First published online 21 January 2004
doi: 10.1242/dev.00973
Development 131, 839-849 (2004)
Published by The Company of Biologists 2004
Mili, a mammalian member of piwi family gene, is essential for spermatogenesis
Satomi Kuramochi-Miyagawa1,
Tohru Kimura1,
Takashi W. Ijiri2,
Taku Isobe3,
Noriko Asada1,
Yukiko Fujita1,
Masahito Ikawa4,
Naomi Iwai1,
Masaru Okabe4,
Wei Deng5,
Haifan Lin5,
Yoichi Matsuda2,3 and
Toru Nakano1,*
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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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).
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© The Company of Biologists Ltd 2004
