Fig 2: DGCR8-knockdown alters global transcriptome of bovine embryos. (A) PCA graph of all 24 libraries. Samples group by day they were collected, although day 5 DGCR8-KD embryos tend to separate from day 5 control and control-injected embryos. PCA key (Top Left) indicates which color represents which treatment and which circle type represents which day samples were collected. (B) Venn diagram identifying overlap of differentially expressed genes in control-injected versus control embryos (yellow) and DGCR8-KD embryos versus both control groups combined (blue) on day 3 (Upper) and day 5 (Lower) of development. (C) Volcano plot of differentially expressed genes in day 3 DGCR8-KD embryos compared with controls. Each point represents a single gene, and points above the red line were considered significant (FDR P < 0.05). (D) Volcano plot of differentially expressed genes in day 5 DGCR8-KD embryos compared with controls. Each point represents a single gene and points above the red line were considered significant (FDR P < 0.05). Four biological replicates were used for developmental stage, and red stars represent notable genes listed to the left. (E) Relative SOX2 mRNA levels measured by qPCR on day 5 of embryo development in control embryos (CO), control-injected embryos (CI), and embryos injected with DGCR8-targeting siRNA and MO (KD). Three biological replicates were used with 20 embryos each. (F) Relative SOX2 protein by immunocytochemistry in CO, CI, and KD embryos. Representative images (40X magnification) (Left) and relative fluorescent intensity (Right). Three biological replicates were used with 10 embryos each. *P < 0.05.

Fig 3: 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 4: Loss of embryonically expressed canonical miRNAs causes developmental arrest at morula stage. (A) Percentage of embryos that developed to blastocyst in control embryos (CO), control-injected embryos (CI), embryos injected with DGCR8-targeting siRNA and MO (KD), embryos injected with DGCR8-targeting siRNA only (siRNA only), and embryos injected with DGCR8-targeting MO only (MO only). Eleven biological replicates were used, with at least 50 embryos per group. Data are presented as mean ± SEM *P < 0.01, **P < 0.00001. (B) Time-lapse data of control embryos, control-injected embryos, or DGCR8-KD embryos cultured in Esco Medical Miri time-lapse incubator for 8 d after IVF. Percentage of embryos that cleaved to each stage of the total embryos cultured. Average time of cleavage of the embryos that developed to that particular stage presented as mean ± SEM. (C) Time-lapse data of control embryos, control-injected embryos, or DGCR8-KD embryos that formed a morula by the end of day 5 cultured in Esco Medical Miri time-lapse incubator for 8 d after IVF. Colors indicate cleavage stage. Change of colors from one to the next indicates cleavage. The x axis is time in hours after IVF. Data were collected during six time-lapse runs.

Fig 5: Venn diagram analysis of gene expression in day 3 DGCR8-KD embryos. Venn diagram representing overlap of up-regulated genes in day 3 DGCR8-KD embryos (red) with up-regulated genes in day 3 a-amanitin–treated embryos compared with controls (blue), down-regulated genes in day 3 embryos compared with MII oocytes (yellow), day 3 a-amanitin–sensitive genes (green), and up-regulated genes in day 3 embryos compared with MII oocytes (purple). Overlap with up-regulated genes in day 3 embryos compared with MII oocytes and down-regulated genes in day 3 a-amanitin compared with controls indicates “embryonic” origin, while overlap with down-regulated genes in day 3 embryos compared with MII oocytes and up-regulated genes in day 3 a-amanitin compared with controls indicates “maternal” origin. See Bogliotti et al. (40).