Molecular Cell, Vol.
6, 975–987, November, 2000, Copyright 2000 by Cell Press
The Mouse Spo11 Gene Is Required
for Meiotic Chromosome Synapsis
Peter J. Romanienko and R. Daniel Camerini-Otero*
Genetics and Biochemistry Branch
National Institute of Diabetes and Digestive
and Kidney Diseases
National Institutes of Health
Bethesda, Maryland 20892
Summary
The Spo11 protein initiates meiotic recombination by
generating DNA double-strand breaks (DSBs) and is
required for meiotic synapsis in S. cerevisiae. Surpris-
ingly, Spo11 homologs are dispensable for synapsis
in C. elegans and Drosophila yet required for meiotic
recombination. Disruption of mouse Spo11 results in
infertility. Spermatocytes arrest prior to pachytene
with little or no synapsis and undergo apoptosis. We
did not detect Rad51/Dmc1 foci in meiotic chromo-
some spreads, indicating DSBs are not formed. Cis-
platin-induced DSBs restored Rad51/Dmc1 foci and
promoted synapsis. Spo11 localizes to discrete foci
during leptotene and to homologously synapsed chro-
mosomes. Other mouse mutants that arrest during
meiotic prophase (Atm ⫺/⫺, Dmc1 ⫺/⫺, mei1, and Morc⫺/⫺)
showed altered Spo11 protein localization and expres-
sion. We speculate that there is an additional role for
Spo11, after it generates DSBs, in synapsis.
Introduction
Sexual reproduction and meiotic recombination are
common to most eukaryotes (Welch and Meselson,
2000). Meiosis is a specialized cell division that reduces
the normal chromosome complement by half in order
to generate haploid gametes. The proper segregation of
chromosomes into gametes generally requires meiotic
recombination. In most organisms, genetic exchange
occurs, and homologous chromosomes undergo synap-
sis and form a synaptonemal complex (SC) (Heyting,
1996). The SC is a proteinaceous structure composed
of lateral elements (LE) and the central element (CE).
The LEs are parallel structures that originate as the axial
elements (AE), one component of which is the protein
Scp3 (Dobson et al., 1994). The AEs are seen at leptotene
during meiotic prophase I and eventually form along
the entire length of each homolog. Synapsis can be
visualized when two AEs of homologous chromosomes
come together during zygotene (Zickler and Kleckner,
1998). The CE is formed when transverse filaments (TFs),
composed of the protein Scp1, bridge the axial elements
during zygotene and pachytene (Schmekel and Dane-
holt, 1998).
Whereas the initiation of meiotic recombination ap-
pears to be highly conserved, some eukaryotes differ
in the molecular requirements for synapsis (Walker and
* To whom correspondence should be addressed (e-mail: camerini@
ncifcrf.gov).
Hawley, 2000). For instance, meiotic recombination is
initiated by DNA DSBs generated by the action of the
Spo11 protein (Spo11p) in S. cerevisiae (Keeney et al.,
1997), and in the absence of Spo11p, DSBs are not
formed and homologous chromosomes do not synapse
at wild-type levels (Giroux et al., 1989). Similarly, the
disruption of SPO11 homologs in S. pombe (Cervantes
et al., 2000), Drosophila (McKim et al., 1998), C. elegans
(Dernburg et al., 1998), and Coprinus cinereus (Celerin
et al., 2000) results in defective gametes, and a reduction
in meiotic recombination has been reported in S. cere-
visiae, S. pombe, Drosophila, and C. elegans. Chromo-
somes still synapse at wild-type levels in spo11⫺/⫺ mu-
tants of the worm and fly but not in Coprinus cinereus.
In addition, meiosis is partially rescued by radiation-
induced DSBs in budding yeast (Thorne and Byers,
1993; Gasior, 1999), worm (Dernburg et al., 1998), fly
(R. Patel and K. McKim, personal communication), and
Coprinus cinereus (Celerin et al., 2000). These rescue
experiments provide evidence for meiotic DSB forma-
tion in eukaryotes other than S. cerevisiae and S. pombe
(Cervantes et al., 2000) and that the requirement for
Spo11 can be partially bypassed. Thus, it appears that
in worm and fly, synapsis can be mediated by pairing
centers that function independently of the need for mei-
otic recombination. If dependence on pairing centers is
seen with an increase in genome complexity, one might
easily imagine that mammals might also utilize pairing
centers.
Spo11-mediated DSBs occur at leptotene in S. cere-
visiae and disappear by zygotene (Roeder, 1997). This
coincides with localization of Rad51 and Dmc1, two
eukaryotic RecA homologs, to discrete foci in mouse
(Tarsounas et al., 1999) and in a Spo11-dependent man-
ner in yeast (Bishop, 1994), the former presumably also
at sites of DSBs. The presence of Rad51 and Dmc1 is
consistent with their biochemical function in DNA strand
exchange at the site of DNA DSBs.
Spo11p is a member of a novel family of type II-like
topoisomerases, and consistent with that, site-directed
mutagenesis of a conserved tyrosine (Y135F) abolishes
meiotic recombination (Bergerat et al., 1997). Homologs
of Spo11p have been isolated from mouse, Spo11,
(Keeney et al., 1999; Romanienko and Camerini-Otero,
1999; Shannon et al., 1999; Metzler-Guillemain and de
Massy, 2000) and man, SPO11, (Romanienko and Cam-
erini-Otero, 1999; Shannon et al., 1999), and their ex-
pression patterns are consistent with having a meiotic
function. Using RT-PCR, many alternatively spliced vari-
ants can be detected in testes, and Spo11 transcripts
are also detected in somatic tissues. Analysis of Spo11
expression in staged mouse spermatocytes showed the
highest level of transcript during pachytene (Shannon
et al., 1999), which is unexpected with Spo11 catalyzing
DNA DSBs during leptotene. This would be more consis-
tent with an additional role for Spo11 when homologous
chromosomes are fully synapsed. In fact, SPO11 tran-
script levels are also at their highest levels during pachy-
tene in yeast (Atcheson et al., 1987; Chu et al., 1998),
but no late function for Spo11p has been proposed.
Molecular Cell
976
with the mouse ␤-actin cDNA. The Spo11 transcript (1.8 kb) was absent in ⫺/⫺ testes and reduced in the heterozygote. The ␤-actin signal
is not relevant to the phenotype and serves as a loading standard. Postmeiotic ␣-actin was absent in ⫺/⫺ testes.
Here, we disrupted the mouse Spo11 gene and deter-
mined it is essential for gametogenesis and meiotic
chromosome synapsis. In addition, we found that Spo11
localizes to discrete foci early in meiosis and later to
regions of homologous chromosome synapsis. Further-
more, Spo11 expression and localization of the protein
is altered in mutant mice that arrest during meiotic pro-
phase I. Finally, we were able to partially rescue the
defect in synapsis in Spo11⫺/⫺ spermatocytes using the
DSB-causing reagent cisplatin.
Results
Targeted Inactivation of Mouse Spo11
The mouse Spo11 gene was inactivated by gene tar-
geting in embryonic stem (ES) cells. A targeting con-
struct was made (Figure 1A) that removed part of the
coding sequence of exon 1 from 3 amino acids after the
second in-frame ATG up to nucleotide 35 of the first
intron (Keeney et al., 1999; Romanienko and Camerini-
Otero, 1999). The removal of most of exon 1 and part
of intron 1 was designed to effectively create a protein
null without greatly disturbing the genomic locus. Tar-
geted clones were analyzed by Southern blotting, and a
correctly targeted cell line was injected into blastocysts.
Southern blot analysis of representative wild-type, het-
erozygotic, and double knockout mice (Figure 1B) is
shown. The ability to generate homozygous knockout
mice indicates Spo11 is not essential for mouse devel-
opment. The mutated Spo11 allele was transmitted to
Figure 1. Generation of Spo11-Deficient
Mice
(A) Gene disruption of the Spo11 locus. The
targeting construct and genomic locus of
Spo11 are shown. The EcoRI site in intron 1
was disrupted, and another EcoRI site was
created at the junction of the neo cassette
and exon 1 of Spo11 to facilitate identification
of targeted clones. The probe was derived
from the Spo11 cDNA encompassing exons
10–13. An EcoRI digest would give a 13 kb
band from wild-type genomic DNA and a 16
kb band from a targeted allele.
(B) Southern blot analysis of representative
genotypes of Spo11 knockout mice. Homo-
zygous knockout, ⫺/⫺; heterozygous, ⫹/⫺;
wild-type, ⫹/⫹. The digest was done with
EcoRI and probed with Spo11 cDNA, exons
10–13. The arrows indicate positions of bands
and their sizes are indicated.
(C) PCR genotyping of Spo11 knockout mice
by PCR. Mice used in experiments were ge-
notyped by PCR. The same mice were used
for Southern blot analysis in (B), indicating
the method is consistent with results from
Southern blotting. The primer locations are
indicated in (A). Targeting at the Spo11 locus
removes the binding site for one Spo11-spe-
cific primer.
(D) Northern analysis of Spo11 knockout
mice. Poly(A)⫹ RNA isolated from testes of
mice with the indicated genotypes was
probed with the mouse coding sequence
cDNA, and the same blot was later probed
offspring in a Mendelian fashion, and homozygotic
knockout mice did not exhibit any gross external mor-
phological defects. Mice used for experiments were ge-
notyped by PCR on genomic DNA (Figure 1C). Northern
blot analysis of mRNA from adult testes from Spo11
⫹/⫹, ⫹/⫺, and ⫺/⫺ mice (Figure 1D) showed that the
transcript was reduced in heterozygotes and absent
from ⫺/⫺ testes. The same blot was probed with mouse
␤-actin as a control for the loading of mRNA. Mouse
␤-actin cross-hybridizes to postmeiotically expressed
␣-actin (Hecht et al., 1984). The absence of ␣-actin in
Spo11⫺/⫺ testes is evidence that there is an arrest prior
to the appearance of postmeiotic cells. These results
show that the Spo11 gene is inactivated in ⫺/⫺ mice.
Spo11⫺/⫺ Male Mice Are Infertile
The first indication of a defect attributable to Spo11
deficiency is the significant decrease in testicular size
in mutant males (Figure 2A). The reduced size of the
testis is similar to results seen in other mouse knockouts
of early meiotic genes (Yoshida et al., 1998; Yuan et al.,
2000). Histological analysis of seminiferous tubule cross
sections from wild-type (Figure 2B) and mutant mice
show many of the tubules in the mutants are barren,
indicating massive cell death or nonproliferation, while
other tubules show clusters of cells morphologically
similar to zygotene spermatocytes (Figure 2C). There
were no cells with pachytene morphology in Spo11⫺/⫺
tubules. Spermatocytes in Spo11⫺/⫺ mice underwent
apoptosis (Figure 2E), while few apoptotic cells were
Mouse Spo11 Is Required for Meiotic Synapsis
977

Figure 2. Histological Examination of Adult Testes from Spo11⫺/⫺ Mice

(A) Reduced testicular size in Spo11⫺/⫺ mice. Wild-type, ⫹/⫹; heterozygote, ⫹/⫺; and homozygote knockout, ⫺/⫺ testes were compared
from 6-week-old male sibs.
(B) Hematoxylin- and eosin-stained cross section of a seminiferous tubule from a wild-type adult mouse. Magnification is 400⫻.
(C) Hematoxylin- and eosin-stained cross sections of seminiferous tubules from a Spo11⫺/⫺ adult mouse. Magnification is 400⫻.
(D) Fluorescent TUNEL assay in wild-type mouse testis section; labeling was detected in some tubules near the basal lamina (not shown).
Magnification is 200⫻.
(E) Fluorescent TUNEL assay in Spo11⫺/⫺ mouse testis section; labeled cells (green fluorescence within tubules) were detected in about 20%
of tubules. Magnification is 200⫻; the arrow indicates positive tubule.

detected in wild-type mice (Figure 2D). Approximately
20% of the tubules in the mutant had clusters of apo-
ptotic cells, while wild-type mice only showed an occa-
sional apoptotic cell.
Spo11⫺/⫺ Female Mice Are Infertile
Histological cross sections of wild-type and mutant
adult ovaries showed a reduction in the number of folli-
cles in the mutant (Figure 3A, wild type; Figure 3B, ⫺/⫺),
indicating there is a severe phenotype in female mice
resulting in few or no mature oocytes. Defects can be
seen as early as 15 dpc in the fetal ovaries of ⫺/⫺ mice
(Figure 3D) that are entering the early stages of meiosis
I. In comparison with wild-type mice (Figure 3C), the
cells in the mutant fetal ovary look homogeneous. Cells
with partially condensed chromatin, indicating zygotene
cells in wild-type mice, were reduced in the absence of
Spo11 (Figure 3D). We were unsuccessful in obtaining
pregnant Spo11⫺/⫺ mice after several months of breed-
ing with wild-type male mice.
Spermatocytes from Spo11⫺/⫺ Mice Arrest Prior
to Pachytene
Spermatocytes from Spo11⫺/⫺ mice proceed normally
through leptotene (Figure 4A), and 40 centromeres are
visualized by staining with CREST antisera, which labels
centromeres (He and Brinkley, 1996). The presence of
40 centromeres at this early stage indicates there is no
disruption of sister chromatid cohesion at the centro-
meres in the absence of Spo11. Most spermatocytes
arrest later at a zygotene-like stage (Figure 4B) where
there is little or no synapsis between chromosomes,
whether homologous or nonhomologous. While the mei-
otic arrest is absolute, the failure to synapse is not.
Approximately 5%–10% of the spread nuclei exhibit par-
tial, and in some instances, considerable synapsis (Fig-
Molecular Cell
978

Figure 3. Histological Examination of Adult and Fetal Ovaries from Spo11⫺/⫺ Mice
(A) Hematoxylin- and eosin-stained adult wild-type ovary. Arrow indicates maturing follicle. Eight to twelve follicles were generally seen per
section. Magnification is 200⫻.
(B) Hematoxylin- and eosin-stained adult Spo11⫺/⫺ ovary. Arrow indicates follicle, but note absence of any other follicles. No more than one
or two follicles were seen per section. Magnification is 200⫻.
(C) Hematoxylin- and eosin-stained wild-type fetal ovary, 15 dpc. The two female fetal mice were from the same dam. Arrows denote cells
with condensed chromatin. Magnification is 1000⫻.
(D) Hematoxylin- and eosin-stained Spo11⫺/⫺ fetal ovary, 15 dpc. Note fewer cells with condensed chromatin morphology as compared to
(C). The number of cells with this morphology was half of those seen in wild type. Magnification is 1000⫻.

ure 4G). The synapsis, indicated by Scp3 staining of
lateral elements, occurs mostly between nonhomologs
since frequent partner switches are observed. Scp3
staining of lateral elements colocalized with Scp1 stain-
ing (unpublished observations).
Rad51 and Dmc1 are RecA-homologs, the latter meio-
sis-specific, that are required for strand invasion (Shino-
hara et al., 1997). Rad51 and Dmc1 are believed to load
onto single-stranded DNA generated at the sites of mei-
otic DSBs. Neither Rad51 nor Dmc1 foci were seen in
Spo11⫺/⫺ spermatocytes (Figures 4D and 4F), but they
were readily detected in wild-type spermatocytes (Fig-
ure 4C and 4E). The ␣Rad51 antibody routinely detected
one or two large signals in Spo11⫺/⫺ spermatocytes.
These signals may be cellular reservoirs of Rad51 or
cross-reactivity to another protein. We do not believe
the latter is true, since we do not see this staining in
wild-type nuclei. The absence of Rad51 and Dmc1 foci
confirms that meiotic DSBs are not formed in the ab-
sence of Spo11.
Localization of Mouse Spo11 in Meiotic Chromosome
Spreads from Spermatocytes
Some meiotic proteins can be visualized with respect
to meiotic chromosomes, and this approach often yields
insights into what role a specific protein may play during
meiosis (Plug et al., 1998; Moens et al., 1999). Spo11
has not been visualized in meiotic nuclei of any organism
previously. We performed indirect immunofluorescence
of meiotic chromosome spreads using an affinity-puri-
fied antibody raised against a peptide (MmPep-1) de-
rived from the Toprim domain (Aravind et al., 1998) of
the mouse Spo11 protein. First, we performed several
controls to make sure the antisera and purified antibody
were Spo11 specific. Preimmune sera did not recognize
SCs in meiotic spreads, but immune serum did. The
signal from crude antiserum and purified antibody was
abolished by incubation in the presence of 10-fold molar
excess of purified MmPep-1 but not in the presence of
100-fold molar excess of an unrelated peptide. Rabbit
␣-mouse Spo11 was able to detect the two major forms
Mouse Spo11 Is Required for Meiotic Synapsis
979

Figure 4. Indirect Immunofluorescence of Spo11⫺/⫺ Spermatocytes

(A) Leptotene stage Spo11⫺/⫺ spermatocyte. Merged image of ␣Scp3 (red) and the centromere antisera CREST (green). All images initially
viewed at 1000⫻ magnification. Scale bar, 10 ␮m. Overlap of red and green signals results in yellow signal.
(B) Zygotene-like stage of arrest in Spo11⫺/⫺ spermatocytes. Merged image of ␣Scp3 (red) and the centromere antisera CREST (green). Three
nuclei are shown.
(C) Zygotene wild-type spermatocyte; merged image of ␣Scp3 (red) and the ␣Rad51 (green).
(D) Zygotene-like Spo11⫺/⫺ spermatocyte; merged image of ␣Scp3 (red) and the ␣Rad51 (green).
(E) Zygotene wild-type spematocyte; merged image of ␣Scp3 (red) and the ␣Dmc1 (green).
(F) Zygotene-like Spo11⫺/⫺ spermatocyte; merged image of ␣Scp3 (red) and the ␣Dmc1 (green).
(G) Zygotene-like Spo11⫺/⫺ spematocyte showing extent of synapsis, indicated by arrows. Stained with ␣Scp3 (red).
Molecular Cell
980

Figure 5. Spo11 Localization in Meiotic Chromosome Spreads

(A) Spo11⫺/⫺ spermatocyte; merged image of ␣Scp3 (red) and the ␣Spo11 (green). Note absence of Spo11 signal. All images were initially
viewed at 1000⫻ magnification. Scale bar, 10 ␮m. Overlap of red and green signals results in yellow signal.
(B) Leptotene spermatocyte; merged image of ␣Scp3 (red) and the ␣Spo11 (green).
(C) Zygotene spermatocyte; merged image of ␣Scp3 (red) and the ␣Spo11 (green). Arrows indicate regions of synapsis.
(D) Pachytene spermatocyte; merged image of ␣Scp3 (red) and the ␣Spo11 (green). Arrow indicates the pseudoautosomal region (PAR).
(E) Magnification of (C); arrow indicates PAR.
(F) Pachytene spermatocyte; merged image of ␣Scp1 (red) and the ␣Spo11 (green). Arrow indicates PAR.
(G) Magnification of (E); arrow indicates PAR.
Mouse Spo11 Is Required for Meiotic Synapsis
981

Figure 6. Spo11 Localization and Expression in Mouse Meiotic Mutants

(A) RT-PCR analysis of Spo11 expression in testes of mutant mice. The two forms of Spo11 (␣ and ␤) are indicated by arrows in the top panel,
and the bottom panel shows control amplification of Aop2 (Pittman et al., 1998). The mutants analyzed are listed across the top. 13.5 dpp
(days postpartum), amplification from testis cDNA from a juvenile mouse; H20, water control in amplification reaction. ␣ and ␤ forms of Spo11
are shown with primer location relative to exon 2, active site region, and Toprim domain. The sizes of putative proteins are indicated on the
right. aa, amino acids.
(B) Localization of Spo11 in Dmc1⫺/⫺ spermatocyte. Merged image of ␣Scp3 (red) and ␣Spo11 (green). Arrowhead indicates Spo11 staining
in synapsed region, arrows indicate ends of axial elements of homologous chromosome participating in homologous synapsis, and asterisks
indicate synapsis between nonhomologs. Images were initially viewed at 1000⫻ magnification. The scale bar is 10 ␮m. Overlap of red and
green signals results in yellow signal.
(C) Localization of Spo11 in mei1 spermatocytes. Merged image of ␣Scp3 (red) and ␣Spo11 (green). Arrow indicates Spo11 staining of fully
synapsed homologs, and asterisks indicate synapsis between nonhomologs. Two nuclei are shown.

of Spo11 expressed from expression vectors trans-
fected into mouse 3T3 cells but did not react with any
other protein from untransfected 3T3 cells (data not
shown). The inability to visualize Spo11 foci or localiza-
tion at partially synapsed regions (Figure 5A) in Spo11⫺/⫺
mice is strong evidence that our antibody to Spo11
is specific.
We first detected Spo11 during the leptotene stage
of meiotic prophase I (Figure 5B). This is the stage when
meiotic DSBs are generated in budding yeast (Roeder,
1997). The majority of Spo11 foci (green) did not colocal-
ize with the antibody marker (␣Scp3, red) we used for
assessing the extent of axial element formation. These
foci most likely represent sites of DNA DSB formation.
The next stage in meiosis is zygotene, during which
homologous chromosomes begin to synapse (Zickler
and Kleckner, 1998). The distribution of Spo11 changes,
and localization is now to the regions of synapsis be-
tween homologs (Figure 5C). Full synapsis of homologs
occurs during pachytene, and Spo11 decorates the
lengths of all autosomes (stained with ␣Scp3) in a dis-
continuous pattern (Figure 5D). ␣Spo11 also stains the
XY chromosome pair only at the pseudoautsomal region
(PAR) (Figure 5D), where the X and Y chromosomes
homologously synapse and recombine in the male (Sori-
ano et al., 1987). A magnification of this region (Figure
5E) shows the PAR and the discontinuous localization
of Spo11 in more detail. Spo11 colocalized with the
Molecular Cell
982
SC, as determined by Scp1 staining (Figure 5F). Similar
results are seen in a magnification of the PAR from
Figure 5F (Figure 5G). Spo11 dissociates from the SC
during diplotene as homologs begin to separate. Before
desynapsis is complete, all Spo11 localization with the
SC vanishes and staining becomes diffuse (data not
shown).
Spo11 Expression and Localization in Spermatocytes
from Other Mutant Mice Arrested
in Meiotic Prophase I
In order to gain further insight into the roles Spo11 may
have in meiosis, we looked at Spo11 expression and
localization in several mutant backgrounds. Atm⫺/⫺ mice
are deficient for the mouse homolog of the gene mutated
in ataxia-telangiectasia (AT) patients that have a defect
in DNA repair and are infertile (Barlow et al., 1996). Atm⫺/⫺
spermatocytes also show mislocalization of Rad51 and
Dmc1 to chromatin and reduced localization of these
proteins to the developing SC (Barlow et al., 1998).
Dmc1⫺/⫺ mice are mutant for the meiosis-specific mouse
RecA homolog, Dmc1 (Pittman et al., 1998), and arrest
but show normal localization of Rad51. The presence
of Rad51 foci in both Atm⫺/⫺ and Dmc1⫺/⫺ spermatocytes
is evidence that meiotic DSBs are made in these mu-
tants. mei1 mice were derived from mutagenized ES
cells and are infertile (Munroe et al., 2000). Morc⫺/⫺ mice
are the result of a random transgene insertion (Watson
et al., 1998), and the mutated gene encodes a protein
with limited homology to GHL (GyraseB, Hsp90, Mut L)
ATPases (Inoue et al., 1999).
In mouse and man there are two predominant, alterna-
tively spliced forms of Spo11 (Romanienko and Camer-
ini-Otero, 1999; Shannon et al., 1999) that code for two
proteins of different sizes (unpublished data). RT-PCR
analysis of Spo11 expression in a panel of mutant mice
revealed a common defect in generation of the predomi-
nant spliced form of the transcript (Figure 6A). All mice
showed a severe reduction in removal of exon 2 (Figure
6A, compare lanes 1–3 with lane 5). The splicing of an-
other transcript, DNA ligase III, was analyzed in these
mutant mice. Splicing of the ␤ form of DNA ligase III
occurs in pachytene spermatocytes (Mackey et al.,
1997), but we saw no difference in the splicing pattern
of this transcript in the mutant mice we analyzed (data
not shown).
The localization of mouse Spo11 was performed in
meiotic chromosome spreads from mouse mutants that
arrest during meiotic prophase I. The mouse mutants
used, for the most part, do not show complete homolo-
gous synapsis. The distribution of Spo11 was different
in Dmc1⫺/⫺ spermatocytes from that of wild-type mice.
Spo11 only localized to a subset of synapsed regions,
and the only consistent observation was that these re-
gions appeared to be homologously synapsed (Figure
6B). In a mei1 mouse, there was considerably more
synapsis, and the fluorescent intensity of Spo11 was just
slightly lower than that seen in normal spermatocytes
(Figure 6C). There appeared to be full synapsis between
homologs (arrow, Figure 6C) as determined by ␣Scp3
staining but there was also some nonhomologous syn-
apsis as well (asterisks, Figure 6C). Morc⫺/⫺ mice un-
dergo very little synapsis, and Spo11 could not be de-
tected in those spermatocytes (data not shown).
Partial Rescue of Meiosis by the DNA Damaging
Agent Cisplatin
Cisplatin is a chemotherapeutic agent that forms many
different adducts with DNA, removal of some of which
would require formation of a DNA DSB (Bhattacharyya et
al., 2000 and references therein). Previous work showed
cisplatin increases the amount of meiotic crossover
products by nearly 2-fold (Hanneman et al., 1997). We
used cisplatin to induce DSBs in arrested spermato-
cytes in our Spo11⫺/⫺ mice to see whether they pro-
gressed beyond the zygotene-like stage we routinely
observed. After cisplatin administration, mice were sac-
rificed at different time points and analyzed. As shown
above, Spo11⫺/⫺ spermatocytes do not have Rad51 and
Dmc1 foci (Figures 4D and 4F, respectively). Three days
after cisplatin administration, we were able to detect
Rad51 (merged with Scp3, Figure 7A) and Dmc1 foci
(merged with Scp3, Figure 7B). Approximately 50% of
the Rad51 and Dmc1 foci colocalized within nuclei (Fig-
ure 7C). We did not see many nuclei with Rad51/Dmc1
foci one day after cisplatin administration (data not
shown), and at five days post cisplatin administration
the results were much like those seen after three days
(data not shown). Seven days after cisplatin administra-
tion, there were very few spermatocytes that stained
with ␣Scp3 or had Rad51 or Dmc1 foci; in fact, the
absolute number of cells isolated from the testis was
severely reduced (unpublished observations).
The extent of synapsis in cisplatin-treated mice was
quantified by counting the number of centromeres in
100 nuclei stained with CREST antisera in cisplatin-
treated and untreated Spo11⫺/⫺ mice (Figure 7D). There
was a significant decrease in nuclei with 40 centromeres
(no synapsis) after cisplatin treatment and an increase
of cells with evidence of considerable synapsis (31–33
CREST foci). The true extent of synapsis was determined
by staining with ␣Scp1 and CREST antisera (Figure 7E).
Regions of synapsis usually extended away from centro-
meres, and cells with fewer staining centromeres (c ⬍
b ⬍ a) had relatively more Scp1. Thus, cisplatin-gener-
ated DSBs promote synapsis in Spo11⫺/⫺ spermato-
cytes. Generally, 30%–50% of nuclei showed consider-
able synapsis (as determined by ␣Scp1 staining) after
cisplatin administration, as compared to 5%–10% in
untreated controls.
Discussion
Spo11 is required for fertility in both male and female
mice, and the meiotic arrest we observed is similar to
that seen in other mouse knockouts that cause infertility.
Spo11⫺/⫺ deficient spermatocytes do not undergo wild-
type levels of synapsis, and the synapsis that does occur
is mostly between nonhomologs. The phenotype in
mouse is more similar to S. cerevisiae and C. cinereus
than to C. elegans and Drosophila, where SC formation
is essentially unaffected in Spo11 mutants. Thus, large
genome size and complexity alone do not imply a need
for a Spo11-independent mechanism for chromosome
synapsis as seen in C. elegans and Drosophila. Our data
on the phenotype of this knockout is in agreement with
that in the accompanying study of Baudat and cowork-
ers (Baudat et al. 2000 [this issue of Molecular Cell]).
Spo11 was first localized to discrete foci during lepto-
Figure 7. Partial Rescue of Meiotic Arrest in Spo11⫺/⫺ Spermatocytes
(A) Induction of Rad51 foci in Spo11⫺/⫺ spermatocytes 3 days after cisplatin administration. Merged image of ␣Scp3 (red) and ␣Rad51 (green).
Cells were in zygotene-like stage. The images were initially viewed at 1000⫻ magnification. Four nuclei are shown; the scale bar is 10 ␮m.
Overlap of red and green signals results in yellow signal.
(B) Induction of Dmc1 foci in Spo11⫺/⫺ spermatocytes 3 days after cisplatin administration. Merged image of ␣Scp3 (red) and ␣Dmc1 (green).
Cells were in zygotene-like stage; six nuclei are shown.
(C) Colocalization of Rad51 and Dmc1 foci in Spo11⫺/⫺ spermatocyte 3 days after cisplatin administration. Merged image of ␣Rad51 (red) and
␣Dmc1 (green).
(D) Graphic representation of increased synapsis seen in Spo11⫺/⫺ spermatocytes after cisplatin administration. One ⫺/⫺ mouse was treated
with cisplatin for 3 days, the other was injected with saline. The mice were from the same litter and ⱖ5 weeks old when injected. One hundred
nuclei were analyzed for each sample, and the number of CREST signals (centromeres) were recorded.
(E) Colocalization of Scp1 and centromeres (CREST antisera) in Spo11⫺/⫺ spermatocytes 3 days after cisplatin administration. Merged image
of ␣Scp1 (red) and CREST (green). Three nuclei are shown, (a) has 33 centromeres; (b), 30, and (c), 28. In many instances, synapsis extends
away from the centromere. Note more ␣Scp1 staining coincident with fewer centromeres.
Molecular Cell
984
tene, when DSBs are believed to occur, and later only
along synapsed regions of homologous chromosomes
during zygotene and pachytene, including the PAR of
the XY bivalent.
The meiotic defect in Spo11⫺/⫺ mice is qualitatively
different from other mouse meiotic mutants that pre-
sumably function downstream in the recombination
process. For example, Atm⫺/⫺ and Dmc1⫺/⫺ spermato-
cytes also arrest during meiotic prophase I, but Atm
plays a more global role in genome surveillance and
the effects are pleiotropic, while the meiotic arrest in
Dmc1⫺/⫺ mice may be due to a checkpoint activated by
unrepaired DSBs.
Our Spo11⫺/⫺ mice were not subjected to an intensive
analysis of somatic tissues to detect effects other than
a disruption of meiosis. Histological analysis of cross
sections from thymus, in which there is a high level
of Spo11 expression in comparison to other somatic
tissues (Romanienko and Camerini-Otero, 1999), did not
indicate any obvious alterations in the absence of Spo11
(unpublished observations). The defect seen in fetal
ovary (Figures 3C and 3D) can be attributed to the mei-
otic function of Spo11, but it would be interesting to
look earlier in gonad development to see if the premei-
otic defect we see in fetal testes (data not shown) can
be detected earlier in primordial germ cells. The morpho-
logical change we saw in Spo11⫺/⫺ fetal testes may be
a reflection of some defect in premeiotic S phase in
Spo11⫺/⫺ mice, as was seen in SPO11-deleted yeast
(Cha et al., 2000).
Spo11 Is Required for Meiotic Chromosome Synapsis
Indirect immunofluorescent analysis of meiotic chromo-
some spreads from Spo11⫺/⫺ male mice showed that
axial elements form but synapsis was severely affected
(Figure 4B). Almost all the synapsis that occurs is be-
tween nonhomologs, as revealed by frequent partner
switches. The lack of synapsis might be attributable to
the absence of meiotic DSBs, but it is also possible that
Spo11 plays a role in earlier homolog pairing (Weiner
and Kleckner, 1994) and/or synapsis itself. Normal syn-
apsis in mouse is also dependent on a number of pro-
teins involved in meiotic recombination (Atm, Barlow et
al., 1998; Dmc1, Pittman et al., 1998; Yoshida et al.,
1998; Msh4, Kneitz et al., 2000; and Msh5, de Vries et
al., 1999; Edelmann et al., 1999). Synapsis was also
defective in Scp3⫺/⫺ males, but female mice were fertile
(Yuan et al., 2000).
The dependence of normal synapsis on recombina-
tion is central to the deficiency seen in Spo11⫺/⫺ sperma-
tocytes. Either the failure to initiate recombination sig-
nals an arrest and normal synapsis does not occur, or
the lack of recombination results in a subsequent failure
to complete synapsis that triggers the arrest. A mutant
mouse that can bypass the requirement for DSB forma-
tion and recombination and permits normal synapsis
would argue for the second model.
Spo11 Localization Is Consistent with Roles
in DSB Formation and Synapsis
Spo11 was localized in discrete foci during leptotene
(Figure 5A) and more intensely at synapsed regions of
homologous chromosomes during zygotene and pachy-
tene in wild-type mice (Figures 5B, 5C, and 5E). Spo11
makes DSBs during leptotene in yeast (Roeder, 1997),
but the temporal events of meiotic recombination have
not been studied in great detail in mouse. The localiza-
tion of mouse Rad51 to discrete foci during leptotene
(Tarsounas et al., 1999) is reasonable evidence that
DSBs occur at this stage. Our demonstration that the
formation of Rad51 foci in mouse is Spo11-dependent,
just as in yeast, is further evidence that DSBs occur
during leptotene. Since we were able to detect Rad51
foci in Spo11⫺/⫺ spermatocytes after administration of
cisplatin, it is therefore reasonable to conclude that
mouse Spo11 generates meiotic DSBs.
The discontinuous pattern of Spo11 on pachytene
chromosomes (Figures 5C and 5E) is unlike that seen
using antibodies against the lateral (Scp3) and central
(Scp1) element components of the SC, and it would be
interesting to see whether the distribution of Spo11 is
specific for each chromosome. That Spo11 is expressed
at its highest level in pachytene spermatocytes in mouse
(Shannon et al., 1999) and during pachytene in S. cere-
visiae (Atcheson et al., 1987; Chu et al., 1998) is consis-
tent with its detection along synapsed regions of paired
homologs, but pachytene localization does not neces-
sarily imply a role in pachytene.
Mutant Mice Reveal a Spo11 Splicing Defect
in Arrested Spermatocytes and Suggest a Role
for Spo11 in Stabilizing Homologous Synapsis
Analysis of Spo11 localization and expression in several
mutant mouse lines allowed us to speculate on the alter-
nate forms of Spo11 and what function they may encode.
The RT-PCR results from the mutant mice show a link
between meiotic progression from zygotene to pachy-
tene and the removal of exon 2 from the Spo11 transcript
(Figure 6A). The predominant form of Spo11 in wild-type
adult testes does not contain exon 2. The simplest model
suggests that the larger form of Spo11 (Spo11␤) is the
catalytic form that generates DSBs, while the smaller
form (Spo11␣) plays a structural role in the developing
SC. Further evidence for this model is the fact that Rad51
foci (marker for DSBs) form in Dmc1⫺/⫺ spermatocytes
(Pittman et al., 1998) (where there is little of the smaller
Spo11␣ transcript) with the same kinetics as wild-type
mice. mei1 mice show more homologous synapsis than
Dmc1 and Atm mutants (data presented above and un-
published observations) and, coincidentally, more splic-
ing to Spo11␣. The finding that DNA ligase III-␤ splicing
is unaffected in the mutant mice we analyzed indicates
there may be multiple checkpoints regulating different
facets of meiotic progression, and that the arrest per
se is not responsible for the splicing defect.
Our interpretation of immunofluorescence results
from Dmc1⫺/⫺ and mei1 spermatocytes is that Spo11
localizes to regions of synapsis between homologs. Very
little staining is seen in regions of incorrect synapsis,
so one can speculate that Spo11 is required for synapsis
between homologs or perhaps stabilizes synapsis be-
tween homologs. It is not clear whether Spo11 can dis-
criminate between homologous and nonhomologous
synapsis or whether the SC formed between nonhomo-
logs is fundamentally different in a way that is not imme-
diately obvious with our cytological criteria.
Mouse Spo11 Is Required for Meiotic Synapsis
985
We note, however, that this picture might be an over-
simplification. Preliminary results indicate that Brca1Co/Co
mice can progress and arrest in pachytene with fully
synapsed chromosomes but still do not splice out exon
2 (unpublished observations).
Partial Meiotic Rescue in Spo11⫺/⫺ Spermatocytes
by Cisplatin Argues for a Role of Spo11
in Generating DSBs
We partially rescued the meiotic arrest seen in our Spo11
knockout by generating DNA damage with the chemo-
therapeutic agent cisplatin. We were able to detect
Rad51/Dmc1 foci, the presence of which is good evi-
dence for formation of cisplatin-induced DSBs. We were
also able to promote synapsis, indicating that DNA DSBs
may influence SC formation, but wild-type levels of syn-
apsis were not observed. In any case, DSBs of any
origin, whether generated by Spo11 or cisplatin, and
recombination are necessary and sufficient to promote
significant levels of synapsis, and the postulated second
role of Spo11 may be in stabilizing homologous syn-
apsis.
Experimental Procedures
Targeted Inactivation of the Mouse Spo11 Gene
The targeting construct was based on ploxPneo (Yang et al., 1998)
(a gift from Chuxia Deng). A 3.3 kb BspEI/ApaI genomic fragment
containing the 5⬘ region of mouse Spo11 was blunted and cloned
into the blunted EcoRI site in ploxPneo between the TK gene and
PGKneo cassette in the opposite orientation of transcription of the
neo gene. This construct was designated pTarget1. A 1.1 kb SmaI/
EcoRV genomic fragment containing intron 1 sequence was cloned
into XhoI-cut, blunted pTarget1 on the other side of the neo cassette.
This step removed the first 35 nucleotides of intron 1, which con-
tained the splice donor site. This effectively deleted 140 bp of the
Spo11 locus and inserted the neo cassette; the construct was desig-
nated pTarget2. A 9 kb EcoRI/AatII genomic fragment containing
exons 2–9 was blunted and cloned into pTarget2 cut with Hpa I,
resulting in the final 20 kb targeting vector, pTarget3. The final clon-
ing step resulted in the deletion of an additional 110 bp from intron
1, including the EcoRI site. PTarget3 was linearized with NotI and
prepared for targeting. Ninety micrograms of linearized pTarget3
was electroporated into 1.8 ⫻ 107 TC1 ES cells (Deng et al., 1996)
(a gift from Chu-Xia Deng) and seeded onto mitomycin C–treated
mouse embryonic feeder cells (Incyte Genomics). Colonies were
selected in media containing LIF (GIBCO–BRL), 350 ␮g/ml G418,
and 5 ␮M gancyclovir (a gift from Rick Proia). Colonies were picked,
expanded, and genomic DNA was extracted for Southern analysis.
The removal of the EcoRI site in intron 1 and insertion of the neo
cassette made the mutated allele 3 kb (16 kb) larger than the wild-
type allele (13 kb). The probe was derived from exons 10–13 of the
mouse Spo11 cDNA. One correctly targeted ES clone was injected
into C57/B6 blastocysts to obtain germline transmission. Mice ob-
tained after germline transmission were screened by PCR on geno-
mic DNA isolated from tail tips. Primers for PCR genotyping were
Spo11-1, 5⬘-TGTCCCGCGGTCAGTGGTGCAG-3⬘ and Spo11-2,
5⬘-TCCAGGGCGTCGAAGAACGAGG-3⬘, which amplified a 150 bp
product from genomic DNA. Spo11-2 lies within the deleted region
and would not be present in the targeted allele. The neo primers were
Neo5⬘-Target, 5⬘-GTACTCGGATGGAAGCCGGTCTT-3⬘ and Neo3⬘-
Target, 5⬘-GCCAAGCTCTTCAGCAATATCACG-3⬘. The amplified
product was 280 bp and was only present if the targeted allele was
present. The PCR protocol was 94⬚C, 2 min; 94⬚C, 15 s; and 68⬚C,
45 s for 32 cycles followed by 72⬚C, 10 min with 100 ng of genomic
DNA in a 20 ␮l reaction using the Advantage cDNA Polymerase Mix
(Clontech).
Analysis of Spo11 Knockout Mice
Northern blot analysis was done on poly(A)⫹ RNA prepared from
testes of the corresponding genotypes using Trizol (GIBCO–BRL)
and the Micro Poly(A) Pure Kit (Ambion). Two micrograms of purified
mRNA was run, transferred, and probed sequentially with the mouse
Spo11 cDNA and the mouse ␤-actin cDNA (Sigma). Tissues for
histological examination were removed and fixed overnight in neu-
tral-buffered 10% formalin (Sigma). Tissue was embedded in paraf-
fin and 6 ␮m sections were cut. Fetal mice were removed from
the mother and placed in fixative. The fetal age of the mice was
determined by comparison of external facial and body morphology,
and their age was placed at 15 dpc (Rugh, 1968). Before fixation, a
piece of tail was removed for genotyping. Male mice were confirmed
by PCR amplification of the mouse male-specific Sry gene (GenBank
accession number U70641) using primers Sry5⬘, 5⬘-GCCCAGCA
GAATCCCAGCATGCA-3⬘ and Sry3⬘, 5⬘-TTTTGTTGAGGCAACTG
CAGGCTG-3⬘. The PCR product was 250 bp, and the cycling proto-
col was 94⬚C, 2 min; 94⬚C, 15 s; and 68⬚C, 45 s for 35 cycles followed
by 72⬚C, 10 min. Sections were stained with hematoxylin and eosin
(H&E) or analyzed for apoptosis using the TACS 2 TdT-In situ Apo-
ptosis Detection Kit (Trevigen).
Antibodies for Immunofluorescence
An antibody was raised against a peptide, MmPep-1 (amino acids
224–242, EKDATFQRLLDDNFCSRMS, encoded in exon 8 of mouse
Spo11 [Romanienko and Camerini-Otero, 1999]), that was synthe-
sized on MAP (Multi-Antigenic Peptide) resin (Novabiochem) on a
PerSeptive Biosystems peptide synthesizer. The crude synthesis
product was used to generate polyclonal antisera in two rabbits
(Covance). Antisera was checked by Western blot analysis of mouse
Spo11:GFP fusion protein expressed in mouse 3T3 cells (ATCC).
␣-Spo11 was affinity purified by passage over a column to which
was coupled HPLC-purified MmPep-1. Peptide coupling was done
using AminoLink Coupling Gel (Pierce), and binding and elution
were done with ImmunoPure Gentle Binding buffer and ImmunoPure
Gentle Elution buffer, respectively (Pierce). The purified antiserum
was dialyzed into Tris-buffered saline, .05% sodium azide, and con-
centrated to approximately 1mg/ml using Slide-A-Lyzer Concentrat-
ing Solution (Pierce). The purified antibody was aliquoted and frozen
at ⫺20⬚C.
Primary antibodies (in addition to rabbit ␣-mouse Spo11 at 1:10
or 1:20) used for immunofluorescence were mouse ␣-rat SCP1 at
1:10 (a gift from Christa Heyting, Wageningen University, The Neth-
erlands), mouse ␣-hamster SCP3 (Cor1) at 1:1000 (a gift from Peter
Moens, York University, Toronto, Canada), CREST human antisera
at 1:200 (a gift from Bill Brinkley, Baylor College, Texas), goat
␣-Dmc1 at 1:200 (C-20) (Santa Cruz Biotechnology), and rabbit
␣-Rad51 at 1:200 (H-92) (Santa Cruz Biotechnology). All secondary
antibodies (Jackson Immunoresearch Laboratories) were used at
1:200. Secondary antibodies were either conjugated with Rhoda-
mine Red-X or FITC and were generated in goats or donkeys.
Meiotic Chromosome Spread Preparation
and Immunofluorescence
Chromosome spreads were prepared as follows: testes were dis-
sected, seminiferous tubules were digested with .5 mg/ml collage-
nase in RPMI 1640 high glucose media supplemented with amino
acids (GIBCO–BRL) for 20 min at 33⬚C with gentle shaking, digested
with .25 mg/ml trypsin, 1 ␮g/ml DNase I for 20 min at 33⬚C with
gentle shaking, then filtered through a 40 ␮m cell strainer (Falcon).
Soybean trypsin inhibitor, SBTI (GIBCO–BRL), was added at 100
␮g/ml, and the cell suspension was centrifuged at 800 ⫻ g for 5
min, washed with media containing SBTI, and recentrifuged. The
supernatant was removed, and cells were resuspended in .5% NaCl
along with any residual media that remained after aspiration. The
cell suspension was pipetted onto glass slides with hydrophobic
rings (Becton Dickinson), and the cells were allowed to swell. When
the cells began to adhere to the slide surface, the slides were fixed
in 2% paraformaldehyde, .03% SDS for 3 min, then in 2% parafor-
maldehyde for 3 min. Slides were washed 3⫻ in .4% Photo-Flo 200
(Kodak) and allowed to air dry.
Slides were washed 2⫻ in PBS, blocking solution (10% serum
from goat or donkey, 3% BSA, .05% Triton X-100 in PBS) was added
to the hydrophobic rings, and slides were incubated for 2 hr in a
humid chamber at room temperature. Primary antibodies were di-
luted in blocking buffer, and slides were incubated in a humid cham-
Molecular Cell
986
ber at 37⬚C for 1 hr. After washing, secondary antibodies were added
for 20 min at room temperature. The slides were washed and allowed
to dry. Vectashield Mounting Medium with DAPI (Vector Labora-
tories) was added and the slides were viewed. The chromosome
spread and antibody incubation procedure was adapted from a
protocol from Peter Moens.
Slides were examined with a Leica fluorescent microscope, and
individual FITC and rhodamine images were captured as eight bit
color images at 1000⫻ magnification using an oil immersion lens.
The images were processed using Adobe Photoshop 4.0 and
merged.
Balb/c mice were used for localization of Spo11. Juvenile male
mice of ages 10–21 days postpartum (dpp) were used to prepare
the spreads. The mutant mice used in this study are as follows,
Atm⫺/⫺ (Barlow et al., 1996) (purchased from Jackson Laboratories),
Dmc1⫺/⫺ (Pittman et al., 1998) and mei1 (Munroe et al., 2000) (a gift
from John Schimenti, Jackson Laboratories, Bar Harbor, Maine),
and Morc⫺/⫺ (Watson et al., 1998) (a gift from Andrew Zinn and
Mark Watson, University of Texas-Southwestern, Dallas, Texas). The
mutant mice were ⱖ5 weeks of age at time of assaying.
Comparison of Spo11 Transcripts in Mutant and Wild-Type
Mice by RT-PCR
The primers used to amplify mouse Spo11 were 5⬘MmSpo, 5⬘-CTG
TTGGCCATGGTGAGAGAGG-3⬘ and MmSpoSeq1, 5⬘-TCCTTGAA
TGTTAGTCGGCACAGC-3⬘. The product with exon 2 is 560 bp and
without exon 2 is 460 bp. The PCR protocol was 94⬚C, 2 min; 94⬚C,
15 s; and 68⬚C, 1 min for 33 cycles followed by 72⬚C, 10 min. The
Aop2 control PCR and primers were as described (Pittman et al.,
1998). Poly(A)⫹ RNA was isolated from mouse testis tissue as de-
scribed earlier, and cDNA synthesis was done as described (Ro-
manienko and Camerini-Otero, 1999).
Cisplatin Administration
Cisplatin (Sigma) was administered as described (Hanneman et al.,
1997). Mice were sacrificed at time points indicated, and testes
were removed for meiotic chromosome spread preparation and/or
histological examination. CREST foci were counted in a methodical
manner with no bias in nuclei selection other than the omission of
leptotene spermatocytes, which are apparently normal in Spo11⫺/⫺
mice. All cells were in a zygotene-like stage. Foci that were partially
overlapped such that there was no space between them were
counted as one focus. Adminsitration of cisplatin was performed
according to ACUC guidelines at the NIH.
Acknowledgments
We are grateful to Chu-Xia Deng and Cuiling Li for blastocyst injec-
tion, generating chimeric mice, and providing ES cells; Rick Proia
and his lab for the gancyclovir and targeting advice; April Anderson
for technical assistance; Linda Robinson for her assistance; and
George Poy for oligo and peptide synthesis. We would like to thank
those who graciously provided the mutant mice, John Schimenti for
the Dmc1⫺/⫺ and mei mice, and Mark Watson and Andrew Zinn for
the Morc⫺/⫺ mice. We are especially grateful to Bill Brinkley for the
␣CREST antisera, Christa Heyting for the ␣SCP1 antibody, and Peter
Moens for the ␣SCP3 antisera and advice on performing meiotic
spreads. Finally, we want to thank Michael Lichten and Rick Proia
for their insightful comments on the manuscript and Chu-Xia Deng
and Kim McKim for communicating unpublished results.
Received August 4, 2000; revised September 19, 2000.
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