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. 2017 Oct 10;27(11):1392–1396. doi: 10.1038/cr.2017.125

Figure 1.

Figure 1

PNLDC1 is required for piRNA 3′ end trimming and male fertility. (A) Schematic diagram of targeting strategy by CRISPR/Cas9. (B-C) Testis size and testis weight of wild-type and Pnldc1−/− mice at 8-week-old. n = 3–4, ***P < 0.001. (D) Periodic acid-Schiff (PAS) staining of testis sections from Pnldc1+/+ and Pnldc1−/− mice at 8 weeks of old. (E) Immunolabeling of SYCP3 (red) and γH2AX (green) was performed on spread nuclei of pachytene spermatocytes from wild-type and Pnldc1−/− testes, and the quantification of γH2AX mislocalization (Pnldc1+/+, n = 105; Pnldc1−/−, n = 90). Scale bar, 10 μm. (F) Electron micrograph analysis of wild-type and Pnldc1-deficient elongated spermatids. Highly condensed chromatin of elongated spermatids in wild-type (white arrows), uncondensed chromatin (white arrowheads) and residual cytoplasm (asterisks) in elongated spermatids from Pnldc1 mutants. Scale bar, 1 μm. (G) Western blot analysis for LINE1 (L1ORF1p) protein levels in P10, P18 and P60 testes. n = 3–4. (H) Immunofluorescence detection of L1ORF1p from P14 and P60 testes. (I) Relative abundance of 233 piRNA generating long single-strand transcripts in P10 Pnldc1−/− testes compared with Pnldc1+/+ testes by transcriptome deep-sequencing (n = 3). (J) Testis lysates from P10 Pnldc1+/+ and Pnldc1−/− mice were subjected to immunoprecipitation with anti-MILI antibody, followed by detection of MILI-associated RNAs through 5′ labeling. n = 10–11. (K-L) Length distribution (K) and the nucleotide preference at the first position (L) of MILI-associated RNAs from P10 mice. (M) The 5′ and 3′ variation analysis of piRNAs derived from the top 19 pre-pachytene piRNA clusters in P10 Pnldc1+/+ and Pnldc1−/− testes. (N) Alignments between mature piRNAs in P10 Pnldc1+/+ testes and longer piRNA species in P10 Pnldc1−/− testes within a 1-kb window of piRNA cluster on chromosome 7. Mature piRNAs showed in black, longer piRNA species showed in red. (O) 5′ labeling of total small RNAs in P18 Pnldc1+/+ and Pnldc1−/− testes. (P-Q) Length distribution (P) and reads mapped to transposon consensus (Q) of total small RNAs reads in P18 Pnldc1+/+ and Pnldc1−/− testes. (R) MILI- or MIWI-associated RNAs were examined by immunoprecipitation and 5′ labeling. n = 3–4. (S-T) Length distribution (S), and the nucleotide composition at the first position (T) of MILI-associated RNAs from P18 mice.