Expression of Arf Tumor Suppressor in Spermatogonia
Facilitates Meiotic Progression in Male Germ Cells
Michelle L. Churchman1,2, Ignasi Roig3,4, Maria Jasin5, Scott Keeney1,3, Charles J. Sherr1,2*
1Howard Hughes Medical Institute, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 2Department of Genetics and Tumor Cell
Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 3Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New
York, New York, United States of America, 4Cytology and Histology Unit, Department of Cell Biology, Physiology, and Immunology, Universitat Autonoma de Barcelona,
Barcelona, Spain, 5Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America
The mammalian Cdkn2a (Ink4a-Arf) locus encodes two tumor suppressor proteins (p16Ink4aand p19Arf) that respectively
enforce the anti-proliferative functions of the retinoblastoma protein (Rb) and the p53 transcription factor in response to
oncogenic stress. Although p19Arfis not normally detected in tissues of young adult mice, a notable exception occurs in the
male germ line, where Arf is expressed in spermatogonia, but not in meiotic spermatocytes arising from them. Unlike other
contexts in which the induction of Arf potently inhibits cell proliferation, expression of p19Arfin spermatogonia does not
interfere with mitotic cell division. Instead, inactivation of Arf triggers germ cell–autonomous, p53-dependent apoptosis of
primary spermatocytes in late meiotic prophase, resulting in reduced sperm production. Arf deficiency also causes
premature, elevated, and persistent accumulation of the phosphorylated histone variant H2AX, reduces numbers of
chromosome-associated complexes of Rad51 and Dmc1 recombinases during meiotic prophase, and yields incompletely
synapsed autosomes during pachynema. Inactivation of Ink4a increases the fraction of spermatogonia in S-phase and
restores sperm numbers in Ink4a-Arf doubly deficient mice but does not abrogate c-H2AX accumulation in spermatocytes
or p53-dependent apoptosis resulting from Arf inactivation. Thus, as opposed to its canonical role as a tumor suppressor in
inducing p53-dependent senescence or apoptosis, Arf expression in spermatogonia instead initiates a salutary feed-forward
program that prevents p53-dependent apoptosis, contributing to the survival of meiotic male germ cells.
Citation: Churchman ML, Roig I, Jasin M, Keeney S, Sherr CJ (2011) Expression of Arf Tumor Suppressor in Spermatogonia Facilitates Meiotic Progression in Male
Germ Cells. PLoS Genet 7(7): e1002157. doi:10.1371/journal.pgen.1002157
Editor: John C. Schimenti, Cornell University, United States of America
Received March 28, 2011; Accepted May 11, 2011; Published July 21, 2011
Copyright: ? 2011 Churchman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Virtually all experimental work was funded by Howard Hughes Medical Institute. Use of core facilities at St. Jude Children’s Research Hospital were
supported in part by NCI Cancer Center Core Grant CA-21765 and by ALSAC, the fund-raising corporation supporting the hospital. MJ and SK are supported by
NIH grant R01 HD-40916. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The Cdkn2a-Cdkn2b gene cluster (also designated Ink4-Arf)
encodes two polypeptide inhibitors (p16Ink4aand p15Ink4b) of
cyclin D-dependent kinases (Cdk4 and Cdk6), as well as a third
protein (p19Arf) that antagonizes the Mdm2 ubiquitin E3 ligase to
activate p53 . Although the Ink4a and Ink4b genes likely arose
through gene duplication, the structure of the Ink4-Arf gene cluster
is highly unusual, as major portions of the p16Ink4aand p19Arf
proteins are encoded by alternative reading frames of a shared
exon . Induction of p16Ink4aand p15Ink4bprevents the
phosphorylation of the retinoblastoma protein (Rb), thereby
maintaining Rb in its growth suppressive state and preventing
entry into the DNA synthetic (S) phase of the cell division cycle. In
contrast, p19Arfexpression elicits a p53-dependent transcription
program that either enforces cell cycle arrest or triggers apoptosis,
depending on cell type, physiologic setting, and collateral
modulating signals . The Ink4-Arf genes prevent cell prolifer-
ation by implementing Rb- and p53-dependent programs that
enforce cellular senescence and inhibit tissue regeneration as
animals age, but their intimate genetic linkage facilitates their
coordinate repression in embryonic and adult tissue stem cells,
thereby allowing self-renewal [3,4]. Deleterious growth-promoting
stimuli conveyed by activated oncogenes induce Ink4-Arf gene
expression and engage both p53 and Rb to counter untoward
cellular proliferation. Not surprisingly, bi-allelic deletion of the
Ink4-Arf gene cluster abrogates this form of tumor suppression and
is one of the more frequent events in human cancer.
Despite its canonical role as an inducer of p53 in response to
oncogene signaling, Arf also has p53-independent tumor suppres-
sive activity. Deletion of Arf together with Mdm2 and p53 expands
the spectrum and decreases the latency of cancers that
spontaneously arise in mice lacking p53, p53 and Mdm2, or Arf
alone . Although highly basic p19Arf(,20% arginine) has been
reported to physically interact with more than 25 different proteins
other than Mdm2, the role of p19Arf, if any, in regulating the
functions of these putative ‘‘target’’ proteins remains controversial
. Indeed, numerous reports that p19Arfregulates such diverse
processes as ribosomal biosynthesis, transcription, DNA repair,
apoptosis and autophagy in a p53-independent manner have
generally relied on experiments performed with cultured cells but
have not been buttressed by more extensive in vivo analyses.
Although the Ink4-Arf locus is not detectably expressed under
most normal physiologic conditions, eye and male germ cell
development provide notable exceptions . Arf is required for
early postnatal regression of the hyaloid vasculature in the
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vitreous, so that Arf-null mice form a retrolenticular mass
predominantly composed of pericytes; the abnormal accumulation
of these cells disrupts the retina and lens and leads to blindness .
Arf inactivation also results in a significant reduction of sperm
production through as yet poorly defined mechanisms, although
young male mice remain fertile . In contrast, Arf-null females
have no discernable reproductive defects.
Spermatogenesis involves a stereotyped sequence of mitotic and
meiotic divisions followed by sperm differentiation . In mice, male
germ cell progenitors (gonocytes) renew in the testis between days 1–7
postpartum (P1–P7) and generate spermatogonia that line the
basement membranes of developing seminiferous tubules [11,12]. At
detach from the basement membrane, are displaced toward the
During the extended prophase of meiosis-I, homologous pairs of
maternal and paternal chromosomes align to form synaptonemal
complexes and exchange genetic information through homologous
recombination . Meiosis-I is completed by P18, and is followed
first mature spermatozoa enter the epididymis. As spermatogenesis
continues throughout life, spermatogonia within mature seminiferous
tubules remain localized on the peripheral tubular basement
membrane, whereas spermatocytes, spermatids, and mature sperm
are arranged in a sequential order from the periphery towards the
Intriguingly, p19Arfis transiently expressed in mitotically
dividing spermatogonia, but not in the meiotic cells that arise
from them . Here, we provide genetic evidence demonstrating
that Arf expression initiates a germ cell autonomous program that
protects meiotic spermatocytes from undergoing p53-dependent
elimination. This physiologic function of p19Arfdirectly contrasts
with its role as a tumor suppressor in inducing p53.
Arf Is Expressed in Mitotically Dividing Spermatogonia
Lineage tracing experiments in the mouse previously revealed that
all viable male germ cells are derived from spermatogonial
progenitors in which transient Arf expression neither inhibits
proliferation nor subsequent meiotic commitment . Underscoring
thesefindings, expression of p19Arfin young adult miceis observed in
all types of spermatogonia, but not in Sox9-expressing Sertoli cells on
the tubular basement membrane or in DAPI-stained intratubular
spermatocytes, spermatids, or sperm (Figure1A). The fact that p19Arf
is not detected in cells that have detached from the basement
membrane implies that Arf expression is extinguished at or near the
primary spermatocyte stage of germ cell differentiation. Consistent
with this interpretation, the Arf protein does not co-localize with
Dmc1 , a meiotic recombinase expressed in leptotene spermato-
cytes. In the mature testis, spermatogenesis occurs in waves along the
length of the seminiferous tubules, so that cross sections capture
the cell cycle. When five month-old mice injected intraperitoneally
with BrdU were sacrificed two hours later, dual immunofluorescence
analysis revealed that many cells on the tubular basement membrane
that had synthesized DNA also expressed p19Arf(Figure 1B).
Similarly, at P12 when the number of mitotically cycling progenitors
exceeds those of more differentiated germ cells, p19Arfwas co-
expressed with cyclin D1, a G1 phase marker of proliferating
spermatogonia  (Figure 1C), and strikingly, was detected during
all stages of mitosis (Figure 1D, 1E). Therefore, in spermatogonia,
p19Arfis expressed throughout the cell division cycle without
interfering with proliferation.
Arf Deficiency Compromises Sperm Production, But Is
Compensated by Ink4a Inactivation
Total body weights of age-matched wild-type, Arf-null, Ink4a-null,
and Ink4a-Arf double-null mice are equivalent, but testis weights of
Arf-null animals were reduced relative to those of wild-type controls
(Figure 2A), and this was associated with a significant reduction in
numbers of mature sperm by the time Arf-null mice were two
months old (Figure 2B). Nonetheless, young Arf-null males remain
fertile, and despite the widespread use of independently derived Arf-
null strains by us and others, there is no suggestion that young fertile
males produce reduced litter sizes. Hence, defects in spermatogen-
esis were not previously appreciated.
Knock-in of a cDNA encoding Cre recombinase in place of the
first Arf exon creates a functionally null Arf allele that expresses Cre
in lieu of p19Arfunder the control of the Arf promoter. Crossing
ArfCre/+females to homozygous males containing Arf alleles
flanked by LoxP sites (‘‘floxed’’ alleles) specifically results in the
inactivation of Arf function in the testis of compound heterozygous
ArfCre/Floxmale offspring. Although penetrance of Cre expression
is not complete, more than 90% of spermatogonia in the
seminiferous tubules of P21 mice had no detectable anti-p19Arf
fluorescence signals . Overall, while p19Arfwas detected in the
testes of haplo-insufficient ArfCre/+mice, any residual levels of the
protein in ArfCre/Floxtestes were too low to be detected by
Figure 3), confirming significant Cre-mediated Arf deletion in this
setting. We therefore used this ‘‘targeted’’ deletion approach to
compare the Arf loss-of-function phenotypes of ArfCre/Floxmales
with those of Arf2/2males. Analysis of testis weights revealed no
differences between those of ArfCre/Floxmice and wild-type
controls (Figure 2C). However, the sperm counts of ArfCre/Flox
animals were reduced to levels approaching those of Arf2/2males
(Figure 2D). Notably, the Arf-Cre or Arf-Flox alleles alone had no
significant effects in limiting sperm production unless coexpressed
in compound heterozygotes. Therefore, tissue-restricted effects of
Arf inactivation independently recapitulated those seen in mice
that completely lack Arf function.
The intimately linked Arf and Ink4a genes, encoded in part
by overlapping reading frames within the Cdkn2a locus,
are induced by oncogenic stress, activating the p53 and Rb
tumor suppressors, respectively, to inhibit proliferation of
incipient cancer cells. As such, expression of the p19Arfand
p16Ink4aproteins is undetected in most normal mouse
tissues. However, p19Arfis physiologically expressed in
mitotically dividing spermatogonia, the progenitor cells
that differentiate to form meiotic spermatocytes in which
Arf expression is extinguished. We show that, instead of
provoking cell cycle arrest or death, Arf expression in
spermatogonia facilitates survival of their meiotic progeny,
ensuring production of normal numbers of mature sperm.
When Arf is ablated, meiotic defects ensue, along with
p53-dependent cell death of spermatocytes, indicating an
unexpected role of p53 in monitoring meiotic progression.
Surprisingly, it is the absence of p19Arfrather than its
induction that enforces p53 expression in this setting. Co-
inactivation of Ink4a compensates for Arf loss by fueling
proliferation of spermatogonial progenitors, but does not
correct meiotic defects triggered by Arf loss. Although the
Arf and Ink4a tumor suppressors are expected to restrain
cellular self-renewal, Arf plays an unexpected role in male
germ cells by facilitating their proper meiotic progression.
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Figure 1. Arf protein expression in mitotically dividing spermatogonia. Protein expression in sections of seminiferous tubules were
determined by immunofluorescence analysis. (A) p19Arf(green, left panel) is expressed in spermatogonia that intervene between Sox9-expressing
Sertoli cells (red, middle panel) in the seminiferous tubules of adult 4 month-old mice. The right panel shows merged images documenting no
overlap in expression of the two proteins. Unlabeled cells within the lumina of the tubules are visualized with DAPI. (B) After a 2 hour in vivo pulse of
bromodeoxyuridine (BrdU) in adult 5 month old mice, p19Arfexpression (green, left panel) was revealed in spermatogonia that had incorporated
BrdU (red, middle panel). The right panel shows merged images documenting co-expression of both markers in many spermatogonia at the tubular
periphery (yellow). (C) In seminiferous tubules from P12 mice, cells expressing p19Arf(green, left panel) co-express cyclin D1 (red, middle panel), a
protein expressed in actively cycling spermatogonia; a merged image is shown at the right. (D, E) Spermatogonia within the seminiferous tubules of
P15 mice express p19Arf(green) during mitosis. Examples of p19Arfexpression during metaphase (D) and telophase (E) are shown. DAPI (blue)
highlights the nuclei of intratubular germ cells and somatic Leydig cells that occupy the intertubular space. Scale bars: (A, D, E) 50 mm; (B, C) 100 mm.
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Hormone signaling networks are involved in the proper control
of spermatogenesis. Key regulatory gonadotrophins include
luteinizing hormone (LH) and follicle-stimulating hormone
(FSH) secreted by the anterior pituitary gland, and testosterone
produced by testicular interstitial Leydig cells. The considerable
day-to-day and even hour-to-hour variation over a 30-fold range
in plasma testosterone levels in age-matched mice of a single strain
precluded accurate measurements of strain-specific differences,
even in a relatively large sample size (Figure 4) . Importantly,
however, no discernable defects in pituitary or Leydig cell
development have been observed in Arf-null, Ink4a-null, or
doubly-deficient mice, and no significant differences were
observed in the ranges of serum FSH and LH among all genotypes
examined (Figure 4). These findings suggest that spermatogenesis
defects in Arf-deficient mice are not a secondary consequence of
Unlike Arf-null males, those lacking functional Ink4a instead
exhibit increased testis weights and produce higher numbers of
sperm than wild type mice (Figure 2A and 2B). Cdk4, the major
target of p16Ink4aprotein inhibition in the adult testis, is expressed
at maximal levels at the earlier stages of spermatogenesis, where
spermatogonia undergoing mitotic cell divisions predominate
[16,17], and Cdk4 inactivation compromises male fertility
[18,19]. We therefore quantified the in vivo incorporation of BrdU
in spermatogonia of young adult wild-type, Arf-null, Ink4a-null,
and Ink4a-Arf-null mice by counting stained cells that had entered
S phase during a two-hour pulse. The S phase fractions of wild-
type and Arf-null spermatogonia did not differ from each other
(Figure 5), implying that the failure of Arf-null mice to produce
normal numbers of sperm reflects a loss of meiotic cells or their
Figure 2. Decreased sperm production in Arf-null mice is compensated by loss of Ink4a or p53. (A, C) Testes were dissected from adult (2--
6 month old) mice of the indicated genotypes and weighed as pairs. (B, D) Caudal epididymides were collected from corresponding mice, and
recovered sperm were enumerated using a hemocytometer. Relative testes weights (A) and sperm counts (B) are reduced in Arf-null males but
increased in Ink4a-null mice. Ink4a-Arf double-null and p532/2; Arf2/2double null mice exhibit increased testes weights (A) and sperm counts (B).
While testes weights (C) are not significantly reduced in ArfCre/Floxmales, reduced sperm counts (D) mimic the Arf loss-of-function phenotype. [N=20–
32 mice (A, B) and 5 mice (C, D)]. Bars represent standard deviations from the mean. P values were determined using a Student’s t-test (*p,0.001,
**p,0.0001) and designate significant differences from the wild type genotype.
Figure 3. Loss of expression of p19Arfin ArfCre/Floxtestes.
Immunoblotting analysis was performed using an antibody against
p19Arf. Whole testis lysates were prepared from two month old mice of
the indicated genotypes. Immunoblotting with an antibody to actin
was used to control for equal protein loading per lane.
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immediate progeny rather than spermatogonia. In contrast, we
observed a significant two-fold increase (p,0.0001, Student’s t-
test) in the relative number of S phase spermatogonia from both
strains that lack Ink4a (Figure 5). Looking only at testis weights and
sperm counts in Ink4a-Arf double-null animals, Ink4a inactivation
appears to compensate for loss of Arf function (Figure 2A and 2B),
presumably by fueling the production of a greater number of
mitotic progenitors. Together, the consequences of these two
independent loss-of-function effects rebalance testis size and sperm
output in the doubly null strain. In this sense, these two ‘‘tumor
suppressor’’ genes play opposing physiologic roles in male germ
Arf Inactivation Leads to p53-Dependent Apoptosis in
Testes from two month-old Arf-null mice exhibited a significant
increase in the numbers of apoptotic (TUNEL-positive) cells when
compared to age-matched wild-type controls (Figure 6A and 6E).
The vast majority of apoptotic cells are spermatocytes as judged by
the topological relation of TUNEL-positive cells to the expression
of the meiosis-specific strand-exchange protein Dmc1, which is
expressed during early prophase-I (Figure 6A). Notably, however,
intratubular TUNEL-positive cells were not stained with antibod-
ies to Dmc1, implying that Arf-null cells die during a later stage of
germ cell development after Dmc1 expression is greatly dimin-
ished. To examine this issue further, we conducted TUNEL
staining of meiotic chromosome spreads. Characteristic stages of
prophase during meiosis-I can be marked by staining chromo-
somes with antibodies to synaptonemal complex proteins, such as
the axial element component SYCP3 , and by several ancillary
criteria (see Materials and Methods). Unlike pachytene cells from
wild-type mice, those from the Arf-null strain exhibited consider-
able TUNEL staining (Figure 6C) with a concomitant reduction in
the fraction of Arf-null diplotene spermatocytes (Figure 6F) that
correlated with decreased sperm production (Figure 2B). Inacti-
vation of Ink4a alone did not trigger spermatocyte apoptosis nor
limit apoptosis in the Arf-null background (Figure 6E) reinforcing
the conclusion that the two closely linked genes play fundamen-
tally different roles within the male germline.
Arf2/2; p532/2doubly-deficient males are even more suscep-
tible to spontaneous tumor development than mice lacking either
Arf or p53 alone . However, the young tumor-free males
produce sufficient viable sperm to remain fertile. Inactivation of
p53 restored testis weights and sperm production in Arf-null males
(Figure 2A and 2B), prevented the apoptotic elimination of Arf-null
germ cells (Figure 6D and 6E), and restored the number of
Figure 4. Hormone levels in mice of different genotypes. Blood
was collected in the afternoon from 15 two to four month-old male
mice of each indicated genotype, and sera were analyzed for hormone
content. P values calculated using a Student’s t-test indicated no
significant differences between samples taken from mice of different
genotypes. Wide day-to-day and even hour-to-hour fluctuations in
plasma testosterone levels yield largely unpredictable changes of as
much as 30-fold between individual samples, as determined by
radioimmunoassay . Error bars indicate standard deviations from
Figure 5. Increased frequency of BrdU-incorporating sper-
matogonia in Ink4a-null mice. Quantification of BrdU-positive
spermatogonia in five month-old wild-type, Arf-null, Ink4a-null, and
doubly Ink4a and Arf-null mice was determined two hours after
intraperitoneal BrdU administration. BrdU-labeled cells were scored in
100 tubules in testis sections from seven different mice. Error bars
indicate standard deviations from the mean. ** p,0.0001 vs wild-type
by Student’s t-test.
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diplotene spermatocytes (Figure 6F). Accordingly, higher levels of
p53 were detected in whole testis lysates from Arf-null mice as
compared to those in wild-type mice (Figure 7). Thus, in direct
contrast to the role of p19Arfin triggering a p53 response following
abnormal hyperproliferative stress in somatic cells, it is instead the
absence of Arf expression in spermatogonial progenitors that
impairs the fidelity of meiotic progression and ultimately leads to
p53-dependent elimination of Arf-null primary spermatocytes.
Inappropriate c-H2AX Accumulation in Arf-Null Germ
Histone H2AX is phosphorylated at serine-139 in response to
DNA strand breaks caused by ionizing radiation , UV
irradiation , replication stress [23,24], failure of nucleotide
excision repair , and at the leptotene stage of meiosis prior to
synaptonemal complex formation . H2AX phosphorylation
also occurs in spermatocytes in a DNA damage-independent
manner during formation of the sex body, a heterochromatic sub-
nuclear domain encompassing the nonhomologous parts of the X
and Y chromosomes . Remarkably, staining of testis sections
and immunoblotting of whole testis lysates revealed a profound
increase in global c-H2AX levels when Arf was inactivated
(Figure 8A and 8B). Again, inactivation of Ink4a neither
recapitulated nor ameliorated this Arf-null defect (Figure 8A and
8B). Germ cells at the periphery of Arf-null seminiferous tubules
exhibited the greatest increase in c-H2AX staining, suggesting that
more immature cells were the ones most affected (Figure 8A).
Microscopic quantification revealed that the number of c-H2AX-
positive spermatogonia, as well as the number of c-H2AX foci per
cell, were increased ,2-fold when Arf was inactivated (Figure 9),
but the greatest increase in c-H2AX staining was observed in
primary Arf-null spermatocytes (Figure 8A and 8B; see below). The
increased c-H2AX in meiotic cells is especially striking because
these cells do not normally express p19Arfwhen the gene is
present (Figure 1).
Figure 6. Arf deficiency leads to increased apoptosis of primary spermatocytes. (A) During spermatogenesis, germ cells move
intraluminally as they differentiate, initially yielding primary meiotic spermatocytes (immunostained for Dmc1, green) that have detached from the
basement membrane (represented by a dashed line). Dmc1-positive cells are visualized in a longitudinal section of a tubule from a two month-old
Arf-null mouse. Spermatocytes within the tubular lumen are TUNEL-positive (red). DAPI (blue) marks the nuclei of all developing germ cells within the
tubule. Neither DAPI-positive spermatogonia residing on the basement membrane nor more mature intraluminal cells express Dmc1 or are TUNEL-
positive. (B–D) Surface spread spermatocytes from three month old wild-type (B), Arf2/2(C), and p532/2; Arf2/2(D) mice were immunostained for
SYCP3 (green) and assayed for TUNEL (red). Although pachytene and diplotene spermatocytes from Arf2/2mice were TUNEL-positive, few such cells
were identified in spreads from age-matched wildtype or p532/2; Arf2/2mice. (E) TUNEL assays were performed on testis sections from 2–3 month
old mice of the indicated genotypes. The total numbers of TUNEL-positive cells were enumerated in 50 tubules within three different sections taken
from 3 different mice of each genotype. (F) One hundred surface spread spermatocytes (n=3 mice) were categorized into the four phases of
prophase I based on SYCP3 and c-H2AX immunostaining patterns, as described in Materials and Methods. Errors bars represent standard deviations
from the mean. ** p,0.0001 and *p,0.003 vs wild-type levels by Student’s t-test.
Figure 7. Levels of p53 detected in wild-type and Arf-null testis.
Immunoblotting analysis was performed using whole testis lysates from
four representative 3–4 month-old mice of each genotype. Actin was
used as a loading control.
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Meiotic double-strand DNA breaks are induced by the Spo11
transesterase and its accessory factors, which are loaded onto
chromatin during the final pre-meiotic S-phase . Autosomal c-
H2AX staining is normally observed during the leptotene and
zygotene phases of meiosis-I, which are the early stages at which
chromatids undergo DNA scission as a prelude to homologous
recombination. In contrast, c-H2AX foci are not normally
detected by early pachytene (except in the sex body) once
homologous synapsis is complete (Figure 8C, top panels) .
Chromosome spreads from meiotic primary spermatocytes from
Arf-null males revealed that 150 of 382 individually enumerated
pachytene cells (39%) exhibited persistent autosomal c-H2AX foci
in addition to normal sex body staining, whereas very few such
cells (6.7%) were detected at the diplotene stage (Figure 8C,
bottom panels). It could be that disappearance of c-H2AX is
delayed until diplonema, or that cells with aberrantly elevated c-
H2AX are preferentially eliminated. The latter interpretation is
supported by the prophase I apoptosis and depletion of diplotene
cells observed in Arf-null mice (Figure 6). Therefore, in the absence
of Arf, c-H2AX accumulates to higher levels than normal starting
in the least mature spermatogonia, continuing into meiotic
prophase I, and persisting past the time when it would normally
disappear from autosomes. Although inactivation of p53 suppress-
es the increased apoptosis of Arf-null spermatocytes, c-H2AX
persists in cells lacking both of these genes (Figure 8A, lower right
panel). In meiotic chromosome spreads from p53; Arf double-null
Figure 8. Arf deficiency provokes elevated and persistent c-H2AX foci. (A) Testis sections from 3 month-old mice of the indicated genotypes
were immunostained for c-H2AX, as visualized here at low magnification to demonstrate overall relative intensities of staining. The brightest c-H2AX-
positive cells are primary spermatocytes. (B) Immunoblotting analysis of whole testis lysates from 3 month-old mice showing an accumulation of c-
H2AX in Arf-deficient testis. (C) Surface spread spermatocytes from three month old mice were immunostained for SYCP3 (red), c-H2AX (green), and
with DAPI to visualize nuclei (blue). Representative spreads from each stage of prophase I are shown for wild type (top) and Arf-null (bottom) strains,
demonstrating persistence of autosome-associated c-H2AX in pachytene spermatocytes from Arf-deficient mice. Staining of sex bodies persists
throughout prophase in both genotypes. Images were captured with the same exposure times using a Zeiss Axioscope fluorescence microscope (A)
or Intelligent Imaging Innovations Marianas spinning disc confocal microscope (C). Scale bars: (A) 100 mm.
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mice, 20.5% (34 of 166) of diplotene spermatocytes display
persistant autosomal c-H2AX immunostaining as compared to
6.7% (5 of 74) of singly Arf-null and less than 1% (1 of 149) of wild-
type diplotene spermatocytes. These data underscore the fact that
the accumulation of c-H2AX in Arf-null spermatocytes is p53-
independent, whereas the elimination of defective spermatocytes
that retain c-H2AX inappropriately is p53-dependent.
Additional Meiotic Prophase Defects in Arf-Null Primary
DNA double-strand breaks induced early in prophase I by
Spo11 serve as substrates for the strand exchange proteins Rad51
and meiosis-specific Dmc1, which are required for double strand
break repair during homologous recombination. Foci of staining
using antibodies to Dmc1 (Figure 10A) and Rad51 (Figure 10B)
were readily observed in zygotene spermatocytes from wild-type
mice (left panels) but were fewer and less prominent in their Arf-
null counterparts (right panels). The number and average
fluorescence intensities of foci in 100 zygotene cells of each
genotype were determined using commercial imaging software. In
wild-type zygotene spermatocytes, the frequency of Dmc1 and
Rad51 foci peaked at 100–125 per cell (Figure 10C and 10D,
respectively, blue bars) and exhibited a broad distribution of
relative intensities over a ,10-fold range (Figure 10E and 10F,
blue bars). In contrast, both the number and intensities of Dmc1/
Rad51 foci were significantly reduced in Arf-null cells (average
number of foci 6 S.D.: 103646 Dmc1 foci in wild-type vs. 44634
in Arf2/2; 108645 Rad51 foci in wild-type vs. 45633 in Arf2/2;
average relative intensities 6 S.D.: 1023964959 Dmc1 foci in
wild-type vs. 661163005 in Arf2/2; 1098065783 Rad51 foci in
wild-type vs. 563763340 in Arf2/2N=100, p,0.0001, Student’s
t-test; Figure 10C–10F). An accumulation of Arf-null spermato-
cytes in zygonema (Figure 6F) suggests that there may be a delay at
this stage before progression to pachytene.
Pachytene cells are normally characterized by well developed
synaptonemal complexes that stretch the length of autosome axes
(Figure 11A). However, 34% of Arf-null cells exhibited defects in
synapsis (quantified in Figure 11D), including forked terminal
ofSYCP3 at telomeres
structures and interstitial bubbles on autosomes (Figure 11B) and
complete asynapsis of sex chromosomes (arrow, Figure 11C). In
addition, interrupted regions of SYCP3 staining (denoted by
arrowheads in Figure 11C) were more frequently observed in
meiotic chromosome spreads from Arf-null cells versus those in
wild-type cells (191 versus 53 such segments, respectively, in 300
pachytene cells of each genotype). Because synapsis was complete
in the majority of Arf-null pachytene cells, we could not distinguish
whether the observed defects arose from regions in which
synaptonemal complexes did not form at all, or where complexes
had formed but subsequently disassembled. Taken together, Arf-
deficiency results in a series of abnormalities during prophase I
that include reduced loading of the Rad51 and Dmc1 recombi-
nases, defects in synapsis, elevated and persistent c-H2AX
expression, and p53-dependent apoptosis, ultimately associated
with diminished production of mature sperm.
With few exceptions, the Arf tumor suppressor is not expressed
in normal tissues of healthy mice but is induced by abnormally
sustained and elevated thresholds of proliferative signals, activating
a p53 response that opposes the deleterious effects of oncogene
activation. Notably, p53 responds to a much wider range of Arf-
independent signal transduction cascades triggered by many other
forms of cellular stress, including acute DNA damage, to which the
Arf promoter does not respond . By converging on p53, these
different signaling pathways inhibit cell cycle progression or trigger
apoptosis, acting to suppress tumor formation.
We now document a physiological role of Arf in mouse male
germ cell development that is distinct from its tumor suppressive
functions in key respects. First, Arf is expressed in spermatogonia,
but not in the primary spermatocytes that arise from them.
Expression of p19Arfneither arrests spermatogonial mitotic
progression nor triggers their p53-dependent apoptosis. However,
the absence of Arf expression in spermatogonia leads to p53-
dependent apoptosis of spermatocytes before they exit meiosis-I.
The defect in spermatogenesis is germ cell autonomous and results
in a significant reduction in sperm counts by the time Arf-null mice
are two months old, although residual sperm production maintains
fertility in young males. Thus, expression of Arf in mitotic
progenitor cells enhances the survival of their meiotic progeny in
which Arf expression is normally extinguished. These features
indicate that Arf expression initiates a salutary, feed-forward
program that facilitates meiotic progression. Indeed, although Arf
and Ink4a are widely viewed to convey tumor suppressive functions
that coordinate the activities of the p53 and Rb signaling
‘‘pathways,’’ inactivation of Arf and Ink4a in the testes leads to
opposing outcomes. Disruption of Ink4a increases the mitotic
activity of spermatogonial progenitors to enhance sperm output
and, in this respect, compensates for Arf loss of function without
eliminating the cellular defects that arise in the Arf-null setting. In
short, loss of Ink4a increases the spermatogonial pool size, but
without Arf expression, spermatocytes undergo increased apopto-
sis, returning the number of mature sperm to normal levels.
Homologous recombination during meiosis exchanges genetic
information between maternally and paternally derived chromo-
somes and also guides proper segregation of chromosome pairs to
maintain correct chromosome numbers in gametes . During
meiosis, in contrast to mitotically diving cells, homologous
chromosomes are favored over sister chromatids as templates for
recombinational DNA repair. Double-strand DNA breaks are
formed by the topoisomerase-II-related transesterase Spo11. This
process activates the Atm kinase and leads to phosphorylation of
Figure 9. Arf deficiency leads to increased c-H2AX foci in
spermatogonia. The number of A and intermediate-type spermato-
gonia with c-H2AX foci in 150 seminiferous tubules and the total
number of foci contained within these cells were quantified in wild-type
and Arf-null mice at P17. Enumeration of c-H2AX foci within 150 tubules
in each of three different sections from each genotype was performed
at high magnification for foci limited to spermatogonia distinguished
by morphology and position along the basement membrane. P values
were determined using a Student’s t- test; **p value,0.0001 vs wild-
type. Error bars indicate standard deviations from the mean.
Arf Tumor Suppressor Regulates Spermatogenesis
PLoS Genetics | www.plosgenetics.org8July 2011 | Volume 7 | Issue 7 | e1002157
the H2AX histone variant near sites of strand breakage during
early prophase I. Binding of the RecA family strand exchange
proteins, Rad51 and meiosis-specific Dmc1, to Spo11-induced
DNA ends generates filaments that search for and invade
homologous duplex DNA molecules, leading to pairing of
homologous chromosomes. Loading of Rad51 and Dmc1 is
normally reversed by early pachytene when chromosomes are fully
synapsed, after which c-H2AX foci are no longer detected.
In the Arf-null setting, a modest but significant increase in c-
H2AX staining was first detected in the least mature spermato-
gonia, and primary spermatocytes displayed accentuated signals
that persisted inappropriately into the pachytene stage. Arf-null
cells also formed fewer Dmc1/Rad51 foci at zygotene and
exhibited focal regions of asynapsis at pachytene. Aberrant Arf-
null spermatocytes underwent apoptosis at pachytene, resulting in
the emergence of fewer diplotene cells and a significant reduction
in sperm output. Importantly, Arf2/2; p532/2
pachytene cells also exhibited persistent c-H2AX staining, but
these cells escaped elimination. Thus, apoptosis was p53-
dependent, but aberrant c-H2AX accumulation was not.
Although the underlying mechanisms remain unknown, we
consider here two plausible interpretations of this apoptotic arrest.
Figure 10. Diminished Dmc1/Rad51 focus formation in Arf-deficient spermatocytes. (A, B) Surface spread spermatocytes from three month
old WT (left panels) and Arf-null (right panels) mice were immunostained for SCP3 (A, B; green) and Dmc1 (A; red) or Rad51 (B; red). Images were
captured with the same exposure time using a Marianas spinning disc confocal microscope. (C, D) Histograms showing the distribution of the
number of Dmc1 (C) and Rad51 (D) foci found in wild-type (blue bars) and Arf-null (red bars) spermatocytes. Foci were enumerated from one hundred
zygotene spermatocytes immunostained for SYCP3 and either Dmc1 (C) or Rad51 (D) using Slidebook 5.0 SDC software. Actual values of foci per cell
are plotted within bin ranges to display the distribution of frequencies. (E, F) Histograms showing the distribution of average intensities of Dmc1 (C)
and Rad51 (D) foci found in wild-type (blue bars) and Arf-null (red bars) spermatocytes analyzed in panels C and D.
Arf Tumor Suppressor Regulates Spermatogenesis
PLoS Genetics | www.plosgenetics.org9 July 2011 | Volume 7 | Issue 7 | e1002157
First, it may be that reduced Rad51/Dmc1 focus formation and
persistent c-H2AX staining in Arf-null male germ cells connote a
defect in DNA repair that then activates p53 through Arf-
independent but Atm/Atr-dependent signaling pathways. In this
scenario, Spo11-induced DSBs would form at normal levels but
Rad51/Dmc1 loading would be impaired such that some DNA
damage would persist into pachytene. This might conceivably
involve the p53-independent ability of p19Arfto promote the
sumoylation of numerous target proteins by inhibiting the
SUMO2/3 protease Senp3 [29–31]. SUMO2/3 accumulates at
sites of DNA damage in mammalian cells [32,33], and various
aspects of DNA repair are regulated by the SUMO conjugation
pathway . There is fragmentary evidence that absence of
p19Arfcompromises nucleotide excision repair in cultured cells
[35,36] raising the possibility that Arf may play an as yet undefined
role in promoting homologous recombination. All meiotic mutants
that cannot properly synapse homologous chromosomes arrest
during pachytene , and accompanying defects in sex body
formation and failure to properly silence transcription of the sex
chromosomes during prophase is itself sufficient to eliminate
pachytene cells [27,38]. However, spermatocytes can also undergo
apoptosis in direct response to unrepaired Spo11-induced breaks
even if sex body formation is normal [27,39]. Where tested,
spermatocyte apoptosis in meiotic mutants with chromosome
synapsis errors has been found to be p53-independent [40–42].
Moreover, Spo11-dependent activated phospho-p53 can be
transiently detected from leptonema and zygonema in wild-type
male mice, and in Drosophila, p53 activity is prolonged in cells
defective for meiotic repair . Thus, it remains a formal
possibility that meiotic recombination defects can trigger p53-
A second, alternative interpretation rests on the idea that the
earlier and less profound accumulation of c-H2AX in Arf-null
spermatogonia might be a symptom of an underlying defect affecting
chromatin structure or Atm/Atr signaling. The appearance of c-
H2AX reflects chromatin modifications that flank sites of DNA
damage rather than strand breaks themselves, so the kinetics of c-
H2AX formation and dissolution do not necessarily coincide with the
appearance and repair of DNA damage [44,45]. Moreover, aberrant
Atm/Atr signalingis itself sufficientto activate p53,whethertriggered
by DNA breaks or not .Thus, it may be that Arf deficiency causes
inappropriate Atm/Atr signaling that provokes p53-dependent
apoptosis in a DNA damage-independent manner. In this view, the
observed meiotic prophase defects in Arf-null spermatocytes may
possibly be a separate downstream consequence of this earlier
anomaly, and may not be the cause of apoptosis. Regardless of which
interpretation is correct, it is important to note that our findings
provide strong evidence that p53-dependent monitoring promotes
proper meiotic maturation, in addition to the previously documented
p53-independent pathway(s). Whatever the underlying mechanisms,
the role of Arf in male germ cell development contrasts with the
general paradigm of p19Arfacting as an activator of p53. Instead, it is
the absence of Arf in spermatogonia that consequently leads to p53-
dependent apoptosis of spermatocytes.
Materials and Methods
No human or non-human primates were studied. All animal
work with mice was performed under established guidelines and
supervision by the St. Jude Children’s Research Hospital’s
Institutional Animal Care and Use Committee (IACUC), as
required by the United States Animal Welfare Act and NIH policy
to ensure proper care and use of laboratory animals for research.
Experiments were undertaken in an accredited facility of the
Association for Assessment of Laboratory Animal Care under the
supervision of trained veterinary personnel and in strict compli-
ance with Howard Hughes Medical Institute, St. Jude Children’s
Research Hospital, and NIH institutional guidelines. The latter
include detailed protocol submission and review of all animal care,
monitoring, and experimental procedures prior to initiation of any
experiments. Ongoing protocols for animal research not necessi-
tating interim amendments are minimally subjected to annual
review by the IACUC. All persons involved in the use of animals
have read and understand all implications of pertinent protocols,
have received training in the execution of relevant animal-related
procedures prior to participation in the protocol, and have
participated in educational or training programs deemed neces-
sary by the IACUC or the Animal Resources Center personnel.
Studies reported herein did not unnecessarily duplicate previous
research, and were undertaken only because suitable non-animal
models were unavailable. The number of animals used was
consistent with good statistical design. Anesthesia, analgesia and
tranquilization were used to relieve pain and distress in accordance
with the IACUC recommendations.
Figure 11. Arf-null pachytene spermatocytes exhibit synaptic
defects. (A–C) Staining of surface spread spermatocytes for SYCP3
marks the lateral elements of bivalents. (A) In wildtype pachytene
spermatocytes, fully synapsed autosomal bivalents are observed, as
judged by continuous SYCP3 staining along the axes. (B–C) Represen-
tative synaptic defects in Arf-null pachytene spermatocytes include (B)
unsynapsed ends (arrow) and interstitial asynaptic ‘‘bubbles’’ (arrow
heads), and (C) asynapsis of the X and Y chromosomes (arrow).
Synapsed X and Y chromosomes are shown in (A, B). Synaptonemal
complexes with segmental disruption of SYCP3 staining were more
frequently observed (p=0.051) in Arf-null cells (C, arrowheads) versus
their wild-type counterparts (191 versus 53 such segments in 300
pachytene chromosomal spreads from each genotype). (D) Quantifica-
tion of synaptic defects observed in wild-type and Arf-null pachytene
spermatocytes. ‘‘Wide open’’ ends are illustrated in (B); less extensive
asynapsis at telomeres was categorized as ‘‘slight’’ open ends. 100
pachytene spermatocytes from each of three mice of different
genotypes yielded highly significant differences in overall defects
(p=0.0017 by Student’s t-test). Error bars indicate standard deviation
from the mean.
Arf Tumor Suppressor Regulates Spermatogenesis
PLoS Genetics | www.plosgenetics.org10 July 2011 | Volume 7 | Issue 7 | e1002157
Arf-null , Arf-GFP , Arf-Flox and Arf-Cre mice  were
generated in the Sherr laboratory. Mouse strains deficient for
Ink4a  and Ink4a-Arf  were generously provided by R.A.
DePinho (Dana Farber Cancer Center). All genetically engineered
mice were backcrossed nine or more times onto a C57Bl/6
background to create isogenic strains. C57Bl/6 mice deficient for
p53 were purchased from Jackson Laboratories (Stock Number
2101). ArfGFP/GFPmice were crossed to p53+/2mice, and
mice functionally null for both genes.
ArfCre/+females were interbred with ArfFlox/Floxmales to generate
Phenotypic Characterization of Mouse Testes and Sperm
Caudal epididymides were harvested before dissection of the
testes. For each male mouse, two cauda were minced into 1 ml of
Dulbecco’s modified Eagle’s medium (DMEM) containing 25 mM
HEPES buffer (pH 7.5) and 4 mg/ml bovine serum albumin and
incubated at 37uC for 20 minutes. Suspensions of sperm were
fixed at a 1:25 dilution in 10% formalin and counted on a
hemocytometer. All sperm counts were performed between 1:00–
3:00 PM. Dissected testes were weighed in pairs.
Immunofluorescence of Testes Sections
Mice were euthanized by CO2asphyxiation, and testes were
removed and fixed overnight at 4uC in 4% paraformaldehyde
followed by saturation in 30% sucrose at 4uC overnight. Tissues
were embedded in TBS Tissue Freezing Medium (Fisher
Scientific, Pittsburg PA), and sliced with a HM500M Cryostat
(Microm International, Walldorf, Germany) into 10 mm sections.
Fixed and frozen samples were sectioned and subjected to antigen
retrieval in 0.1 M Na citrate buffer, pH 6.0, followed by one hour
incubation at room temperature in a blocking solution of 10%
normal goat serum (NGS), 0.1% Triton-X 100 in phosphate-
buffered saline (PBS), and then by overnight incubation at 4uC in
primary antibodies diluted in 3% NGS, 0.1% Triton-X 100 in
PBS. Antibodies were directed to p19Arf
immunoglobulin 5C3-1 , Sox9 (Millipore AB5535, 1:1000),
BrdU (Santa Cruz sc32323, 1:100), cyclin D1 (Santa Cruz 72-
13G, 1:750), Dmc1 (Santa Cruz H-100, 1:750), c-H2AX (Cell
Signaling 2577, 1:200), and SUMO2/3 (Cell Signaling 18H8,
1:300). Slides were washed three times in PBS, and then incubated
for 1 hour at room temperature in 3% NGS, 0.1% Triton-X 100
in PBS containing the relevant secondary antibodies conjugated to
Ig-Alexa Fluor 555 or Ig-Alexa Fluor 488 (1:500 dilutions;
Invitrogen). Slides were washed three times in PBS and mounted
with Vectashield (Vector Labs) containing 49-6-diamidino-2-
phenylindol (DAPI). TUNEL assays were performed using an in
situ cell death detection kit (TMR red, Roche) following the
manufacturer’s protocol. Images of tissue sections were photo-
graphed using a Zeiss Axioscope fluorescence microscope and
assembled using Zeiss Axiovision software.
Analysis and Staging of Meiotic Spreads
Testes were decapsulated and minced in 5 ml of DMEM per
testis and transferred to a 15 ml Falcon tube. After further
dissociation of the tubules by pipeting up and down, large pieces
were allowed to settle to the bottom of the tube by gravity for
10 minutes on ice. One ml of the supernatant, containing a
suspension of spermatocytes, was transferred to a 1.5 ml Eppendorf
tube and centrifuged for five minutes at 58006g. The pellet was
resuspended in 40 ml of a 0.1 M sucrose solution, and 20 ml of
spermatocyte suspension was applied evenly to a slide containing a
thin layer of 1% paraformaldehyde (pH 9.2) containing 0.1%
Triton X-100. Slides were allowed to dry for two hours at room
temperature in a closed humidity chamber before rinsing in Photo-
flo (Kodak 1464510, diluted 1:250 in doubly distilled H2O) and air
dried at room temperature. For immunofluorescence, slides were
incubated in PTBG (0.2% bovine serum albumin, 0.2% gelatin,
0.05% Tween 20 in PBS) for 10 minutes with shaking. Primary
antibodies were diluted in PTBG, applied to the slide, and covered
with parafilm before incubation overnight at 4uC in a humidity
chamber. Antibodies were directed to SYCP3 (Santa Cruz G-3,
1:500) to mark the synaptonemal axial element , to c-H2AX
(Cell Signaling 2577, 1:500) to identify sex body formation and sites
of DNA damage, and to Rad51 (Calbiochem Ab-1, 1:500) and
Dmc1 (Santa Cruz H-100, 1:750) to demonstrate formation of
complexes required for DNA strand exchange during homologous
recombination. Slides were washed three times in PTBG at room
temperature for 3 minutes with shaking. Secondary antibodies, also
diluted in PTBG, were applied to slides which were covered with
parafilm and incubated at 37uC for one hour in a humidity
chamber. Slides were washed three times in PTBG for 3 minute
intervals in the dark with shaking and mounted with Vectashield
(Vector Labs) containing DAPI. Surface spread spermatocytes were
visualized by a Marianas spinning-disc confocal microscope, and
images were assembled and analyzed using Slidebook 5.0 SDC
software (Intelligent Imaging Innovations, Denver CO). Meiotic
spreads from three adult mice (age three months) were analyzed.
One hundred spermatocytes were scored each from mouse.
Distinct staining patterns allow for classification of each stage of
meiotic prophase [51,52]. Leptotene cells were categorized by
short stretches of axial elements accompanied by intense c-H2AX
staining throughout the nucleus and the absence of a distinct sex
body. Zygotene cells also display intense c-H2AX staining
throughout the nucleus and lack a sex body, but can be
distinguished by longer stretches of SYCP3 staining, some of
which are synapsed. Pachytene cells have fully formed and
synapsed axes that appear as thick, continuous SYCP3-stained
threads, while displaying intense c-H2AX staining only in the sex
body. Dmc1 and Rad51 foci are normally present at leptotene and
zygotene, and largely disappear by pachytene. Diplotene cells have
c-H2AX localized only to the sex body, but fully formed axes are
desynapsing and chiasmata are visible.
As previously described , detergent lysates were prepared,
and protein concentration was quantified by bicinchoninic acid
assay (Pierce). Samples (25–75 mg protein per lane) were
electrophoretically separated on 4% to 12% Bis-Tris NuPAGE
gels (Invitrogen), transferred to polyvinylidene fluoride membranes
(Millipore), and detected using antibodies to c-H2AX (Cell
Signaling S139, 1:500), p19Arf(5C3-1; Bertwistle et al. 2004b),
p53 (Cell Signaling 1C12, 1:500), and actin (Santa Cruz C-11,
1:500) to control for protein loading.
We thank Adam Gromley and Frederique Zindy for Arf-Cre and Arf-Flox
mice; Ronald A. DePinho for Ink4a-Arf-null and Ink4a-null mice; Jennifer
Peters in the St. Jude Tissue Imaging Facility for instruction in confocal
microscopy and assistance in capturing and analyzing images; Shelly
Wilkerson, Sarah Gayoso, and Jennifer Craig for assistance with
genotyping mice; Debbie Yons for help with animal care; and members
of the Sherr/Roussel laboratory for helpful criticisms and suggestions
during the course of this work.
Arf Tumor Suppressor Regulates Spermatogenesis
PLoS Genetics | www.plosgenetics.org11 July 2011 | Volume 7 | Issue 7 | e1002157
Conceived and designed the experiments: MLC IR MJ SK CJS.
Performed the experiments: MLC. Analyzed the data: MLC CJS.
Contributed reagents/materials/analysis tools: CJS SK MJ. Wrote the
paper: MLC CJS SK MJ.
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PLoS Genetics | www.plosgenetics.org12 July 2011 | Volume 7 | Issue 7 | e1002157