Conserved Transcriptome Features Define Prepubertal Primate Spermatogonial Stem Cells as Adark Spermatogonia and Identify Unique Regulators
Singh A, Hermann BP, 01.03.2023
Abstract
Antineoplastic treatments for cancer and other non-malignant disorders can result in long-term or permanent male infertility by ablating spermatogonial stem cells (SSCs). SSC transplantation using testicular tissue harvested before a sterilizing treatment is a promising approach for restoring male fertility in these cases, but a lack of exclusive biomarkers to unequivocally identify prepubertal SSCs limits their therapeutic potential. To address this, we performed single-cell RNA-seq on testis cells from immature baboons and macaques and compared these cells with published data from prepubertal human testis cells and functionally-defined mouse SSCs. While we found discrete groups of human spermatogonia, baboon and rhesus spermatogonia appeared less heterogenous. A cross-species analysis revealed cell types analogous to human SSCs in baboon and rhesus germ cells, but a comparison with mouse SSCs revealed significant differences with primate SSCs. Primate-specific SSC genes were enriched for components and regulators of the actin cytoskeleton and participate in cell-adhesion, which may explain why the culture conditions for rodent SSCs are not appropriate for primate SSCs. Furthermore, correlating the molecular definitions of human SSC, progenitor and differentiating spermatogonia with the histological definitions of Adark/Apale spermatogonia indicates that both SSCs and progenitor spermatogonia are Adark, while Apale spermatogonia appear biased towards differentiation. These results resolve the molecular identity of prepubertal human SSCs, define novel pathways that could be leveraged for advancing their selection and propagation in vitro, and confirm that the human SSC pool resides entirely within Adark spermatogonia.
Singh A, Hermann BP. Conserved Transcriptome Features Define Prepubertal Primate Spermatogonial Stem Cells as Adark Spermatogonia and Identify Unique Regulators. Int J Mol Sci. 2023 Mar 1;24(5):4755. doi: 10.3390/ijms24054755. PMID: 36902187; PMCID: PMC10002546.
Publication: https://doi.org/10.3390/ijms24054755
Disclaimer
The publication Conserved Transcriptome Features Define Prepubertal Primate Spermatogonial Stem Cells as Adark Spermatogonia and Identify Unique Regulators by Singh A, Hermann BP is published under an open access license: https://creativecommons.org/licenses/by/4.0/. Permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Curation by the MFGA team Relevant data sets presented in the publication have been identified. If possible, annotations (title, general information, conditions, processed tissue types and processed cell types) have been added based on information from the publication. Data tables and images that provide a good overview on the publication's findings on the data set have been extracted from the publication and/or supplement. If not stated otherwise, images are depicted with title and description exactly as in the publication. Tables have been adjusted to the MFGA table format. Conducted adjustments are explained in the detailed view of the tables. However, titles and descriptions have been adopted from the publication.
Data set 1: Single-cell transcriptomics in prepubertal human, baboon, and macaque testes
Transcriptome: Single-cell RNA-Sequencing
Species
| Species |
|---|
| Human |
| Baboon |
| Rhesus Macaque |
Tissue Types
| BRENDA tissue ontology | Maturity | Description | Species | Replicates |
|---|---|---|---|---|
| BTO_0001363: testis | 2 newborns, 2 infants, 2 juveniles | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Human | 6 |
| BTO_0001363: testis | prepubertal (newborn and 26 months old) | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Baboon | 2 |
| BTO_0001363: testis | prepubertal (newborn and 26 months old) | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Rhesus Macaque | 2 |
Images
Figure 1: Single-cell RNA-seq analyses of testis cells from prepubertal baboons, rhesus macaques, and humans identify major germ and somatic cell types
(A–C) UMAP projections of single-cell transcriptomes from the testes of prepubertal (A) baboons (n = 2), (B) rhesus macaques (n = 2), and (C) humans (n = 6). Each dot represents a single cell and is colored according to unbiased cluster annotation (16 baboon clusters, 21 rhesus macaque clusters, and 23 human clusters). (D–F) UMAP projections showing testicular cells, colored by cell types as determined by differential gene expression, to represent germ and somatic cell types.
Licensed under: https://creativecommons.org/licenses/by/4.0/
Figure 2: Focused single-cell transcriptome analysis of germ cells from prepubertal human, baboon, and rhesus macaque testes
(A–C) UMAP plot visualization of germ cells from (A) human (n = 1451), (B) baboon, (n = 233) and (C) rhesus (n = 599) testes colored by unbiased cluster as noted in the legend for each species. (D–F) Heatmaps show the top 100 significantly differentially expressed genes between each unbiased cell cluster (noted by the numbers at the bottom) and expression of the key markers for each gene cluster (right) for (D) human, I baboon, and (F) rhesus macaque germ cells. Gene lists can be found in Table S2. (G–I) Dot plot showing the expression of the selected markers of undifferentiated and differentiated spermatogonia for unbiased clusters in (G) human, (H) baboon, and (I) rhesus macaque datasets used to identify germ cell subsets. The size of the dot represents the percentage of cells within a cell type, while the color encodes the average expression level across all cells within a cell type (blue is high).
Licensed under: https://creativecommons.org/licenses/by/4.0/
Data set 2: Cross-species comparison of spermatogonia
Transcriptome: Single-cell RNA-Sequencing
Species
| Species |
|---|
| Human |
| Baboon |
| Rhesus Macaque |
Tissue Types
| BRENDA tissue ontology | Maturity | Description | Species | Replicates |
|---|---|---|---|---|
| BTO_0001363: testis | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Human | ||
| BTO_0001363: testis | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Baboon | ||
| BTO_0001363: testis | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Rhesus Macaque |
Cell Types
| Cell ontology | Maturity | Description | Species | Replicates | Cells per replicate |
|---|---|---|---|---|---|
| CL_0000016: male germ line stem cell | Prepubertal | A stem cell that is the precursor of male gametes. | Human | ||
| CL_0000586: germ cell | The reproductive cell in multicellular organisms. | Baboon | |||
| CL_0000586: germ cell | The reproductive cell in multicellular organisms. | Rhesus Macaque |
Images
Figure 3: Cross-species comparison of putative human SSCs revealed analogous cell types in baboon and rhesus and unique markers of primate SSCs
(A) UMAP plot colored by cell type annotation represents human germ cells subsets (n = 1451). (B) Dot plot shows the expression of selected markers for each germ cell subset seen in the UMAP plot. The size of the dot represents the percentage of cells within a cell type, while the color encodes the average expression level across all cells within a cell type (blue is high). (C–D) Correlation heatmaps show the comparison of the human SSC cluster with (C) baboon or (D) rhesus unbiased spermatogonial clusters, colored based on the Pearson correlation coefficient. (E) Correlation heatmap shows the comparison of putative SSC clusters from mouse, human, baboon, and rhesus germ cell datasets, colored based on the Pearson correlation coefficient. (F) Venn diagram shows the comparison of genes differentially expressed in the human SSC cluster (n = 508) with genes differentially expressed in the putative SSC clusters in baboon and rhesus. Thirty-two genes were conserved in humans, baboons, and rhesus macaques and represent markers of putative SSCs.
Licensed under: https://creativecommons.org/licenses/by/4.0/
Data set 3: Pathways analyses reveal unique biological pathways among conserved primate SSC
Transcriptome: Single-cell RNA-Sequencing
Species
| Species |
|---|
| Human |
| Baboon |
| Rhesus Macaque |
Tissue Types
| BRENDA tissue ontology | Maturity | Description | Species | Replicates |
|---|---|---|---|---|
| BTO_0001363: testis | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Human | ||
| BTO_0001363: testis | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Baboon | ||
| BTO_0001363: testis | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Rhesus Macaque |
Cell Types
| Cell ontology | Maturity | Description | Species | Replicates | Cells per replicate |
|---|---|---|---|---|---|
| CL_0000016: male germ line stem cell | A stem cell that is the precursor of male gametes. | Human | |||
| CL_0000016: male germ line stem cell | A stem cell that is the precursor of male gametes. | Baboon | |||
| CL_0000016: male germ line stem cell | A stem cell that is the precursor of male gametes. | Rhesus Macaque |
Images
Figure 4: Pathways analyses reveal unique biological pathways among conserved primate SSC markers
Pathway analysis of the (A) 32 markers of human, baboon, and rhesus SSCs, (B) 105 markers of human and baboon SSCs, (C) 53 markers of human and rhesus SSCs, and (D) 127 markers of mouse SSCs. The lengths of the horizontal bars and their color represent p-values according to the scale.
Licensed under: https://creativecommons.org/licenses/by/4.0/