Fig 2: Molecular characterization of group 3 testes.(a) piRNA length distribution, hematoxylin and eosin (H&E) stained testis sections, and immunohistochemical detection of A-MYB and HIWI for representative samples from groups 1, 2, and 3. (b) Scatter plot of steady-state transcript abundance of transcripts in group 1 versus group 3 testes. Each dot represents mean abundance of an mRNA. (c) Gene ontology analysis of mRNAs detected in group 3 samples and whose abundance changed >3-fold (FDR <0.05) compared to group 1.

Fig 3: Dysregulated expression of A-MYB and HIWI in group 3 adult testes.(a) Change in abundance of HILI-bound 26–27 nt piRNAs and HIWI-bound 30 nt piRNAs mapping to pre-pachytene and pachytene piRNA-producing genes among different groups of adult and juvenile samples. (b) Box plots showing the mRNA abundance among the different groups of adult and juvenile samples for genes encoding piRNA biogenesis factors, 140 additional A-MYB-regulated genes, as well as reproduction-related genes and immune response-related genes. (c, d) A-MYB and HIWI mRNA (c) and protein abundance (d).

Fig 4: Three classes of human post-natal piRNA genes.(a) Heatmap representation of A-MYB occupancy on the promoters of piRNA-producing genes and of the abundance of piRNA precursor transcripts and mature piRNAs from seven adult and one juvenile testis (mean of two technical replicates; Correlation for technical replicates: piRNA sequencing, Spearman’s ? = 0.99, Pearson’s ? = 0.99; RNA sequencing, Spearman’s ? = 0.91, Pearson’s ? = 0.96). Rpkm: reads per million unique mapped reads per thousand nucleotides; rpm: reads per million. (b) Heatmap representation of the abundance of piRNA precursor transcripts and mature piRNAs from a second set of human testis samples. (c) The distance from the nearest A-MYB peak to the transcription start sites (TSS) for each gene class: piRNA-producing, piRNA-biogenesis protein encoding, other protein-coding, or lncRNA-producing. Whiskers show 95% confidence intervals. (d) A-MYB ChIP-seq signal on the promoter of a divergently transcribed human pachytene piRNA gene and of the A-MYB and HIWI genes. Only uniquely mapping reads were analyzed.

Fig 5: Three groups of adult testes defined by length distribution of total piRNAs and A-MYB and HIWI expression.(a) The abundance of piRNAs was normalized to the total number of genome-mapping reads. Relative protein abundance of A-MYB (b) and HIWI (c) in adult testis samples. ACTIN serves as a loading control, while mouse A-Mybrepro9 and Miwi-/- mutant testis lysates provide negative controls. Each lane contained 75 µg protein of testis lysate.