RESEARCH ARTICLE
2417
Coordinating the segregation of sister chromatids
during the first meiotic division: evidence for sexual
dimorphism
Craig A. Hodges, Renée LeMaire-Adkins and Patricia A. Hunt*
Department of Genetics and Center for Human Genetics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland,
Ohio 44106-4955, USA
*Author for correspondence (e-mail: [email protected])
Accepted 5 April 2001
Journal of Cell Science 114, 2417-2426 © The Company of Biologists Ltd
SUMMARY
Errors during the first meiotic division are common in our
species, but virtually all occur during female meiosis. The
reason why oogenesis is more error prone than
spermatogenesis remains unknown. Normal segregation of
homologous chromosomes at the first meiotic division (MI)
requires coordinated behavior of the sister chromatids
of each homolog. Failure of sister kinetochores to act
cooperatively at MI, or precocious sister chromatid
segregation (PSCS), has been postulated to be a major
contributor to human nondisjunction. To investigate the
factors that influence PSCS we utilized the XO mouse, since
the chromatids of the single X chromosome frequently
segregate at MI, and the propensity for PSCS is influenced
by genetic background. Our studies demonstrate that the
strain-specific differences in PSCS are due to the actions
of an autosomal trans-acting factor or factors. Since
components of the synaptonemal complex are thought to
play a role in centromere cohesion and kinetochore
orientation, we evaluated the behavior of the X
chromosome at prophase to determine if this factor
influenced the propensity of the chromosome for selfsynapsis. We were unable to directly correlate synaptic
differences with subsequent segregation behavior.
However, unexpectedly, we uncovered a sexual dimorphism
that may partially explain sex-specific differences in the
fidelity of meiotic chromosome segregation. Specifically, in
the male remnants of the synaptonemal complex remain
associated with the centromeres until anaphase of the
second meiotic division (MII), whereas in the female, all
traces of synaptonemal complex (SC) protein components
are lost from the chromosomes before the onset of the first
meiotic division. This finding suggests a sex-specific
difference in the components used to correctly segregate
chromosomes during meiosis, and may provide a reason for
the high error frequency during female meiosis.
INTRODUCTION
form attachments to the same rather than opposing spindle
poles. The second meiotic division (MII) is similar to a mitotic
cell division, involving the segregation of sister chromatids.
However, because MI and MII occur without an intervening S
phase, successful chromosome segregation at MII requires
a mechanism whereby cohesion is released along the
chromosome arms at anaphase I but maintained between sister
centromeres until anaphase II.
It is commonly assumed that the specialized meiotic
cohesion requirements depend upon the unique events of
meiotic prophase, e.g. synapsis and recombination. The role of
recombination in the disjunction of homologous chromosomes
at MI is well established (e.g. Carpenter, 1994; reviewed in
Hassold et al., 2000; Hawley, 1988; Koehler et al., 1996; Lamb
et al., 1996). The role of synapsis remains less well
characterized, but the inferential evidence is compelling: both
defects in homolog synapsis and the absence of a homologous
partner are associated with an increased frequency of
premature separation of sister chromatids at MI in a variety of
species (reviewed in Moore and Orr-Weaver, 1998; Wolf,
1993). Moreover studies in corn, yeast and mammals provide
evidence for the involvement of components of the
Proper chromosome segregation during mitotic cell division
requires at least two chromosome-associated protein
complexes. At the centromere, functional kinetochores must be
formed on individual sister chromatids to facilitate the
attachment of the chromatids to opposite spindle poles. In
addition, cohesion must be established between the
centromeres and along the arms of the sister chromatids and
maintained until anaphase to prevent their precocious
separation.
By comparison with mitotic cell division, the segregation
behavior of chromosomes during meiotic cell division is
complex, necessitating modification of both the kinetochore
and cohesion complexes. Our understanding of these meiotic
modifications remains rudimentary. The first meiotic division
(MI) involves the segregation of homologs rather than
sister chromatids (Fig. 1A). Thus, successful chromosome
segregation at MI requires specialized cohesion mechanisms
that provide physical connections between homologs (rather
than sister chromatids), and impose constraint on the
centromeres of sister chromatids so that their kinetochores
Key words: Meiosis, Nondisjunction, Sister chromatid cohesion,
Synaptonemal complex, XO mouse
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JOURNAL OF CELL SCIENCE 114 (13)
synaptonemal complex (SC), the proteinaceous structure
involved in homolog synapsis, in MI segregation. In maize, the
analysis of a variety of mutants and structural rearrangements
provides compelling evidence for a relationship between the
SC and sister chromatid cohesion and suggests a direct
correlation between synaptic failure in the centromeric region
and precocious sister chromatid segregation (PSCS) (reviewed
in Maguire, 1995). In yeast, mutations in the meiosis-specific
component of the cohesion complex, Rec8, show an increase
in PSCS at MI (Watanabe and Nurse, 1999). Finally, in
mammals, two pieces of data implicate the involvement of SC
proteins in meiotic sister chromatid cohesion. First, remnants
of the lateral element persist at the centromere until anaphase
II, as revealed by immunolocalization studies using antibodies
to two protein components, SCP3 and SCP2 (Dobson et al.,
1994; Offenberg et al., 1998). Second, studies of the recently
identified cohesin components, SMC1 and SMC3, provide
evidence of interactions with SCP3 and SCP2 (Eijpe et al.,
2000). Thus, there is considerable indirect evidence for the
involvement of SC proteins in the unique behavior of sister
kinetochores at MI, in the maintenance of connections between
homologs, and in the maintenance of cohesion between sister
centromeres until anaphase II. However, the mechanistic
details remain unclear.
These processes are not only of academic interest to students
of meiotic cell division but rather have a dramatic impact on
human life: the meiotic divisions in the human female are
extraordinarily error prone. Estimates of the error frequency
range from one in twenty to one in three human oocytes,
depending upon the age of the woman (reviewed in Hassold et
al., 1996). Despite intensive investigation, the reason for the
high error rate in our species remains unknown. It is clear,
however, that the majority of errors occur during the first
meiotic division, that the propensity for segregation errors is
chromosome-specific, and that the fidelity of chromosome
segregation is influenced by the number and placement of
recombination events along the chromosome arms (reviewed
in Hassold et al., 2000). It has been postulated that PSCS is a
major mechanism responsible for the age-related increase in
segregation errors in our species (Angell, 1997; Angell et al.,
1994; Wolstenholme and Angell, 2000), and the small amount
of data available from direct studies of human oocytes are
consistent with this suggestion (Angell, 1997; Angell et al.,
1994; Mahmood et al., 2000; Volarcik et al., 1998).
Studies of human nondisjunction have been limited both by
the difficulty of obtaining human oocytes and the lack of a
suitable animal model for experimental studies aimed at
understanding the age effect in our species. However, the high
degree of conservation among species in proteins that mediate
some of the specialized meiotic chromosome behaviors
suggests that the mouse may provide valuable mechanistic
insight to human aneuploidy. Accordingly, we have utilized the
female XO mouse to study the factors that influence PSCS at
MI. As in other organisms (reviewed in Wolf, 1993), the sister
chromatids of the univalent X segregate at anaphase of MI in a
proportion of cells (Hunt et al., 1995); however, the frequency
of PSCS versus ‘intact’ segregation (i.e. in which the sister
chromatids of the univalent remain attached) is influenced by
genetic background (LeMaire-Adkins and Hunt, 2000). This
difference in MI segregation on two inbred backgrounds
provides a genetic approach to understanding the factors that
influence the behavior of sister kinetochores at the first meiotic
division. We report here the results of detailed meiotic studies
that exclude X-chromosome specific differences and suggest
that segregation is influenced by the action of an autosomal gene
or genes. We hypothesized that this trans-acting factor(s) would
influence the synaptic behavior of the X chromosome, and that
failure to undergo self-synapsis involving the centromere of the
chromosome would result in the premature separation of X
chromatids at MI. Our observations did not fit this expectation,
but our studies provide new insight to the complexity of the
synaptic process and suggest that inferences about subsequent
meiotic events based on pachytene analysis may be misleading.
More importantly, however, our efforts to correlate segregation
behavior with the retention of synaptonemal complex proteins
revealed a surprising difference in centromere-associated
proteins between oogenesis and spermatogenesis. Differences
in the protein components of the meiotic chromosome cohesion
complex may influence the fidelity of meiotic chromosome
segregation, thus this intriguing sexual dimorphism may
provide a partial explanation for the high chromosome error rate
during human female meiosis.
MATERIALS AND METHODS
Production of XO female mice
XO female mice and XX sibling controls were produced on two
different inbred strain backgrounds using previously described mating
schemes involving males prone to meiotic sex chromosome
nondisjunction. Specifically, C57BL/6 females were mated to
C57BL/6 males carrying the Y* chromosome (Eicher et al., 1982) and
C3H females were mated to C3H males carrying the X-linked Paf
mutation (Lane and Davisson, 1990). Both crosses produce
approximately 20% XO females.
Intercrosses of the two inbred strains were made to generate F1
hybrid females that differed only in the origin of their X chromosome:
C57BL/6 females were mated to C3H males carrying the Paf mutation
to produce XO females with a C57BL/6 X chromosome and C3H
females were mated to C57BL/6 males carrying the Y* mutation to
produce XO females with a C3H X chromosome.
Oocyte collection, culture and fixation
To assess the segregation of the X chromosome at the first meiotic
division, oocytes arrested at metaphase of MII were obtained as
follows: meiotically arrested oocytes at the germinal vesicle stage
were collected from the ovaries of 3.5-week-old females by piercing
the follicles with 26-gauge needles. Oocytes were placed in 10 µl
drops of Waymouth’s MB752/1medium (Gibco BRL) supplemented
with 10% fetal bovine serum and 0.23 mM sodium pyruvate, overlaid
with mineral oil, and incubated overnight at 37°C in an atmosphere
of 5% CO2 in air. After 14-16 hours in culture oocytes were scored
for polar body extrusion, indicating completion of the first meiotic
division and arrest at MII. Oocytes exhibiting polar body formation
were embedded in a fibrin clot attached to a microscope slide as
previously described (Hunt et al., 1995), fixed in 2% formaldehyde,
1% Triton X-100, 0.1 mM Pipes, 5 mM MgCl2, and 2.5 mM EGTA
for 30 minutes at 37°C, washed in 0.1% normal goat serum (NGS) in
PBS for 15 minutes at 37°C, and blocked in 10% NGS containing
0.1% Triton X-100 for 1 hour at 37°C. Fixed oocytes were stored in
10% NGS at 4°C until FISH analysis was performed as described
below.
Synaptonemal complex studies
To assess meiotic pairing we used combined immunofluorescence
Coordinated segregation of sister chromatids
2419
staining (to visualize the SC) and FISH (to identify the X chromosome
and the telomeres). Preparations of oocytes at the pachytene stage
were made from ovaries from 16-21 days post coitus (d.p.c.) fetuses
and newborns according to the method described by Peters et al.
(Peters et al., 1997), and stored at −20°C. To visualize both the SC
and the centromeres, indirect immunofluorescence staining was
performed with combinations of the following antibodies: (1) SCP2
or SCP3, which recognize two different protein components of
the lateral element of the SC, (2) SCP1, which recognizes a
protein component of the central element of the SC and (3) CREST
serum, which recognizes centromere-associated proteins. For
immunostaining, slides were washed in 1% donkey serum in PBS for
1 hour at room temperature, incubated with primary antibody diluted
in 1% donkey serum for 2 hours at 37°C, and washed in 1% donkey
serum for 1 hour at room temperature. The primary antibodies were
detected with an FITC-conjugated donkey anti-goat IgG (Jackson
Immunoresearch) for SCP3, Rhodamine-conjugated donkey antirabbit IgG (Jackson Immunoresearch) for SCP1 and SCP2, and
Rhodamine-conjugated donkey anti-human IgG (Jackson
Immunoresearch) for CREST in PBS for 1 hour at 37°C and washed
in 1% donkey serum for 1 hour at room temperature. Slides were
stored at 4°C in 1% donkey serum until FISH was performed as
described below.
Fluorescence in situ hybridization (FISH)
To evaluate X chromosome segregation at the first meiotic division,
MII arrested cells were hybridized with the X-linked probe,
DXWas70, which recognizes repetitive sequences near the centromere
of the mouse X chromosome. The slides were washed in 2× SSC for
5 minutes at room temperature, blotted and covered with 30 µl of
Hybrisol VII (Oncor) containing 30 ng of digoxigenin-labeled
DXWas70 probe. A coverslip was applied and sealed with rubber
cement, the slides were denatured at 85°C for 10 minutes, and
hybridized overnight at 37°C in a humid chamber. Following
hybridization, slides were washed in 50% formamide/2× SSC at 37°C
for 10 minutes followed by a wash of 2× SSC at 37°C for 5 minutes.
Hybridized slides were washed in PN buffer for 2 minutes, blocked
in PN buffer containing 5% non-fat dry milk and 0.02% sodium azide
for 5 minutes at room temperature, detected with an antidigoxigeninconjugated fluorochrome for 1 hour at 37°C, and washed for 1 hour
in PN buffer. Prior to analysis oocytes were counterstained with 100
ng/ml propidium iodide and mounted with 50% glycerol/PBS
containing 0.1 µg/ml ρ-phenylenediamine and a coverslip. Oocytes
were visualized on a BioRad MRC600 confocal system.
Hybridization of pachytene preparations was essentially the same
as MII arrested oocytes with the following exceptions: (1) in addition
to the DXWas70 probe, 10 µl of a biotin-labeled human pan-telomeric
probe that recognizes mouse telomeres was applied, (2) slides
Fig. 1. Segregation of X chromosome homologs and univalents at MI.
Left: Cartoons depict MI segregation of (A) X homologs to the oocyte
and polar body in control XX females, (B) intact segregation and (C)
precocious segregation of sister chromatids (PSCS) of the univalent X
chromosome in oocytes from XO females (Note that the termed
‘precocious’ refers to the fact that sister segregation occurs at MI
rather than MII; a physical association is maintained between the
sister chromatids until anaphase I). Right: Actual images of oocyte
and polar body chromosomes in MII arrested oocytes from XX and
XO females, hybridized with an X-chromosome specific probe. In all
images, the group of chromosomes (red) segregated to the polar body
at MI is on the right and the group remaining in the oocyte is on the
left. Each FISH signal (yellow) represents a single X chromatid.
(A) Normal MI segregation of homologs results in two X signals (or
one doublet) in both the oocyte and polar body; (B) intact segregation
of the univalent X is evident either as two signals in the egg or in the
polar body (not shown); (C) PSCS of the univalent X chromosome is
evident as a single signal in both the oocyte and the polar body.
were denatured for only 5 minutes and (3) an avidin-conjugated
fluorochrome was added at the detection step to detect the telomere
probe.
Table 1. X chromosome segregation at MI in oocytes from XO females*
Type of segregation
n
Intact
PSCS
369
286 (76%)
83 (24%)
427
194 (45%)
233 (55%)
F1 hybrids
XB6O
209
102 (49%)
107 (51%)
XC3HO
232
122 (53%)
110 (47%)
Inbred strains
C57BL/6
C3H
Significance
χ2=85.06, P<0.001
χ2=0.63, P>0.1
*For inbred strains, data from the present analysis (representing 47 and 205 oocytes from XO females produced on the C57BL/6 and C3H backgrounds,
respectively) were combined with previously reported data (LeMaire-Adkins and Hunt, 2000).
All F1 hybrid data comes from the present study.
n, number of oocytes studied.
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Fig. 2. Analysis of X chromosome synapsis in pachytene stage
oocytes. (A) Pachytene stage oocyte from an XO female mouse
immunostained with an antibody to SCP3 (green) to visualize the
lateral elements of the SC, and hybridized with X-chromosome
specific (purple) and telomere-specific (red) probes. The dispersed X
chromosome signal (arrow) is due to the nature of the probe
(DXWAS70 detects a multicopy sequence close to the centromere of
the X chromosome) and the decondensed state of the chromatin at
this stage. (B-F) Synaptic configurations of the univalent X
chromosome during pachytene, (B) ‘asynapsed’ X chromosome
showing a lateral element with telomeres present at both ends,
(C) ‘fully self-synapsed’ X chromosome forming a hairpin structure
with overlapping telomeres that appear as a single signal, (D) ‘partial
self-synapsis including the centromere’ showing a portion of the
lateral element synapsed with both telomeres overlapping and
appearing as one, (E) ‘partial self-synapsis excluding the centromere’
showing a portion of the lateral element synapsed with two telomeres
distinguishable, (F) ‘X/autosome association’ showing a triradial structure with three telomeres representing the X and an autosomal bivalent.
Chromosome preparations of spermatocytes and oocytes
at diakinesis/MI
Chromosome preparations of spermatocytes at diakinesis/MI were
prepared from testes from 6-week-old males as previously described
(Peters et al., 1997). To obtain comparable preparations in the female,
oocytes from 3.5-week-old females were collected and cultured as
described above. After 4 hours in culture the zona pellucida was
removed by brief incubation in 2% pronase (Calbiochem) in
Waymouth’s MB752/1. Zona-free oocytes were placed on a slide
dipped in 1% paraformaldehyde (pH to 9.2) and dried in a humid
chamber overnight.
RESULTS
At MI, the single X chromosome in XO females can either
segregate intact to one spindle pole or undergo PSCS at
anaphase I, segregating one chromatid to each pole (Fig. 1 and
Hunt et al., 1995). However, previous studies in our laboratory
suggested that the segregation frequencies were markedly
different in XO females produced on C57BL/6 and C3H inbred
strain backgrounds (LeMaire-Adkins and Hunt, 2000). To
confirm this observation, we conducted a new analysis. The
results were not significantly different from the original study,
and the combined data demonstrate a highly significant
Fig. 3. Colocalization of SCP1 and SCP3 on the
univalent X chromosome. (A-C) Combined SCP3 and
SCP1 staining (green and red respectively, with yellow
colocalization) and FISH with X-chromosome specific
(purple) and telomeric (purple) probes. (D-F) SCP1
staining alone. (A,D) ‘Fully self-synapsed’ X
chromosome showing colocalization of SCP3 and SCP1
(yellow) along the length of the SC. (B,E) ‘Asynapsed’ X
chromosome showing no evidence of SCP1 staining.
(C,F) ‘Asynapsed’ X chromosome showing SCP1
staining along the entire length of the SC. Note: the
magnification is the same in all images; however, SC
length is dependent upon the stage of the cell (e.g. B,E
represent early pachytene) and the synaptic behavior of
the X (e.g. the fully self-synapsed X, as in A,D, is half
the length of the asynapsed X, as in C,F).
difference in X chromosome segregation between the two
inbred strains (χ2=85.06, P<0.001), with the incidence of
PSCS significantly higher on the C3H inbred background
(Table 1).
Differences in X chromosome segregation do not
reflect X-chromosome specific differences
To determine if the observed segregation differences were due
to X-chromosome specific differences, we crossed the two
inbred strains to generate F1 hybrid females that were identical
genetically, except that one type of female carried a single X
chromosome of C57BL/6 origin (XB6O females) and the other
an X of C3H (XC3HO females) origin. X chromosome
segregation in the two types of F1 females was virtually
identical (Table 1) and resembled one of the progenitor strains.
That is, segregation in both F1 females was similar to that
observed for the C3H parental strain, but significantly different
from the C57BL/6 strain (e.g. XB6O F1 females versus
C57BL/6 females, χ2=49.82, P<0.001). The altered
segregation pattern of the C57BL/6-derived X chromosome on
an F1 background excludes an X-chromosome specific effect,
and demonstrates that genetic differences in segregation of the
univalent X chromosome are mediated by an autosomal gene
or genes.
Coordinated segregation of sister chromatids
Frequency of oocytes with asynapsed
chromosomes
100%
90%
percent of oocytes
80%
70%
60%
Swiss Albino
C57Bl/6
C3H
50%
40%
30%
20%
10%
0%
16
17
18
19
20
Days post coitus
Fig. 4. Percentage of pachytene oocytes with an asynapsed X
chromosome on successive days of gestation. The two strains used in
this study, C57Bl/6 (squares) and C3H (triangles), exhibit an initial
decline in such cells followed by a slight increase while the Swiss
Albino strain (diamonds) (used by Speed et al., 1986), exhibits a
steady decline.
Does synaptonemal complex formation during
prophase influence segregation?
Coordinated behavior of the centromeres of sister chromatids
at MI (Fig. 1A) is thought to result from events unique to
meiotic prophase (Maguire, 1995). Thus, we hypothesized that
strain-specific segregation differences might reflect differences
in the synaptic behavior of the X chromosome that influence
the deposition or retention of cohesion proteins between sister
centromeres.
The absence of a homolog prevents the
univalent X from undergoing normal synapsis
during meiotic prophase but, in the mouse, the
single X chromosome has been reported to
exhibit frequent non-homologous selfsynapsis (Speed, 1986). To determine if
differences in synaptic behavior were
correlated with subsequent segregation events,
we compared X chromosome synaptic
configurations on the two inbred backgrounds.
Pachytene cells were examined by combining
immunofluorescence to visualize the SC and
two-color FISH to identify the X chromosome
Fig. 5. SCP2/ SCP3 localization at
diakinesis/metaphase I in spermatocytes and
oocytes. (A,C) Chromosomes (blue)
immunostained with CREST antiserum, which
localizes to the centromeres (green).
(B,D) Chromosomes (blue) and SCP3 localization
(red). (A,B) Two spermatocytes at diakinesis/MI
exhibiting SCP3 localization to all centromeres.
(C,D) A comparably fixed oocyte showing CREST
staining but no SCP3 localization. (Note: the
localization pattern of SCP2 is identical.)
2421
and distinguish the centromeric and telomeric ends of the
chromosome. Fig. 2 shows an example of this methodology
and of the different types of X chromosome synaptic
configurations observed. These configurations are similar to
those reported in previous EM studies of oocytes from XO
female mice (Speed, 1986).
To compare synapsis on the two inbred backgrounds,
configurations were categorized as detailed in Fig. 2. Data from
the study of 678 pachytene cells from 17-20 d.p.c. C57BL/6
fetuses and 623 cells from 17-19 d.p.c. C3H fetuses are shown
in Table 2. In addition, a total of 361 control oocytes from XX
females produced on either the C57BL/6 or C3H strain were
scored, and no synaptic defects involving the X bivalent were
observed (data not shown). Inspection of the data from the two
types of XO females revealed no striking difference in the
synaptic behavior of the univalent X chromosome on the two
genetic backgrounds (Table 2). However, to specifically assess
self-synapsis involving the centromeric region of the
chromosome (e.g. to test the hypothesis that PSCS is a
consequence of failure of the X to undergo self-synapsis
involving the centromeric region of the chromosome), we
compared the proportion of cells in which synapsis included
the centromere (e.g. ‘fully self-synapsed’ and ‘partially selfsynapsed including the centromere’; Fig. 2C,D). Contrary to
expectation, the background showing the highest incidence of
centromeric self-synapsis (C3H) was also the background with
the highest incidence of PSCS. Thus, the hypothesis that
nonhomologous synapsis involving the centromeric region of
the X chromosome in some way prevents PSCS at MI was not
supported by the synaptic profiles of the two different inbred
strains.
Because SCP3 only recognizes one component of the SC
(the lateral element), we could not conclude that all cases
scored as self-synapsed actually involved the formation of a
fully mature tripartite SC. To examine this further, we repeated
the analysis using the SCP3 antibody in combination with
SCP1, an antibody that recognizes a component of the central
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JOURNAL OF CELL SCIENCE 114 (13)
Fig. 6. SCP3 localization during the
transition from pachytene to dictyate
arrest. (A,D,G) CREST localization at
the centromeres (red), (B,E,H) SCP3
(green) and (C,F,I) merged images. (AC) Pachytene stage oocyte with SCP3
localization along the length of the SC.
(D-F) Diplotene/early dictyate oocyte
showing clustering of centromeres in
regions of the nucleus and SCP3
localization at most but not all
centromeres. (G-I) Late
diplotene/dictyate cell exhibiting
clustering of centromeres but no SCP3
staining above background levels. (Note:
the temporal localization pattern of SCP2
is similar; see text for details.)
element of the fully formed SC. SCP1
staining was evident on all selfsynapsed X chromosomes (Fig. 3D).
However, unexpectedly, a significant
proportion of cells with an asynapsed
X chromosome exhibited SCP1
staining along the length of the X
chromosome lateral element (Fig.
3F).
Is there a pachytene
checkpoint difference between
backgrounds?
It is generally thought that a
checkpoint mechanism operates to
cull cells with synaptic abnormalities
(Odorisio et al., 1998). Perinatal germ cell loss is dramatically
increased in XO females by comparison with XX siblings
(Burgoyne and Baker, 1985), suggesting that aberrations in X
chromosome synapsis increase germ cell loss at this stage.
Because self-synapsis of the X chromosome has been
suggested to be necessary for progression beyond pachytene
(Speed, 1986), we reasoned that differences in the stringency
of the checkpoint mechanism on the two genetic backgrounds
might account for the observed segregation differences. That
is, the increased incidence of PSCS in the C3H strain might
reflect a strain-specific relaxation in cell cycle control that
allowed a greater number of cells with an asynapsed X
chromosome to bypass the pachytene checkpoint mechanism.
The rapidity of germ cell loss in the perinatal ovary
precludes the analysis of synaptic behavior among apoptotic
cells (e.g. combined tunel assay and immunostaining with
antibodies to SC proteins). Thus, to test the hypothesis that the
survival of cells with an asynapsed X differed on the two
genetic backgrounds, we compared the proportion of cells with
an asynapsed X among late pachytene cells. For this analysis,
we scored pachytene cells in the perinatal ovary at a time at
which, for both strains, greater than 60% of prophase cells had
progressed beyond pachytene (e.g. 20 d.p.c. for C57BL/6 and
19 d.p.c. for C3H). Our expectation was that the accumulation
of pachytene cells with an asynapsed X chromosome (e.g.
pachytene arrest followed by apoptosis) or their rapid
elimination would skew the distribution of synaptic
configurations. However, although temporal fluctuation in the
number of cells with an asynapsed X chromosome was
observed on both backgrounds (see Fig. 4, and Discussion
Table 2. Frequency of different X chromosome synaptic configurations among pachytene cells from XO females produced
on two different inbred backgrounds
Partially self-synapsed
Fetus
C57BL/6
C3H
d.p.c.
n
Asynapsed
Fully
self-synapsed
17
18
19
20
17
18
19
161
223
126
168
233
182
208
107 (66%)
97 (43%)
60 (48%)
87 (52%)
126 (54%)
78 (43%)
94 (45%)
14 (9%)
66 (30%)
34 (27%)
35 (21%)
49 (21%)
58 (32%)
81 (39%)
including
centromere
excluding
centromere
X/autosome
7 (4%)
23 (10%)
8 (6%)
21 (13%)
10 (4%)
7 (4%)
14 (7%)
19 (12%)
23 (10%)
18 (14%)
20 (12%)
23 (10%)
26 (14%)
9 (4%)
14 (9%)
14 (6%)
6 (5%)
5 (8%)
25 (11%)
13 (7%)
10 (5%)
Coordinated segregation of sister chromatids
below), no obvious stain-specific differences were observed; an
asynapsed X chromosome was observed in 87/168 (52%) cells
from C57BL/6 females and 94/208 (45%) of cells from C3H
females (Table 2). Thus, our results provide no evidence
for a strain-specific difference in the synaptic checkpoint
mechanism.
The high incidence of cells with an asynapsed X among late
pachytene cells was unexpected, since these cells should be
strongly selected against by the pachytene checkpoint control
mechanism. As shown in Fig. 4, both XO females produced on
the C57BL/6 and C3H background as well as the previously
studied XO females produced on the Swiss Albino background
by Speed (Speed, 1986) show an initial decline in the number
of pachytene cells with an asynapsed X chromosome.
However, this decline is followed by a slight increase on both
backgrounds in this study (Fig. 4). These data, coupled with
the fact that our analysis of SCP1 stained preparations revealed
positive SCP1 staining on a proportion of asynapsed X
chromosomes, led us to suspect that the synaptic configuration
of the X chromosome was changing over time. That is, we
could not rule out the possibility that cells that escaped the
pachytene checkpoint mechanism via self-synapsis might
subsequently ‘open-out’, appearing asynapsed at late
pachytene. Indeed, a comparison of SCP1 staining between
oocytes at early and late pachytene (e.g. from 17 and 19 d.p.c.
fetuses, respectively) on the C3H background revealed a
significant increase in the number of apparently asynapsed X
chromosomes exhibiting SCP1 staining among late pachytene
cells (Table 3; χ2=13.65, P<0.001). However, there was no
significant difference between backgrounds with respect to
such cells (Table 3; χ2=0.28, P>0.5).
Disappearance of lateral element proteins at the
dictyate stage
Previous immunolocalization studies in mouse spermatocytes
using antibodies directed against the lateral element proteins
SCP2 and SCP3 have demonstrated that remnants of the SC
persist at the centromere until anaphase II (Dobson et al., 1994;
Offenberg et al., 1998). On the basis of these observations,
SCP3 has been postulated to facilitate sister chromatid and
sister kinetochore cohesion (Dobson et al., 1994). Thus, we
reasoned that PSCS might be explained by the premature
disappearance of one or both of these SC proteins from the X
centromere. Accordingly, we conducted immunolocalization
studies of cells at prometaphase of the first division. Our initial
attempts to visualize SCP3 were unsuccessful, and we assumed
that this was a technical artifact resulting from the fixation of
intact oocytes. To overcome this difficulty, we devised a
fixation protocol for fixing and spreading diakinesis stage
oocytes onto a microscope slide while maintaining the
chromosome-associated proteins. Because the fixation
methodology is essentially the same as that used for pachytene
cells, we were confident that any chromosome-associated
SCP3 protein would be visible. To our surprise, we could not
detect either SCP2 or SCP3 in oocytes from either XO or
control females, although the same fixation procedure yielded
strong centromeric staining in the male (e.g. Fig. 5). Similarly,
by western analysis SCP3 was not detectable in samples of 500
diakinesis stage oocytes; further, although a band
corresponding to SCP3 was detected in ovaries from newborn
females, signal intensity diminished with age, becoming
undetectable during the second week post partum (data not
shown).
To determine the timing of the disappearance of SCP2 and
SCP3 from the chromosomes, we analyzed oocytes from 3-, 5and 7-day-old females. This allowed us to assess the
localization of these proteins during the transition from
diplotene to the dictyate arrest stage. To specifically assess
localization at the centromere, preparations were doublelabeled with either the SCP2 or SCP3 antibody and CREST
serum and counterstained with DAPI. As is true at the earlier
stages of meiotic prophase, diplotene and dictyate stage
oocytes could be distinguished from somatic cells on the basis
of size, the dispersed nature of the chromatin and the fact that
all centromeres were replicated. During the course of the
analysis, it became evident that oocytes at the diplotene stage
show a characteristic congregation of centromeres at several
distinct locations within the nucleus, which becomes more
pronounced as the cells enter dictyate arrest (e.g. Fig. 6D,G).
A minimum of 50 cells were scored at each developmental
time point (i.e. 3, 5 and 7 days). A progressive decline in the
number of oocytes exhibiting staining was observed for SCP3,
with nearly 90% of cells from 3-day-old females but only 16%
of cells from 7-day-old females showing discrete SCP3 foci
(χ2=70.72, P<0.001). The disappearance of SCP2 appeared to
be even more rapid, as none of the cells from 7-day-old females
exhibited staining. Moreover, as shown in Fig. 6, although the
latest persisting foci of staining tended to be found at or near
the centromeres, among dictyate cells (i.e. cells exhibiting tight
clustering of centromeres), there was no evidence that either
protein was retained in the centromeric region. Thus, our
analysis suggests that, in contrast to spermatogenesis, neither
SCP2 or SCP3 is present at the centromere during the first
meiotic division in females.
DISCUSSION
Under normal meiotic conditions, where bivalents rather than
univalents are segregated at MI, premature separation of sister
Table 3. SCP1 staining on asynapsed X chromosomes
SCP1 staining
Background
C3H
C57BL/6
Stage (d.p.c.)
n
Present
Absent*
Significance
Early pachytene (17)
46
5 (11%)
41 (89%)
χ2=13.65, P<0.001
Late pachytene (19)
41
19 (46%)
22 (54%)
Late pachytene
30
12 (40%)
18 (60%)
*Includes oocytes with no or only very weak SCP1 staining.
2423
χ2=0.28, P>0.5
2424
JOURNAL OF CELL SCIENCE 114 (13)
chromatids results in the production of aneuploid gametes.
Indeed, our interest in the factors that predispose to this type
of segregation error is motivated by the fact that PSCS has been
postulated to be the major mechanism of age-related
nondisjunction in our species (Angell, 1997; Angell et al.,
1994; Wolstenholme and Angell, 2000). This aberrant MI
segregation behavior requires two events that normally do not
occur until MII: (1) the differentiation of functionally distinct
kinetochores at the centromeres of sister chromatids and (2)
the premature release of cohesion between sister centromeres
at anaphase. The coordinated segregation of sister chromatids
during mammalian female meiosis remains poorly understood.
Hence differences in the propensity for premature segregation
of X chromatids in oocytes from XO females produced on two
different inbred strains provide a genetic tool for understanding
the factors that influence sister chromatid behavior at MI.
An autosomal, trans-acting factor influences sister
chromatid segregation at MI
Based on meiotic studies in lower eukaryotes, it seemed likely
that X chromosome segregation differences would reflect
subtle differences between the X chromosomes on the two
inbred strains. That is, detailed studies of univalent
chromosomes in the grasshopper suggest that the patterns of
meiotic behavior and segregation of univalent chromosomes
reflect chromosome-specific features (Rebollo et al., 1998).
Similarly, in yeast, mutants with a single division meiotic
phenotype exhibit a mixed segregation pattern, with some
chromosomes exhibiting a proclivity for intact and others for
equational segregation. Experiments in which centromeric
sequences were transferred between chromosomes with
different segregation patterns demonstrated that the
segregation phenotype is a property of the centromere
(reviewed in Simchen and Hugerat, 1993).
To test for X-chromosome specific differences, we generated
F1 hybrid females carrying either a single X chromosome
derived from the parental C57BL/6 or the C3H inbred strain.
With the exception of the X chromosome, the two types of F1
females were genetically identical. Meiotic studies revealed no
difference in the segregation pattern of the univalent X
chromosome between the F1 females. Thus, contrary to
expectation, X-chromosome specific differences are not a
plausible explanation for the observed segregation differences.
We conclude that the actions of an autosomal gene or genes
influence X chromosome segregation. Further, since the
segregation phenotypes of both F1 females and one of the
parental strains were identical, this trans-acting genetic effect
appears to be dominant.
Synaptic behavior at pachytene is not a reliable
predictor of segregation
Based on studies of maize mutants, Maguire postulated that the
proteins of the synaptonemal complex confer the specialized
meiotic centromere cohesion requirements necessary for
proper chromosome segregation (reviewed in Maguire, 1995).
Support for this hypothesis can be drawn from a variety of
sources, including (1) mutations with precocious separation of
sister chromatids indicative of complete loss of sister
chromatid cohesion (e.g. in Drosophila, S. pombe and Sordaria
macrospora), which also exhibit defects in synapsis and/or
recombination (reviewed in Moore and Orr-Weaver, 1998), (2)
mutations in the SC components of S. cerevisiae (e.g. red1,
zip1 and hop1), which exhibit a slight increase in PSCS at MI
(Hollingsworth and Byers, 1989; Smith and Roeder, 1997;
Sym and Roeder, 1994), (3) immunolocalization studies in
male mammals, which demonstrate that remnants of the lateral
element of the SC are retained at the centromere until anaphase
II (Dobson et al., 1994; Offenberg et al., 1998) and (4) recent
observations suggesting that the SC components SCP2 and
SCP3 interact with the cohesion proteins SMC1 and SMC3
(Eijpe et al., 2000).
We hypothesized that genetic differences in MI segregation
behavior of the univalent X chromosome might reflect
differences in the synaptic behavior of the X chromosome that
influence the deposition or retention of cohesion proteins at the
centromere. The behavior of the univalent X during prophase
is intriguing; although the X has no homolog, previous studies
have demonstrated that the univalent chromosome frequently
exhibits a fold-back, self-synaptic behavior at pachytene
(Speed, 1986). Indeed, self-synapsis has been suggested to be
essential for germ cell survival, with the high incidence of cells
with an asynapsed X providing an explanation for the increased
perinatal germ cell loss observed in the XO mouse (Burgoyne
and Baker, 1985; Speed, 1986).
Detailed analysis of pachytene stage oocytes revealed no
striking difference in the self-synaptic behavior of the X
chromosome on the two genetic backgrounds. Our expectation
was that the strain with the highest incidence of PSCS would
have more cells in which self-synapsis excluded the
centromeric region of the chromosome. Although we did
observe a small difference between the two genetic
backgrounds, it was the opposite of our prediction; the C3H
background, which showed a higher frequency of PSCS, had a
slightly increased frequency of cells in which the X
chromosome centromeric region was self-synapsed.
The analysis of X chromosome synapsis is complicated by
the fact that cell selection is occurring, and the assumption
that all pachytene configurations are equally likely to survive
is almost certainly not valid. Although the exact timing of the
so called ‘pachytene’ checkpoint control mechanism is not
known, a previous study of XO mice by Speed suggested a
decline in cells with an asynapsed X with advancing
developmental age (Speed, 1986). Thus, we attempted to
enrich for late pachytene cells to test the hypothesis that
selection against cells with an asynapsed X chromosome
might differ on the two backgrounds, and that enhanced
survival of such cells might be associated with an increased
frequency of PSCS. We observed a slight but non-significant
difference in the frequency of pachytene cells with an
asynapsed X chromosome; however, the background with the
higher level of PSCS (C3H) had the lowest frequency of such
cells. Thus, our studies provided no evidence of relaxed cell
selection.
The observation by Speed of a decline in cells with an
asynapsed X chromosome with advancing developmental age
(Speed, 1986), is consistent with selection against this category
of cells. In contrast, we observed an initial decline followed by
a slight increase at the most advanced developmental ages (Fig.
4). Although the difference between Speed’s study and our own
may be due to strain differences, our subsequent studies
suggested that the apparent increase we observed reflected a
change over time in the conformation of the X chromosome.
Coordinated segregation of sister chromatids
That is, in immunolocalization studies using the SCP1
antibody to assess formation of mature SC, we observed SCP1
staining on asynapsed X chromosomes in some cells. This may
simply reflect a pattern of binding similar to that observed on
the unsynapsed portion of the X as occurs during male meiosis
(P. Moens, personal communication). However, a comparison
of early and late pachytene cells demonstrated a significant
increase in such cells with advancing developmental age.
Hence, we suggest that the presence of SCP1 is a remnant of
self-synapsis, and that the apparent asynapsed configuration is
analogous to the synaptic adjustment described in tandem
duplications (Moses and Poorman, 1981), or the residual SCP1
observed on separated cores of autosomal chromosomes at
diplotene (Moens and Spyropoulos, 1995). However, there was
no difference between strains in the frequency of such cells,
hence it does not provide insight to the genetic background
effect on segregation.
Evidence that meiotic centromeric cohesion is
sexually dimorphic
Given the complications of cell selection and the apparent
changes in the synaptic configuration of the X over time,
further studies of meiotic prophase seemed unlikely to provide
insight to the segregation behavior of the X chromosome.
However, previous immunolocalization studies in the male
demonstrated persistence of the lateral element proteins SCP2
and SCP3 at the centromere until anaphase II (Dobson et al.,
1994; Offenberg et al., 1998). Thus we reasoned that PSCS
segregation might be explained by the premature
disappearance of these proteins from the X chromosome
centromere. However, to our surprise, immunolocalization
studies of MI prometaphase cells failed to detect either SCP2
or SCP3 in oocytes from XO or control females. Although
initially we assumed that this was a technical artifact,
subsequent studies demonstrated that this was not the case.
First, using a modified fixation protocol, we were able to
demonstrate SCP2 and SCP3 localization at the centromeres in
spermatocytes but not oocytes at the diakinesis/MI stage.
Second, the SCP3 protein was not detectable in germinal
vesicle stage oocytes by western analysis. Third, an analysis of
fetal oocytes that had progressed beyond pachytene and were
entering dictyate arrest suggested that, as the SC disassembles,
foci of SCP2 and SCP3 proteins remain briefly associated with
the centromere but do not persist throughout the period of
meiotic arrest. Clearly, these studies do not rule out the
possibility that a small amount of these proteins persists but is
below the level of detection by immunostaining and western
analysis. Nevertheless, it seems more likely that the role of
these SC proteins is limited to meiotic prophase in the female.
Thus, the analysis of other meiosis-specific centromereassociated proteins – for example, the recently identified
cohesin component, Rec 8 (Watanabe and Nurse, 1999) or a
homolog of yeast monopolin (Toth et al., 2000), a kinetochore
protein required for normal MI segregation – will be necessary
to understand the segregation behavior of the univalent X
chromosome.
Our observations suggest an important sexual dimorphism
in centromere associated proteins during the first meiotic
division. Disappearance at diplotene of the protein now known
as SCP3 was reported in an early localization study of rat
oocytes (Dietrich et al., 1992). However, this report appears to
2425
have gone unnoticed, as persistence of SCP3 at the centromere
throughout MI is frequently mentioned as though it is a
universal feature of mammalian meiosis. The disappearance of
the two lateral element components, SCP2 and SCP3,
coincident with entry into meiotic arrest suggests that, at least
in the female, these proteins are not essential components of
the cohesion complex that mediates the specialized MI
behavior of sister centromeres. This difference between male
and female meiosis may have functional consequences, e.g. the
disappearance of these proteins prior to the first division may
contribute to the vulnerability of the female meiotic process by
increasing the likelihood of PSCS.
Interestingly, the phenotype of the recently reported SCP3
knockout mouse suggests that sex-specific differences in the
meiotic role of this protein may not be limited to the meiotic
divisions: the SCP3 null mutant male is sterile, exhibiting
synaptic failure and meiotic arrest at the zygotene stage (Yuan
et al., 2000). In contrast, the SCP3 null female is apparently
fertile. Detailed meiotic studies of the mutant female have yet
to be conducted. It will not only be interesting to learn how
homolog synapsis and recombination proceed in the absence
of this protein, but also whether chromosome segregation is
affected. Thus, the SCP3 protein and other SC components
may provide long-sought insight to sex-specific differences in
meiotic cell division in mammals.
We are grateful to T. Hassold and H. F. Willard for helpful
discussions and comments on the manuscript. In addition, we thank
T. Ashley, C. Heyting, R. Jessberger and P. Moens for generous gifts
of the synaptonemal complex antibodies used in these studies. This
work was supported by National Institutes of Health grant R01
HD31866 to P.A.H.
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