Meiosis is a specialized cell division process in germ cells that
reduces the chromosome complement by half and generates
haploid gametes (Page and Hawley, 2004; Zickler and Kleckner,
1999). The integrity of the meiotic process is crucial as errors
may result in chromosomal missegregation and thus contribute
to aneuploidy in mature germ cells and future offspring (Hassold
and Hunt, 2001). Quality assurance mechanisms monitor several
of the developmental transitions that take place during meiosis,
including DNA recombination and repair, synapsis of the
homologous chromosomes and the meiosis I and II divisions
(Homer et al., 2005; Morelli and Cohen, 2005). Synapsis between
homologous chromosomes at the pachytene stage of meiosis is
essential for the formation of crossovers – recombination events
that promote bi-orientation of the chromosomes at the first meiotic
division (Nasmyth and Haering, 2005; Page and Hawley, 2004;
Zickler and Kleckner, 1999). In mammals, the unsynapsed
regions of the XY bivalent in normal pachytene spermatocytes
activate a mechanism known as ‘meiotic sex chromosome
inactivation’ (MSCI), which triggers chromatin changes and
results in transcriptional silencing of sex chromosome-linked
genes (Turner, 2007). The MSCI response is conserved in most
organisms that possess heteromorphic sex chromosomes and a
failure to activate MSCI results in midpachytene apoptosis and
spermatocyte death (Burgoyne et al., 2009; Turner et al., 2005).
The MSCI response depends on the tumor suppressor protein
BRCA1 that accumulates on the unsynapsed axes of the sex
chromosomes (Mahadevaiah et al., 2008; Turner et al., 2005),
but the mechanism for targeting BRCA1 to the axes of unsynapsed
chromosomes is presently unknown. BRCA1 then recruits the
protein kinase ATR (ataxia telangiectasia and Rad3 related),
which phosphorylates histone H2A.X, triggering the MSCI
response (Bellani et al., 2005; Celeste et al., 2002; Mahadevaiah
et al., 2008; Turner et al., 2004). The male-specific role of MSCI
suggests that sex-specific surveillance mechanisms have evolved
to protect the integrity of the germ cells (Morelli and Cohen,
2005). Recently, however, it was proposed that MSCI is a
manifestation of a more general silencing mechanism, known as
MSUC, that targets unsynapsed autosomes at the pachytene stage
of meiosis in male and female germ cells (Baarends et al., 2005;
Burgoyne et al., 2009; Schimenti, 2005; Turner et al., 2005). Our
knowledge of the importance of MSUC as a surveillance
mechanism in germ cells is, as yet, very limited.
In the present study we have explored the role of MSUC in
female mouse germ cells, which given their XX complement do
not undergo MSCI, yet have been shown to mount a MSUC
response. We took advantage of several mouse models that
allowed us to study how different levels of asynapsis affected the
ability of oocytes to trigger an MSUC response. We now show
that MSUC contributes to the elimination of oocytes that contain
asynapsed chromosomes. The MSUC response, however, is
impaired if more than two to three pairs of homologous
chromosomes are asynapsed, due to a limited pool of BRCA1. In
addition, we demonstrate that a structural component of the
prophase meiotic chromosome axis, Sycp3, is essential for
recruitment of BRCA1 to asynapsed chromosomes.
Transcriptional silencing of the sex chromosomes during male
meiosis is regarded as a manifestation of a general mechanism
active in both male and female germ cells, called meiotic
silencing of unsynapsed chromatin (MSUC). MSUC is initiated
by the recruitment of the tumor suppressor protein BRCA1 to
the axes of unsynapsed chromosomes. We now show that Sycp3,
a structural component of the chromosome axis, is required for
localization of BRCA1 to unsynapsed pachytene chromosomes.
Importantly, we find that oocytes carrying an excess of two to
three pairs of asynapsed homologous chromosomes fail to
recruit enough BRCA1 to the asynapsed axes to activate MSUC.
Furthermore, loss of MSUC function only transiently rescues
oocytes from elimination during early postnatal development.
The fact that the BRCA1-dependent synapsis surveillance
system cannot respond to higher degrees of asynapsis and is
dispensable for removal of aberrant oocytes argues that MSUC
has a limited input as a quality control mechanism in female
Supplementary material available online at
Key words: Meiosis, Oocytes, BRCA1, Meiotic silencing of
BRCA1-mediated chromatin silencing is limited to
oocytes with a small number of asynapsed
Anna Kouznetsova1, Hong Wang1, Marina Bellani2, R. Daniel Camerini-Otero2, Rolf Jessberger3and
1Department of Cell and Molecular Biology (CMB), Karolinska Institutet, Stockholm, S-171 77, Sweden
2Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health,
Bethesda, MD 20892, USA
3Institute of Physiological Chemistry, Medical School, MTZ, Dresden University of Technology, Dresden, D-01307, Germany
*Author for correspondence (e-mail: email@example.com)
Accepted 1 April 2009
Journal of Cell Science 122, 2446-2452 Published by The Company of Biologists 2009
Journal of Cell Science
2452Journal of Cell Science 122 (14)
Staging of the oocytes
In determining the developmental stages in Sycp1–/–, Sycp3–/–, Smc1β–/–and
Smc1β–/–Sycp3–/–oocytes, we took advantage of the synchronous development of
oocytes in embryonic ovaries (Dietrich and Mulder, 1983). Therefore we labeled
chromosomal axes and centromeres in oocytes derived from E16.5, E17.5, E18.5 and
E19.5 mutant ovaries and matched the patterns found with wild-type littermates. The
type found only in E16.5 ovaries was classified as ‘early zygotene’, the one prevalent
in E17.5 ovaries as ‘pachytene’, etc.
Ovaries from Smc1β–/–females were collected at day 1-2 after birth, fixed in 4%
formaldehyde for 4 hours, embedded in paraffin and sectioned at 5 mm. To count
the number of oocytes in the ovary, each fifth section was immunostained for
GCNA (Enders and May, 1994), as described previously (Wang and Hoog, 2006).
The dataset published by Novak et al. (Novak et al., 2008) (n=3 for wild type,
n=4 for Smc1β–/–and n=3 for Sycp3–/–Smc1β–/–ovaries) was complemented with
new data (n=3 for wild type, n=2 for Smc1β–/–and n=2 for Sycp3–/–Smc1β–/–
ovaries), and the total dataset was used for statistical analysis of oocyte survival.
Importantly, data for day 1 and day 2 were collected in parallel and analyzed by
the same person. The number of oocytes for each mouse is given in supplementary
material Table S1.
Quantification was performed using ImageJ 1.40g software. Oocytes derived from
mutant animals and their wild-type littermates were spread as described above,
stained with the antibodies of interest plus Sycp2 antibody to assess axis
morphology, and counterstained with DAPI. All slides with one antibody staining
were processed simultaneously to minimize variation; images were taken with the
same exposure times. Only pachytene oocytes with undamaged morphology (as
judged by DAPI staining) and adequate spreading (nucleus diameter between 30
and 50 μm) were processed. The measurements were taken from one image,
representing the focal plane for the whole cell. To measure the total axial BRCA1
fluorescence in Smc1β–/–oocytes, all axes, as determined by Sycp2 staining, were
outlined and the integrated signal density of BRCA1 within these areas was
measured. To measure the total RNA pol II intensity in Sycp1–/–and wild-type
oocytes, the whole nucleus was outlined and the integrated signal density was
measured within this outline. To measure BRCA1 and ubi-H2A intensity on the
asynapsed axes, the whole asynapsed axis (as judged by Sycp2 staining) was
carefully marked and the mean intensity of the signal was measured on this line.
For measurements of γH2A.X, the areas around the asynapsed chromosomes with
an intensity above the threshold were outlined, and the mean intensity of the γH2A.X
signal was measured within this outline. For quantification of RNA pol II signal
in Smc1β–/–oocytes, we measured the mean integrated density around all asynapsed
axes (as judged by intense γH2A.X signal) and the mean integrated density around
five randomly selected synapsed euchromatic regions within the same cell; the
intensity of the γH2A.X signal was also measured in the selected regions. As the
RNA pol II signal showed variation between the cells, we applied nested ANOVA
tests to probe the difference in RNA pol II intensity. The level of asynapsis was
calculated as the proportion of the asynapsed axial element length to the total axial
element length (asynapsed axes plus twice the length of the synapsed regions) for
each nucleus. In our quantification analysis we have assumed that the protein
concentration is directly proportional to the observed intensity of the
Statistical analyses were performed using Excel 2004 and Statistica 7.0 programs.
We thank Ivana Novak for help with experiments on Smc1β–/–mice
and Mary-Rose Hoja for the comments on the manuscript. This work
was supported by grants from the Swedish Cancer Society, the Swedish
Research Council, the Novo Nordisk Foundation and Karolinska
Alton, M., Lau, M. P., Villemure, M. and Taketo, T. (2008). The behavior of the X- and
Y-chromosomes in the oocyte during meiotic prophase in the B6.Y(TIR)sex-reversed
mouse ovary. Reproduction 135, 241-252.
Baarends, W. M., Wassenaar, E., van der Laan, R., Hoogerbrugge, J., Sleddens-Linkels,
E., Hoeijmakers, J. H., de Boer, P. and Grootegoed, J. A.(2005). Silencing of unpaired
chromatin and histone H2A ubiquitination in mammalian meiosis. Mol. Cell. Biol. 25,
Bellani, M. A., Romanienko, P. J., Cairatti, D. A. and Camerini-Otero, R. D. (2005).
SPO11 is required for sex-body formation, and Spo11 heterozygosity rescues the prophase
arrest of Atm–/– spermatocytes. J. Cell Sci. 118, 3233-3245.
Burgoyne, P. S., Mahadevaiah, S. K. and Turner, J. M. (2009). The consequences of
asynapsis for mammalian meiosis. Nat. Rev. Genet. 10, 207-216.
Celeste, A., Petersen, S., Romanienko, P. J., Fernandez-Capetillo, O., Chen, H. T.,
Sedelnikova, O. A., Reina-San-Martin, B., Coppola, V., Meffre, E., Difilippantonio,
M. J. et al. (2002). Genomic instability in mice lacking histone H2AX. Science 296,
de Vries, F. A., de Boer, E., van den Bosch, M., Baarends, W. M., Ooms, M., Yuan,
L., Liu, J. G., van Zeeland, A. A., Heyting, C. and Pastink, A. (2005). Mouse Sycp1
functions in synaptonemal complex assembly, meiotic recombination, and XY body
formation. Genes Dev. 19, 1376-1389.
Di Giacomo, M., Barchi, M., Baudat, F., Edelmann, W., Keeney, S. and Jasin, M.
(2005). Distinct DNA-damage-dependent and -independent responses drive the loss of
oocytes in recombination-defective mouse mutants. Proc. Natl. Acad. Sci. USA 102,
Dietrich, A. J. and Mulder, R. J. (1983). A light- and electron microscopic analysis of
meiotic prophase in female mice. Chromosoma 88, 377-385.
Eijpe, M., Heyting, C., Gross, B. and Jessberger, R. (2000). Association of mammalian
SMC1 and SMC3 proteins with meiotic chromosomes and synaptonemal complexes. J.
Cell Sci. 113, 673-682.
Eijpe, M., Offenberg, H., Jessberger, R., Revenkova, E. and Heyting, C.(2003). Meiotic
cohesin REC8 marks the axial elements of rat synaptonemal complexes before cohesins
SMC1beta and SMC3. J. Cell Biol. 160, 657-670.
Enders, G. C. and May, J. J., 2nd (1994). Developmentally regulated expression of a
mouse germ cell nuclear antigen examined from embryonic day 11 to adult in male and
female mice. Dev. Biol. 163, 331-340.
Garcia-Cruz, R., Roig, I., Robles, P., Scherthan, H. and Garcia Caldes, M. (2009).
ATR, BRCA1 and gammaH2AX localize to unsynapsed chromosomes at the pachytene
stage in human oocytes. Reprod. Biomed. Online 18, 37-44.
Hamer, G., Novak, I., Kouznetsova, A. and Hoog, C. (2008). Disruption of pairing and
synapsis of chromosomes causes stage-specific apoptosis of male meiotic cells.
Theriogenology 69, 333-339.
Hassold, T. and Hunt, P. (2001). To err (meiotically) is human: the genesis of human
aneuploidy. Nat. Rev. Genet. 2, 280-291.
Homer, H. A., McDougall, A., Levasseur, M., Yallop, K., Murdoch, A. P. and Herbert,
M. (2005). Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin
during meiosis I in mouse oocytes. Genes Dev. 19, 202-207.
Kouznetsova, A., Novak, I., Jessberger, R. and Hoog, C. (2005). SYCP2 and SYCP3
are required for cohesin core integrity at diplotene but not for centromere cohesion at
the first meiotic division. J. Cell Sci. 118, 2271-2278.
Kouznetsova, A., Lister, L., Nordenskjold, M., Herbert, M. and Hoog, C. (2007). Bi-
orientation of achiasmatic chromosomes in meiosis I oocytes contributes to aneuploidy
in mice. Nat. Genet. 39, 966-968.
Mahadevaiah, S. K., Bourc’his, D., de Rooij, D. G., Bestor, T. H., Turner, J. M. and
Burgoyne, P. S. (2008). Extensive meiotic asynapsis in mice antagonises meiotic
silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome
inactivation. J. Cell Biol. 182, 263-276.
Morelli, M. A. and Cohen, P. E. (2005). Not all germ cells are created equal: aspects of
sexual dimorphism in mammalian meiosis. Reproduction 130, 761-781.
Nasmyth, K. and Haering, C. H. (2005). The structure and function of SMC and kleisin
complexes. Annu. Rev. Biochem. 74, 595-648.
Novak, I., Wang, H., Revenkova, E., Jessberger, R., Scherthan, H. and Hoog, C.(2008).
Cohesin Smc1beta determines meiotic chromatin axis loop organization. J. Cell Biol.
Page, S. L. and Hawley, R. S. (2004). The genetics and molecular biology of the
synaptonemal complex. Annu. Rev. Cell Dev. Biol. 20, 525-558.
Pelttari, J., Hoja, M. R., Yuan, L., Liu, J. G., Brundell, E., Moens, P., Santucci-
Darmanin, S., Jessberger, R., Barbero, J. L., Heyting, C. et al. (2001). A meiotic
chromosomal core consisting of cohesin complex proteins recruits DNA recombination
proteins and promotes synapsis in the absence of an axial element in mammalian meiotic
cells. Mol. Cell. Biol. 21, 5667-5677.
Peters, A. H., Plug, A. W., van Vugt, M. J. and de Boer, P. (1997). A drying-down
technique for the spreading of mammalian meiocytes from the male and female germline.
Chromosome Res. 5, 66-68.
Revenkova, E. and Jessberger, R. (2006). Shaping meiotic prophase chromosomes:
cohesins and synaptonemal complex proteins. Chromosoma 115, 235-240.
Revenkova, E., Eijpe, M., Heyting, C., Hodges, C. A., Hunt, P. A., Liebe, B., Scherthan,
H. and Jessberger, R. (2004). Cohesin SMC1 beta is required for meiotic chromosome
dynamics, sister chromatid cohesion and DNA recombination. Nat. Cell Biol. 6, 555-562.
Schimenti, J. (2005). Synapsis or silence. Nat. Genet. 37, 11-13.
Turner, J. M.(2007). Meiotic sex chromosome inactivation. Development134, 1823-1831.
Turner, J. M., Aprelikova, O., Xu, X., Wang, R., Kim, S., Chandramouli, G. V., Barrett,
J. C., Burgoyne, P. S. and Deng, C. X.(2004). BRCA1, histone H2AX phosphorylation,
and male meiotic sex chromosome inactivation. Curr. Biol. 14, 2135-2142.
Turner, J. M., Mahadevaiah, S. K., Fernandez-Capetillo, O., Nussenzweig, A., Xu, X.,
Deng, C. X. and Burgoyne, P. S.(2005). Silencing of unsynapsed meiotic chromosomes
in the mouse. Nat. Genet. 37, 41-47.
Wang, H. and Hoog, C. (2006). Structural damage to meiotic chromosomes impairs DNA
recombination and checkpoint control in mammalian oocytes. J. Cell Biol. 173, 485-495.
Yang, F., De La Fuente, R., Leu, N. A., Baumann, C., McLaughlin, K. J. and Wang,
P. J. (2006). Mouse SYCP2 is required for synaptonemal complex assembly and
chromosomal synapsis during male meiosis. J. Cell Biol. 173, 497-507.
Yuan, L., Liu, J. G., Zhao, J., Brundell, E., Daneholt, B. and Hoog, C. (2000). The
murine SCP3 gene is required for synaptonemal complex assembly, chromosome
synapsis, and male fertility. Mol. Cell 5, 73-83.
Yuan, L., Liu, J. G., Hoja, M. R., Wilbertz, J., Nordqvist, K. and Hoog, C. (2002).
Female germ cell aneuploidy and embryo death in mice lacking the meiosis-specific
protein SCP3. Science 296, 1115-1118.
Zickler, D. and Kleckner, N. (1999). Meiotic chromosomes: integrating structure and
function. Annu. Rev. Genet. 33, 603-754.
Journal of Cell Science