Multiomics approach to profiling Sertoli cell maturation during development of the spermatogonial stem cell niche

Abstract

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, a complex process supported by a specialized microenvironment, called the SSC niche. Postnatal development of SSCs is characterized by distinct metabolic transitions from prepubertal to adult stages. An understanding of the niche factors that regulate these maturational events is critical for the clinical application of SSCs in fertility preservation. To investigate the niche maturation events that take place during SSC maturation, we combined different '-omics' technologies. Serial single cell RNA sequencing analysis revealed changes in the transcriptomes indicative of niche maturation that was initiated at 11 years of age in humans and at 8 weeks of age in pigs, as evident by Monocle analysis of Sertoli cells and peritubular myoid cell (PMC) development in humans and Sertoli cell analysis in pigs. Morphological niche maturation was associated with lipid droplet accumulation, a characteristic that was conserved between species. Lipidomic profiling revealed an increase in triglycerides and a decrease in sphingolipids with Sertoli cell maturation in the pig model. Quantitative (phospho-) proteomics analysis detected the activation of distinct pathways with porcine Sertoli cell maturation. We show here that the main aspects of niche maturation coincide with the morphological maturation of SSCs, which is followed by their metabolic maturation. The main aspects are also conserved between the species and can be predicted by changes in the niche lipidome. Overall, this knowledge is pivotal to establishing cell/tissue-based biomarkers that could gauge stem cell maturation to facilitate laboratory techniques that allow for SSC transplantation for restoration of fertility.

Keywords: Sertoli cell maturation; lipid metabolism; lipids; metabolic microenvironment; niche development.

Figure 1.

Global profiling of single-cell transcriptome of human testes and single-cell transcriptome analysis of Sertoli cell lineages. (A) UMAP plot visualization of testicular cells (germ and somatic cells) from combined single-cell RNA sequencing data, displayed by age. (B) UMAP plot visualization of Sertoli cells (n = 7360) distributed into two large, color-coded cell clusters (1 and 2) connected by a small cell cluster (3). (B′) UMAP plot visualization of Sertoli cells from the combined single-cell RNA sequencing dataset split by age. (C) Monocle pseudotime trajectory of Sertoli cells, showing three distinct color-coded states (1, 2, and 3). (C′) Monocle pseudotime trajectory of Sertoli cells, showing the distribution of the three cell states per age. States 1 and 2 are detected in prepubertal stages (1, 2, and 7 years of age), while State 3 is detected in peri/postpubertal stages (11 years of age and older). (D) Focused analysis of Sertoli cells collected from 11-year and older males, representing cells in State 3 (blue cluster in Fig. 1C). The analysis led to the identification of four color-coded distinct states (1–4). (D′) The distribution along the pseudotime trajectory of these four cell states of Sertoli cells by age, with States 3 and 4 mainly detected in 13- to 14-year-old males and adults, respectively. UMAP, uniform manifold approximation projection.

Figure 2.

Pseudotime trajectory analysis of Sertoli cells and peritubular myoid cells from pre- and post-pubertal samples followed by gene enrichment analysis. (A) Heatmap showing the dynamic expression of the top DEG in Sertoli cells (11 years of age and older) between two transition states; 3 and 4 (q value < 1 × 10⁻³) as displayed in Fig. 1 C. Genes are divided into three clusters based on their expression trend along the pseudotime. The color key from blue to red indicates relative expression levels of DEGs from low to high. Pre branch, indicated by State 1, refers to the cells distributed along the trajectory before branch point 2 (shown in Fig. 1D). (B) Functional gene enrichment analysis using Metascape. Heatmap depicting the relative significance of the top enriched hallmarks across gene lists representing the three gene clusters identified by Monocle (Supplementary Fig. S2A). The grey color indicates no enrichment. Scale coloring represents the log P-value for the indicated enriched hallmarks. (C) UMAP plot of PMCs from combined single-cell RNA sequencing data spanning from the prepubertal to the postpubertal stage of testes development (upper panel). Feature plot depicting the expression of ACTA2 in PMCs (bottom panel). (D) Monocle pseudotime trajectory of PMCs showing eight distinct color-coded states (left panel). Monocle pseudotime trajectory of PMCs showing the distribution of these eight cell states per age (right panel). (E) Heatmap showing the dynamic expression of the top DEGs along the pseudotime trajectory (q value < 1 × 10⁻⁵). These genes are divided into four clusters based on their expression trend along the pseudotime. The color key from blue to red indicates relative expression levels of DEGs from low to high. (F) Functional gene enrichment analysis using Metascape. Heatmap depicting the relative significance of the top enriched hallmarks across gene lists representing the four gene clusters identified by Monocle (Fig. 2E). The grey color indicates no enrichment. Scale coloring represents the log P-value for the indicated enriched hallmarks. PMCs, peritubular myoid cells; UMAP, uniform manifold approximation projection; DEG, differentially expressed genes.

Figure 3.

Changes in ultrastructure can be detected at around 7 years of age in humans. (A–L) Human testis from 5 months to 24 years of age showing Sertoli cells (white outlines), seminiferous cords/tubules (black outlines), PMCs (yellow arrowheads), lipid droplets (blue arrows), and Sertoli cell junctions (red arrowheads); scale bar 5 $\mathrm{\mu }$m. BM, basement membrane; PMCs, peritubular myoid cells.

Figure 4.

Development of Sertoli cell junctions during development. (A–H) Human testis from 5 months to 19 years of age, red arrowheads indicate Sertoli cell–cell interactions; scale bar 600 nm. (I–N) Immunohistochemistry of human testis for occludin and AMH; scale bar 10$\mu$m. AMH, anti-Mullerian hormone.

Figure 5.

Ultrastructural changes occurring with maturation of porcine Sertoli cells. (A–G) One-week-old pig testis; (B–H) 8-week-old pig testis. (A, B) HE of 1- and 8-week-old pig testis, showing seminiferous cord (white dotted line), lipid vacuoles (blue arrows); scale bar 10 $\mu$m. (C–H) TEM of 1- and 8-week-old pig testis. (C, D) Overview of the seminiferous cord (black dotted line), lipid droplets (blue arrows); scale bar 10 $\mu$m. (E, F) Sertoli cell nuclei (black outline) within 1- and 8-week-old pig seminiferous cord (black dotted line); scale bar 2 $\mu$m. (G, H) Cell–cell interaction of Sertoli cells in situ. (G) Desmosome-like (Dl) structure. (H) Intermediate filaments (IF) and smooth endoplasmic reticulum (sER) (red arrowheads) adjacent to tight junctions (TJ); scale bar 0.2 $\mu$m. (I) RT-qPCR assessment for maturity and immaturity-related genes, results shown as log 10-fold change relative to earlier Sertoli cell stages (*0.05, **0.01, and ***0.001; P < 0.05, n = 4, Supplementary Table SII). (J) Loss of AMH protein and accumulation of BODIPY⁺ lipid droplets in 8-week-old Sertoli cells; scale bar 100 $\mu$m. (i, iii) One-week-old pig testis, (ii, iv) 8-week-old pig testis. (K) Increase in lipid droplets per Sertoli cell per tubule cross-section (each point is the ratio in 1 cross-section; n = 0.0004, n = 4). (L) Increase in lipid droplet size with maturation (n = 4, P = 0.007). HE, hematoxylin-eosin; TEM, transmission electron microscopy; BODIPY, BODIPY™ 493/503 stain for neutral lipids.

Figure 6.

Changes in the niche lipidome with maturation of pig seminiferous tubules. (A) PCA 2D scores plot with QC injections. (B) Identified and normalized lipid categories, (B′) Identified and normalized lipid subclasses for three main lipid categories. (C) Significantly changed lipids per subclass and age. (D) Heatmap for all samples with the top 50 lipids ranked by P-value. PCA, principal component analysis; 2D, two-dimensional; QC, quality control.

Figure 7.

Upregulation of androgen receptor signaling, cytoskeletal remodeling, and lipid metabolism-associated proteins with maturation of porcine Sertoli cells. (A) String analysis of significantly down- (blue box) and upregulated (red box) proteins and phosphosites; total protein is indicated by a box and a high probability of phosphosite next to protein detected (protein bullets are otherwise randomly distributed and colored); known interactions from curated databases (light blue), experientially detected (pink); predicted interactions from gene neighborhood (green), gene fusions (red), and gene co-expression (blue); and others based on text mining (yellow), co-expression (black), and protein homology (purple). (B) Summary of upregulated and downregulated (transparent) proteins and phosphorylation sites in a cellular context.