Characterization of germ cell differentiation in the male mouse through single-cell RNA sequencing

Abstract

Spermatogenesis in the mouse has been extensively studied for decades. Previous methods, such as histological staining or bulk transcriptome analysis, either lacked resolution at the single-cell level or were focused on a very narrowly defined set of factors. Here, we present the first comprehensive, unbiased single-cell transcriptomic view of mouse spermatogenesis. Our single-cell RNA-seq (scRNA-seq) data on over 2,500 cells from the mouse testis improves upon stage marker detection and validation, capturing the continuity of differentiation rather than artificially chosen stages. scRNA-seq also enables the analysis of rare cell populations masked in bulk sequencing data and reveals new insights into the regulation of sex chromosomes during spermatogenesis. Our data provide the basis for further studies in the field, for the first time providing a high-resolution reference of transcriptional processes during mouse spermatogenesis.

Figure 1

(a and b) T-SNE projection of cells from both replicates, colored by cluster identity obtained from (a) graph-based and (b) K-means clustering with K = 9. Cell types were assigned through the expression of representative marker genes (Supplementary data Table S3, subfigure c) (c). Dot plot of proportion of cells in the respective K-means cluster expressing each marker (dot size), and average expression (color scale). Spg = spermatogonia, SC = spermatocytes, RS = round spermatids, ES = elongating spermatids, CS = condensing spermatids.

Figure 2

(a) SOM portrait indicating metagenes overexpressed at any stage during germ cell differentiation. Examples for gene sets highly enriched in the corresponding metagenes are indicated at the sides of the SOM. (b and c) Correlation spanning tree based on metagene expression with pseudotime (b) or K-means clustering and (c) color coding. (d) SOM portraits for the different K-means clusters indicated in (c), ordered by pseudotime (b). The positioning of the metagenes is identical to that in (a).

Figure 3

Read counts for autosomes and sex chromosomes, centered on the mean for the respective chromosome. Individual cells are represented as circles, with smoothed local regression (Loess) represented as solid lines. Shaded areas indicate the standard error of the regression fit. Meiosis occurs roughly between the 0.925 and the 0.95 mark, while Prm1 transcription and thus histone-to-protamine exchange is initiated at approximately 0.986. The fastest decay of the respective transcripts could be observed at 0.991 (Y chromosome) and 0.992 (X chromosome and autosomes).

Figure 4

(a) Expression values of Pou5f1 and Kit along the pseudotime axis. The black line denotes the smoothed, average expression. (b and c) t-SNE projections with colors indicating the expression of Pou5f1 (b) and Kit (c).