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
PLoS Genetics | www.plosgenetics.org1July 2011 | Volume 7 | Issue 7 | e1002157
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.
data illustrated in
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.
Arf Tumor Suppressor Regulates Spermatogenesis
PLoS Genetics | www.plosgenetics.org2July 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.org 11July 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.
1. Lowe SW, Sherr CJ (2003) Tumor suppression by Ink4a-Arf: progress and
puzzles. Curr Opin Genet Dev 13: 77–83.
2. Quelle DE, Zindy F, Ashmun RA, Sherr CJ (1995) Alternative reading frames of
the INK4a tumor suppressor gene encode two unrelated proteins capable of
inducing cell cycle arrest. Cell 83: 993–1000.
3. Pardal R, Molofsky AV, He S, Morrison SJ (2005) Stem cell self-renewal and
cancer cell proliferation are regulated by common networks that balance the
activation of proto-oncogenes and tumor suppressors. Cold Spr Harb Symp
Quant Biol 70: 177–185.
4. Gil J, Peters G (2006) Regulation of the INK4b-ARF-INK4a tumour suppressor
locus: all for one or one for all. Nat Rev Mol Cell Biol 7: 667–677.
5. Weber JD, Jeffers JR, Rehg JE, Randle DH, Lozano G, et al. (2000) p53-
independent functions of the p19ARFtumor suppressor. Genes Dev 14:
6. Sherr CJ (2006) Divorcing ARF and p53: an unsettled case. Nat Rev Cancer 6:
7. Zindy F, Williams RT, Baudino TA, Rehg JE, Skapek SX, et al. (2003) Arf
tumor suppressor promoter monitors latent oncogenic signals in vivo. Proc Natl
Acad Sci USA 100: 15930–15935.
8. McKeller RN, Fowler JL, Cunningham JJ, Warner N, Smeyne RJ, et al. (2002)
The Arf tumor suppressor gene promotes hyaloid vascular regression during
mouse eye development. Proc Natl Acad Sci USA 99: 3848–3853.
9. Gromley A, Churchman ML, Zindy F, Sherr CJ (2009) Transient expression of
the Arf tumor suppressor during male germ cell and eye development in Arf-Cre
reporter mice. Proc Natl Acad Sci USA 106: 6285–6290.
10. Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED (1990) Histological and
histopathological evaluation of the testis Cache River Press. Clearwater, FL.
11. de Rooij DG (2001) Proliferation and differentiation of spermatogonial stem
cells. Reproduction 121: 347–354.
12. Culty M (2009) Gonocytes, the forgotten cells of the germ cell lineage. Birth
Defects Res (Part C) 87: 1–26.
13. Cole F, Keeney S, Jasin M (2010) Evolutionary conservation of meiotic DSB
proteins: more than just Spo11. Genes Dev 24: 1201–1207.
14. Beumer TL, Roepers-Gajadien HL, Gademan IS, Kal HB, de Rooij DG (2000)
Involvement of the D-type cyclins in germ cell proliferation and differentiation in
the mouse. Biol Reprod 63: 1893–1898.
15. Bartke A, Steele RE, Musto N, Caldwell BV (1973) Fluctuations in plasma
testosterone levels in adult male rats and mice. Endocrinology 92: 1223–1228.
16. Zindy F, den Besten W, Chen B, Rehg JE, Latres E, et al. (2001) Control of
spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c)
and p19(Ink4d). Mol Cell Biol 21: 3244–3255.
17. Bartkova J, Lukas C, Sorenson CS, Meyts ER, Skakkebaek NE, et al. (2003)
Deregulation of the RB pathway in human testicular germ cell tumors. J Pathol
18. Rane SG, Dubus P, Mettus RV, Galbreath EJ, Boden G, et al. (1999) Loss of
Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in
b-islet cell hyperplasia. Nature Genet 22: 44–52.
19. Tsutsui T, Hesabi B, Moons DS, Pandolfi PP, Hansel KS, et al. (1999) Targeted
disruption of CDK4 delays cell cycle entry with enhanced p27Kip1activity. Mol
Cell Biol 19: 7011–7019.
20. Dobson MJ, Pearlman RE, Karaiskakis A, Spyropoulos B, Moens PB (1994)
Synaptonemal complex proteins: occurrence, epitope mapping, and chromo-
some disjunction. J Cell Sci 107: 2749–2760.
21. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-
stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol
Chem 273: 5858–5868.
22. Limoli CL, Giedzinski E, Bonner WM, Cleaver JE (2002) UV-induced
replication arrest in the xeroderma pigmentosum variant leads to DNA
double-strand breaks, c-H2AX formation, and Mre11 relocalization. Proc Natl
Acad Sci USA 99: 233–238.
23. Ward IM, Chen J (2001) Histone H2AX is phosphorylated in an ATR-
dependent manner in response to replicational stress. J Biol Chem 276:
24. Furuta T, Takemura H, Liao ZY, Aune GJ, Redon C, et al. (2003)
Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1
in response to replication-dependent double-strand breaks induced by
mammalian DNA topoisomerase I. J Biol Chem 278: 20303–20312.
25. Marti TM, Hefner E, Feeney L, Natale V, Cleaver JE (2006) H2AX
phosphorylation within the G1phase after UV irradiation depends on nucleotide
excision repair and not DNA double-strand breaks. Proc Natl Acad Sci USA
26. Mahadevaiah SK, Turner JM, Baudet FREP, de Boer P, Blanco-Rodriguez J,
et al. (2001) Recombinational DNA double-strand breaks in mice precedes
synapsis. Nat Genet 27: 271–2716.
27. Burgoyne PS, Mahadevaiah SK, Turner JM (2009) The consequences of
asynapsis for mammalian meiosis. Nat Rev Genet 10: 207–216.
28. Inagaki A, Schoenmakers S, Baarends WM (2010) DNA double strand break
repair, chromosome synapsis and transcriptional silencing in meiosis. Epigenet-
ics 5: 255–266.
29. Haindl M, Harasim T, Eick D, Muller S (2008) The nucleolar SUMO-specific
protease SENP3 reverses SUMO modification of nucleophosmin and is required
for rRNA processing. EMBO Rep 9: 273–279.
30. Kuo M-L, den Besten W, Thomas MC, Sherr CJ (2008) Arf-induced turnover of
the nucleolar nucleophosmin-associated SUMO-2/3 protease Senp3. Cell Cycle
31. Nishida T, Yamada Y (2008) SMT3IP1, a nucleolar SUMO-specific protease,
deconjugates SUMO-2 from nucleolar and cytoplasmic nucleophosmin.
Biochem Biophys Res Commun 374: 382–387.
32. Morris JR, Boutell C, Keppler M, Densham R, Weekes D, et al. (2009) The
SUMO modification pathway is involved in the BRCA1 response to genotoxic
stress. Nature 462: 886–890.
33. Galanty Y, Belotserkovskaya R, Coates J, Polo S, Miller KM, et al. (2009)
Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA
double-strand breaks. Nature 462: 935–939.
34. Bergink S, Jentsch S (2009) Principles of ubiquitin and SUMO modifications in
DNA repair. Nature 458: 461–467.
35. Sarkar-Agarwal P, Vergilis I, Sharpless NE, DePinho RA, Runger TM (2004)
Impaired processing of DNA photoproducts and untraviolet hypermutability
with loss of p16INK4aor p19ARF. J Natl Cancer Inst 96: 1790–1793.
36. Dominguez-Brauer C, Chen Y-J, Brauer PM, Pimkina J, Raychaudhuri P (2009)
ARF stimulates XPC to trigger nucleotide excision repair by regulating the
repressor complex of E2F4. EMBO Rep 10: 1036–1042.
37. de Rooij DG, de Boer P (2003) Specific arrest in spermatogenesis in genetically
modified and mutant mice. Cytogenet Genome Res 103: 267–276.
38. Royo H, Polikiewicz G, Mahadevaiah SK, Prosser H, Mitchell M, et al. (2010)
Evidence that meiotic sex chromosome inactivation is essential for male fertility.
Curr Biol 20: R1022–1024.
39. Roig I, Dowdle JA, Toth A, de Rooij DG, Jasin M, et al. (2010) Mouse TRIP13/
PCH2 is required for recombination and normal higher-order chromosome
structure during meiosis. PLoS Genet 6: e1001062.
40. Odorioso T, Rodriguez TA, Evans EP, Clarke AR, Burgoyne PS (1998) The
meiotic checkpoint monitoring synapsis eliminates spermatocytes via p53-
independent apoptosis. Nat Genet 18: 257–261.
41. Yuan L, Liu JG, Hoja MR, Lightfoot DA, Hoog C (2001) The checkpoint
monitoring chromosomal pairing in male meiotic cells is p53-independent. Cell
Death Differ 8: 316–317.
42. Ashley T, Westphal C, Plug-de Maggio A, de Rooij DG (2004) The mammalian
mid-pachytene checkpoint: meiotic arrest in spermatocytes with a mutation in
Atm alone or in combination with a Trp53 (p53) or Cdkn1a (p21/cip1)
mutation. Cytogenet Genome Res 107: 256–262.
43. Lu WJ, Chapo J, Roig I, Abrams JM (2010) Meiotic recombination provokes
functional activation of the p53 regulatory network. Science 328: 1278–1281.
44. Kastan MB, Bartek J (2004) Cell-cycle checkpoints and cancer. Nature 432:
45. Kinner A, Wu W, Staudt C, Iliakis G (2008) Gamma-H2AX in recognition and
signaling of DNA double-strand breaks in the context of chromatin. Nucl Acids
Res 36: 5678–5694.
46. Bakkenist CJ, Kastan MB (2004) Initiating cellular stress responses. Cell 118:
47. Kamijo T, Zindy F, Roussel MF, Quelle DE, Downing JR, et al. (1997) Tumor
suppression at the mouse INK4a locus mediated by the alternative reading frame
product p19ARF. Cell 91: 649–659.
48. Sharpless NE, Bardeesy N, Lee K-H, Carrasco D, Castrillon DH, et al. (2001)
Loss of p16Ink4awith retention of p19Arfpredisposes mice to tumorigenesis.
Nature 413: 86–91.
49. Serrano M, Lee H-W, Chin L, Cordon-Cardo C, Beach D, et al. (1996) Role of
the INK4a locus in tumor suppression and cell mortality. Cell 85: 27–37.
50. Bertwistle D, Zindy F, Sherr CJ, Roussel MF (2004) Monoclonal antibodies to
the mouse p19Arftumor suppressor protein. Hybridoma and Hybridomics 23:
51. Moens PB, Freire R, Tarsounas M, Spyropoulos B, Jackson SP (2000)
Expression and nuclear localization of BLM, a chromosome stability protein
mutated in Bloom’s syndrome, suggest a role in recombination during meiotic
prophase. J Cell Sci 113: 663–672.
52. Baudat F, Manova K, Yuen JP, Jasin M, Keeney S (2000) Chromosome synapsis
defects and sexually dimorphic meiotic progression in mice lacking Spo11. Mol
Cell 6: 989–998.
53. Zindy F, Eischen CM, Randle DH, Kamijo T, Cleveland JL, et al. (1998) Myc
signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and
immortalization. Genes Dev 12: 2424–2433.
Arf Tumor Suppressor Regulates Spermatogenesis
PLoS Genetics | www.plosgenetics.org12 July 2011 | Volume 7 | Issue 7 | e1002157