Single-cell transcriptomic and cross-species comparison analyses reveal distinct molecular changes of porcine testes during puberty

Single-cell transcriptomic profiles of the developing porcine testes

To delineate the developmental process of the porcine testis, we conducted single-cell transcriptomic profiling on testicular cells isolated from Bama pigs at various postnatal days (PD), including PD9, PD30, PD60, PD90, and PD150, covering the entire period from newborn to adult (Fig.1a). To enhance the representation of testicular somatic cells, additional biological replicates were collected at PD90 and PD150. Following the filtering out of multiplets and low-quality cells, a total of 41,548 cells were retained for downstream analysis (Supplementary Fig. 1a). Cluster analysis enabled the annotation of nine major cell types based on the expression of established cell markers^{14,17,18,19,20}, constructing a temporal gene expression landscape of porcine testis development (Fig. 1b). Each cell exhibited a median expression of 2025 detected genes (Supplementary Fig. 1b). The dataset’s robustness was supported by the specific expression of marker genes for spermatogenic and somatic cell types. Germ cells encompassed spermatogonia (S’gonia, UCHL1+), spermatocytes (S’cyte, SYCP2+), and spermatids (S’tid, TNP1+) (Fig.1c). Testicular niche cells (VIM+) represented a heterogeneous mix of Sertoli cells (SOX9+), myoid/stromal cells (COL1A1+ and/or ACTA2+), Leydig cells (CYP11A1+), vascular cells (VWF+), and immune cells (PTPRC+) (Fig. 1c and Supplementary Fig. 1c). Visualization on the UMAP plot illustrated consistent agreement among samples regarding different postnatal ages, biological replicates, and cell cycle phases (Fig. 1d and Supplementary Fig. 1d, e). The distribution of testicular cell types reflects the developmental progression across the sampled time points (Fig.1e). Notably, the proportion of germ cells increased significantly during spermatogenesis, accompanied by a gradual reduction in the percentage of somatic cells (Fig. 1f and Supplementary Fig. 1f). Furthermore, in contrast to human testes, a limited number of lymphocytes (marked by CCL5 and GZMB) were identified in porcine testes (Supplementary Fig. 1g). In summary, our scRNA-seq datasets effectively captured the major cell types and their dynamic diversity during the testis development of Bama pigs.

a Schematic diagram of the experimental workflow. b UMAP visualization of combined single-cell transcriptome data from porcine testes. Each dot represents a single cell that is color-coded by cell type. c Dot plot depicting selected differentially expressed genes for all cell types identified. d UMAP visualization of distribution of single cells collected from different timepoints. Cells are colored based on the sample of origin. e UMAP visualization of annotated cell types from individual timepoints. f Fraction of cell type per timepoint, exhibiting a progressive increase in cellular complexity throughout development. Source data are provided in Supplementary Data 1.

Full size image

Stepwise establishment of porcine spermatogenesis and comparison with humans and mice

To gain a deeper understanding of the molecular characteristics throughout spermatogenesis, we isolated porcine germ cells from PD9 to PD150, and subsequently categorized them into eight subtypes (Fig. 2a and Supplementary Fig. 2a). By utilizing known markers, we organized two sub-clusters of spermatogonia, four sub-clusters of spermatocytes, and two sub-clusters of spermatids, sequentially representing mitotic, meiotic, and postmeiotic stages (Supplementary Fig. 2b). Through an extensive analysis, we identified 952 genes exhibiting highly dynamic changes along the germ cell differentiation path. This allowed us to explore their associated Gene Ontology (GO) terms (Fig. 2b). As anticipated, biological processes such as ‘ribosome biogenesis’ and ‘cellular respiration’ were notably enriched in spermatogonia, concomitant with upregulation of proliferative genes (e.g., TOP2A) during the transition to differentiating spermatogonia, which may be related to the balance between self-renewal and differentiation. Notably, this analysis unveiled FANCI and FANCD2, components of the core complex in the Fanconi anemia (FA) pathway²¹, as prominent markers for porcine undifferentiated spermatogonia, suggesting potential avenues for future research (Supplementary Fig. 2c). For instance, the confirmed role of FA component in the maintenance of mouse undifferentiated spermatogonia supports the probability of the involvement of this conserved pathway in porcine undifferentiated spermatogonia as well²². Furthermore, in spermatocytes to spermatids, genes implicated in meiosis and spermatogenesis exhibited high expression levels, signifying the most characteristic transcriptomic changes in this phase (Fig. 2b).

a Left: Highlight of germ cells in the UMAP from Fig. 1b, showing the cells selected for focused analysis. Right: UMAP visualization of porcine germ cell types. Each dot represents a single cell that is color-coded by cell type. b Heatmap showing average expression of DEGs for each spermatogenic cell type. Each row represents a gene. The representative gene and enriched GO term of each gene cluster are listed on the right. c Pie plot showing the proportion of spermatogenic cell types at the indicated developmental stages of pigs, humans, and mice. Colors indicate cell types. Source data are provided in Supplementary Data 1. (Data from Guo et al.^15,32, and Zhao et al.¹⁶) d Hematoxylin and eosin (H&E) staining of sections of porcine testes. Arrowhead indicated a specific cell type; SG spermatogonia, SC spermatocyte, rST round spermatid, eST elongated spermatid, ST Sertoli cell, LC Leydig cell, MC myoid cell, BV Blood vessel. Scale bars, 50 μm. e UMAP visualization of sub-clusters of spermatogonia. Each dot represents a single cell that is color-coded by cell type. Arrowhead indicated pseudo-developmental trajectory for spermatogonia. f Dot plot depicting selected differentially expressed genes for each sub-cluster identified in spermatogonia. g Fraction of each sub-cluster of spermatogonia at per timepoint. Source data are provided in Supplementary Data 1. h Heatmap showing the Pearson correlation of the normalized average gene expression between spermatogonia from pigs and humans.

Full size image

Next, we compared germ cell composition across pigs, humans, and mice throughout the infancy to adulthood period. Previous studies have systematically integrated single-cell transcriptomic atlases covering the entire process of male germ cell development in humans and mice^14,15,16. To ensure consistency, we compiled and re-analyzed published data using canonical markers with the same analysis pipeline on our pig data (Supplementary Fig. 2d–g). Remarkably, with sex-maturity normally at 6-months old age in pigs, at PD9, transcriptome-based clustering revealed the emergence of pachytene spermatocytes which is observed only up to the postnatal day (PND) 14 in mice, indicating an earlier onset of meiotic initiation in pigs compared to mice (Fig.2c). In humans, germ cells consist solely of spermatogonia at the period from birth to 7 years afterward, during which the proportion of differentiating spermatogonia increases, and a small number of spermatids appear in the 11-year-old juvenile^15,23. However, histological examination revealed small testis cords lacking apparent spermatocytes in PD9 pig testes (Fig. 2d). This appearance resembles the testis morphology observed in human childhood and fetal/early postnatal mice. Nonetheless, we identified spermatocytes in the seminiferous tubules of porcine testes at PD15 (Fig. 2d). The incomplete consistency between the analysis and histological findings could be due to cells undergoing transcriptional changes preceding the occurrence of morphological transformation. By PD30, a distinct tubular structure with a cavity became progressively apparent, and spermatids were observed in a small proportion of pig tubules (Fig. 2d). Unexpectedly, we discovered that spermatogenesis commences at a very early stage in Bama pigs.

Discovery of a porcine spermatogonial subtype resembling transcriptome state 0 SSCs in humans

Spermatogonia maintain the life-long process of spermatogenesis by balanced self-renewal and differentiation. Our focus turned to Bama pig spermatogonia, given divergent findings between humans and model animals in previous studies²⁴. Through unsupervised clustering, we identified four distinct clusters in porcine Undiff. S’gonia and differentiating spermatogonia: SPG1-SPG4 (Fig. 2e). Pseudotime analysis, as indicated by the arrow, further defined the developmental routine from SPG1 to SPG4 (Fig. 2e and Supplementary Fig. 2g). Here, SPG1 represents the most naïve developmental state of spermatogonia, while SPG4 aggregates at the end of this developmental trajectory. Notably, further analysis of gene expression and cell cycle revealed unique molecular signatures for each subtype (Fig. 2f and Supplementary Fig. 2h). SPG3 expressed markers such as CDK2, CCND1 and MKI67, indicating that this population exhibits active proliferation and appears to share molecular similarities with differentiating spermatogonia in humans and mice. Whereas SPG4 represented the transition to preleptotene cells by upregulating meiotic genes including SYCE3, REC8, and MEIOC (Fig. 2f). Of special note are the porcine SPG1 subgroup, marked by the high expression levels of PIWIL4, EGR4, and FGFR3, resembles human transcriptome state 0 SSCs, which represent undifferentiated and quiescent reserves maintained throughout human male’s lifetime¹⁴. Statistical analysis of germ cell composition supported the stable presence of SPG1 in pig testes from infancy to adulthood (Fig. 2g). We also found a strong correlation in gene expression between porcine SPG1 and human state 0 spermatogonia, with subsequent subtypes showing some degree of similarity (Fig. 2h). Overall, we have uncovered a spermatogonial subtype within porcine testes closely resembling human state 0 SSCs, suggesting their potential as the most naïve germline stem cells. Notably, this marks the first instance of identifying such cells in a species other than primates¹.

Developmental landscape of supporting and interstitial cell lineages

As pivotal constituents of the testicular somatic niche, a substantial number of cells from the supporting lineage (Sertoli cells) and interstitial lineage (myoid/stromal cells and Leydig cells) were captured in the developing testes of Bama pigs (Fig. 1f). To delineate the trajectory of supporting cell development, our focus turned to Sertoli cells across five postnatal stages (Fig. 3a). The clustering plot depicted cellular continuity across ages, initiating from PD9 and progressing towards a uniform cluster after PD60. This observation suggests that Sertoli cells in porcine testes likely remain in uniformed state around three months after birth. Pseudotime analysis revealed the progression of immature Sertoli cells into two distinct states, turning into one from PD30 onwards, ultimately forming terminal Sertoli cells along the trajectory (Supplementary Fig. 3a). Furthermore, we examined the expression dynamics of genes associated with the cell cycle (GO:0007049) and the number of transcripts along the pseudotime of Sertoli cells (Supplementary Fig. 3b). As expected, cell cycle-related genes (e.g., CDC34, GAB1, CDC16) are predominantly expressed in PD9 cells, supporting the more proliferative condition of immature Sertoli cells. We identified differentially expressed genes and GO terms in each sample, offering an additional paradigm for characterizing cell identity (Fig. 3b). At PD30 and PD60, we revealed an absence of SOX9/KI67+ cells, indicating that Sertoli cell proliferation has ceased (Supplementary Fig. 3c). Notably, PD30 marked a potential metabolic shift for Sertoli cells, indicated by the upregulation of genes associated with fatty acid metabolism, including ELOVL2 and FADS2 (Fig. 3c). This metabolic adaptation likely contributes to meeting the energy and morphological requirements for Sertoli cells necessary to support spermatogenesis. Additionally, the synthesis enzyme ALDH1A1, responsible for producing retinoic acid (RA) that induces spermatogonia differentiation, is partially expressed in Sertoli cells since PD9, and significantly elevated from PD30, contributing to the subsequent progression of germ cell development (Fig. 3c). The immunofluorescence staining for ALDH1A1 and SOX9 confirmed this event, demonstrating the specific expression and localization of ALDH1A1 within supporting cells. It also indicated a tendency for ALDH1A1 signals to move towards the basal part of the seminiferous tubule as testis development progresses (Fig. 3d). Also, the expression patterns of genes associated with tight junctions also mirror the developmental timing of Sertoli cells (Fig. 3c). In summary, a focused analysis of porcine Sertoli maturation showcased dynamic changes in molecular features and functional interpretation from newborn to adult.

a Left: Highlight of Sertoli cells in the UMAP from Fig. 1b, showing the cells selected for focused analysis. Right: PCA map visualization of porcine Sertoli cells. Each dot represents a single cell that is color-coded by timepoints. b Heatmap showing average expression of DEGs for timepoints. Each row represents a gene. The representative gene and enriched GO term of each gene cluster are listed on the right. c The gene-normalized dynamics of selected genes along the pseudotime trajectories. Each dot represents a single cell that is color-coded by timepoints. d Immunofluorescence images of ALDH1A1 (green) and SOX9 (red) in porcine testis sections from different timepoints. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. e Immunofluorescence images of ACTA2 (red) and HSD3β (green) in porcine testis sections from different timepoints. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. f Left: Highlight of interstitial cells in the UMAP from Fig. 1b, showing the cells selected for focused analysis. Right: PCA map visualization of porcine interstitial cells. Each dot represents a single cell that is color-coded by timepoints. g Expression of representative marker genes identifying interstitial lineages cast on the PCA plot from Fig. 4f Normalized expression is plotted on a low-to-high scale (gray-purple).

Full size image

We next investigated interstitial cell lineage differentiation during testis development. Immunofluorescence staining revealed the presence of myoid cells (marked by ACTA2) and Leydig cells (marked by HSD3β) within the porcine testis from PD9 to PD150 (Fig. 3e). Upon re-clustering these two cell types, distinct lineages were evident as early as PD9 in Bama pigs (Fig. 3f). The expression of marker genes and enriched pathways affirmed the identity and functionality of myoid cells and Leydig cells in porcine testes, aligning with findings in humans and mice^25,26,27 (Fig. 3g and Supplementary Fig. 3d). However, the origin of these two cell types in Bama pigs from a common interstitial progenitor, as previously demonstrated in human and mouse testes^15,28, remains unknown. This led us to address this question using testicular samples from embryonic pigs.

Identification of a testicular myoid progenitor persisting from the embryonic period to adulthood

To gain a better understanding of interstitial cell lineage specification, we profiled testicular cells from fetal pigs at embryonic day 72 (E72). Additionally, we reanalyzed previously published single-cell transcriptomic data of embryonic testes at E24, E27, E30, and E35 of Bama pigs²⁹ (Supplementary Fig. 4a). After integration and annotation, we successfully isolated somatic cells belonging to the myoid/stromal and Leydig lineages from all samples (Supplementary Fig. 4b, c). Further analysis revealed a notable clustering at the early stage (~E24), followed by transcriptional segregation into two separate lineages around E30, as depicted in the three-dimensional UMAP plot (Fig. 4a). By examining the expression of canonical markers, we identified the upper trajectory characterized by CYP17A1 expression as representing the Leydig lineage, while the lower trajectory marked by ACTA2 expression corresponded to the myoid/stromal lineage (Fig. 4b).

a 3D UMAP visualization of distribution of interstitial cells collected from different developmental stages. Each dot represents a single cell and is colored according to its donor of origin. b Expression patterns of known interstitial marker genes projected onto the UMAP plot from a. c UMAP visualization of reclustered myoid/stromal cells, colored by assignment to four cell types. d Dot plot depicting marker genes for 4 cell types identified from c. e Bar plot showing the cell number of myoid/stromal cell types for each developmental stage (outer). percentage of cell types for myoid/stromal cells from PD60-PD150 testes (inner). Source data are provided in Supplementary Data 1. f Pseudotime trajectory of myoid/stromal cells. Each dot represents a single cell and is colored according to its annotated cell type. g Developmental potency score of each cell projected onto the UMAP plot from c. h Immunofluorescence images of a proliferation marker, KI67 (green) with a myoid cell marker, ACTA2 (red) in porcine testis sections from different timepoints. Nuclei were counterstained with DAPI (blue). Scale bars, 50 µm. i The timeline and proposed model for Bama pig, human and mouse testicular somatic niche cell development at embryonic, fetal, and postnatal stages.

Full size image

This integrated dataset enabled us to explore the origins, emergence, dynamics, and specialization of the interstitial lineage. We isolated myoid/stromal cells for focused clustering analysis (Supplementary Fig. 4d). Due to the expression of genes associated with mesenchymal identity like ACTA2 and COL3A1, we observed the differentiation of myoid cells and stromal cells after birth, respectively (Fig. 4c, d). As development progressed, differentiated cell types eventually occupied the entire proportion within the lineage (Fig. 4e). Notably, we identified one actively proliferating cluster, marked by high expression of TOP2A and MKI67, associated with promoting cellular proliferation (Fig. 4d). Combining this with pseudotime analysis, we distinguished four cell types: proliferating progenitor (Prolif progenitor, marked by TOP2A), the intermediate cluster (Transient, marked by POSTN), and two terminal types mentioned above (Fig. 4c, d and Supplementary Fig. 4e). The resulting branched trajectory tree accurately reflected the differentiation status and expression of known markers (Fig. 4f and Supplementary Fig. 4f). To validate the differentiation potential of the progenitor cells, we compared them with mouse in vitro testis interstitial cell populations³⁰. We found that the progenitor cells in pig testes closely resemble the mice interstitial progenitors distinguished before (Supplementary Fig. 4g)³⁰. Further, using CytoTRACE, we predicted a higher developmental potency for the Prolif progenitors compared to Myoid cells and ACTA2-stromal cells (Fig. 4g).

To further provide functional insights into their characteristics, we calculated the myoid/stromal signatures of these cell types. As anticipated, peritubular myoid cells exhibited higher muscle signatures, while ACTA2– cells showed higher expression of stromal signatures (Supplementary Fig. 4h). Correlation analysis and hierarchical clustering further supported that myoid and stromal cells represent two distinct cell populations with unique functional traits (Supplementary Fig. 4i,j). In contrast, proliferating progenitor cells exist in a more primitive and early state (Fig. 4f and Supplementary Fig. 4j). Considering the known plasticity of the testicular interstitial lineage and the potential proliferative capacity of these cells, we investigated whether the proliferating progenitors annotated were present in the adult testes of Bama pigs. Immunofluorescence staining revealed the presence of a potential proliferating progenitor within the basal lamina of seminiferous tubules, characterized by co-expression of MKI67 and ACTA2 (Fig. 4d, h). These findings demonstrate the existence of actively dividing myoid progenitors in postnatal porcine testes. Despite the prevailing notion suggesting that testicular somatic cells arise post-mitotically, our analysis identified such rare cell subpopulations in Bama pigs, holding the potential to differentiate into either stromal or myoid cells.

The regulatory role of Leydig cell steroidogenesis in developing porcine testes

The sex hormone secreted by Leydig cells plays a pivotal role in male gonadal differentiation and maturation²⁶. We proceeded to analyze the Leydig lineage from Fig. 4a. Re-clustering analysis revealed two major clusters displayed in the PCA plot after regressing out batch effects (Fig. 5a). In line with the recognized roles of fetal Leydig cells in androgen synthesis in humans and mice³¹, we observed that fetal Leydig cells in Bama pigs also exhibited upregulation of terms linked to the steroid biosynthetic process. Conversely, porcine adult Leydig cells showed more significant enrichment of cellular catabolic processes (Supplementary Fig. 5a). As of current, we have delineated the trajectory of testicular somatic differentiation of pigs, and summarized its developmental patterns in relation to human and mouse (Fig. 4i).

a Single cell of Leydig lineage cast on PCA plot, colored by stages of development. b Line plots showing the expression levels of steroidogenic genes in each stage of development. c Box plots showing the concentration of Testosterone (top) or Estrogen (bottom) in serum of postnatal Bama pigs. Boxes represent the interquartile range with the median as a horizontal line, and points are shown as each replicate. Source data are provided in Supplementary Data 1. d Expression patterns of CYP17A1 and CYP19A1 projected onto the PCA plot from (e) GO terms enriched in up-regulated genes of estrogen synthesizing Leydig cells compared to other Leydig cells (log2foldchange > 0.25). f Line plots showing the expression levels of ESR1 and ESRRA in each stage of development. g Clustering analysis of dynamic gene expression during development of supporting lineage. Four modules of genes form two distinct categories according to the expression patterns. Median of normalized gene-expression level of each module (orange line) was shown. h Correlation analysis of ESR1 and ESRRA expression with up-regulated ER genes (left) or down- regulated ER genes (right) using Pearson’s correlation coefficients. Points represent the cells in supporting lineage, colored by developmental stages. AveExpr: average expression. i GO terms enriched in up-regulated and down-regulated ER genes with -log10(p value) colored according to the color key at the bottom.

Full size image

To further explore the steroidogenic activity of Leydig cells, we monitored the expression dynamics of steroid biosynthetic enzymes during development (Fig. 5b). As expected, we observed the upregulation of genes responsible for producing testosterone after PD60, including STAR, CYP11A1, and CYP17A1. Unexpectedly, the expression of estrogen synthase was also found in porcine Leydig cells, reaching a level comparable to testosterone synthase in adulthood, unlike in humans or mice^16,32 (Fig. 5b and Supplementary Fig. 5b). We measured serum testosterone and estrogen levels in Bama pigs aged 0–6 months after birth and observed a gradual rise in estrogen content (Fig. 5c), consistent with previous reports in boar testes^33,34. Notably, the expression of estrogen synthase was concentrated in a subset of Leydig cells termed as “Leydig_type2”, rather than being widely expressed (Fig. 5d and Supplementary Fig. 5c). Compared to other Leydig cells (Leydig_type1), Leydig_type2 exhibits similar LHCGR expression, but higher Androgen Receptor (AR) expression (Supplementary Fig. 5d). Moreover, upregulated GO terms included cholesterol metabolic processes, represented by enzymes functioning in lipid metabolism upstream of sex hormone biosynthesis (e.g., HMGCR) (Fig. 5e and Supplementary Fig. 5d). The downregulated genes included DLK1 (Supplementary Fig. 5d), which is specifically expressed in human fetal Leydig cells³².

Sertoli cells serve as the primary targets of androgens in testes. The androgen receptor signaling pathway governs Sertoli cell proliferation, maturation, and the maintenance of the blood-testis barrier formed between Sertoli cells^{35,36,37,38,39}. Considering this, we hypothesized that estrogen may also play a role in the development of the porcine testicular supporting cell lineage. Indeed, our integrated dataset of supporting cells (from Supplementary Fig. 4b) revealed a significant upregulation of Estrogen Receptor 1 (ESR1) and Estrogen Related Receptor Alpha (ESRRA) at E35 and postnatal stages (Fig. 5f and Supplementary Fig. 5e). Both have been reported as transcription factors responsible for activating or inhibiting the expression of target genes^40,41,42. To identify their target genes, we utilized Cluster-Buster to locate direct binding sites of ESR1 and ESRRA in the promoter regions of all genes in the pig, considering regions with a score greater than 8 (indicating a turning point in the number of regions) as estrogen signaling target genes (Supplementary Fig. 5f). In supporting cells, we identified four clusters of genes exhibiting similar (cluster 1 & 2) or opposite (cluster 3 & 4) expression trends to ESR1 and ESRRA. Among these clusters, 57 and 50 genes, respectively, overlapped with estrogen signaling target genes (Fig. 5g), termed estrogen-regulated genes (ER genes) in supporting cells. The expression of these ER genes positively or negatively correlated with the expression of ESR1 or ESRRA, confirming their reliability as target genes (Fig. 5h). Moreover, upregulated ER genes were enriched in immunity-related GO terms, which were also upregulated in human Sertoli cells during puberty¹⁵, leading to speculation that they may contribute to protecting the testis from infections^43,44,45(Fig. 5i). Conversely, downregulated GO terms enriched in ER genes, such as ‘regulation of cell differentiation’ and ‘cell cycle process’, suggest that estrogen has the potential to regulate porcine supporting cells, guiding them to exit proliferation and commence differentiation into immature Sertoli cells (Fig. 5i). Additionally, estrogen receptors and estrogen-related receptors also exhibited stage-specific expression during germ cell development (Supplementary Fig. 5g–i). Further investigation is needed to determine whether estrogen directly regulates porcine spermatogenesis. In summary, we discovered that a subset of porcine Leydig cells possesses the ability to synthesize estrogens, activating estrogen-mediated signaling pathways in the supporting cell lineage and aiding in the proliferation and maturation of porcine Sertoli cells.

The testicular signaling niche of porcine germ cells

The communication between the niche and germline is crucial for testis development, and understanding how these interactions trigger puberty and spermatogenesis is currently an area of significant interest. Through ligand-receptor analysis using CellChat, we identified cell types actively interacting with porcine germ cells, including myoid/stromal cells, vascular cells, followed by Sertoli cells and Leydig cells (Fig. 6a, b, and Supplementary Fig. 6a). We observed cell type-specific expression of genes encoding ligands and receptors involved in various signaling pathways, such as Pleiotrophin (PTN), KIT, Granulin (GRN), and Laminin (Fig. 6c). Furthermore, the comparative landscape revealed the dynamics of intercellular signaling activity with age, with the strongest cell-cell communication observed at PD9, maintaining nearly consistent levels after PD30 (Supplementary Fig. 6b). In terms of specific signaling pathways, spermatogonia at PD9 receive signals from various somatic cells, such as LAMB1/LAMA1/LAMC3 – DAG1, JAG1/DLL3/DLL4 – NOTCH2, INHABB – ACVR2B, INHABA – ACVR2A, etc (Supplementary Fig. 6c). The interactions gradually diminish as the testes develop (Supplementary Fig. 6c). This finding suggests that regulatory influences from the niche are highly active during the maturation process of porcine testes.

a Circle plots depict the number of interactions (left) or interaction weights/strength (right) in the cell-cell communication network among major cell types. Colors of dot/edges indicated the particular cell type. b Bubble diagram showing ligand-receptor communications between germline (target) and somatic niche (source) of porcine testes. The color of bubbles indicates the means of the communication probability. c Expression of ligands (red) and receptors (blue) of indicated signaling pathways cast on the UMAP plot from Fig. 1b. Normalized expression is plotted on a low-to-high scale (gray-purple). d Dot plot depicting expression of ligands and receptors of GDNF signaling for major cell types of porcine testes. e Schematic model of selected signaling pathways involved in germling-somatic niche interactions in porcine testes.

Full size image

Our data also provides valuable insights into the conservation and diversity of the roles of glial cell line-derived neurotrophic factor (GDNF) signaling in SSCs maintenance across species. Mouse single-cell RNA sequencing (scRNA-seq) data reproduced the expression of the GDNF ligand, Gdnf, primarily in Sertoli cells and myoid/stromal cells, with the receptor Gfra1 highly expressed in spermatogonia (Supplementary Fig. 6d, e). While GFRA1 marks undifferentiated spermatogonia in humans as well, the ligand of GFRA2, NRTN, was detected in human Sertoli cells (Supplementary Fig. 6f, g). In contrast, GDNF was scarcely expressed in the human testicular niche. In light of this, we re-examined the regulatory logic of GDNF signaling in Bama pig using our dataset (Fig. 6d). Enrichment of ligand-receptor genes led us to speculate that NRTN in Sertoli cells, rather than GDNF, plays a central role as a paracrine factor, controlling the fate of porcine spermatogonia. In summary, we outlined the germline-niche interactions in porcine testes, emphasizing the need for further detailed functional investigation (Fig. 6e).

• SEO Powered Content & PR Distribution. Get Amplified Today.
• PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
• PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
• PlatoESG. Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
• PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
• Source: https://www.nature.com/articles/s42003-024-07163-9