Fig 2: DAZL co-localizes with DICER and co-immunoprecipitates with pre-miRNA. (A) Immunostaining of DAZL and DGCR8, and DAZL and DICER of 14-week-old human fetal ovary. Scale bar, 5 µm. (B) Scatterplots of DAZL (green) versus DGCR8 or DICER staining intensity (red) in the merged images of (A). (C) PCC analysis of the co-localization of DAZL-positive staining with DGCR8 or DICER staining. Ten merged regions were quantified, and each contains >30 cells. (D) High resolution 3D imaging of DAZL and DICER immunostaining of 12-week-old human fetal ovary. Scale bar, 5 µm. (E) Schematic diagram showing qPCR analysis of primary and precursor/primary miRNAs. The pink arrow represents the forward primer of primary miRNA, the yellow arrow represents the forward primer of pri-/pre-miRNA, the red arrow represents the reverse primer of primary and pre-miRNA. (F) Relative expression of U6, pri-/precursor and precursor let-7c in the V5-DAZL group (oeDAZL) and DAZL group (CTRL) before and after RIP. Data shown are the means ± standard deviation (SD), n = 3. n.s., no significance. **P <0.01 (Student's t-test, two-sided/unpaired). (G) Heatmap analysis of pre-miRNA expression in CTRL versus shDAZL hPGCLCs. Two sets of shDAZL (shDAZL4 and shDAZL6) were included. The representative up-regulated, down-regulated and unchanged miRNAs were selected based on the results in Figure 1E. Duplicates of each group are shown.

Fig 3: DAZL up-regulates mature miRNA levels in hPGCs. (A) FISH/immunofluorescence of let-7a-5p and DAZL, let-7a-5p and LIN28A of 16-week-old human fetal ovary. Co-staining of let-7a-5p FISH (green) and DAZL immunofluorescence (red) in human fetal ovary. The white arrow indicates DAZL- or LIN28A-positive cells and the arrowhead indicates let-7a-5p-positive cells. Scale bar, 50 µm. (B) Scatterplots of let-7a-5p staining intensity (green) versus DAZL or LIN28A staining intensity (red) in the merged images of (A). (C) PCC analysis of the co-localization of let-7a-5p-positive staining with DAZL or LIN28A stainings. Five merged regions were quantified; each contains ~100 cells. (D) FACS analysis of cells overexpressing DAZL-P2A–eGFP or eGFP (CTRL). Gating represents the GFP+ population and its percentage. APC is an irrelevant channel for detecting the level of autofluorescence. (E) Differentially expressed mature miRNAs after DAZL overexpression (oeDAZL) compared with the control group (CTRL). # represents different biological repeats. (F) Let-7 family members among up-regulated miRNAs in (E). (G) Heatmap analysis of mature miRNA expression in control versus shDAZL hPGCLCs. The representative up-regulated, down-regulated and unchanged miRNAs were selected based on the results in (E). Duplicates of each group are shown. Two sets of shDAZLs (shDAZL4 and shDAZL6) were included. (H) Gene Ontology analysis of biological process enrichment of predicted targets of the DAZL up-regulated miRNAs.

Fig 4: Aberrant germ cell meiosis was observed in Prmt5?/f; Stra8-Cre mice.The germ cells were labeled with Dazl (green) and meiotic germ cells were labeled with Stra8 (red) and Scp3 (red). In control testes, Stra8-positive (A, asterisks) and Scp3-positive (I, asterisks) germ cells were only detected in a small portion of seminiferous tubules at P10. Stra8-positive (C, asterisks) and Scp3-positive (K, asterisks) germ cells were also observed in seminiferous tubules of Prmt5?/f; Stra8-Cre mice at P10, and no statistical difference was noted compared to control mice (R). Stra8-positive (E, asterisks; F, white arrowheads) and Scp3-positive (M, asterisks; N, white arrowheads) germ cells were detected in most of seminiferous tubules of control testes at P12. The number of tubules with Stra8-positive (G) and Scp3-positive (O) germ cells was significantly reduced in Prmt5?/f; Stra8-Cre testes at P12 (S).

Fig 5: Identification of DAZL-bound transcripts in undifferentiated spermatogonia via iCLIP reveals DAZL’s regulation of spermatogonial factors.(A) Schematic of the synchronization of spermatogenesis to obtain undifferentiated spermatogonia via the 2S method for iCLIP (green arrows) and via the 3S method for RNA-seq (purple arrows). WIN 18,446 was used to synchronize spermatogenesis by blocking spermatogonial differentiation, and samples were histologically staged to verify successful synchronization. For 3S samples, germ cells were sorted from synchronized testes. (B) Genomic distribution of DAZL iCLIP peaks identified in three biological replicates (TPM =1; FDR < 0.05). (C) Venn diagram showing overlap of DAZL iCLIP peaks in expressed 3' UTRs (TPM =1) among three biological replicates. Replicated peaks (i.e., present in at least two of three replicates) were identified. After merging replicated peaks that fell on consecutive nucleotides, 11,882 DAZL binding sites (present in at least two of three replicates; highlighted in blue) were identified. These binding sites correspond to 2,633 genes, which are designated as the DAZL-bound genes. (D) Enrichment of factors that regulate the development and differentiation of undifferentiated spermatogonia in DAZL targets compared with all genes expressed in undifferentiated spermatogonia (one-tailed hypergeometric test). Number of genes (n) in each group designated in parentheses in labels along x-axis.****, p<0.0001. (E) DAZL iCLIP, IgG iCLIP, and 3S RNA-seq gene tracks showing exemplary DAZL-bound genes that are required for spermatogonial proliferation and expansion (Lin28a and Sox3) or differentiation (Kit, Foxo1, Rarg, and Sall4). Each iCLIP track represents the crosslinked sites from the sum of unique reads from three biological replicates. The RNA-seq track represents the sum of two biological replicates. The scale of each gene track is marked on the left. 3' UTRs are in light blue.Figure 2—source data 1.Genes that regulate development and differentiation in undifferentiated spermatogonia.Figure 2—source data 2.Replicated DAZL iCLIP peaks and transcript expression levels in 3S undifferentiated spermatogonia.Figure 2—source data 3.Comparison of 3S undifferentiated spermatogonia to previously published spermatogonial datasets.