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First published online March 6, 2009
doi: 10.1242/10.1242/dev.032243
1 Department of Dairy and Animal Sciences, Center for Reproductive Biology and
Health, College of Agricultural Sciences, Pennsylvania State University,
University Park, PA 16802, USA.
2 Department of Animal Biology, School of Veterinary Medicine, University of
Pennsylvania, Philadelphia, PA 19104, USA.
3 Penn Bioinformatics Core, University of Pennsylvania, Philadelphia, PA 19104,
USA.
* Author for correspondence (e-mail: cpope{at}vet.upenn.edu)
Accepted 27 January 2009
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Csf1, Leydig cell, Myoid cell, Niche, Self-renewal, Spermatogonial stem cell
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Huckins and Oakberg, 1978;
Huckins, 1971). Collectively,
As, Apr and Aal spermatogonia are referred to
as proliferating spermatogonia (Russell et
al., 1990; de Rooij and
Russell, 2000). Currently, the mechanisms controlling SSC
functions are poorly understood.
Advancement of knowledge regarding specific characteristics of SSCs has
been hampered by an inability to isolate these cells from the testis of any
mammalian species, mostly due to lack of known specific phenotypic or
molecular markers. However, isolation of SSC-enriched fractions from rodent
testes has been achieved. Recent studies demonstrated that Thy1 (CD90) is
expressed on the surface of mouse SSCs and nearly all SSCs are contained
within the Thy1+ cell fraction
(Kubota et al., 2003;
Kubota et al., 2004a). When
isolated using magnetic activated cell sorting (MACS), the Thy1+
cell fraction is enriched 5- to 30-fold for SSCs compared to the total cell
population in pre-pubertal and adult mouse testes, respectively
(Kubota et al., 2004a). Thus,
isolation of the Thy1+ testis cell fraction provides an
experimental cell population that can be used to study specific
characteristics of mammalian SSCs.
In general, adult stem cell functions are controlled extrinsically from
influence of a niche microenvironment and intrinsically by expression of
specific gene networks. Stem cell niches are composed of both architectural
support and a milieu of growth factors
(Spradling et al., 2001;
Scadden, 2006). These
specialized microenvironments are formed by contributions of surrounding
support cells. In mammalian testes, Sertoli cells are thought to be the major
contributor to the SSC niche (Tadokoro et
al., 2002; Yomogida et al.,
2003), but contributions by other testicular somatic cells
including peritubular myoid and interstitial Leydig cells are also possible.
Knowledge of niche growth factors produced by testicular somatic cells is
limited. Previous studies have established that glial cell line derived
neurotrophic factor (Gdnf) is a major regulator of mouse, rat and hamster SSC
self-renewal in vitro (Kubota et al.,
2004b; Ryu et al.,
2005; Kanatsu-Shinohara et
al., 2008) and essential for normal spermatogenesis in vivo
(Meng et al., 2000;
Naughton et al., 2006).
Additionally, exposure to basic fibroblast growth factor (Fgf2) was shown to
enhance the effects of Gdnf in vitro, but was unable to support SSC
self-renewal alone (Kubota et al.,
2004b). In vivo, both Gdnf and Fgf2 production in the testis has
been localized to Sertoli cells (Tadokoro
et al., 2002; Mullaney and
Skinner, 1991). In long-term cultures of Thy1+ testis
cells supplemented with Gdnf and Fgf2, clumps of germ cells form which are
composed of both SSCs and non-stem cells
(Kubota et al., 2004b;
Kanatsu-Shinohara et al.,
2005). While SSC numbers expand for extended periods of time in
these cultures, their proliferation rate is slow. Additionally, the percentage
of SSCs within the germ cell clumps varies greatly throughout a given culture
period and can be extremely small, estimated to be 0.02% in one instance
(Kanatsu-Shinohara et al.,
2005). These observations suggest that niche growth factors other
than Gdnf and Fgf2 have important roles in supporting SSC self-renewal.
The objective of the current study was to identify genes whose expressions are enriched in the Thy1+ germ cell fraction of mouse testes to provide insights into potential extrinsic and intrinsic regulators of SSC self-renewal. We found that expression of colony stimulating factor 1 receptor (Csf1r) is highly enriched in this fraction and that exposure to the specific ligand for this receptor, the cytokine colony stimulating factor 1 (Csf1) enhances self-renewal of mouse SSCs in vitro. Additionally, we localized Csf1 expression to both interstitial Leydig and peritubular myoid cells in pre-pubertal and adult mouse testes, implicating these cell populations as contributors to the SSC niche.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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; Oatley and
Brinster, 2006). Three replicate samples were collected for each
fraction from independent donor preparations. Viability was >90% for all
isolated cell populations based on assessment of Trypan Blue exclusion. The
SSC contents of these cell fractions were determined using functional germ
cell transplantation and gene expression profiles were evaluated using DNA
microarray analysis.
Germ cell transplantation and analysis
To determine the SSC content of Thy1+ and Thy1-depleted cell
fractions and cultured Thy1+ germ cell clumps we conducted
functional germ cell transplantation analyses using techniques previously
described (Brinster and Avarbock,
1994; Oatley and Brinster,
2006). Briefly, for all transplantations, 8-10 µl of cultured
or MACS isolated cell suspensions originally collected from Rosa
(B6.129S7-Gtrosa26; The Jackson Laboratory) donor males, which express
lacZ in germ cell types, were microinjected into testes of 129
x C57 recipient males that were treated with busulfan (60 mg/kg) at
least 6 weeks earlier to deplete endogenous spermatogenesis. Two months after
transplantation the number of donor-derived colonies of spermatogenesis in
each recipient testis were counted using a dissecting microscope following
staining with X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside).
Because each colony is clonally derived from a single SSC
(Nagano et al., 1999;
Kanatsu-Shinohara et al.,
2006), counting donor-derived colonies of spermatogenesis provides
a relative quantification of SSC number in an injected cell suspension. All
donor cell suspensions were injected at a standard concentration of
1x106 cells/ml.
RNA isolation and DNA microarray processing/analysis
To conduct DNA microarray analyses of gene expression, RNA was isolated
from MACS-isolated Thy1+ and Thy1-depleted cell fractions collected
from 6 days post-partum (dpp) C57BL/6 mouse pups. Highly pure samples of total
RNA were isolated using a hybrid Trizol-DNeasy column protocol previously
described (Oatley et al.,
2006). Each sample was processed and used for hybridization onto
Affymetrix whole genome 430 2.0 GeneChips (Affymetrix, Santa Clara, CA, USA).
Three different replicate samples were collected for each Thy1+ and
Thy1-depleted cell fraction and each was hybridized onto a single array,
totalling six GeneChips. All hybridizations and scanning of GeneChips were
conducted by the University of Pennsylvania Microarray Core facility as
previously described (Oatley et al.,
2006). Briefly, 5 µg of total RNA was reverse transcribed using
Superscript II reverse transcriptase and poly(T) oligomer priming that
incorporated the T7 promoter. In vitro transcription was then conducted,
followed by fragmentation of resultant cRNA and hybridization to Mouse Genome
430 2.0 arrays (Affymetrix). Chips were then washed and stained with
streptavidin-phycoerythrin. A confocal scanner was then used to collect
fluorescence signals, and the average signal from two sequential scans was
calculated for each microarray. Affymetrix microarray suite 5.0 was used to
quantify expression levels for target genes and default values provided by
Affymetrix were applied to all analysis parameters. The number of probe pairs
meeting the default discrimination threshold (t=0.015) was used to
assign a call of absent, present or marginal for each assayed gene, and a
P-value was calculated to reflect confidence in the detection
call.
Microarray data analysis
Affymetrix CEL files for each genechip were imported into Stratagene Array
Assist Lite software version 3.4 to calculate normalized GC Robust Microarray
Analysis (GCRMA) expression levels for each probe set on each array. Data (CHP
files from GCRMA calculations) were then imported into GeneSpring 7.2 software
(Agilent Biotechnology; Santa Clara, CA, USA) to visualize expression patterns
and data were normalized to the median. Filter flags were then applied
including a Present call in three out of six samples and differential
expression in Thy1+ versus Thy1-depleted samples. These parameters
resulted in the filtering out of 26,927 genes from the 45,101 genes present on
the genechips. The GCRMA raw values for these filtered genes were then
exported into Excel for statistical analysis. A two-class paired test using
Statistical Analysis of Microarray version 2.23A tool, which included a 3%
false discovery rate with P-value <0.05, was used to identify
genes with differential expression at the 10-fold level between
Thy1+ and Thy1-depleted cell fractions. All microarray data are
available from the NCBI GEO database
(http://www.ncbi.nlm.nih.gov/geo/),
Accession No. GSE14222.
Quantitative RT-PCR analyses
For quantitative reverse transcriptase PCR (qRT-PCR) analyses 1 µg of
total RNA for each sample was DNase-treated (DNA-free Kit; Invitrogen,
Carlsbad, CA, USA) to remove possible contaminating genomic DNA, and reverse
transcribed using oligo(d)T priming and Superscript II reverse transcriptase
(Invitrogen). Expression levels of specific genes were measured using SYBR
Green assays and an ABI 7300 sequence detection system (Applied Biosystems,
Foster City, CA, USA). Specificity of amplicons was evaluated using melt curve
analyses. Transcript levels for specific genes-of-interest were normalized to
those of ribosomal protein S2 (Rps2) to make quantitative comparisons between
different samples as previously described
(Oatley et al., 2006;
Oatley et al., 2007). All
primer sequences were designed using Primer Express 3.0 (Applied Biosystems)
and the nucleotide sequences were; 5'-TACTTCAAGGCTTCGCCTCTCT-3',
5'-CTACGTGTTCCATCTGCAAATAGG-3' for Bcl6b,
5'-AACTATGTTGTCAAGGGCAATGC-3',
5'-GGACCACACATCACTCTGAACTG-3' for Csf1r,
5'-CAACTTCATGCATATGGCTCTCA-3',
5'-TCTGCTAAAGCACTGGGCTTCT-3' for Gfra1,
5'-CCCAGCTTTCCCGAATCCT-3',
5'-GCGGGACGTAAATAAATAAAATGG-3' for Lhx1, and
5'-CCATGCCTCATCACTTACCCTAT-3',
5'-GTCCGGAAGAGCTTGCAGAA-3' for Rps2.
Thy1+ germ cell cultures and assessment of Csf1r effects on SSC self-renewal in vitro
Germ cell cultures were established from MACS isolated Thy1+
cells collected from 6-dpp Rosa mouse testes using methods previously
described (Kubota et al.,
2004b; Oatley and Brinster,
2006). Germ cells were seeded onto mitotically inactivated STO
feeder monolayers and maintained in chemically defined mouse serum-free medium
(mSFM) (Kubota et al., 2004b)
supplemented with 20 ng/ml recombinant human Gdnf (R&D Systems,
Minneapolis, MN, USA) and 1 ng/ml recombinant human Fgf2 (BD Biosciences; San
Jose, CA, USA). To test its effects on SSC self-renewal, recombinant mouse
Csf1 (R&D Systems) was added to the culture media at a concentration of 10
ng/ml. Control conditions for these Csf1 experiments consisted of
supplementation with Gdnf and Fgf2 alone. To evaluate SSC self-renewing
expansion in the different culture conditions, cells were collected from
culture wells by trypsin-EDTA digestion of entire culture well contents (STO
feeders + germ cell clumps) and an aliquot of the cell suspension was
transplanted into recipient mouse testes to determine SSC content. The
remaining cell suspensions were sub-cultured at a 1:2 ratio. Cells were
transplanted on day 7 of culture and every 2 weeks thereafter throughout a
63-day culture period. Analysis of recipient testes was conducted 2 months
after transplantation by X-Gal staining and using a dissecting microscope to
count the number of blue-stained colonies. These colony numbers were used to
determine SSC content in Csf1-treated and control cultures at each time-point
analysed throughout the 63-day culture period. SSC expansion in Csf1-treated
and control cultures was evaluated by applying the following equation at each
culture time-point analysed:
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For each culture, SSC numbers/105 Thy1+ cells cultured were multiplied by sub-culture ratios in order to determine SSC expansion rates throughout the 63-day culture periods.
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Flow cytometric analysis of Csf1r and F4/80 expression
To determine the percentage of cells expressing Csf1r and the
macrophage-specific marker F4/80 within pup testes and cultured
Thy1+ germ cell clumps we used flow cytometric analysis. For mouse
pup testes, single cell suspensions were generated by trypsin-EDTA digestion
at 37°C for 10 minutes. Approximately 1x106 cells for
each sample were then incubated for 20 minutes on ice in DPBS-S (DPBS with
0.1% FBS, 10 mM HEPES, 10 mM Na pyruvate, 1 mg/ml glucose, and
penicillin/streptomycin) with specific antibodies. Primary antibodies used
were mouse anti-human Thy1 IgG1 (Abcam, Cambridge, MA, USA), PE-conjugated rat
anti-mouse Csf1r IgG1 (AbD Serotec, Raleigh, NC, USA), or APC-conjugated rat
anti-mouse F4/80 IgG2a (Biolegend, San Diego, CA, USA). Detection of Thy1
involved secondary incubation with FITC-conjugated rat anti-mouse IgG1
(Biolegend). Isotype controls consisted of PE conjugated rat IgG1 (Biolegend),
APC conjugated rat IgG2a (Biolegend), and FITC conjugated rat anti-mouse IgG1
(Biolegend). Cells were analysed with a Beckman-Coulter FC500 flow cytometer
(Fullerton, CA USA). For Thy1+ germ cell cultures, clumps were
removed from STO feeders using gentle pipetting, collected into HBSS, and
pelleted at 600 g for 7 minutes. Cell clumps were then
digested with trypsin-EDTA at 37°C for 5 minutes to generate single cell
suspensions. Approximately 1x105 cells for each sample were
then incubated for 20 minutes on ice with rabbit anti-human Csf1r polyclonal
antibody (Santa Cruz Biotechnology) or control immunoglobulin in DPBS-S.
Secondary detection included incubation with Alexa Fluor 488-conjugated goat
anti-rabbit IgG (Invitrogen) on ice for 20 minutes. Propidium iodide (1
µg/ml) was added to each sample just before analysis to distinguish live
and dead cells. The percentage of Csf1r+ cells in each sample was
then evaluated using flow cytometric analysis with a Guava PCA 96 System
(Guava Technologies; Hayward, CA, USA).
Statistical analyses
All statistical analyses were conducted using SPSS v.15 software (SPSS,
Chicago, IL USA). Differences between means for functional germ cell
transplantation analyses were examined using a general linear model (GLM)
univariate ANOVA. Comparisons of total germ cell and SSC expansion rates
between Csf1-treated and control Thy1+ germ cell cultures were
conducted using GLM regression analyses to test for homogeneity of slopes
between growth lines.
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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). Before assaying global gene-expression profiles we used
functional germ cell transplantation with Rosa mice serving as donors to
unequivocally examine relative difference of SSC contents in the cell
fractions (Fig. 1B,C). The
Thy1+ cells produced 228.1±45.9 colonies of donor-derived
spermatogenesis/105 cells injected (mean±s.e.m.,
n=3 individual replicate samples and 24 total testes), whereas
Thy1-depleted cells produced 9.2±3.3 colonies of donor-derived
spermatogenesis/105 cells injected (n=3 replications and
24 total testes). Each colony of donor-derived spermatogenesis is clonally
derived from a single transplanted SSC
(Nagano et al., 1999;
Kanatsu-Shinohara et al.,
2006); thus, SSC content of the MACS isolated Thy1+
cell fraction was approximately 25-fold higher than the Thy1-depleted cell
population.
Identification of differential gene expression between Thy1+ and Thy1-depleted testis cell populations
To identify genes whose expressions are enriched in SSCs we measured global
gene expression profiles in MACS isolated Thy1+ (n=3) and
corresponding Thy1-depleted testis cell populations (n=3) from 6-dpp
inbred C57BL/6 donor mice using DNA microarray analysis. Genes expressed
10-fold or higher in Thy1+ compared with Thy1-depleted fractions
were filtered, resulting in identification of 202 genes (see Table S1 in the
supplementary material). To gain insight into possible biological functions of
these genes they were sorted into 12 functional categories based on Gene
Ontology (GO) Consortium biological process classifications provided by
Affymetrix (Table 1). Genes
without a clearly defined function were classified as other. We confirmed
differential expression for three of these genes using qRT-PCR, including
Bcl6b, Lhx1 and Gfra1 (see Fig. S1 in the supplementary
material). Investigation of surface antigens implicated as SSC markers
(Table 2) revealed that
expression of Thy1, c-Ret (Ret - Mouse Genome Informatics),
Gfra1 and Cdh1 were enriched 10-fold or greater in the
Thy1+ cell fraction. By contrast, expression of Gpr125,
which has been suggested as an SSC marker in adult mouse testes
(Seandel et al., 2007), was
expressed 5.5-fold higher in the Thy1-depleted population than in the
Thy1+ cell fraction. Next, we examined the expression of
transcription regulators implicated as having important roles in SSC functions
(Table 3). Only Bcl6b
and Lhx1, identified in our previous studies as important regulators
of mouse SSC self-renewal (Oatley et al.,
2006; Oatley et al.,
2007), were expressed greater than 10-fold in the Thy1+
cell fraction. Expression of Plzf (Zbtb16) (Bauus et al.,
2004; Costoya et al., 2004) and
Taf4b (Falender et al.,
2005), both of which have been suggested as essential for SSC
self-renewal, were marginally enriched (
2-3-fold) in Thy1+
cells, similar to the expression levels of Ngn3 (Neurog3)
(Yoshida et al., 2004) and
Sohlh1 (Ballow et al.,
2006), which have been implicated as regulators of spermatogonial
differentiation. By contrast to the enriched genes, expression of
n-Myc (Mycn)
(Braydich-Stolle et al., 2007)
and c-Fos (Fos) (He et
al., 2008), which have been suggested as important for
proliferation of testis cell populations with unproven SSC content, were more
highly expressed in the Thy1-depleted testis cell population than in
Thy1+ cells. Overall, identification of differential gene
expression between the Thy1+ and Thy1-depleted cell fractions
provides a database to better understand characteristics of mouse SSCs and
mechanisms regulating their functions.
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). Thus, we
used flow cytometric analysis (FCA) to examine whether enriched Csf1r
expression by the isolated Thy1+ cell fraction from mouse testes
could be due to co-enrichment of macrophages. Examination of the
F4/80+ cell population, a macrophage marker, in 8-dpp mouse pup
testes revealed complete absence of Thy1 expression, indicating that
testicular macrophages are not Thy1+ (see Fig. S2 in the
supplementary material). Thus, we were confident that enriched Csf1r
expression was restricted to the Thy1+ germ cell fraction.
Csf1r is expressed by a sub-population of spermatogonia in mouse pup testes and cultured Thy1+ germ cells
Previous studies indicate that Csf1r is not expressed by any cells within
seminiferous tubules of mouse testes and localized specifically to macrophages
present in the interstitial space and occasionally intermingled with
peritubular myoid cells (Cohen et al.,
1996; Pollard et al.,
1997). Lack of observed Csf1r expression by germ cells in those
studies is not surprising if expression is restricted to SSCs in the
seminiferous epithelium, because their rarity could prove very difficult to
detect. However, studies by Johnson et al. (Johnson et al., 2007) detected
enriched Csf1r transcript expression by spermatogonia within the rat
testis. Using immunofluorescence, we observed Csf1r expression by individual
spermatogonia located on the basement membrane within some seminiferous
tubules of 10-dpp mouse testes that contain the full complement of
spermatogonia (Fig. 3A). These
observations were rare, as only two to three tubules containing
Csf1r+ germ cells were observed per cross-section (n=6
testes examined). Negative controls with normal IgG as the primary antibody
showed minimal background staining (see Fig. S3 in the supplementary
material). Further examination of Csf1r expression using FCA revealed the
presence of an F4/80-/Csf1r+ cell population in testes
of mouse pups (Fig. 3B), and
this population was weakly positive for Thy1
(Fig. 3C), similar to previous
reports of Thy1 expression by the SSC population in mouse pup testes
(Kubota et al., 2004a).
Collectively, these results identify a Csf1r+ spermatogonia
population within mouse testes.
Next, we examined if Csf1r is expressed in cultures of Thy1+
germ cell clumps maintained in serum-free conditions with Gdnf and Fgf2 as the
only growth factor supplements. Self-renewing expansion of SSCs is supported
in these conditions and germ cell clumps are composed of both stem cells and,
to a greater extent, non-stem germ cells. Thus, we reasoned that if Csf1r
expression is restricted to SSCs only a portion of the Thy1+ germ
cells clumps would be Csf1r+. Expression of Csf1r transcript could
be detected using standard RT-PCR analysis
(Fig. 4A) and expression of
Csf1r protein was detected using immunofluorescence
(Fig. 4B). However, staining
was not homogenous throughout germ cell clumps, suggesting that only a portion
of the cells express Csf1r. To further explore this possibility we evaluated
the expression of Csf1r by germ cell clumps using FCA
(Fig. 4C). These analyses
revealed that 2.2% of the cultured Thy1+ germ cells
(n=2 different primary cultures) expressed Csf1r, suggesting that
this characteristic may be specific to SSCs in the heterogeneous cultured
Thy1+ spermatogonial cell population.
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0.05) greater compared with
controls beginning at day 21 of culture and continuing throughout the 63-day
culture period (Fig. 5D). At
day 35 of culture the number of SSCs was determined to be 2.1-fold higher in
Csf1-treated cultures compared with controls, but the total number of germ
cells only differed by a factor of 1.1-fold. This trend continued throughout
the remainder of the culture period, with the number of SSCs being 3.2-fold
higher in Csf1-treated cultures at day 63 when the total number of germ cells
differed by only 1.1-fold. Collectively, these results indicate that exposure
to Csf1 alters the fate decision of self-renewal versus differentiation in
vitro, resulting in greater concentration of SSCs being maintained in the
heterogeneous mix of Thy1+ germ cell clumps, and indicate that Csf1
is an extrinsic stimulator of mouse SSC self-renewal.
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DISCUSSION |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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; Naughton et al.,
2006). SSC content of the Gfra1+ cell fraction is only
marginally enriched (<2-fold) compared to the Gfra1-depleted cell
population in mouse pup testes (Baugeaw et al., 2005), and depleted of SSCs in
the adult testis (Ebata et al.,
2005). Additionally, the c-Ret+ cell fraction contains
fewer SSCs than the total cell population in both pup and adult testes
(Ebata et al., 2005). Also,
expression of Gfra1 and c-Ret is homogenous in Thy1+ germ cell
clumps in vitro, which are composed of both SSCs and non-stem germ cells
(Kubota et al., 2004b). These
observations suggest that Gdnf action is not restricted to SSCs and may
stimulate proliferation of multiple spermatogonia populations. Whereas
proliferation is required for self-renewal, proliferation itself does not
dictate a self-renewal event, because differentiation whether from symmetric
or asymmetric stem cell division also involves mitosis. Thus, it is possible
that Gdnf stimulates general spermatogonial proliferation and cues from other
factors are needed to direct a self-renewal rather than differentiation
pathway. Results of the present study demonstrate that exposure to Csf1
influences SSC self-renewal in vitro without affecting proliferation of
non-SSCs. We observed expression of Csf1r by individual spermatogonia in only
a few seminiferous tubules and could detect its expression on the cell surface
by sub-populations of cells in mouse pup testes and Thy1+ germ cell
clumps, suggesting that Csf1r expression may be restricted to SSCs within
mouse testes. These observations, in conjunction with in vitro analysis of SSC
self-renewal, strongly suggest that at least for mice Csf1 action is
restricted to SSCs in the male germline and does not affect other
spermatogonial populations. Thus, Csf1 may be a specific regulator that
maintains a stem cell phenotype while acting in concert with Gdnf, which
promotes proliferation, and these two influences ultimately stimulate
self-renewing division. Because of the heterogeneous nature of cultured
Thy1+ spermatogonia, consisting of SSCs and non-stem cell
spermatogonia, it is possible that exposure to Csf1 also indirectly effects
SSC self-renewal by acting on non-stem cell spermatogonia. Collectively, our
results suggest that Csf1 is an integral component of the growth factor milieu
that regulates self-renewal of mouse SSCs.
In vivo, interaction of stem cells with their cognate niche
microenvironment is crucial for continual self-renewal and differentiation. In
mammalian testes, the SSC niche is believed to be formed primarily by
contributions of Sertoli cells. To date, production of Gdnf by these cells has
been the only suggested mechanism of the SSC niche
(Tadokoro et al., 2002;
Yomogida et al., 2003).
However, contributions by Leydig and myoid cells are also possible.
Importantly, recent studies by Yoshida et al.
(Yoshida et al., 2007) have
revealed a biased localization of the proliferating spermatogonial population
in regions of seminiferous tubules adjacent to the vascular network and
clusters of Leydig cells. Because SSCs are present in this germ cell
population, these observations suggest that Leydig cells may influence SSC
function. We observed expression of Csf1 by Leydig cells and select myoid
cells in both pre-pubertal and adult mouse testes. In agreement, studies by
Shima et al. (Shima et al.,
2004) identified elevated Csf1 transcript expression by
myoid cells and Ryan et al. (Ryan et al.,
2001) localized Csf1 expression to Leydig cells in adult mouse
testes. While our studies demonstrate that Csf1 influences SSC self-renewal in
vitro, the importance of this factor on SSC functions in vivo remains
undetermined. Studies by Cohen et al.
(Cohen et al., 1996) revealed
an impairment of spermatogenesis in op/op mutant mice that are
deficient for Csf1 expression. In these animals, the number of sperm produced
is approximately 60% fewer than in wild-type males, a phenotype that has been
attributed to the dramatic (86%) reduction of serum testosterone
concentration in op/op mice. Spermatogenesis is sensitive to
intratesticular testosterone concentrations, which is only modestly (28%)
diminished in op/op males. Additionally, in other models of
testosterone deficiency, such as androgen receptor null mice, spermatogenesis
is impaired due to a block in germ cell development during meiosis
(Yeh et al., 2002;
De Gendt et al., 2004), and
this phenotype is not observed in op/op mice. Thus, decreased
testosterone production does not completely explain the dramatic reduction in
sperm production that occurs due to Csf1 deficiency in male op/op
mice, and could be due at least in part to impaired SSC functions. Results of
in vitro experiments in the current study suggest that Csf1 acts in synergy
with Gdnf to regulate SSC self-renewal in vitro. A similar mechanism may also
function in vivo; thus Csf1 deficiency in the testis may result in
sub-fertility rather than dramatic infertility, and it is a sub-fertility
phenotype that may be of greatest importance when relating findings in mice to
men.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/7/1191/DC1
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SUMMARY
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MATERIALS AND METHODS
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DISCUSSION
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Ballow, D., Meistrich, M. L., Matzuk, M. and Rajkovic, A.
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