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
* Author for correspondence (e-mail: tnakano{at}biken.osaka-u.ac.jp)
Accepted 7 November 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Mili, Miwi, piwi, Mvh, Spermatogenesis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Spermatogenesis is one of the most dramatic examples of cell proliferation,
differentiation and morphogenesis. The mouse spermatogenic cycle can be
divided into 12 stages, with each stage consisting of a specific complement of
male germ cells (Russell et al.,
1990). The entire process occurs in three phases: mitosis
(spermatocytogenesis), meiosis and spermiogenesis. A cascade of mitoses, which
are initiated by the self-renewing division of germline stem cells (a subset
of type A spermatogonia), gives rise to the primary spermatocytes.
Subsequently, meiosis of primary spermatocytes leads to the production of
haploid round spermatids. The prophase of the first meiotic division
progresses in the following order: leptotene, zygotene, pachytene, diplotene
and diakinesis. Various genes that are involved in cell cycling, DNA
replication and RNA processing are essential for this process.
One of the germ cell determinant genes in Drosophila, vasa encodes
an ATP-dependent RNA helicase of the DEAD-box protein family that is essential
for the assembly and function of the germ plasm
(Hay et al., 1988;
Hay et al., 1990
;
Lasko and Ashburner, 1988
).
Based on structural conservation data, homologs of vasa have been
identified in many animal species, such as C. elegans, Xenopus,
zebrafish, chicken and mouse (Fujiwara et
al., 1994
; Komiya et al.,
1994
; Olsen et al.,
1997
; Roussell and Bennett,
1993
; Tsunekawa et al.,
2000
; Yoon et al.,
1997
). All of these vasa homologs are expressed
exclusively in the germ lineage. The expression and function of the
Mvh gene were analyzed by immunostaining and gene-targeting analysis,
respectively (Tanaka et al.,
2000
; Toyooka et al.,
2000
). The MVH protein appears initially in PGCs that colonize the
embryonic gonads at 10.5-11.5 dpc, and is maintained in both male and female
germ cells until the development of postmeiotic spermatids and primary
oocytes, respectively. Sperm are absent from the testes of Mvh-null
male mice, in which premeiotic germ cells cease differentiation by the
zygotene stage and undergo apoptotic death.
The piwi gene of Drosophila belongs to a novel class of
evolutionarily conserved genes (the piwi or Argonaute
family) (Benfey, 1999). The
piwi family genes encode basic proteins that contain a highly
conserved PAZ domain of 110-amino acid residues in the middle region of the
proteins and a 300-amino acid Piwi domain in the C-terminal region
(Cerutti et al., 2000
), even
though the function of these domains remains elusive. In Drosophila,
the loss of piwi function leads to the failure of germline stem cell
self-renewal as well as downstream gametogenic functions such as germline cyst
formation, egg polarity and possibly meiosis
(Cox et al., 1998
;
Lin and Spradling, 1997
).
Based on database analyses, three piwi homologs have been identified
in the mouse genome. Two of these genes, Miwi and Mili
(Miwi like; Piwil2 - Mouse Genome Informatics), have been
examined in detail. Miwi-null mice do not complete spermatogenesis,
but arrest occurs at the beginning of the round spermatid stage
(Deng and Lin, 2002
),
significantly downstream of the germline stem cell division stage. To reveal
the functional of Mili, here we report the generation and analysis of
the Mili-null mice. The Mili-null mice showed arrest of
spermatogenesis at the spermatocyte stage, which is reminiscent of the
phenotype of Mvh-null mice. Furthermore, we demonstrate the physical
association between MILI and MVH, which may account for the similarities
between the phenotypes of the Mili- and Mvh-null mice.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The oligonucleotide PCR primers that were used to distinguish the insertion of the neo gene from the wild-type allele were as follows: pPNT-1, 5'-CCTACCCGGTAGAATTGACC-3'; Mili-Int4, 5'-GTCCTGTGTAGAGCCAAG-3'; and Mili-Int5, 5'-TGACAAGGTGCGAGTCT-3'. The pPNT-1 and Mili-Int5 primers gave a 410 bp DNA fragment that identified the targeted allele, while the Mili-Int4 and Mili-Int5 primers yielded the 800 bp fragment of the wild-type allele. The PCR was carried out for 40 cycles of 94°C for 1 minute, 65°C for 1 minute and 72°C for 1 minute.
Antibodies
The GST-Mili-26F plasmid was constructed by inserting the Mili
cDNA fragment that encodes the sequence from Ile357 to Phe502 into the pGEX3X
expression vector (Pharmacia Biotech). The purified GST-fusion proteins were
used to immunize rabbits. The affinity-purified polyclonal antibody directed
against Mili-26F recognized both the MIWI and MILI proteins in western blots.
The anti-MILIN1 and anti-MIWI-C polyclonal antibodies (against MILI and MIWI)
were generated by immunization with the MILI N-terminal peptide
(DPVRPLFRGPTPVHPSQC) and MIWI C-terminal peptide (CHHEPAIQLCGNLFFL),
respectively. The affinity-purified antibodies against the peptides (produced
by MBL, Japan) were used for immunohistochemical analysis. The anti-MVH
(Toyooka et al., 2000) and
anti-SYCP3 (Chuma and Nakatsuji,
2001
) antibodies were donated by Drs. T. Noce and N. Nakatsuji,
respectively. The rabbit B antiserum against whole synaptonemal complexes,
which reacts both COR1 and SYN1 proteins
(Moens and Spyropoulos, 1995
),
were kindly provided by Dr P. B. Moens. The anti-RAD51 (Ab-1, Oncogene, San
Diego, CA), the anti-
-H2AX [anti-phospho-H2A.X(Ser139), Upstate, Lake
Placid, NY], the anti-MYC antibody 9E10 (BIOMOL Research Laboratories,
Plymouth Meeting, PA) and the anti-FLAG M2 antibody (Sigma, St Louis, MO) were
used for the immunohistochemical and immunoprecipitation assays.
Western blotting
The testes were homogenized in TNE buffer [50 mM Tris-HCl (pH 8.0), 1%
Nonidet P-40, 20 mM EDTA] that contained protease inhibitors. Equal amounts of
protein were separated by SDS-PAGE and transferred to a PVDF membrane
(Millipore, Bedford, MA). After blocking, the filters were incubated with the
affinity-purified anti-Mili26F antibody. Peroxidase-conjugated goat
anti-rabbit IgG (Zymed, South San Francisco, CA) was used as the secondary
antibody, and the signals were detected using the ECL kit (Amersham
Pharmacia).
Histological analysis
Littermate embryonic male gonads at 14.5 dpc and postnatal testes were
fixed in 4% paraformaldehyde overnight at 4°C, dehydrated progressively
and embedded in methyl methacrylate. Subsequently, 5 µm rehydrated sections
were used for Hematoxylin-Eosin staining, for the terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) reaction, and for
immunohistochemical staining. For the TUNEL reactions, the rehydrated sections
were treated with methanol that contained 0.3% H2O2 for
10 minutes at room temperature. Apoptotic cells were detected using the In
Situ Cell Death Detection Kit, POD (Roche). After the detection of apoptotic
cells with DAB, the sections were stained with 1% Methyl Green. For the
immunohistochemical analysis, the rehydrated slides were boiled in 10 mM
tri-sodium citrate (pH 6.0) in a microwave oven for 8 minutes, to retrieve the
antigens. After blocking with 5% normal goat serum in PBS, the slides were
incubated with the anti-Mili-N1 (15 µg/ml), anti-Miwi-C (5 µg/ml) and
anti-MVH (1:500) antibodies. For SYCP3, RAD51 and -H2AX staining, the
testes were embedded directly in OCT compound and cut at 10 µm. The
sections were fixed with 2% paraformaldehyde for 20 minutes, permeabilized
with acetone for 20 minutes, and blocked for 1 hour with 3% BSA and 10% normal
goat serum in PBS. The staining was carried out as described previously
(Chuma and Nakatsuji, 2001
). As
negative controls, pre-immune sera or isotype matched immunoglobulins were
used and essentially no positive signals were detected in the control
staining. Positive signals were detected with Alexa568- or Alexa488-conjugated
anti-rabbit IgG (H+L) antibodies (Molecular Probes, Eugene, OR).
Surface spreading methods and chromosome preparation
For immunocytological analysis of SC formation at meiotic prophase was
performed as described by Matsuda et al.
(Matsuda et al., 1992). The
images of immunocytochemical staining were captured with 550CW-QFISH
application program of Leica Microsystems Imaging Solutions (Cambridge, UK)
using a cooled CCD camera (MicroMAX 782Y, Prinston Instruments) mounted on a
Leica DMRA microscope. The preparation of chromosomes and nuclei for light
microscopy of the spermatogenic cells of the Mili-deficient mouse was
performed using the air-drying method
(Imai et al., 1981
), with the
omission of colchicine treatment. The preparations were stained with 4% Giemsa
in phosphate buffer (pH 7.0), followed by counting of the three types of
spermatogenic cells, i.e. spermatogonia, spermatocytes and spermatids, as well
as the Sertoli cells.
RT-PCR analysis
Total RNA samples were isolated from the testes using Sepasol-RNA I
(Nacalai Tesque, Kyoto, Japan). Single-stranded cDNA was prepared from 3 µg
of total RNA using random hexamers and the ThermoScript RT-PCR System
(Invitrogen, Carlsbad, CA). Each PCR reaction was performed with a 1/30
dilution of the RT products and using HotStar Taq (Qiagen, Valencia, CA). The
PCR was carried out for 25-28 cycles of 94°C for 1 minute, 57°C for 1
minute and 72°C for 1 minute. The following primer pairs were used for the
PCR: Mili, 5'-AGTGTGTGGGAGGA-3' and
5'-AGAGCCATCAAAAGCAG-3'; and Miwi,
5'-ATGATCGTGGGCATC-3' and 5'-AGGCCACTGCTGTCATA-3'. The
sequences of the other primers have been described previously
(Tanaka et al., 2000).
Transient transfection and immunoprecipitation
The Myc-tagged Mvh expression plasmid was constructed by
inserting the full-length fragment of Mvh (pQE32-Mvh; donated by Dr
T. Noce) into the pcDNA3 vector. The 293T cells were transfected with plasmid
DNA by the calcium phosphate method, and cultured for 48 hours. The cells were
lysed in TNE buffer that contained protease inhibitors. For anti-MVH
immunoprecipitation, testes were lysed with the same buffer. The lysates were
pre-cleared with protein G Sepharose (Pharmacia), and immunoprecipitated with
the anti-MYC, anti-FLAG or anti-MVH antibody. The immunoprecipitates were
separated by SDS-PAGE and transferred to a PVDF membrane. After blocking, the
filters were incubated with the anti-FLAG, anti-MYC or anti-26F antibody. The
secondary antibody was peroxidase-conjugated goat anti-mouse IgG (Sigma) or
anti-rabbit IgG (Zymed).
GST-pull down assay
The pGEX4T-Mvh plasmid was donated by Dr T. Noce. Deletions of the
GST-Mvh plasmid were produced using the appropriate restriction enzymes, as
shown in Fig. 7B. The
GST-fusion proteins were purified from E. coli lysates using
glutathione-Sepharose 4B beads. The GST-fusion proteins that were bound to the
Sepharose beads were then incubated with testis lysates. The GST precipitates
were separated by SDS-PAGE, and transferred to PVDF membranes. After blocking,
the filters were incubated with the affinity-purified anti-Mili26F
antibody.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Increased apoptosis in Mili-null mice
TUNEL labeling was used to analyze the timing of abnormal cell death in the
first wave of spermatogenesis and the continuous degeneration of early
spermatocytes in adulthood. In mice, spermatogenesis is initiated on day 3
after birth and progresses as a synchronous wave during the first week of
life. The most mature germ cell type observed 8 days after birth is the type B
spermatogonium; the preleptotene/leptotene, zygotene, and pachytene
spermatocytes appear on days 10, 12, and 14, respectively
(Bellve et al., 1977).
TUNEL-positive cells were rarely detected in Mili+/- and
Mili-/- testes on days 7 and 9
(Fig. 3A-D). On days 11 and 14,
there were some TUNEL-positive cells in the heterozygous testes
(Fig. 3E,G). However,
significant numbers of apoptotic cells were observed in the homozygous testes
(Fig. 3F,H). The apoptotic
cells in the homozygous testis were located in the inner layers of the
seminiferous tubules, where the most mature cells exist during the first wave
of spermatogenesis. Virtually no TUNEL-positive cells were detected in the
adult Mili+/- testis. By contrast, in most of the
seminiferous tubules of the Mili-/- testes, significant
numbers of TUNEL-labeling cells were detected in the spermatocyte layers
(Fig. 3I,J). Thus,
spermatogenesis in the Mili-/- mice is blocked during the
early stages of meiosis, probably at the zygotene or early pachytene stage of
the meiotic prophase, and apoptosis occurs subsequently.
|
|
To define further the meiotic defect in the Mili-/-
mice, sections stained with anti-SYCP3, RAD51 and -H2AX (phosphorylated
form of H2AX, a histon H2A variant) antibodies were examined
(Fig. 5). SYCP3 expression in
Mili-/- testis was weaker than that in
Mili+/- testis as expected from RT-PCR
(Fig. 5A,B). Axial core
formation was observed in the SYCP3-expressing meiotic cells in
Mili-/- testis (Fig.
5C,D), suggesting the entry to the first meiotic division;
however, pachytene chromosome formation was not completed in
Mili-/- testis. Expressions of RAD51 and
-H2AX were
essentially same in Mili-/-and Mili+/-
testes (Fig. 5E-P). During
meiotic prophase, the X and Y chromosomes condense to form the sex body or XY
body, and in late zygotene/early pachytene spermatocyte,
-H2AX
accumulates in the sex body (Mahadevaiah
et al., 2001
). Many
-H2AX condensed spots were observed in
Mili-/- testes as well as Mili+/-
testes (Fig. 5K,N arrow).
Furthermore, we analyzed synaptonemal complex formation in more detail using B
antibody that recognizes both COR1 and SYN1 of synaptonemal complex core.
Synaptonemal complex core was detected until early pachytene stage in the
Mili-/- testis (Fig.
6A-E). Similarly, nuclei at and after the mid-pachytene
spermatocyte stage were not detected in the Mili-/- mice
by Giemsa staining (Fig. 6F,G).
The mitotic metaphases of the spermatogonia were observed in
Mili+/- and Mili-/- testes. By
contrast, the first meiotic metaphase of the primary spermatocyte was not
detected in the Mili-/- testis.
Table 1 shows the
quantification of spermatogenic cells and Sertoli cells. Although a lot of
cells can be observed in Fig.
6F,G, the majority of the cells were Sertoli cells as shown in the
Table 1. Nuclei at and after
the mid-pachytene spermatocyte stage were not detected in the
Mili-/- mice. The histological analysis clearly
demonstrates that spermatogenesis after the postmeiotic stage was completely
absent in all the seminiferous tubules of adult Mili-/-
testes. Taken together, the spermatogenesis arrest in adult
Mili-/- testes occurs during the early stages of meiosis,
probably at the zygotene or early pachytene stages of the meiotic
prophase.
|
|
|
To analyze the localization of MILI, MIWI and MVH, immunohistochemical
analyses of the wild-type, Mili-/- and
Miwi-/- mice were carried out with specific antibodies
(Fig. 8). The MILI protein was
detected until pachytene-stage spermatocytes. Although Drosophila
Piwi was localized in the nucleoplasm (Cox
et al., 2000), MILI, like MIWI, was found in the cytoplasm
(Kuramochi-Miyagawa et al.,
2001
). MILI was not detected in the Mili-/-
testes, but its expression was normal in the Miwi-/-
testes. MIWI was not detected in either the Mili-/- or
Miwi-/- testes. The lack of MIWI expression in the
Mili-/- mice is presumably due to the paucity of
MIWI-expressing cells, i.e. midpachytene-stage spermatocytes and round
spermatids. MVH was detected in pre-pachytene spermatocytes in the
Mili-/- testes, which indicates that MILI is not essential
for MVH expression. Although MVH was detected in the
Miwi-/- testes, the subcellular localization of MVH was
different from that in the wild type. Anti-MVH staining showed the
granulo-fibrillar and granular distribution of MVH in spermatocytes and round
spermatids, respectively, in wild-type testes, and anti-MIWI showed the
similar staining pattern. As shown in Fig.
8, MVH was not distributed in a granular pattern in the cytoplasm
of the Miwi-/- round spermatids.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the Mili-/- testis, the expression of genes that are
involved in spermiogenesis was undetectable. The Dmc1 gene, the
expression of which is restricted in leptotene- to zygotene-stage
spermatocytes (Yoshida et al.,
1998), was expressed normally in the Mili-/-
testis. By contrast, the expression levels of Sycp1 and
Sycp3, both of which are transcribed predominantly in the zygotene to
diplotene stages (Dobson et al.,
1994
; Lammers et al.,
1994
; Meuwissen et al.,
1992
), were slightly reduced. A-myb, which is expressed
in early primary spermatocytes (Mettus et
al., 1994
; Trauth et al.,
1994
), showed similar low-level expression. Taken together with
the data from the chromosome analysis, these results suggest that
spermatogenesis in Mili-/- mice is arrested from the
zygotene to early pachytene stages.
Differential expression of MILI and MIWI
Although MILI and MIWI are expressed in germ cells, their expression
kinetics are different (Kuramochi-Miyagawa
et al., 2001). The expression of MILI was detected up to the
pachytene spermatocytes, whereas that of MIWI was detected from the
mid-pachytene stage to the emergence of elongated spermatids. Thus, MILI is
expressed at an earlier stage than MIWI, and the expression of MILI overlaps
somewhat with that of MIWI in the mid-pachytene stage. According to the
kinetics of expression, the stages of spermatogenetic arrest differ between
the Mili-/- and Miwi-/- testes. Arrest
was observed at the early pachytene spermatocyte and round spermatid stages in
Mili-/- and Miwi-/- testes,
respectively.
MILI and MIWI share the molecular characteristics such as binding to RNA and MVH. This raised an interesting question whether molecular function(s) of MILI could be same as that of MIWI. To zero in on this point, we are producing the transgenic mice in which MILI or MIWI is expressed under the promoter of Mili gene and are examining whether these transgenic mice could rescue the Mili-/- phenotype. Our preliminary data of the rescue study suggests that Mili but not Miwi, transgenic mice can rescue the Mili-/- phenotype (data not shown). Thus, the functional differences between MILI and MIWI are not solely dependent on the different expression timing but would be due to their distinctive molecular roles.
Physical association between MILI and MVH
When MILI and MIWI were overexpressed with MVH in 293 cells, the majority
of MVH was co-localized with MILI or MIWI as large perinuclear granule-like
structure (data not shown). The data of overexpression analysis suggested that
MILI and MIWI would define the localization of MVH at least to some extent.
MVH is localized exclusively in the cytoplasm of spermatogenic cells from
spermatogonia to round spermatids, with the highest expression in early
spermatocytes (Toyooka et al.,
2000). The stage of meiotic arrest in the
Mili-/- testis was similar to that in the
Mvh-/- testis, and the gene expression profiles were
essentially the same, as assessed by RT-PCR analysis. MILI and MVH were found
to be cytoplasmic proteins, as discussed below, and MILI and MIWI were
co-expressed with MVH throughout spermatogenesis. These data led us to analyze
the association of MILI and MIWI with MVH. Although physical association does
not necessarily bear functional relevance, the similar phenotypes of
Mili-/- and Mvh-/- spermatogenesis
imply cooperative molecular functions for these molecules.
In round spermatids, MVH is predominantly localized in a single large
granule with a spherical shape that is located in the perinuclear site
(Toyooka et al., 2000). The
perinuclear granule that stained with anti-MVH antibody was the chromatoid
body, which is a perinuclear electron-dense body in the male germ cells of
mammalian testicular germ cells classified as a nuage like structure. The
binding analysis showed that the affinity of MIWI for MVH was presumably
higher than that between MILI and MVH (Fig.
6D). In pull-down analysis of MILI and MIWI with GST-MVH fusion
protein, binding of MILI to MVH was competed out by the excessive amount of
MIWI. The round spermatids showed similar staining patterns with the anti-MIWI
and anti-MVH antibodies. In addition, the round spermatids of the
Miwi-/- mice did not show perinuclear granular staining by
anti-MVH antibody. Given that Miwi is required for the stability of
its target mRNAs (Deng and Lin,
2002
), this further suggests that chromatoid body could be the
subcellular structure essential for controlling the mRNA stability for
spermiogenesis.
Are Mili and Miwi Functional homologs of piwi?
The functions of the C. elegans homologs of piwi, prg-1 and
prg-2, have been studied using RNA interference (RNAi) to be important
for the mitotic ability of the germline nuclei and are essential for germline
proliferation and maintenance (Cox et al.,
1998). In this regard, the prgs are not only structural,
but also functional homologs of piwi. It is intriguing to investigate
whether the mammalian MILI and MIWI are functional homologs of
Drosophila Piwi. A couple of lines of evidence suggest that the two
mammalian structural homologs of Piwi, i.e. MILI and MIWI, only inherit a
subset of piwi functions. One line of evidence comes from the
analysis of gene targeting. In addition to its crucial roles in germline stem
cells, Piwi has less-well characterized roles in early oogenesis and
spermatogenesis, possibly including germline cyst mitosis, meiosis and egg
chamber polarity (Cox et al.,
1998
; Lin and Spradling,
1997
). The expression of MIWI and MILI is restricted to germ
lineages, and the gene-targeted animals show defective spermatogenesis.
However, we have not observed the defect of Mili-/- nor
Miwi-/- mice at the stage of testicular stem cells. Thus,
Mili and Miwi may only represent a subset of piwi
functions.
The other line of evidence is subcellular localization of MIWI and MILI.
The Piwi protein can be localized either to the nucleoplasm in germline stem
cells (Cox et al., 2000) or in
the cytoplasm co-localized with polar granules such as Vasa in early embryos
(D. N. Cox and H. Lin, unpublished), MILI and MIWI are found in the cytoplasm
associated with MVH (Kuramochi-Miyagawa et
al., 2001
). This again reflects only a subset of the Piwi
function. This function is also similar to Aubergine, another
Drosophila Piwi family protein, that is recruited to the posterior
pole in a vasa-dependent manner as a polar granule component
(Findley et al., 2003
).
Interestingly, Aubergine remains exclusively in the cytoplasm even after pole
cell formation. In addition, the levels of homology between MILI or MIWI and
Aubergine (31.0% and 36.6%, respectively) were similar to those seen with Piwi
(32.7% and 37.1%, respectively). Taking these data into consideration, it is
conceivable that MILI and MIWI might be functionally more similar to
Aubergine. Meanwhile, genome analysis has revealed the third mouse homolog of
piwi and aubergine (Accession Number AY135692). It is an
unanswered question whether the third member will represent other functions of
piwi and aubergine.
The piwi family genes, defined by conserved PAZ and Piwi domains
of unknown function, have been implicated in RNAi and related phenomena, such
as post-transcriptional gene silencing (PTGS) and transcriptional gene
silencing (TGS) in several organisms (Doi
et al., 2003; Pal-Bhadra et
al., 2002
; Tijsterman et al.,
2002
; Vaucheret et al.,
2001
). AGO1 (Argonaute) and QDE-2 are required for PTGS
in Arabidopsis and Neurospora, respectively, and RDE-1 is
required for RNAi in C. elegans
(Catalanotto et al., 2000
;
Fagard et al., 2000
;
Tabara et al., 1999
). In
Drosophila, mutations in piwi and aubergine block
RNAi activation during egg maturation and perturb translational control during
oogenesis (Kennerdell et al.,
2002
). Aubergine has the ability to effect the silencing of
Stellate, which is a tandemly repetitive gene
(Schmidt et al., 1999
).
Furthermore, in Drosophila, AGO1 and AGO2 are involved in
RNAi, and piwi is required for PTGS and for TGS, which is induced by
multiple copies of Alcohol dehydrogenase
(Hammond et al., 2001
;
Pal-Bhadra et al., 2002
;
Williams and Rubin, 2002
).
MILI and MIWI may be involved in similar silencing mechanisms required for
spermatogenesis. We are comparing the gene expression profiles between the
control and the Mili-/- testes, which could give some
clues about the function on gene silencing.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baker, S. M., Plug, A. W., Prolla, T. A., Bronner, C. E., Harris, A. C., Yao, X., Christie, D. M., Monell, C., Arnheim, N., Bradley, A. et al. (1996). Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over. Nat. Genet. 13,336 -342.[Medline]
Bellve, A. R., Cavicchia, J. C., Millette, C. F., O'Brien, D.
A., Bhatnagar, Y. M. and Dym, M. (1977). Spermatogenic cells
of the prepuberal mouse. Isolation and morphological characterization.
J. Cell Biol. 74,68
-85.
Benfey, P. N. (1999). Stem cells: A tale of two kingdoms. Curr. Biol. 9,R171 -R172.[CrossRef][Medline]
Catalanotto, C., Azzalin, G., Macino, G. and Cogoni, C. (2000). Gene silencing in worms and fungi. Nature 404,245 .[CrossRef][Medline]
Cerutti, L., Mian, N. and Bateman, A. (2000). Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain. Trends Biochem. Sci. 25,481 -482.[CrossRef][Medline]
Chuma, S. and Nakatsuji, N. (2001). Autonomous transition into meiosis of mouse fetal germ cells in vitro and its inhibition by gp130-mediated signaling. Dev. Biol. 229,468 -479.[CrossRef][Medline]
Cox, D. N., Chao, A., Baker, J., Chang, L., Qiao, D. and Lin,
H. (1998). A novel class of evolutionarily conserved genes
defined by piwi are essential for stem cell self-renewal. Genes
Dev. 12,3715
-3727.
Cox, D. N., Chao, A. and Lin, H. (2000). piwi
encodes a nucleoplasmic factor whose activity modulates the number and
division rate of germline stem cells. Development
127,503
-514.
de Vries, S. S., Baart, E. B., Dekker, M., Siezen, A., de Rooij,
D. G., de Boer, P. and te Riele, H. (1999). Mouse MutS-like
protein Msh5 is required for proper chromosome synapsis in male and female
meiosis. Genes Dev. 13,523
-531.
Deng, W. and Lin, H. (2002). miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev. Cell 2,819 -830.[Medline]
Dix, D. J., Allen, J. W., Collins, B. W., Mori, C., Nakamura,
N., Poorman-Allen, P., Goulding, E. H. and Eddy, E. M.
(1996). Targeted gene disruption of Hsp70-2 results in failed
meiosis, germ cell apoptosis, and male infertility. Proc. Natl.
Acad. Sci. USA 93,3264
-3268.
Dobson, M. J., Pearlman, R. E., Karaiskakis, A., Spyropoulos, B.
and Moens, P. B. (1994). Synaptonemal complex proteins:
occurrence, epitope mapping and chromosome disjunction. J. Cell
Sci. 107,2749
-2760.
Doi, N., Zenno, S., Ueda, R., Ohki-Hamazaki, H., Ui-Tei, K. and Saigo, K. (2003). Short-interfering-RNA-mediated gene silencing in mammalian cells requires dicer and eIF2C translation initiation factors. Curr. Biol. 13,41 -46.[CrossRef][Medline]
Edelmann, W., Cohen, P. E., Kane, M., Lau, K., Morrow, B., Bennett, S., Umar, A., Kunkel, T., Cattoretti, G., Chaganti, R. et al. (1996). Meiotic pachytene arrest in MLH1-deficient mice. Cell 85,1125 -1134.[Medline]
Edelmann, W., Cohen, P. E., Kneitz, B., Winand, N., Lia, M., Heyer, J., Kolodner, R., Pollard, J. W. and Kucherlapati, R. (1999). Mammalian MutS homologue 5 is required for chromosome pairing in meiosis. Nat. Genet. 21,123 -127.[CrossRef][Medline]
Fagard, M., Boutet, S., Morel, J. B., Bellini, C. and Vaucheret,
H. (2000). AGO1, QDE-2, and RDE-1 are related proteins
required for post-transcriptional gene silencing in plants, quelling in fungi,
and RNA interference in animals. Proc. Natl. Acad. Sci.
USA 97,11650
-11654.
Findley, S. D., Tamanaha, M., Clegg, N. J. and Ruohola-Baker,
H. (2003). Maelstrom, a Drosophila spindle-class gene,
encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine,
in nuage. Development
130,859
-871.
Foulkes, N. S., Mellstrom, B., Benusiglio, E. and Sassone-Corsi, P. (1992). Developmental switch of CREM function during spermatogenesis: from antagonist to activator. Nature 355, 80-84.[CrossRef][Medline]
Fujiwara, Y., Komiya, T., Kawabata, H., Sato, M., Fujimoto, H.,
Furusawa, M. and Noce, T. (1994). Isolation of a DEAD-family
protein gene that encodes a murine homolog of Drosophila vasa and its specific
expression in germ cell lineage. Proc. Natl. Acad. Sci.
USA 91,12258
-12262.
Habu, T., Taki, T., West, A., Nishimune, Y. and Morita, T.
(1996). The mouse and human homologs of DMC1, the yeast
meiosis-specific homologous recombination gene, have a common unique form of
exon-skipped transcript in meiosis. Nucleic Acids Res.
24,470
-477.
Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. and
Hannon, G. J. (2001). Argonaute2, a link between genetic and
biochemical analyses of RNAi. Science
293,1146
-1150.
Hay, B., Jan, L. Y. and Jan, Y. N. (1988). A protein component of Drosophila polar granules is encoded by vasa and has extensive sequence similarity to ATP-dependent helicases. Cell 55,577 -587.[Medline]
Hay, B., Jan, L. Y. and Jan, Y. N. (1990). Localization of vasa, a component of Drosophila polar granules, in maternal-effect mutants that alter embryonic anteroposterior polarity. Development 109,425 -433.[Abstract]
Imai, H. T., Matsuda, Y., Shiroishi, T. and Moriwaki, K. (1981). High frequency fo X-Y chromosome dissociation in primary spermatocytes of F1 hybrids between Japanese wild mice (Mus musculus molossinus) and inbred laboratory mice. Cytogenet. Cell Genet. 29,166 -175.[Medline]
Kennerdell, J. R., Yamaguchi, S. and Carthew, R. W.
(2002). RNAi is activated during Drosophila oocyte maturation in
a manner dependent on aubergine and spindle-E. Genes
Dev. 16,1884
-1889.
Kimura, T., Ito, C., Watanabe, S., Takahashi, T., Ikawa, M.,
Yomogida, K., Fujita, Y., Ikeuchi, M., Asada, N., Matsumiya, K. et al.
(2003). Mouse germ cell-less as an essential component for
nuclear integrity. Mol. Cell Biol.
23,1304
-1315.
King, F. J., Szakmary, A., Cox, D. N. and Lin, H. (2001). Yb modulates the divisions of both germline and somatic stem cells through piwi- and hh-mediated mechanisms in the Drosophila ovary. Mol. Cell 7,497 -508.[Medline]
Kneitz, B., Cohen, P. E., Avdievich, E., Zhu, L., Kane, M. F.,
Hou, H., Jr, Kolodner, R. D., Kucherlapati, R., Pollard, J. W. and Edelmann,
W. (2000). MutS homolog 4 localization to meiotic chromosomes
is required for chromosome pairing during meiosis in male and female mice.
Genes Dev. 14,1085
-1097.
Komiya, T., Itoh, K., Ikenishi, K. and Furusawa, M. (1994). Isolation and characterization of a novel gene of the DEAD box protein family which is specifically expressed in germ cells of Xenopus laevis. Dev. Biol. 162,354 -363.[CrossRef][Medline]
Kuramochi-Miyagawa, S., Kimura, T., Yomogida, K., Kuroiwa, A., Tadokoro, Y., Fujita, Y., Sato, M., Matsuda, Y. and Nakano, T. (2001). Two mouse piwi-related genes: miwi and mili. Mech. Dev. 108,121 -133.[CrossRef][Medline]
Lammers, J. H., Offenberg, H. H., van Aalderen, M., Vink, A. C., Dietrich, A. J. and Heyting, C. (1994). The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes. Mol. Cell Biol. 14,1137 -1146.[Abstract]
Lasko, P. F. and Ashburner, M. (1988). The product of the Drosophila gene vasa is very similar to eukaryotic initiation factor-4A. Nature 335,611 -617.[CrossRef][Medline]
Lin, H. and Spradling, A. C. (1997). A novel
group of pumilio mutations affects the asymmetric division of germline stem
cells in the Drosophila ovary. Development
124,2463
-2476.
Lipkin, S. M., Moens, P. B., Wang, V., Lenzi, M., Shanmugarajah, D., Gilgeous, A., Thomas, J., Cheng, J., Touchman, J. W., Green, E. D. et al. (2002). Meiotic arrest and aneuploidy in MLH3-deficient mice. Nat. Genet. 31,385 -390.[CrossRef][Medline]
Liu, D., Matzuk, M. M., Sung, W. K., Guo, Q., Wang, P. and Wolgemuth, D. J. (1998). Cyclin A1 is required for meiosis in the male mouse. Nat. Genet. 20,377 -380.[CrossRef][Medline]
Mahadevaiah, S. K., Turner, J. M., Baudat, F., Rogakou, E. P., de Boer, P., Blanco-Rodriguez, J., Jasin, M., Keeney, S., Bonner, W. M. and Burgoyne, P. S. (2001). Recombinational DNA double-strand breaks in mice precede synapsis. Nat. Genet. 27,271 -276.[CrossRef][Medline]
Matsuda, Y., Moens, P. B. and Chapman, V. M. (1992). Deficiency of X and Y chromosomal pairing at meiotic prophase in spermatocytes of sterile interspecific hybrids between laboratory mice (Mus domesticus) and Mus spretus. Chromosoma 101,483 -492.[Medline]
Mettus, R. V., Litvin, J., Wali, A., Toscani, A., Latham, K., Hatton, K. and Reddy, E. P. (1994). Murine A-myb: evidence for differential splicing and tissue-specific expression. Oncogene 9,3077 -3086.[Medline]
Meuwissen, R. L., Offenberg, H. H., Dietrich, A. J., Riesewijk, A., van Iersel, M. and Heyting, C. (1992). A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes. EMBO J. 11,5091 -5100.[Abstract]
Moens, P. B. and Spyropoulos, B. (1995). Immunocytology of chiasmata and chromosomal disjunction at mouse meiosis. Chromosoma 104,175 -182.[CrossRef][Medline]
Olsen, L. C., Aasland, R. and Fjose, A. (1997). A vasa-like gene in zebrafish identifies putative primordial germ cells. Mech. Dev. 66,95 -105.[CrossRef][Medline]
Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (2002). RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila. Mol. Cell 9,315 -327.[Medline]
Romanienko, P. J. and Camerini-Otero, R. D. (2000). The mouse Spo11 gene is required for meiotic chromosome synapsis. Mol. Cell 6,975 -987.[Medline]
Rongo, C. and Lehmann, R. (1996). Regulated synthesis, transport and assembly of the Drosophila germ plasm. Trends Genet. 12,102 -109.[CrossRef][Medline]
Roussell, D. L. and Bennett, K. L. (1993). glh-1, a germ-line putative RNA helicase from Caenorhabditis, has four zinc fingers. Proc. Natl. Acad. Sci. USA 90,9300 -9304.[Abstract]
Rubin, M. R., Toth, L. E., Patel, M. D., D'Eustachio, P. and Nguyen-Huu, M. C. (1986). A mouse homeo box gene is expressed in spermatocytes and embryos. Science 233,663 -667.[Medline]
Russell, L. D., Ettlin, R. A., Sinha Hihim, A. P. and Clegg, E. D. (1990). In Histrogical and Histpathological Evaluation of the Testis, pp. 119-161. Clearwater, FL: Cache River Press.
Scherthan, H., Jerratsch, M., Dhar, S., Wang, Y. A., Goff, S. P.
and Pandita, T. K. (2000). Meiotic telomere distribution and
Sertoli cell nuclear architecture are altered in Atm- and Atm-p53-deficient
mice. Mol. Cell Biol.
20,7773
-7783.
Schmidt, A., Palumbo, G., Bozzetti, M. P., Tritto, P.,
Pimpinelli, S. and Schafer, U. (1999). Genetic and molecular
characterization of sting, a gene involved in crystal formation and meiotic
drive in the male germ line of Drosophila melanogaster.
Genetics 151,749
-760.
Sweeney, C., Murphy, M., Kubelka, M., Ravnik, S. E., Hawkins, C.
F., Wolgemuth, D. J. and Carrington, M. (1996). A distinct
cyclin A is expressed in germ cells in the mouse.
Development 122,53
-64.
Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A., Timmons, L., Fire, A. and Mello, C. C. (1999). The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99,123 -132.[Medline]
Tanaka, S. S., Toyooka, Y., Akasu, R., Katoh-Fukui, Y.,
Nakahara, Y., Suzuki, R., Yokoyama, M. and Noce, T. (2000).
The mouse homolog of Drosophila Vasa is required for the development of male
germ cells. Genes Dev.
14,841
-853.
Tijsterman, M., Okihara, K. L., Thijssen, K. and Plasterk, R. H. (2002). PPW-1, a PAZ/PIWI protein required for efficient germline RNAi, is defective in a natural isolate of C. elegans. Curr. Biol. 12,1535 -1540.[CrossRef][Medline]
Toscani, A., Mettus, R. V., Coupland, R., Simpkins, H., Litvin, J., Orth, J., Hatton, K. S. and Reddy, E. P. (1997). Arrest of spermatogenesis and defective breast development in mice lacking A-myb. Nature 386,713 -717.[CrossRef][Medline]
Tourtellotte, W. G., Nagarajan, R., Auyeung, A., Mueller, C. and
Milbrandt, J. (1999). Infertility associated with incomplete
spermatogenic arrest and oligozoospermia in Egr4-deficient mice.
Development 126,5061
-5071.
Toyooka, Y., Tsunekawa, N., Takahashi, Y., Matsui, Y., Satoh, M. and Noce, T. (2000). Expression and intracellular localization of mouse Vasahomologue protein during germ cell development. Mech. Dev. 93,139 -149.[CrossRef][Medline]
Trauth, K., Mutschler, B., Jenkins, N. A., Gilbert, D. J., Copeland, N. G. and Klempnauer, K. H. (1994). Mouse A-myb encodes a trans-activator and is expressed in mitotically active cells of the developing central nervous system, adult testis and B lymphocytes. EMBO J. 13,5994 -6005.[Abstract]
Tsunekawa, N., Naito, M., Sakai, Y., Nishida, T. and Noce,
T. (2000). Isolation of chicken vasa homolog gene and tracing
the origin of primordial germ cells. Development
127,2741
-2750.
Tybulewicz, V. L., Crawford, C. E., Jackson, P. K., Bronson, R. T. and Mulligan, R. C. (1991). Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65,1153 -1163.[Medline]
Vaucheret, H., Beclin, C. and Fagard, M.
(2001). Post-transcriptional gene silencing in plants.
J. Cell Sci. 114,3083
-3091.
Watanabe, D., Yamada, K., Nishina, Y., Tajima, Y., Koshimizu,
U., Nagata, A. and Nishimune, Y. (1994). Molecular cloning of
a novel Ca(2+)-binding protein (calmegin) specifically expressed during male
meiotic germ cell development. J. Biol. Chem.
269,7744
-7749.
Williams, R. W. and Rubin, G. M. (2002).
ARGONAUTE1 is required for efficient RNA interference in Drosophila embryos.
Proc. Natl. Acad. Sci. USA
99,6889
-6894.
Wylie, C. (1999). Germ cells. Cell 96,165 -174.[Medline]
Yoon, C., Kawakami, K. and Hopkins, N. (1997).
Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and
4-cell-stage embryos and is expressed in the primordial germ cells.
Development 124,3157
-3165.
Yoshida, K., Kondoh, G., Matsuda, Y., Habu, T., Nishimune, Y. and Morita, T. (1998). The mouse RecA-like gene Dmc1 is required for homologous chromosome synapsis during meiosis. Mol. Cell 1,707 -718.[Medline]
Yuan, L., Liu, J. G., Zhao, J., Brundell, E., Daneholt, B. and Hoog, C. (2000). The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. Mol. Cell 5,73 -83.[Medline]
Zhao, G. Q. and Hogan, B. L. (1996). Evidence that mouse Bmp8a (Op2) and Bmp8b are duplicated genes that play a role in spermatogenesis and placental development. Mech. Dev. 57,159 -168.[CrossRef][Medline]