MOLECULAR AND CELLULAR BIOLOGY, Apr. 2011, p. 1679–1689
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 8
SPATA18, a Spermatogenesis-Associated Gene, Is a Novel
Transcriptional Target of p53 and p63?
Chamutal Bornstein,1† Ran Brosh,1†* Alina Molchadsky,1Shalom Madar,1Ira Kogan-Sakin,1
Ido Goldstein,1Deepavali Chakravarti,2Elsa R. Flores,2Naomi Goldfinger,1
Rachel Sarig,1and Varda Rotter1
Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel,1and Department of Molecular and
Cellular Oncology, Graduate School of Biomedical Sciences, The University of Texas M. D. Anderson Cancer Center,
Houston, Texas 770302
Received 13 September 2010/Returned for modification 31 October 2010/Accepted 29 January 2011
The transcription factor p53 functions not only to suppress tumorigenesis but also to maintain normal
development and homeostasis. Although p53 was implicated in different aspects of fertility, including sper-
matogenesis and implantation, the mechanism underlying p53 involvement in spermatogenesis is poorly
resolved. In this study we describe the identification of a spermatogenesis-associated gene, SPATA18, as a novel
p53 transcriptional target and show that SPATA18 transcription is induced by p53 in a variety of cell types of
both human and mouse origin. p53 binds a consensus DNA motif that resides within the first intron of
SPATA18. We describe the spatiotemporal expression patterns of SPATA18 in mouse seminiferous tubules and
suggest that SPATA18 transcription is regulated in vivo by p53. We also demonstrate the induction of SPATA18
by p63 and suggest that p63 can compensate for the loss of p53 activity in vivo. Our data not only enrich the
known collection of p53 targets but may also provide insights on spermatogenesis defects that are associated
with p53 deficiency.
The p53 (TP53) tumor suppressor gene is a sequence-spe-
cific transcription factor that responds to a broad range of
cellular stress signals and functions as a coordinator of cell fate
decisions (33). Accordingly, p53 activity is attenuated in most
human cancers, usually due to somatic missense mutations (8).
p53 exerts its activity mainly by transactivating target genes; a
few dozen of such targets were identified during the last 3
decades (35). Moreover, p53 can also repress the transcription
of genes (7) and induce a variety of transactivation-indepen-
dent responses (15). The identity of most p53-regulated genes
that mediate processes such as cell cycle arrest, cell death, and
DNA repair have been revealed. However, p53 also affects
processes such as autophagy, differentiation, reproduction, me-
tabolism, and aging (42, 61), but the mediators of these effects
are much less characterized.
The complicated process of spermatogenesis begins with
proliferation of diploid spermatogonia and terminates with the
production of haploid spermatozoa and involves tightly regu-
lated steps of proliferation, meiosis, and differentiation. Sper-
matogenesis occurs within the seminiferous tubules in precisely
timed and highly organized cycles (23). The process starts with
diploid germ cells called spermatogonia that reside on the
basement membrane. After a series of mitotic divisions, dif-
ferentiating spermatogonia divide into spermatocytes, which
can migrate to a more adluminal position of the seminiferous
tubule. Spermatocytes undergo meiosis to form haploid round
spermatids (22), which then undergo spermiogenesis, a process
that includes morphological alterations such as acrosome for-
mation, nuclear condensation, flagellum formation, and extru-
sion of residual cytoplasm, to form mature spermatozoa. Of
note, germ cells in the seminiferous tubules are nursed by
Sertoli cells (34).
The existence of p53 orthologs in lower organisms, such as
worms and flies, which do not develop cancer, implies that
tumor suppression does not represent the original function of
p53. It has been suggested that the role of the p53 ancestral
genes is to ensure the genomic integrity of the germ line and
the fidelity of developmental processes (24). This role was
conserved during evolution as p53 is important for normal
differentiation and development in mammals and other higher
It has been found that p53, like many other proto-oncogenes
and tumor suppressor genes, plays a role in the meiotic process
of spermatogenesis (1). For instance, it was previously re-
ported that during normal spermatogenesis, p53 mRNA and
protein are accumulated in spermatocytes (55), and, similarly,
p21 (CDKN1A), the primary p53 target gene that mediates
cell-cycle arrest (13), is expressed during the prophase of mei-
osis (4). Several studies, including ours, demonstrate specific
roles for p53 in spermatogenesis. For example, transgenic mice
with partial or complete impairment of p53 expression exhibit
a giant-cell degenerative syndrome (52), a phenomenon caused
by an inability of primary tetraploid spermatocytes to undergo
meiotic divisions. Moreover, p53?/?129/Sv mice are infertile
although it is unclear whether this is due to spermatogenesis
defects (19). The p53-dependent DNA damage response was
shown to be highly active throughout spermatogenesis (45),
and accordingly, p53 mediates stress-induced spermatogonial
apoptosis after DNA damage (20). Conversely, p53 deficiency
* Corresponding author. Mailing address: Department of Molecular
Cell Biology, The Weizmann Institute of Science, Rehovot 76100,
Israel. Phone: 972 89343417. Fax: 972 89342398. E-mail: ran.brosh
† C.B. and R.B. contributed equally to this work.
?Published ahead of print on 7 February 2011.
attenuates the regeneration of seminiferous tubules after irra-
diation (21). Moreover, p53 was shown to induce the expres-
sion of Wip1 in mouse testes, and Wip1-null mice display de-
generation of seminiferous tubules, reduced testis size, and
attenuated fertility (10). Besides spermatogenesis, p53 is in-
volved in additional aspects of reproduction, including implan-
tation (25), and in a variety of embryonal development pro-
grams (42). Nevertheless, despite the general reduction in
fecundity in p53-null mice and the increase in developmental
defects, most p53-deficient mice survive, develop normally, and
can reproduce (12). This may indicate an alternative pathway
which can, at least partially, compensate for the loss of p53 in
development and reproduction (51). Notably, it had been
suggested that p53 family members, p63 and p73, can com-
pensate for p53 loss in several processes, including DNA
damage response (65), tumor suppression (14), and devel-
opment (28, 56).
Utilizing RNA interference (RNAi) to knock down p53 in
WI-38 human embryonic fibroblasts, followed by global expres-
sion analysis (9), we identified the gene spermatogenesis-asso-
ciated 18 homolog (SPATA18) as a potential transcriptional
target of p53. Our long interest in the role of p53 in spermato-
genesis (1, 52, 55) and the putative functions of SPATA18 in
this process had driven us to unveil the mechanism by which
p53 regulates SPATA18 expression in vitro and in vivo. In
humans, SPATA18 is localized on chromosome 4q12 and en-
codes a 538-amino acid (aa) protein with two coiled-coil do-
mains. Although the function of SPATA18 in humans or in
mouse is still unknown, its rat homolog, Spetex-1, which shares
82% identity in its protein sequence, was thoroughly charac-
terized as a spermatogenesis-related protein. Specifically, Iida
et al. had identified Spetex-1, using a differential display ap-
proach, as a highly expressed gene in rat testis (27). Testicular
Spetex-1 expression is detected first at 3 weeks of postnatal
development and remains high until adulthood. In situ hybrid-
ization and immunohistochemical analyses revealed that Spe-
tex-1 mRNA and protein are expressed in the cytoplasm of
haploid spermatids and in residual bodies (27). Further sub-
cellular analysis revealed that Spetex-1 protein also localizes at
satellite fibrils associated with outer dense fibers in the middle
piece of the flagella in spermatozoa (26, 30).
Few hypotheses regarding Spetex-1 function were proposed.
Its localization in the cytoplasm of spermatids and in residual
bodies suggests a role in spermiogenesis and, particularly, in
maturation of spermatids into spermatozoa (27). During mat-
uration, elongated spermatids undergo reduction in their cy-
toplasmic volume, which is mediated by engulfment of residual
bodies by Sertoli cells (53). It is assumed that this process
involves apoptotic signals enabling Sertoli cells to recognize
and phagocytose the residual cytoplasm (5). Indeed, it has
been reported that proteins such as caspase-1 (5) and aqua-
porin-7 (59), which mediate apoptosis and volume reduction,
respectively, are restrictively expressed in the cytoplasm of
elongated spermatids. Therefore, it is possible that Spetex-1
might also be involved in this apoptosis-like process (27). In
addition, Iida et al. showed that a portion of Spetex-1 protein
is retained in spermatozoa as a flagellar component after sper-
miation. Thus, Spetex-1 might serve as a structural constituent
in the flagella (26, 30).
Here, we show that the transcription of human and mouse
SPATA18 is directly activated by the p53 tumor suppressor in
a variety of cell types. We demonstrate that p53 binds a con-
sensus DNA motif located in a region corresponding to the
first intron of the SPATA18 gene. We further show that mouse
SPATA18 transcription is regulated in the testes in a unique
spatiotemporal manner, being upregulated primarily by sper-
matids approximately 3 weeks after birth and remaining high
in adults. SPATA18 expression pattern correlates with that
of p21 and might be linked to the accumulation of p53
protein in spermatocytes. Our data imply that p53 transac-
tivates SPATA18 in vivo and that p63 compensates for p53
function in the testes of p53-null mice.
MATERIALS AND METHODS
Cell culture. All cell lines were grown at 37°C in a humidified atmosphere of
5% CO2and were maintained in the following media. Immortalized WI-38
fibroblasts, previously described in Milyavsky et al. (40), as well as IMR-90
fibroblasts, were cultured in minimal essential medium (MEM) supplemented
with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine,
and antibiotics. Mouse embryonic fibroblasts (MEFs) were derived from p53?/?
or p53?/?sibling embryos and maintained in Dulbecco’s modified Eagle’s me-
dium (DMEM) supplemented with 10% FBS and antibiotics. Saos-2, SKOV-3,
HepG2, TM4, and HT-1080 cells were cultured in DMEM with 10% FBS and
antibiotics. LNCaP cells were cultured in RPMI medium with 10% FBS. R1
mouse embryonic stem cells were grown as described by Sarig et al. (54) on a
feeder layer of irradiated MEFs.
Plasmids. The retrovirus encoding GSE56 was described by Milyavsky et al.
(41). Retroviruses harboring short hairpin RNA (shRNA) against human p53
(sh-p53) or mouse NOXA (control shRNA [sh-con] for human cells) were kindly
provided by D. Ginsburg (Bar-Ilan University, Israel). Retroviruses harboring
shRNA against mouse p53 or human pRb (control shRNA for mouse cells) were
kindly provided by S. W. Lowe (Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY). The retrovirus harboring shRNA against mouse p63 (sh-p63) was
generated by subcloning the shRNA cassette from a commercial pSM2 vector
designed to target human p63 (clone V2HS_24249; Open BioSystems) into a
pRetroSuper-hygro vector using XhoI and EcoRI. Note that the mature RNAi
sequence has one mismatch for mouse p63 but is very effective in knocking down
mouse p63 (see Fig. 4E). The p53 expression plasmid pC53-SN3 carrying wild-
type (WT) p53 (2) was kindly provided by B. Vogelstein (Johns Hopkins Uni-
versity School of Medicine, Baltimore, MD), and was used as a template for
site-directed mutagenesis to produce the plasmid carrying p53 with the mutation
R249S (p53R249S), as described by Suad et al. (57). The firefly luciferase reporter
under the regulation of the p21 promoter and the plasmid encoding p63? with
the transactivating domain (TA-p63?) (pCDNA3-TA-p63?) were kindly pro-
vided by M. Oren (Weizmann Institute of Science, Israel). pRL-CMV (where
CMV is cytomegalovirus) plasmid was purchased from Promega.
Construction of SPATA18 first intron luciferase reporters. The luciferase
reporter vector, pGL3-SPATA18-intron1, was generated by cloning a short frag-
ment containing the p53 binding site (BS) from the SPATA18 first intron into a
luciferase reporter plasmid. Briefly, a 495-bp genomic fragment from the first
intron of the human SPATA18 gene, spanning from bp ?114 to bp ?609 relative
to the transcription start site, was PCR-amplified with the primers 5?-TTCGCC
CTCCCATAGGTTC-3? and 5?-CCCGCAACGTTAACAAGTGTC-3?. The
product was ligated into pGEM-T Easy (Promega) and then transferred into
pGL3sb vector (kindly provided by M. Oren) using the restriction enzymes NdeI
and NcoI. Mutations in the p53 binding site were generated using a QuikChange
Site-Directed Mutagenesis kit (Stratagene) with the following primer (only sense
primer is shown; mismatches are underlined): 5?-CGCTGGGGAAGGAAGGA
Retroviral infections. Retroviral infections were described by Milyavsky et al.
(40). Infected cells were selected for 1 week with the following antibiotics: for
human shRNA infections, 200 ?g/ml hygromycin B; for mouse shRNA infec-
tions, 1 ?g/ml puromycin; for GSE56 overexpression, 400 ?g/ml G418. TM4 cells
infected with retroviruses harboring sh-p63 or an shRNA targeting a control
gene (sh-con) were maintained in hygromycin B-containing medium to sustain
Transfections. Saos-2 and SKOV-3 cells were transfected with Fugene HD
(Roche Applied Science), using 0.5 ?g of various p53 expression plasmids, as
1680BORNSTEIN ET AL.MOL. CELL. BIOL.
indicated in the figure legends. HT-1080 cells were transfected with TransIT
(Mirus Bio LLC) using 3.5 ?g of plasmids, as indicated in the figure legends.
Luciferase reporter assay. Saos-2 cells were plated in 24-well plates 24 h
before transfection. Cells were transfected with 300 ng/well of luciferase reporter
construct, 10 ng/well of pRL-CMV for normalization of transfection efficiency, 0
to 30 ng/well p53 expression plasmid, and pBlueScript for a total DNA amount
of 500 ng/well. Luciferase and Renilla activities were determined using commer-
cial reagents and procedures (Promega).
RNA preparation and qRT-PCR. Total RNA was isolated from cultured cells
using a NucleoSpin II kit (Macherey Nagel). Total RNA from testes was isolated
following mechanical homogenization using TRI reagent (Molecular Research
Center). A 2-?g aliquot of the total RNA was reverse transcribed using Moloney
murine leukemia virus reverse transcriptase (MMLV-RT; Bio-RT) and random
hexamer primers. Quantitative real-time PCR (qRT-PCR) was performed using
SYBR green PCR Master Mix (Applied BioSystems) on an ABI 7300 instru-
ment. Human and mouse mRNA levels were normalized to the level of GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) and HPRT (hypoxanthine phos-
phoribosyltransferase), respectively. PCRs were performed in duplicates, and
error bars in charts represent the corresponding standard deviations. For analysis
of mRNA expression in vivo, tissues from at least three mice were collected per
experimental category, and error bars in charts represent standard errors of
mean. Primers for qRT-PCR and semi-qRT-PCR are listed in Table S1 posted
Western blotting. Western blot analyses were performed as described by
Milyavsky et al. (41). The following primary antibodies were used: H47 poly-
clonal anti-human p53 (produced in our laboratory), monoclonal anti-human
p21 (sc-377; Santa Cruz), monoclonal anti-human GAPDH (MAB374; Chemi-
con), monoclonal anti-mouse ?-tubulin (Sigma), and monoclonal anti-human
p63 (sc-8431; Santa Cruz).
Chromatin immunoprecipitation. Chromatin immunoprecipitation analyses
were performed as described by Kalo et al. (29) with the following antibodies.
For human WI-38 cells, polyclonal H47 (produced in our laboratory) was used to
precipitate p53, and polyclonal anti-transforming growth factor ? receptor 2
(TGF-?R2; Santa Cruz) was used for control precipitation. For mouse TM4 cells,
monoclonal 4A4 (sc-8431; Santa Cruz) was used to precipitate p63, and monoclonal
IgG (I-2511; Sigma) was used for control precipitation. Precipitated DNA was
measured by qRT-PCR (primer sequences are listed in Table S2 posted at http:
//www.weizmann.ac.il/mcb/Varda/p53_SPATA18/). Relative DNA levels were calcu-
lated by normalizing to the amount of precipitated nonspecific DNA.
Mice. C57BL/6 p53?/?mice were kindly provided by G. Lozano (University of
Texas M. D. Anderson Cancer Center).
Immunohistochemistry and immunofluorescence analyses. Immunohisto-
chemistry analyses were performed as described by Sarig et al. (54). For immu-
nofluorescence analysis, paraffin-embedded sections were deparaffinized using
xylene and were rehydrated with alcohol series, boiled in a Tris-EDTA, pH 9.0,
buffer, and incubated overnight at room temperature in a humid chamber with
mouse monoclonal 4A4 anti-p63 antibody (sc-8431; Santa Cruz). Sections were
washed three times with phosphate-buffered saline (PBS) and labeled with a Cy3
fluor (Jackson Immunoresearch) at 1:500 for 20 min. To visualize nuclei, sections
were counterstained with 4?,6?-diamidino-2-phenylindole (DAPI; 10 mg/ml) for
5 min and mounted with Elvanol.
In situ hybridization. Probes for in situ hybridization were prepared by PCR
amplification of ?500-bp fragments from the mouse SPATA18 gene, using total
cDNA prepared from p53?/?mouse testes. The following primers were used to
produce two different amplicons: forward primer 1, 5?-CTGCAGACTTCCCTC
AGTTC-3?; reverse primer 1, 5?-GCAGACAGGACAGCTATCTC-3?; forward
primer 2, 5?-ACAGTGGCCAAGATCAGAAG-3?; and reverse primer 2, 5?-A
GACCAAGGGAGCAGTAAAG-3?. Amplicons were ligated into pGEM-T Easy
(Promega). Digoxigenin-labeled riboprobes were produced for sense and antisense
probes by in vitro transcription from the T7 or SP6 promoters using a digoxigenin
RNA labeling kit (Roche Applied Science). Paraffin sections (6 ?m) from mouse
testes were deparaffinized using xylene and were rehydrated with alcohol series.
Sections were treated with 20 ?g/ml proteinase K (Roche Applied Science) at 37°C
for 15 min and incubated with prehybridization buffer (10% dextran sulfate, 50%
formamide, 0.3 M NaCl, 0.1 M Tris [pH 7.5], 1 mM EDTA, 1% blocking reagent, 2
mg/ml torula yeast RNA) at 37°C for 3 h. Sections were hybridized with 400 ng/ml
of each riboprobe at 37°C for 16 h in a humid chamber and serially washed with
washing buffer (30 to 300 mM NaCl, 2 to 20 mM NaH2PO4-H2O, 0.25 to 2.5 mM
EDTA). Hybridized sections were blocked with Genius buffer (100 mM Tris [pH
7.6], 150 mM NaCl) containing 1% BSA and were incubated with an alkaline
phosphatase-conjugated antidigoxigenin antibody at 4°C for 16 h. Sections were
washed with Genius buffer containing 1% BSA and then with Genius buffer con-
taining 2% blocking reagent. Developing was done using nitroblue tetrazolium/5-
bromo-4-chloro-3-indolylphosphate (NBT/BCIP) substrate according to the
manufacturer’s instructions (Roche Applied Science).
Statistical analysis. Statistical significance was evaluated using a one-tailed
unpaired student t test, and asterisks in figures represent P values lower than
5 ? 10?3.
Expression of SPATA18 positively correlates with p53 activ-
ity in human and mouse cells. The aforementioned microarray
experiment pointed at SPATA18 as a putative p53 target gene.
To validate this notion, we utilized WI-38 cells that stably
express a short hairpin RNA (shRNA) targeting either p53
(sh-p53) or a control gene (sh-con). These isogenic cell cul-
tures were treated with Nutlin-3a, a compound which impairs
Mdm2-p53 interaction, thereby stabilizing and activating p53
(60). The protein levels of p53 and p21, a well-known p53
target gene (13), were measured by Western blot analysis (Fig.
1A), revealing a pronounced activation of p53 by Nutlin-3a and
a strong attenuation of p53 activity in sh-p53 cells. Quantitative
real-time PCR (qRT-PCR) measurements were conducted for
p21 and SPATA18 mRNA levels, revealing that they were
significantly downregulated by p53 knockdown and were
strongly induced by Nutlin-3a in a p53-dependent manner (Fig.
1B). Both p21 and SPATA18 were induced relatively fast fol-
lowing Nutlin-3a treatment and reached peak levels after 24 h
(see Fig. S1A posted at http://www.weizmann.ac.il/mcb/Varda
/p53_SPATA18/). The expression of SPATA18 was also mea-
sured in an additional cell type, namely, HT-1080 human fi-
brosarcoma cells, which were infected with either a control
vector or a vector encoding the p53-inactivating peptide
GSE56 (46). In this system we analyzed, in addition to Nutlin-
3a, the effect of various genotoxic drugs known to activate p53.
As depicted in Fig. 1C, the expression of SPATA18 was signif-
icantly elevated upon genotoxic insult in a p53-dependent
manner. The mRNA levels of SPATA18 were further analyzed
in IMR-90 fetal human lung fibroblasts, where knockdown
of p53 or expression of a dominant negative mutant p53
profoundly attenuated SPATA18 expression (see Fig. S1B
and S1C posted at http://www.weizmann.ac.il/mcb/Varda/p53
_SPATA18/). Furthermore, p53-dependent transcriptional ac-
tivation of SPATA18 was also observed in additional human
cell lines, including Saos-2 osteosarcoma (Fig. 2D), SKOV-3
ovarian carcinoma, LNCaP prostate carcinoma, and HepG2
hepatocellular carcinoma (see Fig. S1D to F at the URL men-
SPATA18 is conserved among a wide range of vertebrates,
including chimpanzee, dog, cow, mouse, rat, chicken, and ze-
brafish. We were therefore curious to test whether SPATA18
expression is regulated by p53 in mouse cells. Indeed, following
Nutlin-3a treatment, SPATA18 was upregulated in mouse embry-
onic fibroblasts (MEFs) derived from a p53?/?mouse but not in
MEFs derived from a p53?/?sibling mouse (Fig. 1D) or in
p53?/?MEFs in which p53 was knocked down (see Fig. S1G
Moreover, in mouse embryonic stem cells derived from a p53?/?
mouse, SPATA18 was downregulated following p53 knockdown
by shRNA (Fig. 1E). In sum, SPATA18 transcription is activated
in a p53-dependent manner in a wide variety of human and
mouse cell types and following various types of p53-activating
VOL. 31, 2011 SPATA18 IS TRANSACTIVATED BY p53 AND p631681
p53 directly activates SPATA18 transcription. Next, we
tested whether SPATA18 transcription is directly activated by
p53. To this end, we treated WI-38 cells with Nutlin-3a in
combination with cycloheximide, a compound that inhibits
protein biosynthesis. If SPATA18 induction by Nutlin-3a is
indirect and requires the synthesis of a protein mediator, then
cycloheximide should prevent it. As a positive control we mea-
sured the mRNA and protein levels of p21, which represents a
bona fide direct p53 target. Indeed, p21 transcription was ac-
tivated in a p53-dependent manner following Nutlin-3a treat-
ment, and the addition of cycloheximide did not interfere with
this induction while the accumulation of p21 protein following
Nutlin-3a treatment was prevented by cycloheximide (Fig. 2A
and B). Importantly, SPATA18 expression displayed a pattern
similar to that of p21, indicating that SPATA18 transcription is
most likely activated by p53 directly.
p53 is a transcription factor that specifically binds to DNA
consensus sequences, defined as two copies of the 10-bp motif
5?-PuPuPuC(A/T)(T/A)GPyPyPy-3? (where Pu is a purine and
Py is a pyrimidine) separated by a spacer of 0 to 13 bp (38). We
used MatInspector (48) to search for p53 binding sites in the
human SPATA18 locus. A putative binding site (BS) with high
similarity to the p53 consensus was found in the first intron of
SPATA18. This BS has two perfect core sequences and no
spacer (see Fig. S2A posted at http://www.weizmann.ac.il/mcb
/Varda/p53_SPATA18/), properties associated with strong
binding of p53 (38). To determine whether p53 binds this
putative BS, we carried out a chromatin immunoprecipitation
assay in WI-38 cells and analyzed the amount of DNA precip-
itated with an antibody against p53 or a control antibody, using
specific primers for either the known p53 BS in the promoter
of p21 or the putative BS in the first intron of SPATA18. As
predicted, both of these genomic regions were strongly en-
riched in the p53-immunoprecipitated chromatin (Fig. 2C),
indicating that p53 binds the consensus sequence in the first
intron of SPATA18. Next, we tested the functionality of the
identified p53 BS. To this end we cloned a 495-bp intronic
fragment, which includes the p53 BS, into a luciferase reporter
plasmid and carried out a series of promoter activity assays in
p53-null Saos-2 cells. In these cells, transfection of WT p53,
but not cancer-derived mutant p53R249S, which is incapable of
transactivating p53 target genes (57), induced the transcription
of the endogenous SPATA18 gene (Fig. 2D), as well as of p21
(see Fig. S2B posted at http://www.weizmann.ac.il/mcb/Varda
/p53_SPATA18/). Similarly, cotransfection of WT p53, but not
p53R249S, with luciferase reporter plasmids resulted in activa-
tion of a luciferase reporter under the control of the cloned
SPATA18 intronic fragment while an empty luciferase reporter
was not affected by p53 (Fig. 2E). The activity of a reporter
containing p21 promoter was measured as a positive control
(see Fig. S2C posted at the URL mentioned above). Finally,
mutations of three core nucleotides in the p53 BS within the
cloned fragment abolished its activation by WT p53. Com-
bined, the results presented in Fig. 2 clearly demonstrate that
FIG. 1. Expression of SPATA18 positively correlates with p53 activity in human and mouse cells. WI-38 cells expressing either a control shRNA
(sh-con) or an shRNA targeting p53 (sh-p53) were treated with 10 ?M Nutlin-3a for 48 h. Protein levels of p53, p21, and GAPDH were measured
by Western blot analysis (A). Normalized mRNA levels of p21 and SPATA18 were measured by qRT-PCR (B). (C) qRT-PCR measurements were
conducted for SPATA18 mRNA in HT-1080 fibrosarcoma cells stably expressing either the p53-inactivating peptide GSE56 or a control vector.
Cells were treated with the indicated compounds for 24 h at the following concentrations: Nutlin-3a, 10 ?M; doxorubicin (Dox), 10 ?M;
5-fluorouracil (5-FU), 10 ?M; etoposide (Etop), 100 ?M; cisplatin (Cys), 3 ?M. (D) p53?/?and p53?/?MEFs were treated with 25 ?M Nutlin-3a
for 24 h. qRT-PCR measurements were conducted for SPATA18 mRNA. (E) qRT-PCR measurements were conducted for SPATA18 mRNA in
mouse embryonic stem cells (mESC) expressing either a control shRNA or an shRNA targeting mouse p53. NT, not treated.
1682BORNSTEIN ET AL.MOL. CELL. BIOL.
SPATA18 is a direct transcriptional target of p53 and indicate
that the mechanism of SPATA18 transactivation involves bind-
ing of p53 to a consensus element within its first intron.
SPATA18 is expressed in spermatids in the mouse testes. In
mammals, germ cells called gonocytes populate the seminifer-
ous tubules at birth (31). During the first postnatal week in
mice, the first spermatogonia and primary spermatocytes ap-
pear. At postnatal day 7 the prophase of meiosis begins,
whereas the first round and elongating spermatids are ob-
served at postnatal days 21 and 25, respectively, with sper-
miation commencing at day 34 (37). The synchronous nature
of spermatogenesis in neonatal mice and rats allows accurate
attribution of biological processes to specific cell populations.
Accordingly, the mRNA of Spetex-1, the rat ortholog of
SPATA18, is detected at 3 weeks of postnatal development and
reaches peak levels at 6 weeks (27). The combined results of in
situ hybridization, immunocytochemical analysis, and immuno-
electron microscopy show that Spetex-1 is highly expressed in
elongating spermatids and in spermatozoa (26, 27).
We decided to perform a similar analysis of testicular
SPATA18 levels during mouse postnatal development. qRT-
PCR measurements of total testis RNA revealed that
SPATA18 is profoundly upregulated between postnatal days 21
to 25 and remains high until day 38 (Fig. 3A), as well as in
testes of mature mice (data not shown). We therefore con-
cluded that SPATA18 is expressed primarily in spermatids. We
next analyzed the expression of p21 along the same testicular
development model and found that, similarly to SPATA18, p21
expression is upregulated between postnatal days 21 to 25 (Fig.
3B). Furthermore, the p53 target gene Wip1 was also demon-
strated to be profoundly upregulated in the testis during post-
natal days 20 to 25 (10), further supporting the notion that p53
activity is augmented during this period of postnatal spermato-
To gain more insights on the exact localization of SPATA18
mRNA in the testis, we performed an in situ hybridization
assay. Paraffin sections of mature mouse testes were hybridized
with either an RNA probe complementary to SPATA18
mRNA (antisense) or a sense probe as a negative control.
Using the antisense probe, strong staining was observed in the
inner layer of most of the seminiferous tubules (Fig. 3C and
D), whereas sections hybridized with the sense probe were
FIG. 2. p53 directly activates SPATA18 transcription. Control (sh-con) and p53 knocked-down (sh-p53) WI-38 cells were treated with either
10 ?M Nutlin-3a for 8 h (Nut), 10 ?g/ml cycloheximide for 4 h (Chx), or both Nutlin-3a and cycloheximide (Nut?Chx). In the combined treatment,
Nutlin-3a was applied first, and after 4 h cycloheximide was added. Samples were analyzed for protein levels by Western blotting (A), and mRNA
levels were determined by qRT-PCR (B). (C) Chromatin immunoprecipitation analysis of WI-38 cells using anti-p53 antibody (IP: p53) or a
negative-control antibody against TGF-?R2 (IP: Con). The amount of precipitated DNA was measured by qRT-PCR using specific primers
designed to amplify the known p53 binding sites in p21 promoter or in the first intron of SPATA18. Values were normalized to the amount of
precipitated nonspecific DNA. (D) Saos-2 cells were transfected with control vector (Con) or a pC53-SN3 vector encoding either wild-type p53
(WT) or mutant p53R249S(249S). qRT-PCR was conducted for SPATA18 mRNA 48 h after transfection. (E) Saos-2 cells were transfected with
either an empty luciferase reporter (Empty), a luciferase reporter under the control of a 495-bp DNA fragment from the first intron of SPATA18,
which contains a p53 BS (SPATA18), or the same reporter in which the p53 BS was mutated (SPATA18 mut-p53 BS). pC53-SN3 vector encoding
either wild-type p53 (WT) or mutant p53R249S(249S) was cotransfected in increasing concentrations (0, 3, 10, and 30 ng/well). Luciferase activity
was normalized to the Renilla activity of cotransfected pRL-CMV plasmid. Error bars represent standard deviations between triplicate measure-
VOL. 31, 2011SPATA18 IS TRANSACTIVATED BY p53 AND p631683
devoid of a positive signal (see Fig. S2D posted at http://www
.weizmann.ac.il/mcb/Varda/p53_SPATA18/), attesting to the
specificity of the antisense probe. Careful examination of the
hybridization pattern revealed that SPATA18 mRNA is absent
from both spermatogonia and spermatocytes, which are lo-
cated on the outer layer of the seminiferous tubule, whereas
round and, to a much greater extent, elongating spermatids
express high levels of SPATA18 (Fig. 3D). Interestingly, im-
FIG. 3. Spatiotemporal expression patterns of SPATA18 mRNA and p53 protein. qRT-PCR measurements were conducted for SPATA18 (A) and
p21 (B) mRNAs in mouse testes collected at the indicated postnatal days. For each time point, testes were collected from three mice, and error bars
represent the corresponding standard error of mean. (C) In situ hybridization analysis of SPATA18 mRNA in a testis section from a mature (14 weeks)
mouse. Scale bar, 100 ?m. (D) Digital magnification of the marked area in panel C. (E) Immunohistochemistry analysis of p53 protein in testis from a
25-day-old mouse. Scale bar, 200 mm. (F) Digital magnification of the marked area in panel E. (G) Digital magnifications of immunohistochemistry
analysis of p53 protein in testis from 25-day-old mouse. Arrows indicate stained regions that are populated with elongating spermatids.
1684 BORNSTEIN ET AL.MOL. CELL. BIOL.
munohistochemistry analysis of p53 protein in mouse testes
demonstrated its abundance in the intermediate layer of most
seminiferous tubules, with intense staining in spermatocytes
and round spermatids (Fig. 3E and F). Moreover, in some
seminiferous tubules, we observed strong p53 staining in elon-
gating spermatids (Fig. 3G).
SPATA18 transcription is regulated by p53 and p63 in vivo.
Having shown that SPATA18 is a bona fide p53 target gene in
a variety of cell types of both human and mouse origin and that
SPATA18 is highly expressed in spermatids in vivo, we were
curious whether SPATA18 is activated by p53 in mouse testes.
We therefore compared the mRNA levels of SPATA18 in the
testes of p53?/?, p53?/?, and p53?/?mice. As depicted in Fig.
4A, p53 mRNA levels are strongly reduced in p53?/?mice
compared to levels in p53?/?mice (P value, 2.57 ? 10?5) and
are nondetectable in p53?/?mice. Interestingly, testicular
FIG. 4. SPATA18 transcription is regulated by p53 and p63 in vivo. (A) qRT-PCR analysis was conducted for p53, SPATA18, and p21 mRNA in testes
derived from p53?/?, p53?/?, and p53?/?mice. The number of samples in each group is indicated in parentheses. (B) p63 protein was detected by
immunofluorescent staining of a testis section from a 25-day-old mouse (red). DNA was visualized with DAPI (blue). (C) p63 protein level was measured
by Western blotting of whole-testis lysates derived from either p53?/?or p53?/?mice. Intensities were quantified using ImageJ software, and relative p63
protein levels were calculated by normalizing to ?-tubulin intensities (lower panel). (D) Mouse TM4 Sertoli cells expressing either a control shRNA
(sh-con) or an shRNA targeting mouse p53 (sh-p53) were transfected with pCDNA-TA-p63? (p63) or a control vector (Con). Two days after transfection
shRNA or an shRNA targeting mouse p63 were analyzed for the expression of TA-p63 and SPATA18 by qRT-PCR. (F) ChIP analysis of TA-p63?-
transfected TM4 cells using p63-specific antibody (IP:p63) or control IgG (IP:Con). The amount of precipitated DNA was measured by qRT-PCR using
specific primers designed to amplify a putative p53 binding site in the first intron of mouse SPATA18. Values were normalized to the amount of
precipitated nonspecific DNA. Bars represent mean ? standard deviations from two duplicate immunoprecipitants.
VOL. 31, 2011SPATA18 IS TRANSACTIVATED BY p53 AND p63 1685
SPATA18 mRNA was significantly lower in p53?/?mice than
in p53?/?mice (Fig. 4A) (P value, 3.4 ? 10?4), supporting the
notion that p53 activates SPATA18 transcription in vivo. To
our great surprise, however, testicular SPATA18 expression in
p53?/?was not lower than that in p53?/?mice. Nevertheless,
when the mRNA levels of p21 were analyzed in the same
whole-testis RNA samples, a strikingly similar pattern was
found; i.e., testicular p21 expression was reduced in p53?/?
mice (P value, 1.7 ? 10?3) but not in p53?/?mice (Fig. 4A).
These results led us to hypothesize that in p53?/?mice the
complete absence of p53 is compensated, probably by its family
members, p63 and/or p73. The homology shared by p53, p63,
and p73 suggests that these proteins have similar properties as
transcription factors, and, although each family member has
distinct roles during development and homeostasis, many of
their functions overlap, as well as their DNA binding motifs
and target genes (6, 18, 43). Moreover, all three family mem-
bers were shown to be expressed in mouse testes (17). We
therefore performed immunofluorescence analysis for p63 pro-
tein in mouse testes and found it to localize primarily at the
nuclei of spermatocytes and, to a lower extent, in round sper-
matids (Fig. 4B). This expression pattern partially overlaps
with that of p53 (Fig. 3), supporting the notion that these
paralogs have common functions in spermatogenesis. Impor-
tantly, Western blot analysis of p63 protein in whole-testis
lysates revealed that p63 levels are significantly elevated in
p53?/?mice compared to levels in p53?/?mice (Fig. 4C)
(P value, 1.9 ? 10?4). Of note, p63 protein levels were not
significantly elevated in p53?/?mice compared to levels in
p53?/?mice (data not shown). Moreover, PCR analysis of
whole-testis RNA revealed that the predominant p63 iso-
forms expressed in mouse testes are TA-p63?/? (see Fig.
S3A posted at http://www.weizmann.ac.il/mcb/Varda/p53
_SPATA18/), which encode transactivation-proficient pro-
teins that share functions and target genes with wild-type
p53 (43). Expression of the p63 isoforms lacking the trans-
activation domain (?N-p63) was not detected in the testes
while in kidneys both TA-p63 and ?N-p63 were expressed,
thus serving as a positive control for the detection method.
Next, we analyzed the effect of p63 overexpression in the
mouse Sertoli cell line TM4. Interestingly, overexpression of
p63 in control TM4 cells did not affect SPATA18 expression.
However, when we knocked down p53 in these cells, the
SPATA18 mRNA level was reduced, and p63 overexpression
could restore it (Fig. 4D). These results indicate that p63 is
capable of inducing SPATA18 transcription and imply that, in
the absence of p53, the effect of p63 on SPATA18 transcription
is augmented. Furthermore, knockdown of p63 in TM4 cells
led to a marked reduction of SPATA18 mRNA levels (Fig. 4E),
indicating that the endogenous p63 in these cells activates
SPATA18 transcription. To demonstrate that the effect of p63
on SPATA18 expression is not specific to mice, we overex-
pressed p63, as well as p53 as a positive control, in HT-1080
human fibrosarcoma cells. Indeed, both p53 and p63 induced
SPATA18 expression (see Fig. S3B posted at http://www
.weizmann.ac.il/mcb/Varda/p53_SPATA18/). To support the
notion that p63 can directly transactivate SPATA18, we per-
formed chromatin immunoprecipitation assay in mouse TA-
p63?-transfected TM4 cells with an antibody against p63 or a
control IgG antibody. As p63 shares its consensus DNA-bind-
ing site with p53, we used MatInspector (48) to search for p53
BSs in the mouse SPATA18 locus and found two such BSs,
which reside, similarly to human SPATA18, in the first intron.
One of these BSs, which resides approximately 2 kbp down-
stream from the transcriptional start site and has two perfect
core sequences and no spacer (see Fig. S3C posted at http:
significant binding to p63 (Fig. 4F) (P value, 1.8 ? 10?2).
Therefore, p63 can bind the SPATA18 gene and can activate
its transcription. Combining this result with the observation
that p63 levels are elevated in p53?/?mice, we suggest that
p63 compensates for the absence of p53 in the testes of
p53-null mice. Notably, overexpression of p73 in HT-1080
cells did not induce SPATA18 expression. Moreover, West-
ern blot analysis of p73 in mouse testes did not demonstrate
an increase of p73 levels in p53?/?mice compared to
p53?/?mice (data not shown). Thus, although the evidence
is not conclusive, it appears that testicular p53 deficiency is
primarily compensated by p63 and not p73.
p53 plays central roles in normal cell differentiation and
organismal development (42). Moreover, substantial data im-
plicate p53 in the regulation of spermatogenesis under normal
conditions (1, 52, 55) and following DNA damage (20, 21), as
well as in the regulation of additional aspects of fertility (19,
24, 25). In this study we set out to examine the role of p53 in
the transcriptional regulation of SPATA18, a spermatogenesis-
related gene. We discovered that SPATA18 is a bona fide p53
transcriptional target, and its induction by p53 can be observed
in a variety of normal and cancerous cell types of both human
and mouse origin. Similar to many other p53 target genes (62),
the consensus DNA sequence that mediates p53 binding to the
SPATA18 locus resides within its first intron, probably acting as
Although SPATA18 is known to be expressed primarily in the
and spleen (27). This may explain its expression in normal and
cancerous cells representing a wide variety of tissues, including
human and mouse embryonic fibroblasts and human fibrosar-
coma, osteosarcoma, prostate, ovarian, and hepatocellular carci-
noma cells, as well as in mouse Sertoli and embryonic stem cells
(Fig. 1, 2, and 4; see also Fig. S1 posted at http://www.weizmann
.ac.il/mcb/Varda/p53_SPATA18/). However, as the exact molec-
ular role and biological function of SPATA18 are currently un-
known, the role of its p53-dependent transcriptional induction in
somatic cells is yet to be unveiled.
SPATA18 rat ortholog, Spetex-1, is highly expressed during
spermatogenesis in spermatids, and its protein localizes at de-
fined regions, such as the cytoplasm of spermatids, residual
bodies, and the flagella (26, 27, 30). Accordingly, two distinct
non-mutually exclusive suggestions were raised regarding its
function in spermatogenesis, being involved either in the pro-
cess of spermatid maturation or constituting a flagellar com-
ponent. These possibilities tempted us to speculate that
SPATA18 might mediate, at least partially, the roles exerted by
p53 during spermatogenesis. Our in vivo analyses indicate that
SPATA18 is profoundly upregulated during mouse postnatal
spermatogenesis at a stage corresponding to the appearance of
1686 BORNSTEIN ET AL.MOL. CELL. BIOL.
spermatids. Accordingly, in situ hybridization analysis detected
SPATA18 mRNA primarily in elongating spermatids, as well as
in round spermatids, albeit at a lower level (Fig. 3). This was in
agreement with previous reports on Spetex-1 (26, 27). To fur-
ther support this notion, we utilized global expression analysis
as performed by Namekawa et al. (44), which analyzed mRNA
expression in four enriched germ cell populations from mouse
testes, namely, types A and B spermatozoa, pachytene sper-
matocytes, and round spermatids. Confirming our results,
SPATA18 was expressed almost exclusively in round sperma-
tids (see Fig. S4A posted at http://www.weizmann.ac.il/mcb
We present several lines of evidence that the testicular ex-
pression of SPATA18 is positively regulated by p53. First, the
pattern of p53 accumulation, which typically indicates its func-
tional activation, partially overlaps with that of SPATA18 tran-
scription; i.e., p53 protein is accumulated in spermatocytes,
round spermatids, and occasionally in elongating spermatids
(Fig. 3E to G), while SPATA18 mRNA is induced in round
and, more profoundly, in elongating spermatids (Fig. 3C and
D; see also Fig. S4A posted at http://www.weizmann.ac.il/mcb
/Varda/p53_SPATA18/). Second, p21 is induced between post-
natal days 21 to 25 (Fig. 3B), concomitantly with the appear-
ance of elongating spermatids, indicating functional activation
of p53 at the same stage in which SPATA18 is induced. Of
note, a similar spatiotemporal pattern was previously demon-
strated also for the testicular p53 target gene Wip1 (10). Sup-
porting the notion that SPATA18 is regulated by p53 in vivo, its
expression is significantly attenuated in the testes of p53?/?
mice (Fig. 4A). This attenuation correlates with that of p53 and
p21 mRNA levels. Whether this haploinsufficiency is mani-
fested as fertility- or development-related defects is currently
unclear since the effects of p53 deficiency were analyzed almost
exclusively in p53-null animals. Surprisingly, the expression of
both p21 and SPATA18 is not attenuated in p53?/?mice,
prompting us to hypothesize that the testicular p53 activity is
compensated by its paralogs, p63 and p73, which play impor-
tant and unique roles during development. Indeed, p63 and/or
p73 were reported to compensate for p53 loss in several pro-
cesses, including DNA damage response (65), tumor suppres-
sion (14), and development (28, 56). p63 is expressed in a
highly restricted pattern during embryogenesis and is essential
for limb formation and epidermal morphogenesis. p63-null
mice show profound developmental abnormalities of the skin,
limbs, mammary, prostate, and other epithelial tissues and die
soon after birth (39, 63). In addition, p63 is expressed in mouse
reproductive organs and primordial germ cells (32) and was
suggested to regulate programmed cell death and differentia-
tion of these cells (47). Most importantly, a similar giant-cell
syndrome, which characterizes mice with p53 deficiency (52),
was reported in p63?/?adult mice and in p63?/?cultured fetal
Unlike p63, p73-null mice are viable but are stunted and
have high mortality rates. These mice show profound develop-
mental defects, including hippocampal dysgenesis and hydro-
cephalus (64). Interestingly, while p73-null mice have no struc-
tural abnormalities in their reproductive organs, p73-null
males lack interest in mating, probably due to hormonal or
sensory defects, and thus have low fecundity (64).
Our data indicate that p63 is highly expressed in spermato-
cytes and to a lesser extent in spermatids (Fig. 4B). Supporting
the notion that p63 may compensate for p53 loss, p63 protein
levels were increased in the testes of p53-null mice (Fig. 4C).
Similarly, p63 protein was also shown to be increased in oral-
esophageal epithelia of p53-null mice compared to WT p53
mice (16, 58). It was further demonstrated that p53 can reduce
the stability of TA-p63? (36), perhaps providing a mechanistic
explanation for the accumulation of p63 in p53-deficient tis-
sues. Moreover, we observed an increase in the mRNA level of
TA-p63, but not ?N-p63, following p53 inactivation in WI-38
cells (data not shown), suggesting another mechanism by which
p53 can downregulate p63.
Importantly, we demonstrated that p63 overexpression
induces SPATA18 expression and that knockdown of endog-
enous p63 attenuates SPATA18 levels (Fig. 4; see also Fig.
S3B posted at http://www.weizmann.ac.il/mcb/Varda/p53
_SPATA18/). Moreover, by analyzing publicly available
gene profiling data sets, we found that knockdown of en-
dogenous p63 expression in human keratinocytes or squa-
mous carcinoma cells (3) leads to downregulation of
SPATA18 expression (see Fig. S4D posted at the URL men-
tioned above). We also showed that p63 can directly bind a
regulatory site within the first intron of SPATA18 (Fig. 4F).
Taking together the observations that p63 is elevated in
p53?/?testes and is capable of binding SPATA18 and in-
ducing its transcription, it is likely that the loss of testicular
p53 activity is compensated by p63, resulting in steady tran-
scriptional activity of p53 target genes. Of note, both p63
and p73 were suggested to compensate for p53 loss in sev-
eral processes, including tumor suppression (14) and devel-
opment (28, 56). While both family members can potentially
compensate for testicular p53 function, our data indicate
p63 as the more likely candidate since p73 was not upregu-
lated in the testes of p53?/?mice and was not capable of
transactivating SPATA18 in vitro (data not shown).
It remains highly interesting to investigate the role of
SPATA18 as a p53 target both in the testes and in additional
tissues and cell types. The identification of Spetex-1 protein in
the cytoplasm of elongated spermatids and in residual bodies
engulfed by Sertoli cells (27) implies the possibility that
SPATA18 is involved in the apoptosis-like process of sperma-
tid maturation, which coincides well with the known function
of p53 as an apoptosis inducer. To date, we can only speculate
that SPATA18 mediates p53 functions during spermatogene-
sis. Notably, the developmental effects of p53 deficiency were
analyzed primarily in a homozygous background, in which
SPATA18 expression remains normal. The question of whether
abnormalities in spermatogenesis are present in p53?/?mice is
therefore intriguing. A somewhat equivalent system is repre-
sented by p53 promoter-chloramphenicol acetyltransferase
(CAT)-harboring mice, in which p53 mRNA and protein are
downregulated (52). These mice exhibit a testicular giant-cell
degenerative syndrome, which likely stems from the inability of
tetraploid spermatocytes to complete meiosis. Another impli-
cation of SPATA18 in a spermatogenesis-related pathology
may lie in the observation that SPATA18 is downregulated in
human semen samples collected from individuals with severe
teratozoospermia compared to semen collected from normal
fertile men (P value, 7.4 ? 10?3) (see Fig. S4B posted at
VOL. 31, 2011SPATA18 IS TRANSACTIVATED BY p53 AND p631687
over, expression microarray analysis of testicular biopsy spec-
imens of azoospermia patients reveals that SPATA18 mRNA is
significantly downregulated in biopsy specimens collected from
nonobstructive azoospermia (NOA) patients compared with
obstructive azoospermia (OA) patients (P value, 8.3 ? 10?9)
(see Fig. S4C posted at the URL mentioned above). Com-
bined, these results link attenuated levels of SPATA18 to re-
duced fertility caused by defects in sperm development.
To gain insights into the possible roles of SPATA18 in ad-
ditional tissues or processes, we searched the Oncomine data-
base (49) and found few expression profiling studies that de-
tected downregulation of SPATA18 in cancer samples
compared to levels in the corresponding normal tissues. For
instance, in a study conducted by Richardson et al. (50) com-
paring human ductal breast carcinomas to normal breast sam-
ples, SPATA18 was found to be downregulated approximately
5-fold in the malignant samples (P value, 7.2 ? 10?7). These
data imply a tumor suppressive role for SPATA18, perhaps in
the process of programmed cell death.
Combined, our data enrich the known collection of p53
targets with a gene whose expression and localization imply a
role in spermatogenesis. To the best of our knowledge, besides
Wip1, SPATA18 is currently the only testis-associated p53 tar-
get gene and the only gene proposed to be a structural com-
ponent of the sperm flagella. Our data also provide clues into
the mechanisms underlying spermatogenesis and fertility de-
fects associated with p53 deficiency and highlight the primor-
dial role of p53 as a master regulator of the transmission of
This work was supported by a Center of Excellence grant from the
Flight Attendant Medical Research Institute. V.R. is the incumbent of
the Norman and Helen Asher Professorial Chair Cancer Research at
the Weizmann Institute.
We thank J. Don from Bar-Ilan University for providing scientific
1. Almon, E., et al. 1993. Testicular tissue-specific expression of the p53 sup-
pressor gene. Dev. Biol. 156:107–116.
2. Baker, S. J., S. Markowitz, E. R. Fearon, J. K. Willson, and B. Vogelstein.
1990. Suppression of human colorectal carcinoma cell growth by wild-type
p53. Science 249:912–915.
3. Barbieri, C. E., L. J. Tang, K. A. Brown, and J. A. Pietenpol. 2006. Loss of
p63 leads to increased cell migration and up-regulation of genes involved in
invasion and metastasis. Cancer Res. 66:7589–7597.
4. Beumer, T. L., H. L. Roepers-Gajadien, L. S. Gademan, D. H. Rutgers, and
D. G. de Rooij. 1997. P21(Cip1/WAF1)expression in the mouse testis before
and after X irradiation. Mol. Reprod Dev. 47:240–247.
5. Blanco-Rodriguez, J., and C. Martinez-Garcia. 1999. Apoptosis is physio-
logically restricted to a specialized cytoplasmic compartment in rat sperma-
tids. Biol. Reprod. 61:1541–1547.
6. Brandt, T., M. Petrovich, A. C. Joerger, and D. B. Veprintsev. 2009. Con-
servation of DNA-binding specificity and oligomerisation properties within
the p53 family. BMC Genomics 10:628.
7. Brosh, R., and V. Rotter. 2010. Transcriptional control of the proliferation
cluster by the tumor suppressor p53. Mol. Biosyst. 6:17–29.
8. Brosh, R., and V. Rotter. 2009. When mutants gain new powers: news from
the mutant p53 field. Nat. Rev. Cancer 9:701–713.
9. Brosh, R., et al. 2010. p53-dependent transcriptional regulation of EDA2R
and its involvement in chemotherapy-induced hair loss. FEBS Lett. 584:
10. Choi, J., et al. 2002. Mice deficient for the wild-type p53-induced phospha-
tase gene (Wip1) exhibit defects in reproductive organs, immune function,
and cell cycle control. Mol. Cell. Biol. 22:1094–1105.
11. Reference deleted.
12. Donehower, L. A., et al. 1992. Mice deficient for p53 are developmentally
normal but susceptible to spontaneous tumours. Nature 356:215–221.
13. el-Deiry, W. S., et al. 1993. WAF1, a potential mediator of p53 tumor
suppression. Cell 75:817–825.
14. Flores, E. R., et al. 2005. Tumor predisposition in mice mutant for p63 and
p73: evidence for broader tumor suppressor functions for the p53 family.
Cancer Cell 7:363–373.
15. Green, D. R., and G. Kroemer. 2009. Cytoplasmic functions of the tumour
suppressor p53. Nature 458:1127–1130.
16. Guo, X., et al. 2009. TAp63 induces senescence and suppresses tumorigen-
esis in vivo. Nat. Cell Biol. 11:1451–1457.
17. Hamer, G., I. S. Gademan, H. B. Kal, and D. G. de Rooij. 2001. Role for
c-Abl and p73 in the radiation response of male germ cells. Oncogene
18. Harms, K., S. Nozell, and X. Chen. 2004. The common and distinct target
genes of the p53 family transcription factors. Cell. Mol. Life Sci. 61:822–842.
19. Harvey, M., M. J. McArthur, C. A. Montgomery, Jr., A. Bradley, and L. A.
Donehower. 1993. Genetic background alters the spectrum of tumors that
develop in p53-deficient mice. FASEB J. 7:938–943.
20. Hasegawa, M., Y. Zhang, H. Niibe, N. H. Terry, and M. L. Meistrich. 1998.
Resistance of differentiating spermatogonia to radiation-induced apoptosis
and loss in p53-deficient mice. Radiat. Res. 149:263–270.
21. Hendry, J. H., A. Adeeko, C. S. Potten, and I. D. Morris. 1996. P53 deficiency
produces fewer regenerating spermatogenic tubules after irradiation. Int. J.
Radiat. Biol. 70:677–682.
22. Hess, R. A., and L. Renato de Franca. 2008. Spermatogenesis and cycle of
the seminiferous epithelium. Adv. Exp. Med. Biol. 636:1–15.
23. Hilscher, W., and B. Hilscher. 1976. Kinetics of the male gametogenesis.
24. Hu, W. 2009. The role of p53 gene family in reproduction. Cold Spring Harb.
Perspect. Biol. 1:a001073.
25. Hu, W., Z. Feng, A. K. Teresky, and A. J. Levine. 2007. p53 regulates
maternal reproduction through LIF. Nature 450:721–724.
26. Iida, H., Y. Honda, T. Matsuyama, Y. Shibata, and T. Inai. 2006. Spetex-1:
a new component in the middle piece of flagellum in rodent spermatozoa.
Mol. Reprod. Dev. 73:342–349.
27. Iida, H., J. Ichinose, T. Kaneko, T. Mori, and Y. Shibata. 2004. Comple-
mentary DNA cloning of rat spetex-1, a spermatid-expressing gene-1, en-
coding a 63 kDa cytoplasmic protein of elongate spermatids. Mol. Reprod.
28. Jacobs, W. B., D. R. Kaplan, and F. D. Miller. 2006. The p53 family in
nervous system development and disease. J. Neurochem. 97:1571–1584.
29. Kalo, E., et al. 2007. Mutant p53 attenuates the SMAD-dependent trans-
forming growth factor ?1 (TGF-?1) signaling pathway by repressing the
expression of TGF-? receptor type II. Mol. Cell. Biol. 27:8228–8242.
30. Kaneko, T., E. Murayama, H. Kurio, A. Yamaguchi, and H. Iida. 2010.
Characterization of Spetex-1, a new component of satellite fibrils associated
with outer dense fibers in the middle piece of rodent sperm flagella. Mol.
Reprod. Dev. 77:363–372.
31. Kluin, P. M., and D. G. de Rooij. 1981. A comparison between the morphol-
ogy and cell kinetics of gonocytes and adult type undifferentiated spermato-
gonia in the mouse. Int. J. Androl. 4:475–493.
32. Kurita, T., A. A. Mills, and G. R. Cunha. 2004. Roles of p63 in the diethyl-
stilbestrol-induced cervicovaginal adenosis. Development 131:1639–1649.
33. Lane, D. P. 1992. Cancer p53, guardian of the genome. Nature 358:15–16.
34. Leblond, C. P., and Y. Clermont. 1952. Spermiogenesis of rat, mouse, ham-
ster and guinea pig as revealed by the periodic acid-fuchsin sulfurous acid
technique. Am. J. Anat. 90:167–215.
35. Levine, A. J., and M. Oren. 2009. The first 30 years of p53: growing ever more
complex. Nat. Rev. Cancer. 9:749–758.
36. Li, N., et al. 2006. TA-p63-? regulates expression of ?N-p63 in a manner that
is sensitive to p53. Oncogene 25:2349–2359.
37. Malkov, M., Y. Fisher, and J. Don. 1998. Developmental schedule of the
postnatal rat testis determined by flow cytometry. biology of reproduction.
38. Menendez, D., A. Inga, and M. A. Resnick. 2009. The expanding universe of
p53 targets. Nat. Rev. Cancer. 9:724–737.
39. Mills, A. A., et al. 1999. p63 is a p53 homologue required for limb and
epidermal morphogenesis. Nature 398:708–713.
40. Milyavsky, M., et al. 2003. Prolonged culture of telomerase-immortalized
human fibroblasts leads to a premalignant phenotype. Cancer Res. 63:7147–
41. Milyavsky, M., et al. 2005. Transcriptional programs following genetic alter-
ations in p53, INK4A, and H-Ras genes along defined stages of malignant
transformation. Cancer Res. 65:4530–4543.
42. Molchadsky, A., N. Rivlin, R. Brosh, V. Rotter, and R. Sarig. 2010. p53 is
balancing development, differentiation and de-differentiation to assure can-
cer suppression. Carcinogenesis 31:1501–1508.
43. Moll, U. M., and N. Slade. 2004. p63 and p73: roles in development and
tumor formation. Mol. Cancer Res. 2:371–386.
44. Namekawa, S. H., et al. 2006. Postmeiotic sex chromatin in the male germ-
line of mice. Curr. Biol. 16:660–667.
45. Nicol, C. J., M. L. Harrison, R. R. Laposa, I. L. Gimelshtein, and P. G. Wells.
1688BORNSTEIN ET AL.MOL. CELL. BIOL.
1995. A teratologic suppressor role for p53 in benzo[a]pyrene-treated trans- Download full-text
genic p53-deficient mice. Nat. Genet. 10:181–187.
46. Ossovskaya, V. S., et al. 1996. Use of genetic suppressor elements to dissect
distinct biological effects of separate p53 domains. Proc. Natl. Acad. Sci.
U. S. A. 93:10309–10314.
47. Petre-Lazar, B., et al. 2007. The role of p63 in germ cell apoptosis in the
developing testis. J. Cell. Physiol. 210:87–98.
48. Quandt, K., K. Frech, H. Karas, E. Wingender, and T. Werner. 1995. MatInd
and MatInspector: new fast and versatile tools for detection of consensus
matches in nucleotide sequence data. Nucleic Acids Res. 23:4878–4884.
49. Rhodes, D. R., et al. 2004. ONCOMINE: a cancer microarray database and
integrated data-mining platform. Neoplasia 6:1–6.
50. Richardson, A. L., et al. 2006. X chromosomal abnormalities in basal-like
human breast cancer. Cancer Cell 9:121–132.
51. Rotter, V., et al. 1994. Does wild-type p53 play a role in normal cell differ-
entiation? Semin. Cancer Biol. 5:229–236.
52. Rotter, V., et al. 1993. Mice with reduced levels of p53 protein exhibit the
testicular giant-cell degenerative syndrome. Proc. Natl. Acad. Sci. U. S. A.
53. Russell, L. D. 1979. Spermatid-Sertoli tubulobulbar complexes as devices for
elimination of cytoplasm from the head region late spermatids of the rat.
Anat. Rec. 194:233–246.
54. Sarig, R., et al. 2010. Mutant p53 facilitates somatic cell reprogramming and
augments the malignant potential of reprogrammed cells. J. Exp. Med.
55. Schwartz, D., N. Goldfinger, and V. Rotter. 1993. Expression of p53 protein
in spermatogenesis is confined to the tetraploid pachytene primary sper-
matocytes. Oncogene 8:1487–1494.
56. Stiewe, T. 2007. The p53 family in differentiation and tumorigenesis. Nat.
Rev. Cancer. 7:165–168.
57. Suad, O., et al. 2009. Structural basis of restoring sequence-specific DNA
binding and transactivation to mutant p53 by suppressor mutations. J. Mol.
58. Suliman, Y., et al. 2001. p63 expression is associated with p53 loss in oral-
esophageal epithelia of p53-deficient mice. Cancer Res. 61:6467–6473.
59. Suzuki-Toyota, F., K. Ishibashi, and S. Yuasa. 1999. Immunohistochemical
localization of a water channel, aquaporin 7 (AQP7), in the rat testis. Cell
Tissue Res. 295:279–285.
60. Vassilev, L. T., et al. 2004. In vivo activation of the p53 pathway by small-
molecule antagonists of MDM2. Science 303:844–848.
61. Vousden, K. H., and K. M. Ryan. 2009. p53 and metabolism. Nat. Rev.
62. Wei, C. L., et al. 2006. A global map of p53 transcription-factor binding sites
in the human genome. Cell 124:207–219.
63. Yang, A., et al. 1999. p63 is essential for regenerative proliferation in limb,
craniofacial and epithelial development. Nature 398:714–718.
64. Yang, A., et al. 2000. p73-deficient mice have neurological, pheromonal and
inflammatory defects but lack spontaneous tumours. Nature 404:99–103.
65. Yao, J. Y., and J. K. Chen. 2010. TAp63 plays compensatory roles in p53-
deficient cancer cells under genotoxic stress. Biochem. Biophys. Res. Com-
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