1 Dipartimento di Sanita' Pubblica e Biologia Cellulare, Sezione di Anatomia,
Rome, Italy
2 Istituto Dermopatico dell'Immacolata (IDI, IRCCS), Rome, Italy
3 Dipartimento di Neuroscienze, Universita' di Roma Tor Vergata, Rome,
Italy
* Author for correspondence (e-mail: dolci{at}uniroma2.it )
Accepted 28 January 2002
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Summary |
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Key words: Kitl, Kit, Telomerase, Germ cells, Meiosis, Proliferation, PI3K
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Introduction |
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In the mouse testis, telomerase activity has been reported mainly in
proliferating spermatogonia (type A spermatogonia), and it is downregulated in
the differentiating spermatocytes and spermatids and is no longer present in
spermatozoa (Ravindranath et al.,
1997; Eisenhauer et al.,
1997
). As a result of telomerase activity, sperm cells have long
telomeres, of about 10-20 kb in humans and 50 kb in mice, that apparently do
not shorten with the aging of the organism
(Allsopp et al., 1992
). Type A
spermatogonia are pluripotent stem cells in the testis, which undergo many
rounds of duplications giving rise to type B spermatogonia and to other type A
spermatogonia. The mechanisms of germ cell mitogenesis are poorly understood,
but we have recently shown that stem cell factor (Kit ligand, Kitl) can induce
3H-thymidine incorporation in type A, Kit expressing spermatogonia
in vitro (Rossi et al., 1993
).
In light of the recent reports that spermatogonia lacking telomerase undergo
arrest of mitosis and apoptosis, we investigated whether Kitl is able to
regulate testicular stem cell telomerase activity. Since Kitl is a
survival/proliferation factor for proliferating primordial germ cells (PGCs)
but not for Kit-expressing growing oocytes, we investigated its role in
regulating telomerase activity in these cell types.
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Materials and methods |
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Spermatogonia were obtained from 7-8 days old Swiss CD-1 mice, as
previously reported by Rossi et al. (Rossi
et al., 1993). Briefly, germ cell suspensions were obtained by
sequential collagenase-hyaluronidase-trypsin digestions of freshly withdrawn
testes. A 3 hour period of culture in 10% FCS with E-MEM added was performed
to facilitate adhesion of contaminating somatic cells to the plastic dishes.
At the end of this pre-plating treatment, which was considered to be T0,
enriched germ cell suspensions were rinsed from FCS, and spermatogonia were
then cultured in E-MEM supplemented with 2 mM Na-pyruvate and 1 mM Na-lactate
in the presence or absence of Kitl (100 ng/ml, Genzyme). To obtain mitotic
indexes, nuclear morphologies from at least 103 cells per treatment
were assessed by microscope observations of the cell cultures previously fixed
in 3:1 methanol-acetic acid and stained with Giemsa, according to Meistrich et
al. (Meistrich et al., 1973
).
The purity of the spermatogonia was about 80% after the pre-plating treatment.
During the 24 hours of culture, the contaminating somatic cells attached to
the dish, whereas spermatogonia did not adhere and were recovered in the
suspension at a purity higher than 90%. At different times of culture an equal
number of surviving cells, judged as trypan-blue negative, have been fixed or
frozen to analyse nuclear morphology, telomerase RNA template (TR) and TERT
expression by RT-PCR, in situ hybridization and telomerase activity.
Where indicated, cells were also incubated for 1 hour before Kitl addition with 10 µM LY294002 (#270-038-M005, Alexis).
Probe preparation for in situ hybridization
The plasmid-containing human telomerase RNA template sequences (560 bp) was
kindly provided by Geron Corp., Menlo Park, CA, USA. To generate sense and
antisense riboprobes, the plasmid was linearized by NaeI or
EcoRV, respectively. 35S-UTP-labeled single-stranded RNAs
were synthesized according to the manufacturer's (Stratagene) conditions.
Transcripts were then alkali hydrolyzed to generate probes of an average
length of 200 nucleotides and then ethanol precipitated. The probes were then
dissolved to a final concentration of at least 5x105
cpm/µl in 10 mM DTT.
In situ hybridization
Cells were seeded onto poly-L-lysine-coated slides, fixed in 4%
paraformaldehyde and then air-dried. The hybridization procedure was performed
according to Jannini et al. (Jannini et
al., 1994). Briefly, cells were incubated overnight at 50°C in
hybridization solution containing 5x104 cpm/µl of the
telomerase probe. The day after, slides were incubated in successive
formamide/SSC washes and then dehydrated and exposed to Kodak NTB-2 emulsion
for 2 weeks. The microautoradiographs were then developed and counter-stained
with Toluidine blue.
TRAP assay
This method is based on PCR amplification of telomerase extension products
and was performed as previously described (Piatyszek et al., 1995). Briefly,
cells were lysed in 400 µl of ice-cold extraction buffer (0.5%
3-(3-cholamidopropyl)-dimethyl ammonio- 1 -propanesulfonate, 10 mM Tris-HCl pH
7.5, 1 mM MgCl2, 1 mM EGTA, 5 mM mercaptoethanol, 0.1 mM
4-(2-aminoethyl)-benzene-sulfonyl fluoride hydrochlorine and 10% glycerol). To
increase the extraction strength of the lysis buffer, experiments performed
with fetal germ cells were carried out using a modified extraction buffer
according to Norton et al. (Norton et al.,
1998). Preliminary assays were carried out in order to assess the
relationship between the concentration of cell extracts and the amount of TRAP
product obtained, which was not at saturation using 200 cells. 4 µl of the
cell extracts, corresponding to 200 viable cells, was then used for the
spermatogonia and fetal male germ cells TRAP assays in 50 µl of the
reaction mixture consisting of 20 mM Tris-HCl (pH 8.3), 68 mM KCl, 1.5 mM
MgCl2, 1 mM EGTA, 0.05% Tween 20, 0.1 g of TS (AATCCGTCGAGCAGAGTT)
primer, 0.5 M T4 gene 32 protein, 50 µM of each deoxyribonucleotide
triphosphate, 2 units of Taq DNA polymerase and 0.2 µl of 32P
dCTP (3000 Ci/mmol, Dupont NEN Research Products, Boston, MA). Each reaction
was carried out in a single PCR tube containing 100 ng of CX-Ext
oligonucleotide (5'-GTGCCCTTACCCTTACCCTTACCCTTA) sealed at the bottom of
the tube by a wax barrier. Samples were incubated at room temperature for 20
minutes to allow telomerase to extend TS primer, followed by 31 cycles of PCR
amplification of the telomeric products. 45 µl of the reactions was loaded
on a 10% non-denaturing polyacrylamide gel and identified by autoradiography.
6 µg/ml of RNase A (Boehringer Mannheim) was added to the reactions to
confirm the specificity of the telomerase products, which disappeared in the
presence of RNase A. The TRAP assay on oocytes was performed according to
Eisenhauer et al. (Eisenhauer et al.,
1997
). Cell extracts corresponding to 15 oocytes for each
treatment were used in each assay.
An assay of alkaline phosphatase activity as an internal control for the
quality of the cell extracts was performed using a commercially available kit
(SIGMA) as previously described (Pyiatszek
et al., 1995).
RT-PCR experiments
1 µg of total RNA, obtained from an equal number of viable spermatogonia
after 24 hours of culture, in the presence or absence of 100 ng/ml of Kitl,
was reverse transcribed by Mo-MuLV reverse transcriptase using random hexamers
in 20 µl of reaction mixture (ImpromII reverse transcription system,
Promega). To discriminate bands originating from contaminating DNA, another
set of reactions was performed omitting the reverse transcriptase. Prelimirary
RT-PCR experiments were performed for each set of primers in order to evaluate
the conditions under which PCR amplification was in the logarithmic phase.
Step cycle amplifications were performed diluting 1 µl of the cDNAs in 50
µl of PCR mixture (Taq polymerase, Promega) using TERT and TR murine
primers (mTERT and mTR respectively) designed on the basis of the published
sequences: mTERT A (5'-CTCTGCTGCGCAGCCGATACC3'); mTERT B
(5'-AGCTGAAGG CACACTTGTA-CT-3') (Greenberg et al., 1998); mTR A
(5'-ACTTCCAGCGGGCCAGGAAGCT-3'); and mTR B
(5'-CTGACAGAGGCGAGCTCTTC (Blasco et
al., 1998). Hprt (Rossi et
al., 1993
) and Kit-specific primers
(5'-TATGGACATGAAGCCTGGCGT; 3'-CATTCCTGATGTCTCTGGCTAGC; the
expected size of the amplified band is 293 bp) were used as internal controls
of gene expression. The conditions for amplification of mTERT were 35 cycles
at 94°C for 1 minute, 58°C for 30 seconds and 72°C for 45 seconds,
and the expected size of the amplified band is 776 bp; The conditions for
amplification of mTR were 35 cycles at 94°C for 1 minute, 52°C for 30
seconds and 72°C for 30 seconds; expected size of the amplified band is
183 bp). Conditions for Hprt and Kit amplifications were 30 cycles at 94°C
for 1 minute, 58°C for 30 seconds and 72°C for 45 seconds.
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Results |
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To verify whether Kitl was also influencing telomerase at the expression level, RNA from spermatogonia cultured for 24 hours with or without Kitl was analysed by RT-PCR using mTR- and mTERT-specific primers. A 183 bp band corresponding to the mTR amplification product was amplified only from Kitl-treated cell; omission of RT in the reverse transcription step resulted in no signals being obtained (Fig. 2A). PCR amplifications using mTERT-specific primers showed a strong increase in the corresponding 776 bp amplified band in the Kitl-treated sample compared with the control (Fig. 2B,C). RNA integrity was assessed by Hprt amplification (Fig. 2B). Kit primers were used to show that equal amounts of RNA extracted from spermatogonia were analysed (Fig. 2C).
|
mTR expression was also studied by in situ hybridization on spermatogonia immediately following pre-plating (T0) and after 24 hours of culture in the presence or absence of 100 ng/ml of Kitl. The riboprobe was synthesized from a human TR cDNA, which shares 65% sequence homology with the murine gene. In Fig. 3A,E, it is shown that mTR template is strongly expressed in spermatogonia but that such expression is downregulated after in vitro culture (B,F). Kitl addition completely restores mTR template expression (C,G). Panels D and H shows a negative control with a sense probe. The increase of silver grain density in the Kitl-treated cells further confirms that the cells were Kit-expressing spermatogonia, even though, owing to the in situ hybridization procedure, cellular morphology could not be well preserved.
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Telomerase activity is modulated by the P13-K pathway in mitotic
spermatogonia
Activation of the Kit receptor in mouse spermatogonia has been shown to be
mediated by the PI-3K-Akt signal transduction pathway
(Blume-Jensen et al., 2000;
Kissel et al., 2000
).
Therefore we studied the effect of the PI-3K inhibitor LY294002 on telomerase
activity. Spermatogonia were preincubated for 1 hour in the presence of 10
µM LY294002 and then cultured for 24 hours in the presence or absence of
100 ng/ml Kitl. The presence of the inhibitor slightly influenced the basal
levels of telomerase activity; however it blocked the induction mediated by
Kitl (Fig. 4) as well as
spermatogonia proliferation (10 µM LY294002=1.5%±0.9 versus 10 µM
LY294002+Kitl=1.9±0.7) in agreement with our results
(Dolci et al., 2001
) and those
by Feng et al. (Feng et al.,
2000
).
|
Kitl induces telomerase activity in male primordial germ cells
Prospermatogonia develop within the fetal testis from PGCs. After gonadal
colonization, PGCs continue to proliferate, and by 13.5 dpc in the male they
enter mitotic arrest, which lasts until few days after birth, and
differentiate into prospermatogonia
(McLaren, 1983). PGCs express
high levels of Kitl receptor (Kit), whereas prospermatogonia do not express
Kit until they have differentiated in spermatogonia
(Manova et al., 1990
). Since
telomerase activity is present in mitotically active prepuberal spermatogonia
and it is induced by Kitl, we investigated whether mitotically active male
PGCs and mitotically arrested prospermatogonia showed telomerase activity.
TRAP assays were performed on cell extracts corresponding to 200
trypan-blue-negative germ cells from 12.5 dpc male gonads (mitotic germ
cells), either freshly isolated (T0) or after 24 hours of culture in the
absence or presence of 100 ng/ml Kitl, and on germ cell extracts from 14.5 and
15.5 dpc male gonads (arrested prospermatogonia). In
Fig. 5 it is shown that
telomerase activity is present in mitotic germ cells (12.5 dpc) at the
beginning of culture (lane 1), that it drops following 24 hours (lane 3) and
that the activity is restored after addition of Kitl (lane 2). On the
contrary, mitotically arrested prospermatogonia from 14.5 and 15.5 dpc embryos
show low levels of enzyme activity (lanes 5 and 6, respectively), which is not
influenced by Kitl addition (not shown).
|
Telomerase activity is not modulated in Kit-positive oocytes
Kit is also re-expressed in female fetal germ cells when oocytes reach the
diplotene stage, throughout oocyte growth and acquisition of meiotic
competence (Manova et al.,
1993). The function(s) of Kit/Kitl during this period have not
been fully understood, although some results indicate that it can play a role
in oocyte growth (Manova et al.,
1993
; Ismail et al.,
1996
). To test whether telomerase activity in growing oocytes was
coupled to Kit activation, a TRAP assay was performed in isolated oocytes
freshly collected or cultured for 24 hours in the presence or absence of 100
ng/ml of Kitl. Growing oocytes collected from 10 dpn females were chosen,
since at this age they respond to Kitl by increasing their diameter (Packer et
al., 1994) but are unable to undergo spontaneous meiotic resumption (GVBD).
During this period of culture, Kitl did not influence growing oocyte
viability, and cell survival in the two groups was comparable (data not
shown). Telomerase activity was present in freshly isolated oocytes, and its
levels were stably maintained during 24 hours of culture
(Fig. 6). The enzyme activity
was not influenced by the addition of Kitl, and all the oocytes were at the GV
stage (data not shown).
|
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Discussion |
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Recently, it has been shown that the activation of one of the PI3K
downstream targets, the protein-kinase Akt, enhances human telomerase activity
through the phosphorylation of the TERT subunit
(Kang et al., 1999). In
addition, induction of telomerase activity and proliferation by antigen
stimulation in B lymphocytes can be suppressed by the PI3K inhibitor
whortmannin (Igarashi and Sakaguchi,
1997
). The possibility that Kitl might influence telomerase
activity through Kit-mediated PI3K pathway activation in mitotic spermatogonia
was evaluated. Treatment of the spermatogonia cultures with the PI3K inhibitor
LY42000 did not modify the basal levels of telomerase activity, instead it
blocked the increase of enzyme activity induced by Kitl as well as
proliferation, indicating that Kit activation of the PI3K downstream signaling
can regulate telomerase activity. It is interesting to note that the targeted
mutation of the PI3K docking site in the Kit receptor completely blocks
spermatogonia proliferation 8 days postnatum
(Blume-Jensen et al., 2000
;
Kissel et al., 2000
).
Genetic evidence has shown that mutations in the Steel locus (Sl) encoding
Kitl causes multiple stem cell defects in the homozygous mice. On of these
effects germ cell deficiency is always present during
embryonic development. It has in fact been demonstrated that Kitl plays an
important role in the establishment of the mouse germline during embryogenesis
(Godin et al., 1991;
Dolci et al., 1991
). Here we
show that telomerase activity is also present in the precursors of
spermatogonia, that is, in proliferating primordial germ cells, and that such
activity is induced by Kitl treatment. At the entry in G1 mitotic arrest, Kit
is downregulated and is no longer induced by Kitl in prospermatogonia.
Germline stem cells play a critical role in the reproductive process. Their
divisions rely on the regulatory control of several extrinsic signals,
including Kitl, which plays a crucial role in germ cell development.
Kitl-induced upregulation of telomerase correlates with the stimulation of
germ cell proliferation, which is the basis of the self-renewing ability of
these cells. This effect appears to be germline specific, since PGCs also
upregulate telomerase activity upon Kitl stimulation, whereas primitive human
hematopoietic stem cells, which are responsive to Kitl, do not increase
telomerase activity after Kitl treatment
(Yui et al., 1998). Kitl
induction of telomerase activity is also developmentally regulated. In fact,
Kit-expressing growing oocytes, which are blocked in the first meiotic
prophase, do not show any increase in telomerase activity upon Kitl treatment,
and Kit activation does not lead to meiotic progression, suggesting that
Kit-activated signal transduction and telomerase are not coupled in meiotic
cells. The PI3K-Akt pathway is probably not required or is redundant in these
cells, since targeted mutation of the specific PI3K-docking site of Kit
receptor does not affect (Blume-Jensen et
al., 2000
), or only partly affects (Kisser et al., 2000) female
germ cells, although it blocks spermatogonia proliferation. The results we
obtained show a strong correlation between mitosis and telomerase activity in
the male germline and identify telomerase as a molecular target of a
PI3K-dependent pathway induced by Kit activation in mitotic germ cells from
neonatal testis. It will be interesting to investigate whether a
PI3K-dependent downstream effector can also regulate telomerase at the
expression level. To study this, regulatory sequences have been recently
identified in the mouse telomerase gene
(Greenberg et al., 1999
), and
it has been shown that, at least in the human gene, c-myc binds to two
regulatory regions located within 300 bp 5' upstream to the ORF
(Greenberg et al., 1999
) and
induces TERT expression (Wuang et al.,
1998
; Wu et al.,
1999
).
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Acknowledgments |
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