice while it corresponds to the Apale to B transition in monkeys and humans8.Therefore, identification and direct comparison of equivalent cell types and states is currently lacking in the field, and as a result, there has been limited success in applying knowledge from mice to inform the study of spermatogenesis and fertility in higher primates.

Although the basic cellular events underlying spermatogenesis and the ultimate goal of gamete production remain the same, there are numerous interspecies differences, including the histological organization of the testis, duration of the seminiferous epithelium cycle, dynamics of stem cell renewal and differentiation, and the quantity and morphology of sperm produced (reviewed in9-11). Therefore, to molecularly dissect the spermatogenesis process, translate knowledge gained between species, and to better make use of the diverse tools available in genetic and physiologic models like mice and macaques we needed a comprehensive and unbiased analysis of the gametogenesis process. Using the power of single cell transcriptomics, we sought to better discern molecularly analogous populations in order to identify shared and divergent properties of spermatogenesis between species, within both the germline and the soma.

Specifically, we analyzed ~14K adult human and ~21K macaque testicular cells, and combined these new datasets with our previously published mouse single cell data12. This allowed us to directly define homologous cell types across species using hundreds to thousands of , and uncovered both known and underrepresented cell types, including transient cell states too rare to be detected with low-throughput approaches. As a result, we produced a high-resolution three-species atlas with aligned analogous somatic and germ cell types/states. First, we describe six molecularly defined consensus spermatogonia states (SPG states) that are conserved in mice, monkeys and humans. While the early terminology used to describe undifferentiated spermatogonia in rodents (As, Apr, Aal) are different than monkeys or humans (Adark, Apale), the SPG cluster identities now provide a harmonizing vocabulary to facilitate cross- species comparisons. Furthermore, we demonstrate that TSPAN33, a cell surface marker of SPG1, can be used to isolate and enrich transplantable human spermatogonia. However, a substantial number of transplantable human spermatogonia were also recovered in the TSPAN33 negative fraction, indicating that cells with colonizing potential in the transplant assay are heterogeneous, at least with respect to TSPAN33 expression. Additionally, we generate the first mammalian spermatogenesis pseudotime map, and describe several conserved and diverged molecular pathways operating within or across species. Finally, we describe between- species differences in somatic cell type relatedness/plasticity, functional roles, and their bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

potential communications with the germline. Overall, this study provides the first single-cell comparative analysis of the spermatogenesis program between primates and rodents. Such a new resource is expected to improve our knowledge base for future studies of germ cell development in primates, and ultimately improve our understanding of the intrinsic and extrinsic evolutionary changes of the gametogenesis program. Knowledge gained from these data will inform fertility restoration efforts, including SSC culture and in vitro gametogenesis.

Results

Single-cell sequencing identifies major germ and somatic cell types of adult human and macaque testes. Using the Drop-seq platform, we generated single cell transcriptome data from ~14K and ~22K adult human and rhesus macaque testes, respectively. Using these datasets, our overall strategy was to first identify major cell types and states in the adult human and rhesus macaque testes separately, then combine them, along with our previous reported mouse data12, to define evolutionarily conserved or diverged programs within germ cells and the somatic cells of the testis (Figure 1A). The doublet rates for the human and macaque datasets is estimated to be <2%, therefore, allowing reliable analysis of individual cells (Figure S1A). After QC filtering (see Methods), 13,837 and 21,574 cells were retained from human and macaque, respectively, with an average of 3,210 unique molecular identifiers (UMIs) per cell, and 1,304 genes detected per cell. This sequencing depth and the number of detected genes was sufficient to define major cell types13,14. Systematic comparisons of technical batches (such as Human 1.1-1.5, Figure S1B-C) and biological replicates (4 humans and 5 macaques, Figure S1D-E) confirmed high batch-to-batch or individual-to-individual concordance, despite the variable proportions of cell types in different samples, likely due to differences in processing or storage of the tissue fragments analyzed.

By unsupervised clustering of human and macaque cells separately, we defined 11 major cell types in both species. Using the expression pattern of previously known cell type-specific markers, we identified somatic cells and the four major germ cell types: spermatogonia (SPG), meiotic spermatocytes (SCytes), post-meiotic haploid round spermatids (STids) and elongating spermatids (Elong) (Figure 1B-C, Supplemental Table 1). Focused re-clustering of somatic cells defined seven major somatic cell types: macrophages, T cells, endothelial cells, peritubular myoid cells, two pericyte subpopulations (with either smooth muscle or fibroblast properties), and a Leydig cell precursor population (Figure 1B insert, 1C, Supplemental Table 1). Altogether, our atlas captures the majority of cell types in both species and allows us to directly compare testis cell types/states across human, macaque, and mouse to define recurrent or divergent molecular programs spanning 85 million years of mammalian evolution.

Between-species differences of cellular attributes defined by transcriptomic properties. To better understand the global molecular and programmatic differences between primate and rodent germ and somatic cells, we compared several cellular attributes extracted from our data using transcriptome-defined indices (Figure 1D).

Previous comparisons of bulk RNAseq data across tissues have shown that testis, and particularly germ cells, express a relatively high number of detected genes15,16. To what extent this diverse expression reflects truly higher levels of unique transcripts per cell or rather a greater heterogeneity of distinct cell types in primate testis was not clear. Our scRNAseq data revealed that individual germ cells tend to express a larger number of genes ("nGene" in Figure 1D) than somatic cells in both macaque and human testis, consistent with our early findings in mice12. Interestingly, the largest diversity of expressed genes is variable across species: highest in spermatocytes and spermatids of macaques and mice, and in spermatogonia for humans bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(Figure 1D). Therefore, germ cell pervasive transcription occurs in different spermatogenic stages across species, although the functional significance of this germline attribute remains to be determined.

As germ cells tend to have higher number of total transcripts ("nUMI" in Figure 1D) sequenced per cell, this elevated "library size factor" could partially account for the higher number of genes detected. To discern the distribution pattern of transcripts within individual cells, we calculated a Gini Index for each cell and compared across cell types in all three species. This analysis quantified the level of transcriptional inequality among genes, determining whether a cell expresses many distinct genes at comparable levels or concentrates most transcripts on a small number of genes. At a given nUMI, a larger Gini index indicates a more biased distribution. Similar to what we previously observed in mice, the somatic cells and the spermatogonia population in human and macaque have the lowest Gini index, which increases progressively in the remaining germ cell populations, reaching the highest level (i.e., the most uneven distribution) in the elongated spermatids (Figure S1F-G). This trend indicates that germ cells along the differentiation trajectory devote an increasingly higher fraction of their transcriptome to a narrower set of genes, which likely reflects the focus on increasingly specific biological functions17,18.

During meiosis, sex become transcriptionally silenced via meiotic sex inactivation (MSCI)19. In all three species, X-linked genes account for 2-5% of the cell's total transcripts in the somatic cells and spermatogonia, yet this fraction is markedly lower in spermatocytes, and partially recovers in the round and elongated spermatids, although to varying degrees in different species (“%X” in Figure 1D). Furthermore, the suppression of sex chromosome in spermatocytes appears to be less complete in primates than in mice ((“%X” in Figure 1D, Figure S1H), and is truly limited to X-chromosome genes since non- average gene expression in spermatocytes and the average X- chromosome gene expression in germ cell states proceeding MSCI are comparable (Figure S1H). To generate a comprehensive list of genes reliably "expressed" in spermatocytes that escape MSCI in either species, we used the following two selection criteria: (1) absolute expression level cutoff: nUMI>0.5 in spermatocytes, and (2) two fold higher expression in spermatocytes than in spermatogonia to ensure that the escapee genes are being actively transcribed, rather than being protected from degradation (Figure 1E). Using this selection criteria, we identified 16 human, 14 macaque genes that escape MSCI, whereas the same threshold in mice identified a single gene Tsga8. These predicted escapees in each species do not enrich in the pseudo autosomal regions, rather they are scattered along both arms of the X-chromosome and lack an expressed Y-chromosome homologue (Supplemental Table 1, see Discussion). Of the genes that escape MSCI in primate spermatocytes, 7 are shared between the two species (Supplemental Table 1, see Discussion). To experimentally test whether these genes can truly escape MSCI, we designed Intron-seq fluorescent in situ hybridization probes for RIBC1 (a predicted escapee in humans and macaque), and show that RIBC1 is indeed transcribed in the XY body of pachytene spermatocytes of macaques (Figure 1F). This incomplete silencing of sex chromosomes observed in our human/macaque single cell data and now in macaque tissue cross-section is consistent with earlier cytological data showing that XY body in human pachytene spermatocytes maintain low levels of Cot1 and tritiated uridine staining in humans, whereas, Cot1 staining was undetectable in mice20,21. Therefore, our single cell data extend these cytological observations and identify candidate genes escaping MSCI.

Taken together, our global molecular analysis of germ cells and somatic highlights important differences in organization and/or regulation of the spermatogenic program among mammals. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Iterative clustering of germ cells independently in each species reveals analogous patterns of discrete and continuous developmental trajectories. Previously, we re-clustered of 20K mouse germ cells and identified a single discrete developmental transition after spermatogonia (SPG) and a continuous developmental trajectory from spermatocytes to elongated spermatids12. Here, we extract 6,015 germ cells in human and 12,799 germ cells in macaque for focused re-clustering separately in each species, and similarly observed the clear partition between SPG and non-SPG germ cells for both species using either principal component analysis (PCA) (Figure S1I-J) or rank correlation among the seven clusters (Figure S1K-L). A provisional clustering analysis divides the germ cells into seven clusters for each species (hGC1-7 and mGC1-7) (Figure S1H, I). Using the expression of known cell state markers, we annotate hGC1-2, mGC1-2 as spermatogonia, hGC3 and mGC3-4 as meiotic spermatocytes, hGC4-5, mGC5 as post-meiotic round, and hGC6-7, mGC6-7 as elongated spermatids (Supplemental Table 2). While this seven-cluster solution will be immediately updated by finer partitions when we zoom in to the SPGs and non-SPG germ cells separately (see below), this mid-level analysis of germ cells provides independent catalogs of cell types/states and comprehensive lists of molecular markers for human and macaque that have not been restricted to 1:1:1 orthologous genes.

Provisional clustering of SPGs from each species identifies four spermatogonial states. To get a cursory view of spermatogonia subtypes we further divided human SPGs (cells in hGC1-2) into finer groups by re-clustering, which uncovers four transcriptionally distinct states, hSPG1-4 (Figure S1I, inset, Supplemental Table 2). These four molecular states are highly correlated to the transcriptional states 0-4 (r = 0.83 - 0.93, Figure S2A) recently described by Guo et al and others22-24 Similarly, re-clustering of macaque spermatogonia (mGC1-2) finds four states referred to as mSPG1-4 (Figure S1J, inset, Supplemental Table 2). A similar analysis of our mouse spermatogonia also identifies four molecular states. The repeated identification of four states in each individual species raises the immediate question as to whether the four provisionally divided spermatogonial states, identified independently in each of the three species, map to each other in a one-to-one fashion. In the past, a common practice was to align cellular states across species based on a small selection of markers. However, here we have the opportunity to perform transcriptome-wide alignment to identify equivalent cellular states.

Joint analysis of human, macaque, and mouse spermatogonia uncovers six analogous molecularly states. To directly identify comparable molecular states across species we used 1- 1-1 homologous genes to merge the 1688 human SPGs, 747 macaque SPGs, and 2174 mouse SPGs. Compared to single-species analyses, this three-species merging significantly increases the total number of SPG cells, allowing more sensitive detection of rarer cell states that may have been missed in individual species. The re-clustering of ~4500 spermatogonia cells identifies six consensus states, ordered as SPG1-6 (Figure 2A, S2B). Expression patterns of previously established conserved marker genes suggest that SPG1/2 are undifferentiated spermatogonia, while SPG3-5 represent progressive stages of differentiation, up to SPG6 which consists of cells preparing for meiotic entry (Supplemental Table 3).

A closer examination of the composition of SPG1-6 states shows that the three species are not distributed evenly across the six consensus states (Figure 2B, S2C, Figure S2D upper panels). By tallying the cells' initial assignment to the four SPG states defined in each species and to the six consensus states, we see that while the cells largely maintain their developmental ordering, there are notable between-species differences of state occupancy (Figure S2D lower panels). Notably, we find that the majority of cells in the undifferentiated SPG1 and SPG2 states are from macaque and human while fewer mouse cells occupy those states. In contrast, the majority of cells in differentiating SPG3 and SPG4 states are from mouse and fewer bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

macaque and human cells occupy those states. All three species are represented in SPG5 and SPG6. The between-species shifts in state occupancy reflect differences in the relative size of the stem/progenitor pool and differing number of transit amplifying divisions (Figure S2C-E), as previously observed by histology (reviewed in 9).

Consensus SPG1-2. Although cells in SPG1 and SPG2 are similar to each other (Figure S2B) and at first glance express many of the well-established canonical markers of undifferentiated spermatogonia, (e.g., GFRA1, ZBTB16 (PLZF), ITGA6, ID4, UCHL1, LIN28, DPPA4 (macaque), FGFR3 and UTF1 (human) (Fig 2D, S2F, Table S3) 9,25-28 the cells within each cluster enrich for a unique subset of canonical and noncanonical markers that can be used to clearly distinguish these two clusters (Figure 2C,D,E). Specifically, cells in SPG1 are largely contributed by human and macaque (Figure 2B, S2C), and are enriched for MORC1, MSL3, ZBTB43, TSPAN33, TCF3, FMR1, MAGEB2 and CDK17 (Figure 2E (TSPAN33, PIWIL4, MORC1; see Table S3 for complete list of shared and species-specific markers). Interestingly, PIWIL4 is a significant SPG1 state-specific marker for all three species (Figure 2E; Table S3). However, mouse contributes very few cells to SPG1 (~40 cells; Figure S2C), accounting for ~2% of all mouse spermatogonia, which are too rare to have clustered independently in our earlier work. This observation is consistent with the understanding that the stem/progenitor pool of spermatogonia in mice is much smaller than in primates (reviewed in 9.) In SPG2 cells, the noncanonical markers enriched in SPG1 (described above) are diminished, whereas, certain canonical undifferentiated spermatogonia markers (i.e. ZBTB16 (mouse and human only), GFRA1 (all three species)) increased in expression, as did several noncanonical markers (DUSP6, TCF4, L1TD1 (macaque and human only))(Figure 2D,E (L1TD1 shown); Table S3). Given these distinct markers, we conclude that consensus clusters SPG1&2 correspond to two distinct undifferentiated spermatogonia populations.

To validate newly-identified noncanonical primate markers expressed by SPG1 and SPG2 undifferentiated spermatogonia (TCF3, MAGEB2, CDK17, MORC1, DNAJB6, PIWIL4 and FMR1), we co-stained with UCHL1, a broad undifferentiated spermatogonia marker (Figure 3A- D, S3A-E, upper panels), or CKIT, a differentiating spermatogonia marker (Figure 3A-D, S3A- E, lower panels) in human testes tissue. TCF3 and PIWIL4 have been described in previous papers22,23,28, while MAGEB2, CDK17, MORC1, DNAJB6, and FMR1 are new markers described in this study. Consistent with our scRNAseq data predictions, our markers exhibit overlap with UCHL1+ spermatogonia cells lining the of the human testis (Figure 3A-D, upper panels), but less overlap with cKIT+ differentiating spermatogonia (Fig 3A-D, lower panels); confirming that SPG1 and SPG2 are undifferentiated spermatogonial cell populations.

Next, we explored whether SPG1 and SPG2 represent cells in different phases of the mitotic cycle. We used ~500 genes known to vary with cell cycle29 to determine each cell's likely phase (Figure S3F-G) and found that the clusters similarly enriched for cells in M/G1 and G1/S (Figure S3H). Thus, state does not account for the difference between SPG1 and 2, but rather these molecular clusters define two transcriptionally distinct states of undifferentiated spermatogonia.

Consensus SPG3-5. A combination of markers is often used to identify differentiating spermatogonia populations across species9,28. States SPG3-SPG5 are more similar to each other than to SPG1-2 or SPG6 (Figure S2B). Human and macaque testes contain cells that express many similar molecular markers to mouse SPG3 such as DMRT1, KIT, STRA8, SOHLH1 and SOHLH2 (possibly equivalent to Aaligned - A1 spermatogonia in mice, Apale - B1 macaque; Apale - B human), but many of these markers are not restricted to specific bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

developmental state – rather are broadly expressed in multiple differentiating SPG clusters and are often temporally uncoupled across species (Figure S2F, summarized in Figure 2G). For example, KIT positive cells are sporadic in SPG1-3 and then increase in frequency in human SPG4 (Macaque KIT not annotated), while STRA8 is absent in SPG3, but sharply upregulated in human and macaque SPG5-6. MKI67 is upregulated in SPG3 of mice and macaques, but not until SPG4 in humans (Figure S2F; Table S3). Cells in macaque and human SPG4 expressed markers such as MKI67, PCNA, numerous cyclins, DMRT1, DMRTB1 and KIT (unannotated in macaque), indicating that this population is actively proliferating, and appears to be molecularly analogous to cells transitioning from Type A1-4/ AIntermediate to B spermatogonia in mouse (see Table S3, Figure S2F)30. This transition has a distinct cell cycle program depicted in Figure S3F and G, which appears to have either extended S/G2-phase or more transient G1 phase, possibly reducing the likelihood of premature meiotic entry of germ cells. The macaque and human SPG5 population continue to express DMRT1 and DMRTB1 (unannotated in monkey) while turning down KIT and turning on STRA8 as well as the meiotic gene REC8 (See Table S3, Figure S2F). Together, these data suggest that consensus SPG4 and SPG5 may represent two transcriptionally distinct Type B spermatogonia populations – referred to here as early (SPG4) and late (SPG5) Type B – in all three species. Classical descriptions of spermatogenic lineage development describes six populations of differentiating spermatogonia in mice (A1-4, Intermediate, B); four in monkeys (B1-4) and one in humans (B).

Consensus SPG6. In SPG6, all species turn off DMRT1 while maintaining DMRTB1 expression (not annotated in macaque), which is a specific signature of mouse Type B spermatogonia12,30. In addition, SPG6, all species begin to express many markers previously identified in mouse preleptotene spermatocytes including Tex101, Ly6k and many established meiotic genes such as Sycp2/3, Syce1/2, Meiob, Prdm9, Spata22, and DNAJB11. Therefore, SPG6 marks a transition from type B spermatogonia to early meiotic preleptotene cells in all three species. However, we identify many new genes that are either expressed in all three species (i.e. FMR1NB, ZCWPW1, DPEP3, IQBP1, CALR), in primates only (BEND2, ZRANB2, PAGE4, ZNFX1, HLTF, ZBED5) or singularly expressed in mouse (Swt1, Tgfbr1, Rhox13, PhTF1, Fmr1), macaque (ZNF850, MAGEB17, ZMYM1, TCEA1) and human (PRDM7, full VCX family, ERBB3, ERVK13-1, SSX3, TRIML2). While functional significance for many of these species- specific genes in meiosis remain unknown, these genes may provide new insights into the evolution of meiotic entry program (see Table S3A for comprehensive list of markers that are shared or species unique genes).

Nomenclature that has evolved to describe spermatogenic lineage development in rodents is different than monkeys or humans, which makes it difficult to translate the implications of data produced in one species to the other species. Therefore, in an effort to unify our transcriptionally distinct populations with classical morphometric descriptors, we updated a model comparing spermatogenic lineage development across species Figure S2D 9. Briefly, SPG1 and SPG2 represent two distinct populations of undifferentiated spermatogonia which do not strictly segregate with classic descriptions of Adark and Apale spermatogonia but are comparable to the mouse undifferentiated spermatogonia populations (As-Apaired-AAligned) (see below). SPG3 contains cells that are transitioning from undifferentiated (Aal mouse; Ad/p monkey and human) to differentiated (A1 mouse; B1 macaque; B human). SPG4 and SPG5 are transcriptionally distinct sub-populations of transit-amplifying differentiating spermatogonia (A2-4, Intermediate mouse, and Type B; B2-B4 monkey and B human). SPG6 cells have upregulated the meiotic program and represent Type B to preleptotene spermatocytes in all three species.

Altogether, our analysis identifies six molecularly defined SPG states which are comparable across species, but not based on a few preselected marker genes. The equivalency of the SPG bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

states across species is confirmed by the average cell-cell Jaccard distances between clusters (Figure 2F). On a global scale, cluster-cluster similarities between species are weaker in earlier SPG states when compared to SPG5-6 across species, suggesting that the start states and molecular pathways for early spermatogonia differentiation are variable across species, but these cells ultimately reach a more conserved molecular identity, or experience a convergence in differentiation program prior to meiosis. Our analysis also reveals both similarities and differences between species in marker gene identity and timing of expression (summarized Figure 2G). While previous studies assumed that well known markers are transferable across species, we now provide a foundational set of universal and species-specific markers that can be used to isolate and analyze equivalent cellular states in human, monkey and mouse.

Molecular states SPG1 and SPG2 do not correlate with Adark and Apale histological states in human. Historically, the undifferentiated spermatogonia in humans and macaques has been described by intensity of nuclear staining with PAS hematoxylin as Adark and Apale spermatogonia5,31,32. Based on in vivo mitotic labeling with 3H-thymidine, Clermont proposed that Adark were quiescent (reserve) stem cells and Apale are active stem cells that maintain steady state spermatogenesis7. To determine whether SPG1 corresponds to either histological state, we combined PAS and hematoxylin staining with immunohistochemistry of human testis cross sections for SPG1 markers PIWIL4, MORC1 and TCF3. PIWIL4 and MORC1 are enriched in SPG1, while TCF3 is more equally distribute between SPG1 and SPG2 (Figure 2E-F). All three markers were localized to the basement membrane of human seminiferous tubules and were expressed by both Adark and Apale spermatogonia (Figure 3E-H). However, TCF3, which is expressed by cells in both SPG1 and SPG2, was skewed more toward Apale spermatogonia (Figure 3G). Thus, SPG1 contains both Adark and Apale spermatogonia, indicating that these descriptions of nuclear morphology do not necessarily segregate transcriptionally. However, there may be a subpopulation of TCF3+ Apale with a distinct transcriptional program (SPG2).

Human spermatogonial stem cell activity is enriched in, but not exclusive to SPG1 While it is tempting to predict stem cell potential for a population based on developmental ordering of single cell data or expression of markers, the only way to truly test spermatogonial stem cell (SSC) potential is by transplanting cells into autologous/homologous recipient testis to reconstitute spermatogenesis33. Autologous/homologous transplantation in humans for experimentation is not possible. For humans, the gold standard is xenotransplantation into the testes of busulfan-treated immunodeficient mice (NCr nu/nu; Taconic)34,35. Although hSSCs do not complete spermatogenesis in this context, they do migrate and colonize the basement membrane of the seminiferous tubule, where they proliferate and produce characteristic chains of spermatogonia that survive for months (Figure 3I).

In order to functionally characterize the colonization potential of the SPG1 population, we enriched for these cells by FACS using the SPG1 cell surface marker TSPAN33 (Figure 2E, Figure 3J) that was also described in the human undifferentiated spermatogonia clusters of two previous studies22,23. Unsorted, TSPAN33-negative, and TSPAN33-positive cells were xenotransplanted into recipient nude mice (Figure 3I-J). Analysis of recipient testes two months after transplantation revealed that stem cell concentration was significantly enriched in the TSPAN33 fraction (46 ± 15 colonies/105 cells transplanted) compared with the Unsorted (10.7 ± 3 colonies/105 cells) and TSPAN33-negative fraction (3.2 ± 0.9 colonies/105 cells) (Figure 3K). This confirms that the TSPAN33+ population (merged SPG1) is enriched for transplantable human spermatogonia, but it does not capture the entire population. In fact, when adjusting for the total number of cells in each fraction, it becomes clear that almost half of colonizing activity resides outside of the TSPAN33+ fraction (Figure 3L). Thus, transplantable human spermatogonia are transcriptionally heterogeneous, at least with respect to TSPAN33 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

expression. Future studies using combinatorial staining, sorting, and transplantation experiments will be needed to determine the phenotype(s) of TSPAN33-negative human SSCs. Molecular heterogeneity in the pool of undifferentiated stem/progenitor spermatogonia may allow spermatogonia to respond to continuously changing niche signals and maintain tissue homeostasis, as suggested by Guo and colleagues22.

Generation of a universal pseudo-timescale for mammalian spermatogenesis. The time required to complete a full spermatogenic cycle is variable across species (~35d in mouse36,37 vs. ~42d in macaque38-40 vs. ~74d in human37,41). Histological analysis of pulse labelling and cell ablation have been used to determine the length of major spermatogenic processes, but to what extent germ cell trajectories and molecular states align across species is poorly understood. To address this gap, we used our scRNA-seq data to reveal heterochrony in the spermatogenesis process and generate a universal spermatogenesis pseudotime comprised of aligned cellular states across species. which will allow us for the first time to define conserved and diverged cellular and molecular processes.

To align the three species, we collapsed the spermatogonia populations to a single group, and ordered the remainder of non-SPG germ cells (N=18537 mouse, N=12016 macaque, N=4293 human) along a linear trajectory using 2,000 highly expressed and highly variable genes selected independently for each species. We then defined distinct pseudotime-points by using Monocle2, which divided cells into 200 ordered "bins", each corresponding to a biological state. To compare across pseudotimes between species, we calculated a rank correlation of each of the 200 states between each species pair, using only a shared subset of highly variable 1:1:1 orthologs detected in all three species (highly variable genes/species = 2000, Shared N=247 genes) (Figure 4A). Therefore, if the differentiation process started and ended in equivalent states between two species and advanced at the same pace through intervening states, a perfect diagonal line would be observed. However, heterochrony occurs between species as deviations from the diagonal are observed (Figure 4A), suggesting that the biological steps defined separately for each species - using the current standard method - proceed at different rates, even when the comparison is based on the same set of 247 genes. We subsequently confirmed that this pseudotime-warping pattern is not driven by the use of 247 common genes in the initial cell assignment, because when we reassign cells to an alternative set of 200 bins, defined for each species separately by using their own highly variable 2000 genes (see Methods), there is little difference in the between-species alignment patterns (Figure S4A).

Although the Human and macaque pseudotimes were largely aligned, the final ~1/3 of the macaque trajectory maps to the final ~1/5 of the human trajectory, suggesting that most mature or terminal state (elongating spermatids) in human corresponded biologically to a wider span of the mature states in macaque (Figure 4A, left panel). Furthermore, we found that the last ~10% of molecular states in macaque appear to extend further beyond the final states of either human or mouse, suggesting that macaque spermatids progress molecularly further than humans or mouse (Figure 4B). Targeted analysis of these overhanging states in macaque revealed no unique markers (data not shown), but rather progressive loss of pre-existing transcripts regardless of UMI depth (Figure S4B-C). This observation is consistent with the previously reported RNA degradation in human sperm and mouse sperm 42-44. Whether these differences are reflected in the transcript content of mature sperm or paternal contribution of transcripts to the zygote remains to be determined.

As expected, more distantly related species showed greater mis-alignments of their pseudotime (Figure 4A). For example, human-mouse and macaque-mouse comparisons showed heterochrony at both the start and the end of the trajectories (Figure 4A, middle, right panels). bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

To directly compare equivalent molecular states across human, macaque, and mouse we developed a common pseudotime shared by the three species by fitting the three individual species pseudotime-series to a single principle curve in PC1-PC3 space (see Methods) (Figure 4B). Cells were assigned to the nearest point on this consensus trajectory, now divided to 20 bins. The final states of the macaque trajectory that did not have an equivalent state in human or mouse (Figure 4B), were added as a single group at the end of the aligned 20-bin trajectory, representing the 21st bin in macaque (Figure 4D). As expected, the 20 centroids, obtained for each of the three-species using the universal coordinates now showed a straight diagonal in the 20-by-20 distance matrix (Figure 4C), confirming their synchrony after warping to the universal time.

With analogous states properly matched, we compared cell occupancy within the 20 (mouse and human) or 21 (macaque) post-mitotic germ cell differentiation states, along with the six spermatogonia states defined above for each species (Figure 4D). Based on the temporal profiles of several conserved marker genes (Figure 4E), we mapped the 26 (27 in macaque) molecular states in each species to major spermatogenesis processes (Figure 4D): mitosis (6 spermatogonia bins as defined in the SPG section above), meiosis (~bins 7-11), and spermiogenesis (~bins 12-20 round spermatids and ~21-26 elongating spermatids). Macaque has an extra bin (the 27th) accounting for its unique terminal state in elongating spermatids. The number of cells per bin can reflect the rate of traversing developmental states and/or the balance of cell division and death. For example, humans and macaques, unlike mice, have a larger pool of undifferentiated spermatogonia (bins 1 and 2; Figure 4C) that undergo a limited number of transit amplifying divisions, whereas, mice have a much smaller population of undifferentiated spermatogonia that undergo a series of mitotic amplification divisions (bins 1 and 2; Figure 4C) (see Spermatogonia section). The cell numbers in bins 5 and 6 (SPG populations) in macaque and human were maintained relatively constant, but a reduction was observed in mouse, consistent with a theoretical 50% reduction in spermatogonia estimated to occur in vivo3,45. Furthermore, in bins 7-10, which represent the transition to meiosis and meiotic divisions, the mouse and macaque states expectedly display an expansion of cells, whereas, humans appeared to initially face a significant bottle-neck at the transition to meiosis. However, only a portion of this reduction can be accounted for by previous histological estimations of degeneration rates in humans46, suggesting that human germ cells may uniquely proceed more quickly through initial stages of meiosis. Finally, we find a greater number of cells towards the end of the trajectory in primates, likely reflecting molecular differences in spermatid differentiation, as there are no cell divisions occurring post-meiotically. Thus, our cross-species alignement of spermatogenesis supports previous histological estimations but also identifies divergence in the amount of molecular states allocated to certain processes, suggesting unique regulation.

In summary, our dataset establishes the first molecular coordinate system for the temporal progression of male germ cell differentiation for three mammalian species and provides markers for individual steps for each species (Table S4) which will be a valuable resource for the community for future experiments.

Species-specific changes in gene expression across the germ cell developmental trajectory. To identify modules of genes with distinct phases of activation in each species, we employed k-means clustering of 2000 highly variable 1-1-1 orthologs expressed across aligned pseudo-time points independently in mouse, human, and macaque (Figure 5A). This unbiased gene clustering analysis revealed successive waves of gene expression corresponding to major biological processes of mitosis (K=1), meiosis (K2-3), and spermiogenesis (K4-6) confirmed by (Table S5). To determine if genes were activated at the same or different phases bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

across species, we used the union of highly variable 1-1-1 orthologs in each of the species pair, ordered them by the six K-means gene group designations, and linked the orthologous genes by lines (Figure 5A). Genes expressed “in phase” (I.e. in K1 of both human and macaque) in two species are linked by horizontal lines and are considered to have conserved temporal expression patterns, whereas, genes that have “shifted” expression patterns are not temporally conserved and are linked by diagonal lines. Finally, genes without a connecting line are those that have “unique” expression patterns, such that their ortholog is not detectable or not dynamically expressed in the other species, and thus cannot be assigned to a K-means group.

As expected, more closely related species displayed higher phase conservation (Figure 5A, S5A). For instance, a comparison of human-macaque showed more in-phase genes (40-56%) than phase-shifted (25-38%), whereas human-mouse showed the opposite trend (20-38% in phase vs. 31-48% shifted), as did macaque-mouse (26-45% vs. 36-49%) (Figure S5A). The shifted genes may reflect changes in the functionality or regulation of genes over the course of evolution. The shifted genes were found to be involved in diverse biological processes such as DNA repair, RNA splicing/processing/binding, and transcription regulation. The direction of shift, revealed some separation of functionality (Figure 5B). For example, human genes that lagged in expression compared to either macaque or mouse, included numerous genes involved in mRNA export and splicing including genes such as RNPC3, NONO, PRPF8, and SNRPB. Broadly, many RNA-binding exhibit shifts in expression across species, including dead- box genes, an evolutionarily conserved family of RNA-helicases. DDX4 (Vasa) is specifically expressed in germ cells from fruit flies to mammals47, whereas many additional members of this family are also expressed during spermatogenesis with greater variation among species. For example, while DDX18 and DDX46 were expressed early in K1 in humans, these genes were not expressed until K2 and 3, respectively in mouse. Reciprocally, DDX23 is expressed early (K1) in mouse, but later in human and macaque (K2), while yet another member, DDX52 is expressed meiotically in (K2) in mouse, but significantly later in human spermiogenesis in K5. The precise functions of these DDX family members have not been determined, but their expression patterns illustrate potential diversification of functions in spermatogenesis. Future analyses are needed to determine whether the increase in out-of-phase genes reflects overall reduced conservation of gene expression during evolution, or programmatic differences in the regulation of spermatogenesis.

Additionally, we find that the number of genes with unique species-specific expression increased progressively across the different k-means groups across all pairwise comparisons. For example, the human vs. macaque, K=1 gene group has 10% unique genes whereas K=6 has ~25% unique (Figure S5A). Therefore, this analysis identifies numerous species-specific transcripts (unique or shifted transcripts) that may be important for the germ cell developmental program.

Overall divergence in expression increases across spermatogenesis in mammals While it is known that testis is one of the most rapidly evolving tissues on both a sequence level and transcriptome level48, our study offers a new opportunity to compare the degree of gene expression conservation across different stages of spermatogenesis. Using 1-1-1orthologs we calculated the rank correlation between gene expression centroids for pairs of species, for each of the 26 ordered stages: 6 for SPG and 20 for the germ cells. The between-species correlation is highest in spermatogonia and decreases progressively through differentiation to spermatids (Figure 5C, solid lines). The correlation values for 835 transcription factors (TF) showed similar decreasing patterns, albeit with lower levels than for all orthologs (Figure 5C, dotted lines). To assess if the lower correlations for TFs than for all orthologs are partly due to the lower expression levels of TFs, we re-calculated the correlations for randomly subsampled bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

orthologs of the same number (835) and matched gene expression distribution as the TFs (Figure 5D). After this resampling-based correction, the TF correlations are no longer lower for meiotic and post-meiotic germ cell states but remained lower than the matched orthologs in SPG states. This result suggests a greater between-species programmatic divergence for transcription factors than for non-TF genes, and this effect is mainly observed in SPG stages.

Many TFs may be expressed at low levels in scRNA-seq data, yet their functional effects can be estimated by the concerted changes of their downstream target genes, i.e., each TF's "regulon". By using SCENIC49 we obtained the activity scores for 82 and 88 regulons with 10+ target genes for human and mouse germ cell centroids, respectively (Table X). We ranked their degree of conservation by the human-mouse correlation of regulon scores across the 26 stages, and summarized the top 17 most conserved and top 5 most diverged between human and mouse (Fig S5C). (This analysis was not performed in macaque since a corresponding TF database is not available). Importantly, many of the top TF regulons (e.g. EZH2, E2F1, YY1 and Sox6) we identify have been previously implicated in spermatogenesis50-54. Second, the phasic pattern we report here is consistent with previously described functional dynamics in mice50-54 . However, our results also include several novel regulons that have not been previously explored in the context of spermatogenesis (e.g., UBTF, GABPA, BRG1, GABPB1, BACH1 ) and may play a role in germ cell epigenetic memory or transcriptional regulation.

While this comparison takes into account only orthologous genes, it is possible that other species-specific genes may have temporal bias in expression and thus contribute to observed divergence in the spermatogenesis program. Next we examined the bulk gene expression levels for genes of various ortholog classes (Fig 5E, S5B). Macaque appeared largely unchanged in the amount of 1-1-1 ortholog expression, and macaque-specific genes contributed little to total expression, likely a reflection of low annotation quality of the macaque where the vast majority of genes are orthologs (Figure 5E, S5B). This is further underscored by the high number of orthologs overlapping in human and mouse only (Fig S5B), many of which likely have currently unannotated genes in macaque. Focusing instead on categories with greater annotation confidence, several interesting patterns are observed. Human and mouse show a decrease in the degree of ortholog expression across spermatogenesis, which is complemented by an increase in species specific (lacking an ortholog in mouse or macaque) gene expression (Fig 5E). The amount of primate specific gene expression (present exclusively in both human and macaque, regardless of copy number) peaks at the entry into meiosis. Interestingly, these primate specific genes contain a large number of C2H2 zinc-finger containing proteins (227/875 human orthologs), about half of which have been characterized as having testis-enriched expression relative to all other tissues in the Human Protein Atlas. Most of these genes (198) are KRAB domain-containing, the largest subgroup of C2H2 zinc-finger proteins which have been shown to bind endogenous retro elements (ERE)55. Indeed many of these genes are capable of binding at least one ERE in vitro, with most binding ERVs (Figure S5D,E).These genes may then aid in identifying novel primate-specific regulators and mechanisms of entry into meiosis.

Species specific transcriptomic differences in interstitial somatic cells suggest functional divergence between rodents and primates Germ cell maturation requires intricate communication between the germline and soma. The importance of germline-soma compatibility has been elegantly underscored in vivo by cross-species transplantation experiments, where rat SSCs can complete spermatogenesis in the mouse testis56, whereas macaque or human SSC colonize the mouse testis but fail to initiate meiosis57. The incompatibility between SSCs and somatic compartments of distant organisms may reflect changes in signaling pathways or cell types in the microenvironment that alter regulatory programs within SSCs. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

To create a new atlas of the somatic compartment we performed focused re-clustering of 3,722 human and 2,098 macaque somatic cells, yielding 7 clusters for each (Figure S6A-B). Using well-established markers, we identified myoid cells, endothelial cells, Leydig cell precursors, and macrophages, and several rarer cell types not extensively studied in testis, such as T cells and two types of pericytes (Figure 6A-B, S6C). However, unlike mouse our human and macaque datasets failed to capture Sertoli cells, Innate Lymphoid cells, or mature Leydig cells12, likely due to the mechanical or physiological stress of the cryopreservation and dissociation methods.

One of the most abundant populations captured in primates was peritubular myoid cells, defined by the expression of MYH11, ACTA2, , , (VCAN), and (Figure 1C, 6A, S6C, Table S6). This overrepresentation of myoid cells in primates, as compared to mouse12, reflects structural difference in seminiferous tubule basement membrane, which consists of multiple layers of myoid cells in human/macaque, but a single layer in mouse (reviewed in 58) (Fig 6C). Myoid cells are contractile and are involved in the transport of spermatozoa and testicular fluid in the tubule59. Recent studies in mice have suggested that myoid cells can also secrete a number of substances, including components (in vitro) or growth factors that may modulate the function of neighboring somatic cells (i.e. TGFB or Activin A) or spermatogonial cell proliferation (i.e. GDNF, in vivo). Consistent with these findings, human and macaque myoid cells expressed many genes involved in muscle contraction, as well as, some conserved (fibronectin and collagens) or unique extracellular matrix (ECM) proteins such as decorin and (Table S6), which are believed to be absent from the mouse testis under normal cellular homeostasis60. Unlike mouse myoid cells, the human and macaque myoid cells don’t produce the spermatogonial stem cell proliferative factor GDNF, but expressed components of other signaling pathways like PDGFRA, EGFR, PTCH1, and PPARA which are not detected in the analogous mouse population (Supplemental Table 6). Therefore, although both mouse and primate myoid cells have contractile properties, they have acquired species-specific signaling and molecular functions.

A second major population identified in the human and macaque is immature Leydig cells, which is observed in neonatal rodent testis, but thought to be absent in adult mouse testis (reviewed in 61,62) and was lacking in our adult mouse testis data12. This human and macaque population expressed multiple markers previously shown to be restricted to spindle-shaped putative progenitor Leydig cells63 and are critical for Leydig cell differentiation64,65 (e.g. DLK1 (unannotated in macaque), IGF1/2, CFD (Adipsin), SFRP, PTCH2, and IGFB3 (Figure 6A, Figure S6C, Table S6)), whereas their expression of steroidogenic enzymes (STAR and Cytochrome P450 genes) were very low and limited to a small subset of cells (data not shown). These cells also expressed ECM genes such as decorin and collagens. Additionally, we found that the immature Leydig cell transcriptome is highly similar to that of myoid cells (Figure S6B), suggesting that myoid and Leydig cell lineages may share a common progenitor in human and macaque, as seen in mice66,67.

The somatic compartments also contained an endothelial cell (VWF, NOSTRIN, AQP1, CD34 (human)) population and a heterogeneous population of cells known as pericytes, which together interact to form the mural wall of small blood vessels throughout tissues68,69. Pericytes express MCAM, RGS5, PDGFRB, NG2, CCL2, ABCC9, and NOTCH3, consistent with recently defined markers in brain and lung datasets70,71 (Fig 6A, S6C, Supplemental Table 6). Furthermore, the testis pericyte population splits into two distinct sub-clusters of pericytes that can be uniquely separated into a muscular (m-pericytes) or fibroblastic (f-pericytes) types based on their expression profiles (m-pericytes: MCAM (higher expression), CRIP1/2, RERGL, ADIRF (unannotated in macaque), and several myosins MYL9, PTM1/2) (f-pericytes: STEAP4, bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

GUCY1A1/2, ITGA1, CD36 (human), CD44(human) and several collagens and laminins) (Figure 6B, S6C, Table S6). Using immunohistochemistry data available from the Human Protein Atlas, we find that several markers of m-pericytes (MCAM, CRIP1, ADIRF) are more restricted to cells surrounding blood vessels; whereas f-pericyte markers (CD36, CD44, ITGA1) are present both around blood vessels and in the interstitial space (Figure S6D). Despite the high correlation between the two pericyte populations (Figure S6B), the individual species cluster-cluster centroid correlations suggested that the transcriptome of m-pericytes was more highly correlated to myoid and immature Leydig cells, whereas f-pericytes were more distinct (Figure S6B). These findings suggest that f- and m-pericytes might arise from different cells of origin in the testis.

Finally, we identified several types of immune cells. The majority were macrophages (CD163, HLA-DRA/MAMU-DRA, LYZ, TYROBP) (Figure 6B, S6C, Table S6). Although rare, lymphocytes were also present in the testis. In rodents, CD8+ (cytotoxic) T cells are the predominant lymphocyte population72, but in humans, the frequency and location of lymphocytes is poorly understood73-75. Here we identified T cells (CD3D, TRAC, TRBC2, CD69) (Figure 6B, S6C, Table S6) with some CD8 expression (23% of T cells in human, 10% in macaque), indicating that CD8+ lymphocytes are indeed present in adult human and macaque testis.

Taken together, we identified many of the expected major or rarer somatic cell populations of the testis that express shared or species-specific factors. Given the several noted differences in the marker genes of primate vs. mouse somatic cells, we sought to compare somatic cells more globally to uncover how these somatic populations have changed over the course of mammalian evolution. Briefly, we merged the somatic cells from all three species and performed principal component analysis (Figure 6D) and rank correlation of the cell type cluster centroids (Figure 6E) using 1-1-1 orthologs (see Methods). In the merged PCA plot, PC1 and PC2 segregated the immune cell populations (macrophages and Tcell/ILCII) from the remaining somatic cells, confirming that the widest distinction is driven by the rapid evolution of cell type differences, rather than species differences (Figure 6D). Although, we did not previously identify T cells in mice, mouse innate lymphoid cells had the highest correlation with primate T cells, as expected based on their similar origin from a common lymphoid progenitor and shared functions76. Endothelial and Macrophage cell transcriptional programs appeared to be largely conserved as they have higher correlation values across species ( r=0.6-0.8; Figure 6E, upper half of matrix).

However, there are notable between-species differences in the remaining somatic cells. For example, mouse myoid cells appeared to be more transcriptionally similar to primate m- pericytes, than primate myoid cells. Similarly, although Leydig precursor cells in primates somewhat resembled adult mouse Leydig cells, they were more similar to our previously unexpected mesenchymal cell population in the mouse testis12. Thus, many of the somatic cells within the testis have morphed transcriptionally across species. To confirm that the unexpected cellular associations are not driven by orthologous gene selection, we performed clustering using the union of marker (Figure S6E; lower half of matrix) or the highly variable genes from each of the three species (Figure S6E; upper half of matrix), and show similar cellular association and correlation patterns. Using ~300 genes encoding known ligands, we found a nearly identical pattern of correlation among the cell types and across species (Figure 6E, lower half of matrix), indicating that the observed shifts in cell identity relationships is likely due to genuine global programmatic changes in the transcriptome, signaling pathways and possibly function. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Finally, as expected, the strength of the cell type correlations across species decreases with increasing evolutionary distance. How these changes impact spermatogenesis are unclear, but they may contribute to the incompatibility observed between germ cells and somatic cells of distant species.

Evolutionary differences in the germ cell - soma cell communication. Our scRNA-seq data also provides an unprecedented opportunity to begin exploring the poorly understood cell-to-cell communication between the soma and the germline in the testis. To comprehensively catalogue the cell-to-cell communication we filtered known ligand–receptor interaction pairs in public interaction databases 77 by focusing on those with 1) ligands detected in somatic cell clusters and receptors detected in germ cell clusters, and 2) either the ligand and receptor were highly variable in at least one species. This led to 1059, 529, and 785 L-R pairs in human, monkey, and mouse, respectively (Table S7). By calculating the Interaction Scores for each L-R pair (Methods) across all 182 cell type pairs (7 somatic and 26 germ cell types), we created a somatic-to-germ cell interaction map that depicts the number of putative strong interactions (defined in Methods) for each cell pairs (Figure 7A). This global analysis reveals that many somatic cells do have the potential (i.e., the expression of ligands) to communicate with germ cells, supporting an open niche model for the mammalian testis. In humans and monkeys, the immature Leydig cells, Myoid and f-pericytes have the most extensive putative interactions than other somatic cell types detected in our dataset. Meanwhile, Sertoli and Leydig cells – two central niche cell populations - have the least number of outgoing signaling interactions in mouse. Instead, the Tcf21+ interstitial population, myoid, macrophage, and endothelial cells exhibit the greatest number of interactions (Figure 7A). Among the germ cells, the spermatogonia populations (SPG1-6) tend to have more putative L-R interactions than the germ cells, consistent with the role of the blood-testis barrier (BTB) that prevents meiotic and post meiotic germ cells from directly communicating with neighboring somatic cells. A possible exception may be when the BTB breaks (stages VIII-IX), giving differentiating germ cell populations the opportunity to communicate with surrounding somatic cells and possibly explaining the weak and few interactions detected between somatic cells and later germ cell stages.

Given the significant signaling differences observed across cell types, we explored the conservation of individual signaling pairs. For each L-R pair we calculated its between-species correlations across the 182 cell type pairs and used such a measure to rank the degree of conservation of signaling pathways (see Table S7 for the full list). Globally, Fig S7A-B shows the 30 most conserved and 30 least conserved L-R pairs, from which we highlight examples for several classes of conservation patterns (Fig 7B, S7C). First, the target cell is conserved but the source cell is not (FGF2-FGFR1, COL4A1-ITGB1, WNT5A-RYK; CXCL12-ITGB1), while for some pairs there is better conservation within primates specifically (FGF2-SDC2; BMP4- BMPR2). Second, the source cell of the ligand is conserved but the target cell is not (FGF2- SDC2). Third, both the source and target cell type vary across species (SLIT2-ROBO; APOE- SCARB1; WNT5A-ROR1; BMP4-BMPR1B; FGF2-SDC4; JAG1-NOTCH2; FGF7-FGFR3). These patterns suggest that while some pathways may be employed in multiple mammalian species to regulate similar stages of spermatogenesis, the timing of activation and the origin or target of the signal may have diverged. In sum, we leveraged ligand and receptor expression levels in specific cell types to generate testable hypotheses regarding intercellular signaling patterns and potential evolutionary shifts. Future perturbation and spatial transcriptomics and proteomics experiments will be needed to validate them.

Discussion bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Germ cell differentiation is a highly regulated process reliant on both intrinsic and extrinsic signals. Although the basic cellular events - mitosis, meiosis, spermiogenesis- are largely conserved across species, there is notable divergence in the cellular dynamics, structural organization, hierarchical patterns, and output of this differentiation process9,11. Molecular and genetic analyses of mouse testes have provided substantial insights into the process of spermatogenesis and are often used to guide parallel studies in primate testes. However, most comparative studies to date are reliant on a handful of markers developed in histological analyses, which are used to define presumably equivalent cellular states across species. These approaches may overlook important functional divergence between species, and may yield an incomplete, or even incorrect, picture of primate spermatogenesis. Therefore, an unbiased comparative analysis of germ cell development and somatic microenvironment is needed to elucidate similarities and differences across mammalian spermatogenesis. We addressed this challenge by employing single-cell RNA-seq analysis of normal adult human and macaque testes, and comparing with our previous mouse dataset to provide a new generation of cellular atlases within each species and across species

The first contribution of this study is the complete differentiation trajectory of the spermatogenic process in human and macaque, along with molecular markers for major germ cell and somatic cell types, which we provide separately for each species (Table S1). On a global scale, we observe instances of inter-species difference in transcription regulation, suggesting evolutionary changes of some aspects of spermatogenesis program. For example, we find that meiotic sex chromosome inactivation (MSCI) is incomplete in primates; we estimate at least 16 genes in human and 14 genes in macaque escape sex chromosome repression in spermatocytes. Several of the identified escapees appear to be critical for normal germ cell development, e.g. DYNLT3 is involved in meiotic chromosome segregation78,79, while AKAP14, AKAP4, CYLC1, and PIH1D3 are important for sperm structure and motility80-85. Several escapees (SPANXN3, SPANXN5, SPANXD) are members of the primate ampliconic SPANX gene family86. Although the function of these genes is unknown, copy number variants involving SPANXN5 have been linked to human infertility 87-89, indicating this family may have critical roles in spermatogenesis.

To directly compare germ cell states, we combined and re-clustered spermatogonia from all three species, allowing us to identify six consensus states. While different vocabulary has been developed to describe spermatogonial development in rodents (As, Apr, Aal, A1-4, Intermediate, B) and in monkeys and humans (Adark, Apale, B1-4 (only one generation of B in human)), our comparative analysis of spermatogonia to transitioning preleptotene spermatocytes suggest that spermatogonial stem cells traverse through molecularly comparable states across species. Furthermore, we find that the proportion of cells contributed by each consensus state varies by species, reflecting inherent differences in the size of the stem/progenitor pool and the number of transit-amplifying divisions in the differentiating spermatogonia compartment across species. In these six states (SPG1-SPG6), we identify two molecularly distinct undifferentiated spermatogonial cell states (SPG1: TSPAN33, PIWIL4, CDK17, MORC1, ID4, ZBTB16, and SPG2: L1TD1, ID4, GFRA1, and ZBTB16). Immunohistochemistry analysis of these markers shows that SPG1 and SPG2 do not have a direct, one-to-one correspondence to the two histologically defined primate spermatogonial stem cell populations in the human testis known as Adark or Apale. Rather, together SPG1 and 2 represent a heterogeneous pool of undifferentiated spermatogonia. Furthermore, we used human-to-nude mouse xenotransplantation to demonstrate that stem cell activity is enriched in the TSPAN33-positive fraction of SPG1, but was not restricted to that fraction as roughly half of total stem cell activity was recovered in the TSPAN33-negative fraction. Future studies using additional markers are needed to resolve the cellular heterogeneity, stem cell potential, and the phenotype(s) of the bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

TSPAN33-negative stem cells. Based on the current data, it is not possible to know whether stem cell activity in the TSPAN33-negative fraction is from TSPAN33-negative cells in SPG1 or might also include cells in SPG2. Nonetheless, these data clearly indicate that the transplantable hSSCs are heterogeneous, at least with respect to the TSPAN33 cell surface marker.

Although SPG1 cells in our dataset mainly come from macaque and human samples, they do contain a small number of PIWIL4 (a.k.a. Miwi2) expressing cells from mouse. They account for <2% of mouse spermatogonia, too small a fraction to be detected in our earlier work. Curiously, Miwi2 knockout mice exhibit progressive loss of germ cells90,91. With a Miwi2tdtomato reporter, MIWI2 expression is detectable in a small number of Asingle but is more prevalent in Apaired and Aaligned undifferentiated spermatogonia. These Miwi2-expressing cells possess SSC activity as demonstrated by transplantation92, and are required for efficient regeneration of spermatogenesis after injury in the adult mouse testis 93. Together, these findings suggest that a rare population of early-stage undifferentiated SPG cells may exist in mouse that are analogous to SPG1 cells in primates. In mice, these cells may represent a rare population of As, Apr, Aal undifferentiated spermatogonia that were missed in our previous analysis due to its smaller number of cells.

The consensus SPG3-5 contains cells that are transitioning from undifferentiated (Aal mouse; Ad/p monkey and human) to differentiating (A1-4, Aintermediate and B spermatogonia mouse; B1-4 macaque; B human). We link these molecular states with morphometric states described across species and identify two molecularly distinct Type B molecular states. Further, SPG6 includes type spermatogonial cells transitioning to preleptotene spermatocytes based on the expression of conserved meiotic genes, such as PRDM9, ZCWPW1, and REC8. In addition to the known, classic meiotic genes, our dataset uncovers many primate-specific genes (i.e., lacking mouse orthologs) that are active in human and macaque SPG6, including the simian- specific Variably Charged (VC) gene family (VCX, VCX2, VCX3A, VCX3B)94, which is an X chromosome multi-copy gene family theorized to play a role in meiotic drive94,95. Recent studies have shown that VCX may be regulated by PRDM996, further underscoring its potential role in meiosis. Although the function of this gene family is not yet directly tested, CNVs involving VCX have been associated with nonobstructive azospermia97. In addition to the VC-family, many primate-specific zinc-finger containing proteins peak in expression at the SPG6 stage. Unlike other transcriptional regulators, zinc-finger proteins have undergone bursts of duplication in , including the primate lineage98,99, and are rapidly evolving (dN/dS >1)100, indicating they may have evolved specific functions in primate spermatogenesis. Specifically, KRAB-ZFPs are rapidly expanding and evolving ZF family101, and several lines of experimental evidence demonstrate that endogenous retro elements are the primary target sequences, suggesting this diversity may stem from dynamic competition between TEs and KRAB-ZFPs (reviewed in 102,103). Based on the timing of expression of the primate-specific KRAB-ZF genes shown here, these genes may be activated in order to protect against the sequelae of active or inactive EREs, which are often also species-specific.

Taken together, our analysis suggests that many mammalian species traverse through comparable molecular states, which share some conserved features, while diverging on other features, reflecting evolving molecular pathways that execute the germ cell differentiation process.

Although the post-SPG portion of the spermatogenesis program (meiosis and spermiogenesis) is largely continuous, our multi-species comparisons of germ cell trajectories suggest that bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

different species do not always follow the same progression through transcriptional intermediates during germ cell differentiation. Rather they exhibit heterochrony in the alignment of cellular states, both in the beginning (meiosis) or end (spermiogenesis) stages and in the pace of progression in the middle of the germ cell differentiation process. This apparent meandering in the comparison of germ cell trajectories is more pronounce with increasing evolutionary distance (human vs. mouse). To directly compare molecular states across species, we constructed a universal spermatogenesis “pseudotime” as the consensus of the three species and aligned molecularly analogous populations across species. This universal pseudo- temporal map represents the first common molecular atlas of mammalian germ cell differentiation and provides state-specific markers in analogous populations in each species. With cells from all three species now mapped on the same scale, we were able to directly compare molecular states to reveal similarities and differences in the transcriptome and temporal shifts in gene expression. Interestingly, many of the genes experiencing shifted phase across species include transcription factors and RNA binding proteins.

Finally, we also discovered interspecies differences in the somatic cells that provide surrounding niche for the developing germ cells. The new catalog of somatic cells are accompanied by their molecular markers, expected functions, and likely communications among distinct cell types. A direct comparison of the somatic cell populations across species shows that the transcriptome of endothelial cells and immune cells are highly conserved across species, whereas other interstitial cells show greater divergence. For example, mouse myoid cells do not have the highest correlation to the human myoid cells, but instead bear a high transcriptional resemblance to human m-pericytes. To gain a preliminary view of the soma-germ cell communication and its potentially altered function across species, we examined known ligand- receptor pairs with predicted signaling from somatic cell populations to germ cells. We find that although certain pathways are likely employed to regulate similar stages of spermatogenesis, many other ligands/receptor pairs have either changed the source (cells of secretion) or the target (cells bearing the receptor), or both, generating many interesting hypotheses regarding the evolution of germ cell – soma communication in mammals. One example is the FGF signaling pathway which provides regulatory cues for spermatogonial dynamics 104,105. FGF signaling depends on , such as syndecans, to bind and transfer FGFs to receptor tyrosine kinases (FGFRs) on target cells. In mouse spermatogonia, SDC4 promotes the consumption of FGFs105. We find that this interaction appears to be conserved in human and macaque, and there is an additional syndecan (SDC2) expressed in primates which can bind FGFs secreted by various somatic cells. Therefore, germline-soma communications have changed over the course of evolution through both the receptor and the ligand.

Altogether, our analyses provide the first reference system for integrated interspecies comparisons of spermatogenesis, highlighting multiple ways by which this process has evolved while maintaining the capacity to reach a common goal – male gamete production. Furthermore, we identify molecularly equivalent states between human, macaque, and mouse, so that despite the many apparent differences, mechanistic parallels can be drawn between species to enable future studies that make joint use of these complementary model systems. This knowledge enhances our ability to interpret contemporary discoveries in the context of the classic descriptions of cell morphology from the past. It is expected to reduce barriers to studying human infertility, and accelerate the progress towards developing novel therapies, such as in vitro gametogenesis.

Acknowledgements bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

We thank members of the Hammoud, Li, Orwig, and Yamashita Labs for scientific discussions and manuscript comments. This research was supported by National Institute of Health (NIH) grants 1R21HD090371-01A1 (S.S.H. and J.Z.L.), 1DP2HD091949-01 (S.S.H.), P01 HD075795, R01 HD076412 (K.E.O), R01 HD092084 (K.E.O., S.S.H), F30HD097961 (A.N.S), training grants 5T32HD079342 (A.N.S.), 5T32GM007863 (A.N.S.), T32HD0871494 (S.K.M.), and Michigan Institute for Data Science (MIDAS) grant for Health Sciences Challenge Award (J.Z.L. and S.S.H.), Open Philanthropy Grant 2019-199327 (5384).

Author contributions

S.S.H. and A.N.S. overall project design. J.Z.L. oversaw the computational analysis. K.E.O oversaw functional experiments. A.N.S., S.K.M., C.D.G., and M.S. performed experiments. X.Z., Q.M., and A.N.S. analyzed data. A.N.S and S.S.H. wrote the manuscript with input from J.Z.L. and K.E.O. Comments from all authors were provided.

Declaration of interests

The authors have no competing interests. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.994509; this version posted March 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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% nGene # Markers nUMI ChrX Fig E D bioRxiv preprint C u 501 10 10 10 2K 4K 8K re 0 4 8 351 280 274 521 202 251 216 263 160 266 251 A 3 4 5 4 Humans Major Cell Type Clusters Type Cell Major

SPG 1 Germline Iterative Re 1e+0 T cell 1e 1e 1e Single cell RNA cell Single T cell (which wasnotcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. - - -

3 2 1 Macrophage Cross 1e Endothelial Macrophage Sub - Analysis 4 ~25 mya ~25 Endothelial doi:

m-Pericyte - clusters - Species SCyte 1e F-Pericyte m-Pericyte - 5 Macaques 5 clustering https://doi.org/10.1101/2020.03.17.994509 - 2 Myoid F-Pericyte Somatic

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ImmLeydig seq ~85 mya ~85 1e+0 SPG ImmLeydig SCyte SPG STid SCyte Ma & Green Elongating STid Elongating

SPG TNP1, PRM1/2, SPATA3, TSSK6 SPACA3/4 H1FNT, BRDT, ACRV1, YBX2, PIWIL1 SYCP1/2, DMRT1, MORC1, DAZL, SYCP3 BEND4, NR6A1, ZBTB43, ID4, SFRP1, NR2F2 DLK1, IGF1/2,CFD, DCN, ACTA2, COL4A1, NR2F2, CXCL12 STEAP4, IGFBP5/7, ITGA1, TAGLN MCAM, MYL9, ADIRF, CRIP1, VWF, TIE1 CD34, CD74, CD52,

1e+0 T cell 1e 1e 1e et al, 2018 - - - UCHL1, L1TD1, FMR1,

3 2 1 Macrophage Representative Representative B 1e LYZ, TYROBP, CSF1R CD69, CD3D, TRAC, ID2

Endothelial MYL6 MYH11, - 4 1e CENP1, SPATA16, m-Pericyte Markers

SCyte F-Pericyte - 2 1e+0 Myoid ImmLeydig SPG Soma

SCyte ; this versionpostedMarch18,2020. STid Elongating # Markers Spermatid Elongating Innate Lymphoid SPG Spermatogonia Spermatocyte 1e+0 1e 1e 1e 1e Macrophage - - - - 4 3 2 1

Endothelial Spermatid 273 206 822 846 301 347 304 344 297 380 260 Round 1e Myoid - 4 1e TCF21+ Int

SCyte T cell Leydig Macrophage -

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Endothelial ImmLeydig Myoid f m Endothelial Macrophage cellT - Pericyte SPG - m-Pericyte Pericyte SCyte F-Pericyte STid Myoid The copyrightholderforthispreprint Elongating ImmLeydig SPG

F SCyte Soma

STid Spermatogonia Elongating TNP2, SPATA19 ACRV1, PIWIL1 SYCP1/2, DAZL, SYCP3 NR6A1, BEND4, DMTR1, MORC1, FMR1, L1TD1, UCHL1, ID4, NR2F2 IGF1/2, ACTA2, COL4A1, NR2F2, CXCL12 STEAP4, MYL6/9, VWF, SELE CD83,LYZ, TYROBP CD52, CD3D, Representative Representative TPPP2, SPATA3, TSSK6 SPATA3, TPPP2, DCN, MYH11, H1FNT, BRDT, TAGLN, CRIP2 TAGLN, SPARC, Spermatocyte CENPU, YBX2, Markers CFD, SFRP1/2, Spermatid CD69, ID2 Spermatid Elongating Round MYL6 IGFBP7, ITGA1, ITGA1, IGFBP7, CHD5, Figure 2

A 3 Species Consensus SPG B

● ●● ●● ●●● ● ●● ● ● ● ●●●● ●●●● ● ●●●●●● ● ●● ●● ● ●● ● ● ●●●● ● ● ●●●● ●● ● ●● ●● ●●● ●● ●● ●● ● ●● ● ●● ●● ● ● ● ●● ●● ● ●● ● ●● ●●● ●● ●●● ●●● ●●● ●●● ●●● ● ●●● ● ●● ●●● ● ●●● ●● ●●● ● ●● ● ● ●● ●●●● ●●●●●● ●●●●●● ●●●●● ●●● ●● ●● ● ● ●●●●●● ● ● ● ● ● ●● ●● ●● ●●●●●●●●● ●●●● ● ●●● ●●● ●● ● ● ● ●●●●●● ●● ● ●●● ●●●●● ●● ● ● ●●●●● ● ● ● ●● ● ●●●●● ●●●●● ● ●●●●●● ●● ●●● ● ● ●●●●●● ● ● ●●●●● ● ● ●●● ● ●●● ● ● ●●●● ● ●●●● ●●●●●● ●● ● ●● ●●●● ●●●●●●●● ● ●●●●●●●●● ● ● ● ● ●● ● ●●●●●● ●● ● ● ●● ● ●●● ● ●●● ●●●●● ●● ●●●● ● ● ●●● ●●●●●●● ●● ●●● ●● ● ● ● ●●●● ● ● ●● ● ●● ● ● ● ●●●● ● ● ● ●● ●●● ●●●● ●● ●● ● ●●●● ● ●●●●●● ● ●● ●● ● ● ●● ●● ●● ●● ●●● ●● ●● ●● ● ● ●● ● ● ●● ● ●●● ● ●●●● ●● ● ● ● ●● ●●●● ● ●● ● ●● ● ● ●●●●● ●● ● ● ●● ●●●● ●●●●● ● ● ● ●●● ●●●●●●● ●●● ●● ●●●● 20 ● ●● ● ● ● ●● ● ● ●● ●● ● ● ● ●● ●●● ●●● ●● ● ● ●● ●●● ● ●●●●●● ● ●●●●●●●●●● ● ●● ● ● ●●●●● ●● ●●●● ●● ●● ●● ●●●●●●●● ●● ●●●●● ●● ●●●●●● ● ●●●●●●●● ● ● ●● ●●●●●●● ●●● ●● ●● ● ●● ● ●●●●●●● ● ● ●●●●●●● ●●●●●●● ●● ●●● ●●● ● ● ●●●●●●●●●● ● ●● ● ●●●● ●●●● ● ●● ● ●●● ●●●● ●● ●●●●●●●●●● ●● ●● ●● ●●●● ● ● ●● ● ● ● ● ●● ● ● ● ● ●●● ●●●● ●● ● ● ● ● ●●●●●●●●● ● ● ● ●● ●● ● ● ●● ●●●●●● ●●● ● ● ● ●● ● ●● ●● ● ●● ● ● ● ● ● ●●● ●● ● ●●●● ●● ● ●●●●● ●●●●● ● ● ●●● ●● ● ● ● ● ●● ● ● ●● ●●● ● ●● ● ●●● ●●●●●●●●●●● ●●●●●●● ●●● ● ● ●●●●● ● ●●●●●● ● ●●● ●●●●● ● ● ● ●●●● ●● ● ● ●●●●●●● ● ●●●●● ●● ● ●●●●●●●● ●●●●● ●●● ● ● ●●● ● ●●● ●●●● ●● ●● ●● ● ●●● ● ●●●●●●●● ● ●●●● ● ●●●●● ●●●●● ● ●●●●●● ● ●●● ● ●●●● ●● ● ●● ●●● ● ● ●● ●●● ● ●●●●● ●● ●●●● ●● ●●●● ● ● ●●● ●● ●●● ●● ● ●●● ●●●● ●●●●●● ● ●● ●●● ●● ● ● ●●●●●●● ● ● ● ●● ●● ● ●● ●● ●●●●● ● ● ●●● ● ●●● ●●●● ● ● ●● ●●● ● ●● ●●●● ●●●●●●●●● ● ● ●● ●●●● ● ●● ●● ● ●● ●●● ● ● ●●● ●●●●● ●●● ●●● ●● ●● ●●● ● ● ● ●●●●● ● ●● ● ●●● ● ●●●●● ● ●● ●● ● ●●● ● ●●●● ● ● ● ● ●● ●● ●● ●●●●● ●● ●● ● ● ●● ●●● ●● ●● ●● ●●● ● ● ● ●●● ● ●● ●●●●●●● ● ●●●● ●●●● ●● ●● ●● ●● ● ● ● ● ●● ●●●●● ●●● ● ● ● ● ● ● ● ● ●● ●●● ●●●●● ●● ● ● ● ● ● ●●●● ● ●●● ● ● ● ●●●●●●●●● ●● ● ●● 1 ● ●●●●●●● ●●● ● ● ● ●● ●●● ● ● ● ● 3●●● ● ●● ● ●●● ●●● ● ●●●●●●●●● ●● ● ● ●● ● ●●●●● ● ●● ● ●●● ● ●●●●● ●● ●● ● ● ● ● ● ● ●● ● ● ●●● ● ● ●●● ● ● ● ●●●● ● ● ●●●● ● ●● ●●●● ● ● ● ● ● ●●● ● ●● ●●●● ●●●●● ●2● ●●●● ●●● ● ● ●● ●●●●● ●● ●●●● ●● ●● ●● ●●● ● ● ● ● ● ● ● ● ● ● ●●● ● ●●●● ● ● ● ●●●●● ● ●●●●●●●● ● ● ● ● ●●● ● ●● ● ●●●●●●● ●●●● ●●●●●●●●●● ● ●●●● ●● ● ●●●●●● ●●● ● ● ●●● ●●● ●● ● ●● ● ● ●● ● ●●●● ● ● ●● ●● ●●●●● ● ●●● ● ●● ●●●● ●● ●●● ●●● ● ● ● ● ● ●● ● ●●●●● ● ●●●● ● ●●●●● ● ●● ●● ● ●● ● ●●●●●● ● ●● ●● ●●●●● ● ●● ● ● ● ●●● ● ● ●●●● ●●● ●● ●● ●● ● ●●●● ●●● ●●●●● ●●●●●● ●●●● ●● ● ● ● ● ●● ●●● ●●●●●● ● ● ●● ●● ●●●●● ● 2 ●●● ●● ●● ● ●●● ●●●●●●●●● ●● ●●● ● ● ● ●●●●●●●●● ●● ●● ● ●●●● ● ● ● ●●●● ● ●●●●●●●● ●● ● ●●● ● ●●●● ● ● ●● ● ● ● ●● ●● ● ● ● ●●● ● ● ● ● ● ● ●● ●●● ● ● ●● ● ● ● ● ●●●●● ●● ● ●● ●●● ●● ●●●● ●● ●●● ● ● ●● ●●●●●● ●●●● ● ● ●● ●●● ●●●●●● ●●● ● ●●● ● ●● ● ●●●● ● ●● ●●● ●● ●●●●●● ● ● ●● ●●● ●● ●● ●●●●● ● ● ● ●●●●●● ● ●●●● ●● ●● ● ●●●●● ● ●● ●●● ●●● ● ●● ● ● ●●●●●●●● ●●● ●● ●●●● ● Human ● ●●●● ● ●● ● ●●●●●● ●● ●● ●●● ●● ● ● ● ●●● ● ● ●● ●● ●●●●● 0 ●● ● ●● ● ● ●● ●● ●●● ● ●●●● ●● ●● ● ●●● ● ● ●●●●●● ●●● ● ●●●●●● ● ●●● ●●●● ●●●●●●● ●●●●● ● ●●● ●●● ● ●●●● ●●●●●●●●● ● ● ●● ● ●● ● ● ●● ● ● ● ● ● ●● ●●●●● ●● ●● ●●●●●●●●● ●●● ●● ●●●●● ●●●● ● 3 ●● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ●4●●● ● ●●● ● ●●●●●●●● ● ● ●●●● ● ● ● ● ● ● ● ●● ●● ● ● ●● ● ●● ● ●●● ● ● ●● ● ● ● ●● ● ●●● ● ●● ● ●● ● ● ●● ●● ●● ● ● ●●● ● ● ●●● ●● ● ● ●●●●●●● ●●●●● ● ● ● ● ● ●●● ●●● ● ●●●● ● ● ●●● ●● ●● ● ● ●● ●●● ●● 1 ● ● ●●●●● ●● ● ● ● ● ●● ●●● ● ● ● ● ●● ● ●●● ●● ● ●● ● ●●● ●● ●●● ● ● Macaque ● ●● ●●●●● ● ● ● ●●● ●●● ●●●●● ● ● ● ●● ● ● ● ●●●● ●● ● ● ● ● ●●● ●● 4 ● ● ● ●●●● ● ● ●●●● ● ●● ● ●●● ● ● ● ● ●● ● ●● ● ●●●●●●● ●●●● ● ●●●● ●● ● ● ● ●●● tSNE_2 ● ● ●● ●●● ● ●●● ●● ● ● ● ●●● ● ● ●●● ●● ● ● ●●●●●●● ●●● ● ●●● ●●● ● ●● ●● ● ●● ● ●● ● ● ● ● ● ●● ●● ●●● ● ●●● ● ● ● ●●●● ●● ● ●● ● ● ● ●● ● ●●●●● ● ●●● ● ● ● 5 Mouse ● ● ● ● ● ●● ● ● ●●● ● ● ● ●● ● ●●●●●●● ●●●●●● ● ● ● ● ● ● ● ● ●●● ● ● ●●● ● ●● ● ● ●● ●● ●●●●● ● ● ● ●●●●●● ●●●● ●●●● ●● ● ●●●●●●● ● ●●●●●●● ●●●●●●●●●●● ● ●● ●● ●● ● ●●●●● ●●● ●● ● ● ● ● ● ●●● ●● ● ● ● ● ●●●●●●●● ●● ●● ●● ● ●● ● ●●●●●●● 6 ●● ●● ● ●● ●●● ● ●●●● ●●●● ● ●●● ●●●● ●● ● ●●●● ●●● ● ● ●● ●● ● ● ● ●●●●●● ●● ● ● ●●● 5● ● ●●●●● −20 ●●● ●● ●● ●● ● ●●● ●● ●●● ● ●● ●● ● ● ● ● ●● ●●●● ● ●●● ●●● ●● ●● ●● ●●●●●● ●●● ● ● ● ● ●● ●●●● ● ●●●● ●● ●●●●● ●● ●●●●●●●●● ● ●●●● ●● ●●●●●● ● ●● ●●●● ● ● ●●●●●●●●●●● ● ● ●●● ● ●●●● ●●● ● ●●●●●● ● ● ●●●●●●● ● ●●●● ●●● ● ● ●●●●●●● ● ● ●● ● ●● ● ●●●● ●●●● ●● ●●● ●● ●●●● ● ●●●●●●●●●●●● ● ●●●● ●● ●● ● ● ●●●● ● ● ●●●●●●●●● ● ●●●●● ● ●● ●● ● ● ● ● ●●● ● ●●●● ●●● ●● ●● ● ●●●● ●●● ●●● ● ● ● ● ●●●●● ●●●●● ●● ●●● ●●●● ●●● ● ● ●●●●● ●●●● ●● ●● ●●●●● ● ● ●● ●● ●● ●●● ●●●● ●●●●● ●●●●●● ● ●● ● ● ●● ●●●●●●●●●●●● ●●●●●●● ● ●●● ●●●● ●● ●● ● ●●●● ● ●●●●●●●●●●●●●● ●● ●●● ●●●● ●●● ●●● ● ●●● ●●● ●● ●●●●●●●●●● ● ●● ●●● ● ●●●● ● ● ● ●●●● ● ●● ● ●●●●● ●●●●● ●● ● ●●● ● ● ● ●●●●● ●● ●●●● ●● ●●●● ●●● ●●● ● ●● 6 −40 ● −20 0 20 40 tSNE_1

Undifferentiated SPG Differentiating SPG Preleptotene SC C ID4 ITGA6 ZBTB16 GFRA1 LIN28A UCHL1 DMRT1 STRA8 MKI67 SYCP2 HORMAD1 TEX10 LY6K Est. Conserved Undifferentiated SPG Preleptotene SC D TSPAN33 PIWIL4 MORC1 TCF3 CDK17 MAGEB2 FMR1 L1TD1 DNAJB6 VCX VCX2 VCX3A VCX3B

E Noncanonical

HumanF Macaque MouseMouse G Human Human

Human matrix_63 Macaque 0.03 Mouse 0.02 0.01 0 Shared Macaque

Macaque Unnanotated Macaque Macaque * Mouse Figure 3 A TCF3 MERGE UCHL1 B MAGEB2 MERGE UCHL1

TCF3 MERGE KIT MAGEB2 MERGE KIT

MERGE UCHL1 MORC1 MERGE C CDK17 D UCHL1

CDK17 MERGE KIT MORC1 MERGE KIT

50µm

E F PIWIL4 MORC1 Adark Adark

Apale

Apale cells/tubule PIWIL4+ 50µm MORC1+ cells/tubule -type Adark Apale B S’cytes -type G H Adark Apale B TCF3 CONTROL S’cytes

Apale TCF3+ cells/tubule

Adark type Adark Apale - B S’cytes Busulfan-treated Donor Fluorescent activated Analyze colonization I cell sorting nude mouse recipient activity

P1 P2

P3

50µm I J J Tspan33- K ns Unsorted control L 95.98% 100% Tspan33+ 3.71% 1000) 1000) - - X X ns H( H( - - SSC SSC Colonies/fraction Colonies/10^5 cells Colonies/10^5

- -

Tspan33 Negative PE Tspan33 PE Unsorted Tspan33+ Tspan33 Tspan33+ Figure 4

A Pseudo-time Pt Distance SC 1 Distance Distance Alignment - Pre 200 Elong Pseudo-time Pt 1 200

B D Mitosis Meiosis Spermiogenesis Species SPG SC RStid Elong Human Macaque Mouse Mouse # Cells # Cells 20 40

PC3 10 Macaque 20 60

30 Species 40 80

Human GC1 GC10 SPG1 SPG6 GC20 PC1 Aligned Cell State

C Pseudo-time Corr Corr Pt Corr 1 Alignment - Post 20

Pseudo-time 20 Pt 1

E

Human Macaque Mouse GC1 GC1 GC1 GC1 SPG1 GC20 SPG1 GC20 SPG1 SPG1 SPG6 GC20 GC20 SPG6 SPG6 SPG6 Aligned Cell State Figure 5

Relative Expression

A Z-: -4 -2 0 2 4 #Genes 2286 2228 2288 2490 2745 2490 Mitosis Meiosis Spermiogenesis

mRNA Export/Splicing Mitosis/Meiosis mRNA Export/Splicing B DNA Repair Gene Silencing Cell Cycle Regulation

507 585 372

230 480 779 #Genes

Protein Folding mRNA Splicing mRNA Export/Splicing mRNA Destabilization Mitosis Transcriptional Regulation DNA Repair

C Expression Correlation with Human E 1-1-1 Orthologs (δ) Human Macaque Mouse 75 α1 70 E Gene Set β1 γ β3 All Orthologs 65 TFs in Orthologs δ 60 Species α2 α3 Total Expression % Macaque β2

Rank Correlation Mouse GC1 SPG1 SPG6 GC20

Species Specific Genes (α1-3) Primate Specific Genes (β1) GC1

GC10 12 D SPG1 SPG6 GC20 9 5

6 4 Gene Set Subset Orthologs 3 Total Expression % TFs in Orthologs Total Expression % 3

Species GC1 GC1 SPG1 SPG6 GC20 SPG1 SPG6 Macaque GC20

Rank Correlation Mouse GC1 GC10 SPG1 SPG6 GC20 Aligned Cell State Figure 6

A T cell Macrophage Endothelial m-Pericyte f-Pericyte Myoid immLeydig CD3D LYZ VWF MCAM STEAP4 ACTA2 IGF2

UMAP2 UMAP1

B C

Macrophage Human MacaqueMaca MouseMouse

T cell Tubule Seminiferous m-Pericyte Myoid f-Pericyte Imm Leydig Endothelial

E Intersection of Highly Variable Genes D

1 T cell Macrophage 0.5 Endothelial 0 m-Pericyte

Human Macaque mouse f-Pericyte H M m -0.5 Myoid ImmLeydig T cell Macrophage Endothelial m-Pericyte f-Pericyte Myoid ImmLeydig InnateLymphoid Macrophage Endothelial Myoid TCF21+ Int Leydig Signaling Ligands FGF2−FGFR1 CTHRC1−FZD3 C3−CD81 COL4A1−ITGB1 Figure 7 SPG1 SPG1 SPG1 SPG1 SPG2 SPG2 SPG2 SPG2 Global InteractionSPG3 Map SPG3 SPG3 SPG3 SPG4 SPG4 SPG4 SPG4 SPG5 SPG5 SPG5 SPG5 SPG6 SPG6 SPG6 SPG6 FGF2−FGFR1 CTHRC1−FZD3 C3−CD81 FGF2−FGFR1COL4A1−ITGB1 GC1 CTHRC1−FZD3 GC1 C3−CD81 GC1 COL4A1−ITGB1 GC1 GC2 GC2 GC2 GC2 GC3 GC3 GC3 GC3 Tcell GC4 Tcell GC4 Tcell GC4 Tcell GC4 SPG1 A SPG1 SPG1 Macrophage SPG1 SPG1GC5 Macrophage SPG1 GC5 Macrophage SPG1 GC5 Macrophage SPG1 GC5 SPG2 SPG2 SPG2 Endothelial SPG2 SPG2GC6 Endothelial SPG2 GC6 Endothelial SPG2 GC6 Endothelial SPG2 GC6 SPG3 SPG3 SPG3 m.Pericyte SPG3 SPG3GC7 m.Pericyte SPG3 GC7 m.Pericyte SPG3 GC7 m.Pericyte SPG3 GC7 SPG4 SPG4 SPG4 f.Pericyte SPG4 SPG4GC8 f.Pericyte SPG4 GC8 f.Pericyte SPG4 GC8 f.Pericyte SPG4 GC8 SPG5 SPG5 SPG5 Myoid SPG5 SPG5GC9 Myoid SPG5 GC9 Myoid SPG5 GC9 Myoid SPG5 GC9 SPG6 SPG6 SPG6 ImmLeydig SPG6 SPG6GC10 ImmLeydig SPG6 GC10 ImmLeydig SPG6 GC10 ImmLeydig SPG6 GC10 GC1 GC1 GC1 GC1 GC1GC11 GC1 GC11 GC1 GC11 GC1 GC11 GC2 GC2 GC2 GC2 GC2GC12 GC2 GC12 GC2 GC12 GC2 GC12 GC3 GC3 GC3 GC3 GC3GC13 GC3 GC13 GC3 GC13 GC3 GC13 Tcell GC4 Tcell GC4 Tcell GC4 Tcell Tcell GC4 GC4GC14 Tcell GC4 GC14 Tcell GC4 GC14 Tcell GC4 GC14 Macrophage GC5 Macrophage GC5 Macrophage GC5 Macrophage Macrophage GC5 GC5GC15 Macrophage GC5 GC15 Macrophage GC5 GC15 Macrophage GC5 GC15 Endothelial GC6 Endothelial GC6 Endothelial GC6 Endothelial Endothelial GC6 GC6GC16 Endothelial GC6 GC16 Endothelial GC6 GC16 Endothelial GC6 GC16 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte m.Pericyte GC7 GC7GC17 m.Pericyte GC7 GC17 m.Pericyte GC7 GC17 m.Pericyte GC7 GC17 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte f.Pericyte GC8 GC8GC18 f.Pericyte InnateLymphoidGC8 GC18 f.Pericyte GC8 GC18 f.Pericyte GC8 GC18 Myoid GC9 Myoid GC9 Myoid GC9 Myoid Myoid GC9 GC9GC19 Myoid GC9 GC19 Myoid GC9 GC19 Myoid GC9 GC19 ImmLeydig GC10 ImmLeydig GC10 ImmLeydig GC10 ImmLeydig ImmLeydig GC10 GC10GC20 ImmLeydig GC10 GC20 ImmLeydig GC10 GC20 ImmLeydig GC10 GC20 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC14 GC14 Fgf2−Fgfr1 GC14 Cthrc1−GC14Fzd3 GC14 C3−Cd81GC14 Col4a1−GC14Itgb1 GC14 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC18 GC18 GC18SPG1 GC18 GC18SPG1 GC18 SPG1 GC18 SPG1 GC18 GC19 GC19 GC19SPG2 GC19 GC19SPG2 GC19 SPG2 GC19 SPG2 GC19 GC20 GC20 GC20SPG3 GC20 GC20SPG3 GC20 SPG3 GC20 SPG3 GC20 SPG4 SPG4 SPG4 SPG4 FGF2−FGFR1 SPG5 FGF2−FGFR1 CTHRC1−SLIT2FZD3−ROBO2 SPG5 CTHRC1−FZD3 C3−CD81APOE−SCARB1 SPG5 C3−CD81 COL4A1−VEGFAITGB1 −EGFR SPG5 COL4A1−ITGB1 GDNF−GFRA1 SPG6 SPG6 SPG6 SPG6 GC1 GC1 GC1 GC1 GC2 GC2 GC2 GC2 SPG1 GC3 SPG1 SPG1 SPG1GC3 SPG1 SPG1 SPG1GC3 SPG1 SPG1 SPG1GC3 SPG1 SPG1 InnateLymphoid SPG2 GC4 SPG2 InnateLymphoid SPG2 SPG2GC4 SPG2 InnateLymphoid SPG2 SPG2GC4 SPG2 InnateLymphoid SPG2 SPG2GC4 SPG2 SPG2 Macrophage SPG3 GC5 SPG3 Macrophage SPG3 SPG3GC5 SPG3 Macrophage SPG3 SPG3GC5 SPG3 Macrophage SPG3 SPG3GC5 SPG3 SPG3 Endothelial SPG4 GC6 SPG4 Endothelial SPG4 SPG4GC6 SPG4 Endothelial SPG4 SPG4GC6 SPG4 Endothelial SPG4 SPG4GC6 SPG4 SPG4 Myoid SPG5 GC7 SPG5 Myoid SPG5 SPG5GC7 SPG5 Myoid SPG5 SPG5GC7 SPG5 Myoid SPG5 SPG5GC7 SPG5 SPG5 FGF2−FGFR1 CTHRC1−FZD3 C3−CD81 Leydig SPG6 GC8 COL4A1−ITGB1 SPG6 Leydig SPG6 FGF2SPG6GC8−FGFR1 FGF2SPG6−FGFR1Leydig SPG6 CTHRC1SPG6GC8 −FZD3 CTHRC1SPG6 −FZD3Leydig SPG6 C3SPG6GC8−CD81 C3SPG6−CD81 COL4A1SPG6 −ITGB1 COL4A1−ITGB1 SLIT2−ROBO2 APOE−SCARB1 Sertoli VEGFA−EGFRGC1 GC9 GC1 SLIT2Sertoli −ROBO2FGF2−SDC2GC1 GC1GC9 GC1 APOESertoli −SCARB1 GC1 GC1GC9 GC1 VEGFASertoli −EGFR GC1 GC1GC9 GC1 FGF2−SDC2 GC1 Tcf21+Interstitial GC2 GC10 GC2 Tcf21+Interstitial GC2 GC2GC10 GC2 Tcf21+Interstitial GC2 GC2GC10 GC2 Tcf21+Interstitial GC2 GC2GC10 GC2 GC2 GC3 GC11 GC3 GC3 GC3GC11 GC3 GC3 GC3GC11 GC3 GC3 GC3GC11 GC3 GC3 Tcell GC4 TcellGC12 GC4 Tcell Tcell GC4 TcellGC4GC12 GC4 Tcell Tcell GC4 TcellGC4GC12 GC4 Tcell Tcell GC4 TcellGC4GC12 GC4 Tcell GC4 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 Macrophage GC5MacrophageSPG1GC13 MacrophageGC5 Macrophage SPG1GC5MacrophageSPG1GC5GC13 MacrophageGC5 Macrophage SPG1GC5MacrophageGC5GC13 MacrophageGC5 Macrophage SPG1GC5MacrophageGC5GC13 GC5 Macrophage SPG1 GC5 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 Endothelial GC6EndothelialSPG2GC14 EndothelialGC6 Endothelial SPG2GC6EndothelialSPG2GC6GC14 EndothelialGC6 Endothelial SPG2GC6EndothelialGC6GC14 EndothelialGC6 Endothelial SPG2GC6EndothelialGC6GC14 GC6 Endothelial SPG2 GC6 SPG3 SPG3 SPG3 SPG3 SPG3 Tcf21+InterstitialSPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 m.Pericyte GC7m.PericyteSPG3GC15 GC7m.Pericyte m.Pericyte SPG3GC7m.PericyteSPG3GC7GC15 GC7m.Pericyte m.Pericyte SPG3GC7m.PericyteGC7GC15 GC7m.Pericyte m.Pericyte SPG3GC7m.PericyteGC7GC15 GC7 m.Pericyte SPG3 GC7 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 f.Pericyte GC8 f.PericyteSPG4GC16 GC8f.Pericyte f.Pericyte SPG4GC8 f.PericyteSPG4GC8GC16 GC8f.Pericyte f.Pericyte SPG4GC8 f.PericyteGC8GC16 GC8f.Pericyte f.Pericyte SPG4GC8 f.PericyteGC8GC16 GC8 f.Pericyte SPG4 GC8 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 Myoid GC9 MyoidSPG5GC17 GC9Myoid Myoid SPG5GC9 MyoidSPG5GC9GC17 GC9Myoid Myoid SPG5GC9 MyoidGC9GC17 GC9Myoid Myoid SPG5GC9 MyoidGC9GC17 GC9 Myoid SPG5 GC9 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 ImmLeydig GC10ImmLeydigSPG6GC18 ImmLeydigGC10 ImmLeydig SPG6GC10ImmLeydigSPG6GC10GC18 ImmLeydigGC10 ImmLeydig SPG6GC10ImmLeydigGC10GC18 ImmLeydigGC10 ImmLeydig SPG6GC10ImmLeydigGC10GC18 GC10 ImmLeydig SPG6 GC10 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC11 GC1GC19 GC11 GC1GC11 GC1GC11GC19 GC11 GC1GC11 GC11GC19 GC11 GC1GC11 GC11GC19 GC11 GC1 GC11 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC12 GC2GC20 GC12 GC2GC12 GC2GC12GC20 GC12 GC2GC12 GC12GC20 GC12 GC2GC12 GC12GC20 GC12 GC2 GC12 GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC13 GC3 GC13 GC3GC13 GC3GC13 GC13 GC3GC13 GC13 GC13 GC3GC13 GC13 GC13 GC3 GC13 Tcell GC4 Tcell GC4 Tcell GC4 Tcell GC4 Tcell TcellGC4 GC4 Tcell TcellGC4 GC4 Tcell TcellGC4 GC4 Tcell TcellGC4 GC4 Tcell GC4 Tcell GC4 Tcell GC14 GC4 GC14Tcell Tcell GC4GC14 GC4GC14 GC14Tcell GC4GC14 GC14 GC14Tcell GC4GC14 GC14 GC14Tcell GC4 GC14 Macrophage GC5 Macrophage GC5 Macrophage GC5 Macrophage GC5 Macrophage MacrophageGC5 GC5 Macrophage MacrophageGC5 GC5 Macrophage MacrophageGC5 GC5 Macrophage MacrophageGC5 GC5 Macrophage GC5 Macrophage GC5 Macrophage GC15 GC5 MacrophageGC15 Macrophage GC5GC15 GC5GC15 MacrophageGC15 GC5GC15 GC15 MacrophageGC15 GC5GC15 GC15 MacrophageGC15 GC5 GC15 Endothelial GC6 Endothelial GC6 Endothelial GC6 Endothelial GC6 Endothelial EndothelialGC6 GC6 Endothelial EndothelialGC6 GC6 Endothelial EndothelialGC6 GC6 Endothelial EndothelialGC6 GC6 Endothelial GC6 Endothelial GC6 Endothelial GC16 GC6 EndothelialGC16 Endothelial GC6GC16 GC6GC16 EndothelialGC16 GC6GC16 GC16 EndothelialGC16 GC6GC16 GC16 EndothelialGC16 GC6 GC16 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte m.PericyteGC7 GC7 m.Pericyte m.PericyteGC7 GC7 m.Pericyte m.PericyteGC7 GC7 m.Pericyte m.PericyteGC7 GC7 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte GC17 GC7 m.PericyteGC17 m.Pericyte GC7GC17 GC7GC17 m.PericyteGC17 GC7GC17 GC17 m.PericyteGC17 GC7GC17 GC17 m.PericyteGC17 GC7 GC17 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte f.PericyteGC8 GC8 f.Pericyte f.PericyteGC8 GC8 f.Pericyte f.PericyteGC8 GC8 f.Pericyte f.PericyteGC8 GC8 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte GC18 GC8 GC18f.Pericyte f.Pericyte GC8GC18 GC8GC18 GC18f.Pericyte GC8GC18 GC18 GC18f.Pericyte GC8GC18 GC18 GC18f.Pericyte GC8 GC18 Myoid GC9 Myoid GC9 Myoid GC9 Myoid GC9 Myoid MyoidGC9 GC9 Myoid MyoidGC9 GC9 Myoid MyoidGC9 GC9 Myoid MyoidGC9 GC9 Myoid GC9 Myoid GC9 Myoid GC19 GC9 GC19Myoid Myoid GC9GC19 GC9GC19 GC19Myoid GC9GC19 GC19 GC19Myoid GC9GC19 GC19 GC19Myoid GC9 GC19 ImmLeydig GC10 ImmLeydig GC10 ImmLeydig GC10 ImmLeydig GC10 ImmLeydig ImmLeydigGC10 GC10 ImmLeydig ImmLeydigGC10 GC10 ImmLeydig ImmLeydigGC10 GC10 ImmLeydig ImmLeydigGC10 GC10 ImmLeydig GC10 ImmLeydig GC10 ImmLeydig GC20 GC10 ImmLeydigGC20 ImmLeydig GC10GC20 GC10GC20 ImmLeydigGC20 GC10GC20 GC20 ImmLeydigGC20 GC10GC20 GC20 ImmLeydigGC20 GC10 GC20 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC13 GC13 GC13 GC13 GC13 GC13 Fgf2−Fgfr1 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC14 GC14 GC14 GC14 GC14 Fgf2−Fgfr1Slit2−Robo2 GC14 GC14 Cthrc1−Fzd3Apoe−Scarb1GC14 GC14 Cthrc1−GC14Fzd3 C3GC14−Cd81Vegfa−GC14Egfr C3−Cd81GC14 Col4a1GC14 −Itgb1Gdnf−Gfra1GC14 Col4a1−GC14Itgb1 GC14 GC14 GC14 GC14 GC15 GC15 GC15 B GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC18 GC18 GC18 GC18 GC18 SPG1 GC18SPG1 GC18SPG1 GC18SPG1 GC18SPG1 GC18 SPG1 GC18 GC18SPG1 SPG1 GC18 SPG1 GC18 GC18SPG1 SPG1 GC18 SPG1 GC18 GC18 GC18 GC18 GC19 GC19 GC19 GC19 GC19 SPG2 GC19SPG2 GC19SPG2 GC19SPG2 GC19SPG2 GC19 SPG2 GC19 GC19SPG2 SPG2 GC19 SPG2 GC19 GC19SPG2 SPG2 GC19 SPG2 GC19 GC19 GC19 GC19 GC20 GC20 GC20 GC20 GC20 SPG3 GC20SPG3 GC20SPG3 GC20SPG3 GC20SPG3 GC20 SPG3 GC20 GC20SPG3 SPG3 GC20 SPG3 GC20 GC20SPG3 SPG3 GC20 SPG3 GC20 GC20 GC20 GC20 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SLIT2−ROBO2 SPG5 SPG5 SLIT2−ROBO2 SPG5 APOE−SCARB1FGF2−SDC2SPG5 FGF2SPG5−FGFR1APOE−SCARB1FGF2SPG5−FGFR1VEGFA−JAG1EGFR−NOTCH2SPG5 CTHRC1SPG5 −VEGFAFZD3 −EGFRCTHRC1SPG5 −FZD3GDNF−GFRA1WNT5A−SPG5RYK C3SPG5−CD81GDNF−GFRA1 C3SPG5−CD81 WNT5A−ROR1COL4A1−ITGB1 COL4A1−ITGB1 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 GC1 GC1 GC1Individual InteractingGC1 GC1 Pairs GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 SPG1GC3 GC3 SPG1GC3 SPG1GC3 SPG1GC3 SPG1 SPG1GC3 SPG1 SPG1GC3 SPG1GC3 SPG1 SPG1GC3 SPG1 SPG1GC3 SPG1GC3 SPG1 SPG1GC3 SPG1 SPG1 SPG1 SPG1 InnateLymphoidInnateLymphoid InnateLymphoidSPG2GC4 GC4 InnateLymphoidSPG2GC4 InnateLymphoid InnateLymphoidSPG2GC4 SPG2GC4 SPG2 InnateLymphoidSPG2GC4 InnateLymphoidSPG2 InnateLymphoidSPG2GC4 SPG2GC4 SPG2 InnateLymphoidSPG2GC4 InnateLymphoidSPG2 InnateLymphoidSPG2GC4 SPG2GC4 SPG2 SPG2GC4 SPG2 SPG2 SPG2 SPG2 Macrophage Macrophage SPG3GC5MacrophageGC5 MacrophageSPG3GC5 Macrophage SPG3GC5MacrophageSPG3GC5 SPG3 MacrophageSPG3GC5 MacrophageSPG3 SPG3GC5MacrophageSPG3GC5 SPG3 MacrophageSPG3GC5 MacrophageSPG3 SPG3GC5MacrophageSPG3GC5 SPG3 SPG3GC5 SPG3 SPG3 SPG3 SPG3 Endothelial Endothelial SPG4GC6EndothelialGC6 EndothelialSPG4GC6 Endothelial SPG4GC6EndothelialSPG4GC6 SPG4 EndothelialSPG4GC6 EndothelialSPG4 SPG4GC6EndothelialSPG4GC6 SPG4 EndothelialSPG4GC6 EndothelialSPG4 SPG4GC6EndothelialSPG4GC6 SPG4 SPG4GC6 SPG4 SPG4 SPG4 SPG4 Myoid Myoid SPG5GC7 MyoidGC7 SPG5GC7Myoid Myoid SPG5GC7 MyoidSPG5GC7 SPG5 SPG5GC7Myoid MyoidSPG5 SPG5GC7 MyoidSPG5GC7 SPG5 SPG5GC7Myoid MyoidSPG5 SPG5GC7 MyoidSPG5GC7 SPG5 SPG5GC7 SPG5 SPG5 SPG5 SPG5 FGF2−FGFR1 SLIT2−ROBO2 CTHRC1−FZD3 APOE−SCARB1 C3−CD81 FGF2−FGFR1VEGFA−EGFR LeydigCOL4A1Leydig−ITGB1 SPG6GC8CTHRC1LeydigGC8−FZD3FGF2−SDC2 SPG6GC8Leydig Leydig SPG6GC8 C3SLIT2Leydig−CD81SPG6GC8−ROBO2 SPG6 SLIT2SPG6GC8Leydig−ROBO2LeydigSPG6 SPG6GC8COL4A1APOELeydigSPG6GC8−ITGB1−SCARB1 SPG6 APOESPG6GC8−LeydigSCARB1LeydigSPG6 SPG6GC8 VEGFALeydigSPG6GC8 −EGFR SPG6 VEGFASPG6GC8 −EGFR SPG6 FGF2SPG6−SDC2 SPG6 FGF2−SDC2 SPG6 JAG1−NOTCH2 WNT5AFGF2−RYK -FGFR1 COL4A1Sertoli Sertoli-ITGB1WNT5A − GC1GC9ROR1 Sertoli GC9 FGF2-SDC2 GC1 GC9 Sertoli JAG1 Sertoli BMP4−NOTCH2BMP4-−BMPR2BMPR2GC1GC9 SertoliGC1GC9 GC1 SLIT2GC1GC9Sertoli-ROBOSertoliWNT5AGC1 − RYK GC1GC9APOESertoliGC1GC9-SCARB.GC1 GC1GC9 Sertoli WNT5AWNT5ASertoliGC1 −-ROR1 GC1GC9 SertoliGC1GC9 GC1 GC1GC9 BMP4GC1−BMPR2 GC1 GC1 GC1 Tcf21+InterstitialTcf21+Interstitial Tcf21+InterstitialGC2GC10 GC10 Tcf21+InterstitialGC2GC10 Tcf21+Interstitial Tcf21+InterstitialGC2GC10 GC2GC10 GC2 Tcf21+InterstitialGC2GC10 Tcf21+InterstitialGC2 Tcf21+InterstitialGC2GC10 GC2GC10 GC2 Tcf21+InterstitialGC2GC10 Tcf21+InterstitialGC2 Tcf21+InterstitialGC2GC10 GC2GC10 GC2 GC2GC10 GC2 GC2 GC2 GC2 GC3GC11 GC11 GC3GC11 GC3GC11 GC3GC11 GC3 GC3GC11 GC3 GC3GC11 GC3GC11 GC3 GC3GC11 GC3 GC3GC11 GC3GC11 GC3 GC3GC11 GC3 GC3 GC3 GC3 Tcell GC4GC12 TcellGC12 GC4GC12Tcell Tcell TcellGC4GC12 TcellGC4GC12 GC4 GC4GC12Tcell Tcell TcellGC4GC12 TcellGC4GC12 GC4 GC4GC12Tcell Tcell TcellGC4GC12 TcellGC4GC12 GC4 GC4GC12 Tcell Tcell GC4 GC4 SPG1 SPG1 SPG1 SPG1 TcellSPG1 GC4SPG1 TcellSPG1 GC4SPG1 TcellSPG1 GC4SPG1 TcellSPG1 GC4SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 Macrophage SPG1 GC5GC13MacrophageSPG1GC13 SPG1 MacrophageGC5GC13 Macrophage MacrophageSPG1GC5GC13MacrophageSPG1GC5GC13 SPG1GC5 MacrophageGC5GC13 Macrophage MacrophageSPG1GC5GC13MacrophageGC5GC13 SPG1GC5 MacrophageGC5GC13 Macrophage MacrophageSPG1GC5GC13MacrophageGC5GC13 GC5 GC5GC13 Macrophage MacrophageSPG1 GC5 GC5 SPG2 SPG2 SPG2 SPG2 MacrophageSPG2 GC5SPG2 MacrophageSPG2 GC5SPG2 MacrophageSPG2 GC5SPG2 MacrophageSPG2 GC5SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 Endothelial SPG2 GC6GC14EndothelialSPG2GC14 SPG2 EndothelialGC6GC14 Endothelial EndothelialSPG2GC6GC14EndothelialSPG2GC6GC14 SPG2GC6 EndothelialGC6GC14 Endothelial EndothelialSPG2GC6GC14EndothelialGC6GC14 SPG2GC6 EndothelialGC6GC14 Endothelial EndothelialSPG2GC6GC14EndothelialGC6GC14 GC6 GC6GC14 Endothelial EndothelialSPG2 GC6 GC6 SPG3 SPG3 SPG3 SPG3 EndothelialSPG3 GC6SPG3 EndothelialSPG3 GC6SPG3 EndothelialSPG3 GC6SPG3 EndothelialSPG3 GC6SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 m.Pericyte SPG3 GC7GC15m.PericyteSPG3GC15 SPG3 GC7m.PericyteGC15 m.Pericyte m.PericyteSPG3GC7GC15m.PericyteSPG3GC7GC15 SPG3GC7 GC7m.PericyteGC15 m.Pericyte m.PericyteSPG3GC7GC15m.PericyteGC7GC15 SPG3GC7 GC7m.PericyteGC15 m.Pericyte m.PericyteSPG3GC7GC15m.PericyteGC7GC15 GC7 GC7GC15 m.Pericyte m.PericyteSPG3 GC7 GC7 SPG4 SPG4 SPG4 SPG4 m.PericyteSPG4 GC7SPG4 m.PericyteSPG4 GC7SPG4 m.PericyteSPG4 GC7SPG4 m.PericyteSPG4 GC7SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 f.Pericyte SPG4 GC8GC16f.PericyteSPG4GC16 SPG4 GC8GC16f.Pericyte f.Pericyte f.PericyteSPG4GC8GC16f.PericyteSPG4GC8GC16 SPG4GC8 GC8GC16f.Pericyte f.Pericyte f.PericyteSPG4GC8GC16f.PericyteGC8GC16 SPG4GC8 GC8GC16f.Pericyte f.Pericyte f.PericyteSPG4GC8GC16f.PericyteGC8GC16 GC8 GC8GC16 f.Pericyte f.PericyteSPG4 GC8 GC8 SPG5 SPG5 SPG5 SPG5 f.PericyteSPG5 GC8SPG5 f.PericyteSPG5 GC8SPG5 f.PericyteSPG5 GC8SPG5 f.PericyteSPG5 GC8SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 Myoid SPG5 GC9GC17 MyoidSPG5GC17 SPG5 GC9GC17Myoid Myoid MyoidSPG5GC9GC17 MyoidSPG5GC9GC17 SPG5GC9 GC9GC17Myoid Myoid MyoidSPG5GC9GC17 MyoidGC9GC17 SPG5GC9 GC9GC17Myoid Myoid MyoidSPG5GC9GC17 MyoidGC9GC17 GC9 GC9GC17 Myoid MyoidSPG5 GC9 GC9 SPG6 SPG6 SPG6 SPG6 MyoidSPG6 GC9SPG6 MyoidSPG6 GC9SPG6 MyoidSPG6 GC9SPG6 MyoidSPG6 GC9SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 ImmLeydig SPG6 GC10GC18ImmLeydigSPG6GC18 SPG6 ImmLeydigGC10GC18 ImmLeydig ImmLeydigSPG6GC10GC18ImmLeydigSPG6GC10GC18 SPG6GC10 ImmLeydigGC10GC18 ImmLeydig ImmLeydigSPG6GC10GC18ImmLeydigGC10GC18 SPG6GC10 ImmLeydigGC10GC18 ImmLeydig ImmLeydigSPG6GC10GC18ImmLeydigGC10GC18 GC10 GC10GC18 ImmLeydig ImmLeydigSPG6 GC10 GC10 GC1 GC1 GC1 GC1 ImmLeydigGC1 GC10GC1 ImmLeydigGC1 GC10GC1 ImmLeydigGC1 GC10GC1 ImmLeydigGC1 GC10GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC11GC19 GC1GC19 GC1 GC11GC19 GC1GC11GC19 GC1GC11GC19 GC1 GC11 GC11GC19 GC1GC11GC19 GC11GC19 GC1 GC11 GC11GC19 GC1GC11GC19 GC11GC19 GC11 GC11GC19 GC1 GC11 GC11 GC2 GC2 GC2 GC2 GC2 GC11GC2 GC2 GC11GC2 GC2 GC11GC2 GC2 GC11GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC12GC20 GC2GC20 GC2 GC12GC20 GC2GC12GC20 GC2GC12GC20 GC2 GC12 GC12GC20 GC2GC12GC20 GC12GC20 GC2 GC12 GC12GC20 GC2GC12GC20 GC12GC20 GC12 GC12GC20 GC2 GC12 GC12 GC3 GC3 GC3 GC3 GC3 GC12GC3 GC3 GC12GC3 GC3 GC12GC3 GC3 GC12GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC13 GC3 GC3 GC13 GC3GC13 GC3GC13 GC3 GC13 GC13 GC3GC13 GC13 GC3 GC13 GC13 GC3GC13 GC13 GC13 GC13 GC3 GC13 GC13 Tcell GC4 Tcell GC4 Tcell GC4 Tcell GC4 Tcell TcellGC4 GC13GC4 Tcell TcellGC4 GC13GC4 Tcell TcellGC4 GC13GC4 Tcell TcellGC4 GC13GC4 Tcell GC4 Tcell Tcell GC4 GC4 Tcell Tcell TcellGC4 GC4 GC4 Tcell Tcell TcellGC4 GC14 GC4 GC4 GC14Tcell Tcell Tcell GC4GC14 GC4GC14 GC4 GC14 GC14Tcell Tcell GC4GC14 GC14 GC4 GC14 GC14Tcell GC4GC14 GC14 GC14 GC14Tcell GC4 GC14 GC14 Macrophage GC5 Macrophage GC5 Macrophage GC5 Macrophage GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage GC5 Macrophage Macrophage GC5 GC5 Macrophage Macrophage MacrophageGC5 GC5 GC5 Macrophage Macrophage MacrophageGC5 GC15 GC5 GC5 MacrophageGC15 Macrophage Macrophage GC5GC15 GC5GC15 GC5 GC15 MacrophageGC15 Macrophage GC5GC15 GC15 GC5 GC15 MacrophageGC15 GC5GC15 GC15 GC15 MacrophageGC15 GC5 GC15 GC15 Endothelial GC6 Endothelial GC6 Endothelial GC6 Endothelial GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial GC6 Endothelial Endothelial GC6 GC6 Endothelial Endothelial EndothelialGC6 GC6 GC6 Endothelial Endothelial EndothelialGC6 GC16 GC6 GC6 EndothelialGC16 Endothelial Endothelial GC6GC16 GC6GC16 GC6 GC16 EndothelialGC16 Endothelial GC6GC16 GC16 GC6 GC16 EndothelialGC16 GC6GC16 GC16 GC16 EndothelialGC16 GC6 GC16 GC16 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte GC7 m.Pericyte m.Pericyte GC7 GC7 m.Pericyte m.Pericyte m.PericyteGC7 GC7 GC7 m.Pericyte m.Pericyte m.PericyteGC7 GC17 GC7 GC7 m.PericyteGC17 m.Pericyte m.Pericyte GC7GC17 GC7GC17 GC7 GC17 m.PericyteGC17 m.Pericyte GC7GC17 GC17 GC7 GC17 m.PericyteGC17 GC7GC17 GC17 GC17 m.PericyteGC17 GC7 GC17 GC17 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte GC8 f.Pericyte f.Pericyte GC8 GC8 f.Pericyte f.Pericyte f.PericyteGC8 GC8 GC8 f.Pericyte f.Pericyte f.PericyteGC8 GC18 GC8 GC8 GC18f.Pericyte f.Pericyte f.Pericyte GC8GC18 GC8GC18 GC8 GC18 GC18f.Pericyte f.Pericyte GC8GC18 GC18 GC8 GC18 GC18f.Pericyte GC8GC18 GC18 GC18 GC18f.Pericyte GC8 GC18 GC18 Myoid GC9 Myoid GC9 Myoid GC9 Myoid GC9 Myoid MyoidGC9 GC18GC9 Myoid MyoidGC9 GC18GC9 Myoid MyoidGC9 GC18GC9 Myoid MyoidGC9 GC18GC9 Myoid GC9 Myoid Myoid GC9 GC9 Myoid Myoid MyoidGC9 GC9 GC9 Myoid Myoid MyoidGC9 GC19 GC9 GC9 GC19Myoid Myoid Myoid GC9GC19 GC9GC19 GC9 GC19 GC19Myoid Myoid GC9GC19 GC19 GC9 GC19 GC19Myoid GC9GC19 GC19 GC19 GC19Myoid GC9 GC19 GC19 ImmLeydig GC10 ImmLeydig ImmLeydigGC10 GC10 ImmLeydig ImmLeydigGC10 ImmLeydigGC10 ImmLeydig GC10 ImmLeydigGC10 ImmLeydigGC10 ImmLeydig GC10 GC10 ImmLeydigImmLeydig ImmLeydigGC10 GC10 GC19GC10 ImmLeydigImmLeydig ImmLeydigGC10 GC10 GC19GC10 ImmLeydig ImmLeydigGC10 GC19GC10 ImmLeydig ImmLeydigGC10 GC19GC10 ImmLeydig GC10 ImmLeydig GC10 ImmLeydig GC20 GC10 ImmLeydigGC20 ImmLeydig GC10GC20 GC10GC20 GC20 ImmLeydigGC20 GC20 GC10GC20 GC20 GC20 ImmLeydigGC20 GC20 GC10GC20 GC20 GC20 ImmLeydigGC20 GC20 GC10 GC20 GC20 GC20 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 VegfaGC13−GC13Egfr GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC14 GC14 GC14 GC14 GC14 GC14 GC14 GC14 GC14 Slit2−Robo2Fgf2GC14−Sdc2 GC14 Slit2GC14−Robo2 GC14 Apoe−Scarb1Jag1−Notch2GC14 Fgf2GC14−Fgfr1ApoeGC14−Scarb1GC14 Fgf2−Fgfr1 VegfaGC14 −EgfrWnt5a−GC14Ryk Cthrc1−Fzd3GC14 GC14 Cthrc1−Fzd3 GdnfGC14−Gfra1Wnt5a−GC14Ror1 C3−Cd81Gdnf−Gfra1GC14 C3−Cd81 GC14 GC14 Col4a1−Itgb1 GC14 Col4a1−Itgb1 GC14 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC18 GC18 GC18 GC18 GC18 GC18 GC18 GC18 GC18 GC18 SPG1 GC18SPG1 GC18 GC18SPG1 GC18SPG1 GC18SPG1 GC18 GC18SPG1 SPG1 SPG1GC18 GC18SPG1 SPG1 GC18 GC18SPG1 SPG1 SPG1GC18 GC18SPG1 SPG1 GC18SPG1 SPG1 SPG1GC18 GC18 GC18SPG1 SPG1GC18 GC19 GC19 GC19 GC19 GC19 GC19 GC19 GC19 GC19 GC19 SPG2 GC19SPG2 GC19 GC19SPG2 GC19SPG2 GC19SPG2 GC19 GC19SPG2 SPG2 SPG2GC19 GC19SPG2 SPG2 GC19 GC19SPG2 SPG2 SPG2GC19 GC19SPG2 SPG2 GC19SPG2 SPG2 SPG2GC19 GC19 GC19SPG2 SPG2GC19 GC20 GC20 GC20 GC20 GC20 GC20 GC20 GC20 GC20 GC20 SPG3 GC20SPG3 GC20 GC20SPG3 GC20SPG3 GC20SPG3 GC20 GC20SPG3 SPG3 SPG3GC20 GC20SPG3 SPG3 GC20 GC20SPG3 SPG3 SPG3GC20 GC20SPG3 SPG3 GC20SPG3 SPG3 SPG3GC20 GC20 GC20SPG3 SPG3GC20 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 FGF2−FGFR1 CTHRC1−FZD3 C3−CD81 FGF2−FGFR1 COL4A1FGF2−ITGB1−SDC2 SPG5CTHRC1SPG5−FZD3FGF2−SDC2 SPG5 JAG1−NOTCH2BMP4−BMPR2SPG5C3SLIT2−CD81SPG5−ROBO2JAG1−NOTCH2SPG5 SLIT2SPG5−ROBO2WNT5ASPG5 BMP4−RYK−BMPR1BSPG5COL4A1APOESPG5−ITGB1−SCARB1WNT5A−SPG5RYKAPOESPG5−SCARB1WNT5ASPG5 −ROR1FGF2−SDC4SPG5 VEGFASPG5−EGFRWNT5A−ROR1SPG5 VEGFASPG5 −EGFR SPG5 CXCL12−ITGB1GDNF−GFRA1 SPG5 GDNF−GFRA1 SPG5 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1GC3 GC3 SPG1 SPG1GC3 SPG1GC3 SPG1GC3 SPG1SPG1GC3 SPG1GC3 GC3SPG1 SPG1GC3 SPG1GC3 SPG1SPG1GC3 SPG1GC3 GC3SPG1 SPG1GC3 SPG1GC3 SPG1GC3 SPG1GC3 GC3SPG1 SPG1 SPG1GC3 GC3SPG1 SPG2 SPG2 SPG2 SPG2 InnateLymphoidInnateLymphoid SPG2 InnateLymphoidSPG2GC4 GC4 SPG2 InnateLymphoidSPG2GC4 InnateLymphoid InnateLymphoidInnateLymphoidSPG2GC4 SPG2GC4 InnateLymphoidSPG2SPG2GC4 InnateLymphoidSPG2GC4 InnateLymphoidGC4SPG2 InnateLymphoidInnateLymphoidSPG2GC4 SPG2GC4 InnateLymphoidSPG2SPG2GC4 InnateLymphoidSPG2GC4 InnateLymphoidGC4SPG2 InnateLymphoidInnateLymphoidSPG2GC4 SPG2GC4 InnateLymphoidSPG2GC4 SPG2GC4 GC4SPG2 InnateLymphoid SPG2 InnateLymphoidSPG2GC4 GC4SPG2 SPG3 SPG3 SPG3 SPG3 Macrophage Macrophage SPG3 SPG3GC5MacrophageGC5 SPG3 MacrophageSPG3GC5 Macrophage MacrophageSPG3GC5MacrophageSPG3GC5 MacrophageSPG3SPG3GC5 MacrophageSPG3GC5 MacrophageGC5SPG3 MacrophageSPG3GC5MacrophageSPG3GC5 MacrophageSPG3SPG3GC5 MacrophageSPG3GC5 MacrophageGC5SPG3 MacrophageSPG3GC5MacrophageSPG3GC5 MacrophageSPG3GC5 SPG3GC5 GC5SPG3 Macrophage SPG3 MacrophageSPG3GC5 GC5SPG3 SPG4 SPG4 SPG4 SPG4 Endothelial Endothelial SPG4 SPG4GC6EndothelialGC6 SPG4 EndothelialSPG4GC6 Endothelial EndothelialSPG4GC6EndothelialSPG4GC6 EndothelialSPG4SPG4GC6 EndothelialSPG4GC6 EndothelialGC6SPG4 EndothelialSPG4GC6EndothelialSPG4GC6 EndothelialSPG4SPG4GC6 EndothelialSPG4GC6 EndothelialGC6SPG4 EndothelialSPG4GC6EndothelialSPG4GC6 EndothelialSPG4GC6 SPG4GC6 GC6SPG4 Endothelial SPG4 EndothelialSPG4GC6 GC6SPG4 SPG5 SPG5 SPG5 SPG5 Myoid Myoid SPG5 SPG5GC7 MyoidGC7 SPG5 SPG5GC7Myoid Myoid MyoidSPG5GC7 MyoidSPG5GC7 SPG5MyoidSPG5GC7 SPG5GC7Myoid MyoidGC7SPG5 MyoidSPG5GC7 MyoidSPG5GC7 SPG5MyoidSPG5GC7 SPG5GC7Myoid MyoidGC7SPG5 MyoidSPG5GC7 MyoidSPG5GC7 MyoidSPG5GC7 SPG5GC7 GC7SPG5 Myoid SPG5 MyoidSPG5GC7 GC7SPG5 SLIT2−ROBO2 SPG6 JAG1−NOTCH2 APOE−SCARB1 SPG6 WNT5A−RYK VEGFA−EGFR SPG6 SLIT2−ROBO2WNT5ASPG6−ROR1 Leydig FGF2Leydig−SDC2 SPG6 SPG6GC8APOELeydig−SCARB1GC8 BMP4SPG6−BMPR2 SPG6GC8Leydig Leydig LeydigSPG6GC8VEGFAJAG1Leydig−SPG6GC8EGFR−NOTCH2SPG6LeydigSPG6GC8 JAG1SPG6GC8−LeydigNOTCH2LeydigGC8SPG6 LeydigSPG6GC8FGF2WNT5ALeydig−SPG6GC8SDC2−RYKSPG6LeydigSPG6GC8 WNT5ASPG6GC8Leydig−RYKLeydigGC8SPG6 LeydigSPG6GC8 WNT5ALeydigSPG6GC8 −ROR1LeydigSPG6GC8 WNT5ASPG6GC8 −ROR1 GC8SPG6 Leydig BMP4SPG6−BMPR2LeydigSPG6GC8 BMP4−BMPR2 GC8SPG6 GC1 BMP4GC1−BMPR1B FGF7GC1−FGFR3 GC1 Sertoli Sertoli FGF2GC1−SDC4GC1GC9 SertoliGC9 GC1 GC1GC9Sertoli BMP4Sertoli −BMPR1BCXCL12Sertoli−GC1GC9ITGB1SertoliGC1GC9 GC1SertoliGC1GC9 GC1GC9Sertoli FGF7SertoliGC9GC1−FGFR3SertoliGC1GC9 SertoliGC1GC9 GC1SertoliGC1GC9 GC1GC9Sertoli SertoliFGF2GC9GC1 −SDC4 SertoliGC1GC9 SertoliGC1GC9 SertoliGC1GC9 GC1GC9 CXCL12GC9GC1 −ITGB1Sertoli GC1 SertoliGC1GC9 GC9GC1 GC2 GC2 GC2 GC2 Tcf21+InterstitialTcf21+Interstitial GC2 Tcf21+InterstitialGC2GC10 GC10 GC2 Tcf21+InterstitialGC2GC10 Tcf21+Interstitial Tcf21+InterstitialTcf21+InterstitialGC2GC10 GC2GC10 Tcf21+InterstitialGC2 GC2GC10 Tcf21+InterstitialGC2GC10 Tcf21+InterstitialGC10GC2 Tcf21+InterstitialTcf21+InterstitialGC2GC10 GC2GC10 Tcf21+InterstitialGC2 GC2GC10 Tcf21+InterstitialGC2GC10 Tcf21+InterstitialGC10GC2 Tcf21+InterstitialTcf21+InterstitialGC2GC10 GC2GC10 Tcf21+InterstitialGC2GC10 GC2GC10 GC10GC2 Tcf21+Interstitial GC2 Tcf21+InterstitialGC2GC10 GC10GC2 GC3 GC3 GC3 GC3 GC3 GC3GC11 GC11 GC3 GC3GC11 GC3GC11 GC3GC11 GC3 GC3GC11 GC3GC11 GC11GC3 GC3GC11 GC3GC11 GC3 GC3GC11 GC3GC11 GC11GC3 GC3GC11 GC3GC11 GC3GC11 GC3GC11 GC11GC3 GC3 GC3GC11 GC11GC3 Tcell GC4 Tcell Tcell GC4GC12 TcellGC12 GC4 GC4GC12Tcell Tcell Tcell TcellGC4GC12 TcellGC4GC12 GC4 GC4GC12 GC4GC12Tcell TcellGC12 Tcell TcellGC4GC12 TcellGC4GC12 GC4 GC4GC12 GC4GC12Tcell TcellGC12 TcellGC4GC12 TcellGC4GC12 GC4GC12 GC4GC12 TcellGC12 Tcell GC4 GC4GC12 GC12 Tcell GC4 TcellSPG1 GC4 TcellSPG1 GC4 TcellSPG1 GC4 SPG1 TcellSPG1 GC4SPG1 TcellSPG1 GC4SPG1 TcellSPG1 GC4SPG1 TcellSPG1 GC4SPG1 SPG1 SPG1 SPG1 MacrophageSPG1 SPG1 SPG1GC5 Macrophage MacrophageSPG1 GC5GC13MacrophageSPG1GC13 SPG1GC5 MacrophageGC5GC13 Macrophage MacrophageMacrophageSPG1GC5GC13MacrophageSPG1GC5GC13 SPG1GC5 GC5GC13 MacrophageGC5GC13 MacrophageGC13MacrophageMacrophageSPG1GC5GC13MacrophageGC5GC13 SPG1GC5 GC5GC13 MacrophageGC5GC13 MacrophageGC13 MacrophageSPG1GC5GC13MacrophageGC5GC13 GC5GC13 GC5GC13 MacrophageGC13 MacrophageSPG1 GC5 GC5GC13 GC13 Macrophage GC5 MacrophageSPG2 GC5 MacrophageSPG2 GC5 MacrophageSPG2 GC5 SPG2 MacrophageSPG2 GC5SPG2 MacrophageSPG2 GC5SPG2 MacrophageSPG2 GC5SPG2 MacrophageSPG2 GC5SPG2 SPG2 SPG2 SPG2 EndothelialSPG2 SPG2 SPG2GC6 Endothelial EndothelialSPG2 GC6GC14EndothelialSPG2GC14 SPG2GC6 EndothelialGC6GC14 Endothelial EndothelialEndothelialSPG2GC6GC14EndothelialSPG2GC6GC14 SPG2GC6 GC6GC14 EndothelialGC6GC14 EndothelialGC14 EndothelialEndothelialSPG2GC6GC14EndothelialGC6GC14 SPG2GC6 GC6GC14 EndothelialGC6GC14 EndothelialGC14 EndothelialSPG2GC6GC14EndothelialGC6GC14 GC6GC14 GC6GC14 EndothelialGC14 EndothelialSPG2 GC6 GC6GC14 GC14 Endothelial GC6 EndothelialSPG3 GC6 EndothelialSPG3 GC6 EndothelialSPG3 GC6 SPG3 EndothelialSPG3 GC6SPG3 EndothelialSPG3 GC6SPG3 EndothelialSPG3 GC6SPG3 EndothelialSPG3 GC6SPG3 SPG3 SPG3 SPG3 m.PericyteSPG3 SPG3 SPG3GC7 m.Pericyte m.PericyteSPG3 GC7GC15m.PericyteSPG3GC15 SPG3GC7 GC7m.PericyteGC15 m.Pericyte m.Pericytem.PericyteSPG3GC7GC15m.PericyteSPG3GC7GC15 SPG3GC7 GC7GC15 GC7m.PericyteGC15 m.PericyteGC15 m.Pericytem.PericyteSPG3GC7GC15m.PericyteGC7GC15 SPG3GC7 GC7GC15 GC7m.PericyteGC15 m.PericyteGC15 m.PericyteSPG3GC7GC15m.PericyteGC7GC15 GC7GC15 GC7GC15 m.PericyteGC15 m.PericyteSPG3 GC7 GC7GC15 GC15 m.Pericyte GC7 m.PericyteSPG4 GC7 m.PericyteSPG4 GC7 m.PericyteSPG4 GC7 SPG4 m.PericyteSPG4 GC7SPG4 m.PericyteSPG4 GC7SPG4 m.PericyteSPG4 GC7SPG4 m.PericyteSPG4 GC7SPG4 SPG4 SPG4 SPG4 f.PericyteSPG4 SPG4 SPG4GC8 f.Pericyte f.PericyteSPG4 GC8GC16f.PericyteSPG4GC16 SPG4GC8 GC8GC16f.Pericyte f.Pericyte f.Pericytef.PericyteSPG4GC8GC16f.PericyteSPG4GC8GC16 SPG4GC8 GC8GC16 GC8GC16f.Pericyte f.PericyteGC16 f.Pericytef.PericyteSPG4GC8GC16f.PericyteGC8GC16 SPG4GC8 GC8GC16 GC8GC16f.Pericyte f.PericyteGC16 f.PericyteSPG4GC8GC16f.PericyteGC8GC16 GC8GC16 GC8GC16 f.PericyteGC16 f.PericyteSPG4 GC8 GC8GC16 GC16 f.Pericyte GC8 f.PericyteSPG5 GC8 f.PericyteSPG5 GC8 f.PericyteSPG5 GC8 SPG5 f.PericyteSPG5 GC8SPG5 f.PericyteSPG5 GC8SPG5 f.PericyteSPG5 GC8SPG5 f.PericyteSPG5 GC8SPG5 SPG5 SPG5 SPG5 MyoidSPG5 SPG5 SPG5GC9 Myoid MyoidSPG5 GC9GC17 MyoidSPG5GC17 SPG5GC9 GC9GC17Myoid Myoid MyoidMyoidSPG5GC9GC17 MyoidSPG5GC9GC17 SPG5GC9 GC9GC17 GC9GC17Myoid MyoidGC17 MyoidMyoidSPG5GC9GC17 MyoidGC9GC17 SPG5GC9 GC9GC17 GC9GC17Myoid MyoidGC17 MyoidSPG5GC9GC17 MyoidGC9GC17 GC9GC17 GC9GC17 MyoidGC17 MyoidSPG5 GC9 GC9GC17 GC17 Myoid GC9 MyoidSPG6 GC9 MyoidSPG6 GC9 MyoidSPG6 GC9 SPG6 MyoidSPG6 GC9SPG6 MyoidSPG6 GC9SPG6 MyoidSPG6 GC9SPG6 MyoidSPG6 GC9SPG6 SPG6 SPG6 SPG6 ImmLeydigSPG6 SPG6 SPG6GC10 ImmLeydig ImmLeydigSPG6 GC10GC18ImmLeydigSPG6GC18 SPG6GC10 ImmLeydigGC10GC18 ImmLeydig ImmLeydigImmLeydigSPG6GC10GC18ImmLeydigSPG6GC10GC18 SPG6GC10GC10GC18 ImmLeydigGC10GC18 ImmLeydigGC18 ImmLeydigImmLeydigSPG6GC10GC18ImmLeydigGC10GC18 SPG6GC10GC10GC18 ImmLeydigGC10GC18 ImmLeydigGC18 ImmLeydigSPG6GC10GC18ImmLeydigGC10GC18 GC10GC18 GC10GC18 ImmLeydigGC18 ImmLeydigSPG6 GC10 GC10GC18 GC18 ImmLeydig GC10 ImmLeydigGC1 GC10 ImmLeydigGC1 GC10 ImmLeydigGC1 GC10 GC1 ImmLeydigGC1 GC10GC1 ImmLeydigGC1 GC10GC1 ImmLeydigGC1 GC10GC1 ImmLeydigGC1 GC10GC1 GC1 GC1 GC1 GC1 GC1 GC1GC11 GC1 GC11GC19 GC1GC19 GC1GC11 GC11GC19 GC1GC11GC19 GC1GC11GC19 GC1GC11GC11GC19 GC11GC19 GC19 GC1GC11GC19 GC11GC19 GC1GC11GC11GC19 GC11GC19 GC19 GC1GC11GC19 GC11GC19 GC11GC19 GC11GC19 GC19 GC1 GC11 GC11GC19 GC19 GC11 GC2 GC11 GC2 GC11 GC2 GC11 GC2 GC2 GC11GC2 GC2 GC11GC2 GC2 GC11GC2 GC2 GC11GC2 GC2 GC2 GC2 GC2 GC2 GC2GC12 GC2 GC12GC20 GC2GC20 GC2GC12 GC12GC20 GC2GC12GC20 GC2GC12GC20 GC2GC12GC12GC20 GC12GC20 GC20 GC2GC12GC20 GC12GC20 GC2GC12GC12GC20 GC12GC20 GC20 GC2GC12GC20 GC12GC20 GC12GC20 GC12GC20 GC20 GC2 GC12 GC12GC20 GC20 GC12 GC3 GC12 GC3 GC12 GC3 GC12 GC3 GC3 GC12GC3 GC3 GC12GC3 GC3 GC12GC3 GC3 GC12GC3 GC3 GC3 GC3 GC3 GC3 GC3GC13 GC3 GC13 GC3 GC3GC13 GC13 GC3GC13 GC3GC13 GC3GC13GC13 GC13 GC3GC13 GC13 GC3GC13GC13 GC13 GC3GC13 GC13 GC13 GC13 GC3 GC13 GC13 GC13 Tcell GC4 GC13 Tcell GC4 GC13 Tcell GC4 GC13 Tcell GC4 Tcell TcellGC4 GC13GC4 Tcell TcellGC4 GC13GC4 Tcell TcellGC4 GC13GC4 Tcell TcellGC4 GC13GC4 Tcell GC4 Tcell Tcell GC4 GC4 Tcell Tcell TcellGC4 GC4 GC4GC14 Tcell Tcell TcellGC4 GC14 GC4 GC4GC14 GC14Tcell Tcell Tcell GC4GC14 GC4GC14 GC4GC14GC14 GC14Tcell Tcell GC4GC14 GC14 GC4GC14GC14 GC14Tcell GC4GC14 GC14 GC14 GC14Tcell GC4 GC14 GC14 GC14 Macrophage GC5 GC14 Macrophage GC5 GC14 Macrophage GC5 GC14 Macrophage GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage GC5 Macrophage Macrophage GC5 GC5 Macrophage Macrophage MacrophageGC5 GC5 GC5GC15 Macrophage Macrophage MacrophageGC5 GC15 GC5 GC5GC15 MacrophageGC15 Macrophage Macrophage GC5GC15 GC5GC15 GC5GC15GC15 MacrophageGC15 Macrophage GC5GC15 GC15 GC5GC15GC15 MacrophageGC15 GC5GC15 GC15 GC15 MacrophageGC15 GC5 GC15 GC15 GC15 Endothelial GC6 GC15 Endothelial GC6 GC15 Endothelial GC6 GC15 Endothelial GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial GC6 Endothelial Endothelial GC6 GC6 Endothelial Endothelial EndothelialGC6 GC6 GC6GC16 Endothelial Endothelial EndothelialGC6 GC16 GC6 GC6GC16 EndothelialGC16 Endothelial Endothelial GC6GC16 GC6GC16 GC6GC16GC16 EndothelialGC16 Endothelial GC6GC16 GC16 GC6GC16GC16 EndothelialGC16 GC6GC16 GC16 GC16 EndothelialGC16 GC6 GC16 GC16 GC16 m.Pericyte GC7 GC16 m.Pericyte GC7 GC16 m.Pericyte GC7 GC16 m.Pericyte GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte GC7 m.Pericyte m.Pericyte GC7 GC7 m.Pericyte m.Pericyte m.PericyteGC7 GC7 GC7GC17 m.Pericyte m.Pericyte m.PericyteGC7 GC17 GC7 GC7GC17 m.PericyteGC17 m.Pericyte m.Pericyte GC7GC17 GC7GC17 GC7GC17GC17 m.PericyteGC17 m.Pericyte GC7GC17 GC17 GC7GC17GC17 m.PericyteGC17 GC7GC17 GC17 GC17 m.PericyteGC17 GC7 GC17 GC17 GC17 f.Pericyte GC8 GC17 f.Pericyte GC8 GC17 f.Pericyte GC8 GC17 f.Pericyte GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte GC8 f.Pericyte f.Pericyte GC8 GC8 f.Pericyte f.Pericyte f.PericyteGC8 GC8 GC8GC18 f.Pericyte f.Pericyte f.PericyteGC8 GC18 GC8 GC8GC18 GC18f.Pericyte f.Pericyte f.Pericyte GC8GC18 GC8GC18 GC8GC18GC18 GC18f.Pericyte f.Pericyte GC8GC18 GC18 GC8GC18GC18 GC18f.Pericyte GC8GC18 GC18 GC18 GC18f.Pericyte GC8 GC18 GC18 GC18 Myoid GC9 GC18 Myoid GC9 GC18 Myoid GC9 GC18 Myoid GC9 Myoid MyoidGC9 GC18GC9 Myoid MyoidGC9 GC18GC9 Myoid MyoidGC9 GC18GC9 Myoid MyoidGC9 GC18GC9 Myoid GC9 Myoid Myoid GC9 GC9 Myoid Myoid MyoidGC9 GC9 GC9GC19 Myoid Myoid MyoidGC9 GC19 GC9 GC9GC19 GC19Myoid Myoid Myoid GC9GC19 GC9GC19 GC9GC19GC19 GC19Myoid Myoid GC9GC19 GC19 GC9GC19GC19 GC19Myoid GC9GC19 GC19 GC19 GC19Myoid GC9 GC19 GC19 ImmLeydig GC19GC10 ImmLeydig ImmLeydigGC10 GC19GC10 ImmLeydig ImmLeydigGC10 ImmLeydigGC19GC10 ImmLeydig GC10 ImmLeydigGC10 ImmLeydigGC19GC10 ImmLeydig GC10 GC10 ImmLeydigImmLeydig ImmLeydigGC10 GC10 GC19GC10 ImmLeydigImmLeydig ImmLeydigGC10 GC10 GC19GC10 ImmLeydig ImmLeydigGC10 GC19GC10 ImmLeydig ImmLeydigGC10 GC19GC10 GC20 ImmLeydig GC20 GC10 ImmLeydig GC20 GC10 GC20 ImmLeydig GC20 GC20 GC10 GC20 ImmLeydigGC20 ImmLeydig GC10GC20 GC10GC20 GC20GC20 ImmLeydigGC20 GC20 GC10GC20 GC20 GC20GC20 ImmLeydigGC20 GC20 GC10GC20 GC20 GC20 ImmLeydigGC20 GC20 GC10 GC20 GC20 GC20 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 Fgf2−Fgfr1 GC13 GC13 Cthrc1−Fzd3 GC13 GC13 GC13 C3−Cd81 GC13 Fgf2−GC13Fgfr1 GC13 GC13 Col4a1−Itgb1 GC13 Cthrc1GC13−Fzd3 Fgf2GC13−Sdc2 GC13 GC13 C3−Cd81GC13 Jag1GC13−Notch2GC13 GC13 GC13Col4a1−Itgb1 Wnt5aGC13 GC13−Ryk GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC14 GC14 GC14 GC14 GC14 GC14 GC14 GC14 GC14 Fgf2−Sdc2Bmp4GC14−Bmpr2 GC14 GC14 GC14 Jag1−Notch2Bmp4−Bmpr1bGC14 Slit2GC14−Robo2GC14 GC14 Slit2−Robo2 Wnt5aGC14 −RykFgf7−Fgfr3GC14 Apoe−Scarb1GC14 GC14 Apoe−Scarb1 Wnt5aGC14 −Ror1Fgf2−Sdc4GC14 Vegfa−EgfrWnt5a−GC14Ror1 Vegfa−Egfr GC14 GC14 Gdnf−Gfra1 GC14 Gdnf−Gfra1 GC14 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17SPG1 GC17 GC17 GC17 GC17SPG1 GC17 GC17 GC17 GC17SPG1GC17 GC17 GC17 GC17SPG1GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 GC18 GC18 GC18 GC18 GC18 GC18 GC18 GC18SPG2 GC18 GC18 SPG1 GC18 GC18SPG2 GC18 GC18SPG1 GC18 GC18SPG2GC18SPG1 SPG1GC18 GC18SPG1 GC18SPG2GC18SPG1 SPG1GC18 GC18SPG1 GC18SPG1 SPG1GC18 GC18 GC18SPG1 SPG1GC18 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 GC19 GC19 GC19 GC19 GC19 GC19 GC19 GC19SPG3 GC19 GC19 SPG2 GC19 GC19SPG3 GC19 GC19SPG2 GC19 GC19SPG3GC19SPG2 SPG2GC19 GC19SPG2 GC19SPG3GC19SPG2 SPG2GC19 GC19SPG2 GC19SPG2 SPG2GC19 GC19 GC19SPG2 SPG2GC19 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 GC20 GC20 GC20 GC20 GC20 GC20 GC20 GC20SPG4 GC20 GC20 SPG3 GC20 GC20SPG4 GC20 GC20SPG3 GC20 GC20SPG4GC20SPG3 SPG3GC20 GC20SPG3 GC20SPG4GC20SPG3 SPG3GC20 GC20SPG3 GC20SPG3 SPG3GC20 GC20 GC20SPG3 SPG3GC20 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG5 SPG4 SPG5 SPG4 SPG5 SPG4 SPG4 SPG4 SPG5 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG5 SPG5 SPG5 SLIT2−ROBO2 SPG5 APOE−SCARB1SPG5 SPG5 VEGFA−SPG5EGFR SPG5 GDNF−SPG5GFRA1 SPG5 SPG5 SPG5 SLIT2−ROBO2 APOE−SCARB1 VEGFA−EGFR SPG6 GDNFBMP4−GFRA1−BMPR2 SPG5 BMP4SPG6−BMPR2 BMP4−BMPR1B SPG5 FGF2−SDC2BMP4SPG6−BMPR1BSPG5 FGF2−SDC2 FGF2SPG5−SDC4 SPG5 JAG1−NOTCH2FGF2SPG6−SDC4SPG5 JAG1−NOTCH2CXCL12SPG5 −ITGB1 SPG5 WNT5ACXCL12−RYK −SPG5ITGB1WNT5A−RYK SPG5 WNT5A−ROR1 SPG5 WNT5A−ROR1 SPG5 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 GC1 SPG6 GC1 SPG6 GC1 SPG6 SPG6 SPG6 GC1 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC2 GC1 GC2 GC1 GC2 GC1 GC1 GC1 GC2 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC3 GC2 GC3 GC2 GC3 GC2 GC2 GC2 GC3 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC3 GC3 GC3 SPG1 GC3 GC3 SPG1 GC3 GC3 SPG1 GC3 GC3 SPG1 GC3 GC3 GC3 SPG1 SPG1 InnateLymphoidSPG1 GC4 InnateLymphoidSPG1 SPG1GC3 GC4 SPG1 InnateLymphoid SPG1GC3 GC4 SPG1GC3 SPG1 GC3SPG1InnateLymphoid SPG1GC3 GC4 SPG1GC3 SPG1 GC3SPG1 SPG1GC3 SPG1GC3 SPG1 GC3SPG1 SPG1GC3 GC3SPG1 InnateLymphoid GC4 InnateLymphoid GC4 InnateLymphoid GC4 SPG2 InnateLymphoid InnateLymphoid GC4 InnateLymphoidGC4 SPG2 GC4 InnateLymphoid InnateLymphoidGC4 SPG2 GC4 InnateLymphoid InnateLymphoidGC4 SPG2 GC4 InnateLymphoid InnateLymphoidGC4 GC4 SPG2 SPG2 MacrophageSPG2 GC5 InnateLymphoid MacrophageSPG2 SPG2GC4 GC5 InnateLymphoidSPG2 MacrophageInnateLymphoidSPG2GC4 InnateLymphoidGC5 SPG2GC4 InnateLymphoidSPG2 GC4SPG2MacrophageInnateLymphoidSPG2GC4 InnateLymphoidGC5 SPG2GC4 InnateLymphoidSPG2 GC4SPG2 InnateLymphoidSPG2GC4 InnateLymphoidSPG2GC4 SPG2 GC4SPG2 InnateLymphoid InnateLymphoidSPG2GC4 GC4SPG2 Macrophage GC5 Macrophage GC5 Macrophage GC5 SPG3 Macrophage Macrophage GC5 MacrophageGC5 SPG3 GC5 Macrophage MacrophageGC5 SPG3 GC5 Macrophage MacrophageGC5 SPG3 GC5 Macrophage MacrophageGC5 GC5 SPG3 SPG3 EndothelialSPG3 GC6 Macrophage EndothelialSPG3 SPG3GC5 GC6 MacrophageSPG3 EndothelialMacrophageSPG3GC5 MacrophageGC6 SPG3GC5 MacrophageSPG3 GC5SPG3 EndothelialMacrophageSPG3GC5 MacrophageGC6 SPG3GC5 MacrophageSPG3 GC5SPG3 MacrophageSPG3GC5 MacrophageSPG3GC5 SPG3 GC5SPG3 Macrophage MacrophageSPG3GC5 GC5SPG3 Endothelial GC6 Endothelial GC6 Endothelial GC6 SPG4 Endothelial Endothelial GC6 EndothelialGC6 SPG4 GC6 Endothelial EndothelialGC6 SPG4 GC6 Endothelial EndothelialGC6 SPG4 GC6 Endothelial EndothelialGC6 GC6 SPG4 SPG4 MyoidSPG4 GC7 Endothelial MyoidSPG4 SPG4GC6 GC7 EndothelialSPG4 MyoidEndothelialSPG4GC6 EndothelialGC7 SPG4GC6 EndothelialSPG4 GC6SPG4 MyoidEndothelialSPG4GC6 EndothelialGC7 SPG4GC6 EndothelialSPG4 GC6SPG4 EndothelialSPG4GC6 EndothelialSPG4GC6 SPG4 GC6SPG4 Endothelial EndothelialSPG4GC6 GC6SPG4 Myoid GC7 Myoid GC7 Myoid GC7 SPG5 Myoid Myoid GC7 MyoidGC7 SPG5 GC7 Myoid MyoidGC7 SPG5 GC7 Myoid MyoidGC7 SPG5 GC7 Myoid MyoidGC7 GC7 SPG5 SPG5 LeydigSPG5 GC8 Myoid LeydigSPG5 SPG5GC7 GC8 SPG5Myoid Leydig MyoidSPG5GC7 GC8MyoidSPG5GC7 SPG5Myoid GC7SPG5 Leydig MyoidSPG5GC7 GC8MyoidSPG5GC7 SPG5Myoid GC7SPG5 MyoidSPG5GC7 MyoidSPG5GC7 SPG5 GC7SPG5 Myoid MyoidSPG5GC7 GC7SPG5 Leydig GC8 Leydig GC8 Leydig GC8 SPG6 Leydig Leydig GC8 LeydigGC8 SPG6 GC8 Leydig LeydigGC8 SPG6 GC8 Leydig LeydigGC8 SPG6 GC8 Leydig LeydigGC8 GC8 SPG6 SPG6 SertoliSPG6 GC9 Leydig SertoliSPG6 SPG6GC8 GC9 SPG6Leydig SertoliLeydigSPG6GC8 GC9LeydigSPG6GC8 SPG6Leydig GC8SPG6 SertoliLeydigSPG6GC8 GC9LeydigSPG6GC8 SPG6Leydig GC8SPG6 LeydigSPG6GC8 LeydigSPG6GC8 SPG6 GC8SPG6 Leydig LeydigSPG6GC8 GC8SPG6 Sertoli JAG1−NOTCH2 GC9 BMP4−BMPR1BSertoli WNT5A−RYK GC9 FGF7−FGFR3 Sertoli WNT5A−ROR1 GC9 JAG1−NOTCH2FGF2GC1−SDC4 Sertoli BMP4Sertoli−BMPR2 GC9 WNT5ASertoliGC9−RYKCXCL12GC1−ITGB1 GC9 Sertoli WNT5ABMP4SertoliGC9−ROR1−BMPR1BGC1 BMP4GC9−BMPR1BSertoli BMP4FGF7Sertoli−BMPR2GC9−FGFR3GC1 FGF7GC9−FGFR3Sertoli SertoliFGF2GC9−SDC4 FGF2GC9 −SDC4 CXCL12−ITGB1 CXCL12−ITGB1 GC1 GC1 Tcf21+InterstitialGC1 GC10 Sertoli Tcf21+InterstitialGC1 GC1GC9 GC10 GC1Sertoli Tcf21+InterstitialSertoliGC1GC9 GC10SertoliGC1GC9 GC1Sertoli GC9GC1Tcf21+InterstitialSertoliGC1GC9 GC10SertoliGC1GC9 GC1Sertoli GC9GC1 SertoliGC1GC9 SertoliGC1GC9 GC1 GC9GC1 Sertoli SertoliGC1GC9 GC9GC1 Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 GC2 Tcf21+Interstitial Tcf21+Interstitial GC10 Tcf21+InterstitialGC10 GC2 GC10 Tcf21+Interstitial Tcf21+InterstitialGC10 GC2 GC10 Tcf21+Interstitial Tcf21+InterstitialGC10 GC2 GC10 Tcf21+Interstitial Tcf21+InterstitialGC10 GC10 GC2 GC2 GC2 GC11 Tcf21+Interstitial GC2 GC2GC10 GC11 Tcf21+InterstitialGC2 Tcf21+InterstitialGC2GC10 Tcf21+InterstitialGC11GC2GC10 Tcf21+InterstitialGC2 GC10GC2 Tcf21+InterstitialGC2GC10 Tcf21+InterstitialGC11GC2GC10 Tcf21+InterstitialGC2 GC10GC2 Tcf21+InterstitialGC2GC10 Tcf21+InterstitialGC2GC10 GC2 GC10GC2 Tcf21+Interstitial Tcf21+InterstitialGC2GC10 GC10GC2 GC11 GC11 GC11 GC3 GC11 GC11 GC3 GC11 GC11 GC3 GC11 GC11 GC3 GC11 GC11 GC11 GC3 GC3 GC3 GC12 GC3 GC3GC11 GC12 GC3 GC3GC11 GC12GC3GC11 GC3 GC11GC3 GC3GC11 GC12GC3GC11 GC3 GC11GC3 GC3GC11 GC3GC11 GC3 GC11GC3 GC3GC11 GC11GC3 GC12 GC12 TcellGC12 GC4 Tcell TcellGC12 GC4GC12 TcellGC12 GC4 GC4GC12Tcell Tcell TcellGC4GC12 TcellGC12 GC4 GC4GC12 GC4GC12Tcell GC12 Tcell TcellGC4GC12 TcellGC12 GC4 GC4GC12 GC4GC12Tcell GC12 TcellGC4GC12 TcellGC12 GC4GC12 GC4GC12 GC12 Tcell GC4GC12 GC12 Tcell GC4 TcellSPG1 GC4 TcellSPG1 GC4 GC13 TcellSPG1 GC4 GC13 SPG1 GC13TcellSPG1 GC4SPG1 GC13TcellSPG1 GC4SPG1 TcellSPG1 GC4SPG1 TcellSPG1 GC4SPG1 SPG1GC13 SPG1GC13 MacrophageSPG1GC13 SPG1GC5 Macrophage MacrophageSPG1GC13 GC5GC13MacrophageGC13 SPG1GC5 MacrophageGC5GC13 MacrophageMacrophageGC5GC13MacrophageGC13 SPG1GC5 GC5GC13 MacrophageGC5GC13 GC13MacrophageMacrophageGC5GC13MacrophageGC13 SPG1GC5 GC5GC13 MacrophageGC5GC13 GC13 MacrophageGC5GC13MacrophageGC13 GC5GC13 GC5GC13 GC13 Macrophage GC5GC13 GC13 Macrophage GC5 MacrophageSPG2 GC5 MacrophageSPG2 GC5 GC14 MacrophageSPG2 GC5 GC14 SPG2 MacrophageGC14SPG2 GC5SPG2 MacrophageGC14SPG2 GC5SPG2 MacrophageSPG2 GC5SPG2 MacrophageSPG2 GC5SPG2 SPG2GC14 SPG2GC14 EndothelialSPG2GC14 SPG2GC6 Endothelial EndothelialSPG2GC14 GC6GC14EndothelialGC14 SPG2GC6 EndothelialGC6GC14 EndothelialEndothelialGC6GC14EndothelialGC14 SPG2GC6 GC6GC14 EndothelialGC6GC14 GC14 EndothelialEndothelialGC6GC14EndothelialGC14 SPG2GC6 GC6GC14 EndothelialGC6GC14 GC14 EndothelialGC6GC14EndothelialGC14 GC6GC14 GC6GC14 GC14 Endothelial GC6GC14 GC14 Endothelial GC6 EndothelialSPG3 GC6 EndothelialSPG3 GC6 GC15 EndothelialSPG3 GC6 GC15 SPG3 EndothelialGC15SPG3 GC6SPG3 EndothelialGC15SPG3 GC6SPG3 EndothelialSPG3 GC6SPG3 EndothelialSPG3 GC6SPG3 SPG3GC15 SPG3GC15 m.PericyteSPG3GC15 SPG3GC7 m.Pericyte m.PericyteSPG3GC15 GC7GC15m.PericyteGC15 SPG3GC7 GC7m.PericyteGC15 m.Pericytem.PericyteGC7GC15m.PericyteGC15 SPG3GC7 GC7GC15 GC7m.PericyteGC15 GC15 m.Pericytem.PericyteGC7GC15m.PericyteGC15 SPG3GC7 GC7GC15 GC7m.PericyteGC15 GC15 m.PericyteGC7GC15m.PericyteGC15 GC7GC15 GC7GC15 GC15 m.Pericyte GC7GC15 GC15 m.Pericyte GC7 m.PericyteSPG4 GC7 m.PericyteSPG4 GC7 GC16 m.PericyteSPG4 GC7 GC16 SPG4 m.PericyteGC16SPG4 GC7SPG4 m.PericyteGC16SPG4 GC7SPG4 m.PericyteSPG4 GC7SPG4 m.PericyteSPG4 GC7SPG4 SPG4GC16 SPG4GC16 f.PericyteSPG4GC16 SPG4GC8 f.Pericyte f.PericyteSPG4GC16 GC8GC16f.PericyteGC16 SPG4GC8 GC8GC16f.Pericyte f.Pericytef.PericyteGC8GC16f.PericyteGC16 SPG4GC8 GC8GC16 GC8GC16f.Pericyte GC16 f.Pericytef.PericyteGC8GC16f.PericyteGC16 SPG4GC8 GC8GC16 GC8GC16f.Pericyte GC16 f.PericyteGC8GC16f.PericyteGC16 GC8GC16 GC8GC16 GC16 f.Pericyte GC8GC16 GC16 f.Pericyte GC8 f.PericyteSPG5 GC8 f.PericyteSPG5 GC8 GC17 f.PericyteSPG5 GC8 GC17 SPG5 f.PericyteGC17SPG5 GC8SPG5 f.PericyteGC17SPG5 GC8SPG5 f.PericyteSPG5 GC8SPG5 f.PericyteSPG5 GC8SPG5 SPG5GC17 SPG5GC17 MyoidSPG5GC17 SPG5GC9 Myoid MyoidSPG5GC17 GC9GC17 MyoidGC17 SPG5GC9 GC9GC17Myoid MyoidMyoidGC9GC17 MyoidGC17 SPG5GC9 GC9GC17 GC9GC17Myoid GC17 MyoidMyoidGC9GC17 MyoidGC17 SPG5GC9 GC9GC17 GC9GC17Myoid GC17 MyoidGC9GC17 MyoidGC17 GC9GC17 GC9GC17 GC17 Myoid GC9GC17 GC17 Myoid GC9 MyoidSPG6 GC9 MyoidSPG6 GC9 GC18 MyoidSPG6 GC9 GC18 SPG6 GC18MyoidSPG6 GC9SPG6 GC18MyoidSPG6 GC9SPG6 MyoidSPG6 GC9SPG6 MyoidSPG6 GC9SPG6 SPG6GC18 SPG6GC18 ImmLeydigSPG6GC18 SPG6GC10 ImmLeydig ImmLeydigSPG6GC18 GC10GC18ImmLeydigGC18 SPG6GC10 ImmLeydigGC10GC18 ImmLeydigImmLeydigGC10GC18ImmLeydigGC18 SPG6GC10GC10GC18 ImmLeydigGC10GC18 GC18 ImmLeydigImmLeydigGC10GC18ImmLeydigGC18 SPG6GC10GC10GC18 ImmLeydigGC10GC18 GC18 ImmLeydigGC10GC18ImmLeydigGC18 GC10GC18 GC10GC18 GC18 ImmLeydig GC10GC18 GC18 ImmLeydig GC10 ImmLeydigGC1 GC10 ImmLeydigGC1 GC10 GC19 ImmLeydigGC1 GC10 GC19 GC1 ImmLeydigGC19GC1 GC10GC1 ImmLeydigGC19GC1 GC10GC1 ImmLeydigGC1 GC10GC1 ImmLeydigGC1 GC10GC1 GC1GC19 GC1GC19 GC1GC19 GC1GC11 GC1GC19 GC11GC19 GC19 GC1GC11 GC11GC19 GC11GC19 GC19 GC1GC11GC11GC19 GC11GC19 GC19 GC11GC19 GC19 GC1GC11GC11GC19 GC11GC19 GC19 GC11GC19 GC19 GC11GC19 GC11GC19 GC19 GC11GC19 GC19 GC11 GC2 GC11 GC2 GC11 GC20 GC2 GC11 GC20 GC2 GC20GC2 GC11GC2 GC20GC2 GC11GC2 GC2 GC11GC2 GC2 GC11GC2 GC2GC20 GC2GC20 GC2GC20 GC2GC12 GC2GC20 GC12GC20 GC20 GC2GC12 GC12GC20 GC12GC20 GC20 GC2GC12GC12GC20 GC12GC20 GC20 GC12GC20 GC20 GC2GC12GC12GC20 GC12GC20 GC20 GC12GC20 GC20 GC12GC20 GC12GC20 GC20 GC12GC20 GC20 GC12 GC3 GC12 GC3 GC12 GC3 GC12 GC3 GC3 GC12GC3 GC3 GC12GC3 GC3 GC12GC3 GC3 GC12GC3 GC3 GC3 GC3 GC3GC13 GC3 GC13 GC3GC13 GC13 GC13 GC3GC13GC13 GC13 GC13 GC3GC13GC13 GC13 GC13 GC13 GC13 GC13 GC13 Tcell GC4 GC13 Tcell GC4 GC13 Tcell GC4 GC13 Tcell GC4 Tcell TcellGC4 GC13GC4 Tcell TcellGC4 GC13GC4 Tcell TcellGC4 GC13GC4 Tcell TcellGC4 GC13GC4 Tcell GC4 Tcell GC4 Tcell TcellGC4 GC4GC14 Tcell TcellGC4 GC14 GC4GC14 GC14 Tcell GC14 GC4GC14GC14 GC14 Tcell GC14 GC4GC14GC14 GC14 GC14 GC14 GC14 GC14 GC14 Macrophage GC5 GC14 Macrophage GC5 GC14 Macrophage GC5 GC14 Macrophage GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage MacrophageGC5 GC14GC5 Macrophage GC5 Macrophage GC5 Macrophage MacrophageGC5 GC5GC15 Macrophage MacrophageGC5 GC15 GC5GC15 GC15 Macrophage GC15 GC5GC15GC15 GC15 Macrophage GC15 GC5GC15GC15 GC15 GC15 GC15 GC15 GC15 GC15 Endothelial GC6 GC15 Endothelial GC6 GC15 Endothelial GC6 GC15 Endothelial GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial EndothelialGC6 GC15GC6 Endothelial GC6 Endothelial GC6 Endothelial EndothelialGC6 GC6GC16 Endothelial EndothelialGC6 GC16 GC6GC16 GC16 Endothelial GC16 GC6GC16GC16 GC16 Endothelial GC16 GC6GC16GC16 GC16 GC16 GC16 GC16 GC16 GC16 m.Pericyte GC7 GC16 m.Pericyte GC7 GC16 m.Pericyte GC7 GC16 m.Pericyte GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte m.PericyteGC7 GC16GC7 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte m.PericyteGC7 GC7GC17 m.Pericyte m.PericyteGC7 GC17 GC7GC17 GC17 m.Pericyte GC17 GC7GC17GC17 GC17 m.Pericyte GC17 GC7GC17GC17 GC17 GC17 GC17 GC17 GC17 GC17 f.Pericyte GC8 GC17 f.Pericyte GC8 GC17 f.Pericyte GC8 GC17 f.Pericyte GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte f.PericyteGC8 GC17GC8 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte f.PericyteGC8 GC8GC18 f.Pericyte f.PericyteGC8 GC18 GC8GC18 GC18 f.Pericyte GC18 GC8GC18GC18 GC18 f.Pericyte GC18 GC8GC18GC18 GC18 GC18 GC18 GC18 GC18 GC18 Myoid GC9 GC18 Myoid GC9 GC18 Myoid GC9 GC18 Myoid GC9 Myoid MyoidGC9 GC18GC9 Myoid MyoidGC9 GC18GC9 Myoid MyoidGC9 GC18GC9 Myoid MyoidGC9 GC18GC9 Myoid GC9 Myoid GC9 Myoid MyoidGC9 GC9GC19 Myoid MyoidGC9 GC19 GC9GC19 GC19 Myoid GC19 GC9GC19GC19 GC19 Myoid GC19 GC9GC19GC19 GC19 GC19 GC19 GC19 GC19 GC19 ImmLeydig GC10 GC19 ImmLeydig GC10 GC19 ImmLeydig GC10 GC19 ImmLeydig GC10 ImmLeydig ImmLeydigGC10 GC19GC10 ImmLeydig ImmLeydigGC10 GC19GC10 ImmLeydig ImmLeydigGC10 GC19GC10 ImmLeydig ImmLeydigGC10 GC19GC10 ImmLeydig GC10 ImmLeydig GC10 ImmLeydig ImmLeydigGC10 GC10GC20 ImmLeydig ImmLeydigGC10 GC20 GC10GC20 GC20 ImmLeydig GC20 GC10GC20GC20 GC20 ImmLeydig GC20 GC10GC20GC20 GC20 GC20 GC20 GC20 GC20 GC20GC11 GC11 GC20GC11 GC11 GC20GC11 GC11 GC11 GC20GC11 GC11 GC11 GC11 GC11 GC20GC11 GC11 GC11 GC20GC11 GC11 GC20GC11 GC11 GC20GC11 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC13 GC13 GC13 GC13 Vegfa−Egfr GC13 Slit2−Robo2 GC13 GC13 GC13 Apoe−Scarb1 GC13 GC13 Vegfa−Egfr GC13 GC13 GC13 Gdnf−Gfra1 GC13 GC13 GC13 GC13 GC13 GC13 GC13 Slit2−Robo2 GC14 GC14 Apoe−Scarb1 GC14 GC14 GC14 GC14 GC14 GdnfBmp4−Gfra1−Bmpr2Cxcl12GC14−Itgb1 Bmp4GC14−Bmpr2 GC14 Bmp4−Bmpr1b Fgf2−Sdc2Bmp4GC14−Bmpr1bGC14 Fgf2−Sdc2 Fgf7GC14−Fgfr3 Jag1−Notch2Fgf7GC14−Fgfr3GC14 Jag1−Notch2 Fgf2GC14−Sdc4 Wnt5a−RykFgf2−Sdc4GC14 Wnt5a−Ryk GC14 Wnt5a−Ror1 GC14 Wnt5a−Ror1 GC14 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC17 GC17 GC17 GC17 GC17 GC17SPG1 GC17 GC17 GC17SPG1 GC17 GC17SPG1GC17 GC17 GC17SPG1GC17 GC17 GC17 GC17 GC17 GC17 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 GC18 GC18 GC18 GC18 GC18 GC18SPG2 GC18 GC18 SPG1 GC18SPG2 GC18 SPG1 GC18SPG2GC18SPG1 SPG1GC18 SPG1 GC18SPG2GC18SPG1 SPG1GC18 SPG1 GC18SPG1 SPG1GC18 GC18SPG1 SPG1GC18 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 GC19 GC19 GC19 GC19 GC19 GC19SPG3 GC19 GC19 SPG2 GC19SPG3 GC19 SPG2 GC19SPG3GC19SPG2 SPG2GC19 SPG2 GC19SPG3GC19SPG2 SPG2GC19 SPG2 GC19SPG2 SPG2GC19 GC19SPG2 SPG2GC19 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 GC20 GC20 GC20 GC20 GC20 GC20SPG4 GC20 GC20 SPG3 GC20SPG4 GC20 SPG3 GC20SPG4GC20SPG3 SPG3GC20 SPG3 GC20SPG4GC20SPG3 SPG3GC20 SPG3 GC20SPG3 SPG3GC20 GC20SPG3 SPG3GC20 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG5 SPG4 SPG5 SPG4 SPG5 SPG4 SPG4 SPG4 SPG5 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG5 SPG5 SPG5 FGF2−SDC2 SPG5 JAG1−NOTCH2SPG5 SPG5 WNT5A−RYK SPG5 WNT5A−ROR1 SPG5 SPG5 FGF2−SDC2 JAG1−NOTCH2 WNT5A−RYK SPG6 WNT5A−ROR1 SPG5 SPG6 SPG5 BMP4−BMPR2SPG6 SPG5 BMP4−BMPR2 SPG5 SPG5 BMP4−BMPR1BSPG6 SPG5 BMP4−BMPR1B SPG5 SPG5 FGF2−SDC4 SPG5 FGF2−SDC4 SPG5 CXCL12−ITGB1 SPG5 CXCL12−ITGB1 SPG5 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 GC1 SPG6 GC1 SPG6 GC1 SPG6 SPG6 SPG6 GC1 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC2 GC1 GC2 GC1 GC2 GC1 GC1 GC1 GC2 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC3 GC2 GC3 GC2 GC3 GC2 GC2 GC2 GC3 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC3 GC3 GC3 SPG1 GC3 GC3 SPG1 GC3 SPG1 GC3 SPG1 GC3 GC3 SPG1 SPG1 InnateLymphoidSPG1 GC4 InnateLymphoidSPG1 GC3 GC4 InnateLymphoid GC3 GC4 SPG1GC3 GC3SPG1InnateLymphoid GC3 GC4 SPG1GC3 GC3SPG1 GC3 SPG1GC3 GC3SPG1 SPG1GC3 GC3SPG1 InnateLymphoid GC4 InnateLymphoid GC4 InnateLymphoid GC4 SPG2 InnateLymphoid InnateLymphoid GC4 InnateLymphoidGC4 SPG2 GC4 InnateLymphoid SPG2 GC4 InnateLymphoid SPG2 GC4 InnateLymphoid GC4 SPG2 SPG2 MacrophageSPG2 GC5 InnateLymphoid MacrophageSPG2 GC4 GC5 InnateLymphoid MacrophageInnateLymphoidGC4 InnateLymphoidGC5 SPG2GC4 InnateLymphoid GC4SPG2MacrophageInnateLymphoidGC4 InnateLymphoidGC5 SPG2GC4 InnateLymphoid GC4SPG2 InnateLymphoidGC4 InnateLymphoidSPG2GC4 GC4SPG2 InnateLymphoid InnateLymphoidSPG2GC4 GC4SPG2 Macrophage GC5 Macrophage GC5 Macrophage GC5 SPG3 Macrophage Macrophage GC5 MacrophageGC5 SPG3 GC5 Macrophage SPG3 GC5 Macrophage SPG3 GC5 Macrophage GC5 SPG3 SPG3 EndothelialSPG3 GC6 Macrophage EndothelialSPG3 GC5 GC6 Macrophage EndothelialMacrophageGC5 MacrophageGC6 SPG3GC5 Macrophage GC5SPG3 EndothelialMacrophageGC5 MacrophageGC6 SPG3GC5 Macrophage GC5SPG3 MacrophageGC5 MacrophageSPG3GC5 GC5SPG3 Macrophage MacrophageSPG3GC5 GC5SPG3 Endothelial GC6 Endothelial GC6 Endothelial GC6 SPG4 Endothelial Endothelial GC6 EndothelialGC6 SPG4 GC6 Endothelial SPG4 GC6 Endothelial SPG4 GC6 Endothelial GC6 SPG4 SPG4 MyoidSPG4 GC7 Endothelial MyoidSPG4 GC6 GC7 Endothelial MyoidEndothelialGC6 EndothelialGC7 SPG4GC6 Endothelial GC6SPG4 MyoidEndothelialGC6 EndothelialGC7 SPG4GC6 Endothelial GC6SPG4 EndothelialGC6 EndothelialSPG4GC6 GC6SPG4 Endothelial EndothelialSPG4GC6 GC6SPG4 Myoid GC7 Myoid GC7 Myoid GC7 SPG5 Myoid Myoid GC7 MyoidGC7 SPG5 GC7 Myoid SPG5 GC7 Myoid SPG5 GC7 Myoid GC7 SPG5 SPG5 LeydigSPG5 GC8 Myoid LeydigSPG5 GC7 GC8 Myoid Leydig MyoidGC7 GC8MyoidSPG5GC7 Myoid GC7SPG5 Leydig MyoidGC7 GC8MyoidSPG5GC7 Myoid GC7SPG5 MyoidGC7 MyoidSPG5GC7 GC7SPG5 Myoid MyoidSPG5GC7 GC7SPG5 Leydig GC8 Leydig GC8 Leydig GC8 SPG6 Leydig Leydig GC8 LeydigGC8 SPG6 GC8 Leydig SPG6 GC8 Leydig SPG6 GC8 Leydig GC8 SPG6 SPG6 SertoliSPG6 GC9 Leydig SertoliSPG6 GC8 GC9 Leydig SertoliLeydigGC8 GC9LeydigSPG6GC8 Leydig GC8SPG6 SertoliLeydigGC8 GC9LeydigSPG6GC8 Leydig GC8SPG6 LeydigGC8 LeydigSPG6GC8 GC8SPG6 Leydig LeydigSPG6GC8 GC8SPG6 Sertoli BMP4−BMPR1B GC9 Sertoli FGF7−FGFR3 GC9 Sertoli FGF2−SDC4 GC9 BMP4−BMPR1B GC1 Sertoli CXCL12Sertoli−ITGB1 GC9 FGF7Sertoli−FGFR3GC9 GC1 GC9 FGF2Sertoli−SDC4 GC1 GC9 CXCL12Sertoli−ITGB1 GC1 GC9 Sertoli GC9 GC1 GC1 Tcf21+InterstitialGC1 GC10 Sertoli Tcf21+InterstitialGC1 GC9 GC10 Sertoli Tcf21+InterstitialSertoliGC9 GC10SertoliGC1GC9 Sertoli GC9GC1Tcf21+InterstitialSertoliGC9 GC10SertoliGC1GC9 Sertoli GC9GC1 SertoliGC9 SertoliGC1GC9 GC9GC1 Sertoli SertoliGC1GC9 GC9GC1 Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 GC2 Tcf21+Interstitial Tcf21+Interstitial GC10 Tcf21+InterstitialGC10 GC2 GC10 Tcf21+Interstitial GC2 GC10 Tcf21+Interstitial GC2 GC10 Tcf21+Interstitial GC10 GC2 GC2 GC2 GC11 Tcf21+Interstitial GC2 GC10 GC11 Tcf21+Interstitial Tcf21+InterstitialGC10 Tcf21+InterstitialGC11GC2GC10 Tcf21+Interstitial GC10GC2 Tcf21+InterstitialGC10 Tcf21+InterstitialGC11GC2GC10 Tcf21+Interstitial GC10GC2 Tcf21+InterstitialGC10 Tcf21+InterstitialGC2GC10 GC10GC2 Tcf21+Interstitial Tcf21+InterstitialGC2GC10 GC10GC2 GC11 GC11 GC11 GC3 GC11 GC11 GC3 GC11 GC3 GC11 GC3 GC11 GC11 GC3 GC3 GC3 GC12 GC3 GC11 GC12 GC11 GC12GC3GC11 GC11GC3 GC11 GC12GC3GC11 GC11GC3 GC11 GC3GC11 GC11GC3 GC3GC11 GC11GC3 GC12 GC12 TcellGC12 GC4 TcellGC12 GC12 GC4 GC12 Tcell GC4 GC12 Tcell GC4 GC12 GC12 Tcell GC4 Tcell GC4 Tcell GC4 GC13 Tcell GC4 GC12 GC13 TcellGC12 GC13TcellGC4GC12 GC12GC4 TcellGC12 GC13TcellGC4GC12 GC12GC4 TcellGC12 TcellGC4GC12 GC12GC4 Tcell TcellGC4GC12 GC12GC4 SPG1GC13 SPG1GC13 MacrophageSPG1GC13 SPG1GC5 MacrophageSPG1GC13 GC13 SPG1GC5 GC13 Macrophage SPG1GC5 GC13 Macrophage SPG1GC5 GC13 GC13 Macrophage GC5 Macrophage GC5 Macrophage GC5 GC14 Macrophage GC5 GC13 GC14 MacrophageGC13 MacrophageGC14GC5GC13 GC13GC5 MacrophageGC13 MacrophageGC14GC5GC13 GC13GC5 MacrophageGC13 MacrophageGC5GC13 GC13GC5 Macrophage MacrophageGC5GC13 GC13GC5 SPG2GC14 SPG2GC14 EndothelialSPG2GC14 SPG2GC6 EndothelialSPG2GC14 GC14 SPG2GC6 GC14 Endothelial SPG2GC6 GC14 Endothelial SPG2GC6 GC14 GC14 Endothelial GC6 Endothelial GC6 Endothelial GC6 GC15 Endothelial GC6 GC14 GC15 EndothelialGC14 EndothelialGC15GC6GC14 GC14GC6 EndothelialGC14 EndothelialGC15GC6GC14 GC14GC6 EndothelialGC14 EndothelialGC6GC14 GC14GC6 Endothelial EndothelialGC6GC14 GC14GC6 SPG3GC15 SPG3GC15 m.PericyteSPG3GC15 SPG3GC7 m.PericyteSPG3GC15 GC15 SPG3GC7 GC15 m.Pericyte SPG3GC7 GC15 m.Pericyte SPG3GC7 GC15 GC15 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte GC7 GC16 m.Pericyte GC7 GC15 GC16 m.PericyteGC15 m.PericyteGC16GC7GC15 GC15GC7 m.PericyteGC15 m.PericyteGC16GC7GC15 GC15GC7 m.PericyteGC15 m.PericyteGC7GC15 GC15GC7 m.Pericyte m.PericyteGC7GC15 GC15GC7 SPG4GC16 SPG4GC16 f.PericyteSPG4GC16 SPG4GC8 f.PericyteSPG4GC16 GC16 SPG4GC8 GC16 f.Pericyte SPG4GC8 GC16 f.Pericyte SPG4GC8 GC16 GC16 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte GC8 GC17 f.Pericyte GC8 GC16 GC17 f.PericyteGC16 f.PericyteGC17GC8GC16 GC16GC8 f.PericyteGC16 f.PericyteGC17GC8GC16 GC16GC8 f.PericyteGC16 f.PericyteGC8GC16 GC16GC8 f.Pericyte f.PericyteGC8GC16 GC16GC8 SPG5GC17 SPG5GC17 MyoidSPG5GC17 SPG5GC9 MyoidSPG5GC17 GC17 SPG5GC9 GC17 Myoid SPG5GC9 GC17 Myoid SPG5GC9 GC17 GC17 Myoid GC9 Myoid GC9 Myoid GC9 GC18 Myoid GC9 GC17 GC18 MyoidGC17 GC18MyoidGC9GC17 GC17GC9 MyoidGC17 GC18MyoidGC9GC17 GC17GC9 MyoidGC17 MyoidGC9GC17 GC17GC9 Myoid MyoidGC9GC17 GC17GC9 SPG6GC18 SPG6GC18 ImmLeydigSPG6GC18 SPG6GC10 ImmLeydigSPG6GC18 GC18 SPG6GC10 GC18 ImmLeydig SPG6GC10 GC18 ImmLeydig SPG6GC10 GC18 GC18 ImmLeydig GC10 ImmLeydig GC10 ImmLeydig GC10 GC19 ImmLeydig GC10 GC18 GC19 ImmLeydigGC18 ImmLeydigGC19GC10GC18 GC18GC10 ImmLeydigGC18 ImmLeydigGC19GC10GC18 GC18GC10 ImmLeydigGC18 ImmLeydigGC10GC18 GC18GC10 ImmLeydig ImmLeydigGC10GC18 GC18GC10 GC1GC19 GC1GC19 GC1GC19 GC1GC11 GC1GC19 GC19 GC1GC11 GC19 GC1GC11 GC19 GC1GC11 GC19 GC19 GC11 GC11 GC11 GC20 GC11 GC19 GC20 GC19 GC20GC11GC19 GC19GC11 GC19 GC20GC11GC19 GC19GC11 GC19 GC11GC19 GC19GC11 GC11GC19 GC19GC11 GC2GC20 GC2GC20 GC2GC20 GC2GC12 GC2GC20 GC20 GC20 GC2GC12 GC20 GC20 GC2GC12GC12GC20 GC20 GC20 GC20 GC2GC12GC12GC20 GC20 GC20 GC20 GC12GC20 GC20 GC20 GC12GC20 GC20 GC12GC3 GC12GC3 GC12GC3 GC3 GC12GC3 GC3 GC3 GC12 GC3 GC12 GC12 GC12 GC13 GC13 GC13 GC13 GC13 GC13 GC13GC13 GC13 GC13GC13 GC13 GC13 GC13 GC13 GC13 Tcell GC4 Tcell GC4 Tcell TcellGC4 GC4GC14 Tcell TcellGC4 GC4GC14 Tcell GC4GC14GC14 Tcell GC4GC14GC14 GC14 GC14 Macrophage GC14GC5 Macrophage GC14GC5 Macrophage MacrophageGC14GC5 GC5 Macrophage MacrophageGC14GC5 GC5 Macrophage GC5 GC14Macrophage GC5 GC14 GC14 GC14 GC15 GC15 GC15 GC15 GC15 GC15 GC15GC15 GC15 GC15GC15 GC15 GC15 GC15 GC15 GC15 Endothelial GC6 Endothelial GC6 Endothelial EndothelialGC6 GC6GC16 Endothelial EndothelialGC6 GC6GC16 Endothelial GC6GC16GC16 Endothelial GC6GC16GC16 GC16 GC16 m.Pericyte GC16GC7 m.Pericyte GC16GC7 m.Pericyte m.PericyteGC16GC7 GC7 m.Pericyte m.PericyteGC16GC7 GC7 m.Pericyte GC7 GC16 m.Pericyte GC7 GC16 GC16 GC16 GC17 GC17 GC17 GC17 GC17 GC17 GC17GC17 GC17 GC17GC17 GC17 GC17 GC17 GC17 GC17 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte f.PericyteGC8 GC8GC18 f.Pericyte f.PericyteGC8 GC8GC18 f.Pericyte GC8GC18GC18 f.Pericyte GC8GC18GC18 GC18 GC18 Myoid GC18GC9 Myoid GC18GC9 Myoid MyoidGC18GC9 GC9 Myoid MyoidGC18GC9 GC9 Myoid GC9 GC18 Myoid GC9 GC18 GC18 GC18 GC19 GC19 GC19 GC19 GC19 GC19 GC19GC19 GC19 GC19GC19 GC19 GC19 GC19 GC19 GC19 ImmLeydig GC10 ImmLeydig GC10 ImmLeydig ImmLeydigGC10 GC10GC20 ImmLeydig ImmLeydigGC10 GC10GC20 ImmLeydig GC10GC20GC20 ImmLeydig GC10GC20GC20 GC20 GC20 GC20GC11 GC20GC11 GC20GC11 GC11 GC20GC11 GC11 GC11 GC20 GC11 GC20 GC20 GC20 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 Fgf2−Sdc2 GC13 Jag1−Notch2 GC13 Wnt5a−Ryk GC13 Fgf2−Sdc2 GC13 GC13 Jag1−Notch2 GC13 Wnt5a−Ryk GC13 Wnt5a−Ror1 GC13 GC14 GC14 GC14 GC14 Wnt5aCxcl12−Ror1−Itgb1GC14 Cxcl12GC14−Itgb1 Bmp4−Bmpr2GC14 Bmp4−Bmpr2 Bmp4−Bmpr1bGC14 Bmp4−Bmpr1b Fgf7−Fgfr3 Fgf7−Fgfr3 Fgf2−Sdc4 Fgf2−Sdc4 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 GC18 GC18 GC18 GC18SPG2 GC18 SPG1 GC18SPG2 GC18SPG2 SPG1 SPG1 GC18SPG2 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 GC19SPG2 GC19SPG2 GC19SPG2 GC19 GC19SPG2 SPG2 GC19 SPG2 GC19 SPG2 SPG2 GC19 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 GC20 GC20 GC20 GC20SPG4 GC20 SPG3 GC20SPG4 GC20SPG4 SPG3 SPG3 GC20SPG4 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG5 SPG5 SPG5 BMP4−BMPR2 SPG5 SPG5 BMP4−BMPR1B SPG5 SPG5 FGF2−SDC4 SPG5 CXCL12−ITGB1 SPG5 BMP4−BMPR2 BMP4−BMPR1B FGF2−SDC4 SPG6 CXCL12−ITGB1 SPG5 SPG6 SPG6 SPG5 SPG5 SPG6 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC2 GC1 GC2 GC2 GC1 GC1 GC2 GC1 GC1 GC1 GC1 GC1 GC1 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC3 SPG1 SPG1 SPG1 SPG1 SPG1 GC3 SPG1 GC3 SPG1 GC3 GC3 SPG1 GC3 GC3 GC3 GC3 GC3 GC3 InnateLymphoid GC4 InnateLymphoid GC4 InnateLymphoid InnateLymphoidGC4 GC4 InnateLymphoid InnateLymphoidGC4 GC4 InnateLymphoid GC4 InnateLymphoid GC4 SPG2 SPG2 SPG2 SPG2 InnateLymphoid SPG2 InnateLymphoidGC4 SPG2 GC4 InnateLymphoid InnateLymphoidSPG2 GC4 GC4 InnateLymphoid InnateLymphoidSPG2 GC4 GC4 InnateLymphoid InnateLymphoidGC4 GC4 InnateLymphoid InnateLymphoidGC4 GC4 Macrophage GC5 Macrophage GC5 Macrophage MacrophageGC5 GC5 Macrophage MacrophageGC5 GC5 Macrophage GC5 Macrophage GC5 SPG3 SPG3 SPG3 SPG3 Macrophage SPG3 GC5Macrophage SPG3 GC5 Macrophage MacrophageSPG3 GC5 GC5 Macrophage MacrophageSPG3 GC5 GC5 Macrophage MacrophageGC5 GC5 Macrophage MacrophageGC5 GC5 Endothelial GC6 Endothelial GC6 Endothelial EndothelialGC6 GC6 Endothelial EndothelialGC6 GC6 Endothelial GC6 Endothelial GC6 SPG4 SPG4 SPG4 SPG4 Endothelial SPG4 GC6Endothelial SPG4 GC6 Endothelial EndothelialSPG4 GC6 GC6 Endothelial EndothelialSPG4 GC6 GC6 Endothelial EndothelialGC6 GC6 Endothelial EndothelialGC6 GC6 Myoid GC7 Myoid GC7 Myoid MyoidGC7 GC7 Myoid MyoidGC7 GC7 Myoid GC7 Myoid GC7 SPG5 SPG5 SPG5 SPG5 Myoid SPG5 GC7 Myoid SPG5 GC7 Myoid SPG5MyoidGC7 GC7 Myoid SPG5MyoidGC7 GC7 Myoid MyoidGC7 GC7 Myoid MyoidGC7 GC7 Leydig GC8 Leydig GC8 Leydig LeydigGC8 GC8 Leydig LeydigGC8 GC8 Leydig GC8 Leydig GC8 SPG6 SPG6 SPG6 SPG6 Leydig SPG6 GC8 Leydig SPG6 GC8 Leydig SPG6LeydigGC8 GC8 Leydig SPG6LeydigGC8 GC8 Leydig LeydigGC8 GC8 Leydig LeydigGC8 GC8 Sertoli GC9 Sertoli GC9 Sertoli SertoliGC9 GC9 Sertoli SertoliGC9 GC9 Sertoli GC9 Sertoli GC9 GC1 GC1 GC1 GC1 Sertoli GC1 GC9 Sertoli GC1 GC9 Sertoli GC1SertoliGC9 GC9 Sertoli GC1SertoliGC9 GC9 Sertoli SertoliGC9 GC9 Sertoli SertoliGC9 GC9 Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 Tcf21+Interstitial Tcf21+InterstitialGC10 GC10 Tcf21+Interstitial Tcf21+InterstitialGC10 GC10 Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 GC2 GC2 GC2 GC2 Tcf21+Interstitial GC2 Tcf21+InterstitialGC10 GC2 GC10 Tcf21+Interstitial Tcf21+InterstitialGC2 GC10 GC10 Tcf21+Interstitial Tcf21+InterstitialGC2 GC10 GC10 Tcf21+Interstitial Tcf21+InterstitialGC10 GC10 Tcf21+Interstitial Tcf21+InterstitialGC10 GC10 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC3 GC3 GC3 GC3 GC3 GC11 GC3 GC11 GC3 GC11 GC11 GC3 GC11 GC11 GC11 GC11 GC11 GC11 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC12 Tcell GC4 Tcell GC4 Tcell TcellGC4 GC4 Tcell TcellGC4 GC12 GC4 GC12 Tcell GC4 GC12 GC12 Tcell GC4 GC12 GC12 GC12 GC12 GC12 GC12 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 Macrophage GC5 Macrophage GC5 Macrophage MacrophageGC5 GC5 Macrophage MacrophageGC5 GC13 GC5 GC13 Macrophage GC5 GC13 GC13Macrophage GC5 GC13 GC13 GC13 GC13 GC13 GC13 GC14 GC14 GC14 GC14 GC14 GC14 GC14 GC14 Endothelial GC6 Endothelial GC6 Endothelial EndothelialGC6 GC6 Endothelial EndothelialGC6 GC14 GC6 GC14 Endothelial GC6 GC14 GC14 Endothelial GC6 GC14 GC14 GC14 GC14 GC14 GC14 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 m.Pericyte GC7 m.Pericyte GC7 m.Pericyte m.PericyteGC7 GC7 m.Pericyte m.PericyteGC7 GC15 GC7 GC15 m.Pericyte GC7 GC15 GC15 m.Pericyte GC7 GC15 GC15 GC15 GC15 GC15 GC15 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 f.Pericyte GC8 f.Pericyte GC8 f.Pericyte f.PericyteGC8 GC8 f.Pericyte f.PericyteGC8 GC16 GC8 GC16 f.Pericyte GC8 GC16 GC16 f.Pericyte GC8 GC16 GC16 GC16 GC16 GC16 GC16 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 Myoid GC9 Myoid GC9 Myoid MyoidGC9 GC9 Myoid MyoidGC9 GC17 GC9 GC17 Myoid GC9 GC17 GC17 Myoid GC9 GC17 GC17 GC17 GC17 GC17 GC17 GC18 GC18 GC18 GC18 GC18 GC18 GC18 GC18 ImmLeydig GC10 ImmLeydig GC10 ImmLeydig ImmLeydigGC10 GC10 ImmLeydig ImmLeydigGC10 GC18 GC10 GC18 ImmLeydig GC10 GC18 GC18 ImmLeydig GC10 GC18 GC18 GC18 GC18 GC18 GC18 GC19 GC19 GC19 GC19 GC19 GC19 GC19 GC19 GC11 GC11 GC11 GC11 GC11 GC19 GC11 GC19 GC11 GC19 GC19 GC11 GC19 GC19 GC19 GC19 GC19 GC19 GC20 GC20 GC20 GC20 GC20 GC20 GC20 GC20 GC12 GC12 GC12 GC12 GC12 GC20 GC12 GC20 GC12 GC20 GC20 GC12 GC20 GC20 GC20 GC20 GC20 GC20 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC14 GC14 GC14 GC14 GC14 GC14 GC14 GC14 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC18 GC18 GC18 GC18 GC18 GC18 GC18 GC18 GC19 GC19 GC19 GC19 GC19 GC19 GC19 GC19 GC20 GC20 GC20 GC20 GC20 GC20 GC20 GC20

Bmp4−Bmpr2 Bmp4−Bmpr1b Fgf7−Fgfr3 Bmp4−Bmpr2 Fgf2−Sdc4 Bmp4−Bmpr1b Fgf7Cxcl12−Fgfr3−Itgb1 Cxcl12−Itgb1 Fgf2−Sdc4

SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG1 SPG2 SPG2 SPG2 SPG1 SPG1 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG2 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG3 SPG4 SPG4 SPG4 SPG3 SPG3 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG4 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG5 SPG6 SPG6 SPG6 SPG5 SPG5 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 SPG6 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC1 GC2 GC2 GC2 GC1 GC1 GC2 GC2 GC2 GC2 GC2 GC2 GC2 GC3 GC3 GC3 GC3 GC3 GC3 GC3 GC3 InnateLymphoid GC4 InnateLymphoid GC4 InnateLymphoid GC4 GC3 GC3InnateLymphoid GC4 InnateLymphoid GC4 InnateLymphoid GC4 InnateLymphoid GC4 InnateLymphoid GC4 InnateLymphoid InnateLymphoidGC4 GC4 Macrophage GC5 Macrophage GC5 Macrophage MacrophageGC5 GC5 Macrophage MacrophageGC5 GC5 Macrophage GC5 Macrophage GC5 Endothelial GC6 Endothelial GC6 EndothelialMacrophage MacrophageGC6 GC5 GC5 Endothelial GC6 Endothelial GC6 Endothelial GC6 Endothelial GC6 Endothelial GC6 Endothelial EndothelialGC6 GC6 Myoid GC7 Myoid GC7 Myoid MyoidGC7 GC7 Myoid MyoidGC7 GC7 Myoid GC7 Myoid GC7 Leydig GC8 Leydig GC8 Leydig Myoid GC8MyoidGC7 GC7 Leydig GC8 Leydig GC8 Leydig GC8 Leydig GC8 Leydig GC8 Leydig LeydigGC8 GC8 Sertoli GC9 Sertoli GC9 Sertoli SertoliGC9 GC9 Sertoli SertoliGC9 GC9 Sertoli GC9 Sertoli GC9 Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 Tcf21+InterstitialSertoli GC10SertoliGC9 GC9Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 Tcf21+Interstitial Tcf21+InterstitialGC10 GC10 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC11 GC12 GC12 GC12 GC11 GC11 GC12 GC12 GC12 GC12 GC12 GC12 GC12 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC13 GC14 GC14 GC14 GC13 GC13 GC14 GC14 GC14 GC14 GC14 GC14 GC14 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC15 GC16 GC16 GC16 GC15 GC15 GC16 GC16 GC16 GC16 GC16 GC16 GC16 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC17 GC18 GC18 GC18 GC17 GC17 GC18 GC18 GC18 GC18 GC18 GC18 GC18 GC19 GC19 GC19 GC19 GC19 GC19 GC19 GC19 GC20 GC20 GC20 GC19 GC19 GC20 GC20 GC20 GC20 GC20 GC20 GC20

Cxcl12−Itgb1 Cxcl12−Itgb1

SPG1 SPG1 SPG2 SPG2 SPG3 SPG3 SPG4 SPG4 SPG5 SPG5 SPG6 SPG6 GC1 GC1 GC2 GC2 GC3 GC3 InnateLymphoid GC4 InnateLymphoid GC4 Macrophage GC5 Macrophage GC5 Endothelial GC6 Endothelial GC6 Myoid GC7 Myoid GC7 Leydig GC8 Leydig GC8 Sertoli GC9 Sertoli GC9 Tcf21+Interstitial GC10 Tcf21+Interstitial GC10 GC11 GC11 GC12 GC12 GC13 GC13 GC14 GC14 GC15 GC15 GC16 GC16 GC17 GC17 GC18 GC18 GC19 GC19 GC20 GC20