Understanding testicular single cell transcriptional atlas: from developmental complications to male infertility

Abstract

Spermatogenesis is a multi-step biological process where mitotically active diploid (2n) spermatogonia differentiate into haploid (n) spermatozoa via regulated meiotic programming. The alarming rise in male infertility has become a global concern during the past decade thereby demanding an extensive profiling of testicular gene expression. Advancements in Next-Generation Sequencing (NGS) technologies have revolutionized our empathy towards complex biological events including spermatogenesis. However, despite multiple attempts made in the past to reveal the testicular transcriptional signature(s) either with bulk tissues or at the single-cell, level, comprehensive reviews on testicular transcriptomics and associated disorders are limited. Notably, technologies explicating the genome-wide gene expression patterns during various stages of spermatogenic progression provide the dynamic molecular landscape of testicular transcription. Our review discusses the advantages of single-cell RNA-sequencing (Sc-RNA-seq) over bulk RNA-seq concerning testicular tissues. Additionally, we highlight the cellular heterogeneity, spatial transcriptomics, dynamic gene expression and cell-to-cell interactions with distinct cell populations within the testes including germ cells (Gc), Sertoli cells (Sc), Peritubular cells (PTc), Leydig cells (Lc), etc. Furthermore, we provide a summary of key finding of single-cell transcriptomic studies that have shed light on developmental mechanisms implicated in testicular disorders and male infertility. These insights emphasize the pivotal roles of Sc-RNA-seq in advancing our knowledge regarding testicular transcriptional landscape and may serve as a potential resource to formulate future clinical interventions for male reproductive health.

Keywords: male infertility; single-cell RNA-sequencing (Sc-RNA-seq); spatial transcriptomics; spermatogenesis; testis.

Figure 1

Testicular tissue architecture and Spermatogenic Development. (A) Testis Anatomy: Cross-section illustrating the testis with internal cellular composition and associated structures: the epididymis and vas deferens. (B) Seminiferous Tubule: Histological image showing the transverse section of a seminiferous tubule where spermatogenesis occurs. (C) Spermatogenesis Overview: Schematic representation of spermatogenesis within the seminiferous tubule, from spermatogonial stem cells (SSC) to mature spermatozoa. (D) Sperm Maturation: Detailed progression of sperm development from type A spermatogonium through meiosis to form mature sperm. (E) Human vs. Rodent Spermatogenesis: Comparative stages of spermatogenesis between humans and rodents, highlighting the differentiation of spermatogonial cells. (F) Sperm Cell Morphology: Detailed diagram of a motile sperm cell, showcasing its various structural components from head to tail.

Figure 2

Illustrating fundamental difference between bulk and single-cell RNA sequencing (Sc-RNA-seq). (A) Bulk Processing: The left diagram illustrates the enzymatic and mechanical dissociation of testicular tissue into a heterogeneous mixture of cells including Sertoli cells, Leydig cells, spermatogonia, spermatocytes, spermatids etc. Following dissociation, cells are processed in bulk, as shown by the central test tube graphic, including DNA barcoding and sequencing to yield average gene expression profiles. (B) Single Cell-Suspension and Enrichment: The top middle flow diagram transitions from an unselected testicular cell suspension to cell type enrichment via methods like transgenic reporters (e.g., GFP) and antibody labeling etc. The bottom middle panel depicts the conversion of the unselected cellular mixture into a single-cell suspension, followed by cell sorting based on type-specific markers, with each cell type represented by a different color. The right side shows the cell clustering outcome, visualized by a t-SNE plot, and the subsequent sequencing of individual cells resulting in a detailed gene expression heatmap, with colors indicating expression levels from high (red) to low (green).

Figure 3

Comprehensive platforms of Single-Cell Transcriptomics. (A) 10× Genomics Chromium: The workflow initiates with encapsulation of individual cells with barcoded primer gel beads into oil droplets to generate Gel bead-in-emulsions (GEMs), ready for collecting and processing. Subsequent steps include sequencing, which generates data for proportion of cell type bar charts, a volcano plot showing differential gene expression, and a violin plot depicting quantification of average gene expression across cell types. (B) Fluigidm C1: Individual cells are physically captured on microfluidic chips. (C) Data Integration and Analysis: The t-SNE plot illustrates cell cycle analysis of individual cells. UMAP with pseudotime calculation provides insights into the developmental trajectory of the cells. The chord diagram highlights cell-to-cell interaction analysis, elucidating the complex interplay between different cell types.

Figure 4

Molecular Profiling of Spatial transcriptomics. (A) In situ Capture: Cells are captured on a chip with poly-T barcoded beads, allowing for mRNA binding and subsequent steps of RNA extraction, cDNA synthesis, and library preparation, followed by sequencing. (B) Laser Capture Micro-dissection: This panel demonstrates the isolation of specific cells or tissue regions using a laser beam, with subsequent lysis, RNA extraction, reverse transcription (RT), and library preparation for sequencing and alignment. (C) In situ Sequencing: Rolling amplification is performed on tissue sections, followed by sequencing, imaging, and alignment. The heatmap represents gene expression levels in specific spots or cells, which is used for reconstitution, mapping, and visualization of cell types and states. (D) In situ Hybridization: Hybridization probes target specific RNA in tissue sections, followed by decoding through microscopy, which leads to the identification of mRNA transcripts and allows for 3D reconstruction of spatial architecture within the tissue.

Figure 5

Single-cell analysis of spermatogenesis reveals developmental trajectories and gene expression profiles. (A) UMAP visualization of cell clusters from testicular single-cell RNA sequencing: Cell Differentiation Trajectories: [i] Spermatogonia clusters 1-4 (Progenitor to Late Differentiated), [ii]. Spermatocytes stages 5-8 (Preleptotene to Diplotene), [iii]. Spermatids 9-12 (Early to Late Round), [iv]. Adult Testis Cells 13-16 (Undifferentiated to Perivascular). (B) Gene Expression Markers throughout spermatogenic progression: Sequential expression from self-renewal (GFRAL) to spermiogenesis (PRM1).

Figure 6

Gene expression profiles during human spermatogenesis. (A) Gene expression heatmap of spermatogonia: The heatmap illustrates the gene expression levels at different steps of spermatogonial development, from pre-meiotic to SSCs. (B) Gene expression heatmap of spermatocytes: Displays the gene expression patterns during the meiotic stages (spermatocytes, from pre-leptotene to post-meiotic). (C) Gene expression heatmap of spermatids: Depicts variations of gene expression in spermatids, highlighting the transition from early to late spermatid stages. (D) Comparative expression heatmap of adult human testis: Compares gene expression across different cell types and stages of sperm development, including leptotene and zygotene spermatocytes and diplotene to secondary spermatocytes.

Figure 7

Single-cell transcriptomic analysis of testicular cells at different developmental age groups. (A) UMAP visualizations representing the cellular composition of testicular cells at (i) embryonic days 18.5 (E18.5), (ii) postnatal days 2 (P2D), and (iii) postnatal days 7 (P7D). Each dot represents a single cell, color-coded by identified cell types: stroma (cyan), Leydig cells (yellow), Sertoli cells (dark blue), endothelial cells (green), innate lymphoid cells (purple), peritubular myoid cells (red), macrophages (light blue), and germ cells (orange). (B) UMAP plots displaying cell cycle phases for testicular cells on E18.5, P2D, and P7D. Cells are color-coded according to their phase in the cell cycle: G1 (blue), S (red), G2/M (green), and cells not in cycle (black). Arrows indicate the direction of the developmental trajectory. (C) Heatmaps illustrating the expression profiles of selected marker genes across different cell types on E18.5, P2D, and P7D. Gene expression levels are represented by a gradient from low (blue) to high (red) expression. Beneath each UMAP plot in B, stacked bar charts show the proportion of cells in each cell cycle phase for two replicates at each developmental stage (EMB1, EMB2, P2D1, P2D2, P7D1, P7D2).

Figure 8

Trends in publication on testicular Sc-RNA-seq studies. The bar graph illustrates the number of articles published by year, from 2015 to 2024 available at PubMed. A noticeable peak is observed in 2022, followed by a decline in subsequent years. The x-axis represents the years, while the y-axis quantifies the number of articles published. The data suggests a period of heightened research activity in 2022, with a gradual decrease in the volume of publications thereafter.

Figure 9

Overview of application of Sc-RNA-seq data. (A) Single-cell RNA sequencing approach: Depicts the workflow from human testicular tissue sample to single-cell suspension, followed by library preparation for RNA sequencing, cell partitioning, and subsequent cell-type identification using clustering analysis. (B) Preclinical study: Represents the preclinical research progression, beginning with in vitro experiments, advancing through in vivo studies in rodents and large animals, and culminating in analysis within a human population. (C) Diagnostic and prognostic study: Illustrates the process of collecting blood and semen samples, performing in vitro assays, utilizing various detection methods, and integrating findings with medical records for the advancement of healthy male reproduction diagnostics and prognostics.