1 Laboratoire de Méiose et de Maturation Gamétique, DRR / DSV/ CEA
U566 INSERM Université Paris 7, Fontenay aux Roses,
France
2 Laboratoire de Radiosensibilité des Cellules Germinales, DRR / DSV/ CEA
U566 INSERM Université Paris 7, Fontenay aux Roses,
France
3 CNRS FRE 2358, Génétique Expérimentale et
Moléculaire, Orléans, France
* Author for correspondence (e-mail: pfouchet{at}armoise.saclay.cea.fr)
Accepted 13 October 2003
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SUMMARY |
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Key words: Spermatogenesis, Stem cells, BCRP1/ABCG2, Transplantation
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Introduction |
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The presence of stem cells as for all self-renewing tissues
among spermatogonia is already clearly established and several markers have
been recently identified. The location of undifferentiated spermatogonia close
to the basement membrane of seminiferous tubules suggests that extracellular
matrix receptors may be expressed on stem cells. Using functional assays, the
ability of the candidate stem cells positive for receptor expression
to recolonize in vivo sterile testis has been established. Screening
for the integrin receptors of laminin has demonstrated that the cell
populations expressing ß1 or 6 chains
include stem cells and that the
6-integrin positive cell
population is more highly enriched in stem cells
(Shinohara et al., 1999
;
Shinohara et al., 2000b
).
These subunits are also stem cell markers in some other epithelia [hepatocytes
(Suzuki et al., 2000
),
keratinocytes (reviewed by Watt,
2002
)]. Based on
v expression, the
6hiSSClo
v-
fraction of cryptorchid mouse testicular cells is greatly enriched in germ
stem cells (Shinohara et al.,
2000b
). Among the genes expressed early during spermatogenesis,
another stem cell marker has been characterized: the Stra8 gene is
expressed in spermatogonia
(Oulad-Abdelghani et al.,
1996
) and the activity of its regulatory sequences enables the
purification of germinal stem cells in transgenic mice
(Giuili et al., 2002
).
In different lineages [hematopoietic
(Goodell et al., 1996),
skeletal muscle (Jackson et al.,
1999
; Gussoni et al.,
1999
), neural system (Hulspas
and Quesenberry, 2000
)], stem cells share a phenotype based on the
efflux of vital fluorescent dyes. The use of DNA-dye staining [i.e.
bis-benzimide Hoechst 33342 (Ho)] coupled to flow cytometry analysis
on these lineages revealed a population exhibiting a low Ho fluorescence and
highly enriched in stem cells: the `Side Population' (SP). Some members (MDR1,
BCRP1/ABCG2) of the ATP-binding cassette (ABC) transporter family contribute
to the dye efflux component of the SP phenotype (reviewed by
Gottesman et al., 2002
;
Bunting, 2002
). BCRP1 (Abcg2
Unigene cluster Mm. 196728) was recently reported to be responsible for Ho
efflux, at least in hematopoietic stem cells, as described in
Bcrp1/ mice
(Zhou et al., 2002
).
Evidence that stem cells of different somatic lineages share similar
molecular properties (Lowell,
2000) led us to address the question concerning the occurrence of
SP in the male germinal lineage of adult mice. We have demonstrated that
testicular cells also display an SP population and that the expression of
markers of germinal stem cells is, partly (
6-integrin) or
even totally (Stra8), restricted to SP cells. In addition, transplantation of
testicular SP cells demonstrated that spermatogonial stem cells are present in
testicular SP of adult mice.
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Materials and methods |
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Testicular single-cell suspensions
Cells were isolated from 3-month-old male mice according to a modification
of the protocol of Vincent et al. (Vincent
et al., 1998). The albuginea was removed and the seminiferous
tubules were dissociated using enzymatic digestion by collagenase type I at
100 U/ml for 25 minutes at 32°C in Hanks' balanced salt solution (HBSS)
supplemented with 20 mM HEPES pH 7.2, 1.2 mM MgSO47H2O,
1.3 mM CaCl22H2O, 6.6 mM sodium pyruvate, 0.05% lactate.
After an HBSS wash and centrifugation, the pelleted tubules were further
incubated in Cell Dissociation Buffer (In Vitrogen) for 25 minutes at
32°C. The resulting whole cell suspension was successively filtered
through a 40 µm nylon mesh and through a 20 µm nylon mesh to remove cell
clumps. After an HBSS wash, the cell pellet was resuspended in incubation
buffer (HBSS supplemented with 20 mM HEPES pH 7.2, 1.2 mM
MgSO47H2O, 1.3 mM CaCl22H2O, 6.6
mM sodium pyruvate, 0.05% lactate, glutamine and 1% fetal calf serum) and
further incubated at 32°C in a water bath. Cell concentrations were
estimated using Trypan Blue staining (>95% viable cells).
To discard the interstitial cells, a filtration step with a 40 µm nylon mesh was added after the collagenase digest, and the seminiferous tubules were subsequently treated as previously described.
Flow cytometry analysis and cell sorting
Two million cells were diluted in 2 ml incubation buffer and stained with
Hoechst 33342 (5 µg/ml) for 1 hour at 32°C. Before analysis, propidium
iodide (PI at 2 µg/ml) was added to exclude dead cells. For analysis of
CD45 expression, cells were incubated 20 minutes at 4°C with 1 µg of
Cychrome anti-mouse CD45 clone 30-F11 (Pharmingen) in incubation buffer. Cells
were then washed, resuspended in incubation buffer with PI and maintained at
4°C before analysis. Cychrome rat IgG2b (Pharmingen) isotype was used as
control.
Analysis and cell sorting were performed on a dual-laser FACStar Plus flow cytometer (Becton Dickinson) equipped with a 360 nm UV argon laser and a 488 nm argon laser. Hoechst blue and red fluorescence emissions were collected using a combination of 400 nm long pass and 505 nm short pass filters, and a 630/30 band pass filter. Cychrome fluorescence was collected with 695/35 band pass filter. EGFP and TruCount microbeads fluorescences were collected using a short pass 560 nm dichroic mirror, and 530/30 and 695/35 band pass filters.
6-integrin magnetic cell sorting (MACS)
This procedure was modified from a previously published method
(Shinohara et al., 2000b). To
purify
6-integrin positive cells, 40x106
testicular cells were suspended in 60 µl of incubation buffer and labeled
with 40 µl of PE conjugated anti-
6-integrin antibodies
(GoH3, BD Biosciences) for 15 minutes at 4°C. Cells were washed and
resuspended in HBSS supplemented with 1 mM HEPES pH 7.2 and 0.5% bovine serum
albumin (HBSS/HEPES/BSA). Labeled cells were then sorted with anti-PE antibody
microbeads (Miltenyi Biotech) according to the manufacturer's procedure. The
6-integrin-positive and negative cellular fractions were
collected and resuspended at 106 cells/ml in incubation medium. For
transplantation, SP cells were sorted in tubes coated with 3% BSA containing
DMEM supplemented with 10% FBS.
Hoechst efflux inhibition
Hoechst efflux inhibition was performed by preincubating
6-integrin-positive cells (106 cells/ml) for 30
minutes at 32°C in incubation medium supplemented either with
2-deoxyglucose and sodium azide (Sigma) at 50 mM and 15 mM final
concentrations respectively, or verapamil (Sigma) at 25 and 75 µg/ml
(Scharenberg et al., 2002
), or
specific BCRP1 inhibitor Ko143 at 200 nM
(van der Pol et al., 2003
).
Cells were further stained with Ho in the presence of the inhibitor as
previously described. They were then washed in ice-cold incubation buffer and
resuspended in cold incubation medium containing 2 µg/ml PI before flow
cytometry analysis.
RNA extraction and RT-PCR
For RNA purification, from 50,000 to 100,000 cells were sorted in silicone
treated tubes containing PBS and 20 U recombinant RNasin® ribonuclease
inhibitor (Promega). After centrifugation of the cells, total RNA was purified
using the RNeasy® Mini Kit according to the manufacturer's instructions
(Qiagen). RNA concentrations were quantified using the RiboGreen® RNA
quantification kit (Molecular Probes). The first strand of cDNA was
synthesized from 100 ng of total RNA, in 6.7 mM MgCl2, 67 mM
Tris-HCl pH 8.8, 16.6 mM (NH4)2SO4 with 5
µM pdN6 and 1.25 mM each dNTP. After 5 minutes denaturation at
70°C, 200 U of M-MLV RT were added and the reaction mix (20 µl final)
was further incubated for 45 minutes at 42°C
(Ory et al., 2001). The cDNA
was then diluted to 150 µl and 10 µl were used in PCR (5 µl for
ß-actin). All PCR reactions were classically performed using the primers
shown in Table 1.
|
Testis cell transplantation and analysis of recipient mice
Total unstained testicular single-cell suspensions, used as control, and
sorted testis SP cells both obtained from EGFP transgenic males were
resuspended in Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum, and 100 µg/ml DNAse Type I (Sigma) and 0.5% Trypan Blue, and
maintained at 4°C throughout the procedure. Cell concentrations were
adjusted ranging from 10 to 50x106 cells/ml and 1.6 to
2.5x106 cells/ml for total and SP sorted cell populations,
respectively. Donor testis cell populations were transplanted into
immunologically compatible C57BL6 recipient mice that were previously treated
with busulfan (40 mg/kg, Sigma) to destroy endogenous spermatogenesis
(Brinster and Zimmermann, 1994;
Ohta et al., 2000
). Busulfan
was intraperitoneally injected at 4-6 weeks of age and the mice were used as
recipients 4 weeks later. In addition recipient mice received a single 7.6
mg/kg i.p. injection of leuprolide (gonadotropin-releasing hormone agonist,
Sigma) prior transplantation; treatment is known to enhance colonization after
spermatogonial transplantation (Ogawa et
al., 1998
). Recipient mice were anesthetized by Avertin injection
(640 mg/kg, i.p.). A volume of 10 µl of donor cell suspension was
introduced in each recipient testis via the efferent ductules according to the
method of Ogawa et al. (Ogawa et al.,
1997
). Approximately 70-90% of seminiferous tubules were filled
with the donor cell suspension as monitored by Trypan Blue staining.
Analysis of transplanted testis was performed 10 weeks after donor cell transplantation. Recipient mice were sacrificed and the removed testes were observed under an Olympus epifluorescent microscope to detect the presence of EGFP fluorescent seminiferous tubules. Single cell suspension from recipient testis were prepared and stained with Ho. To determine the repopulation efficiency, the EGFP-positive cell number per recipient testis was measured by flow cytometry using the TruCountTM (BD Biosciences) methodology on testicular cell suspension according to manufacturer's instructions. Briefly, recipient testis cellular suspension (0.5 ml) with 2 µg/ml PI was added into BD TruCount tube. Microbeads lyophilized pellet dissolved, releasing a known number of fluorescent beads. After flow analysis, EGFP-positive cell number per testis could be calculated by dividing the number of EGFP cells by the number of fluorescent beads, then multiplying by the bead concentration, dilution factor and cellular suspension volume. To compare repopulation efficiency of SP and total cells, EGFP-positive cell number per testis was normalized to 105 cell injected. Statistical analysis was performed by Student's t-test.
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Results |
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The SP population includes spermatogonial cells
To identify further the different subpopulations, the Ho staining pattern
of normal cells was compared to those of two well-defined models of altered
spermatogenesis: the W/Wv mutant and the cryptorchid male.
Infertile W/Wv adult males display an early block in
spermatogenesis, owing to mutations in the Kit gene resulting in a
defective Kit/Kit ligand pathway. The cryptorchidism induces by heat shock a
depletion in differentiating germ cells. In addition to somatic cells, the
seminiferous tubules of W/Wv mutants therefore contain
rare spermatogonia (Brinster and
Zimmermann, 1994), and proliferating early spermatogonia are
present in the cryptorchid testis (de Rooij
et al., 1999
).
Analysis of single cell suspensions purified from W/Wv and cryptorchid whole testis gave similar fluorescence profiles (Fig. 1C,D, respectively). Compared with the analysis of normal cells (Fig. 1A), subpopulations 3, 4 and 5 were totally absent while subpopulations 1 and 2 were conserved in the two models. This suggests that on the normal pattern the subpopulations 3 (DNA content 4n) and 4 (2n) were meiotic, and population 5 (n) was postmeiotic corresponding to spermatids. The SP fraction (1) and the diploid population (2), common to the three profiles, might include premeiotic cells and somatic cells.
To assign somatic interstitial cells to subpopulations 1 and/or 2, a filtration step was added (see Materials and methods) to collect on the one hand a fraction enriched in interstitial cells and on the other hand a tubular and germinal cell suspension. The comparison of the two Ho profiles (Fig. 1E,F) strongly suggested that the diploid subpopulation 2 was mainly composed of interstitial cells. Some SP cells were detected in the interstitial cell-enriched fraction (Fig. 1E). This fraction contained 76% cells positive for CD45 marker (data not shown), demonstrating that they were hematopoietic cells. A contamination by haploid cells was present in the interstitial cell-enriched fraction. In the seminiferous tubules fraction (Fig. 1F), the SP population was present as well as subpopulations 3, 4 and 5. In this fraction, 93% of SP cells were negative for the CD45 marker and confirmed that this population contained premeiotic spermatogonial cells.
In conclusion, in the Ho profile of whole testicular cells (Fig. 1B) were clearly defined: the diploid interstitial population (2), the SP fraction including spermatogonial cells (1), the meiotic populations tetraploid spermatocytes I (3) and diploid population 4 (probably spermatocytes II) and the postmeiotic spermatids (5).
RT-PCR analysis of differentiation markers on flow-sorted subpopulations
Further characterization was achieved by analysis of differentiation marker
expression on sorted cell populations. A W/Wv cell
suspension was used as a control because few spermatogonia are present and it
is also enriched in SP cells (Fig.
1C).
Each subpopulation was tested for the expression of genes transcribed
before the first meiotic division (Fig.
2): Dazl and Kit genes
(Ruggiu et al., 1997;
Vincent et al., 1998
). Both
genes were expressed only in the SP cells and in the tetraploid cell
population (4n), confirming that these subpopulations corresponded to
spermatogonia and spermatocytes I, respectively. This result was emphasized by
H1t RNA analysis, whose level of expression was higher in
spermatocytes I (4n) than in premeiotic SP cells
(Drabent et al., 1998
). The
Kit gene mutations have little effect on the levels of Kit
RNA in W/Wv cells
(Nocka et al., 1990
).
|
Spermatogonial stem cell markers are expressed in SP cells
As the SP fraction was a phenotype common to normal,
W/Wv and cryptorchid testicular cells
(Fig. 1), it might include
undifferentiated spermatogonia. The expression of 6-integrin
and Stra8 markers of germinal stem cells was analyzed
(Shinohara et al., 1999
;
Giuili et al., 2002
).
First, as previously mentioned, the 6-integrin-positive
population is highly enriched in stem cells. We took advantage of this
property to enrich for
6-integrin-positive cells by
immunomagnetic purification prior to DNA staining and FACS analysis. The
selected
6-integrin-positive cells
(Fig. 3A), representing 2-4% of
the total population, was principally composed of SP cells and spermatids
compared with the
6-integrin negative cell fraction
(Fig. 3B). Nine percent of
6-integrin-positive cells displayed an SP phenotype.
Excluding the contaminating haploid spermatids (subpopulation 5) from
analysis, the SP population represented 60% of
6-integrin-positive cells, showing that the stem
cell-enriched
6-integrin-positive fraction was highly
enriched in SP cells.
|
The germinal SP phenotype depends on ABC transporter activity and SP cells express Bcrp1/Abcg2 gene
The SP phenotype is caused by an active Ho efflux via members of the ABC
transporter superfamily. This mechanism can be inhibited either by ATP
depletion or specifically using ABC transporter inhibitors
(Zhou et al., 2001;
Scharenberg et al., 2002
). To
determine whether ABC transporter activity was involved in the SP phenotype of
spermatogonial cells, the effects of sodium azide and deoxyglucose, and
verapamil, were assessed in the
6-integrin-positive
fraction. The SP population was markedly reduced in the presence of sodium
azide and deoxyglucose (Fig.
4B, 3.4%) compared with controls
(Fig. 4A, 11.5%) showing that
this phenotype in germinal cells was energy-dependent. Verapamil an
inhibitor of PgP at 25 µg/ml moderately inhibited the Ho efflux
(Fig. 4C, 8.8%) suggesting that
PgP was not the major ABC transporter involved in this process. However, an
increase in the efflux inhibition (Fig.
4D, 3.6%) was observed at higher verapamil concentration (75
µg/ml), as already described for the SP population in bone marrow or
carcinoma cell line (Goodell et al.,
1996
; Scharenberg et al.,
2002
).
|
In vivo repopulation assay in busulfan-treated recipient mice show that the testis SP is enriched for stem cell activity
To determine stem cell activity, donor EGFP germ cells were used for
transplantation into seminiferous tubules of busulfan-treated recipient normal
mice. EGFP germ cells were obtained from EGFP transgenic mice whose
spermatogenesis is normal (Okabe et al.,
1997). As we have shown that
6-integrin-positive
fraction is highly enriched in SP cells, testis cells were firstly enriched
for
6-integrin-positive cells by immunomagnetic
purification. The testis EGFP-positive SP was then purified by flow sorting
(Fig. 5A) and transplanted into
recipient testis.
|
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Discussion |
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The nature of each subpopulation has been identified. The lower diploid one
(2) is present only in whole cell suspensions and is composed of somatic
interstitial cells. This population may include Leydig cells, endothelial and
blood cells which are released during collagenase treatment
(Bellve, 1993). The meiotic
origin of the tetraploid (3) and upper diploid (4) populations was assigned by
comparison with two models of early blocked
spermatogenesisW/Wv mutants and cryptorchid males
in which these cells are completely absent. This is confirmed by
RT-PCR studies: these two cell populations express meiotic differentiation
markers (Crem
and Hsp70-2) highly but express
spermatogonial markers weakly (Kit and Dazl) or not at all
(Stra8). Consequently, these populations can be identified as
spermatocytes I (tetraploid cells) and spermatocytes II (diploid cells). The
red Hoechst fluorescence shift of the spermatocytes II (upper diploid) versus
interstitial cells (lower diploid) might result from differences in their
chromatin structure. Finally, spermatids are identified, as population 5, by
their haploid DNA content and their expression of Crem
and
Hsp70-2 genes.
The SP phenotype after Ho staining is one of the molecular properties
shared by a number of somatic cells [hematopoietic, muscle and neuronal cells,
as well as ES cell lines (for a review, see
Bunting, 2002)] in various
species (mouse, pig, monkey and human)
(Goodell et al., 1997
). Our
results clearly show the presence of an SP fraction among adult male germ
cells. This SP fraction represents 1% of the total viable cells (or 0.6% of
total testicular cells), which is on the order of magnitude of somatic SP
population sizes (Goodell et al.,
1996
; Zhou et al.,
2002
). In addition, the germinal SP fraction described here
exhibits properties of somatic SP populations
(Zhou et al., 2001
;
Scharenberg et al., 2002
): (1)
the SP phenotype corresponds to an active Ho exclusion that can be inhibited
by ATP depletion; and (2) the SP phenotype is specifically sensitive to
inhibitors of ABC pumps. The SP population includes spermatogonial cells as
confirmed by the RNA expression of differentiation markers, in particular
Kit and Dazl. Their expression is known to initiate in the A
spermatogonia the Kit gene
(Manova et al., 1990
) and in
the more differentiated B spermatogonia the Dazl gene
(Niederberger et al., 1997
),
which is consistent with our RT-PCR data.
The screening of two markers of germinal stem cells strongly suggests that
the SP population also includes the spermatogonial stem cells. The
6-integrin-positive cells are enriched in stem cells and
their Ho profile demonstrates that the majority of
6-integrin-positive cells exhibit a SP phenotype. In
addition, Stra8 gene expression is restricted to the SP population,
confirming the presence of spermatogonia and stem cells. We have also observed
that the experimental cryptorchid mouse model, highly enriched in
spermatogonial stem cells, is also highly enriched in SP population.
To confirm that the stem cell population is present in the testis SP, its
functional activity was studied by an in vivo assay, namely the establishment
of a spermatogenesis after transplantation in sterile testis
(Brinster and Zimmermann,
1994). Donor EGFP germ cells from adult males were used for
transplantation into seminiferous tubules of busulfan-treated recipient mice.
In order to improve SP cell sorting, testis cells were first enriched for
6-integrin-positive cells, before flow sorting of the testis
SP and transplantation into recipient testis. Results showed that this testis
SP fraction colonized seminiferous tubules of recipient testis as observed 10
weeks after transplantation. Testis SP cells can proliferate and
differentiate. In addition, the testis SP was 15-fold enriched in stem cell
activity compared with total cells, demonstrating that the testis SP in adult
mouse contain male germinal stem cells. A recent study reported that, although
the SP phenotype was detected in testis, the SP fraction did not display
spermatogonial stem cell activity by transplantation assay
(Kubota et al., 2003
). Those
data, which are in contradiction with our results, were obtained using the
cryptorchid mouse model as a SP donor a model in which spermatogenesis
is early blocked by heat shock. By contrast, our experiments involve normal
transgenic testicular cells as a SP donor. The apparent discrepancy might
result from the use of those two different models. Heat shock could modify the
regulation of ABC transporter gene expression. Consequently the cellular
composition of the testis SP could be modified between both models, although
SP is observed. Additional works need to be carried out to investigate this
discrepancy.
At least in hematopoietic lineages, Ho exclusion is directly caused by
BCRP1/ABCG2 protein activity as demonstrated in Bcrp1-null mice
(Zhou et al., 2002). We have
shown that Bcrp1 RNA is detected in the germinal SP population cells
and that Hoechst efflux can be inhibited by BCRP1 specific inhibitor. Thus,
BRCP1 activity is a determinant of the germinal SP phenotype reported here.
The Bcrp1/Abcg2 gene is a member of the superfamily of the ABC
transporters that are key elements of the blood-testis and blood-brain
barriers. The blood-testis barrier is composed of capillary endothelial cells
and Leydig cells, and by the myoid layer of the seminiferous tubules and
Sertoli cells. Those somatic cells express specific ABC transporters, such as
P-gP/MDR1 (endothelial and myoid cells), and MRP1 (Sertoli and Leydig cells)
(Wijnholds et al., 1998
;
Bart et al., 2002
). Therefore,
we cannot exclude that somatic cells might be present in the SP fraction,
especially for myoid cells expressing P-gP. In the germinal lineage, P-gP/MDR1
expression has been recently detected in postmeiotic cells (late spermatids)
(Melaine et al., 2002
). As
demonstrated here, Bcrp1 RNA is also present in spermatids, but
Hoechst efflux was not observed for spermatids, suggesting an RNA storage
process (Kleene, 2001
).
Concerning spermatogonial and male germinal stem cells, BRCP1 is the first ABC
transporter detected in these cells with an active efflux phenotype. Such a
pattern of Bcrp1 transcription regulation has been described in the
hematopoietic differentiation process. The Bcrp1 gene is expressed in
hematopoietic stem cells, downregulated during differentiation commitment and
finally reinduced in specific lineages
(Scharenberg et al., 2002
).
BCRP1 has been reported to confer on hematopoietic stem cells a resistance to
mitoxantrone, a chemotherapeutic drug
(Miyake et al., 1999
;
Allen et al., 1999
), suggesting
that its physiological role is to protect stem cells from cytotoxic
substrates. In this context, it is not surprising that germinal stem cells
express such ABC transporters as these cells are involved in the integrity and
in the good transmission of genetic material to progeny. This self-protection
mechanism may constitute the last barrier against genotoxic stress. If stem
cells were hit by mutagenesis, hereditary risk would become continuous during
the lifespan of the individual. In this way it is important to note that
mitoxantrone a specific substrate of ABCG2 is a potentially
mutagenic DNA intercalating drug.
Whether Bcrp1 is a common marker of stem cells in various tissues raises
the question about the significance of ABC transporters in stem cell biology
and their presumed role in the regulation of stem cell self-renewal and
differentiation processes. Steady-state hematopoiesis is functionally normal
in Bcpr1-null mice, despite the absence of SP phenotype, and Bcrp1
does not appear to play a major role in the hematopoietic differentiation
process. The same observations were also made in Mdr1a/Mdr1b-null
mice (Zhou et al., 2002). In
the same way, Bcrp1-null mice are fertile and spermatogenesis appears
normal (Zhou et al., 2002
;
Jonker et al., 2002
). These
results suggest that ABCG2 is not essential for spermatogenesis, especially
for spermatogonial and stem cell differentiation in testis. Those observations
confirm the idea of the protective role against genotoxicity. However,
redundancy between different ABC transporter pathways in stem cell biology
cannot be ruled out, which could explain the lack of impairment in
hematopoiesis and spermatogenesis in Bcrp1-null mice.
The testis SP in male adult mice was confirmed to be highly enriched in
male germ stem cells by specific germ stem cell marker analysis and by in vivo
repopulation assay after transplantation. Hence, the SP phenotype should be a
common marker of germinal and somatic stem cells, although somatic and
germinal lineages are separated with distinct fates very early in the
development of the mouse embryo (from 6-7 days post-coitum onwards)
(Zhao and Garbers, 2002). SP
sorting constitutes an alternative strategy to isolate a highly enriched
germinal stem cell population. It might be possible to enrich SP cells using a
specific antibody-based procedure Bcrp1 being a cell surface marker.
The SP phenotype has been described in hematopoietic stem cells of various
species (mouse, monkey, human) (Goodell et
al., 1997
), suggesting that this phenotypic marker is highly
conserved between species in stem cell from somatic lineages. In the same way,
we suggest that the SP germinal male stem cell phenotype could be conserved
between different species. It would constitute an interesting strategy to
isolate highly enriched germinal stem cell populations without having to know
their stem cell-specific surface markers.
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allen, J. D., Brinkhuis, R. F., Wijnholds, J. and Schinkel, A.
H. (1999). The mouse Bcrp1/Mxr/Abcp gene:
amplification and overexpression in cell lines selected for resistance to
topotecan, mitoxantrone, or doxorubicin. Cancer Res.
59,4237
-4241.
Allen, J. D., van Loevezijn, A., Lakhai, J. M., van der Valk, M., van Tellingen, O., Reid, G., Schellens, J. H., Koomen, G. J. and Schinkel, A. H. (2002). Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol. Cancer Ther. 6,417 -425.
Bart, J., Groen, H. J., van der Graaf, W. T., Hollema, H., Hendrikse, N. H., Vaalburg, W., Sleijfer, D. T. and de Vries, E. G. (2002). An oncological view on the blood-testis barrier. Lancet Oncol. 3,357 -363.[CrossRef][Medline]
Bellve, A. R. (1993). Purification, culture, and fractionation of spermatogenic cells. Methods Enzymol. 225,84 -113.[Medline]
Brinster, R. L. and Zimmermann, J. W. (1994).
Spermatogenesis following male germ-cell transplantation. Proc.
Natl. Acad. Sci. USA 91,11298
-11302.
Bunting, K. (2002). ABC Transporters as
Phenotypic Markers and Functional Regulators of Stem Cells. Stem
Cells 20,11
-20.
Chiarini-Garcia, H. and Russell, L. D. (2001).
High-resolution light microscopic characterization of mouse spermatogonia.
Biol. Reprod. 65,1170
-1178.
de Rooij, D. G., Okabe, M. and Nishimune, Y.
(1999). Arrest of spermatogonial differentiation in jsd/jsd,
Sl17H/Sl17H, and cryptorchid mice. Biol. Reprod.
61,842
-847.
Dix, D. J., Rosario-Herrle, M., Gotoh, H., Mori, C., Goulding, E. H., Barrett, C. V. and Eddy, E. M. (1996). Developmentally regulated expression of Hsp70-2 and a Hsp70-2/lacZ transgene during spermatogenesis. Dev. Biol. 174,310 -321.[CrossRef][Medline]
Drabent, B., Bode, C., Miosge, N., Herken, R. and Doenecke, D. (1998). Expression of the mouse histone gene H1t begins at premeiotic stages of spermatogenesis. Cell Tissue Res. 291,127 -132.[CrossRef][Medline]
Escalier, D. (2001). Impact of genetic
engineering on the understanding of spermatogenesis. Hum. Reprod.
Update 7,191
-210.
Foulkes, N. S., Schlotter, F., Pevet, P. and Sassone-Corsi, P. (1993). Pituitary hormone FSH directs the CREM functional switch during spermatogenesis. Nature 362,264 -267.[CrossRef][Medline]
Giuili, G., Tomljenovic, A., Labrecque, N., Oulad-Abdelghani,
M., Rassoulzadegan, M. and Cuzin, F. (2002). Murine
spermatogonial stem cells: targeted transgene expression and purification in
an active state. EMBO Rep.
3, 753-759.
Goodell, M. A., Brose, K., Paradis, G., Conner, A. S. and Mulligan, R. C. (1996). Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183,1797 -1806.[Abstract]
Goodell, M. A., Rosenzweig, M., Kim, H., Marks, D. F., DeMaria, M., Paradis, G., Grupp, S. A., Sieff, C. A., Mulligan, R. C. and Johnson, R. P. (1997). Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat. Med. 3,1337 -1345.[Medline]
Gottesman, M. M., Fojo, T. and Bates, S. E. (2002). Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2, 48-58.[CrossRef][Medline]
Gussoni, E., Soneoka, Y., Strickland, C. D., Buzney, E. A., Khan, M. K., Flint, A. F., Kunkel, L. M. and Mulligan, R. C. (1999). Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401,390 -394.[CrossRef][Medline]
Hulspas, R. and Quesenberry, P. J. (2000). Characterization of neurosphere cell phenotypes by flow cytometry. Cytometry 40,245 -250.[CrossRef][Medline]
Jackson, K. A., Mi, T. and Goodell, M. A.
(1999). Hematopoietic potential of stem cells isolated from
murine skeletal muscle. Proc. Natl. Acad. Sci. USA
96,14482
-14486.
Jonker, J. W., Buitelaar, M., Wagenaar, E., van Der Valk, M. A.,
Scheffer, G. L., Scheper, R. J., Plosch, T., Kuipers, F., Elferink, R. P.,
Rosing, H. et al. (2002). The breast cancer resistance
protein protects against a major chlorophyll-derived dietary phototoxin and
protoporphyria. Proc. Natl. Acad. Sci. USA
99,15649
-15654.
Kleene, K. C. (2001). A possible meiotic function of the peculiar patterns of gene expression in mammalian spermatogenic cells. Mech. Dev. 106, 3-23.[CrossRef][Medline]
Kubota, H., Avarbock, M. R., Brinster, R. L.
(2003). Spermatogonial stem cells share some, but not all,
phenotypic and functional characteristics with other stem cells.
Proc. Natl. Acad. Sci. USA
100,6487
-6492.
Lowell, S. (2000). Stem cells show their potential. Trends Cell Biol. 10,210 -211.[CrossRef][Medline]
Manova, K., Nocka, K., Besmer, P. and Bachvarova, R. F. (1990). Gonadal expression of c-kit encoded at the W locus of the mouse. Development 110,1057 -1069.[Abstract]
Melaine, N., Lienard, M. O., Dorval, I., le Goascogne, C.,
Lejeune, H. and Jegou, B. (2002). Multidrug resistance genes
and p-glycoprotein in the testis of the rat, mouse, Guinea pig, and human.
Biol. Reprod. 67,1699
-1707.
Miyake, K., Mickley, L., Litman, T., Zhan, Z., Robey, R.,
Cristensen, B., Brangi, M., Greenberger, L., Dean, M., Fojo, T. et al.
(1999). Molecular cloning of cDNAs which are highly overexpressed
in mitoxantrone-resistant cells: demonstration of homology to ABC transport
genes. Cancer Res. 59,8
-13.
Niederberger, C., Agulnik, A. I., Cho, Y., Lamb, D. and Bishop, C. E. (1997). In situ hybridization shows that Dazla expression in mouse testis is restricted to premeiotic stages IV-VI of spermatogenesis. Mamm. Genome 8, 277-278.[CrossRef][Medline]
Nishimune, Y., Aizawa, S. and Komatsu, T. (1978). Testicular germ cell differentiation in vivo. Fertil. Steril. 29,95 -102.[Medline]
Nocka, K., Tan, J. C., Chiu, E., Chu, T. Y., Ray, P., Traktman, P. and Besmer, P. (1990). Molecular bases of dominant negative and loss of function mutations at the murine c-kit/white spotting locus: W37, Wv, W41 and W. EMBO J. 9,1805 -1813.[Abstract]
Ogawa, T., Arechaga, J. M., Avarbock, M. R. and Brinster, R. L. (1997). Transplantation of testis germinal cells into mouse seminiferous tubules. Int. J. Dev. Biol. 41,111 -122.[Medline]
Ogawa, T., Dobrinski, I., Avarbock, M. R. and Brinster, R. L. (1998). Leuprolide, a gonadotropin-releasing hormone agonist, enhances colonization after spermatogonial transplantation into mouse testes. Tissue Cell. 30,583 -588.[Medline]
Ohta, H., Yomogida, K., Yamada, S., Okabe, M. and Nishimune, Y. (2000). Real-time observation ot transplanted `green germ cells': proliferation and differentiation of stem cells. Dev. Growth Differ. 42,105 -112.[CrossRef][Medline]
Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. and Nishimune, Y. (1997). `Green mice' as a source of ubiquitous green cells. FEBS Lett. 407,313 -319.[CrossRef][Medline]
Ory, K., Lebeau, J., Levalois, C., Bishay, K., Fouchet, P., Allemand, I., Therwath, A. and Chevillard, S. (2001). Apoptosis inhibition mediated by medroxyprogesterone acetate treatment of breast cancer cell lines. Breast Cancer Res. Treat. 68,187 -198.[CrossRef][Medline]
Oulad-Abdelghani, M., Bouillet, P., Decimo, D., Gansmuller, A., Heyberger, S., Dolle, P., Bronner, S., Lutz, Y. and Chambon, P. (1996). Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene. J. Cell Biol. 135,469 -477.[Abstract]
Ruggiu, M., Speed, R., Taggart, M., McKay, S. J., Kilanowski, F., Saunders, P., Dorin, J. and Cooke, H. J. (1997). The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 389, 73-77.[CrossRef][Medline]
Russell, L., Ettlin, A., Sinha Hikim, A. and Clegg, E. (1990). Histological and Histopathological Evaluation of the Testis. Clearwater, FL: Cache River Press.
Scharenberg, C. W., Harkey, M. A. and Torok-Storb, B.
(2002). The ABCG2 transporter is an efficient Hoechst 33342
efflux pump and is preferentially expressed by immature human hematopoietic
progenitors. Blood 99,507
-512.
Shinohara, T., Avarbock, M. and Brinster, R. L.
(1999). ß1- and 6-integrin are
surface markers on mouse spermatogonial stem cells. Proc. Natl.
Acad. Sci. USA 96,5504
-5509.
Shinohara, T., Avarbock, M. and Brinster, R. L. (2000a). Functional analysis of spermatogonial stem cells in Steel and cryptorchid infertile mouse models. Dev. Biol. 220,401 -411.[CrossRef][Medline]
Shinohara, T., Orwig, K. E., Avarbock, M. R and Brinster, R.
L. (2000b). Spermatogonial stem cell enrichment by
multiparameter selection of mouse testis cells. Proc. Natl. Acad.
Sci. USA 97,8346
-8351.
Suzuki, A., Zheng, Y., Kondo, R., Kusakabe, M., Takada, Y., Fukao, K., Nakauchi, H. and Taniguchi, H. (2000). Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver. Hepatology 32,1230 -1239.[Medline]
van der Pol, M. A., Broxterman, H. J., Pater, J. M., Feller, N., van der Maas, M., Weijers, G. W., Scheffer, G. L., Allen, J. D., Scheper, R. J., van Loevezijn, A., Ossenkoppele, G. J. and Schuurhuis, G. J. (2003). Function of the ABC transporters, P-glycoprotein, multidrug resistance protein and breast cancer resistance protein, in minimal residual disease in acute myeloid leukemia. Haematologica 88,134 -147.[Medline]
Vincent, S., Segretain, D., Nishikawa, S., Nishikawa, S. I.,
Sage, J., Cuzin, F. and Rassoulzadegan, M. (1998).
Stage-specific expression of the Kit receptor and its ligand (KL) during male
gametogenesis in the mouse: a Kit-KL interaction critical for meiosis.
Development 125,4585
-4593.
Wang, P. J., McCarrey, J. R., Yang, F. and Page, D. C. (2001). An abundance of X-linked genes expressed in spermatogonia. Nat. Genet. 27,422 -426.[CrossRef][Medline]
Watt, F. M. (2002). Role of integrins in
regulating epidermal adhesion, growth and differentiation. EMBO
J. 21,3919
-3926.
Wijnholds, J., Scheffer, G. L., van der Valk, M., van der Valk,
P., Beijnen, J. H., Scheper, R. J. and Borst, P. (1998).
Multidrug resistance protein 1 protects the oropharyngeal mucosal layer and
the testicular tubules against drug-induced damage. J. Exp.
Med. 188,797
-808.
Zakeri, Z. F., Wolgemuth, D. J. and Hunt, C. R. (1988). Identification and sequence analysis of a new member of the mouse HSP70 gene family and characterization of its unique cellular and developmental pattern of expression in the male germ line. Mol. Cell. Biol. 8,2925 -2932.[Medline]
Zhao, G. Q. and Garbers, D. L. (2002). Male germ cell specification and differentiation. Dev. Cell 2, 537-547.[Medline]
Zhou, S., Schuetz, J. D., Bunting, K. D., Colapietro, A. M., Sampath, J., Morris, J. J., Lagutina, I., Grosveld, G. C., Osawa, M., Nakauchi, H. et al. (2001). The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat. Med. 7,1028 -1034.[CrossRef][Medline]
Zhou, S., Morris, J. J., Barnes, Y., Lan, L., Schuetz, J. D. and
Sorrentino, B. P. (2002). Bcrp1 gene expression is
required for normal numbers of side population stem cells in mice, and confers
relative protection to mitoxantrone in hematopoietic cells in vivo.
Proc. Natl. Acad. Sci. USA
99,12339
-12344.