1 Department of Pharmacology and Cancer Biology, Duke University Medical Center,
Durham, NC 27710, USA
2 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710,
USA
* Author for correspondence (e-mail: means001{at}mc.duke.edu)
Accepted 5 February 2003
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SUMMARY |
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Key words: Primordial germ cells, Pin1, Proliferation, Cell cycle, Knockout mice
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INTRODUCTION |
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Pin1 catalyses the cis-trans isomerization of phosphorylated
serine/threonine-proline bonds in phosphoproteins, thereby altering
conformation leading to a change in protein stability or function
(Lu et al., 2002;
Stukenberg and Kirschner,
2001
; Yaffe et al.,
1997
). Many studies in cultured cells have implicated Pin1 as a
regulator of cell cycle progression, as well as the DNA replication and DNA
damage checkpoints (Lu et al.,
1996
; Lu et al.,
2002
; Winkler et al.,
2000
; Zacchi et al.,
2002
; Zheng et al.,
2002
). Pin1 has been shown to interact with, and suggested to
regulate, crucial cell signaling proteins such as Jun, cyclin D1, Cdc25,
ß-catenin and p53 (Crenshaw et al.,
1998
; Liou et al.,
2002
; Ryo et al.,
2001
; Wulf et al.,
2001
; Zacchi et al.,
2002
; Zheng et al.,
2002
). In addition, Pin1 plays a role in dorsoventral patterning
of the developing egg chamber in Drosophila by regulating the
stability of a transcription factor, Cf2, via the MAPK pathway
(Hsu et al., 2001
). Depletion
of Pin1 in HeLa cells and budding yeast has been reported to cause mitotic
arrest and nuclear fragmentation in those cells
(Lu et al., 1996
). In spite of
the numerous critical cellular roles attributed to Pin1
(Lu et al., 2002
), adult mice
homozygous for the targeted deletion of the Pin1 gene were reported
to exhibit only mild defects in the mammary gland, and in testis and retina of
very old animals (Liou et al.,
2002
). However, these studies were carried out on a mixed genetic
background. To explore further the in vivo function of Pin1, we used
marker-assisted speed congenic breeding to backcross the mutation into an
inbred C57BL/6J background, in which the studies described below were
conducted (see Fig. S1 at
http://dev.biologists.org/supplemental/).
In this report, we found that male and female Pin1-/- mice
were born with fewer germ cells, resulting in severe fertility defects in both
genders. We examined the development of PGCs in Pin1-/-
embryos, and identified Pin1 as a regulator of the timing of PGC
proliferation.
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MATERIALS AND METHODS |
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Fertility studies
Continuous mating studies were carried out to assess fertility as described
by others (Jeffs et al.,
2001). Briefly, males around 12 weeks of age were housed
individually with females around 7 weeks of age. Mounting behavior and
copulatory plugs were observed to confirm normal mating behavior. Six pairs of
each mating combination (wild-type males and females,
Pin1-/- males and females, Pin1-/-
males and wild-type females, Pin1-/- females and wild-type
males) were followed for six months. The number of litters produced by each
pair and the number of pups per litter were recorded and summed for each
mating group.
Histology and immunohistochemistry
Postnatal testes and ovaries were fixed in Bouin's fixative and embedded in
paraffin wax. Testes and ovaries were sectioned at 7 µm intervals. Testis
sections were stained with periodic acid-Schiff reagent and Hematoxylin
(PAS-H) (Polyscientific). Ovary sections were stained with Hematoxylin and
Eosin. Testis sections were also processed for immunohistochemistry as
previously described (Enders and May,
1994). Briefly, sections were deparaffinized, rehydrated, followed
by antigen retrieval in 10 mM sodium citrate buffer. Sections were blocked in
normal goat serum in PBS for 1 hour, and incubated with primary antibody at
4°C overnight. After washing in PBS, secondary antibody was applied for 1
hour and followed by washing in PBS. Staining was visualized using VectaStain
(Vector Laboratories). Gonocytes were detected using the anti-GCNA1 antibody
(a generous gift from G. C. Enders). Pin1 protein was detected using the
rabbit polyclonal anti-Pin1 antibody 1:100 generated previously in our
laboratory (Winkler et al.,
2000
). Specificity for the Pin1 immunostaining were confirmed in
negative controls using the Pin1-/- tissues or
pre-absorbed anti-Pin1 antibodies with GST-Pin1 proteins described previously
in our laboratory (Winkler et al.,
2000
), and both provided similar negative staining results. Pin1
immunostaining in gonads was carried out in wholemounts as described below.
For Pin1 immunostaining in 7.5, 8.5 and 9.5 dpc embryos, 10 µm embryo
cryosections (see below) were blocked in blocking buffer (10% heat-inactivated
goat serum, 3% BSA, 0.1% TritonX-100 in PBS) at room temperature for 2 hours,
then incubated in anti-Pin1 antibodies (1:100 diluted in blocking buffer)
overnight at 4°C. Sections were washed in wash buffer (1% heat-inactivated
goat serum, 3% BSA, 0.1% TritonX-100 in PBS) three times for 10 minutes, and
incubated in FITC-conjugated donkey anti-rabbit secondary antibodies 1:500
(Jackson ImmunoResearch) for 1 hour at room temperature. Sections were washed
in wash buffer three times for 10 minutes with
4',6-diamidino-2-phenylindole dihydrochloride (DAPI) 1 µg/ml (Sigma)
added in the last wash to identify the nuclei. The adjacent serial sections
were processed for alkaline phosphatase assays to detect PGCs in those stages
as described below.
PGC detection in embryos and gonads
Embryos were obtained from pregnant females 7.5, 8.5, 9.5, 11.5, 12.5 and
13.5 dpc, with the first day of vaginal plug identification defined as 0.5
dpc. Embryos and gonads were dissected in PBS and fixed in 4% paraformaldehyde
overnight at 4°C, followed by washing in PBS. Embryos and gonads were
ready to be processed as wholemounts after washing in PBS. For embryo
sections, embryos were further dehydrated in 10% and 15% sucrose for 15
minutes each, and in 20% sucrose for 1 hour, then in 1:1 20% sucrose:OCT
compound (Tissue-Tek) overnight at 4°C. Embryos were then embedded in 1:3
20% sucrose:OCT compound and cryosectioned at 10 µm. PGCs were detected in
7.5, 8.5 and 9.5 dpc embryos and in embryo sections using alkaline phosphatase
assays as described by others (Lawson et
al., 1999). Briefly, whole embryos or embryo sections were placed
in 70% ethanol at 4°C for 1 hour, then washed once in PBS and stained with
Fast Red TR and
-napthyl phosphate (Sigma) to detect alkaline
phosphatase-positive cells. PGCs in 11.5, 12.5 and 13.5 wholemount gonads were
detected using anti-PECAM 1:500 (Pharmingen) as previously described
(Schmahl et al., 2000
;
Yao et al., 2002
). Briefly,
wholemount gonads were blocked for 3-4 hours in blocking buffer (1%
heat-inactivated goat serum, 5% BSA, 0.1% TritonX-100 in PBS) at room
temperature, then incubated in primary antibodies diluted in blocking buffer
overnight at 4°C. Gonads were washed in wash buffer (0.1% TritonX-100 in
PBS) three times for 1 hour at room temperature, and incubated in secondary
antibodies in blocking buffer overnight at 4°C, followed by washing three
times for 1 hour in wash buffer at room temperature and mounted on slides
using imaging spacers (Sigma).
Apoptosis assay
Apoptotic cells were detected using LysoTracker Red (Molecular Probes) in
wholemount gonads as previously described
(Yao et al., 2002;
Zucker et al., 1999
). Briefly,
12.5 dpc gonads were dissected in sterile PBS, cultured in 500 µl DMEM
medium with 2 µl/ml LysoTracker Red for 30 minutes in a 37°C, 5%
CO2 incubator. Gonads were washed in PBS, fixed in 4%
paraformaldehyde overnight at 4°C and processed for whole-mount
immunohistochemistry.
BrdU pulse labeling and antibodies
BrdU pulse labeling and detection were carried out using procedures
previously described (Schmahl et al.,
2000). Briefly, heterozygous pregnant females at 12.5 dpc received
an i.p. injection of 50 mg/kg of BrdU (Sigma) and pulsed for 30 minutes.
Gonads were dissected from embryos and processed for whole-mount
immunohistochemistry as in Schmahl et al.
(Schmahl et al., 2000
).
Anti-BrdU (Roche) 1:100, anti-Ki67 (Pharmingen) 1:100, anti-phosphohistone H3
(Upstate Biotechnology) 1:200 and anti-laminin (a generous gift from
H.Erickson) 1:200 were used accordingly. All conjugated secondary antibodies
(Jackson ImmunoResearch) were used at 1:500 dilutions.
PGC quantitation
Embryos and gonads were mounted on slides using imaging spacers (Sigma) and
cover slips after each experimental procedure. All PGCs in 8.5 and 9.5 dpc
whole embryos were counted under a light microscope. PGCs in gonads were
counted using methods in Schmahl et al.
(Schmahl et al., 2000).
Briefly, PGCs in the interior middle third segment of each gonad were counted
using the 40x objective of a Zeiss LSM 410 confocal microscope, with
images spaced at 15 µm intervals to avoid counting the same cells. The BrdU
index was the ratio of BrdU-positive PGCs and all counted PGCs in each
gonad.
Statistics
PGC numbers from each genotype, age and sex group were log-transformed
because of heterogeneous variance across ages, and analysed using three-factor
ANOVA (genotype, age, sex) in StatView. P-values were used to
determine statistical significance. The BrdU index was analysed using unpaired
Student's t-test. N for each category ranged from 6 to 19. Bar graphs
were plotted in MS Excel.
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RESULTS |
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Absence of cell cycle arrest and apoptosis in Pin1-/-
PGCs
Because mouse embryo fibroblasts from Pin1-/- embryos
have been shown to have difficulty entering into the cell cycle from G0 arrest
(Fujimori et al., 1999;
You et al., 2002
), we
investigated the cell cycle status of Pin1-/- PGCs using
antibodies against Ki67, a protein expressed in all phases of the cell cycle
but absent in G0 cells (Scholzen and
Gerdes, 2000
). Almost all PGCs in the wild-type and
Pin1-/- male and female gonads at 12.5 dpc were positive
for the Ki67 antigen (Fig.
4A,B, male shown), indicating that all PGCs were actively cycling
at this stage, and that the absence of Pin1 did not cause G0 arrest
in PGCs. As antisense depletion of Pin1 in HeLa cells, and deletion of the
Pin1 homolog Ess1 in budding yeast induced mitotic arrest
(Lu et al., 1996
), we also
analysed Pin1-/- PGCs using antibodies against
phosphohistone H3, a mitosis marker (Fig.
4C,D, male shown). If PGCs in Pin1-/- gonads
were arrested in mitosis, we would expect to observe a large accumulation of
phosphohistone H3-positive cells. However, quantification revealed that the
percentage of Pin1-/- PGCs in mitosis (6.4%) was not
significantly different from wild type (5.8%), indicating that mammalian
Pin1-deficient PGCs progressed through mitosis. To investigate
whether Pin1 deficiency affected PGC survival, we analysed the
presence of apoptotic cells in whole-mount gonads
(Yao et al., 2002
;
Zucker et al., 1999
). In 12.5
dpc wild-type gonads, some cells stained positive for the apoptosis marker
LysoTracker (red), but little if any overlap was seen with the PGC marker
PECAM (green, round cells, Fig.
4E, male shown), consistent with published literature that few
PGCs undergo apoptosis at this stage in vivo
(Coucouvanis et al., 1993
;
Yao et al., 2002
). Similarly,
no apoptotic PGCs were seen in Pin1-/- gonads
(Fig. 4F, male shown). These
findings indicated that unlike cultured cells and budding yeast
(Lu et al., 1996
), PGCs did
not undergo cell cycle arrest and cell death in the absence of
Pin1.
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DISCUSSION |
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We have discovered that Pin1 is required for proper cell cycle progression
and proliferation of mammalian primordial germ cells in vivo. Interestingly,
embryonic somatic cell proliferation did not require Pin1, despite
the fact that somatic cells also express the Pin1 protein, raising the
possibility that embryonic somatic cells possessed an additional compensatory
prolyl-isomerase. Recently, it has been reported that another prolyl-isomerase
in the parvulin family to which Pin1 belongs, Par14, was upregulated about
threefold in Pin1-/- MEFs, and inhibitors of both Pin1 and
Par14 decreased cell proliferation. Thus, Par14 may function as a compensatory
prolyl-isomerase in the absence of Pin1. It is possible that PGCs lack Par14
or that Par14 is not upregulated in PGCs, contributing to the selective germ
cell phenotype in Pin1-/- mice. Availability of Par14
antibodies would help to investigate this possibility in the future.
Alternatively, mammalian PGCs may be particularly sensitive to the loss of
Pin1. Current evidence suggests that gonadal somatic cells have a
sex-specific proliferation pattern that is influenced by the male factor
Sry, while primordial germ cells appear to have an intrinsic
proliferation program that operates in a non-sex-specific manner
(Schmahl et al., 2000;
Tilmann and Capel, 2002
). The
fact that Pin1 regulates both male and female PGC proliferation
similarly at 12.5 dpc, after the onset of Sry, is consistent with an
intracellular function for Pin1 in germ cells, rather than an indirect effect
through somatic cells. This view is supported by the high expression of the
Pin1 protein in germ cells.
How Pin1 regulates primordial germ cell proliferation and cell cycle
progression is currently unknown. Pin1 has a well-recognized function as a
mitotic regulator, particularly in cultured transformed cells and in budding
yeast (Lu et al., 1996), but
its absence did not cause mitotic arrest in mammalian PGCs. However, the
mitotic index of Pin1-/- PGCs was somewhat higher than
wild type, although non-significant with our sampling size. This raises the
possibility that the M phase length was prolonged in
Pin1-/- PGCs, and, if so, Pin1 may be required
for proper progression of PGCs through mitosis. Nevertheless, we favor the
idea that PGCs have a prolonged cell cycle as a result of defective G1/S
progression in the absence of Pin1. Pin1 has been reported to increase the
transcriptional activity of Jun and the stability of cyclinD1, both of which
regulate G1/S progression (Liou et al.,
2002
; Wulf et al.,
2001
). These studies imply that impaired Jun activity and
decreased cyclinD1 levels may contribute to G1/S delays and a lengthening of
the cell cycle in Pin1-deficient PGCs. However, mice null for
phosphorylated Jun (the form that binds Pin1) or cyclinD1 are viable and
fertile (Behrens et al., 1999
;
Fantl et al., 1995
), revealing
that neither protein can be the sole target responsible for the PGC phenotype
and infertility in Pin1-/- mice. Therefore, it is possible
that Pin1 acts on multiple targets to achieve a combinatorial effect in its
regulation of PGC proliferation. Alternatively, an intriguing possibility is
that Pin1 may regulate a factor unique to proliferating PGCs. In this regard,
in Drosophila where Pin1 plays a role in developmental signaling, it
regulates the stability of a transcription factor Cf2 in follicle cells in
response to growth factor receptor-activated MAPK signaling
(Hsu et al., 2001
). Of the
molecules proposed to influence PGC proliferation, including Scf, Kit and Fgf,
many are extracellular growth factors and growth factor receptors capable of
activating the MAPK cascade (De Miguel et
al., 2002
; Godin et al.,
1991
; Matsui et al.,
1992
; Resnick et al.,
1992
; Zhao and Garbers,
2002
). Therefore, Pin1 may act as an intracellular signal
responder in a growth factor-activated MAPK pathway in primordial germ cells
in vivo, ensuring efficient cell cycle progression and facilitating their
proliferation to establish the male and female germline.
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ACKNOWLEDGMENTS |
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Footnotes |
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