UHRF1 interacts with snRNAs and regulates alternative splicing in mouse spermatogonial stem cells

Abstract

Life-long male fertility relies on exquisite homeostasis and the development of spermatogonial stem cells (SSCs); however, the underlying molecular genetic and epigenetic regulation in this equilibrium process remains unclear. Here, we document that UHRF1 interacts with snRNAs to regulate pre-mRNA alternative splicing in SSCs and is required for the homeostasis of SSCs in mice. Genetic deficiency of UHRF1 in mouse prospermatogonia results in gradual loss of spermatogonial stem cells, eventually leading to Sertoli-cell-only syndrome (SCOS) and male infertility. Comparative RNA-seq data provide evidence that Uhrf1 ablation dysregulates previously reported SSC maintenance- and differentiation-related genes. We further found that UHRF1 could act as an alternative RNA splicing regulator and interact with Tle3 transcripts to regulate its splicing event in spermatogonia. Collectively, our data reveal a multifunctional role for UHRF1 in regulating gene expression programs and alternative splicing during SSC homeostasis, which may provide clues for treating human male infertility.

Keywords: Sertoli-cell-only syndrome; UHRF1; alternative splicing; snRNA; spermatogenesis; spermatogonial stem cell.

Graphical abstract

Figure 1

Dynamic expression of UHRF1 in mouse spermatogonia (A and B) Representative co-immunofluorescence images of PLZF (green) and UHRF1(red) on postnatal day 1 (P1) (A) and adult testis (B) sections. Scale bars, 50 μm. (C) Representative immunofluorescence images of seminiferous tubules at P1 stained for GFRα1 (green) and UHRF1(red). Scale bar, 50 μm. (D) Violin plots of published scRNA-seq data showing the gene expression patterns of Uhrf1 and selected spermatogonia marker genes (Plzf and Gfrα1).

Figure 2

Uhrf1 deletion in prospermatogonia results in germ cell degeneration (A) Schematic diagram of the targeting strategy for generating a floxed Uhrf1 allele through homologous recombination in murine embryonic stem cells. Exon 4 will be deleted after Cre-mediated recombination. (B–D) Representative RT-qPCR, western blots, and immunofluorescence depicting the mRNA and protein levels of Uhrf1 in Uhrf1^flox/−;Vasa-Cre mouse testes compared with those of controls. Blots are representative of n = 3 independent experiments. Scale bars, 50 μm. (E) Uhrf1^flox/−;Vasa-Cre, Uhrf1^+/flox;Vasa-Cre, and Uhrf1^flox/flox male and female mice were mated with more than three pairs to perform fertility tests. Mean litter sizes are shown with the indicated genotypes. (F) Testis growth curve indicates that the Uhrf1^flox/−;Vasa-Cre testes were significantly decreased from P5. Data are presented as the mean ± SEM; n = 3; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (G) Periodic acid-Schiff (PAS) staining of adult testis and epididymis (cauda and corpus) sections from Uhrf1^flox/−;Vasa-Cre and control mice. Scale bars, 50 μm.

Figure 3

Critical role of UHRF1 in spermatogonial differentiation (A) Co-immunofluorescence staining for γ-H2AX (red) and STRA8 (green) on control and Uhrf1^flox/−;Vasa-Cre testis sections at P5, P7, P12, and P15. Scale bar, 50 μm. White dashed lines indicate the single seminiferous tubule. (B and C) Quantification of STRA8-positive cells (B) and γ-H2AX-positive cells (C) per cross section in (A). Data are presented as the mean ± SEM; n = 3; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (D) The co-immunofluorescence staining of γ-H2AX (red) and STRA8 (green) on control and Uhrf1^flox/−;Vasa-Cre testis sections at P15. The asterisks (^∗) mark tubules that lack γ-H2AX and STRA8 signals. Scale bar, 50 μm. (E) Quantification of the percentage of STRA8- or γ-H2AX-negative tubules for (D) is shown. Data are presented as the mean ± SEM; n = 3.

Figure 4

UHRF1 is essential for maintenance of undifferentiated spermatogonia (A) Co-immunofluorescence staining for DDX4 (red) and PLZF (green) on control and Uhrf1^flox/−;Vasa-Cre testis sections at P5, P7, P12, and P15. Scale bars, 50 μm. (B and C) Quantification of PLZF-positive cells (B) and DDX4-positive cells (C) per tubule for (A). Data are presented as the mean ± SEM; n = 3; ^∗∗p < 0.01, ^∗∗∗p < 0.001. (D) Co-immunofluorescence staining for DDX4 on control and Uhrf1^flox/−;Vasa-Cre testis sections at P1 and P3. Scale bars, 50 μm. White dashed lines indicate the single seminiferous tubule. (E) Histogram showing the RNA-seq results of selected transcripts (log₂-fold change) associated with SSC maintenance and differentiation. (F) Gene ontology of downregulated genes in P7 Uhrf1^flox/−;Vasa-Cre testes.

Figure 5

UHRF1 interacts with snRNA and spermatogonial maintenance-associated transcripts (A) The schematic diagram shows the isolation of SSCs via MACS (magnetic-activated cell sorting) and the identification of interacting proteins. (B) Gene ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of UHRF1-bound proteins in isolated mouse SSCs are shown. BP, biological process; MF, molecular function; CC, cellular component. (C) Gene ontology analysis of 116 RNA splicing-related proteins identified by UHRF1 IP-MS. The p value was obtained from the data by GOrilla analysis. (D and E) Anti-UHRF1 RNA immunoprecipitation (RIP) coupled with qPCR (D) and PCR (E) assays showed significant U1, U2, and U4 snRNA enrichment but not that of U5 and U6 snRNAs. (F and G) Anti-UHRF1 RIP coupled with qPCR (F) and PCR (G) showed UHRF1 binding to Foxo1, Bcl6b, and Fgfr3 transcripts in mouse testis.

Figure 6

UHRF1 regulates alternative splicing in spermatogonia (A) The differential alternative splicing events identified in Uhrf1^flox/−;Vasa-Cre testes at P7 are summarized. The numbers of individual alternative splicing events in each category upon UHRF1 ablation are indicated. (B) Visualization of differential splicing analysis of RNA-sequencing data comparing control and Uhrf1^flox/−;Vasa-Cre testes at P7. (Left) Representative examples of a skipped exon (SE) in Apbb1, Senp7, Tle3, and Zfp808. (Right) RT-PCR validation of SEs by ethidium bromide-stained agarose gels. (C) Bar chart shows the quantification of percentage spliced in (PSI); ^∗p < 0.05, ^∗∗p < 0.01. (D) RNA immunoprecipitation (RIP) using UHRF1 antibody followed by PCR and gel electrophoresis for differentially spliced candidates (Apbb1, Senp7, Tle3, and Zfp808). (E) A schematic model of UHRF1-mediated gene regulation in spermatogonia is shown. UHRF1 plays a critical role in post-transcriptional gene regulation. UHRF1 contributes to the fidelity of mRNA splicing in mouse spermatogonia.