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First published online 17 December 2008
doi: 10.1242/dev.030858
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1 The State Key Laboratory of Molecular Biology, Institute of Biochemistry and
Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, 320 Yueyang Road, Shanghai 200031, China.
2 The Key Laboratory of Cell Biology, Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, 320 Yueyang Road, Shanghai 200031, China.
3 Department of Cell Biology, Institute of Basic Medical Sciences, Chinese
Academy of Medical Sciences, 5 Dong Dan San Tiao, Beijing 100005, China.
4 Max Planck Institute of Biophysical Chemistry, Department of Molecular Cell
Biology, Am Fassberg, 37077 Goettingen, Germany.
Author for correspondence (e-mail:
glxu{at}sibs.ac.cn)
Accepted 19 November 2008
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: RIM-BP3 (Rimbp3), Hook1, Spermatid manchette, Spermiogenesis, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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).
Spermatid development requires the function of genes expressed under
stringent temporal and spatial regulation. Using gene targeting technology,
around 20 male germ cell-specific genes have been identified to play important
roles during spermiogenesis (Matzuk and
Lamb, 2002). These genes are involved in the regulation of
acrosome biogenesis (Hrb, Gopc and Csnk2a2)
(Escalier et al., 2003;
Kang-Decker et al., 2001;
Yao et al., 2002), tail
formation (Tektin-t, Vdac3, Sepp1, Akap4 and Spag6)
(Olson et al., 2005;
Sampson et al., 2001;
Sapiro et al., 2002;
Shirohzu et al., 2002;
Tanaka et al., 2004) and
chromosomal packaging (Prm1, Prm2, Tnp1, Tnp2 and H1t2)
(Fuhrmann et al., 2001;
Martianov et al., 2005;
Tanaka et al., 2005;
Yu et al., 2000), among
others. Despite the identification of genes involved in various aspects of
spermiogenesis, the mechanism of nuclear shaping is still poorly
understood.
The manchette as a transient microtubular structure assembles concurrently
with the elongation and condensation of spermatid nucleus and growth of the
centrosome-derived axoneme (Kierszenbaum,
2002). It has been proposed that the microtubular manchette, which
could provide a track for vesicles to mobilize, contributes to the transport
of molecules to spermatid tail and facilitates nucleocytoplasmic transport
across the relocated nuclear pore complexes
(Kierszenbaum and Tres, 2004).
Vesicles might remain linked to the microtubules in a `holding pattern' until
microtubule-based motors recruit them for transport. A candidate
vesicle-microtubule adaptor is Hook1, a manchette-associated protein that is
truncated in the azh mutant mouse, which is characterized by abnormal
sperm heads (Mendoza-Lujambio et al.,
2002). Murine Hook1 belongs to the Hook protein family, which is
conserved from fly to mammals (Kramer and
Phistry, 1999). Previous work in Drosophila suggests that
the Hook protein plays a role in the endocytosis of transmembrane ligands or
their transport to multivesicular bodies
(Kramer and Phistry, 1996;
Kramer and Phistry, 1999).
Murine Hook1 is located in the manchette of developing spermatids
(Mendoza-Lujambio et al.,
2002). The loss of Hook1 function results in ectopic positioning
of microtubular structures within the spermatid
(Mendoza-Lujambio et al.,
2002), causing the mouse azh phenotype, which is
characterized by abnormal head shape (Cole
et al., 1988), head dislocation and spermatid tail coiling
(Mochida et al., 1999). Based
on these studies, Hook1 was proposed to be essential for the manchette
development and function.
We have characterized RIM-BP3 (Rimbp3 - Mouse Genome Informatics), a novel
manchette-associated protein. It belongs to the RIM-binding protein (RIM-BP)
family (Mittelstaedt and Schoch,
2007). RIM-BPs have been proposed to function as adaptors in the
process of vesicle fusion and release
(Hibino et al., 2002). RIM-BP1
and RIM-BP2 were identified as binding partners of the presynaptic active zone
proteins RIM1 and RIM2 as well as for voltage-gated Ca2+-channels
(Wang et al., 2000;
Hibino et al., 2002). All
three RIM-BP members are large multidomain proteins containing three
SH3-domains, two or three contiguous fibronectin type III domains, and
paralog-specific regions at the N terminus and between the clustered domains
(Mittelstaedt and Schoch,
2007). These signature domains and known interactions with
Ca2+-channels and RIM proteins suggest that RIM-BPs provide a
scaffold for other regulatory proteins. The RIM-BP proteins are particularly
conserved in invertebrates and vertebrates throughout the SH3- and FNIII
domains during evolution. Although invertebrates have one RIM-BP protein,
vertebrates have at least two RIM-BPs (RIM-BP1 and RIM-BP2), with RIM-BP3 only
found in therian mammals. In contrast to the predominant expression of RIM-BP1
and RIM-BP2 in the brain, RIM-BP3 is expressed almost exclusively in the
testis. Except for bioinformatic analysis data
(Mittelstaedt and Schoch,
2007), little is known about the function of RIM-BP3. Here, we
report that RIM-BP3 is associated with the manchette in elongating and
elongated spermatids, and is essential for normal sperm morphology and male
fertility. Furthermore, we find that RIM-BP3 forms complex with Hook1, which
is mutated in azh mice.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Antibodies
For the generation of RIM-BP3 specific antibodies, PCR fragments for
antigen regions (Fig. 1A) were
cloned into pET-41b (+) vector (Novagen) to produce glutathione
S-transferase (GST) fusion proteins. Antisera were raised and
affinity purified as described previously
(Ge et al., 2004). The
monoclonal anti-
-tubulin, anti-β-actin, anti-Flag and anti-HA
antibodies were from Sigma-Aldrich. The polyclonal anti-human Hook1 was from
Proteintech Group. The Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor
546 goat anti-mouse IgG were purchased from Molecular Probes.
Germ cell purification
Spermatogenic cells were isolated from adult mouse testes as described
previously (Bellve, 1993). When
required, discrete populations of germ cells were isolated using unit gravity
sedimentation velocity in 2-4% BSA gradient
(Bellve, 1993). The purity of
the isolated germ cells was monitored by phase-contrast microscopy.
Western blotting analysis
Mouse tissues and purified germ cells were solubilized in the mild tissue
lysis buffer as described (Herrada and
Wolgemuth, 1997). The protein concentration of supernatants was
determined with BioRad Assay Reagent (Bio-Rad), using BSA as the standard.
Extracts were diluted in SDS-loading buffer and analyzed by standard SDS-PAGE
and western blotting.
Construction of the RIM-BP3 targeting vector
To generate the RIM-BP3 targeting vector, DNA fragments for the
5' and 3' homology arms were amplified from mouse (129/SvEv)
genomic DNA by PCR. The left arm consists of a 1.2 kb 5' coding region
(nucleotides 73-1248 of the RIM-BP3 ORF) and the right arm is a 3.2
kb fragment covering the 3' coding region of 2759 bp and the downstream
UTR of 435 bp. The two arms were cloned into the pPNT vector in
NotI-XhoI and BamHI-EcoRI sites,
respectively, and confirmed by sequencing.
Generation of RIM-BP3 knockout mice
The targeting vector linearized with NotI was electroporated into
MPI-II ES cells (129Sv/Pas derived), which were subsequently cultured in the
presence of G418 and ganciclovir on mitotically inactivated MEF cells.
Resistant ES cell clones were picked 
6-8 days after drug selection. Two
targeted ES clones were identified from 225 clones by Southern blotting
analysis using the 3' external probe. The 3' probe was prepared by
PCR (forward, 5'-GAATATTTCGGGAGTTAAAGCATGGC-3'; reverse,
5'-CTTACAAAGCATCATGGGAACACCAG-3'). Positive ES clones identified
by Southern blotting were aggregated with CD1 (ICR) morulae for 24 hours and
then transferred into pseudopregnant foster mothers. Male chimeras were bred
first with ICR females to obtain mutant mice on 129SvxICR mixed
background. The chimeras that produced high percentages of offspring with
agouti fur were also bred with 129Sv/Pas females to obtain inbred mice.
Heterozygotes were intercrossed to produce homozygous offspring. Mouse
genotypes were identified by Southern analysis of tail DNA using 3'
external probe and by PCR using primers as follows: RIM-BP3 (+756),
5'-ATCTTTGGCAACAGCACATTCCT-3'; and RIM-BP3 (-3253),
5'-TTTCTTGGCTTGCGGTTTGGAGT-3'. Mice on mixed 129SvxICR
genetic background were used except where indicated.
Fertility test and sperm counts
RIM-BP3-/- males at 8-9 weeks of age were housed with
6- to 8-week-old virgin ICR females for 2 months. The numbers and sizes of
litters were recorded. Wild-type and heterozygous males were used for
comparison. Females paired with homozygous males that never produced litters
were subsequently placed with males of proven fertility and, if they produced
a litter, were considered to be fertile and included in the study; otherwise,
they were excluded. Student's t-test was used to compare averages in
different experimental groups and P<0.05 was considered to be
significant.
For isolation of sonication-resistant spermatid nuclei, which represent
step 12 to 16 spermatids, we followed the procedure as described previously
(Yu et al., 2000) with slight
modifications. Briefly, the testes from two mice were homogenized in 4 ml of
water containing protease inhibitors (1 mM PMSF, 1 µg/ml leupeptin and 1
µg/ml pepstatin A) using a glass-Teflon homogenizer and then sonicated for
6 minutes of total elapsed time (15 seconds on, 15 seconds off) to remove
sonication-sensitive cells. Spermatid nuclei were separated from tissue debris
by passing the mixture through an 80 µm mesh filter. All counts were
performed using a hemacytometer.
Immunofluorescence microscopy
Squashed samples were prepared as described
(Kotaja et al., 2004). Samples
were then fixed sequentially in cold methanol for 10 minutes, in acetone for
30 seconds and then in PBS containing 4% paraformaldehyde for 15 minutes.
After fixation, samples were permeablized with 0.5% Triton X-100 for 15
minutes and blocked for at least 1 hour at room temperature with blocking
solution (PBS with 0.3% Triton X-100 and 1% BSA). Indirect immunofluorescence
staining to study the localization of RIM-BP3, Hook1 and -tubulin in
testicular germ cells was carried out as described for cultured cells
(Ge et al., 2004) and images
of stained cells were captured with a Leica TCS SP2 laser confocal
microscope.
Electron microscopy
Testes and cauda epididymides from wild-type and
RIM-BP3-/- mice were examined with electron microscopy.
For scanning electron microscopy of mature sperm, cauda epididymides were
dissected and minced in 0.1 M phosphate buffer (pH 7.4), allowing the sperm to
be released into the supernatant. The sperm were fixed in 2.5% glutaraldehyde
solution in phosphate buffer, collected on poly-L-lysine-coated glass cover
slips, post-fixed in osmium tetroxide, dehydrated in a graded ethanol series,
subjected to critical point drying and then coated with gold/palladium.
Samples were examined with a JEOL JSM-6360LV Scanning Electron Microscope. For
transmission electron microscopy, samples were fixed in 4% paraformaldehyde
containing 0.05% glutaraldehyde in 0.1 M phosphate buffer, and then post-fixed
in 1% osmium tetroxide. Dehydration was carried out in ethanol and the samples
were embedded in Epon 812. Ultrathin sections were counterstained with uranyl
acetate and lead citrate, and examined with a JEOL JEM-1230 transmission
electron microscope.
Affinity purification of RIM-BP3-associated proteins
Affinity purification was carried out as described
(Li et al., 2007a;
Li et al., 2007b) with slight
modifications. Spermatids were purified from wild-type and
RIM-BP3-/- testes and lysed in lysis buffer [50 mM
Tris-HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% (v/v) NP-40, 0.25% (w/v) sodium
deoxycholate, 1 mM DTT, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml
pepstatin A and 1 mM PMSF]. After brief sonication, the lysates were
centrifuged at 20,000 g at 4°C for 10 minutes. The
supernatants were incubated with anti-RIM-BP3 (N) and anti-RIM-BP3 (C)
antibodies, respectively, for 4 hours at 4°C with gentle rotation. The
immuno-complex was captured by incubation with Protein A-agarose beads (GE
Healthcare) at 4°C overnight with gentle rotation. The beads were washed
four times with lysis buffer. Bound proteins were analyzed by SDS-PAGE and
silver staining. Specific bands were excised and the protein sequence was
determined using electrospray ionization LTQ tandem mass spectrometry at the
Research Center for Proteome Analysis, Shanghai Institutes for Biological
Sciences.
Co-immunoprecipitation
Decapsulated testes or HEK-293T cells transfected with pFlag-CMV2-RIM-BP3
and pcDNA3-HA-Hook1 or pcDNA3-HA-Hook1azh were lysed in lysis
buffer. Co-immunoprecipitation of RIM-BP3 and Hook1 was carried out as
previously described (Xie et al.,
2006). The immuno-complex was analyzed by SDS-PAGE and standard
western analysis.
Yeast two-hybrid assay
For mapping the interaction regions in RIM-BP3 and Hook1, a yeast
two-hybrid assay was carried out as previously described
(Li et al., 2007c). RIM-BP3
fragments were cloned in-frame with the GAL4 activation domain (GAL4AD) on the
prey vector pGADT7 (Clontech) and Hook1 fragments were fused with the GAL4 DNA
binding domain (GAL4BD) on the bait vector pGBKT7. The bait and prey
constructs were co-transformed into the yeast strain AH109 and colonies were
selected and assayed on SD medium with or without adenine, His, Leu, Trp
according to the recommended protocol (Clontech).
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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). In the N-terminal half, RIM-BP3 has a unique SbcC domain,
which may have ATPase activity (Connelly
et al., 1998; Connelly and
Leach, 1996).
We examined the expression of RIM-BP3 in adult mouse tissues by
northern blotting analysis. A RIM-BP3 transcript of about 5 kb was
detected exclusively in the testis (Fig.
1B). The size of the transcript is consistent with a previous
report (Mittelstaedt and Schoch,
2007). We then determined the expression of the RIM-BP3 protein in
different tissues by western blotting analysis using polyclonal antibodies
with validated specificity (see Fig. S1 in the supplementary material).
Consistent with the northern blotting result, the RIM-BP3 protein was also
detected exclusively in the testis (Fig.
1C).
In murine testis, the seminiferous epithelium undergoes postnatal
development (Bellve et al.,
1977; Malkov et al.,
1998). We then examined the expression pattern of RIM-BP3 at
various stages of this process by western analysis using testicular extracts
from juvenile and adult mice (Fig.
1D). The earliest stage at which RIM-BP3 was detected was
postnatal day 20, when spermatids first appear. Thereafter, the RIM-BP3
protein level increased until adulthood. Thus, RIM-BP3 is expressed most
abundantly in postmeiotic germ cells.
We further investigated the types of postmeiotic germ cells that express RIM-BP3 by performing western analysis on protein extracts from purified germ cells. The purity of each type of germ cells was determined to be at least 90% by microscopic analysis (data not shown). Western blotting analysis shows that RIM-BP3 is highly expressed in elongate spermatids and weakly expressed in pachytene spermatocytes and round spermatids (Fig. 1E). Interestingly, it was detectable in residual bodies but not in mature sperm. These data show that RIM-BP3 is a testis-specific protein predominantly expressed at the haploid stage and suggest that it plays a specialized role in spermiogenesis.
The RIM-BP3 protein is associated with the manchette in developing spermatids
We then investigated subcellular distribution of RIM-BP3 in developing
spermatids by performing indirect immunofluorescence analysis using squashed
samples of seminiferous tubules (Kotaja et
al., 2004). The expression of RIM-BP3 was first evident in step 9
spermatids, persisted until step 13 and finally disappears at step 16
(Fig. 2). In these spermatids,
an extensive colocalization with the manchette was observed by
co-immunostaining with anti -tubulin antibody. The RIM-BP3 antibody is
specific, as shown by the absence of signal in the knockout mouse samples (see
Fig. S2 in the supplementary material). This expression pattern of RIM-BP3
correlates well with the dynamic changes of the manchette
(Clermont et al., 1993),
suggesting a specialized role for RIM-BP3 in the development of late-stage
spermatids.
Targeted disruption of the RIM-BP3 gene results in male infertility
To investigate the role of RIM-BP3 during spermiogenesis, we disrupted its
function in mice by gene targeting. In the targeted allele, the entire
sequence encoding the SbcC domain was replaced by a cassette of the
PGK-neomycin resistance gene (Fig.
3A). The generation of mutant mice was confirmed by PCR and
southern blotting analysis of tail genomic DNA
(Fig. 3B). Western analysis
with two different antibodies confirmed the absence of the RIM-BP3 protein in
the testis of mutant mice (Fig.
3C).
Interbreeding of heterozygous mice yielded an expected Mendelian ratio
(39:92:44) of RIM-BP3+/+, RIM-BP3+/-
and RIM-BP3-/- in F2 offspring on 129SvxICR genetic
background, suggesting that the RIM-BP3 deficiency caused no
embryonic lethality. Adult mutant mice were normal in appearance, health and
developed with no identifiable anatomical or behavioral abnormalities. Given
the expression of RIM-BP3 in the testis, we studied fertility in
RIM-BP3-null mice by mating them with wild-type females. The mutant
males were found to be infertile, although they were sexually active and
produced vaginal plugs in female partners. Over a 2-month period of continuous
mating, only one female became pregnant and gave birth to one pup
(Table 1). The heterozygous
males showed normal fertility and produced normal litter sizes
(Table 1 and data not shown).
To determine whether the poor reproductive performance of homozygous males was
due to a failure of fertilization, they were mated with superovulated normal
females. Eggs were collected from the ampullae of the oviducts of mated
females and scored for fertilization based on the presence of a male
pronucleus (Borghei et al.,
2006). We observed that 92.4% eggs harvested from the females
plugged by wild-type males were fertilized. By contrast, only 9.3% eggs
harvested from the females plugged by RIM-BP3-/- males
were fertilized (see Table S1 in the supplementary material). These data
reveal that the ability of RIM-BP3-/- males to fertilize
eggs in vivo was severely compromised. In addition, this abnormality was not
dependent on genetic background, as a lower fertilization rate (2%) by the
mutant males was also observed in pure 129Sv strains.
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Spermiogenesis is impaired in RIM-BP3 mutant mice
Disruption of RIM-BP3 in mice caused male infertility owing to
abnormal sperm heads. To characterize the morphological defects in the
developing germ cells in detail, we performed transmission electron
microscopic study of testis sections. The histological analysis of the
seminiferous tubules above revealed spermatogonia, spermatocytes and Sertoli
cells with normal morphology, whereas spermatids of step 12 displayed deformed
nuclei (see Fig. S3 in the supplementary material). This observation was
confirmed by transmission electron microscopic analysis. The spermatids from
RIM-BP3-deficient mice displayed various defects in the acrosome,
acroplaxome, manchette and nucleus. The earliest abnormality was seen
occasionally in step 5 round spermatids. Proacrosomal vesicles derived from
Golgi apparatus could not fuse to form a single large acrosomal vesicle and
the acroplaxome was bent at several sites
(Fig. 5B). However, from a
structural point of view, elongating and elongated spermatids were the most
affected with a variety of abnormalities. About 10% of elongate spermatids
showed discontinuity of acrosome in the plane of section (arrowhead in
Fig. 5D,F,K). This occurrence
was a consequence of a deficient fusion of proacrosomal vesicles during the
early stages of acrosome biogenesis. Moreover, the acroplaxome was severely
deformed. As a consequence, the acrosome was misplaced and the outline of the
nucleus under the acroplaxome was irregular
(Fig. 5F,G). As spermatid
elongation proceeded across step 10/11, abnormal development of the manchette
could be clearly seen. The manchette in RIM-BP3-null spermatids
presented symmetric development with a conical shape
(Fig. 5F) and, in appropriate
sections, invagination of membrane-bound microtubules could be observed
(Fig. 5H). Some elongated
spermatids also showed an abnormal positioning of the perinuclear ring
anterior to the marginal ring of the acroplaxome. Consequently, the manchette
covers a part of the acrosome (Fig.
5I). At the end of spermiogenesis, the majority of condensed
spermatids exhibited detached acrosome, deformed nucleus and expanded
perinuclear space with some unidentified materials
(Fig. 5K,L).
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-tubulin antibody. Consistent
with TEM analysis, the manchette became abnormal in elongating
RIM-BP3-deficient spermatids (see Fig. S4, left in the supplementary
material). At the early stage of differentiation (step 9), the spermatids of
mutant mice had a normal bell-shaped manchette covering a typical ovoid-shaped
nucleus. However, by step 11/12, the manchette and nucleus of most mutant
spermatids had conically symmetric caudal ends that were different from the
asymmetric ones of wild-type spermatids. In addition, the manchette oriented
parallel to the longitudinal axis of the mutant spermatid head, whereas in the
normal spermatids the manchette oriented at 45° to the principal axis of
the spermatid head (see Fig. S4, right in the supplementary material).
The RIM-BP3 protein interacts with Hook1
In order to uncover the molecular consequences of the lack of RIM-BP3 in
spermiogenesis, we set out to identify RIM-BP3-associated protein(s) in
spermatids. Immunoprecipitation with two different RIM-BP3 antibodies pulled
down a prominent protein (about 100 kDa) co-purified from wild-type
spermatids, but not from RIM-BP3-deficient spermatids
(Fig. 6A). Mass spectrometry
analysis revealed that the co-purified protein is Hook1, a
manchette-associated protein in developing spermatids
(Mendoza-Lujambio et al.,
2002). The confirmation of Hook1 as an interacting protein was
provided by performing a co-immunoprecipitation (Co-IP) assay on whole testis
extracts. Hook1 was detected by western analysis in the immunoprecipitated
proteins obtained with anti-RIM-BP3 antibody from wild-type but not from the
RIM-BP3-deficient testis extract
(Fig. 6B).
To define domains responsible for the interaction, we constructed a series of deletion mutants of both proteins for yeast two-hybrid assays. We found that yeast expressing the C-terminal part (amino acids 830-1606) of RIM-BP3 could grow on the selective medium (Fig. 6C), indicating that this region containing the SH3 and FNIII domains is required for the interaction with Hook1. Similarly, we mapped the interaction region in Hook1. We found that only the coiled-coil domain (amino acids 165-661) enabled yeast cells to grow on the selective medium, though more slowly than the yeast expressing the full-length Hook1 (Fig. 6D). These findings indicate that a large part of Hook1, including the coiled-coil domain is involved in the interaction with RIM-BP3.
Mutation of Hook1 in azh mice disrupts interaction with RIM-BP3
Electron microscopy and immunostaining analyses revealed aberrant
manchettes in RIM-BP3 deficient spermatids
(Fig. 5 and see Fig. S4 in the
supplementary material). These abnormalities are reminiscent of those observed
in azh mutant mice, in which a deletion of a 2 kb genomic sequence of
the Hook1 gene leads to truncation of the entire C-terminal part
(Cole et al., 1988;
Mendoza-Lujambio et al., 2002)
(Fig. 7A). Based on the
physical association of RIM-BP3 and Hook1 in vivo and in vitro, we reasoned
that the truncated Hook1azh might lose interaction with RIM-BP3,
which could be an underlying mechanism for the ectopic positioning of the
manchette. Therefore, co-immunoprecipitation was carried out with the two
proteins expressed in transfected 293T cells. Indeed, we found that the mutant
Hook1 had lost its ability to interact with RIM-BP3
(Fig. 7B).
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Spermiogenesis is an intricate process by which a round haploid spermatid
cell differentiates into a mature elongated spermatozoon. There are multiple
mechanisms proposed to ensure the correct formation and organization of
specialized structures of a mature spermatozoon
(Meistrich, 1993;
Toshimori and Ito, 2003). The
acrosome-acroplaxome-manchette complex was proposed to play a major role in
nuclear shaping (Kierszenbaum and Tres,
2004). In wild-type spermatids, the acrosome is tightly bound to
the acroplaxome plate, which in turn is anchored to the nuclear envelope of
the elongating spermatid nucleus. The acroplaxome may provide a scaffold to
modulate exogenous constriction forces generated by Sertoli cell F-actin hoops
during spermatid head elongation. The manchette is composed of bundles of
microtubules that emerge from the perinuclear ring, which is organized
subjacent to the marginal ring of the acroplaxome. Both the marginal ring and
the perinuclear ring reduce their diameter along with the elongation of the
spermatid nucleus (Kierszenbaum and Tres,
2004). Our finding that targeted disruption of RIM-BP3
resulted in complex abnormalities in the acrosome-acroplaxome-manchette
complex in association with sperm head deformity underscores the importance of
this complex in spermiogenesis. The variable anomalies include: (1) deficient
tethering and fusion of proacrosomal vesicles in round spermatids, which
results in the discontinuities of the acrosome in elongating and elongated
spermatids; (2) distorted acroplaxome plate and misplaced acrosome; (3)
malformed manchette in elongate spermatids such as the appearance in a
symmetric conical shape, invagination into the nucleus and the ectopic
positioning of the perinuclear ring anterior to the marginal ring. These
perturbations in acrosome-acroplaxome-manchette complex are conceivably the
major causes for malformation of spermatid heads.
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) and
CLIP-170, a microtubule plus-end-tracking protein
(Akhmanova et al., 2005) as
well as in animals treated with microtubule-disrupting agents such as
colchicine and carbendazim (Handel,
1979; Nakai et al.,
1998).
The role of RIM-BP3 in the control of microtubule organization might be
mediated by Hook1. We have demonstrated that Hook1 is stably associated with
RIM-BP3 in elongate spermatids. Hook1 contains a microtubule-binding domain in
its N-terminal part and an organelle-binding domain in the C-terminal region.
This domain organization may allow Hook1 to establish a link between the
manchette microtubules and other cellular organelles, e.g. the nuclear
envelope (Mendoza-Lujambio et al.,
2002). RIM-BP3-deficient spermatids often show the
ectopic positioning of manchette, whereas Hook1 remains associated to the
malformed manchette (Fig. 5;
see Fig. S4 in the supplementary material). This suggests that RIM-BP3 has no
effect on the binding of Hook1 to the manchette. Rather, it may modulate the
interaction of Hook1 with certain organelles to which the manchette should be
anchored. Interestingly, the phenotypes of RIM-BP3-deficient mice,
such as abnormal sperm heads, detached tails and aberrant manchette
positioning, are very similar to those of azh/azh mice, which are
caused by loss of Hook1 function (Cole et
al., 1988; Mendoza-Lujambio et
al., 2002; Mochida et al.,
1999). In azh/azh mice, exons 10 and 11 in the
Hook1 gene are deleted, resulting in a truncated protein containing
only the microtubule-binding domain and a small part of the coiled-coil domain
(Mendoza-Lujambio et al.,
2002). Consistent with requirement of the coiled-coil domain for
the interaction with RIM-BP3 (Fig.
6D), the truncated Hook1azh mutant protein has lost the
ability to interact with RIM-BP3 (Fig.
7). As the microtubule-binding domain is intact in
Hook1azh, we assume that the azh/azh phenotype related to
the manchette might arise due to abolished interaction of Hook1 with RIM-BP3.
The overall phenotypic similarity of azh/azh and
RIM-BP3-/- mice could reflect the importance of the
interaction between Hook1 and RIM-BP3 in positioning the manchette along the
nuclear surface. As an adaptor molecule, RIM-BP3 might link with the manchette
through Hook1 to ensure correct positioning of the manchette. The disruption
of a common pathway involving Hook1 and RIM-BP3 might be responsible for the
abnormal spermatid development observed in both azh and
RIM-BP3 mutant mice.
|
). KIFC1,
another kinesin member that associates with a complex containing nucleoporin
NUP62, has been implicated in acrosome/manchette transport along the nuclear
membrane (Yang et al., 2006).
Further circumstantial evidence comes from the fact that both RIM-BP1 and
RIM-BP2, the two highly conserved parologs that are expressed in the brain,
are involved in vesicle transport in neuronal cells
(Hibino et al., 2002).
Finally, both RIM-BP3 interacting partners Hook1 and KIF3B have been connected
with Rab proteins involved in endocytosis
(Imamura et al., 2003;
Luiro et al., 2004). All these
proteins, including RIM-BP3, Hook1, Kinesins, RIMs and Rabs, seem to be
networked for complex intracellular trafficking and transport.
The highly conserved human RIM-BP3 protein is expected to have similar
function in spermiogenesis. Human male infertility is often related to
abnormal spermatozoon head shape (Baccetti
et al., 1989; Baccetti et al.,
1984; Toyama et al.,
2000; Toyama et al.,
1995). Although so far there is no report of infertile men with
mutations on chromosome 22q11.21, where the three copies of the human
RIM-BP3 gene are located, our study in the mouse suggests that the
human RIM-BP3 could serve as a candidate gene for mutational analysis
in infertile individuals with teratozoospermia.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/3/373/DC1
|
Footnotes |
|---|
* These authors contributed equally to this work 
Present address: Ludwig Maximilians University Munich, Department of
Biology II, 82152 Martinsried, Germany
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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