Genomic location, sequence alignment and evolution conservation of the X-linked miR-506 family.

A. Genomic location of the X-linked miR-506 family miRNAs (black bars) and the two flanking coding genes, Slitrk2 (green blocks) and Fmr1 (red blocks). The number of miRNAs within each cluster is indicated underneath the miRNA clusters.

B. Evolution conservation of X-linked miR-506 family based on Multiz Alignment & Conservation using the human genome as a reference. Positive PhyloP scores indicate conservation and vice versa. PhastCons has a score between 0∼1, and the higher the score, the more conserved the DNA region is. C-D. Comparison of mean phyloP (C) phastCons (D) scores among CDS of Slitrk2 and Fmr1, intergenic region (IGR), pachytene piRNAs, SmiR, FmiR, and all miRNAs. **, ***, **** indicate adjusted p-value < 0.01, 0.001, and 0.0001, respectively. ns, not significant. Kruskal-Wallis test was used for statistical analyses. E-F. Comparison of derived allele (E) and mean nucleotide (F) frequencies among pachytene piRNAs, SmiR, FmiR, and all miRNAs. *, ** and **** indicate adjusted p-value < 0.05, 0.01 and 0.0001, respectively. ns, not significant. Kruskal-Wallis test was used for statistical analyses.

Evolutionary history of the X-linked miR-506 family.

A. Sequences alignment of FmiRs from various species using human MER91C DNA transposon as the reference. The first line is the human MER91C DNA transposon, and below are the miRNAs of various species. Mismatched nucleotides are highlighted with various colors. B. A phylogenetic tree of the MER91C DNA transposons and the X-linked miR-506 family miRNAs. The MER91C DNA transposons are labeled in purple. C. RNA structure of the MER91C DNA transposon-derived miRNA (hsa-miR-513c). D. sRNA-seq reads (lower panel) of the MER91C DNA transposon-derived miRNA, hsa-miR-513c. E. Representative gel images showing expression levels of the MER91C DNA transposon-derived miRNA (hsa-miR-513a1) in HEK293T cells. The asterisk (*) indicates the expected miRNA size. U6 was used as the loading control. F. qPCR analyses of expression levels of MER91C DNA transposon-derived miRNA (hsa-miR-513a1) in HEK293T cells. *, **, **** indicate adjusted p-value < 0.05, 0.01, 0.0001, respectively. One-way ANOVA was used for statistical analyses.

Expression profiles of X-linked miR-506 family in mammalian testes and male germ cells.

A. Heatmaps showing the miR-506 family expression in the testis, pachytene spermatocytes, round spermatids and sperm in mice. Biological triplicates of the testis samples (n=3) and duplicates of pachytene spermatocytes, round spermatids and sperm samples isolated from 2-4 mice were used for sRNA-seq. *, **, ***, and **** indicate FDR < 0.05, 0.01, 0.001, and 0.0001, respectively, when comparing round spermatids to pachytene spermatocytes. #, ##, ###, and #### indicate FDR < 0.05, 0.01, 0.001, and 0.0001, respectively, when comparing cauda sperm to pachytene spermatocytes. ns and na indicate not significantly and not not applicable, respectively.B-C. LogCPM bar graphs showing the miR-506 family expression in the testis of humans (B) and marmosets (C). D. LogCPM bar graph showing the miR-506 family expression in sexually immature and mature horse testes. n=3. *, **, ***, and **** indicate FDR < 0.05, 0.01, 0.001, and 0.0001, respectively.

Ablation of X-linked miR-506 family miRNAs compromised sperm competitiveness and reproductive fitness in male mice.

A. Schematics showing the strategy used to generate six lines of KO mice lacking individual or combined miRNA clusters within the miR-506 family using CRISPR-Cas9. B-C. Litter size (B) and litter interval (C) of six miR-506 KO lines, at least 10 litters from 3 different breeding pairs for each KO line were counted. Dunnett’s multiple comparisons test as the posthoc test following one-way ANOVA was used for the statistical analysis. ns, not significant. * and **indicate adjusted p-value <0.05 and 0.01, respectively. D-F. Analyses of testis weight (D), sperm counts (E) and sperm motility (F) in four miR-506 KO lines. n ≥ 3 and Dunnett’s multiple comparisons test as the posthoc test following one-way ANOVA was used for the statistical analysis. G. Testicular histology of WT and four miR-506 KO lines showing largely normal spermatogenesis. Scale bars = 50 µm. H. Representative genotyping results of the sequential mating experiments. I. Sequential mating of WT female mice with mTmG and quinKO males. Upper panel, an overview of the polyandrous mating scheme. “mTmG V.S. quinKO”: mTmG male mice mated first; “quinKO V.S. mTmG”: quinKO male mice mated first. J. Percentage of mTmG blastocysts obtained from IVF using WT MII oocytes and mixed sperm from mTmG (control) and quinKO males at different ratios. Data were based on three independent IVF experiments. The expected ratio was indicated as the blue line. Chi-squared test was used for statistical analyses. * and **** indicate p<0.05 and 0.0001, respectively. K. Percentage of mTmG embryos obtained from co-artificial insemination using different ratios of mTmG and quinKO sperm. Data were based on 9 and 3 independent AI experiments for the 1:1 and 1:4 sperm ratio (mTmG: quinKO), respectively. The expected ratio was indicated as the blue line. Chi-squared test was used for statistical analyses. *, p<0.05.

Target genes and genetic compensation of the X-linked miR-506 family miRNAs.

A. Intersections of the DETs among different KO testes. B. GO term enrichment analyses of the 431 DETs shared among the four different miR-506 KO testes. C. MA plots showing the expression levels of the miR-506 family miRNAs in WT, sKO, tKO, quadKO, and quinKO testes. Three biological replicates (n=3) were used for sRNA-seq analyses.

Rapid evolution of the X-linked miR-506 family miRNAs correlates with increased complexity of genetic networks that regulate spermatogenesis across mammalian species.

A. Overlap between the dysregulated targets in mice and the predicted targets in humans and rats. B. Comparison of the cumulative distribution between the miR-506 family targeting sites and the other regions in humans. *: p< 0.05; t-test was used for statistical analyses. C. Comparison of the cumulative distribution between the miR-506 family targeting sites and the other regions in mice. *: p< 0.05; t-test was used for statistical analyses. D. Paired comparison of the PhyloP score between the miR-506 family targeting sites and the other regions in humans. E. Paired comparison of the PhyloP score between the miR-506 family targeting sites and the other regions in mice. F. Comparison of the number of the targets per miRNA for the X-linked miR-506 family in mice and humans. *: p< 0.05; t-test was used for statistical analyses. G. The number of target sites within individual target mRNAs in both humans and mice. H. Schematics show that human miR-506 family miRNAs have more targets relative to those of mice during evolution.