CDK2 kinase activity is a regulator of male germ cell fate

Abstract

The ability of men to remain fertile throughout their lives depends upon establishment of a spermatogonial stem cell (SSC) pool from gonocyte progenitors, and thereafter balancing SSC renewal versus terminal differentiation. Here, we report that precise regulation of the cell cycle is crucial for this balance. Whereas cyclin-dependent kinase 2 (Cdk2) is not necessary for mouse viability or gametogenesis stages prior to meiotic prophase I, mice bearing a deregulated allele (Cdk2^Y15S ) are severely deficient in spermatogonial differentiation. This allele disrupts an inhibitory phosphorylation site (Tyr15) for the kinase WEE1. Remarkably, Cdk2^Y15S/Y15S mice possess abnormal clusters of mitotically active SSC-like cells, but these are eventually removed by apoptosis after failing to differentiate properly. Analyses of lineage markers, germ cell proliferation over time, and single cell RNA-seq data revealed delayed and defective differentiation of gonocytes into SSCs. Biochemical and genetic data demonstrated that Cdk2^Y15S is a gain-of-function allele causing elevated kinase activity, which underlies these differentiation defects. Our results demonstrate that precise regulation of CDK2 kinase activity in male germ cell development is crucial for the gonocyte-to-spermatogonia transition and long-term spermatogenic homeostasis.

Keywords: Cell cycle; Gonocytes; Mouse; Spermatogonia.

Fig. 1.

Cdk2^Y15S/Y15S mice are born with a normal germ cell complement, but exhibit defective spermatogonial differentiation. (A) IHC of newborn testis sections showing germ cells labeled with the germ cell marker MVH. (B) Quantification of the data shown in A. ns, not significant; YS, Cdk2^Y15S. (C) IHC on adult testis sections labeled with the spermatogonia marker LIN28 and DNA-binding dye DAPI. Scale bars: 50 μm (A); 100 μm (C).

Fig. 2.

Cdk2^Y15S/Y15S mice have a defective gonocyte-to-spermatogonia transition. (A) Immunolabeling of testis histological sections. Arrows and arrowheads indicate germ cells with cytoplasmic or nuclear FOXO1, respectively. The locations of some seminiferous tubule boundaries are indicated by dashed lines. (B) Percentage of cytoplasmic FOXO1 in P30 and P90 tubule sections. YS, Cdk2^Y15S/Y15S. Scale bar: 50 µm.

Fig. 3.

Cdk2^Y15S/Y15S seminiferous tubules contain increased undifferentiated spermatogonia. (A,B) Confocal images of P15 seminiferous tubules showing the density of GFRA1⁺ spermatogonia (A) and of P90 tubules immunolabeled for markers of undifferentiated spermatogonia (B). The outermost two or three optical sections are projected in A and B. Dotted lines indicate the edges of a tubule. Scale bars: 50 μm (A); 200 μm (B). (C,D) Density of total (C) and A_s, A_pr and A_cluster (D) GFRA1⁺ spermatogonia in >8-mm-long tubule segments. n≥3 for each genotype (except n=2 for P90 WT). P-values: a<0.0001, b=0.0001, c=0.0006, d=0.0002, e=0.0039. Data are mean±s.d. (E) Distribution of GFRA1⁺ subpopulations. cl, cluster. (F) Quantification of A_s spermatogonia subtypes. YS, Cdk2^Y15S/Y15S. Two or more tubules (6-8 mm length) from three different animals were quantified. Boxes represent minimum and maximum values, and horizontal lines represent median values.

Fig. 4.

Mutation of the CDK2^Tyr15 phosphorylation site impedes differentiation of GFRA1⁺ A_s spermatogonia. (A) Seminiferous tubule (P15) whole mounts showing abnormally long clusters of GFRA1⁺ and EdU⁺ spermatogonia. A_cl, A_cluster. (B) DNA replication in FOXO1⁺ germ cells. Arrowheads indicate examples of double-positive cells. FOXO1 is mostly nuclear in WT, and cytoplasmic in mutants (insets). (C) Quantification of the data shown in A (mean±s.d.). (D) Quantification of replicating spermatogonial subtypes (mean±s.d., n>3 for each group). Boxes represent values in the 10-90 percentiles, and horizontal lines represent median values. (E) Replicating FOXO1⁺ cells at P90. Over 100 and 55 tubules were analyzed from three different animals of each. Error bars represent s.d. (F) Quantification of replicating LIN28⁺ spermatogonia. Error bars represent s.d. YS, Cdk2^Y15S/Y15S. Scale bars: 50 µm (A,B); 25 μm (inset in B).

Fig. 5.

Single cell RNA sequencing of P3.5 testes reveals abnormal differentiation of Cdk2^Y15S/Y15S gonocytes. (A) A t-SNE (t-distributed stochastic neighbor analysis) plot of 14,422 testicular cells with K-means clustering. Cell types were classified by expression of key marker genes (e.g. Myh11 for myoid cells). Inset shows MVH⁺ germ cells (263 in total; WT=106, Cdk2^Y15S/Y15S=99, Cdk2^Y15S/+=58), color coded by genotype. YS, Cdk2^Y15S. (B) Identification of germ cell clusters and distribution by genotype. In panels B-D, the WT and Cdk2^Y15S/Y15S germ cells shown in A were filtered for those that had >10,000 UMIs/cell, leaving in an expression matrix of 10,451 genes across 141 cells (WT=69; Cdk2^Y15S/Y15S=72). 734 highly variably expressed genes were used to identify five distinct clusters, called A-E. (C) Violin plots showing log2-transformed reads per million (RPM) of key genes in cells from each of the five clusters (see color-coded legend) defined in panel B. (D) Pseudotime trajectory analysis plots, showing transitions from one cell state to another, as predicted based on expression of the aforementioned 734 most variable genes. The cells are colored by genotype or clusters A-E. The black circles represent branch nodes as predicted by Monacle. (E) RNA velocity plotted in tSNE space. Shaded circles represent cells. Arrows indicate their estimated differentiation trends. The lower left area is enriched for cells moving towards a WT cluster A identity, and the upper right towards mutant (clusters C-E). (F) Schematic summary of cell states and transitions. The germ cell clusters identified in panel B can be classified into three states: normal SSCs (cluster A), intermediate (clusters B and C) and mutant (clusters D and E). However, based on pseudotime and RNA velocity analyses, the WT cells in clusters B and C (blue) are predisposed to differentiate towards normal SSCs (cluster A; green), and the mutant cells towards abnormal gonocytes (clusters D and E; red). Ab. gono, abnormal gonocytes; Gono, gonocytes; YS, Cdk2^Y15S/Y15S.

Fig. 6.

Downstream transcriptional effects of CDK2^Y15S. (A) Heat map showing expression of CDK2 activity signature genes (McCurdy et al., 2017) in indicated clusters of WT and Cdk2^Y15S/Y15S germ cells. In A and E, color key shows log₂-transformed RPM expression. (B) GSEA enrichment plots for CDK2 activity signature genes. (C) CDK2 activity scores defined as median of normalized expression of CDK2 target genes per cell, across indicated clusters. (Student's t-test P-values: a=5.0×10⁻¹¹; b=4.5×10⁻¹²; c=5.7×10⁻¹⁹; d=1.2×10⁻¹¹). YS, Cdk2^Y15S/Y15S. (D) Enrichment analysis for GO-slim ‘biological process’ terms in genes upregulated in clusters A versus E. (E) Heat map showing expression of FOXO1 targets in indicated germ cell clusters and genotypes. (F,G) GSEA enrichment score plots for E2F and FOXO1 target-genes (MSigDb) in clusters A versus E. (H) FOXO1 activity defined as median of z-score normalized expression of FOXO1 target genes per cell (Student's t-test P-values: e=8.9×10⁻⁵; f=6.2×10⁻⁵; g=1.5×10⁻¹³; h=3.7×10⁻¹⁹). In C and H, boxes represent data points between the first and third quartiles of the distribution, whiskers depict minimum and maximum values, with outliers shown as black dots, and black horizontal lines indicate the median value.

Fig. 7.

Phenotypic effects of CDK2 Tyr15 and Thr160 phosphorylation during spermatogenesis. (A,B) Images (A) and weights (B) of P120 testes. (C) Sperm counts of age-matched genotypes at P120. (D) Hematoxylin & Eosin-stained testis cross-sections from adult animals. Abnormal degenerating cells, possibly multinucleate, are indicated by the arrowheads. Scale bar: 100 μm. YS, Cdk2^Y15S; TA, Cdk2^T160A. −/− corresponds to a putative null allele of Cdk2 generated by CRISPR mutagenesis as described in Materials and Methods. The formal name is Cdk2^em3Jcs.

Fig. 8.

Model for regulation of SSC differentiation by CDK2 phosphorylation.