1 Department of Genetics, Biology and Biochemistry, Via Santena 5 bis, Torino,
Italy
2 Department of Histology and Medical Embryology, University of Rome "La
Sapienza", Rome, Italy
Author for correspondence (e-mail:
ferdinando.dicunto{at}unito.it)
Accepted 5 September 2002
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Summary |
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Key words: Spermatogenesis, Cytokinesis, Rho GTPases, Cytoskeleton, Apoptosis
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Introduction |
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In the past few years, remarkable progress has been made in understanding
how cytokinesis is controlled in animal cells
(Glotzer, 2001;
Hales et al., 1999
). In
particular, small GTPases of the Rho family have been recognized as playing a
crucial role in this process (Glotzer,
2001
; Hales et al.,
1999
). Rho GTPases are molecular switches that control
cytoskeletal dynamics, cycling between an active GTP-bound, and an inactive,
GDP-bound state (Hall, 1998
;
Van Aelst and Souza-Schorey,
1997
). This conformational transition is tightly regulated by
guanine-nucleotide exchange factors (GEFs), which stimulate the binding to
GTP, and GTPase-activating proteins (GAPs), which increase their basal GTPase
activity (Van Aelst and Souza-Schorey,
1997
). Signal transduction by Rho GTPases is mediated by a complex
network of effector molecules, which specifically bind to the active
conformation and change their biological properties upon binding
(Bishop and Hall, 2000
).
Cytokinesis is blocked in different model organisms by inhibition of Rho
signaling (Drechsel et al.,
1997
; Kishi et al.,
1993
; Nakano et al.,
1997
). Moreover, Rho regulators and effectors specifically
involved in cytokinesis have been recently isolated
(Hirose et al., 2001
;
Jantsch-Plunger et al., 2000
;
Kosako et al., 1999
;
Lehner, 1992
;
Madaule et al., 1998
;
Tatsumoto et al., 1999
).
Citron kinase (CIT-K) is a myotonin-related protein highly enriched in the
cleavage furrow and in the midbody of dividing cells
(Madaule et al., 1998
). The
first in vitro studies on this molecule suggested that it could be one of the
principal ubiquitous downstream effectors of Rho during cytokinesis
(Madaule et al., 1998
).
However, the subsequent production of knockout (-/-) mice showed
that CIT-K is functionally required for cytokinesis only by specialized cell
populations, like proliferating neuronal precursors
(Di Cunto et al., 2000
).
Here we report that CIT-K is required for cytokinesis by mouse spermatogenic precursors, which become multinucleated and display increased apoptosis in CIT-K-/- animals. As a consequence, the seminiferous tubules of these mice are characterized by progressive loss of undifferentiated germ cells and complete absence of spermatocyte differentiation. Despite this severe testicular phenotype, the ovogenesis of CIT-K mutant females is apparently preserved. Our results strongly suggest that CIT-K is a key regulator of the cytokinesis endgame in developing and differentiating testicular germ cells.
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Materials and Methods |
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Histology
The genotype of mice was determined by PCR as previously described
(Di Cunto et al., 2000). The
sex of 12.5-day-old (E12.5) embryos was determined by PCR with SRY-specific
oligonucleotides (Kunieda et al.,
1992
). For light microscopy, embryos were fixed by immersion in
fixative solution (4% paraformaldehyde in PBS), whereas postnatal mice were
deeply anaesthetized and transcardially perfused with fixative solution.
Testes and ovaries were removed, postfixed for at least 12 hours, embedded in
paraffin, and sectioned at 5 µm. Sections were stained with
hematoxylin-eosin. For electron microscopy, testes were fixed with 2.5%
glutaraldehyde in 0.1 M cacodylate buffer, pH. 7.4, postfixed in osmium
tetroxide, dehydrated in ethanol and embedded in epoxy resin. Semi-thin
sections were stained with toluidine blue. Ultra-thin sections were viewed
using an Hitachi 7000 transmission electron microscope.
Alkaline phosphatase staining
Detection of endogenous alkaline phosphatase (AP) activity was carried out
as described previously (Zackson and
Steinberg, 1988), with minor modifications. Briefly, embryos were
fixed by immersion in fixative solution, embedded in paraffin and sectioned at
5 µm. After rehydration sections were permeabilized for 10 minutes with
0.1% Triton in TBS and incubated for 40 minutes in AP buffer (100 mM NaCl, 5
mM MgCl2, 100 mM Tris pH 9.5) at room temperature. Two chromogenic
substrate systems were employed to visualize the activity: 165 µg/ml
5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) and 330 µg/ml nitro blue
tetrazolium (NBT, Sigma) were added to AP buffer. The reaction was performed
in the dark at room temperature and stopped in PBS.
Immunohistochemistry
The TUNEL assay was performed as previously described
(Migheli et al., 1999). For
BrdU immunostaining, mice were labeled with a single injection of BrdU (Sigma)
at 100 mg/kg and sacrificed after 1 hour. Tissues were fixed in Carnoy,
embedded in paraffin and sectioned at 5 µm. After rehydration, sections
were treated for 20 minutes with 1N HCl. Anti BrdU antibodies (Chemicon) were
used at a 1:100 dilution. For detection of CIT-K, sections were treated for 30
minutes with trypsin 0.1% in PBS, rinsed in HCl 0.5N at 4°C and then
incubated with affinity-purified rabbit polyclonal anti-Citron antibodies at
1:200. The other immunostainings were performed as described previously
(Migheli et al., 1999
) using
the following antibodies: anti-activated Caspase-3 (1:500, New England
Biolabs); anti-Cyclin D1 (1:1000, Santa Cruz). In every case, sections were
counterstained with hematoxylin.
Quantitative analysis
All the quantitative analyses were performed on inbred 129Sv
CIT-K+/+ and -/- mice. Numbers represent the average of
at least three independent animals per genotype. For the embryonic gonads, the
total number of germ cells was determined by counting all the
alkaline-phosphatase-positive cells on serial sections. For post-natal testes,
the volume of gonads was estimated on serial sections using the Image Pro Plus
program (Olympus). The average density of gonocytes and undifferentiated
spermatogonia per surface area was determined on at least six sections and
then referred to the estimated volume. The proliferative and apoptotic indices
were determined at E14.5 and P4 as the ratio between the number of tubular
immunohistochemistry-positive cells and the sum of gonocytes and
spermatogonia, counted on at least six sections.
The percentage of polyploid germ cells was determined at P4 on semi-thin sections of seminiferous tubules; the total number of analyzed tubules was 274 for +/+ and 296 for -/- testes.
For determination of the percentage of binucleated apoptotic cells, caspase-3-positive intratubular elements at E14.5 and P4 were scored at high power as early or late apoptotic cells on the basis of the absence/presence of nuclear condensation and/or fragmentation. Early apoptotic cells were then scored as mononucleate or binucleate.
In every case, the statistical significance of differences between CIT-K and control samples was assessed by the Student t-test.
Reverse transcription (RT)-PCR
Total RNA was isolated from 8- and 14-day-old (P8 and P14) testes of
CIT-K+/+ and -/- 129Sv inbred mice (three animals per
age per genotype) using the Rneasy kit (Qiagen). Single-strand cDNAs were
prepared by reverse transcription of 2 µg of testis total RNA with MMLV
reverse transcriptase (GIBCO/BRL). Each PCR amplification was performed in a
50 µl reaction volume using as a 5 µl template of the room temperature
reaction. Amplification primers were synthesized as previously described
(Tanaka et al., 2000). The
linear amplification range was determined for each primer pair, and control
for equal loading was performed using the 18S primers of the QuantumRNA kit
(Ambion), according to the manufacturer's specifications.
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Results |
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Inactivation of CIT-K results in specific depletion of testicular
germ cells
Mice heterozygous for a null mutation of the CIT-K gene display
normal life span and fertility. By contrast, CIT-K-/- mice are
characterized by reduced growth and die during the first three postnatal weeks
due to lethal epilepsy (Di Cunto et al.,
2000). At P14, if compared with +/+ and +/-
controls, the body weight of -/- mice is reduced by approximately
25% (Di Cunto et al., 2000
).
Autopsy performed at the same age revealed that most somatic organs, with the
only exception being the brain, display a similar size reduction but maintain
a normal histological structure (Di Cunto
et al., 2000
). However, the volume of CIT-K-/- testes
was reduced by approximately 70% (Fig.
2A), thus suggesting a specific impairment. Using histological
examination in control animals, mature spermatocytes appeared to differentiate
in the innermost layer of the seminiferous tubules
(Fig. 2B). By contrast, testes
of the knockout mice were severely depleted of proliferating and
differentiating spermatogenic precursors
(Fig. 2C). The testicular
epithelium was dramatically simplified and appeared to consist mostly of a
single row of Sertoli cells (Fig.
2C). A comparable number of Leydig cells was detected in the
interstitial stroma of-/- and control animals. Other reproductive
organs such the vas deferens and epididymus appeared to be normal in the
knockouts (data not shown). Strikingly, histological analysis of P14 ovaries
from knockout and control females did not show gross morphological differences
among genotypes (Fig. 2D,E). In
particular, several resting and growing follicles were detected throughout the
ovarian cortex of-/- animals
(Fig. 2D,E). Since no
significant differences were observed between +/+ and
+/- controls, all the subsequent analysis were performed only on
+/+ and -/- animals.
|
Analysis of the spermatogenic block in CIT-K-/-
testes
In order to elucidate whether postnatal testes of CIT-K knockout mice still
contain spermatogenic cells and to establish at which stage of differentiation
the observed depletion occurs, we carried out semi-quantitative RT-PCR of
different spermatogenic markers (Tanaka et
al., 2000). At P8, analysis of transcripts that appear in early
spermatogenic cells before they reach the spermatocyte stage, such as the
mouse Vasa homologue (Mvh) and A-Myb
(Mettus et al., 1994
;
Tanaka et al., 2000
), showed
no differences among genotypes (Fig.
3A). By contrast, the mRNAs of Dmc1 and Mlh1, which are expressed
in spermatocytes before they reach the pachytene stage
(Baker et al., 1996
;
Habu et al., 1996
), were
dramatically reduced in the knockout samples
(Fig. 3A). Strikingly, the
expression of Calmegin, which normally starts in pachytene spermatocytes
(Watanabe et al., 1994
), was
not detectable in the knockouts (Fig.
3A). At P14 the relative levels of spermatocyte marker expression
was comparable to P8 samples (data not shown), and Mvh and A-Myb mRNAs were
still detectable in the knockout testes
(Fig. 3B). However, if compared
with controls, their expression levels were clearly reduced in the
-/- samples (Fig.
3B). Taken together, these observations indicate that the
simplified testicular epithelium of CIT-K-/- mice still contains
germinal cells, which are completely unable to differentiate as meiotic
spermatocytes and undergo progressive depletion.
|
In order to address whether male germ cells of CIT-K knockout mice are
already compromised during embryonic development, serial sections
of+/+ and-/- male embryos were analyzed at different
developmental stages by histology and alkaline phosphatase histochemistry. As
shown in Fig. 3C,D PGCs were
easily detected in the genital ridges of both control and knockout embryos,
and no ectopic alkaline-phosphatase-positive cells were observed, thus
indicating that the specification and migration of spermatogenic precursors
are not severely affected by the absence of CIT-K. However, their number was
significantly reduced in the knockout mice as early as E12.5
(Fig. 3C,D). Accordingly, at
E14.5 the number of gonocytes in the lumen of CIT-K-/- seminiferous
cords was reduced to approximately 60% of the controls
(Fig. 3G). After birth, cells
displaying the morphological features of gonocytes, and type A spermatogonia
were easily detectable on histological sections of knockout testes (data not
shown), in good agreement with the RT-PCR analysis. To further confirm the
presence of these cells, we performed on the same samples immunohistochemical
detection of cyclinD1, which has been recently recognized as a specific marker
(Beumer et al., 2000). As shown
in Fig. 3F, cyclinD1-positive
cells were detected in the tubular lumen and on the basal membrane of the
knockout tubules, further indicating that both gonocytes and type A
spermatogonia were present. However, if compared with the embryonic testes,
the relative number of germinal cells was further decreased, being only 30% of
the control at P4 (Fig. 3E,G). These data indicate that, besides the differentiating spermatogonia, even the
PGCs and the gonocytes are significantly compromised by the absence of
CIT-K.
Apoptotic loss of CIT-K-/- spermatogenic precursors
To investigate if the observed depletion of testicular germ cell precursors
was caused by reduced proliferation or increased cell death, the apoptotic and
proliferative indices of testicular tubular cells were determined in both
embryonic and postnatal testes of wild-type and knockout animals by
anti-activated Caspase-3 immunostaining and BrdU labeling, respectively.
As shown in Fig. 4A,B, the number of apoptotic cells detected in the lumen of seminiferous cords at E14.5 was clearly increased in sections of -/- versus +/+ embryos. The relative increase was even more significant when the absolute counts were normalized for the reduced number of gonocytes observed in the knockouts (Fig. 4E).
|
Strikingly, despite the reduced numbers of gonocytes, the BrdU labeling index of knockout seminiferous cords was increased by approximately 2.5 fold (Fig. 4C,E). Similar results were obtained at P4 (data not shown).
Taken together, the above observations indicate that the striking depletion of testicular germ cells occurring in CIT-K knockout mice is caused by progressive apoptotic loss of embryonic and post-natal precursors.
Defective cytokinesis in CIT-K-/- spermatogenic
precursors
CIT-K has been previously shown to be required for cytokinesis in specific
populations of the developing central nervous system (CNS) but not in the
other somatic tissues that express it at significant levels
(Di Cunto et al., 2000). In
order to address whether the increased cell death of CIT-K-/-
testicular precursors is correlated with a specific impairment of this
process, we asked whether an increase in germinal cells displaying a
binucleate or multinucleate morphology could be detected in the knockouts.
Examination of semi-thin sections obtained from postnatal testes revealed that
the number of gonocytes and spermatogonia with at least two nuclei was
increased by approximately four-fold in the -/- samples
(Fig. 5A,C). Electron
microscopy of ultra-thin sections, obtained from the same samples,
demonstrated that these cells were not adjacent mononuclear elements but real
syncitia (Fig. 5D), thus
indicating a cytokinesis block. Strikingly, many cells with more than two
nuclei were observed in the CIT-K-/- tubules, but never in the
control samples (Fig. 5A,B,D),
indicating that cytokinesis-deficient cells are able to re-enter the cell
cycle and to undergo multiple rounds of DNA synthesis.
|
Interestingly, in cyclinD1 immunohistochemistry we noticed that many of the binucleate and multinucleate gonocytes and spermatogonia display high immunoreactivity for this protein (Fig. 5E).
To establish a direct link between the cytokinesis block and the induction
of apoptosis in the knockout testicular germ cells, we first attempted to
quantify the percentage of polyploid cells undergoing apoptosis by FACS
analysis after double labeling with propidium iodide and anti-Caspase-3
antibodies, as previously reported for the brain
(Di Cunto et al., 2000).
However, probably because of the low ratio between germinal and support cells
at the analyzed stages (P4 and P8), no significant results were obtained (data
not shown). Therefore, to address the same point, we tried to establish the
percentage of activated Caspase-3-positive cells clearly displaying
mononucleate versus binucleate or multinucleate morphology. One potential
complication for this kind of analysis is that cells at late stages of the
apoptotic process often undergo nuclear condensation and fragmentation
(Walker et al., 1988
), making
it impossible to assess the original number of nuclei. Therefore, cells
displaying condensed and fragmented nuclei were excluded from the counts.
Using these criteria, we were not able to identify early apoptotic cells with
more than one nucleus in wild-type sections (total number of scored cells=92
on 50 sections). By contrast, approximately 10% of the Caspase-3-positive
cells displayed a binucleate or multinucleate morphology in the knockouts
(Fig. 5F, total number of
scored cells=250 on 25 sections).
Taken together, these observations indicate that the absence of CIT-K results in abnormal cytokinesis and polyploidization of spermatogenic precursors, which can be followed by re-entering in the cell cycle or by induction of programmed cell death.
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Discussion |
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The Rho effector CIT-K is the first protein that has been shown to be
required in vivo for cytokinesis of specialized mammalian cell types
(Di Cunto et al., 2000;
Glotzer, 2001
). Indeed,
inactivation of this molecule by gene targeting resulted in a dramatic
malformative syndrome of the CNS, caused by cytokinesis failure and apoptosis
of specific neuronal precursors, whereas no abnormalities were observed in the
other somatic tissues (Di Cunto et al.,
2000
). In this report we have shown that, in addition,
CIT-K-/- mice display a dramatic testicular phenotype,
characterized by progressive loss of germ cells during development.
The number of spermatogenic precursors of CIT-K-/- mice was significantly reduced as early as E12.5, even though the specification and migration of PGCs appeared to be conserved. In the first postnatal days, gonocytes and type A spermatogonia were still present in CIT-K-/- testes, although their number was severely reduced. At later developmental stages, the presence of undifferentiated germ cells was confirmed by the expression of the spermatogenic markers Mvh and A-Myb, even though they were not easily identified by morphological analysis. Interestingly, the expression levels of the two markers were higher than expected from the morphological data because no difference between +/+ and -/- samples was detected at P8, and only a partial reduction was observed at P14. Since the density of the other testicular elements was not dramatically changed, a possible explanation for this observation could be that the Mvh and A-Myb are upregulated in the residual spermatogenic precursors of knockout animals. By contrast, both morphological and molecular analysis consistently showed that production of mature spermatocytes is completely blocked in CIT-K-/- testes.
Taken together, these observations indicate that CIT-K is necessary for the normal expansion of undifferentiated male germ cell precursors and is absolutely required to ensure the transition from spermatogonia to spermatocytes. The most likely cause of this phenotype is increased apoptosis rather than reduced proliferation.
The complex brain malformations of CIT-K-/- mice, which lead
them to death in the first postnatal weeks
(Di Cunto et al., 2000),
raised the possibility that this testicular phenotype could be a secondary
consequence of neuro-endocrine defects. In particular, among their distinctive
features, CIT-K knockout mice display a dramatic cellular depletion in the
olfactory subventricular stream (Di Cunto
et al., 2000
) involved in the migration of GnRH-producing cells
(Kim et al., 1999
). However,
the requirement for CIT-K in gametogenesis appears to be exquisitely sex
specific, as the ovaries of knockout females were histologically comparable to
their littermate controls. This finding strongly argues against the hormonal
mechanism, since GnRH deficiency results in both male and female hypogonadism
(Halpin et al., 1986
;
Hargreave, 2000
;
Kendall et al., 1991
).
Moreover, the good correlation between the expression pattern and the defects
observed in the knockouts strongly suggests that CIT-K acts in these cells
through a cell-autonomous mechanism.
Histological and EM analysis clearly showed that gonocytes and spermatogonia with multiple nuclei are produced in the seminiferous tubules of CIT-K-/- mice, thus indicating a cytokinesis block. To our knowledge, CIT-K is the first molecule that has been shown to be specifically required for cytokinesis of mammalian testicular stem cells.
It is remarkable that, although CIT-K is expressed in many proliferating
tissues and was initially identified as a crucial, generic player of
cytokinesis (Madaule et al.,
1998), the in vivo requirement for its function is restricted to
male germ cells and specific neuronal precursors. One explanation for this
specificity could be that the affected cell types are physiologically devoid
of the molecule(s) that is able to compensate for the absence of CIT-K in the
non-affected cell types of the knockouts. Extensive homology search of the
mouse and human genome sequence databases performed using the BLAST programs
(Altschul et al., 1997
)
revealed that CIT-K is encoded by a unique gene, as no closely related
paralogs could be identified (data not shown). Among the other members of the
myotonin protein kinase subfamily, Rho-kinases (ROCKs) and
myotonin-related-CDC42-binding kinases (MRCKs) displayed the highest
similarity to CIT-K, as previously reported
(Leung et al., 1998
). However
these molecules are very unlikely to compensate for CIT-K loss. Indeed,
although the ROCKs have been involved as Rho effectors in cytokinesis
(Kosako et al., 1999
), they
are ubiquitous proteins and are very well expressed in the testis
(Nakagawa et al., 1996
) and in
the proliferating neuroblasts affected by the absence of CIT-K
(Di Cunto et al., 2000
). On
the other hand, the MRCKs display a relatively tissue-specific expression
pattern but have been reported to bind only to CDC42 and have not been
localized to the cleavage furrow and the midbody of dividing cells
(Leung et al., 1998
).
An attractive alternative possibility could be that the cytokinesis machinery of the affected cell types shares important and tissue-specific molecular details, which require CIT-K in order to act properly.
The characterization of molecules capable of physically and functionally interacting with CIT-K in the affected versus spared cell types will be very important to clarify these points.
Despite the impossibility of rigorously establishing a direct link between
abnormal cytokinesis and increased apoptosis, our results strongly suggest
that, as in the developing brain, in the testes of CIT-K knockout mice the
induction of programmed cell death is secondary to the cytokinesis block. The
mechanisms responsible for activation of the apoptotic pathway in these cells
are still unknown. Interestingly, we found that multinucleate cells in
post-natal testes of CIT-K knockout mice display elevated levels of cyclinD1.
This observation could be simply explained by the ability of the
cytokinesis-deficient gonocytes and spermatogonia to re-enter the G1 phase of
the cell cycle. However, the available evidence suggests that the expression
of cyclinD1 is directly linked to activation of the apoptotic pathway. Indeed,
it has been previously shown that overexpression of cyclinD1 is necessary and
sufficient for apoptotic induction in neuronal cells
(Kranenburg et al., 1996).
Moreover, elevated levels of this protein were previously detected in the
apoptotic brain regions of CIT-K-/- mice
(Di Cunto et al., 2000
).
Finally, activation of the cyclinD-dependent CDKs by double inactivation of
the proteins p18(Ink4c) and p19(Ink4d) resulted in increased apoptosis of
testicular germ cells (Zindy et al.,
2001
).
The results of this study are consistent with the idea that proper temporal
regulation of cytokinesis is essential for normal differentiation of the
testicular epithelium. Indeed, the incomplete cytokinesis of gonocytes and
undifferentiated spermatogonia occurring in CIT-K-/- testes does
not result in the anticipated formation of ICBs but produces multinucleated
cells, which are unable to further differentiate and are eventually lost by
programmed cell death. The mechanisms by which committed spermatogenic cells
arrest their cytokinesis at the midbody stage and then assemble stable ICBs
(Kojima, 1992) remain obscure.
In particular, it is not understood whether the incomplete cell division of
differentiating male germ cells is caused by downregulation of molecules
required for completion of cytokinesis, by upregulation of specific
cytokinesis inhibitors or from a combination of both. Since CIT-K is necessary
for cytokinesis of the basal spermatogonia and its expression is downmodulated
at later stages of differentiation, it is tempting to speculate that its
inactivation could play a role in this process. Although no direct evidence in
support of this model has been so far obtained, a testable prediction would be
that the ectopic overexpression of CIT-K in differentiating spermatogenic
precursors should be able to interfere with the establishment of their
ICBs.
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Acknowledgments |
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Footnotes |
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