Blockade of pachytene piRNA biogenesis reveals a novel requirement for maintaining post-meiotic germline genome integrity

Abstract

Piwi-interacting RNAs are a diverse class of small non-coding RNAs implicated in the silencing of transposable elements and the safeguarding of genome integrity. In mammals, male germ cells express two genetically and developmentally distinct populations of piRNAs at the pre-pachytene and pachytene stages of meiosis, respectively. Pre-pachytene piRNAs are mostly derived from retrotransposons and required for their silencing. In contrast, pachytene piRNAs originate from ~3,000 genomic clusters, and their biogenesis and function remain enigmatic. Here, we report that conditional inactivation of the putative RNA helicase MOV10L1 in mouse spermatocytes produces a specific loss of pachytene piRNAs, significant accumulation of pachytene piRNA precursor transcripts, and unusual polar conglomeration of Piwi proteins with mitochondria. Pachytene piRNA-deficient spermatocytes progress through meiosis without derepression of LINE1 retrotransposons, but become arrested at the post-meiotic round spermatid stage with massive DNA damage. Our results demonstrate that MOV10L1 acts upstream of Piwi proteins in the primary processing of pachytene piRNAs and suggest that, distinct from pre-pachytene piRNAs, pachytene piRNAs fulfill a unique function in maintaining post-meiotic genome integrity.

Figure 1. Post-natal disruption of Mov10l1 leads to post-meiotic spermiogenic arrest.

(A) Timeline of mouse spermatogenesis, with blue lines and tan histograms representing developmental expression patterns of three mouse Piwi proteins (MIWI, MIWI2, MILI) and MOV10L1, and pre-pachytene and pachytene piRNA populations, respectively. Crosses on lines mark the time point of spermatogenic arrest in the respective mouse mutant (4 ubiquitous null mutants and 2 Mov10l1 conditional mutants). Arrows on the timeline bar indicate the onset of Cre expression in the different Mov10l1 mutants generated. Disruption of Mov10l1 by Prm-Cre did not cause spermiogenic arrest (Table S1 and Figure S3). (B–D) Histology of testes from adult wild-type (B), Mov10l1 ^fl/- Neurog3-Cre (C), and Mov10l1 ^fl/- Hspa2-Cre (D) mice. H&E staining was performed on testis sections as described in the Materials and Methods. Abbreviations: Pa, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids.

Figure 2. Mov10l1 is required for biogenesis of pachytene piRNAs.

(A) Depletion of pachytene piRNAs in mutant testes from Mov10l1 ^fl/- Neurog3-Cre adult mice. Total RNAs were ³²P-end-labelled and separated by denaturing polyacrylamide gel electrophoresis. 18S and 28S ribosomal RNAs served as a loading control. (B) MILI is devoid of pachytene piRNAs in Mov10l1 ^fl/- Neurog3-Cre testes. MILI was immunoprecipitated from testicular extracts. One-tenth of the immunoprecipitated material was used for detection of associated piRNAs, and the remainder was used for Western blotting (WB) analysis of MILI. (C) MIWI immunoprecipitation on Mov10l1 ^fl/- Neurog3-Cre and wild-type (+/+) testes, and serial dilutions (1∶2) of MIWI IP complexes. (D) Depletion of pachytene piRNAs in mutant testes from Mov10l1 ^fl/- Hspa2-Cre adult mice. (E) MILI is loaded with pre-pachytene piRNAs in postnatal day 10 Mov10l1 ^fl/- Neurog3-Cre testes.

Figure 3. Northern blot analysis of individual pachytene piRNAs in conditional Mov10l1 mutant testes.

RNAs were prepared from adult testes and hybridized with radiolabeled probes specific for pachytene piR1, piR2, piR3, and a pre-pachytene piRNA. Hybridization with a let7g miRNA probe served as a loading control. The three individual pachytene piRNAs were absent in Mov10l1 ^fl/- Neurog3-Cre testes, sharply reduced in abundance in Mov10l1 ^fl/- Hspa2-Cre testes, and present in Mov10l1 ^fl/- Prm-Cre testes, while the level of the pre-pachytene piRNA was not affected in either of these mutant testes.

Figure 4. Polar conglomerate of piRNA pathway protein components and mitochondria in Mov10l1-deficient pachytene spermatocytes.

Adult testis sections were immunostained with antibodies (in green) against MILI (A, B), MIWI (C, D), TDRD1 (E, F), and GASZ (G, H). DAPI staining of DNA is shown in blue. (I) EM analysis of a wild-type pachytene spermatocyte. Note the random distribution of mitochondria (indicated by arrows). (J) EM image of a Mov10l1-deficient pachytene spermatocyte with a single mitochondrial cluster. Mito, mitochondria. Scale bars: A–H, 25 µm; I–J, 2 µm.

Figure 5. Massive DNA damage in piRNA-deficient round spermatids.

(A) Schematic representation of the expression of several proteins involved in chromatin remodeling during mouse spermiogenesis. (B) Phosphorylation of histone H2AX during normal spermatogenesis. Note the presence of γH2AX in three distinct types of germ cells: leptotene/zygotene spermatocytes (due to meiotic recombination), pachytene spermatocytes (XY body only, due to sex chromosome silencing), and elongating spermatids (due to chromatin remodeling). (C) Presence of γH2AX in round spermatids from Mov10l1^{fl /-} Neurog3-Cre testes. (D, E) Double staining of γH2AX and TOP2B in seminiferous tubules from wild-type and Mov10l1^{fl /-} Neurog3-Cre testes. (F, G) Double staining of γH2AX and PRM2 in seminiferous tubules from wild-type and Mov10l1^{fl /-} Neurog3-Cre testes. Red channels and DAPI staining are also shown in separate panels (D–G). Pa, pachytene spermatocytes; RS, round spermatids; ES, elongating spermatids. Scale bar, 25 µm.

Figure 6. MOV10L1 is essential for the primary processing of pachytene piRNA precursors.

(A) The RNA helicase domain in MOV10L1 is required for interaction with MILI and MIWI. Input was 1/50^th of extracts used for IP. TDRD1 is complexed with MILI and MOV10L1 and thus served as a positive control for immunoprecipitation. The same results were obtained from the repeat of IP experiments using independent samples. (B) Blockade of pachytene piRNA precursor processing in Mov10l1 ^fl/- Neurog3-Cre testes. Total RNAs were pre-treated with DNase I. RT-PCR produced no products in controls without reverse transcriptase (data not shown). PCR primers and product sizes are listed in Table S2. (C) Quantitative RT-PCR analysis of piRNA precursor transcripts in adult testes. Numbers on the vertical axis represent fold increase of levels in mutant testis compared to levels in wild-type testes defined as 1. (D) Proposed model for the essential role of MOV10L1 in the primary processing of pachytene piRNAs. Single strand RNAs are transcribed from the piRNA clusters and are digested by an unknown nuclease into putative piRNA intermediate like molecules (piR ILs). Such piR ILs have been reported in Drosophila but not yet in mammals . MOV10L1 and Piwi proteins select and bind to piR ILs for further processing into mature primary piRNAs through 3′ trimming .