MOV10L1 is associated with Piwi proteins and piRNAs in testis. (A) Validation of protein associations by coimmunoprecipitation (co-IP) and Western blotting. Note that nuage components MVH and RNF17 are not associated with MOV10L1. (B) Reciprocal IP confirms the association between MOV10L1 and MILI. (C) MOV10L1 interacts with MILI, MIWI, and MIWI2 in cotransfected 293T cells. (D) Analysis of piRNAs in immunoprecipitated MILI, MIWI, and MOV10L1 complexes from adult mouse testes. (E) Length distribution of small RNA reads in three small RNA libraries prepared from adult mouse testes. (F and G) Adjacent testis sections from 2-mo-old (P60) mice were immunostained with anti-MOV10L1 (F, green) and anti-MILI (G, red) antibodies. Strong interstitial signal is due to autofluorescence of Leydig cells. (H and I) Expression of MOV10L1 in gonocytes (Gc) from newborn (P0) testes. In contrast to nuclei of Sertoli cells, gonocyte nuclei contain little heterochromatin (H). Spg, spermatogonium; Spc, spermatocyte; Spt, spermatid. (Scale bar, 25 μm.)

Mov10l1 is essential for spermatogenesis and chromosomal synapsis. (A) Western blot analysis of adult wild-type, Mov10l1^+/−, and Mov10l1^−/− testes. The mutant MOV10L1 protein (1,061 aa; MOV10L1Δ) is indicated. (B) Dramatic size reduction in 7-wk-old Mov10l1^−/− testis. (C and D) In contrast to wild-type tubules with full spermatogenesis (C), Mov10l1^−/− tubules from adult mice exhibited early meiotic arrest (D). (E) Normal pachytene spermatocyte with 19 pairs of fully synapsed autosomes and the partially synapsed sex chromosomes. (F) Zygotene-like spermatocytes from Mov10l1^−/− adult testes.

Binary derepression of LINE1 and IAP retrotransposons in mitotic vs. meiotic germ cells. (A) Quantitative RT-PCR analysis. (B) Northern blot analysis. (C) Western blotting analysis. (D and E) Immuofluorescence analysis of LINE1 ORF1p in 2-mo-old (P60) Mov10l1^+/− and Mov10l1^−/− testes. (F) Immunofluorescence analysis of IAP in Mov10l1^−/− testes. Spc, spermatocytes; Spg, spermatogonia. (G) Methylation-sensitive Southern blot analysis of testis genomic DNA. Genomic DNA was digested with methylation sensitive (HpaII, H) and methylation insensitive (MspI, M) enzymes. Arrows indicate the position of methylation-sensitive (H) restriction products in the Mov10l1^−/− testes. (Scale bar in F, 25 μm for D–F.)

Biogenesis blockade of both prepachytene and perinatal piRNAs in Mov10l1^−/− testes. (A) MILI is unloaded in P10 and P14 Mov10l1^−/− testes. One tenth of the immunoprecipitated material was used for detection of associated RNAs, whereas the remaining was used for Western blotting (WB) to detect MILI. (B) MILI immunoprecipitations with P10 Mov10l1^−/−, Mov10l1^+/−, and serial dilutions (1:2) of P10 wild-type testicular extracts. (C) Profile of read lengths in total small RNA (18–32 nt) libraries from P10 Mov10l1^+/− and Mov10l1^−/− testes. (D) MIWI2 is localized to the cytoplasm in Mov10l1^−/− perinatal (P0) gonocytes (arrows). (E) MILI and MIWI2 are devoid of piRNAs in Mov10l1^−/− newborn (P0) testes.