QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 21 January 2004
doi: 10.1242/dev.00973

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kuramochi-Miyagawa, S. Articles by Nakano, T. Search for Related Content PubMed PubMed Citation Articles by Kuramochi-Miyagawa, S. Articles by Nakano, T.

Mili, a mammalian member of piwi family gene, is essential for spermatogenesis

¹ Department of Molecular Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita-shi, Osaka 565-0871, Japan
² Laboratory of Cytogenetics, Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan
³ Laboratory of Animal Cytogenetics, Center for Advanced Science and Technology, Hokkaido University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan
⁴ Department of Laboratory Sciences for Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita-shi, Osaka 565-0871, Japan
⁵ Department of Cell Biology, Duke University Medical Center, PO Box 3709, DUMC, Durham, NC 27710, USA

View larger version (49K): [in a new window]   Fig. 7. Association of MVH with MILI and MIWI. (A) Co-immunoprecipitation of MILI and MIWI with MVH. The 293T cells were transfected with plasmids that expressed FLAG-tagged MILI or MIWI and either Myc-tagged MVH or mock plasmids. The lysates were immunoprecipitated with the anti-Myc or anti-FLAG antibodies and detected with the anti-FLAG or anti-Myc antibodies. (B) Co-immunoprecipitation of MILI and MIWI with MVH in the testis lysate. Testis lysate of normal mice was immunoprecipitated with pre-immune serum or anti-MVH antibody, and detected with the anti-MILI-26F antibody recognizing both MILI and MIWI. (C) Schematic structure of the truncated MVH protein. Broken boxes show the helicase domains. E, EcoRI; H, HindIII; Xh, XhoI. (D) CBB staining of the GST-fusion proteins. GST-fusion proteins that were purified with glutathione-Sepharose 4B were loaded on 4-20% SDS-PAGE. (E) GST pull-down assay. Testis lysates were incubated with GST or the various GST-MVH fusion proteins. The MILI and MIWI proteins that were pulled down with glutathione-Sepharose were detected by western blotting using antibody 26F.

View larger version (37K): [in a new window]   Fig. 1. Targeted disruption of the Mili gene. (A) Schematic representation of the wild-type allele, the targeting vector and the mutated alleles. The numbered boxes (1-6) denote the 5'-end non-coding exon (exon 1) and the five coding exons (exons 2-5). The targeting vector includes the PGK-neo gene (neo) and the thymidine kinase gene (TK). (B) Southern blot analysis of representative offspring from heterozygous mating. The wild-type allele produces a 10.1 kb EcoRV product, while the disrupted allele gives rise to a 5.5 kb band with the 5'-end hybridization probe. (C) Western blot analysis of the MILI and MIWI proteins from testes using antibody 26F, which recognizes both MILI and MIWI. Lysates (10 µg of protein in each lane) of the testes were loaded on the gel. The +/+, +/- and -/- designations indicate samples from wild-type, heterozygous and homozygous mutant testes, respectively. The 293T cells that were transfected with Mili- or Miwi-expressing plasmids and mock plasmids are shown as the controls.

View larger version (95K): [in a new window]   Fig. 2. Defective spermatogenesis in Mili-deficient mice. (A) Immunostaining of 14.5-dpc gonads with the anti-MVH antibody. Sections from heterozygous (+/-) and homozygous mutant (-/-) littermate embryos were stained with the anti-MVH antibody (I, II) or DAPI (III, IV). (B) Comparison of the testes from 10-week-old wild type (+/+), heterozygous (+/-) and homozygous mutant (-/-) mice. The testis from the Mili-/- mouse is smaller. (C) Weights of testes from wild-type (+/+), heterozygous (+/-) and homozygous mutant (-/-) mice. (D) Hematoxylin and Eosin-stained sections of testes from 10-week-old mice. Sections from the heterozygous (I, III) and homozygous mutant (II, IV) mouse testes are shown.

View larger version (102K): [in a new window]   Fig. 3. Apoptosis in the Mili+/- and Mili-/- testes. TUNEL-labeled testes during the first wave of spermatogenesis (A-H) and in adulthood (I,J). Testes from heterozygous (A,C,E,G,I) and homozygous mutant (B,D,F,H,J) littermates were stained using the TUNEL technique. Testis sections are shown from 7-day-old (A,B), 9-day-old (C,D), 11-day-old (E,F), 14-day-old (G,H) and adult (I,J) mice. The nuclei were counterstained with Methyl Green.

View larger version (34K): [in a new window]   Fig. 4. Analysis of gene expression in Mili-/- mouse testes. The expression of stage-specific genes during spermatogenesis was analyzed by RT-PCR. G3PDH was used as the standard. Total testis RNA samples were prepared from 3-week-old and 12-week-old wild-type (+/+), heterozygous (+/-) and homozygous mutant (-/-) mice.

View larger version (79K): [in a new window]   Fig. 5. Expression of SYCP3, RAD51 and -H2AX in Mili-/- spermatocytes. Anti-SYCP3 immunostaining (A-D), anti-RAD51 staining (E,H), anti--H2AX staining (K,N) and DAPI staining (F,I,L,O), and the merged images of RAD51 or -H2AX and DAPI (G,J,M,P) in Mili+/- (upper panels of each pair) and Mili-/- (lower panels of each pare) testes. Arrows show the -H2AX restricted to the sex bodies in early pachytene spermatocyte. All images except A and C were observed by confocal microscopy. Scale bars: 10 µm.

View larger version (104K): [in a new window]   Fig. 6. No mid-pachytene spermatocyte in the Mili-/- testes. Merged image of synaptonemal complexes stained with the B antiserum recognizing both COR1 and SYN1 (green) and DNA stained with DAPI (blue) (A-E) in mid-zygotene spermatocytes (A,D), early-pachytene spermatocytes (B,E) and mid-pachytene spermatocyte (C). The images of FITC-stained synaptonemal complexes and DAPI-stained nuclei were captured with L5 and A4 filters, respectively. The surface spreading samples were prepared from control testes (A-C) and the homozygous mutant testes (D,E). The micrographs of spermatogenic cell nuclei in Giemsa-stained preparations of heterozygous (F) and homozygous mutant (G) mice are shown. The arrows indicate mitotic metaphases of spermatogonia, and the arrowhead indicates the first meiotic metaphase of a primary spermatocyte.

View larger version (130K): [in a new window]   Fig. 8. Immunohistochemical localization of the MILI, MIWI and MVH proteins. Sections from 10-week-old wild-type (A-C), Mili-/- (D-F) and Miwi-/- (G-I) mouse testes were stained with the anti-MILI (A,D,G), anti-MIWI (B,E,H), and anti-MVH (C,F,I) antibodies. MILI expression was detected in the cytoplasm of zygotene to pachytene spermatocytes in the wild-type and Miwi+/- testes (A,G). MIWI expression in wild-type testis was from pachytene to the round spermatid stage, and granular staining of MIWI was detected in the round spermatids (B). No MIWI-positive cells were observed in the Mili-/- testis (E). MVH protein was detected in the zygotene spermatocyte to round spermatid stages of the wild-type testis (C). Although granular distribution of MIWI was detectable in the wild-type testis (C), this granular pattern was not detectable in the Miwi-/- testis (I).