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First published online 3 October 2007
doi: 10.1242/dev.003467
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1 Inserm U636, F-06108, Nice, France.
2 Université de Nice-Sophia Antipolis, Laboratoire de
Génétique du Développement Normal et Pathologique,
F-06108 Nice, France.
3 Institut de Biologie Moléculaire et Cellulaire CNRS-INSERM-ULP,
Illkirch, F-67404, France.
Author for correspondence (e-mail:
minoo{at}unice.fr)
Accepted 7 August 2007
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MATERIALS AND METHODS
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DISCUSSION
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Key words: Spermatogonial stem cells, Mouse development, Blastocyst, Spermatogenesis, Transcriptome
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INTRODUCTION |
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SUMMARY
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MATERIALS AND METHODS
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DISCUSSION
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). During embryonic development, the primordial germ
cells (PGCs) specified in the epiblast at embryonic day (E) 7.5 divide and
colonize the gonadal ridge, where they become associated with somatic cells in
the cord structures characteristic of the fetal testis. At the end of prenatal
development, the germ cells (gonocytes) stop dividing. The spermatogonial stem
cells (SSCs), which resume proliferation during the first days after birth
(de Rooij and Grootegoed,
1998), were functionally defined by their ability to reconstitute
a functional seminiferous epithelium after transplantation into a sterile host
(Brinster, 2002;
Brinster and Zimmermann, 1994).
The cyclic process of spermatogenesis initiated from SSCs will thereafter be
maintained throughout the whole of adult life.
The two critical turning points in this process are the specification of
PGCs in the early embryo and the appearance of dividing SSCs shortly after
birth. Genes expressed at the earliest stages of specification of the germ
line have been identified recently. They include fragilis (Ifitm3)
(Saitou et al., 2002;
Tanaka et al., 2005), stella
(PGC7, Dppa3) (Bortvin et al.,
2004; Nakamura et al.,
2007; Payer et al.,
2003; Saitou et al.,
2002; Sato et al.,
2002) and Blimp1 (Prdm1)
(Ohinata et al., 2005;
Saitou et al., 2005;
Vincent et al., 2005). PGCs
appear first in the proximal epiblast, in a region in which fragilis is
upregulated at E6.5. This restricted PGC population (approximately 40 cells)
expresses Blimp1 (E7.25) and thereafter (E7.5) stella. After birth,
only a limited number of genes crucial for establishment and self-renewal of
SSCs have been identified. Requirement for the Ret receptor was
postulated based on the requirement for its ligand GDNF
(Meng et al., 2000), and a
partial requirement for Bcl6b was described recently
(Oatley et al., 2006).
Our starting point was the establishment of a transcriptome profile of
purified preparations of SSCs, on the assumption that crucial genes would
appear among those expressed at elevated levels in the stem cells. Work on
adult SSCs is made difficult by their small number. We previously reported an
efficient procedure, in which a truncated version of the Stra8
promoter is used to express in SSCs a neutral heterologous surface protein
including two domains of the human CD4 antigen (huCD4)
(Giuili et al., 2002). Once
fractionated by immunomagnetic sorting, the huCD4-positive fraction is
homogeneous for the expression of known SSC markers and highly efficient in
the colonization of a sterile recipient. Hybridization on DNA arrays provided
a shortlist of transcripts upregulated in the purified fraction.
Interestingly, the list included PU.1 (Sfpi1), a member of
the Ets family, which has been extensively studied for its important
regulatory functions in the renewal of progenitor cells and in early
differentiation, so far exclusively in the various lineages of hematopoietic
differentiation (reviewed by Lloberas et
al., 1999; Metcalf et al.,
2006). The availability of a null mutation generated by insertion
of the green fluorescent protein (GFP) reporter gene in the first exon
(Back et al., 2004), allowed us
to demonstrate here a strict requirement for PU.1 in the fetal
differentiation of the male germ line and to show promoter activity starting
at the earliest embryonic stages.
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MATERIALS AND METHODS |
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DISCUSSION
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Transgenic mice
A truncated version of the mouse Stra8 promoter was used as
described previously (Giuili et al.,
2002) to direct expression to the surface of SSCs of a fusion
protein including two domains of the human CD4 protein (Stra8-CD4HAglo mice).
Progeny were genotyped by PCR amplification of tail DNA using primers hCD4F
and hCD4R (Table 1).
PU.1G/+ and PU.1G/G mice
(Back et al., 2004) were
genotyped by PCR with primers VP102 (wild-type exon 1), VP104 (mutated exon 1)
and WF189 (intron 1) (Table 1).
Amplified fragments were 650 bp for the PU.1+ allele and
900 bp for PU.1G. Families were maintained in the
C57BL/6xDBA/2 F1 genetic background. Investigations were conducted in
accordance with French and European rules for the care and use of laboratory
animals.
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Immunomagnetic cell-sorting
Total germ cells were prepared as described previously
(Vidal et al., 2001).
Immunomagnetic isolation of huCD4-positive cells from total testicular cells
of Stra8-CD4HAglo mice was performed using the CD4-positive Isolation Kit
(Dynal, Oslo, Norway) according to the manufacturer's instructions. For each
SSC preparation, 50 transgenic males were used for a final yield of
2x106 cells corresponding to the expected number of 20,000
stem cells per testis (de Rooij and
Grootegoed, 1998).
Purification of spermatids and pachytene spermatocytes
Total germ cells prepared from 20 mice were loaded in the Beckman (Palo
Alto, CA, USA) elutriation rotor JE-5.0, at a flow rate of 7 ml/minute and a
constant speed of 2000 rpm. Cells were collected in 11 fractions of 400 ml
each, obtained by changing the flow rate from 7 to 50 ml/minute at constant
speed. Fraction purity was checked by microscopic analysis after Hoechst 33253
staining.
RNA preparation
Total RNA was extracted from cell fractions using Trizol (Invitrogen, Cergy
Pontoise, France) according to the manufacturer's instructions. Ten to 20
µg of stem cell RNA was obtained per SSC preparation. Quality of RNA was
checked by agarose gel electrophoresis and using an Agilent bioanalyser.
Histology and immunohistochemistry
Histology and immunohistochemistry analyses were performed according to
published protocols (Wagner et al.,
2005). Briefly, the samples were fixed overnight at 4°C in
paraformaldehyde (3% in PBS) and embedded in paraffin. Routine histological
examination was performed after Hematoxylin staining. Sections (3 µm) were
incubated (16 hours, 4°C) with primary antibodies against PU.1 (T-21,
sc-352, Santa Cruz Biotechnology, Santa Cruz, CA), PCNA (PC10, sc-56, Santa
Cruz Biotechnology), each diluted 1:100 in PBS, 0.1% Triton X-100, 3% BSA.
Antigen detection was performed with a biotinylated goat anti-rabbit antibody
(Vector Laboratories, Burlingame, CA), followed by incubation with
peroxidase-coupled streptavidin (Sigma, St Louis, MO). Visualization was
achieved with DAB substrate (SK-4100, Vector Laboratories)
(Wagner et al., 2006). For
PCNA staining, the subsequent antigen detection was performed using the M.O.M.
Kit (PK-2200, Vector laboratories). ES cells were stained with the PU.1
antibody as described for U2OS cells
(Wagner et al., 2005).
Omission of the first antibody served as a negative control; additionally,
blocking peptide against PU.1 (sc-352P, Santa Cruz Biotechnology) was used to
confirm the specificity of staining.
Tunel labeling of apoptotic cells
Apoptotic cells were detected by Tunel staining of paraffin sections using
the In Situ Cell Death Detection Kit (Roche Molecular Biochemicals) as
described previously (Wagner et al.,
2005). Three sections from three different animals were
analyzed.
Confocal microscopy
Distribution of GFP fluorescence within the embryo was determined by
confocal microscopy with a Leica CLSM microscope equipped with an
argon-krypton ion laser emitting light at 488 and 514 nm and producing an
excitation wavelength of 515 and 580 nm, and with a UV laser (excitation 352
nm, emission 405 nm). A series of optical sections of approximately 0.95 µm
was recorded through the z-axis, using an objective of 43x and
a zoom of 1.99.
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Microarray data analysis
RNA preparations were analyzed by in vitro amplification and hybridization
to DNA microarrays containing about 14,000 genes (Mouse Genome 430A 2.0,
Affymetrix, Santa Clara, CA, USA). Absolute and comparison analyses of the
mouse oligonucleotide arrays were conducted using the statistics-based
Affymetrix software MAS-5.0 (GeneChip Software) with the default settings. We
used the MAS5 algorithm (Affymetrix) to determine the `present' and `absent'
calls for each probe set. Quantitative expression values were obtained with
the robust microarray analysis (RMA) algorithm, which uses a method based on
quantile normalization to calculate an expression value for each probe set.
The complete microarray data are available in the NCBI GEO repository
(http://www.ncbi.nlm.nih.gov/geo/).
Quantitative real-time PCR
cDNA samples from SSC and total fractions were analyzed by real-time PCR
using the qPCR MasterMix Plus for SYBR Green (Eurogentec, Seraing, Belgium)
and an ABI Prism 7000 sequence detector according to the manufacturer's
protocol (Applied Biosystems, Foster City, CA). Amplification was performed by
applying the comparative Ct method, as described in the manufacturer's
protocol. Sequences of oligonucleotide primers are shown in
Table 1.
BrdU incorporation
Pregnant female mice received 50 mg/kg of 5-bromo-2'-deoxyuridine
(BrdU, Roche Molecular Biochemicals) by intraperitoneal injection and were
sacrificed 5 hours later. Paraffin sections were stained by immunolabeling
with anti-BrdU antibody (Roche Molecular Biochemicals) as described above.
Testis transplantation
The donor testes were dissected at E17-19. For transplantation, the
recipient testis (6-week-old ICR nu/nu mice, Charles River, Wilmington, MA)
was exteriorized through a midline abdominal incision after anaesthetizing the
recipient mouse. Using fine forceps, a small cut was made in the tunica
albuginea, and the testis graft inserted into the testicular parenchyma. One
week to 1 month after transplantation, the recipient testis with the
transplant was embedded in paraffin and histological sections were cut at
8-µm intervals from each testis piece and stained with Hematoxylin and
Eosin for microscopic examination.
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RESULTS |
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DISCUSSION
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), a
neutral surface marker is expressed under control of a truncated version (411
bp) of the promoter of the Stra8 gene. Unlike the wild-type promoter,
the truncated version directs transgene expression to a minor population of
premeiotic cells, positively identified as SSCs on the basis of expression of
marker genes and of their high efficiency of establishment upon
transplantation. Among other markers routinely used to ascertain the purity of
the fractions (expression of cyclin A2, but not A1, of integrins ß1 and
6), the two most characteristic complementary criteria are shown in
Fig. 1A, namely expression of
the endogenous Stra8 gene, characteristic of both SSCs and
differentiated spermatogonia
(Oulad-Abdelghani et al.,
1996), and absence of Kit transcripts, which are
expressed only in differentiated spermatogonia
(Shinohara et al., 2000).
Contamination by cells engaged in the germinal differentiation process was
minimal, as shown by the negative results registered for Sycp1, which
is characteristic of the pachytene spermatocytes
(Meuwissen et al., 1992) and
Prm1, a marker of spermatids
(Peschon et al., 1987).
Possible contamination by postmeiotic cells was in addition limited by
starting the purification process from young males (3-4 weeks), in which the
first wave of spermatogenesis would have just passed the second meiotic
division (Bellve et al., 1977).
Yields were 
20,000 cells per testis, in agreement with previous estimates
(de Rooij and Grootegoed,
1998).
A shortlist of `increased in SSCs' transcripts includes PU.1 (Sfpi1), a pleiotropic regulator of hematopoiesis
Three independent preparations of 20-40 µg SSC RNA were analyzed by
hybridization on DNA microarrays (14,000 genes, Affymetrix Mouse Genome 430A
2.0). In order to identify genes selectively expressed in SSCs, we selected
genes that were detected as `present' in all three huCD4+ samples
and `absent' in all three huCD4- samples. We further imposed that
the measured detection values exhibit at least a 2-fold difference between
huCD4+ and huCD4- samples, with a P-value
(Student's t-test) of less than 0.05. These combined criteria led to
a list of 84 genes (Table 2).
In addition, to estimate the `background' of non-specific genes within that
list, we performed similar analyses between permuted combinations of samples,
in which two samples had been switched between the positive and negative
groups. In each case, less then ten genes were found, suggesting that most of
the selected genes are indeed differentially expressed between SSCs and
non-SSCs. Differential expression of four genes randomly taken in the list
(Cd14, Cd97, Nek7, Sfpi1) was also confirmed by quantitative RT-PCR
(data not shown). We also noted that a number of genes, although excluded from
this most stringent list because of low but significant expression in at least
one of the huCD4- samples, showed higher expression levels in the
positive fraction. RT-PCR assays of five transcripts in this category
(Cd81, Nptn, Fos, Cdc42, Lyn) again showed a differential expression
in the two fractions (data not shown).
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),
Elk3 encoding an Ets-domain factor
(Chung et al., 2006) and
Rbpsuh (Rbpj) encoding the transcriptional mediator of Notch
signaling (Vasyutina et al.,
2007).
Although all of these genes might play important regulatory roles in
controlling SSC gene expression, we were especially intrigued by the presence
of PU.1 (Sfpi1). This finding warranted further analysis
because the published literature (reviewed by
Lloberas et al., 1999;
Metcalf et al., 2006)
consistently refers to PU.1 as a transcription regulator that acts
exclusively in the progenitors of blood cells. Both PU.1 RNA and the
protein were unequivocally detected in huCD4+ cells by quantitative
RT-PCR and by immunolabeling (Fig.
1B,C).
PU.1 expression in the adult testis
Immunodetection of PU.1 protein in adult testis sections revealed a complex
pattern. As expected from analysis of the huCD4+ fraction, the
protein was detected in a small number of spermatogonia in the most peripheral
region of the tubule (Fig. 2A).
Among these cells, the paired (Apaired) and aligned
(Aaligned) cells characteristic of the early spermatogonial stages
(de Rooij and Grootegoed,
1998) were clearly identified
(Fig. 2G-I). Whereas the bulk
of differentiated spermatogonia were negative, expression of the protein was
also evidenced in meiotic spermatocytes
(Fig. 2A, asterisk). This
finding was somewhat unexpected, a contamination by pachytene spermatocytes of
the huCD4+ fraction being made unlikely by the absence of the
characteristic meiotic transcripts Sycp1
(Fig. 1A) and Ccna2
(cyclin A2, not shown) (see Giuili et al.,
2002). Meiotic expression was nevertheless confirmed by in situ
analysis in the testis of 3-week-old males, in which the first synchronous
wave of spermatogenesis leads to the accumulation of meiotic cells, which were
uniformly and strongly labeled by anti-PU.1 immunostaining
(Fig. 2C-E). This was further
confirmed by PCR assays performed on elutriation-purified spermatocytes
(Fig. 2F). The quantitatively
minor representation of pachytene spermatocytes in the total huCD4-
fraction accounts for the initial detection of higher levels of PU.1
transcripts in huCD4+ cells.
PU.1 activity during development is required in germinal differentiation
In the embryonic testis, PU.1 was detected in the germinal compartment, at
least from E12.5 onward (Fig.
3). Further studies took advantage of a mouse mutant,
PU.1G, generated by insertion of a GFP cassette into the
first exon of the gene to provide both a reporter for promoter activity and a
null mutation (Back et al.,
2004). PU.1G/G homozygotes die shortly after
birth and heterozygotes are healthy and fertile. Embryos of crosses between
heterozygotes were individually genotyped and testis development was analyzed
in the PU.1G/G progeny, with their G/+ and +/+
littermates used as controls. Histological analysis of homozygotes at E15.5
and E17.5 seemed at first to indicate that the testis was normally
constituted, with a myoid cell layer surrounding internal cells in a structure
similar to the testis cords of the wild type
(Fig. 4A,B). The number of
gonocytes per section was, however, smaller than in the controls. Among a
series of genes characteristic of prenatal germinal differentiation, RT-PCR
analysis detected transcripts of Oct4
(Yoshimizu et al., 1999),
Dazl (Lin and Page,
2005), Taf4b
(Falender et al., 2005) and
vasa (Tanaka et al., 2000),
but expression of Plzf (Zbtb16)
(Buaas et al., 2004;
Costoya et al., 2004) and
stella (Saitou et al., 2002)
was strongly reduced or absent in the null mutant
(Fig. 4C and
Fig. 5).
The defect in spermatogenesis of the PU.1G/G mutant was
related to a decrease in cell proliferation, as evidenced by decreased BrdU
incorporation during development (Fig.
6). The number of germ cells incorporating BrdU was significantly
decreased at E12.5 and E13.5. By contrast, BrdU incorporation in the somatic
component of the testis cords (essentially Sertoli cells) was decreased to a
lesser extent and only at later times, an observation that will be addressed
in future studies. At postnatal stages, a functional defect of
PU.1G/G testes was evidenced by testis graft experiments,
an approach made necessary by the early death of the mutant homozygotes.
Testes dissected shortly before birth (E18.5) were grafted under the tunica
albuginea of recipient nude mice. Spermatogenesis is known to proceed normally
in such grafts up to the production of functional sperm
(Honaramooz et al., 2002).
Grafts of the PU.1G/+ testes showed, after 1 week, a
strong proliferative activity as evidenced by PCNA immunolabeling
(Fig. 7Ae,g), leading, after 1
month, to the development of a structurally normal seminiferous epithelium
(Fig. 7Ba,c). By contrast,
grafts of their PU.1G/G littermates showed, 1 week after
transplantation, only a limited proliferative activity of Sertoli cells in the
periphery of the tubules (Fig.
7f,h), which were essentially empty after 2 weeks (not shown) and
4 weeks (Fig. 7Bb,d). Thus, not
only was the number of gonocytes reduced owing to a decreased proliferation
rate, but the surviving germ cells were unable to enter a normal
differentiation pathway.
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), stella
is the earliest marker of embryonic differentiation; it is already expressed
in the blastocyst and later restricted to the germinal lineage
(Saitou et al., 2002;
Sato et al., 2002) (our
unpublished results). The correlation between PU.1 and stella
expression at the late fetal stage led us to check whether, as is stella
itself, PU.1 is a marker of early embryonic development. Confocal
microscopy detected GFP fluorescence in PU.1G/+
heterozygotes as early as the blastocyst stage (E3.5), at which the inner cell
mass showed heterogeneous but strong labeling
(Fig. 8A). ES cells were
accordingly checked and found positive by immunolabeling with anti-PU.1
antibodies (Fig. 8B). In the
E6.5 postimplantation embryo (Fig.
8C), GFP fluorescence indicated extensive expression in the
epiblast and extraembryonic ectoderm, with a marked concentration in the
general area of the node, which was maintained during the following days in a
variety of structures. Although both genes are expressed at the same early
stage, we could exclude the possibility that the PU.1 protein directly
controls stella transcription, as transcripts of the latter were clearly
detected in the early PU.1G/G embryo (E10.5, data not
shown). The pattern of PU.1 expression is in fact distinct and more
extensive than that of stella, Blimp1 and fragilis, currently
considered as the relevant markers of germ line specification and similarly
expressed at very early stages (Saitou et
al., 2002).
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; Metcalf et al.,
2006). The picture must now be expanded to include other classes
of stem cells, at least the SSCs of the adult mammalian testis and embryonic
PGCs. The list of progenitors under PU.1 control might not yet be complete,
considering the widespread expression of the gene during embryogenesis.
Considering the lineages for which sufficient data exist, namely the
lymphopoietic and erythropoietic progenitors
(Back et al., 2004;
Iwasaki et al., 2005) and now
the germinal lineage, a common picture seems to be emerging, in which the gene
would act in the maintenance of cells in the undifferentiated state. In the germinal differentiation process, our initial finding was a regulated expression of PU.1 RNA in the adult testis, first in SSCs and early spermatogonia, and later in meiotic spermatocytes. The availability of the null mutant PU.1G was initially thought to provide the right tool to unravel its role in these cell types. It turned out, however, that expression of the locus starts at a much earlier stage of germinal differentiation - in fact, at the time of the first specification of germ cells and even before. Furthermore, although the structure of the fetal testis appeared at least grossly normal, it became clear that proliferation of the germ cells was strongly affected, resulting in a much reduced number of gonocytes at the end of fetal development. The remaining cells were in fact unable to enter the normal differentiation pathway, the lack of recognizable germinal progression in the grafted testes defining an absolute requirement for PU.1 expression in the fetal differentiation of germ cells.
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), and we also noted that the widespread expression of
PU.1 in the postimplantation embryo does not correspond to that of
stella, which is rapidly restricted to the germinal lineage.
The finding that PU.1 is required for the embryonic differentiation of germ
cells leaves open the question of its role in the adult testis. An analysis
would require a conditional mutation in which gene expression would be
arrested exclusively after birth. An intriguing and complex pattern, with
expression in SSCs and possibly early spermatogonia, extinguished in
differentiated spermatogonia and starting again in meiotic cells, might
actually suggest distinct activities in these different cell types.
Altogether, a relatively extended domain of expression, starting at the
earliest stages of development and required in at least two independent
lineages, suggests a common function in the maintenance of embryonic and adult
stem cells and in the control of their differentiation. Given the multiplicity
of protein variants encoded by the PU.1 gene
(Lloberas et al., 1999), it
remains to be determined whether they exert distinct functions in different
lineages.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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90%) in pachytene spermatocytes (lane 1) and
elongated and round spermatids (lanes 2 and 3, respectively). (G-I)
Expression in single (G), paired (H) and aligned (I) cells (arrows) in the
peripheral compartment of the seminiferous tubule (Asingle,
Apaired and Aaligned spermatogonia). Scale bars: 50
µm in A-E; 25 µm in G-I.
