Hormad1 Mutation Disrupts Synaptonemal Complex
Formation, Recombination, and Chromosome
Segregation in Mammalian Meiosis
Yong-Hyun Shin1,2., Youngsok Choi3., Serpil Uckac Erdin1, Svetlana A. Yatsenko4, Malgorzata Kloc5,
Fang Yang6, P. Jeremy Wang6, Marvin L. Meistrich7, Aleksandar Rajkovic1,2*
1Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas, United States of America, 2Department of Obstetrics and Gynecology and
Reproductive Sciences, Magee Women’s Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 3Fertility Center of CHA General
Hospital, CHA Research Institute, CHA University, Seoul, Korea, 4Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States
of America, 5Department of Surgery, The Methodist Hospital and The Methodist Hospital Research Institute, Houston, Texas, United States of America, 6Department of
Animal Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 7Department of Experimental Radiation Oncology, The University of
Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
Meiosis is unique to germ cells and essential for reproduction. During the first meiotic division, homologous chromosomes
pair, recombine, and form chiasmata. The homologues connect via axial elements and numerous transverse filaments to
form the synaptonemal complex. The synaptonemal complex is a critical component for chromosome pairing, segregation,
and recombination. We previously identified a novel germ cell–specific HORMA domain encoding gene, Hormad1, a
member of the synaptonemal complex and a mammalian counterpart to the yeast meiotic HORMA domain protein Hop1.
Hormad1 is essential for mammalian gametogenesis as knockout male and female mice are infertile. Hormad1 deficient
(Hormad12/2) testes exhibit meiotic arrest in the early pachytene stage, and synaptonemal complexes cannot be visualized
by electron microscopy. Hormad1 deficiency does not affect localization of other synaptonemal complex proteins, SYCP2
and SYCP3, but disrupts homologous chromosome pairing. Double stranded break formation and early recombination
events are disrupted in Hormad12/2testes and ovaries as shown by the drastic decrease in the cH2AX, DMC1, RAD51, and
RPA foci. HORMAD1 co-localizes with cH2AX to the sex body during pachytene. BRCA1, ATR, and cH2AX co-localize to the
sex body and participate in meiotic sex chromosome inactivation and transcriptional silencing. Hormad1 deficiency
abolishes cH2AX, ATR, and BRCA1 localization to the sex chromosomes and causes transcriptional de-repression on the X
chromosome. Unlike testes, Hormad12/2ovaries have seemingly normal ovarian folliculogenesis after puberty. However,
embryos generated from Hormad12/2oocytes are hyper- and hypodiploid at the 2 cell and 8 cell stage, and they arrest at
the blastocyst stage. HORMAD1 is therefore a critical component of the synaptonemal complex that affects synapsis,
recombination, and meiotic sex chromosome inactivation and transcriptional silencing.
Citation: Shin Y-H, Choi Y, Uckac Erdin S, Yatsenko SA, Kloc M, et al. (2010) Hormad1 Mutation Disrupts Synaptonemal Complex Formation, Recombination, and
Chromosome Segregation in Mammalian Meiosis. PLoS Genet 6(11): e1001190. doi:10.1371/journal.pgen.1001190
Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America
Received April 3, 2010; Accepted September 30, 2010; Published November 4, 2010
Copyright: ? 2010 Shin 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: This work was supported in part by the National Institutes of Health Grant HD054829 to AR (http://grants.nih.gov/grants/oer.htm). The funder 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: firstname.lastname@example.org
. These authors contributed equally to this work.
Mammalian meiosis is unique to germ cells and a critical step in
sexual reproduction. Meiosis reduces the chromosome comple-
ment to haploidy in preparation for fertilization. The first meiotic
division is unique in pairing of homologous chromosomes,
homologous recombination, and formation of chiasmata. The
reduction in chromosome numbers happens when homologous
chromosomes segregate to opposite poles during the first meiotic
division. Proper disjunction (separation) requires crossovers
(manifested cytologically as chiasmata). The sister chromatids
organize along structures called axial elements (AEs) and
transverse elements connect AEs to form the synaptonemal
complex (SC) . SC is a proteinaceous structure that connects
paired homologous chromosomes during prophase I of meiosis,
and SC is critical for wild-type levels of crossovers to occur during
meiosis. AEs are critical part of the SCs and mutations in proteins
that form AEs disrupt sister chromatid cohesion, recombination,
and chromosome segregation [2–4]. Proteins with HORMA
domain are critical components of the axial elements .
HORMA domain proteins are predicted to form globular
structure that may sense specialized chromatin states, such as
those associated with double strand breaks (DSBs) or other forms
of DNA damage . Several mammalian proteins that contain
HORMA domain, such as mitotic arrest deficient protein 2,
MAD2, are essential for mitosis [6–7]. Mice lacking MAD2
unsurprisingly die during early embryogenesis . In lower
organisms, several meiotic specific HORMA proteins are known
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and all are critical for meiosis. These HORMA proteins are: Hop1
 and Red1  in yeast; Him-3  in nematodes; and Asy1
 in plants. Him-3 localizes to the axial cores of both synapsed
and unsynapsed chromosomes. C. elegans Him-3 mutants are
deficient in chromosome pairing, synapsis, and the regulation of
double strand break repair [10,12–13]. Synapsis in both male and
female Asy1 mutants is disrupted [14–15]. In yeast, plants, and
nematodes, HORMA domain proteins are critical components of
the synaptonemal complex and essential for meiosis I. Others and
we identified a previously uncharacterized gene that we named
Nohma, later re-named to Hormad1 [16–18]. Hormad1 encodes a
protein that contains a HORMA domain, and unlike Mad2,
Hormad1 expression is germ cell–specific . Mouse and human
HORMAD1 are highly conserved and share 77% amino acid
identity overall, and share 89% amino acid identity in the
HORMA domain. Moreover, mouse and human HORMAD1
HORMA domains share 28% amino acid identity with Hop1
HORMA domain. Hop1 in yeast appears to bind near or at the
sites of DSB formation and may modulate the initial DSB cleavage
. Hop1 mutants in yeast have reduced number of DSBs ,
and Hop1 may participate in recruiting DMC1, RAD51 and other
proteins that are required for DNA repair during meiotic synapsis
and recombination [19–20]. Phosphorylation of Hop1 by Mec1/
Tel1 yeast kinases is important for interhomologue recombination
and prevents DMC1-independent repair of meiotic DSBs .
Here we report that HORMAD1 is likely the mammalian
counterpart of Hop1, and that HORMAD1 deficiency disrupts
mammalian synaptonemal complex formation, meiotic recombi-
nation, and chromosome segregation.
HORMAD1 is essential for spermatogenesis
We previously showed that Hormad1 RNA expression in testes
began at postnatal day 10, with little expression detected at birth
or postnatal day 5 . Hormad1 RNA expression pattern
coincided with the onset of meiosis, and appearance of primary
spermatocytes in the developing testes. In situ hybridization with
anti-sense Hormad1 riboprobe revealed that Hormad1 expression
was confined to germ cells, and specifically spermatocytes, with no
signal detected in spermatogonia or sertoli cells . We
generated antibodies against HORMAD1 and studied its protein
localization pattern in testes. HORMAD1 localized exclusively in
germ cells, specifically in zygotene, and early pachytene sper-
matocytes as previously described for the RNA expression .
Since HORMAD1 protein showed localization consistent with
its potential role in meiosis I and contains the HORMA domain,
we disrupted the Hormad1 gene to examine its requirement for
germ cell development and meiosis in mouse. Hormad1 is located
on chromosome 3 and composed of sixteen exons. We deleted
exons 4 and 5 (Figure S1A), and this mutation is predicted to
remove 33 amino acids from the highly conserved HORMA
domain and to cause a frame shift mutation. Small amounts of
truncated Hormad1 RNA transcripts were detectable on RT-PCR,
and Western blots on testes extracts showed absence of
HORMAD1 protein in knockout mice as expected (Figure S1B
Female and male heterozygote matings produced expected
Mendelian ratios, averaged 8.162 pups per litter (n=20 breeding
pairs) over a 6-month period, and remained fertile for at least 9
months. The litter size was statistically not significantly different
from the wild-type average (8.462 pups per litter). Male and
female mice heterozygous for the mutation (Hormad1+/2) were
fertile with grossly normal male and female gonadal morphology
and histology. However, both Hormad12/2males and females
were infertile with no pups produced over a period of 6 months
from mating with wild-type female and male mice, respectively.
While ovaries showed no gross morphologic differences between
the knockout and wild-type mice, Hormad1 knockout adult testes
were significantly smaller than the wild-type testes (Figure S2A).
Testes in the 7-day-old Hormad12/2mice were grossly normal and
weighed 8.062.0 mg/pair and did not significantly differ from the
wild-type, 9.063.0 mg/pair of testes. By 4 weeks of postnatal life,
the knockout testes (4766 mg/pair) were 50% of the wild-type
weight (9461.7 mg/pair), and by 8 weeks the knockout testes
(22562.7 mg/pair) (Figure S2B).
Histology at 6 weeks showed hypocellular seminiferous tubules
with clumps of sertoli cells in the lumen. We observed
spermatogonia and early spermatocytes, but no post-meiotic germ
cells such as spermatids or spermatozoa (Figure 1A–1H). We
therefore carefully examined spermatogenesis in Hormad12/2
mice. Spermatogenesis is a complex process that involves
differentiating and proliferating self-renewing spermatogonia that
differentiate into spermatozoa. Type A spermatogonia self-renew
and can initiate differentiation into Type B spermatogonia which
in turn differentiate into primary spermatocytes. Primary sper-
matocytes undergo meiosis I to form secondary spermatocytes.
Secondary spermatocytes enter meiosis II and divide to produce
haploid spermatids. We examined Hormad1 knockout testes
histology during gonadal development to determine the stage at
which spermatogenesis is disrupted. Identical testes weights at
postnatal day 7, and similar histology between Hormad12/2and
wild-type testes argue that pre-spermatogonia in Hormad12/2
testes proliferate into Type A spermatogonia without major
against PLZF and SOHLH1, markers that identify self-renewing
(PLZF) and differentiating spermatogonia (SOHLH1), showed the
presence of both proteins in the wild-type as well as the knockout
animals, confirming that spermatogonia are unaffected (Figure 1J,
1K, 1N, 1O). At postnatal day 10, testes contain preleptotene/
leptotene primary spermatocytes, and there was no gross
difference between wild-type and Hormad12/2testes. At 14 days,
testes contain pachytene spermatocytes, and there was a significant
difference between the wild-type and Hormad12/2testes, with
many apoptotic cells and few pachytene spermatocytes in
The biology of germ cells is intimately intertwined with
meiosis. Meiosis I is a unique biological event, when
chromosomes pair, recombine, and segregate. The synap-
tonemal complex is a protein lattice that enables
chromosome pairing and recombination and is unique to
meiosis I. Meiosis I requires a subset of factors that are
unique to germ cells and meiosis. Germ cell–specific
factors are known to play crucial roles during formation of
the synaptonemal complex and include synaptonemal
complex proteins SYCP1, SYCP2, and SYCP3, among
others. We discovered a mouse HORMA domain contain-
ing protein, Hormad1 (Nohma), which is germ cell–specific
and essential for male and female fertility. Mice deficient in
Hormad1 have severe defects in early recombination,
synapsis, and segregation—functions attributed to yeast
HORMA domain containing protein, Hop1. Moreover,
Hormad1 is likely a germ cell–specific component of the
meiotic sex chromosome inactivation and transcriptional
HORMAD1 Is Essential for Male and Female Meiosis
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Hormad12/2testes (Figure S3A, S3B, S3C, S3D, S3E, S3F, S3G,
S3H, S3I, S3J), and rising apoptotic index with age in the
knockout as compared to the wild-type (Figure S3K). We counted
leptotene, zygotene and pachytene spermatocytes in 6 week old
wild-type and Hormad12/2testes. Hormad12/2testes showed
declining number of spermatocytes beginning in stages II-III with
2868 spermatocytes as compared to 52612 in the wild-type
(Figure 1A and 1E). No spermatocytes were noted in stages IV-IX
in the Hormad12/2testes (Figure 1F and 1G), and no significant
difference was noted in the spermatocyte number in stages X-XII
between the wild-type and knockout testes (Figure 1D and 1H).
These results indicate that Hormad1 deficiency in the male gonad
caused meiotic arrest at the pachytene stage.
HORMAD1 is critical for chromosome synapsis
Previous studies on HORMA domain proteins indicate their
specific involvement in cell division. MAD2 is a ubiquitously
expressed mammalian HORMA domain protein involved in both
meiosis and mitosis , while yeast HOP1, RED1, nematode
HIM3 and plant ASY1 genes are specifically involved in meiotic
segregation, synapsis and recombination [2,10–11,13,15,22]. No
mammalian counterparts to Hop1, Red1, Him3 and Asy1 have
been functionally evaluated up to date. We previously hypothe-
sized that HORMAD1 is a functional counterpart to Hop1, Him3
and Asy1 . Critical components of the synaptonemal complex
include meiosis specific SYCP1, SYCP2 and SYCP3 proteins.
SYCP1 is a major component of the transverse filaments, while
both SYCP2 and SYCP3 are components of the axial lateral
elements [23–26]. To determine HORMAD1 localization during
meiosis, and whether HORMAD1 localizes to the axial elements,
or transverse filaments, we used antibodies against SYCP1,
SYCP2 and SYCP3 to study their respective co-localization with
HORMAD1. HORMAD1 co-localized with SYCP3 and SYCP2
but did not co-localize with SYCP1, which indicates that
Figure 1. HORMAD1 is required for spermatogenesis. (A–H). Periodic acid/Schiff reagent (PAS) stained cross sections from 6 weeks old wild-type
testes (A-D) andHormad12/2(E-H) testes.Seminiferous tubulestages areshownabove the panels. Lack of mature sperm andarrest in spermatogenesis
is shown at different tubular stages. (I-P). Immunohistochemistry (IHC) of 6 weeks old wild type and Hormad12/2testes with anti HORMAD1, SOHLH1,
PLZF and DDX4 antibody. IHC with anti-HORMAD1 antibody shows HORMAD1 expression (brown) in wild-type stage IV pachytene, and stage XI
zygotenespermatocytes (I), while noexpression wasobservedin theknockout(M). SOHLH1is expressedin differentiatingspermatogonia andis present
in wild-type and Hormad12/2testes as shown by arrows (J and N). PLZF identifies differentiating and self-renewing spermatogonia and is expressed in
both wild-type and Hormad12/2testes as shown by arrows (K and O). DDX4 is an RNA binding protein specific for germ cells, and is under-expressed in
Hormad12/2spermatocytes (L and P). Arrows point to Spermatogonia (Sg) and Spermatocytes 1(Sc). Scale bars: 50 mm.
HORMAD1 Is Essential for Male and Female Meiosis
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HORMAD1 is located along the axial elements (Figure 2A, 2C,
and 2E). Recent studies also show that HORMAD1 localizes to
the axial elements [17–18]. We also studied whether absence of
SYCP2 affected HORMAD1 localization along the chromosomes.
HORMAD1 localization is independent of major germ cell–
specific components of the axial elements of the synaptonemal
complex because neither Sycp2 (Figure 3A–3F) nor Sycp3 mutation
affected HORMAD1 localization to the axial elements .
We examined localization of germ cell–specific synaptonemal
complex proteins SYCP1, SYCP2 and SYCP3 in Hormad12/2
testes. SYCP1 is a major component of the transverse filaments
and is known to form fibrillar structures in Sycp3 mutant
spermatocytes . However, the fibers are truncated, contain
axial gaps, and do not associate with the centromeres of the
meiotic chromosomes. We also observed truncated fibers with
anti-SYCP1 antibodies in Hormad12/2mice (Figure 2B). HOR-
MAD1 is therefore not necessary for SYCP1 binding to the
chromatin. We also examined localization of SYCP2 and SYCP3
proteins in Hormad12/2mice. The localization of SYCP2 and
SYCP3 to the chromatin was not significantly affected by the lack
of HORMAD1 (Figure 2D and 2F). These results indicate that
HORMAD1 is not necessary for SYCP2 and SYCP3 localization
to the axial elements.
The deficiency in synaptonemal complex proteins such as
SYCP3 is known to affect chromosome synapsis . In order to
determine the effect of HORMAD1 deficiency on chromosome
synapsis during meiosis I, we utilized CREST sera. CREST sera
labels centromeres and allows the determination of the pairing
status during meiosis . In the wild type spermatocytes, prior
to the synapsis, 40 centromeres are usually observed in the
leptotene stage. The number of visible centromeres become
reduced as the synapsis of homologues progresses. At the
completion of the synapsis in pachytene, 20 centromeric foci
are usually observed corresponding to 19 autosomal homologues
and partially paired X-Y chromosomes. We examined CREST
foci formation in Hormad12/2spermatocytes. Examination of
over 100 Hormad12/2spermatocytes and oocytes in meiosis I,
revealed greater than 20 centromeric foci in both male and
female germ cells, most containing 40 CREST foci (Figure 4A,
4D, and data not shown). These results indicate that Hormad1
deficient germ cells cannot complete homologous chromosome
pairing, and Hormad1 is therefore critical for chromosome
synapsis during meiosis.
Our experimental evidence strongly suggests that HORMAD1
localizes to the axial core and is yet another critical component of
the synaptonemal complex. To determine the effect of Hormad1
Figure2. HORMAD1 co-localizes with SYCP2 and SYCP3, but not with SYCP1. Chromosomal spread assay in wild-type (+/+) andHormad12/2
(2/2) zygotene stage spermatocytes. (A–B) Immunofluorescence staining with anti-SYCP1 (Green) and anti-HORMAD1 (Red) antibody. HORMAD1
preferentially localizes to unsynapsed regions of chromosome axes (arrow head) and sex body (arrow) but does not co-localize with SYCP1 (A-A’’).
Hormad1 deficiency does not affect SYCP1 localization to the axes (B). HORMAD1 co-localizes with SYCP2 on the unsynapsed chromosome axes (C-C’’),
but HORMAD1 deficiency does not affect SYCP2 localization to the axes (D). SYCP3, another integral and critical component of the synaptonemal
complex co-localizes with HORMAD1 on unsynapsed axes (E-E’’). Similar to SYCP1 and SYCP2, SYCP3 localizes to the axes despite HORMAD1 deficiency
(F). DNA was stained with DAPI. Scale bars: 10 mm.
HORMAD1 Is Essential for Male and Female Meiosis
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deficiency on the structure of the synaptonemal complex, we
visualized synaptonemal complexes during meiosis I in wild-type
and Hormad12/2spermatocytes using electron microscopy. In the
wild-type, synaptonemal complexes were well visualized during
the pachytene stage of meiosis (Figure 4B and 4C). Electron
microscopy examination of one hundred and ten Hormad12/2
spermatocytes from three independent experiments revealed the
lack of the typical tripartite synaptonemal complex structure
(Figure 4E and 4F). Persistence of pre-synaptic number of
centromeric foci (CREST staining) in Hormad12/2spermatocytes,
as well as non-visualization of the tripartite synaptonemal complex
structure by electron microscopy, demonstrate that HORMAD1 is
essential for chromosomal synapsis.
Hormad1 deficiency disrupts localization of proteins
important in early recombination
Previous studies have indicated that Sycp3 deficiency has subtle
effects on meiotic recombination . Early recombination events
do not seem to be disrupted in Sycp3, as similar number of DMC1
foci are present in Sycp3 mutant and wild-type meiosis .
DMC1 is a meiotic specific recombinase that together with
ubiquitously expressed RAD51 catalyzes homologous pairing and
Figure 3. HORMAD1 localization in Sycp2 mutant spermatocytes. Chromosome spread assay was performed on the wild-type (+/+) (A–C) and
Sycp22/2(D–F) zygotene spermatocytes with anti-HORMAD1 (green) and anti-SYCP2 (red) antibodies. The truncated SYCP2 protein (SYCP2t) in
Sycp22/2mice is still made and localizes to axial chromosomal cores. SYCP2t lacks the domain necessary to bind SYCP3. Scale bars: 10 mm.
Figure 4. HORMAD1 is required for chromosome synapsis during male meiosis. (A and D) Immunofluorescence with CREST (red) and anti-
SYCP3 (green) antibodies. Anti-CREST antibody recognizes chromosome centromeres. Synapsed wild-type zygotene spermatocytes contain about 20
CREST foci (n=50) (A-A’). However, Hormad12/2spermatocytes contain approximately 40 CREST foci (n=50), an indication that synapsis is disrupted
(D-D’). Arrow indicates chromosome synapses in the wild-type. Scale bars: 10 mm. (B–C, E–F) Electron microscopy analysis of synaptonemal complexes
in 2 week old wild-type and Hormad12/2spermatocyte at different magnifications. The typical, tripartite structure of the synaptonemal complex
consists of one central element (CE) that connects with two axial elements (AE) (C). We did not identify normal tripartite synaptonemal complex
structure in Hormad12/2spermatocytes. Scale bars: 1 mm.
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DNA strand exchange [30–31]. These early steps in recombina-
tion are critical for establishing the physical connections between
homologous chromosomes during meiosis. Hop1 has been
implicated in modulating the formation and processing of double
stranded breaks . We examined formation of DMC1, RAD51,
and RPA foci in zygotene stage Hormad12/2spermatocytes. There
is a dramatic decrease in the number of DMC1 foci as compared
to the wild-type (Figure 5A and 5B). We counted a total of
98.9628.2 DMC1 foci in the wild-type spermatocytes (n=50) and
9.2863.9 DMC1 foci in the Hormad12/2spermatocytes (n=45).
The number of RAD51 foci was also decreased from 189.3631.8
in the wild-type spermatocytes (n=50), to 69.3634.5 in the
Hormad12/2spermatocytes (n=40) (Figure 5C and 5D).
Following double-strand breaks formation by SPO11, RPA is
recruited together with RAD51 to the single stranded DNA regions
. We counted a total of 194.5664.5 RPA foci in the wild-type
spermatocytes (n=30) as compared to 70.1638.5 foci in the
Hormad12/2spermatocytes (n=20) (Figure 5E and 5F). These
results indicate that early meiotic recombinational events were
disrupted and not surprisingly, MLH1, a protein that forms foci in
later stages of recombination and required for the formation of most
of the crossovers(chiasmata) observed inmice, was dramatically
reduced in Hormad12/2spermatocytes (data not shown).
We also examined DMC1, RAD51 and RPA foci formation in
female meiocytes at embryonic day 15.5 (E15.5). Embryonic
ovaries contain zygotene to early pachytene oocytes at E15.5 .
We counted a total of 208.76117.1 DMC1 foci in the wild-type
E15.5 oocytes (n=50), and 79.1681.5 foci in the Hormad12/2
oocytes (n=30) (Figure 6A and 6B), a total of 197.9646.0 RAD51
foci in the wild-type oocytes (n=50) versus 85.1637.6 foci in the
Hormad12/2oocytes (n=40) (Figure 6C and 6D) and a total of
317.166135.3 RPA in the wild-type oocytes (n=50) and
51.7648.8 in the Hormad12/2oocytes (n=50) (Figure 6E and
6F). DMC1, RAD51 and RPA foci are therefore, similar to our
observations in spermatocytes, significantly decreased in Hormad1
deficient female meiocytes.
These results indicate that homologous recombination is
significantly affected in Hormad12/2mammalian germ cells, as
previously reported for HOP1 . We also observed effects of
Hormad1 deficiency on cH2AX staining (a phosphorylated form of
histone H2AX), a well known surrogate marker for DSB formation
. In the leptotene stage, phosphorylation of H2AX is induced
by SPO11 catalyzed DSBs in meiotic DNA, and cH2AX appears
as large, cloud-like patterns thatdisappearat the pachytene stage
. At the leptotene stage, cH2AX staining in Hormad12/2
spermatocytes was significantly decreased (76% decrease in signal
Figure 5. Hormad2/2spermatocytes are defective in early recombination. (A-B’) Immunofluorescence assay with anti-SYCP2 (Green) and
anti-DMC1 (Red) antibody in zygotene spermatocytes. (C-D’) Immunofluorescence assay with anti-RAD51 (Red) and anti-SYCP2 (Green) antibody in
zygotene spermatocytes. (E-F’) Immunofluorescence assay with anti-RPA (Green) and anti-SYCP2 (Red) antibody in zygotene spermatocytes. DMC1
catalyzes DNA strand exchange during recombination, and DMC1, RAD51 and RPA foci mark early recombination events. DMC1, RAD51 and RPA foci
are drastically decreased in Hormad12/2spermatocytes. Scale bars: 10 mm.
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intensity) as compared to the wild-type (Figure 7A–7D). cH2AX
staining was also significantly decreased in Hormad12/2fetal
oocytes (71% decrease in signal intensity) (Figure 6G and 6H).
These results suggest that similar to Hop1 mutants, DSBs do not
efficiently form in Hormad1 mutants.
HORMAD1 is a germ cell–specific component of the
meiotic sex chromosome inactivation complex
HORMAD1 protein localization along autosomes was previ-
ously shown to be transient  (Figure 7B and 7F). HORMAD1
staining was highest along unsynapsed chromosome axis in the
zygotene to pachytene stage and diminished significantly along
autosomes in the pachytene [17–18] (Figure 7F and 7I).
Interestingly, HORMAD1 localized strongly along desynapsed
autosomes in diplotene meiocytes . During the pachytene
stage, HORMAD1 is faintly visible along the autosomes, but co-
localizes strongly with cH2AX on the XY chromosomes, and
specifically along the axial elements [17–18]. cH2AX is a
phosphorylated form of histone H2AX, and a marker for DSBs
[36,38]. H2AX is phosphorylated throughout the chromatin in
leptotene spermatocytes and by the pachytene stage [17–18],
cH2AX staining is undetectable on autosomes and restricted to
the sex body (Figure 7E and 7F) . H2ax knockout shows the
essential role for H2AX in sex body formation and meiotic sex
Figure 6. Hormad2/2fetal oocytes are defective in early recombination. Chromosome spread assay in the wild-type and Hormad12/2E15.5
fetal ovary. A-E represent zygotene stage, G and H represent leptotene stage. (A-B’) Immunofluorescence assay with anti-SYCP2 (Red) and anti-DMC1
(Green) antibody. (C-D’) Immunofluorescence assay with anti-SYCP2 (Red) and anti-RAD51 (Green) antibody. (E-F’) Immunofluorescence assay with
anti-SYCP2 (Red) and anti-RPA (Green) antibody. (G-H’) Immunofluorescence assay with anti-SYCP2 (Green) and anti-cH2AX (Red) antibody. DMC1,
RAD51, RPA and cH2AX signals were significantly decreased in Hormad12/2fetal oocyte. DNA was stained with DAPI (Blue). Scale bars: 10 mm.
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chromosome inactivation . Meiotic sex chromosome inactiva-
tion also involves ATR and BRCA1 dependent phosphorylation of
H2AX [18,40]. Interestingly, Hormad1 deficiency, similar to H2ax
deficiency, abolishes the formation of the sex body (Figure 7G and
7H, and data not shown). The lack of sex body formation is most
likely due to the disruption of Hormad12/2spermatocytes prior to
the pachytene. We examined the cH2AX localization in the wild-
type and Hormad12/2spermatocytes. Chromatin in Hormad12/2
spermatocytes stained with anti-cH2AXantibodies, but no prefer-
ential localization to the sex chromosomes was observed (Figure 7G
and 7H). BRCA1 and HORMAD1 have recently been shown to
co-localize in the sex body . We determined whether BRCA1
localization is dependent on HORMAD1. BRCA1 protein could
not be localized in Hormad12/2spermatocytes (Figure 7I–7J, 7M).
This finding is unlike H2ax knockout, where BRCA1 is still
detected on the sex chromosomes despite the lack of the sex body
. We also examined ATR localization in wild-type and
Hormad12/2testes. In wild-type testes, HORMAD1 and ATR co-
localized in the sex body, but we could not detect ATR in
Hormad12/2spermatocytes (Figure 7K–7L, 7N). Above data
suggest that HORMAD1 may be involved in the recruitment of
BRCA1, ATR and cH2AX to the sex chromosome.
Figure 7. HORMAD1 disrupts cH2AX, BRCA1, and ATR localization to the XY chromosomes. Chromosome spread assay was performed
on wild-type and Hormad12/2leptotene (A-D), zygotene (E-H) and pachytene (I-L) spermatocytes to determine effects of HORMAD1 deficiency on
cH2AX, BRCA1 and ATR localization to the sex chromosomes. (A-D) Immunofluorescence assay with anti-SYCP2 (Green) or anti-HORMAD1 (Green)
and anti-cH2AX (Red). (E-H) Immunofluorescence assay with anti-SYCP1 (Green) or anti-HORMAD1 (Green) and anti-cH2AX (Red) antibody. (C and D)
At the leptotene stage, cH2AX and SYCP2 localization to chromatin can be detected in Hormad12/2spermatocytes, however, cH2AX is substantially
reduced. (F and H) cH2AX staining localizes preferentially to the sex chromosome in the wild-type pachytene stage spermatocytes but no preferential
localization to the sex chromosomes was observed in the Hormad12/2spermatocytes, which is not surprising since sex body does not form in
Hormad1 mutants. (G) Arrow head indicates truncated axial fiber. (I, M-M’’) BRCA1 and HORMAD1 co-localize to the sex chromosomes, but Hormad1
deficiency disrupts BRCA1 localization (J). Similarly, ATR co-localizes with HORMAD1 to the XY chromosomes (K, N-N’’), and Hormad1 deficiency
disrupts ATR localization (L). These results indicate that HORMAD1 is upstream of the currently known critical components of the meiotic sex
chromosome inactivation complex, cH2AX, BRCA1 and ATR. DNA was stained with DAPI (Blue), and arrows indicate sex chromosomes. Scale bars:
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Since ATR, BRCA1 and cH2AX are involved in the
transcriptional silencing of sex chromosomes, we examined
whether Hormad1 deficiency affects transcriptional repression.
Previous studies have shown that H2ax and Brca1 deficiencies
individually, lead to the over-expression of genes exclusively
expressed from the X or Y chromosome . We examined
whether X-linked germ cell–specific genes were over-expressed in
Hormad12/2testes compared to wild-type testes. We performed
quantitative real-time PCR on select autosomal genes (Hormad2,
Rnh2 and Mov10l1) as well as X-chromosome derived genes
(Usp26, Fthl17, Pramel3, Tex11, and Tex13) on wild-type and
Hormad12/2testes (Figure 8A–8H). Rnh2 and Mov10l1 are germ
cell–specific transcripts, derived from autosomes, and were not
differentially expressed between wild-type and Hormad12/2testes
(Figure 8B and 8C). In contrast, all of the germ cell–specific
transcripts transcribed from the X chromosome were significantly
elevated in Hormad12/2testes over the wild-type. These include
Usp26 (4.5 fold increase), Fthl17 (6.5 fold increase), Pramel3 (3.5
fold increase), Tex11 (2.2 fold increase), and Tex13 (2.8 fold
increase) (Figure 8D–8H). Moreover, RNA expression microarray
analyses comparing two week old Hormad1 deficient testes with
corresponding wild-type, indicate that almost 20% of the up-
regulated genes derive from the X chromosome (Figure 8I). Our
results are remarkably similar to transcriptional de-repression
observed in H2ax and Brca1 mutants [39–40], and indicate that
HORMAD1 is a germ cell and meiosis specific factor critical in
meiotic sex chromosome inactivation and transcriptional silencing.
Hormad1 deficiency abrogates ATM autophosphorylation
HORMAD1 is a likely mammalian counterpart to the yeast
HORMA domain meiotic protein, Hop1. Hop1 is phosphorylated
by Mec1/Tel1, the budding yeast homologue to the mammalian
ATR and ATM kinases, and this phosphorylation is thought to
play an important role in inter-homologous recombination .
We therefore examined HORMAD1 expression in Atm deficient
mice as well as ATM expression in Hormad12/2animals. ATM is a
serine/threonine-specific protein kinase that has been associated
with cell cycle regulation, apoptosis, and response to DNA damage
repair. ATM kinase activation is associated with increased auto-
phosphorylation of ATM at multiple sites including serine 1981
. We examined HORMAD1 protein expression in testes
between postnatal days 5–21 (Figure 9A). Eight to ten day old
testes contain spermatogonial cells as well as preleptotene/
leptotene spermatocytes . At postnatal day 14–18, testes
contain pachytene spermatocytes and visible sex bodies .
HORMAD1 is known to be phosphorylated  (Figure 9B).
Western blot analysis on testes extracts detected phospho-
HORMAD1 beginning at postnatal day 14 (Figure 9A). Phos-
pho-HORMAD1 protein was decreased in postnatal day 18 and
21 wild-type testes (Figure 9A). Phospho-HORMAD1 appearance
correlates temporally with sex body formation. HORMAD1
phosphorylation was not affected by Atm deficiency (Figure 9A).
These results indicate that ATM is not responsible for HOR-
MAD1 phosphorylation. We also examined HORMAD1 locali-
zation in Atm2/2spermatocytes. We observed HORMAD1
localization to chromosomal axes in Atm deficient spermatocytes,
as previously described by others  (Figure 9C and 9D). Since
synaptonemal complexes do not form in Atm mutants, HOR-
MAD1 association with unsynapsed chromosomes does not
require ATM. The anti-ATM phospho-S1981 antibody did not
detect phosphorylated ATM in Hormad12/2testes (Figure 9E and
9F). These results suggest that HORMAD1 is upstream of ATM
auto-phosphorylation, and therefore likely upstream of ATM
Hormad1 deficiency does not affect gross ovarian
We have previously shown that Hormad1 RNA expression in the
ovary was confined to the germ cell . Meiosis I in the female
gonad commences circa E13.5 and most oocytes arrest at the
dictyate stage by the time of birth. Antibodies against HORMAD1
recognized HORMAD1 protein at E14.5 (leptotene) and E18.5
(arrest in diplotene) oocytes (Figure 10A and 10B), but little
HORMAD1 protein was detected in the newborn ovary oocytes
(Figure 10C), at the time when oocytes are arrested in diplotene.
Deficiency in genes critical in meiosis can disrupt early ovarian
development, as is the case for Dmc1, Msh5, Spo11 and Atm
We therefore examined ovarian development in Hormad12/2
females. Antibodies against germ cell–specific transcriptional
regulator SOHLH1  stained wild-type and knockout oocytes
throughout embryonic gonadal development with no significant
differences noted (Figure 10A–10F). Moreover, the numbers of
primordial, primary and secondary follicle counts did not
significantly differ between the mutant and wild-type ovaries at
post-natal day 8 (Figure S4). These results indicate that Hormad1
deficiency does not affect embryonic ovarian development, germ
cell cyst breakdown, and primordial follicle formation. We also
examined the histology of wild-type and Hormad12/2ovaries
between 2 and 30 weeks of life. Mice reach sexual maturity around
6 weeks of life, and mouse ovaries at this time consist of all of the
follicular types including corpora lutea, an indication that the
ovaries are ovulating. Postnatal Hormad12/2ovaries were grossly
indistinguishable from wild-type mice between 2 and 30 weeks of
life, with abundant corpora lutea in the Hormad12/2ovaries
indicating that the normal process of oocyte maturation was not
disrupted, and ovulation has occurred (Figure 11A–11H). We
induced superovulation in knockout and wild-type mice with
exogenous gonadotropins to determine whether subtler ovarian
defects contributed to infertility in Hormad12/2mice. Hormad12/2
females super-ovulated 28611 eggs (n=22), while wild-type
animals superovulated 29614 eggs (n=13). We therefore did
not observe significant difference between the number of eggs
superovulated from wild-type versus knockout mice. These results
indicate that ovarian development is grossly normal in Hormad12/2
mice, and that ovarian defects are unlikely to account for observed
Hormad1 deficient eggs fertilize and embryonic
development arrests at blastocyst stage due to
We studied early embryonic development of fertilized Hor-
mad12/2oocytes because ovarian defects were unlikely to explain
observed infertility. We recovered embryos from wild-type male
matings with Hormad12/2females at E0.5, E1.5, E2.5, and E3.5.
Comparable numbers of morphologically indistinct 1-cell zygotes
were recovered from oviducts of control and mutant female mice
at E0.5 (Figure 12A–12C), and little difference was noted at the 2-
cell stage, except for an increased number of 1-cell embryos in the
knockouts, indicating a lag in the progression from the 1-cell to 2-
cell stage (Figure 12D–12F). By E2.5, the number of normal
appearing 8-cell stage embryos was significantly less in Hormad12/2
fertilized eggs as opposed to the wild-type (Figure 12G–12I), and
no morphologically normal blastocysts were observed in the
Hormad12/2fertilized eggs at E3.5. It is interesting to note that at
E3.5, a significant number of 4 and 8 cell stage embryos were
observedinthe knockoutwhileonlyblastocysts wereobservedinthe
wild-type E3.5 embryos (Figure 12J–12L). We also tested, using
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Figure 8. Sex chromosome expressed transcripts escape meiotic sex chromosome inactivation in Hormad2 2/ /2 2spermatocytes. (A–H)
Quantitative real time PCR analyses show that expression of testes specific genes derived from the autosome, Hormad2, Rnh2, and Mov10l1, did not
significantly differ between the wild-type and the Hormad12/2testes (A-C). However, X chromosome linked, testis specific genes, Usp26, Fthl17,
Pramel3, Tex11 and Tex13 transcript were 2–6 fold increased (D-H). (I) Microarray analysis shows preponderance of X and Y derived transcripts among
the top 38 up-regulated genes in Hormad2/2testes. Data are normalized to b-actin expression and presented as the mean relative quantity
(compared to wild-type) with error bars representing the standard error of mean. Student’s t test was used to calculate the P values.
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Chicago Sky Blue 6B dye (Sigma, MO, USA) injection into the tail
vein , whether blastocysts derived from Hormad12/2fertilized
eggs could implant. We did not detect implantation of Hormad12/2
fertilized eggs (Figure 12M). These results indicate that a defect in
early embryogenesis led to premature loss of embryos.
Hormad1 deficiency causes aneuploidy and early
We hypothesized that aneuploidy is the major cause of embryo
wastage in Hormad12/2females. Unlike meiosis in testes, errors
during oocyte meiosis have milder effect on oocyte loss and
apoptosis, as has been observed in Sycp3 and Smc1b knockouts
[27,29,47]. The presence of growing oocytes in the Hormad12/2
ovaries, as opposed to early germ cell loss observed in testes during
pachytene stage of meiosis, indicates greater tolerance of Hormad1
deficiency in the oocytes as compared to spermatocytes. The whole
range of ovarian follicle types are present in the Hormad12/2
ovaries, including primordial follicles, primary, secondary and
antral follicles. In contrast to other critical genes during meiosis,
such as Dmc1, Msh5, Spo11 and Atm that cause early ovarian failure
Hormad1 deficiency does not activate apoptotic pathways and does
not lead to gross premature loss of oocytes. We used chromosome
specific mouse BAC (bacterial artificial chromosome) clones to
perform fluorescent in situ hybridization (FISH) on germinal vesicle
(GV) oocytes, 2 cell and 8 cell stage embryos to determine whether
aneuploidy is significantly higher in Hormad12/2embryos. Wild-
type GV oocytes are arrested in meiosis I and contain bivalent
chromosomes that consist of four chromatids. Examination of 58
GV oocytes from three independent knockout animals and 112
GV oocytes from three wild-type animals with BAC probes
specific for chromosomes 19, 18 and X, revealed no significant
differences, with presence of four chromatids for each of the
chromosome examined as expected (Figure 13A–13C). Following
fertilization and completion of meiosis II, each cell in the
developing embryo should contain two chromosomes except for
the sex chromosomes. We examined by FISH, mouse chromo-
somes 6 and X in the 2-cell wild-type and Hormad12/2embryos.
Sixty three cells examined for chromosome 6 in the Hormad12/22-
cell embryos revealed that 22% of the cells had 4 signals
corresponding to chromosome 6, 35% had 3 signals corresponding
to chromosome 6, 24% had 1 signal, and 5% had no signal. Wild-
type 2-cell embryos were also examined by chromosome specific
FISH, and 44 cells examined out of 46 showed only 2 signals, as
expected. In 2-cell Hormad12/2embryos, out of sixty three cells
examined for chromosome X, 44% showed four, three or no
signal. Wild-type 2-cell stage embryos, as expected, showed only 2
or 1 signal, consistent with either XX or XY sex of the cell. These
results indicate widespread hypo and hyperdiploidy in 2-cell
embryos (Figure 13D–13F).
We also examined cells from the 8-cell stage embryos. A total of
84 cells from the Hormad12/2embryos were examined with FISH
probes specific for chromosomes 11, X and 2. Hormad12/2cells
from the 8-cell stage, hybridized with BAC specific to chromosome
11, showed 4 signals in 10% of the cells, and 3 signals in 55% of
the cells. Therefore, a total of 65% of the cells were either
monosomic or trisomic for chromosome 11. In the wild-type 8 cell
stage embryo, a total of 96 cells were scored for chromosome 11,
and only 2 out of 96 (2%) cells showed a single signal, while 94 out
of 96 cells (98%) showed two signals as expected. Similar results
were obtained for chromosome 2 (Figure 13G–13I). Our results
indicate widespread aneuploidy involving different chromosomes
in both 2-cell and 8-cell embryos. Such aneuploidy causes early
embryo demise and failure of implantation.
We also visualized second metaphase (M2) spindle using anti-b-
tubulin antibodies in Hormad12/2oocytes. During M2, sister
chromatids migrate to the opposite pole to form functional,
haploid gametes. Sister chromatids are bi-oriented on the spindle
and congregate in the center of the spindle prior to separation
(Figure 13J). Each sister kinetochore in a pair is attached to the
opposite spindle, and such arrangement generates sufficient
tension to cause proper segregation. M2 in Hormad12/2oocytes
is grossly disrupted with mis-orientation of chromatid attachment
to the spindle observed in all M2 oocytes examined (Figure 13K)
Figure 9. HORMAD1 phosphorylation is unaffected by Atm
deficiency while Hormad1 deficiency disrupts ATM autopho-
sphorylation. (A) Western blots analyses with anti-HORMAD1 specific
antibodies show that HORMAD1 protein and its phosphorylated form
(*) appear circa post-natal day 14. Atm1 deficient testes (Atm2/2)
express both forms of HORMAD1 and HORMAD1 is therefore unlikely to
be ATM1 substrate. (B) The presumed phosphorylated form of
HORMAD1 (higher molecular weight band indicated by asterisk)
decreases in intensity after protein phosphatase I (PPI) treatment. (C
and D) Chromosome spread assay in wild-type (Atm+/+), and Atm
deficient spermatocytes (Atm2/2). HORMAD1 preferentially localizes to
unsynapsed regions of chromosome axes (C), and HORMAD1 antibody
stains unsynapsed chromosome axes in Atm2/2spermatocytes (D)
more intensely than in the wild-type spermatocytes. Scale bars: 10 mm.
(E and F) Immunohistochemistry analysis in 6 week old wild-type and
Hormad12/2testes with anti-phospho ATM-S1981 antibody. Phospho-
ATM S1981 was detected in wild-type zygotene, but not detected in
Hormad12/2zygotene spermatocytes. Scale bars: 50 mm.
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(n=50). These results indicate importance of Hormad1 in proper
spindle formation and segregation.
We discovered Hormad1 (Nohma), using in silico screen to identify
germ cell–specific transcripts critical for gonadal development .
We showedthat Hormad1 expression was confined to germcells, and
that Hormad1 transcripts were mostly concentrated in the pachytene
spermatocytes and early oocytes. Here we show that Hormad1 is
essential for both male and female fertility in the mouse. HORMA
domain-containing proteins interact with chromatin, particularly
chromatin associated with DNA adducts, and are critical mitotic
spindle checkpoint proteins . HORMA domain-containing
proteins are critical regulatory proteins for mitosis as exemplified
by MAD2 protein, and meiosis as exemplified by Hop1p , and
Red1p  in yeast, Him-3  in nematodes, and Asy1  in
plants. The 205-aa HORMA domain in the mouse HORMAD1,
shares highest similarity to the Saccharomyces cerevisiae Hop1 protein
(28% amino acid similarity). Yeast Hop1 mutants have defects in
chromosome condensation, synapsis and recombination [4,8], and
Hop1p binds DSBs during meiotic prophase and appears to play an
important role in interhomologue recombination. We previously
hypothesized, based on homology to Hop1 in yeast, that Hormad1
Figure 10. HORMAD1 is expressed in embryonic but not post-natal oocytes. (A–C) HORMAD1 protein was detected by
immunofluorescence in the embryonic ovary at E14.5 (A), E18.5 (B), but not in the newborn ovary (C). (D–F) Oocytes in Hormad12/2ovary (2/2)
were stained with antibodies against the germ cell–specific transcriptional regulator SOHLH1 at E14.5 (D), E18.5 (E), and newborn ovary (F). DNA was
stained with DAPI (Blue). Scale bars: 20 mm.
Figure 11. Histological analysis of wild-type and Hormad1–/–
does not show gross histological differences. Wild-type and Hormad12/2ovaries are shown at different post-natal ages that show the full range of
ovarian follicles: primordial follicles (PF), primary follicles (PrF), secondary follicles (SF), antral follicles (AF) and corpus luteum (CL). Scale bars: 50 mm.
–/–ovaries. (A–H) PAS staining of wild-type (+/+) and Hormad12/2(2/2) ovaries
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Figure 12. Hormad1–/–
involving Hormad12/2oocytes fertilized by wild-type males (M+/+-F2/2), were collected at E0.5, E1.5, E2.5 and E3.5 for the analysis of the early
embryonic development. (A-C) No significant differences were noted between wild-type and Hormad12/2at E 0.5, with normal appearing one cell
embryos and polar bodies visible in both. (D-F) At E1.5, there is an excess of 1 cell embryos in Hormad12/2fertilized oocytes, indicating a lag in
embryo development at this stage. (G-I) At E2.5 there is a significantly less number of eight cell embryos formed in Hormad12/2fertilized oocytes as
opposed to the wild-type. A large proportion of 8-cell embryos appear disorganized and apoptotic. (J-L) Blastocysts form at E3.5 in wild-type but very
few normal blastocysts are visible in Hormad12/2embryos. The count only includes morphologically normal blastocysts (L). Error bars represent
standard error of the mean. Student’s t test was used to calculate P values (*:P,0.5, **:P,0.001). (M) Embryos derived from Hormad12/2fertilized
oocytes can not implant, as visualized by Sky Blue dye injected into the tail vein of pregnant females at E6.5 gestational age.
–/–oocyte derived embryos arrest at blastocyst stage. Embryos derived from wild-type matings (M+/+-F+/+) and matings
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plays an important role in mammalian meiosis. Our results indicate
that HORMAD1 is the mammalian homologue to the Hop1
protein in yeast.
Our current studies, and those of others, have shown that
HORMAD1 protein is confined to germ cells and co-localizes with
synaptonemal complex proteins SYCP2 and SYCP3 on the
chromatin as part of the axial core [27,48]. Synaptonemal
complex is a complex structure composed of multiple germ cell–
specific and ubiquitously expressed proteins that connect paired
homologous chromosomes. Similar to railroad tracks, the
Figure 13. Hormad1–/–
have replicated to produce identical sister chromatids, which are visualized as closely located double signals for examined chromosomes 18, 19 and
X. (C) Histogram of individual chromosomes, does not show significant difference between the wild type and mutants. Fifty eight knockout GV
oocytes and 112 wild type GV oocytes were scored. Abbreviations stand for the following: tetra-Tetrasomy (blue), tri-Trisomy (red), di-Disomy (green),
mono-Monosomy (purple), zero-Zerosomy (grey). (D-F) E1.5 day (2-cell stage embryo) FISH assay using chromosomes 6 (green) and X-specific DNA
probes (red). The wild type embryos were diploid for 6 and X (D). Trisomy for chromosome 6 (arrows) is noted in the knockout (E). In addition,
chromosomes 6 and X demonstrate double hybridization signals, the same FISH pattern as observed during the GV stage, and indicative of sister
chromatids. (F) Histograms for individual chromosomes show high prevalence of chromosomal aneuploidies in embryos derived from mutant
oocytes. We scored 63 knockout and 46 wild type cells. (G-I) E2.5 day (8-cell stage embryo) FISH assay with chromosomes X (red), 11 (red) and 2
(yellow) specific probes. (G) A hybridization pattern of a normal diploid number of chromosomes in the wild type embryo. (H) Trisomy for
chromosome X (arrow), as well as double signals likely produced by sister chromatids on chromosome 2 and 11 were observed in embryos derived
from knockout eggs. (I) Histogram shows substantial number of trisomies and monosomies in the knockout as compared to the wild type. We scored
84 wild type and 96 knockout cells. (J and K) Immunofluorescence with b-tublin antibody (red) in wild-type and Hormad2/2MII stage oocytes.
Hormad2/2oocytes are grossly disrupted with mis-orientation of the chromatid attachment to the spindle. DNA was stained with DAPI (Blue).
–/–fertilized oocytes show widespread hypo- and hyperdiploidy. (A-B) In GV stage oocytes, parental chromosomes
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synaptonemal complex axial lateral elements (SYCP2, SYCP3) are
connected to each other by proteins known as transverse filaments
(SYCP1, TEX12) . The axial/lateral elements play critical roles
in chromosome condensation, pairing, and repress recombination
pathways that involve sister chromatids . Synaptonemal
complex formation is associated with HORMAD1 depletion from
the axes [17–18]. Our data show that HORMAD1 phosphory-
lation peaks at the time when HORMAD1 localization shifts to
the sex body. ATM and ATR do not appear to be involved in
HORMAD1 phosphorylation and binding to chromosomal axes.
Previous study showed that Trip13, a homologue of yeast Pch2
kinase, may be involved in HORMAD1 depletion from synapsed
chromosome axes . Kinases, such as Mec1/Tel1 in yeast are
important to effect inter-homologous recombination  by
phosphorylating Hop1, but not much is known regarding
HORMAD1. Future studies are necessary to understand HOR-
MAD1 phosphorylation sites and responsible kinases.
Although HORMAD1 is not essential for the binding of well-
characterized germ cell–specific synaptonemal complex proteins
such as SYCP1, SYCP2 and SYCP3, HORMAD1 deficient
spermatocytes are defective in synapsis and do not form
recognizable synaptonemal complexes. Hormad1 deficiency causes
a male meiotic arrest, that is similar to male meiotic arrests
observed in other components of the synaptonemal complex and
recombination including: Atm, Spo11, Sycp1, Sycp2, Sycp3 and Dmc1
[27,30–31,48–50]. These findings are not surprising as HOR-
MAD1 co-immunoprecipitates with known axial proteins, SYCP2,
SYCP3, REC8, and SMC1b . Whether these interactions are
biologically significant, and whether HORMAD1 interacts with
other members of the synaptonemal complex, including newly
discovered HORMAD 2 protein, is unclear. Interestingly, Hop1
interacts with another phosphoprotein, Red1 [35,51], and whether
HORMAD2 is the mammalian counterpart of Red1 remains to be
Like its counterpart in yeast, Hop1, Hormad1 deficiency disrupts
early and later stages of recombination as shown by the drastic
diminution in the formation of DMC1, RAD51, RPA and MLH1
foci. These findings differ from the Sycp3 mutant, which affects
later stages of recombination . DMC1 is a meiotic specific
recombinase that together with ubiquitously expressed RAD51,
catalyzes homologous pairing and DNA strand exchange, and
marks earlier stages of recombination than MLH1 foci .
HORMAD1 expression is not significantly affected in Dmc1
mutant spermatocytes . These results indicate that unrepaired
DSBs and synaptonemal complexes between non-homologous
chromosomes seen in Dmc1 mutants, do not affect HORMAD1
localization. In Hop1 mutants, DMC1 foci are only faintly detected
when compared to controls . These findings have led to a
suggestion that Hop1 binds at or near the sites of DNA double
strand break and modulates the action and perhaps recruitment of
recombinases such as DMC1 and RAD51 . Moreover, Hop1
appears to be part of the meiosis specific surveillance system that
monitors meiotic double stranded break repair . Our
observations support the possibility that HORMAD1 affects early
recombination and may therefore perform similar functions in the
During mammalian meiosis, X and Y chromosomes are
inactivated and transcriptionally silenced (meiotic sex chromosome
inactivation, MSCI) and chromatin condenses to form a sex (XY)
body. The epigenetics of MSCI involves cH2AX, BRCA1 and
ATR at a minimum. The most intriguing finding regarding
HORMAD1 is its co-localization with BRCA1, ATR and cH2AX
in the sex chromosomes during pachytene . Hormad1
deficiency, similar to H2ax deficiency, abrogates formation of the
sex body, as well as cH2AX localization to the sex chromosomes.
Hormad1 deficiency also abrogates BRCA1 and ATR localization
to the sex chromosomes. Interestingly, H2ax deficiency does not
affect BRCA1 localization to the sex chromosome, despite
anomalous sex body, while BRCA1 deficiency does affect both
ATR and cH2AX localization to the sex body. These findings
indicate that HORMAD1 is involved in the initial recruitment of
BRCA1, ATR and cH2AX to the sex body. The mechanism
whereby HORMAD1 affects ATR, cH2AX and BRCA1
localization is unclear, but our results are consistent with the
conclusion that HORMAD1 is upstream of ATR, cH2AX and
BRCA1, and establishes HORMAD1 as an essential component
of the MSCI complex. Moreover, we have shown here that
Hormad1 deficient testes preferentially over-express X-linked genes
as observed in mice deficient for other components of the MSCI
complex such as cH2AX and BRCA1 . Almost 20% of genes
preferentially up-regulated from the Hormad12/2testes are derived
from the X chromosome. Hormad1 is therefore the first germ cell–
specific component known to play a role in MSCI.
Unlike males, meiosis I in females arrests during the embryonic
development at the diplotene stage and is completed upon
ovulation. The lack of dramatic germ cell apoptosis during
ovarian follicle development has also been observed for Sycp2 and
Sycp3 deficient mice [48,52], and reiterates the laxity of oocytes to
early meiotic errors, as opposed to spermatogenesis, where meiotic
check point causes the dramatic phenotype observed in male
gonads. Hormad1 deficiency does not affect folliculogenesis nor
ovulation. These findings are unusual, given that Hormad1 affects
recombination, and recombination associated proteins DMC1 and
MLH1 are important in early oogenesis. Unrepaired recombina-
tion intermediates or defects in homologous chromosome pairing
and synapsis are likely causes of early oocyte loss . Spo11
deficient animals can ameliorate observed oocyte losses in
recombination defective mutants, presumably due to the failure
of Spo112/2deficient mice to form DSBs that initiate meiotic
recombination. Spo11 deficiency does not affect HORMAD1
association with the chromosome axes , a finding similarly
observed for Hop1 localization in Spo112/2yeast . Moreover,
Hop1 mutants are required for full levels of DSBs formation
[37,54]. It is therefore possible that early loss of oocytes in Hormad1
deficient animals does not occur in part because unrepaired DSBs
do not form. Reduced staining of cH2AX in Hormad1 mutant
leptotene stage oocytes argues that unrepaired DSB formation is
indeed reduced. However, unlike Spo112/2ovaries that have
abnormal folliculogenesis, Hormad12/2ovaries are not defective in
folliculogenesis and other mechanisms must protect Hormad1
mutant oocytes from early loss.
Infertility in Hormad12/2females is due to early embryo demise.
This is in stark contrast to males, where spermatocytes are
eliminated due to the pachytene defect. The greater ability of
oocytes to tolerate meiosis I errors is well known , and
exemplified by Sycp3 and Smc1b knockouts [52,56], which have
defects in synapsis and recombination. Mechanisms that underlie
oocyte’s insensitivity to such errors are not well understood .
Embryos derived from Hormad12/2oocytes fertilized with wild-
type sperm, cannot implant. Examination of post-fertilization
events in fertilized Hormad12/2
abnormalities in early embryo development, including growth lag,
and severe disruption in development prior to the blastocyst stage.
Very few blastocysts were produced in fertilized Hormad12/2
oocytes, and most were abnormal in morphology. We utilized
FISH hybridization against specific chromosomes to show wide-
development. These results are reminiscent of Sycp3 and Smc1b
oocytes, showed significant
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knockout embryos. SYCP3 and SMC1b are involved in the
synaptonemal complex formation, are germ cell–specific, and
implicated in human aneuploidy. Sycp3 deficient females are
subfertile, but one third of the embryos die in utero due to
aneuploidy . Smc1b encodes a meiosis specific component of
the cohesin complex, important in sister chromatid cohesion and
recombination . Smc1b deficient females are sterile, like
Hormad12/2females, and meiosis continues until metaphase II
However, unlike Smc1b2/2, Hormad12/2ovaries do not lose
oocytes by 6 months of age, and postnatal oocyte counts do not
differ significantly from the wild type. Sycp32/2ovaries show
significant decline in the primordial follicle pool at postnatal day 8,
indicating accelerated apoptosis of oocytes as germ cell clusters
break down to form primordial follicles . DSBs, as indicated
by the assembly of the histone variant cH2AX, form in Smc1b2/2
and Sycp32/2meiocytes and may lead to higher rate of oocyte loss
in these mutants [47,52]. Hormad1 deficient ovaries do not display
loss of primordial follicles. Hormad12/2ovaries may not be losing
oocytes at an accelerated rate due to our observations that DSBs,
as assessed by cH2AX staining, are substantially reduced in
Hormad1 mutants. Similar to Hop1 mutants in yeast, lack of
HORMAD1 may derepress Dmc1-independent inter-sister repair
pathway, resultinginefficient DNAbreakrepairs . The reduced
DSBs in Hormad12/2meiocytes, and de-repression of Dmc1-
independent inter-sister repair pathways, may not affect regular
apoptotic pathways that eliminate substantial number of oocytes
during germ cell clusters breakdown to form primordial follicles at
the time of birth, resulting in no decrease in Hormad12/2primordial
Materials and Methods
All murine experiments were carried out on the 129S7/
SvEvBrd x C57BL/6 hybrid background. Litters were weaned at 3
weeks and breeding pairs set up at 6 weeks of age. One mating
pair was placed per cage and inspected daily for presence of litters.
All experimental and surgical procedures complied with the Guide
for the Care and Use of Laboratory Animals and were approved
by the Institutional Agricultural Animal Care and Use of
Committee of Baylor College of Medicine and the Institutional
Animal Care and Use of Committee of University of Pittsburgh.
Targeting construct, generation of transgenic mice, and
Targeting construct, electroporation and ES cell selection was
done as previously described [60–61]. Exons 4 and 5 of the mouse
Hormad1 gene were replaced with a neomycin resistance gene
flanked by 5.3 kb (59) and 6.2 kb (39) homologous sequences
(Figure S1A). Targeted ES cells were verified by Southern blotting,
and microinjected into the C57BL/6 blastocysts to produce
chimeric mice that carried the mutation into the germ line. These
mice were mated with C57BL/6 wild-type mice to generate
Hormad1 heterozygous animals that were subsequently crossed to
produce F2offspring for functional analysis. PCR genotyping was
performed using the following primers: WT-forward (59- TCAA-
GACCAACCTGGGCTAC -39) and WT-reverse (59- CCA-
TGTGGGTTGTAGGGAGT -39) to amplify a 196-nucleotide
wild-type band, and KO- forward (59- TCAAGACCAAC-
CTGGGCTAC -39) and KO- reverse (59- GGGGAACTTCCT-
GACTAGGG -39) to amplify a 505-nucleotide mutant band
Histology and immunohistochemistry analysis
Testes were fixed in Bouin’s solution (Sigma-Aldrich, MO,
USA) and ovaries were fixed in 10% buffered formalin or 4%
paraformaldehyde. Fixed tissues were embedded in paraffin,
serially sectioned (5 mm) and stained with hematoxylin and eosin
or with Periodic Acid Schiff (PAS). At least five pairs of testes and
ovaries of each genotype were subjected to gross and microscopic
analyses for each time point. Germ cell cysts, primordial, primary,
and secondary follicles were defined as described . We used
antibodies against SOHLH1 , PLZF ( sc-22839, Santa Cruz,
CA,USA), DDX4 (ab13840, Abcam, MA, USA), ATM phospho-
S1981 (05-740, Millipore, CA, USA), and HORMAD1 proteins.
Affinity purified, anti-HORMAD1 guinea pig and rabbit anti-
bodies, were generated against part of HORMAD1 protein
(amino acids 23-373) at Cocalico Biologicals (Lancaster, PA,
Meiotic chromosome analysis and immunostaining
Oocytes and spermatocytes for chromosome analyses were
prepared essentially as described previously [62–63]. Ovaries and
testes were incubated in trypsin-EDTA solution at 37uC for
15 min and washed briefly in PBS. Trypsinized ovaries and testes
were pipetted repeatedly and centrifuged, followed by resuspen-
sion in PBS. Cell suspensions were placed on poly-L-lysine-coated
slides containing 120 mM sucrose solution and 0.05% Triton X-
100. The slides were fixed in 2% paraformaldehyde and 0.02%
SDS for 1 hour at room temperature, washed in distilled water
and air dried, and stored at 280uC before use. Immunostaining
was preformed as described [62–63]. Slides were incubated with
the primary antibody overnight at 4uC, washed with PBS and
incubated with Alexa-488 and Alexa-595 (Invitrogen, CA, USA)
secondary antibody (1:300 dilutions) for 1 hour at room
temperature. After washing with PBS, slides were mounted using
VECTASHIELD medium with 4,6-diamidino-2-phenylindole
(DAPI) (Vector Laboratories, CA, USA). SYCP2 rabbit and
guinea pig immunoaffinity-purified antibody , DMC1 (sc-
8973, Santa Cruz, CA, USA), RAD51 (ab1837, Abcam, CA,
USA) and RPA (ab87272, Abcam, CA, USA) were used. Dr.
Christer Ho ¨o ¨g (Karolinska Institutet, Sweden) kindly provided
SYCP1 and SYCP3 rabbit immunoaffinity-purified antibodies.
Dr. William R. Brinkley (Baylor College of Medicine, TX, USA)
kindly provided anti-CREST human serum.
Quantification of the immunofluorescence signal
Wild-type and mutant oocyte and spermatocyte spreads were
stained at the same time with the same mixture of antibodies. In
each experiment, when comparing wild type and mutants,
imaging of the cells was performed on the same day with the
same microscope and camera settings. PerkinElmer Volocity
software 5.3 was used to control for possible changes in
illumination during the course of imaging and measurement of
the immunofluorescence. We measured total immunofluorescence
of cH2AX in identical-sized rectangles that were placed over the
cell boundaries. Fifty individual leptotene stage spreads from wild
type, mutant spermatocytes and oocytes were subjected to
immunofluorescence analysis. A two-tailed non-parametric Wil-
coxon–Mann–Whitney two-sample rank-sum test was used for
Electron microscopy analysis
Testes were dissected into ,0.25 cm pieces and fixed in 1x PBS
with 2% formaldehyde and 3% glutaraldehyde (Ted Pella Inc.,
CA, USA), for 2 hr at room temperature. Samples were treated
HORMAD1 Is Essential for Male and Female Meiosis
PLoS Genetics | www.plosgenetics.org16November 2010 | Volume 6 | Issue 11 | e1001190
with 0.5% uranyl acetate and osmium tetraoxide, dehydrated with
ethanol, and embedded in LX-112 medium (Ladd Research
Industries, VT, USA). The samples were polymerized in a 70uC
oven for 2 days. Ultrathin sections (70 –100 nm) were cut in a
Leica Ultracut microtome (Leica, IL, USA), stained for 5 min in
1% aqueous uranyl acetate and 2 min in 1% aqueous lead citrate
at room temperature in a Leica EM Stainer, and examined by a
JEM 1010 transmission electron microscope (JEOL, MA, USA) at
an accelerating voltage of 80 kV. Digital images were obtained
using AMT Imaging System (Advanced Microscopy Techniques
Corp, MA, USA).
Western blot analysis and phosphatase treatment
Western blot analysis was performed essentially as described
previously . Postnatal day 5, 8, 14, 18 and 21 testes were
collected and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1%
NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM
EDTA with or without protease inhibitor cocktail (Roche)). Total
protein (20 mg) was loaded onto the SDS–PAGE gel, transferred to
nitrocellulose membrane, and immunoblotted with HORMAD1
rabbit antibody (1:5000 dilutions). Varying amounts of protein
phosphatase I (Sigma P7937, MO, USA) were incubated with
20 mg of 18 day old wild-type total testes lysates, for 30 min on ice.
Phosphatase treated testes lysates were electrophoresed, trans-
ferred to nitrocellulose membrane and immunoblotted with anti-
HORMAD1 rabbit antibody.
RNA isolation, RT-PCR, quantitative real-time PCR (Q-
PCR), and microarray analysis
RT-PCR and quantitative real time PCR were performed as
described previously [45,65]. We used previously published
oligonucleotide sequences corresponding to Rnh2, Mov10l1,
Pramel3, Usp26, Fthl17, Tex11, Tex13 , and Hormad1 (forward
59 CTGCTGACACCAAGAAAGCA 39- and reverse 59- CC-
TGGTGGTTGGTAATCTGG -39) and Hormad2 (forward 59
GCTCATCAGGGGCTAGACTG 39- and reverse 59- TGG-
TTCGCTGACCTTCTTCT -39) primers. Q-PCR was per-
formed using the SyberGreen PCR Master Mix (Bio-Red, CA,
USA) and probe set specific for each gene under investigation.
Each sample was analyzed in triplicate from at least three
independent wild type and Hormad12/2testes cDNA samples. The
relative amount of transcripts was calculated by the DDCT
method as described by Applied Biosystems, and normalized to b-
actin. The average and standard errors were calculated for the
triplicate measurements, and the relative amount of target gene
expression for each sample was plotted. Student’s t test was used to
compute the P values. Significance was defined as a P value,0.05.
RNA expression arrays on wild-type and Hormad12/22 week
old testes were performed on the Illumina BeadChip MouseWG-6
2.0 arrays and analyzed as previously described (GEO accession
numbers, GSE21524) [67–68].
Superovulation and oocyte collection in mouse
Mouse oocytes and pre-implantation embryos were collected by
using standard protocols for timed mating . Briefly, 4 to 6
weeks female mice were superovulated by injection of 5 IU of
pregnant mare serum gonadotropin (PMSG) (Prospec, Rehovot,
Israel) and 48 h later 5 IU of human chorionic gonadotropin
(hCG) (Sigma, MO, USA). Females were placed individually with
10-week old males immediately after the injection of hCG.
Collection of one cell, two cell, four cell, eight cell, and blastocyst
stages was performed at 24 h, 42 h, 68 h, and 96 h after hCG
injection, by flushing the oviducts and uterine horns with HEPES-
buffered media under the microscope. Removal of the cumulus
cells was achieved in HEPES-buffered media containing 1 mg/ml
hyaluronidase (Sigma, MO, USA).
Oocytes and embryos were exposed to a hypotonic solution
containing 0.1% sodium citrate, 0.1% bovine serum albumin for
about 15 min and gently transferred onto the slides. Cells were
fixed with fresh methanol: acetic acid (3:1) solution dispensed
carefully over the surface of the slide. The slides were air-dried and
dehydrated at room temperature in a series of 70%, 85% and
100% ethanol solutions before FISH analysis.
Twenty BAC (bacterial artificial chromosomes) clones from a
mouse RPCI-23 library representing sequences unique to specific
chromosomes were selected from the UCSC Genome browser
(http://genome.ucsc.edu) and NCBI (http://www.ncbi.nlm.nih.
gov) databases (Table S1). Probe DNA extraction was performed
according to the standard alkaline lysis method and labeled by
standard nick translation with Spectrum Orange- or Spectrum
Green-dUTP using a commercially available kit (Abbott/Vysis,
IL, USA). Sequential fluorescence in situ hybridization experiments
using a mixture of three probes were performed according to
manufacturer’s instructions (Abbott/Vysis, IL, USA). Each
mixture contained a red, green and yellow (combined green/
red) fluorescent probes. Probes were applied to slides, hybridized
for 20 hr at 37uC, washed with 0.46SSC/0.3% NP-40 for 2
minutes at 73uC and with 26SSC/0.1% NP-40 for 1 minute at
room temperature, and counterstained with DAPI. Digital FISH
images were captured by a Power Macintosh G3 System using
MacProbe software version 4.4 (Applied Imaging, CA, USA).
the Hormad1 locus. The targeting construct and schematized
genomic locus of Hormad1 are shown. The neo cassette replaced
HORMA domain encoding highly conserved exons 4 and 5, and
introduced an Xba I restriction enzyme site into the locus. This
Xba I site was used as a diagnostic for Southern blot analysis of ES
cells electroporated with the targeting vector (data not shown).
Arrows indicate genotyping primers used to distinguish wild-type
and mutant alleles in the transgenic animals. (B) RT-PCR analysis
of Hormad1 knockout mice shows lack of transcript corresponding
to Hormad1 in the knockout animals. A faintly visible lower
molecular weight band in the knockout (2/2), corresponds to a
mutant transcript without exons 4 and 5. Removal of exons 4 and
5 causes a frameshift mutation. (C) Total protein was isolated from
testes of 2 week old wild-type (+/+), Hormad12/2(2/2) and
Hormad1+/2(+/2) mice and Western blot analysis was performed
with anti-HORMAD1 specific antibody. No significant amount of
HORMAD1 was detected. The b-actin signal serves as a loading
Found at: doi:10.1371/journal.pgen.1001190.s001 (0.18 MB TIF)
Generation of Hormad12/2Mice. (A) Disruption of
Reduced testicular size and weight in Hormad12/2(KO) as
compared to the wild-type (WT) testes from 4 week old male
siblings. (B) Ratio of testes/body weight from wild-type (WT) and
Hormad12/2(KO) testes. Error bars represent the standard error
of mean. Student’s t test was used to calculate P values.
Found at: doi:10.1371/journal.pgen.1001190.s002 (0.18 MB TIF)
Adult testes are atrophied in Hormad12/2Mice. (A)
Hormad12/2testes. (A-E) TUNEL assay in 1, 2, 3, 4, and 6 week
old wild-type mouse testes. (F-J) TUNEL assay in 1, 2, 3, 4, and 6
Excess apoptosis and increased apoptotic index in
HORMAD1 Is Essential for Male and Female Meiosis
PLoS Genetics | www.plosgenetics.org 17 November 2010 | Volume 6 | Issue 11 | e1001190
week old Hormad12/2mouse testis. (K) Apoptotic index was
calculated in wild-type (WT) and Hormad12/2mouse testis (KO)
mice. Knockout testes had significantly higher apoptotic index at 2
weeks of post-natal life and beyond. Error bars represent the
standard error of the mean. Error bars represent the standard
error of the mean. Student’s t test was used to calculate P values. P
value was less than 0.001 at every time point except one week.
Scale bar: 100 mM.
Found at: doi:10.1371/journal.pgen.1001190.s003 (3.02 MB TIF)
natal day 8, primordial, primary, and secondary follicles were
detected in wild-type (A) and the Hormad12/2ovaries (B). Anti-
Lhx8 antibody was used to detect germ cells (brown stain).
Histogram represents primordial follicles (PF), primary follicles
(PrF), and a total number of secondary follicles (SF) in the wild-
type (WT) and mutants (KO) (C). Every fifth section of wild-type
(n=6) and Hormad12/2ovaries (n=5) were counted. Error bars
represent the standard error of the mean. Fisher’s exact t test was
used to calculate P values. P value between mutant and wild-type
No germ cell loss in the Hormad12/2ovary. At post-
primordial follicles, primary, and secondary follicles was .0.5, and
therefore not statistically significant. Bars, 50 mm.
Found at: doi:10.1371/journal.pgen.1001190.s004 (1.85 MB TIF)
Found at: doi:10.1371/journal.pgen.1001190.s005 (0.03 MB
BAC clones used in FISH experiments.
We thank Hyo won Ahn for technical assistance, Hitomi Suzuki and
Krishna Jagarlamudi for critical thinking, Tianjiao Chu for statistical
analysis of the RNA expression array data, and Christer Ho ¨o ¨g and William
R. Brinkley for supplying antibodies.
Conceived and designed the experiments: YHS YC PJW MLM AR.
Performed the experiments: YHS YC SUE SAY MK FY. Analyzed the
data: YHS SUE SAY MK FY PJW MLM AR. Contributed reagents/
materials/analysis tools: YC PJW AR. Wrote the paper: YHS SUE SAY
1. Page SL, Hawley RS (2004) The genetics and molecular biology of the
synaptonemal complex. Annu Rev Cell Dev Biol 20: 525–558.
2. Bailis JM, Roeder GS (1998) Synaptonemal complex morphogenesis and sister-
chromatid cohesion require Mek1-dependent phosphorylation of a meiotic
chromosomal protein. Genes Dev 12: 3551–3563.
3. Klein F, Mahr P, Galova M, Buonomo SB, Michaelis C, et al. (1999) A central
role for cohesins in sister chromatid cohesion, formation of axial elements, and
recombination during yeast meiosis. Cell 98: 91–103.
4. Loidl J, Klein F, Scherthan H (1994) Homologous pairing is reduced but not
abolished in asynaptic mutants of yeast. J Cell Biol 125: 1191–1200.
5. Aravind L, Koonin EV (1998) The HORMA domain: a common structural
denominator in mitotic checkpoints, chromosome synapsis and DNA repair.
Trends Biochem Sci 23: 284–286.
6. Michel LS, Liberal V, Chatterjee A, Kirchwegger R, Pasche B, et al. (2001)
MAD2 haplo-insufficiency causes premature anaphase and chromosome
instability in mammalian cells. Nature 409: 355–359.
7. Dobles M, Liberal V, Scott ML, Benezra R, Sorger PK (2000) Chromosome
missegregation and apoptosis in mice lacking the mitotic checkpoint protein
Mad2. Cell 101: 635–645.
8. Hollingsworth NM, Goetsch L, Byers B (1990) The HOP1 gene encodes a
meiosis-specific component of yeast chromosomes. Cell 61: 73–84.
9. Smith AV, Roeder GS (1997) The yeast Red1 protein localizes to the cores of
meiotic chromosomes. J Cell Biol 136: 957–967.
10. Zetka MC, Kawasaki I, Strome S, Muller F (1999) Synapsis and chiasma
formation in Caenorhabditis elegans require HIM-3, a meiotic chromosome
core component that functions in chromosome segregation. Genes Dev 13:
11. Armstrong SJ, Caryl AP, Jones GH, Franklin FC (2002) Asy1, a protein required
for meiotic chromosome synapsis, localizes to axis-associated chromatin in
Arabidopsis and Brassica. J Cell Sci 115: 3645–3655.
12. Colaiacovo MP, MacQueen AJ, Martinez-Perez E, McDonald K, Adamo A,
et al. (2003) Synaptonemal complex assembly in C. elegans is dispensable for
loading strand-exchange proteins but critical for proper completion of
recombination. Dev Cell 5: 463–474.
13. Couteau F, Nabeshima K, Villeneuve A, Zetka M (2004) A component of C.
elegans meiotic chromosome axes at the interface of homolog alignment,
synapsis, nuclear reorganization, and recombination. Curr Biol 14: 585–
14. Ross KJ, Fransz P, Armstrong SJ, Vizir I, Mulligan B, et al. (1997) Cytological
characterization of four meiotic mutants of Arabidopsis isolated from T-DNA-
transformed lines. Chromosome Res 5: 551–559.
15. Caryl AP, Armstrong SJ, Jones GH, Franklin FC (2000) A homologue of the
yeast HOP1 gene is inactivated in the Arabidopsis meiotic mutant asy1.
Chromosoma 109: 62–71.
16. Pangas SA, Yan W, Matzuk MM, Rajkovic A (2004) Restricted germ cell
expression of a gene encoding a novel mammalian HORMA domain-containing
protein. Gene Expr Patterns 5: 257–263.
17. Wojtasz L, Daniel K, Roig I, Bolcun-Filas E, Xu H, et al. (2009) Mouse
HORMAD1 and HORMAD2, two conserved meiotic chromosomal proteins,
are depleted from synapsed chromosome axes with the help of TRIP13 AAA-
ATPase. PLoS Genet 5: e1000702. doi:10.1371/journal.pgen.1000702.
18. Fukuda T, Daniel K, Wojtasz L, Toth A, Hoog C (2010) A novel mammalian
HORMA domain-containing protein, HORMAD1, preferentially associates
with unsynapsed meiotic chromosomes. Exp Cell Res 316: 158–171.
19. Kironmai KM, Muniyappa K, Friedman DB, Hollingsworth NM, Byers B
(1998) DNA-binding activities of Hop1 protein, a synaptonemal complex
component from Saccharomyces cerevisiae. Mol Cell Biol 18: 1424–1435.
20. Mao-Draayer Y, Galbraith AM, Pittman DL, Cool M, Malone RE (1996)
Analysis of meiotic recombination pathways in the yeast Saccharomyces
cerevisiae. Genetics 144: 71–86.
21. Carballo JA, Johnson AL, Sedgwick SG, Cha RS (2008) Phosphorylation of the
axial element protein Hop1 by Mec1/Tel1 ensures meiotic interhomolog
recombination. Cell 132: 758–770.
22. Bailis JM, Smith AV, Roeder GS (2000) Bypass of a meiotic checkpoint by
overproduction of meiotic chromosomal proteins. Mol Cell Biol 20: 4838–4848.
23. Meuwissen RL, Offenberg HH, Dietrich AJ, Riesewijk A, van Iersel M, et al.
(1992) A coiled-coil related protein specific for synapsed regions of meiotic
prophase chromosomes. EMBO J 11: 5091–5100.
24. Dobson MJ, Pearlman RE, Karaiskakis A, Spyropoulos B, Moens PB (1994)
Synaptonemal complex proteins: occurrence, epitope mapping and chromosome
disjunction. J Cell Sci 107(Pt 10): 2749–2760.
25. Lammers JH, Offenberg HH, van Aalderen M, Vink AC, Dietrich AJ, et al.
(1994) The gene encoding a major component of the lateral elements of
synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated
genes. Mol Cell Biol 14: 1137–1146.
26. Offenberg HH, Schalk JA, Meuwissen RL, van Aalderen M, Kester HA, et al.
(1998) SCP2: a major protein component of the axial elements of synaptonemal
complexes of the rat. Nucleic Acids Res 26: 2572–2579.
27. Yuan L, Liu JG, Zhao J, Brundell E, Daneholt B, et al. (2000) The murine SCP3
gene is required for synaptonemal complex assembly, chromosome synapsis, and
male fertility. Mol Cell 5: 73–83.
28. del Mazo J, Kremer L, Avila J (1987) Centromeric proteins recognized by
CREST sera and meiotic chromosome segregation. Chromosoma 96: 55–59.
29. Yuan L, Liu JG, Hoja MR, Wilbertz J, Nordqvist K, et al. (2002) Female germ
cell aneuploidy and embryo death in mice lacking the meiosis-specific protein
SCP3. Science 296: 1115–1118.
30. Pittman DL, Cobb J, Schimenti KJ, Wilson LA, Cooper DM, et al. (1998)
Meiotic prophase arrest with failure of chromosome synapsis in mice deficient
for Dmc1, a germline-specific RecA homolog. Mol Cell 1: 697–705.
31. de Vries FA, de Boer E, van den Bosch M, Baarends WM, Ooms M, et al. (2005)
Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombi-
nation, and XY body formation. Genes Dev 19: 1376–1389.
32. Gasior SL, Wong AK, Kora Y, Shinohara A, Bishop DK (1998) Rad52
associates with RPA and functions with rad55 and rad57 to assemble meiotic
recombination complexes. Genes Dev 12: 2208–2221.
33. Edelmann W, Cohen PE, Kane M, Lau K, Morrow B, et al. (1996) Meiotic
pachytene arrest in MLH1-deficient mice. Cell 85: 1125–1134.
34. Kolas NK, Marcon E, Crackower MA, Hoog C, Penninger JM, et al. (2005)
Mutant meiotic chromosome core components in mice can cause apparent
sexual dimorphic endpoints at prophase or X-Y defective male-specific sterility.
Chromosoma 114: 92–102.
35. Woltering D, Baumgartner B, Bagchi S, Larkin B, Loidl J, et al. (2000) Meiotic
segregation, synapsis, and recombination checkpoint functions require physical
interaction between the chromosomal proteins Red1p and Hop1p. Mol Cell Biol
36. Ward IM, Chen J (2001) Histone H2AX is phosphorylated in an ATR-
dependent manner in response to replicational stress. J Biol Chem 276:
HORMAD1 Is Essential for Male and Female Meiosis
PLoS Genetics | www.plosgenetics.org18 November 2010 | Volume 6 | Issue 11 | e1001190
37. Prieler S, Penkner A, Borde V, Klein F (2005) The control of Spo11’s interaction Download full-text
with meiotic recombination hotspots. Genes Dev 19: 255–269.
38. Nakamura TM, Du LL, Redon C, Russell P (2004) Histone H2A phosphor-
ylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest,
and influences DNA repair in fission yeast. Mol Cell Biol 24: 6215–6230.
39. Fernandez-Capetillo O, Mahadevaiah SK, Celeste A, Romanienko PJ,
Camerini-Otero RD, et al. (2003) H2AX is required for chromatin remodeling
and inactivation of sex chromosomes in male mouse meiosis. Dev Cell 4:
40. Turner JM, Aprelikova O, Xu X, Wang R, Kim S, et al. (2004) BRCA1, histone
H2AX phosphorylation, and male meiotic sex chromosome inactivation. Curr
Biol 14: 2135–2142.
41. Kozlov SV, Graham ME, Peng C, Chen P, Robinson PJ, et al. (2006)
Involvement of novel autophosphorylation sites in ATM activation. EMBO J 25:
42. Yoshida K, Kondoh G, Matsuda Y, Habu T, Nishimune Y, et al. (1998) The
mouse RecA-like gene Dmc1 is required for homologous chromosome synapsis
during meiosis. Mol Cell 1: 707–718.
43. Edelmann W, Cohen PE, Kneitz B, Winand N, Lia M, et al. (1999) Mammalian
MutS homologue 5 is required for chromosome pairing in meiosis. Nat Genet
44. Barlow C, Liyanage M, Moens PB, Tarsounas M, Nagashima K, et al. (1998)
Atm deficiency results in severe meiotic disruption as early as leptonema of
prophase I. Development 125: 4007–4017.
45. Pangas SA, Choi Y, Ballow DJ, Zhao Y, Westphal H, et al. (2006) Oogenesis
requires germ cell-specific transcriptional regulators Sohlh1 and Lhx8. Proc Natl
Acad Sci U S A 103: 8090–8095.
46. Deb K, Reese J, Paria BC (2006) Methodologies to study implantation in mice.
Methods Mol Med 121: 9–34.
47. Revenkova E, Eijpe M, Heyting C, Hodges CA, Hunt PA, et al. (2004) Cohesin
SMC1 beta is required for meiotic chromosome dynamics, sister chromatid
cohesion and DNA recombination. Nat Cell Biol 6: 555–562.
48. Yang F, De La Fuente R, Leu NA, Baumann C, McLaughlin KJ, et al. (2006)
Mouse SYCP2 is required for synaptonemal complex assembly and chromo-
somal synapsis during male meiosis. J Cell Biol 173: 497–507.
49. Xu Y, Ashley T, Brainerd EE, Bronson RT, Meyn MS, et al. (1996) Targeted
disruption of ATM leads to growth retardation, chromosomal fragmentation
during meiosis, immune defects, and thymic lymphoma. Genes Dev 10:
50. Romanienko PJ, Camerini-Otero RD (2000) The mouse Spo11 gene is required
for meiotic chromosome synapsis. Mol Cell 6: 975–987.
51. de los Santos T, Hollingsworth NM (1999) Red1p, a MEK1-dependent
phosphoprotein that physically interacts with Hop1p during meiosis in yeast.
J Biol Chem 274: 1783–1790.
52. Wang H, Hoog C (2006) Structural damage to meiotic chromosomes impairs
DNA recombination and checkpoint control in mammalian oocytes. J Cell Biol
53. Di Giacomo M, Barchi M, Baudat F, Edelmann W, Keeney S, et al. (2005)
Distinct DNA-damage-dependent and -independent responses drive the loss of
oocytes in recombination-defective mouse mutants. Proc Natl Acad Sci U S A
54. Hollingsworth NM, Ponte L (1997) Genetic interactions between HOP1, RED1
and MEK1 suggest that MEK1 regulates assembly of axial element components
during meiosis in the yeast Saccharomyces cerevisiae. Genetics 147: 33–42.
55. Hassold T, Hunt P (2001) To err (meiotically) is human: the genesis of human
aneuploidy. Nat Rev Genet 2: 280–291.
56. Hodges CA, Revenkova E, Jessberger R, Hassold TJ, Hunt PA (2005)
SMC1beta-deficient female mice provide evidence that cohesins are a missing
link in age-related nondisjunction. Nat Genet 37: 1351–1355.
57. Handel MA, Schimenti JC (2010) Genetics of mammalian meiosis: regulation,
dynamics and impact on fertility. Nat Rev Genet 11: 124–136.
58. Revenkova E, Eijpe M, Heyting C, Gross B, Jessberger R (2001) Novel meiosis-
specific isoform of mammalian SMC1. Mol Cell Biol 21: 6984–6998.
59. Goate A, Chartier-Harlin MC, Mullan M, Brown J, Crawford F, et al. (1991)
Segregation of a missense mutation in the amyloid precursor protein gene with
familial Alzheimer’s disease. Nature 349: 704–706.
60. Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG (2005) Simple
and highly efficient BAC recombineering using galK selection. Nucleic Acids
Res 33: e36.
61. Rajkovic A, Pangas SA, Ballow D, Suzumori N, Matzuk MM (2004) NOBOX
deficiency disrupts early folliculogenesis and oocyte-specific gene expression.
Science 305: 1157–1159.
62. Baltus AE, Menke DB, Hu YC, Goodheart ML, Carpenter AE, et al. (2006) In
germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes
premeiotic DNA replication. Nat Genet 38: 1430–1434.
63. Choi Y, Ballow DJ, Xin Y, Rajkovic A (2008) Lim homeobox gene, lhx8, is
essential for mouse oocyte differentiation and survival. Biol Reprod 79: 442–449.
64. Li M, Shin YH, Hou L, Huang X, Wei Z, et al. (2008) The adaptor protein of
the anaphase promoting complex Cdh1 is essential in maintaining replicative
lifespan and in learning and memory. Nat Cell Biol.
65. Rajkovic A, Yan MSC, Klysik M, Matzuk M (2001) Discovery of germ cell-
specific transcripts by expressed sequence tag database analysis. Fertil Steril 76:
66. Wang PJ, McCarrey JR, Yang F, Page DC (2001) An abundance of X-linked
genes expressed in spermatogonia. Nat Genet 27: 422–426.
67. Mouillet JF, Chu T, Nelson DM, Mishima T, Sadovsky Y (2010) MiR-205
silences MED1 in hypoxic primary human trophoblasts. FASEB J.
68. Smyth GK (2004) Linear models and empirical bayes methods for assessing
differential expression in microarray experiments. Stat Appl Genet Mol Biol 3:
69. Seli E, Lalioti MD, Flaherty SM, Sakkas D, Terzi N, et al. (2005) An embryonic
poly(A)-binding protein (ePAB) is expressed in mouse oocytes and early
preimplantation embryos. Proc Natl Acad Sci U S A 102: 367–372.
HORMAD1 Is Essential for Male and Female Meiosis
PLoS Genetics | www.plosgenetics.org19 November 2010 | Volume 6 | Issue 11 | e1001190