Localization of MILI and MIWI2 in the MVH-deficient fetal germ cells. (A) Immunofluorescence staining of wild-type and mutant testes. Sections of E16.5 testes were stained with anti-MVH, anti-MILI, anti-TDRD1, anti-MIWI2, and anti-TDRD9 antibodies, control IgG. (B) Electron microscopic analyses of MVH-, MILI-, and MIWI2-deficient and control E17.5 testes. Arrowheads indicate intermitochondrial cement.

piRNA expression in the MVH-deficient fetal testis. (A) Microarray analysis of the repeat-associated piRNAs and miRNAs. A microarray containing 672 types of repeat-associated piRNA and 150 types of miRNA, both of which were expressed in fetal testes, was produced. Testis RNA samples isolated from E16.5 MVH-deficient and heterozygous mice were used. The values normalized with control probes are plotted, and diagonal lines indicate a 1.5-fold difference in expression. (B) Binding of piRNA with MILI, MIWI2, and MVH. The immunoprecipitated RNAs from E16.5 wild-type testicular lysates were ³²P-end-labeled and separated in 15% denaturing urea–polyacrylamide gels. (C) MILI- and MIWI2-bound piRNAs in the mutant embryonic testes. The immunoprecipitated RNAs from E16.5 wild-type and mutant testicular lysates were ³²P-end-labeled and separated in 15% denaturing urea–polyacrylamide gels. The numbers of testes used for the immunoprecipitation assays are indicated at the bottom.

piRNA expression analysis in the MVH-deficient fetal testis by deep sequencing. (A) The size distribution of the total small RNAs in E16.5 testes. Total numbers of small RNAs sequenced from wild-type and MVH-deficient testis libraries were 2,090,035 and 2,812,945, respectively. (B) Ratio of total and repeat-associated piRNA to miRNA. (C,D) Ratio of the first (C) and 10th (D) nucleotides of the repeat-associated piRNAs. (E) Expression of piRNA cluster-derived piRNAs in the MVH-deficient testes. The data of piRNAs derived from the large 19 piRNA clusters are shown. The ratios of piRNAs in the MVH-deficient testes per those in wild-type testes are shown. (Supplemental Table 2) Number 1 represents the data of the largest piRNA cluster, and the following are ordered according to the sizes of the clusters.

Expression of the IAP and Line-1 retrotransposons and DNA methylation of their regulatory regions in the MVH-deficient testes. (A) Northern blotting analysis of IAP and Line-1 retrotransposons in the testes of 2-wk-old and 2-mo-old MVH- and MILI-deficient mice. The 3′ noncoding region of IAP and the 5′ noncoding regions of Line-1 were used as probes. (B) Methylation-sensitive Southern blotting analysis of the Line-1 promoter region. DNA was extracted from the testes of 2-wk-old heterozygous and homozygous mice. The probe was the same as that used in A. (C,D) Bisulfite sequencing of IAP and Line-1. Germ cells were collected from p10 testis (C) and fetal testis (D) using EGFP as a marker. The LTR region from the IΔI-type IAP in chromosomes 3qD and the 5′ noncoding regions of Line-1 were analyzed. Filled and open circles represent methylated and unmethylated CpGs, respectively. The percentages of methylated CpGs are shown in parentheses.