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First published online 23 January 2008
doi: 10.1242/dev.013474
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1 Department of Pathology, Graduate School of Medicine, Osaka University, 2-2
Yamada-oka, Suita, Osaka 565-0871, Japan.
2 Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka,
Suita, Osaka 565-0871, Japan.
3 Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1,
Bunkyo-ku, Tokyo 113-8657, Japan.
* Author for correspondence (e-mail: tnakano{at}patho.med.osaka-u.ac.jp)
Accepted 11 December 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: AKT, Primordial germ cells, EG cells, Pluripotency, Stem cells, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wylie, 2000). PGCs emerge from
precursor cells in the proximal epiblast at embryonic day 7.25 (E7.25) in
mice. PGCs migrate through the hindgut and dorsal mesentery to colonize the
genital ridges by E11.5. The PGCs proliferate from about 45 founder cells to
25,000 cells between E7.25 and E13.5. In males, the cells enter mitotic arrest
at E13.5, and spermatogonia resume proliferation and undergo spermatogenesis
after birth. By contrast, female PGCs enter meiosis at E13.5, and oocytes
mature to ovulation after birth.
Although the developmental potency of PGCs is restricted to the germ
lineage in normal development, two lines of evidence suggest that mammalian
PGCs can dedifferentiate into cells with broader differentiation potential
(Kimura et al., 2005). First,
when PGCs are cultured in the presence of leukemia inhibitory factor (LIF),
stem cell factor (SCF) and basic fibroblast growth factor (bFGF), they give
rise to embryonic germ (EG) cells (Matsui
et al., 1992; Resnick et al.,
1992). EG cells possess pluripotency similar to that of embryonic
stem (ES) cells, as EG cells contribute to the somatic and germ lineages after
being introduced into blastocysts (Labosky
et al., 1994; Stewart et al.,
1994). However, freshly isolated PGCs do not contribute to any
tissues upon transfer into blastocysts, which indicates that some
reprogramming events are necessary for the development of EG cells from PGCs
(Durcova-Hills et al., 2006).
Second, germ cell tumors, called teratocarcinomas, contain a range of
differentiated cell types, including more than two germ layers and a
population of undifferentiated embryonic cells, known as embryonal carcinoma
(EC) cells. The tumors have been shown experimentally to originate from PGCs
(Stevens, 1967).
Phosphoinositide 3-kinase (PI3K), which is activated by a number of growth
factors, cell adhesion molecules and chemokines, produces the second messenger
molecule phosphatidylinositol (3,4,5)-triphosphate
(PtdIns(3,4,5)P3) from PtdIns(4,5)P2
(Cantley, 2002).
PtdIns(3,4,5)P3 then transmits the signal via various
downstream effectors, including the serine/threonine kinase AKT, and regulates
proliferation, survival, migration, metabolism and tumorigenesis
(Brazil et al., 2004). By
contrast, the tumor suppressor PTEN dephosphorylates
PtdIns(3,4,5)P3 to PtdIns(4,5)P2,
thereby antagonizing the physiological and pathological actions of PI3K
signaling (Kishimoto et al.,
2003; Stiles et al.,
2004). An analysis of PGC-specific Pten-deficient mice
has shown that PI3K signaling promotes the dedifferentiation of PGCs
(Kimura et al., 2003;
Moe-Behrens et al., 2003). In
particular, the deletion of Pten increased the cloning efficiency of
EG cells and led inevitably to the development of testicular teratomas. PI3K
signaling exerts its biological effects through the activation of various
downstream molecules, which include AKT and the GTPases RAC and CDC42
(Brazil et al., 2004;
Cantley, 2002). It remains to
be elucidated which PI3K downstream molecules mediate the effects of
Pten deletion in the dedifferentiation of PGCs.
AKT is hyperphosphorylated in PGCs and early teratomatous foci in
PGC-specific Pten-deficient mice
(Kimura et al., 2003), and
PI3K/AKT signaling plays an important role in the regulation of ES cell
pluripotency (Ivanova et al.,
2006; Paling et al.,
2004; Watanabe et al.,
2006). We examined the effect of activating AKT signaling on EG
cell derivation and found that AKT activation considerably increased EG cell
formation from E11.5 PGCs in the presence of LIF, SCF and bFGF, as had been
seen for the Pten- deficient PGCs. In addition, AKT activation
efficiently promoted EG cell derivation, even in the absence of bFGF. However,
the signal did not promote the establishment of EG cells from germ cells after
E15.5, by which time mitotic arrest and meiosis had occurred in the male and
female germ cells, respectively. Our study indicates that the PI3K/AKT
signaling axis controls the derivation of EG cells from PGCs.
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|
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
prokaryotic sequences were excised from the plasmid, and the gel-isolated DNA
fragment was injected into the pronuclei of BDF1 fertilized zygotes. Founder
mice were screened by PCR on tail DNA and were bred in the BDF1 background
(Murayama et al., 2007). The
morning on which a copulation plug was noted was defined as embryonic day 0.5
(E0.5). The p53-deficient mice with mixed background (C57BL/6, CBA2 and ICR)
were described previously (Tsukada et al.,
1993). Animal care procedures were in accordance with the
guidelines of Osaka University.
PGC culture
Gonads were dissociated into single cells by incubation in 0.05% trypsin
and 0.02% EDTA in PBS for 10 minutes. Dispersed suspensions of germ
cell-containing tissues were cultured on Sl/Sl4-m220 feeder cells
in 24-well plates with DMEM that was supplemented with 15% KNOCKOUT Serum
Replacement (Invitrogen, Carlsbad, CA), 2 mM glutamine, 1 mM sodium pyruvate,
and nonessential amino acids, in the presence or absence of 1000 U/ml LIF, 20
ng/ml bFGF (R&D Systems, Minneapolis, MN), and 10 µM forskolin
(Sigma-Aldrich, St Louis, MO) (Koshimizu
et al., 1996; Matsui et al.,
1992). The primary cultures were passaged to
Sl/Sl4-m220 feeder cells (secondary cultures) and subsequently to
mouse embryonic fibroblasts (MEFs; tertiary cultures) every 5 days (see
Fig. 2A). For the secondary and
tertiary cultures, 4OHT and bFGF were omitted from the medium. When PGCs were
cultured with LIF alone, the cell suspensions were seeded onto MEFs in
six-well plates and passaged to MEFs until tertiary culture.
The Sl/Sl4-m220 cells were treated with 5 µg/ml mitomycin C
for 1 hour and plated at 4x105 cells/well in 24-well plates
one day before use. The MEFs were treated with 10 µg/ml mitomycin C for 2
hours and plated at 1.1x105 cells/well in 24-well plates or
at 6.3x105 cells/well in six-well plates. PGCs and EG cells
were fixed using 4% paraformaldehyde (PFA) and visualized using an alkaline
phosphatase staining kit (Sigma). The number of adherent PGCs at 8 hours
post-seeding was defined as the number of seeded PGCs. Multilayered colonies
with more than 20 cells at day 5 of primary culture were considered to be
primary EG cell colonies, as described
(Kimura et al., 2003;
Koshimizu et al., 1996;
Moe-Behrens et al., 2003).
Immunostaining and flow cytometry
PGC cultures were fixed with 4% PFA and stained as described
(Kimura et al., 2006). The
primary antibodies used were anti-Ser473-phosphorylated AKT antibody (1:50
dilution; Cell Signaling Technology, Beverly, MA),
anti-phospho-GSK3
/β antibody (1:50; Cell Signaling),
anti-Ser20-phopho-p53 antibody (1:25; Cell Signaling), MDM2 (1:50; Santa Cruz,
CA, USA), PGC7/Stella (1:1000; DPPA3 - Mouse Genome Informatics)
(Sato et al., 2002), and
SSEA-1 (1:50; Kyowa Hakko, Tokyo, Japan; FUT4 - Mouse Genome Informatics).
Genital ridge suspensions were stained with the SSEA-1 antibody and were
analyzed using a FACSCalibur (BD Biosciences, Franklin Lakes, NJ).
Apoptosis and proliferation index
Apoptotic cells were stained with fluorescently labeled caspase inhibitor
(SR-VAD-FMK; BIOMOL, Plymouth Meeting, PA), which specifically binds to
activated caspases, for 1 hour before fixation. PGCs were detected by
immunostaining with SSEA-1. Mitotic cells were visualized by staining
chromosomal DNA with DAPI (4',6-diamidino-2-phenylindole).
|
). For the teratoma formation assay,
5x106 EG cells were injected subcutaneously into nude mice
(Japan SLC, Hamamatsu, Japan). After 3 weeks, the tumors were fixed with 4%
PFA and embedded in OCT compound (Sakura Finetek, Tokyo). Frozen sections were
cut and stained with Hematoxylin and Eosin. EG cells established by AKT
activation were injected into blastocyst-stage embryos from C57BL/6 strain
mice. The incorporation of cells into the chimeric mice was monitored by EGF
fluorescence. |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
kinase activity of AKT-MER can be regulated by the addition or removal of the
synthetic ligand of MER, 4-hydroxytamoxifen (4OHT). The fusion protein is in
an inactive form in the absence of 4OHT but is activated rapidly by the
addition of 4OHT (Kohn et al.,
1998; Watanabe et al.,
2006). We used a cytomegalovirus enhancer linked to the β-actin promoter to drive the expression of AKT-MER. Because Akt-Mer cDNA fuses to IRES-EGFP (internal ribosomal entry site linked to enhanced green fluorescent protein), the transgenic mice can be identified by EGFP fluorescence (Fig. 1A). Flow cytometry analysis of the E11.5 gonad suspensions revealed that the majority of the SSEA-1-positive PGCs were positive for EGFP, showing that AKT-MER was efficiently expressed in the PGCs (Fig. 1B).
The level of AKT activation in the cultured PGCs was analyzed by immunostaining using a phospho-AKT-specific antibody (Fig. 1C). When wild-type PGCs were cultured without bFGF, the AKT phosphorylation level was comparable to background fluorescence level. However, phosphorylation was induced by bFGF treatment. In the transgenic PGCs, the addition of 4OHT induced a much stronger signal, regardless of bFGF treatment. A weak but significant phospho-AKT signal was also detected in the transgenic PGCs in the absence of 4OHT, suggesting leakiness of the kinase activity of the AKT-MER fusion protein without 4OHT.
Increased derivation of EG cells by AKT signaling activation
We cultured PGCs from E11.5 embryos on SCF-expressing
Sl/Sl4-m220 cells in the presence of LIF and bFGF, which is the
standard culture procedure for establishing EG cell lines
(Fig. 2A)
(Matsui et al., 1992;
Resnick et al., 1992). We used
a mixture of gonads from male and female mice, as the proliferation rates and
EG cell-forming efficiencies of the PGCs from the two sexes at E11.5 were
equal and the Pten deletion enhanced PGC proliferation and EG cell
production regardless of sex (Kimura et
al., 2003). Although E11.5 PGCs rapidly undergo apoptosis in
vitro, SCF, LIF and bFGF act as survival and growth factors
(De Felici and Dolci, 1991;
Godin et al., 1991;
Koshimizu et al., 1995;
Matsui et al., 1991). When
seeded onto the feeder layer, morphology and adhesion were not altered by AKT
hyperactivation (data not shown). We then examined the rates of apoptosis and
mitosis by staining with the fluorescently labeled caspase inhibitor
SR-VAD-FMK and with DAPI, respectively. Apoptotic PGCs significantly decreased
but mitotic cells significantly increased in 4OHT-treated transgenic PGC
cultures (Table 1). As a
result, the number of PGCs derived from the AKT-MER transgenic mice was
significantly enhanced by adding 4OHT after 3 days of culture
(Fig. 2C). Similarly, the
formation of primary EG cell colonies, defined as the alkaline
phosphatase-positive, multilayered colonies that appeared on day 5 of culture
(Fig. 2B), was significantly
enhanced by the presence of 4OHT in the transgenic PGC cultures
(Fig. 2D). By contrast, no
significant increase in EG cell colony formation was observed for the control
PGCs.
|
|
). As
summarized in Table 2A, the
efficiency of EG cell establishment from the transgenic PGCs was significantly
enhanced when 4OHT was added to the primary culture. EG cell derivation was
also enhanced when the migratory-phase PGCs at E8.5 were cultured with 4OHT
(Table 2B). Conversely, the
inhibition of PI3K signaling abrogated EG cell derivation from E11.5 PGCs
(Table 3). However, AKT
activation rescued EG cell derivation in the presence of PI3K inhibitor. These
results indicate that PI3K/AKT signaling promotes the establishment of EG cell
lines.
|
|
;
Resnick et al., 1992). For
this purpose, PGCs from E11.5 embryos were cultured in the presence of SCF and
LIF, but in the absence of bFGF, during primary cultures. The percentage of
apoptotic cells was significantly decreased in 4OHT-treated, transgenic PGC
cultures (Table 1). Although
mitotic PGCs were hardly detectable in the control cultures, mitotic cells
were consistently observed in 4OHT-treated transgenic cultures
(Table 1). At day 3 of primary
culture, although the number of wild-type PGCs had decreased to less than 20%,
the cultured PGCs of transgenic mice were restored by adding 4OHT
(Fig. 3A). Similarly, although
no EG cell colonies were detected for the wild-type PGCs, about 10% of the
transgenic PGCs gave rise to primary EG cell colonies in the presence of 4OHT
at day 5 of culture (Fig.
3B,C). A few primary EG cell colonies emerged from the transgenic
PGCs in the absence of 4OHT, presumably due to the leakiness of AKT-MER fusion
protein activity in the absence of 4OHT
(Fig. 1C).
|
It has been reported that treatment with bFGF during the first 24 hours of
primary culture was sufficient for EG cell derivation
(Durcova-Hills et al., 2006).
To determine the effective time window of AKT signaling on EG cell derivation
from transgenic mouse PGCs, AKT signaling was activated for 1 day only at
various time points of primary culture, in the absence of bFGF (data not
shown). When 4OHT was added for the first day and then removed for the
remaining 4 days, EG cell lines were established efficiently from E11.5
transgenic PGCs. This efficiency was comparable to that achieved when 4OHT was
added for the entire 5 days. As the level of AKT phosphorylation decreased to
basal level within 24 hours of the removal of 4OHT (data not shown), we
conclude that AKT activation during the first 48 hours of primary culture was
sufficient for EG cell derivation.
To examine whether AKT signaling could replace SCF signaling in the derivation of EG cells, we seeded E11.5 PGCs onto MEFs in the presence of LIF alone. As shown in Table 2A, although the efficiency was low, EG cell lines were established reproducibly from AKT-MER-expressing PGCs when 4OHT was added to the primary culture (Fig. 3E). By contrast, no EG cell colonies developed from wild-type PGCs or transgenic PGCs in the absence of 4OHT. The EG cell lines established without bFGF or SCF could be propagated indefinitely on MEFs in the presence of LIF.
Given that AKT-MER is expressed in germ cells and somatic cells, the enhanced EG cell derivation could be attributable to the supporting effects of gonadal somatic cells with high AKT activity. To exclude this possibility, mixtures of cells obtained from wild-type and transgenic gonads were cultured in the presence of SCF and LIF without bFGF. As shown in Fig. 3F, all of the established EG cells were transgenic PGC-derived, EGFP-positive cells. Therefore, enhanced EG cell establishment was the result of cell-autonomous effects of AKT signaling in the PGCs.
A unique property of pluripotent stem cells is the ability to differentiate
into multiple cell lineages. Therefore, we investigated whether pluripotency
was maintained in EG cells established from E11.5 PGCs by AKT activation
instead of by bFGF and/or SCF signaling. When the EG cells were transplanted
into nude mice, these cells produced teratomas that were composed of various
differentiated cells (Fig.
4A,B). In an in vitro hematopoietic differentiation system using
OP9 stromal cells, these cells formed mesodermal colonies and subsequently
produced a variety of hematopoietic cells on day 12 after the induction of
differentiation (Fig. 4C).
Furthermore, we injected the EG cells into the blastocoel to examine whether
they had the ability to contribute to chimeric mice. When the EG cells
established without bFGF were used, EGFP-positive EG cell-derived cells were
detectable in the whole bodies of the E12.5 embryos, and live chimeric mice
were born (Fig. 4D). The EG
cells derived without bFGF or SCF also contributed to embryonic tissues, but
live chimeras were not born (Fig.
4E). Some chimeras showed fetal overgrowth and were dead at birth
(Table 4). The EG cells were
not transmitted through the germ line. It is worthwhile to mention that
germ-line transmission was reported in the literature for EG cells derived
from E8.5 PGCs but not those derived from E11.5-E12.5 PGCs
(Labosky et al., 1994;
Stewart et al., 1994;
Tada et al., 1998). Thus, AKT
signaling produces EG cells with pluripotency from PGCs, in the absence of
bFGF and SCF.
|
;
Wylie, 2000). EG cell
production from E13.5 transgenic germ cells cultured under standard conditions
was enhanced by 4OHT in males but not in females
(Table 5). Co-stimulation with
bFGF and forskolin enhances EG cell formation
(Koshimizu et al., 1996). In
the presence of forskolin, 4OHT enhanced the efficiency of EG cell
establishment from E13.5 PGCs of both males and females.
|
|
). We next examined the status of possible downstream proteins
in the cultured PGCs. The activity of glycogen synthase kinase 3 (GSK3) is
inhibited by AKT via direct phosphorylation. Treatment with a GSK3 inhibitor,
BIO (6-bromoindirubin-3'-oxime), supports ES cell pluripotency and
improves the efficiency of EG cell derivation
(Sato et al., 2004;
Umehara et al., 2007). As
shown in Fig. 5A, the
percentage of transgenic PGCs stained with anti-phospho-GSK3 antibody was
increased by AKT activation regardless of the bFGF treatment. However, the
treatment of wild-type PGCs with BIO did not enhance EG cell derivation
(Table 6A).
|
;
Mayo and Donner, 2001;
Zhou et al., 2001). As shown
in Fig. 5B, only a faint MDM2
signal was detected in the cytoplasm of transgenic PGCs in the absence of
4OHT. However, the percentage of PGCs that showed stronger MDM2 staining, in
both the cytoplasm and nucleus, increased in 4OHT-treated transgenic PGCs.
Second, the Ser20 phosphorylation of p53 by checkpoint kinase CHK1/CHK2
(CHEK1/CHEK2 - Mouse Genome Informatics) is required to induce cell cycle
arrest and apoptosis at the G2/M phase transition
(Hirao et al., 2000;
Shieh et al., 1999). It has
been reported that PI3K/AKT signaling inhibits both the basal and the DNA
damage-induced activity of CHK1/2 (Hirose
et al., 2005; Shtivelman et
al., 2002). In the absence of bFGF and 4OHT, about 6% of
transgenic PGCs were stained with the anti-Ser20-phospho-p53 antibody
(Fig. 5C). However, the
percentage of phopho-p53-positive PGCs decreased after the addition of bFGF
and/or 4OHT in the transgenic cultures
(Fig. 5C). These results
suggest that AKT signaling inhibited p53 activity in transgenic PGCs. To
examine the effects of p53 inhibition on EG cell derivation, we used
p53-deficient mice with a mixed background (C57BL/6, CBA2 and ICR). In the
presence of bFGF, the efficiency of EG cell derivation from p53-deficient
embryos was significantly higher than that from wild-type and heterozygous
mice (Table 6B,
Fig. 5D). In addition, EG cell
lines were reproducibly established from p53-deficient mice even in the
absence of bFGF. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Furthermore, EG cell colony formation from E11.5 PGCs is
enhanced in PGC-specific Pten-deficient mice, and by treatment with
an antisense oligonucleotide against Pten, in the presence of three
growth factors (Kimura et al.,
2003; Moe-Behrens et al.,
2003). The EG cell-forming efficiency of AKT-MER-expressing PGCs
in the presence of 4OHT was comparable to that of PGC-specific Pten-
deficient mice (Fig. 2C).
Therefore, it is conceivable that AKT is the main downstream effector of PI3K
that causes the phenotypes of Pten-deficient mice.
EG cell derivation from the PGCs of either Pten-deficient or
wild-type mice requires bFGF (T.K. and T.N., unpublished), which suggests that
the PI3K signaling activated by bFGF is crucial for EG cell derivation. Our
finding that 4OHT-treated AKT-MER-expressing PGCs generate EG cell colonies
without bFGF as efficiently as do wild-type PGCs cultured with bFGF
(Table 2,
Fig. 2D,
Fig. 3B) demonstrates that AKT
signaling activation can functionally replace bFGF. However, the EG
cell-forming efficiency was lower than that of AKT-MER-expressing PGCs
cultured in the presence of 4OHT and bFGF, or Pten-deficient PGCs
cultured in the presence of bFGF. In addition, the effects of AKT activation
in our system would be greater than those caused at a physiological level,
because AKT phosphorylation in AKT-MER transgenic PGCs was higher than that in
bFGF-treated wild-type PGCs (Fig.
1C). These results suggest that the PI3K/AKT signal cooperates
with some other signaling pathways in EG cell derivation. Besides PI3K/AKT
signaling, bFGF activates several intracellular signaling components, such as
mitogen-activated protein kinase (MAPK) and phospholipase C
(Klint and Claesson-Welsh,
1999). In addition, the proliferation of PGCs in culture is
supported by MAPK signaling (De Miguel et
al., 2002), which raises the possibility that the PI3K/AKT and
MAPK signaling pathways promote EG cell derivation in a cooperative
fashion.
The mitotic activity and survival of PGCs strongly correlate with the
efficiency of EG cell formation and the incidence of testicular teratomas.
Forskolin and retinoic acid, which can substitute for bFGF in EG cell
derivation, are reported to act as mitogenic and survival factors for PGCs
(Koshimizu et al., 1996;
Koshimizu et al., 1995).
Similarly, AKT activation also promoted proliferation and inhibited apoptosis
in the cultured AKT-MER transgenic PGCs
(Table 1). Despite the fact
that male PGCs enter mitotic arrest at E13.5 in most strains of mice, the germ
cells continue to proliferate until after E14.5 in mouse strains that are
susceptible to testicular teratoma, such as 129/Sv-Ter/Ter
and Pten-deficient mice (Kimura
et al., 2003; Noguchi and
Stevens, 1982). Although the majority of mitotic PGCs are
eventually lost by apoptosis in these mouse strains, a population of PGCs
survives to generate testicular teratomas. Thus, sustained proliferation and
subsequent survival of PGCs may be a prerequisite for EG cell
establishment.
The potential to develop EG cells from germ cells is completely lost at
E14.5, which is coincident with the emergence of mitotic arrest and meiosis of
male and female germ cells, respectively. Responsiveness to AKT signaling for
EG cell establishment was not detectable after E14.5 and E15.5 in females and
males, respectively (Table 5).
In contrast to the E11.5 PGCs, the E15.5 transgenic germ cells could not
resume proliferation following AKT activation (data not shown). The effects of
AKT signaling diminished earlier in females than in males, which could be due
to the small number of mitotic germ cells that remain in E14.5 male gonads
(Kimura et al., 2003). Thus,
mitotic responsiveness to AKT signaling may be crucial for enhancing EG cell
establishment. At the same time, AKT signaling promoted self-renewing cell
division of spermatogonial stem cells but not derivation of pluripotent cells
from spermatogonial stem cells (Lee et
al., 2007). These results show that AKT provoked distinct
responses in germ cells depending on the developmental stage.
PI3K/AKT signaling regulates a variety of downstream molecules, some of
which play crucial roles in various stem cell systems. In this study, we have
shown that AKT signaling negatively regulated p53 in the cultured PGCs by
enhanced MDM2 function, and inhibited phosphorylation of p53. We also revealed
that EG cell derivation was promoted by the absence of p53. Thus, AKT-mediated
p53 inhibition would be important to promote the EG cell derivation.
Similarly, derivation of pluripotent cells from spermatogonial stem cells is
enhanced by the p53 deficiency
(Kanatsu-Shinohara et al.,
2004). Therefore, suppression of p53 may be crucial for germ cells
to acquire pluripotency. Consistently, the activity and nuclear translocation
of p53 is suppressed in the pluripotent ES cells
(Aladjem et al., 1998), and
differentiation of ES cells is induced by p53 through suppression of
Nanog expression (Lin et al.,
2005). The p53 deficiency in the cultured germ cells may promote
the acquisition of undifferentiated states by altering the expression of the
target genes of p53. Meanwhile, considering that AKT signaling regulates other
downstream molecules, such as mTOR complex 1, it is likely that AKT signaling
promotes EG cell derivation through multiple downstream molecules. Further
analysis would not only provide an efficient means for establishing germ
cell-derived stem cells, but also give valuable insights into the
developmental plasticity of the germ cells.
|
ACKNOWLEDGMENTS |
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|
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2 test), than in wild type.
The percentage of phospho-p53-positive PGCs cultured without 4OHT and bFGF was
significantly higher than that of other groups
(*P<0.005,