Cdc20 Is Critical for Meiosis I and Fertility of Female Mice
Fang Jin1, Masakazu Hamada2, Liviu Malureanu1, Karthik B. Jeganathan1, Wei Zhou1, Dean E. Morbeck3,
Jan M. van Deursen1,2*
1 Department of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota, United States of America, 2 Department of Biochemistry and
Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota, United States of America, 3 Department of Obstetrics and Gynecology, Mayo Clinic College of
Medicine, Rochester, Minnesota, United States of America
Abstract
Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital
birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely
unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C), initiates sister-chromatid
separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from
metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at
present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we
generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but
females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with
wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they
become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast
majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome
misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in
metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective
threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome
missegregation and formation of aneuploid gametes.
Citation: Jin F, Hamada M, Malureanu L, Jeganathan KB, Zhou W, et al. (2010) Cdc20 Is Critical for Meiosis I and Fertility of Female Mice. PLoS Genet 6(9):
e1001147. doi:10.1371/journal.pgen.1001147
Editor: Orna Cohen-Fix, National Institute of Diabetes and Digestive and Kidney Diseases, United States of America
Received February 22, 2010; Accepted September 1, 2010; Published September 30, 2010
Copyright: ß 2010 Jin 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 by NIH grant CA96985. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: vandeursen.jan@mayo.edu
holds sister chomatids together, inducing the physical separation of
sister chromatids by spindle forces [7,8].
A thorough assessment of the role of mitotic checkpoint genes in
gametogenesis and infertility has not been possible because
complete inactivation of mammalian mitotic checkpoint genes
invariably disrupts the chromosome segregation process so
severely that cells cannot survive [2,9]. In vitro studies of primary
mouse oocytes in which key mitotic checkpoint proteins were
depleted by morpholinos or RNA interference have pointed to an
importance of several mitotic checkpoint proteins during the first
meiotic division. For instance, sustained prophase I arrest of
primary oocytes depends on stabilization of the Cdc20-related
APC/C coactivator Cdh1 by BubR1 [10]. BubR1 retains control
of Cdh1 stability after hormone-induced resumption of meiosis,
thereby allowing APC/CCdh1-mediated securin degradation and
progression through prometaphase I. Interestingly, BubR1 protein
levels have been shown to decline in ovary and testis as normal
mice age, which combined with the observation that mutant mice
with low amounts of BubR1 are infertile, has led to speculation
that BubR1 might be a key determinant of age-related meiotic
errors in germ cells [11]. While APC/CCdh1 regulates early
meiotic events in mice [10,12], Cdc20 knockdown experiments in
primary oocytes indicate that APC/CCdc20 is active in late meiosis
I [10], where it is responsible for driving oocytes into anaphase via
the destruction of cyclin B1and securin, much like mitosis in
Introduction
Mitotic checkpoint genes are believed to be prime targets for
deregulation in human infertility [1]. The mitotic checkpoint
constitutes an intricate molecular network that ensures accurate
chromosome segregation by coordinating metaphase-to-anaphase
progression with the establishment of bipolar spindle attachment
and metaphase plate alignment of all mitotic chromosome pairs
[2]. At early stages of mitosis, various mitotic checkpoint proteins,
including members of the Bub and Mad protein families,
concentrate at unattached kinetochores to generate a diffusible
signal that inhibits the anaphase-promoting complex or cyclosome
(APC/C), a large E3 ubiquitin ligase that drives metaphase-toanaphase transition by catalyzing the ubiquitination and degradation of cyclin B1 and securin [3]. Although the exact
composition of the inhibitory signal remains a major subject of
investigation, it is believed to contain Bub3-bound BubR1 and
Mad2 that has been primed by kinetochore-associated Mad1Mad2 to stably interact with the APC/C activating subunit Cdc20
[4,5,6]. Upon attachment and alignment of the last chromosome
pair, the inhibitory signal is quenched and APC/C activated
through release of Cdc20 inhibition, triggering the ubiquitination
and destruction of cyclin B1 and securin. Separase, a protease that
is held in an inactive state by securin and cyclin B1/Cdk1, is then
allowed to cleave the Scc1 subunit of the cohesin complex that
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The Female Germline Is Sensitive to Cdc20 Loss
alleles (Figure 1A–1D). The Cdc20H allele was produced by
targeted insertion of a neomycin phosphotransferase II (neo) gene
cassette into the third intron of the Cdc20 gene (Figure 1A). The
neo gene contains a cryptic exon with stop codons in all three
reading frames, thereby considerably reducing the amount of wildtype protein produced by targeted allele [11,20,21,22]. The
Cdc202 allele was from gene trap mouse embryonic stem (ES) cell
clone XE368 (Figure 1B). Previously, it has been shown that this
gene trap allele is the equivalent of a null allele and that embryos
that are homozygous for this allele arrest and die at the two-cell
stage of development [23]. In contrast, Cdc20+/H, Cdc20+/2,
Cdc20H/H and Cdc202/H mice were viable and had no overt
phenotypes. Western blot analysis demonstrated that Cdc20+/H,
Cdc20+/2, Cdc20H/H and Cdc202/H ovary and testes had a graded
reduction of Cdc20 protein (Figure 1E and 1F). Western blot
analysis of spleen, bone marrow, and mouse embryonic fibroblast
extracts of Cdc20+/+ and Cdc202/H mice suggested that the
observed Cdc20 protein reductions are universal, irrespective of
tissue or cell type (Figure 1G, and data not shown).
Author Summary
Aneuploidy, an abnormal number of chromosomes, is a
common defect in sperm and egg cells that is responsible
for human infertility, miscarriage, and congenital birth
defects. Although these developmental outcomes are
prevalent in human reproduction, little is known about
the molecular defects that may cause aneuploidy in germ
cells. In this study, we identify Cdc20, a critical activator of
the APC/C E3 ubiquitin ligase that initiates sister chromosome separation by ordering the destruction of cyclin B1
and securin, as a female infertility gene. We show that
female mice with low amounts of Cdc20 have normal
fitness but almost exclusively produce aneuploid embryos
that fail to thrive and die early in development. The
aneuploidy primarily results from chromosome segregation errors in primary oocytes that may be caused by
inefficient APC/C-mediated destruction of mitotic cyclins
and securin during metaphase I. Thus, our studies reveal
that primary oocytes are highly dependent on Cdc20 for
accurate chromosome segregation and raise the possibility
that Cdc20 insufficiency may be a cause of infertility in
otherwise healthy women.
Cdc20 Hypomorphic Females Are Infertile or Subfertile
Despite Normal Oogenesis
While establishing cohorts of Cdc20 mutant mice for long-term
observation, we noticed that Cdc202/H females yielded little or no
offspring, which prompted us to measure the impact of graded
reduction in Cdc20 expression on female fertility. Two-month-old
Cdc20+/+, Cdc20+/H, Cdc20+/2, Cdc20H/H and Cdc202/H mice
were bred to Cdc20+/+ males of the same age and the number of
litters and pups produced per female was recorded for three
months. Despite normal copulation rates (Figure 2A), Cdc202/H
females produced on average about 4-fold fewer litters than
females of the other genotypes (Figure 2B), while the average
number of pups was about 15-fold lower (Figure 2C). Notably, of
the seven Cdc202/H females in the study, four failed to produce
any offspring (Figure 2D). Only Cdc20+/2 and Cdc20+/H embryos
can be produced by Cdc202/H females bred to Cdc20+/+ males.
Importantly, pups of these genotypes were produced at normal
rates when Cdc20+/2, Cdc20+/H and Cdc20H/H females were bred
to Cdc20+/+ males (Figure 2C and 2D), indicating that the failure
of Cdc202/H females to produce offspring with Cdc20+/+ males was
not due to the genotype of the embryos produced. Together, the
above data demonstrate that Cdc202/H females are either infertile
or severely subfertile. The Cdc20 threshold level for fertility
problems is remarkably sharp because Cdc20H/H females, which
produce slightly more Cdc20 than Cdc202/H females, have normal
fertility (Figure 2A–2D). Ten of 10 Cdc202/H males were fertile
and produced on average 7 pups per litter (data not shown),
indicating that gametogenesis in male mice has a lower
dependence on Cdc20 than the female reproductive system.
To study how Cdc20 deficiency impedes female fertility, we
screened hematoxylin-eosin ovary sections of sexually mature
Cdc202/H females for overt defects in oogenesis. However, no
apparent morphological differences were found (Figure 2E).
Cdc202/H and Cdc20+/+ ovary sections contained similar amounts
of primordial, primary, secondary and antral follicles, as well as
similar numbers of mature oocytes and corpora lutea (Figure 2E and
2F). These data indicated that the fertility problem of Cdc202/H
females is not due to a failure to produce, mature or ovulate oocytes.
Cdc20
somatic cells [13]. Coordination of APC/C
activation with
proper kinetochore-microtubule attachment in meiosis I is
dependent on the mitotic checkpoint proteins Mad2 and Bub1,
as depletion or expression of dominant-negative mutants of these
proteins in primary mouse oocytes causes chromosome missegregation [14,15,16,17].
Whereas the depletion studies in primary mouse oocytes identify
Cdc20 and Cdh1 as critical regulators of the first meiotic division,
testing whether the functions unveiled in vitro operate in vivo
remains an important challenge. Furthermore, it remains
unknown whether Cdc20 and Cdh1 are also important for male
meiosis I or stages of male and female gametogenesis other than
meiosis I. Importantly, for Cdc20 and Cdh1 to be candidate
infertility genes, one would expect their dysfunction to reduce
fertility without compromising overall health and viability.
Addressing these issues has been hampered by the embryonic
lethality caused by inactivation of Cdh1 and Cdc20 in mice, with
Cdh1-null embryos dying at mid-gestation due to placental defects
[18,19] and Cdc20-null embryos at the two-cell stage due to
permanent metaphase arrest [18].
In the present study, we bypassed the problem of early
embryonic lethality of Cdc20 knockout mice by generating mutant
mouse strains in which the dose of Cdc20 is reduced in graded
fashion, enabling us to examine the physiological relevance of this
APC/C cofactor. Our findings reveal that the threshold for
pathophysiology is lowest in the female germline. We demonstrate
that while both mitotic and meiotic divisions of male and female
germ cells are characterized by inaccurate chromosome segregation and aneuploidization, only female meiosis I is so severely
affected that almost exclusively aneuploid mature eggs are
generated. We show that these eggs fertilize normally, but that
the resulting zygotes die after the first few embryogenic divisions.
Results
Generation of Mutant Mice with Graded Reduction of
Cdc20
Fertilized Eggs from Cdc20 Hypomorphic Females Fail to
Develop into Blastocysts
A series of mutant mouse strains in which expression of Cdc20 is
gradually reduced was generated by using various combinations of
wild-type (Cdc20+), hypomorphic (Cdc20H) and knockout (Cdc202)
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To explore preimplantation embryonic development, Cdc202/H
and Cdc20+/+ females were naturally mated with Cdc20+/+ males
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The Female Germline Is Sensitive to Cdc20 Loss
Figure 1. Generation of mice with graded reduction in Cdc20 dosage. (A) Schematic representation of the primary Cdc20 gene targeting
strategy. Part of the Cdc20 locus (+), the targeting vector, the hypomorphic allele (Cdc20H), EcoR1 restriction sites and the Southern probe are
indicated. (B) Schematic representation of the Cdc202allele was from gene trap mouse embryonic stem (ES) cell clone XE368. (C) Southern-blot
analysis of mice with indicated Cdc20 genotypes. (D) PCR-based genotype analysis of Cdc20 mutant mice. Positions of PCR primers (a–e) are indicated
in (A,B). (E–G) Western blot analysis of whole ovary (E), testis (F), spleen and bone marrow (G) extracts of the indicated genotypes for Cdc20. Actin and
tubulin served as loading controls. Cdc20 protein signals were quantified using ImageJ software and normalized to background and either actin or
tubulin. For details see materials and methods.
doi:10.1371/journal.pgen.1001147.g001
and embryos were collected at day 3.5 of development (E3.5).
While 93% of embryos collected from Cdc20+/+ females were at
the expected blastocyst stage, only 15% of Cdc202/H females had
reached this stage (Figure 3A and 3B). The remaining embryos
were either in the one- to four-cell stage or completely
degenerated. Notably, the total number of embryos produced by
Cdc20+/+ and Cdc202/H females was the same (Figure 3B),
indicating Cdc202/H females had normal fertilization rates and
were capable of ovulating normal numbers of mature oocytes.
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Furthermore, the number of normal blastocysts produced by
Cdc202/H females is similar to the number of live born pups these
females produce, indicating that embryos that attain the blastocyst
stage were capable of developing into healthy animals.
The above data indicated that the majority of eggs produced by
Cdc202/H females stop proliferating after the first cell divisions of the
preimplantation period. To confirm this and to characterize
preimplantation embryo development, we collected one-cell stage
embryos from Cdc20+/+ and Cdc202/H females crossed with Cdc20+/+
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Figure 2. Female mice with low amounts of Cdc20 have poor fertility. (A) Average number of vaginal plugs per female for the indicated
genotypes (during 3 months of breeding). (B) Average number of litters per female for the indicated genotypes (during 3 months of breeding). Data
presented in (A,B) are mean 6 SEM. (C) Average number of pups per female (during 3 months of breeding). Chart legend is as in (A). Asterisks indicate
statistical significance (one way ANOVA p,0.0001) between Cdc202/H and the other genotypes. (D) Percentages of subfertile and infertile females per
genotype. (E) H/E-stained ovary sections from Cdc20+/+ and Cdc202/H females (5 mm paraffin sections). A = antral follicles; CL = corpus luteum. Bars in
top and bottom panels are 400 mm and 100 mm, respectively. (F) Quantification of various follicles and corpora lutea in H/E sections of Cdc20+/+ and
Cdc202/H ovaries. Error bars represent mean 6 SEM.
doi:10.1371/journal.pgen.1001147.g002
males and monitored their development in vitro. As expected, most
embryos from Cdc20+/+ females developed to the blastocyst stage
within four days (Figure 3C and 3D). In contrast, none of the embryos
from Cdc202/H females developed beyond the 4-cell stage, with the
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majority of embryos remaining at the one cell stage. This growth
phenotype is remarkably different from that of Cdc202/2 embryos,
which typically arrest in metaphase at the two-cell stage due to
inability to degrade cyclin B1 and securin in the absence of Cdc20
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The Female Germline Is Sensitive to Cdc20 Loss
Figure 3. Cdc202/H eggs fertilized by Cdc20 +/+ males rarely develop into blastocysts. (A) In vivo development of E3.5 embryos from Cdc20+/+
and Cdc202/H females crossed with Cdc20+/+ males. (B) Quantitation of the in vivo developmental defects. (C) In vitro development of E0.5 embryos from
Cdc20+/+ and Cdc202/H females fertilized by Cdc20+/+ males. Images were collected at 24 h intervals (day 1 is the day of embryo collection, which
corresponds to E0.5). Note that none of the embryos from Cdc202/H females developed beyond the 4-cell stage. Yellow and red arrowheads highlight
malformed two- and four-cell stage embryos, respectively. (D) Quantitation of the in vitro developmental defects. Note that 62% of the embryos from
Cdc202/H females crossed with Cdc20+/+ males failed to develop beyond the one-cell stage.
doi:10.1371/journal.pgen.1001147.g003
[23]. Importantly, one cell stage embryos from Cdc202/H females are
either Cdc20+/2 or Cdc20+/H. Embryos of these genotypes show
normal survival rates when derived from Cdc20+/2 and Cdc20+/H
females and Cdc20+/+ males (see Figure 2B and 2C). Together, these
data suggested that the early death of the embryos produced by
Cdc202/H females is due to defects introduced during oogenesis.
collected one-cell stage embryos from Cdc20+/+, Cdc20H/H and
Cdc202/H females mated with Cdc20+/+ males and prepared
metaphase spreads for chromosome counts. We found that 11% of
embryos from Cdc20+/+ females were aneuploid compared to 27%
and 78% of embryos from Cdc20H/H and Cdc202/H females,
respectively (Figure 4A). Aneuploidy was strongly biased toward
loss of chromosomes, irrespective of Cdc20 genotype. Importantly,
nearly 30% of aneuploid embryos from Cdc202/H females had 14
to 19 extra chromosomes (Figure 4A and 4D). We noted that these
embryos contained a very high proportion of chromosome pairs
(Figure 4D), which suggested that they originated from mature
oocytes that had failed to complete meiosis II after fertilization.
Cdc20 Hypomorphic Females Produce Aneuploid
Oocytes and Embryos
We hypothesized that Cdc20 hypomorphism promotes chromosome missegregation during oogenesis, resulting in production
of aneuploid embryos that fail to thrive. To test this idea, we
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Figure 4. Oocytes and embryos from Cdc202/H females have abnormal chromosome numbers. (A) Fertilized eggs from Cdc202/H females
x Cdc20+/+ males show near-diploid or near-triploid aneuploidy. (B) Mitotic divisions that establish oogonia during fetal development are aneuploidy
prone if Cdc20 levels are low. (C) Meiosis I is a prominent source of aneuploidy in Cdc202/H females. (D) Image of a chromosome spread of a fertilized
egg with probable meiosis II failure. Arrowheads mark examples of duplicated chromosomes (most likely oocyte derived). (E) Image of an aneuploid
metaphase I of a Cdc202/H primary oocyte. (F) Image of an aneuploid metaphase II oocyte from a Cdc202/H female.
doi:10.1371/journal.pgen.1001147.g004
harvested from ovaries of Cdc20+/+, Cdc20H/H and Cdc202/H
females. In mice, primary oocytes normally have 20 paired
chromosomes, called bivalents. Primary oocytes from Cdc20+/+
and Cdc20H/H females had abnormal numbers of bivalents in 10%
and 13% of spreads, respectively (Figure 4B). In contrast, primary
oocytes from Cdc202/H females had considerably more aneuploidy, with 29% of spreads showing abnormal numbers of bivalents
(Figure 4B and 4E). These spreads showed no evidence of
Next, we determined whether Cdc20 hypomorphism also leads
to erroneous chromosome segregation at earlier stages of
oogenesis. During embryogenesis, primordial germ cells migrate
to the developing gonad to form oogonia, which expand in
number through a series of mitotic divisions before differentiating
into primary oocytes that arrest in prophase of meiosis I. To
determine whether the early mitotic divisions might contribute to
the aneuploidy seen in fertilized eggs, primary oocytes were
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The Female Germline Is Sensitive to Cdc20 Loss
precocious separation of bivalents, indicating that formation of
chiasmata was intact at low Cdc20 levels.
Although Cdc20 insufficiency causes aneuploidy during the
early mitotic divisions of oogenesis, aneuploidy rates of primary
oocytes were substantially lower than those of fertilized eggs. To
explore whether additional aneuploidy occurred during meiosis I,
we prepared metaphase spreads from secondary oocytes of
Cdc20+/+, Cdc20H/H and Cdc202/H females and counted chromosomes. We found that aneuploidy rates of secondary oocytes from
Cdc20+/+ and Cdc20H/H females increased modestly to 23% and
22%, respectively (Figure 4C). This verified that the level of Cdc20
protein in oocytes from Cdc20H/H females was enough to let the
chromosomes separate correctly at meiosis I. In contrast, a much
more dramatic increase was recorded for secondary oocytes from
Cdc202/H females, with 90% of spreads showing numerical
chromosome abnormalities (Figure 4C and 4F).
To obtain direct evidence for chromosome missegregation
during the first meiotic division of Cdc20 insufficient oocytes, we
monitored chromosome movements of Cdc20+/+ and Cdc202/H
primary oocytes during meiosis I using time-lapse fluorescence
imaging (Figure 5A). To visualize chromosomes we injected in
vitro transcribed H2B-mRFP mRNA into the oocytes. In this
setup, oocytes from Cdc202/H females displayed much higher rates
of chromosome missegregation than oocytes from Cdc20+/+
females (Figure 5B). The two types of errors that were observed
are congression failure and chromosome lagging, of which the
latter defect was clearly most frequent. Particularly, chromosome
lagging incidents involving three or more lagging chromosomes
occurred at much higher rates in Cdc202/H oocytes (Figure 5B and
5C, and Video S1 and Video S2). Thus, consistent with our
chromosome counts on secondary oocytes, chromosome segregation errors during meiosis I contribute considerably to the
infertility phenotype of Cdc202/H females.
were coinjected with H2B-mRFP mRNA to accurately assess the
timing of cyclin B1-EGFP degradation. As illustrated in Figure 7A
and 7B, Cdc20+/+ oocytes degraded most of their cyclin B1-EGFP
during late prometaphase and early metaphase. Cdc202/H oocytes
entered metaphase I around the same time as Cdc20+/+ oocytes.
However, they did so with relatively high cyclin B1-EGFP protein
levels and completed substrate degradation ,2 h later than
Cdc20+/+ oocytes. To confirm that cyclin B1 degradation was
delayed, we used indirect immunofluorescence to measure
endogenously expressed cyclin B1 levels of Cdc20+/+ and
Cdc202/H oocytes in metaphase I. As shown in Figure 7C and
7D, cyclin B1 levels were indeed higher in Cdc202/H oocytes than
in Cdc20+/+ oocytes. Importantly, these oocytes also showed
elevated levels of phosphorylated Cdk substrates (Figure 7C and
7D), suggesting that the rise in cyclin B1 expression resulted in
increased cyclin B1-Cdk1 activity in metaphase I.
Next, we coinjected securin-EYFP [24] and H2B-mRFP mRNA
into Cdc202/H and Cdc20+/+ primary oocytes. We noticed that
expression of securin-EYFP protein markedly inhibited PBE even
in Cdc20+/+ oocytes (data not shown), but were able to control this
problem by reducing the concentration of the injected securinEYFP mRNA. In Cdc20+/+ oocytes, onset of securin-EYFP
degradation typically coincided with metaphase entry and then
rapidly progressed until anaphase onset (Figure 8). In Cdc202/H
oocytes, however, securin-EYFP protein degradation did not start
until mid metaphase. Degradation not only started later, but was
also less efficient, resulting in anaphase entry with higher than
normal levels of securin-EYFP. In a recent study, McGuinness et
al. demonstrated that the timing of cyclin A2 degradation in
primary oocytes is similar to that of securin [17], which is
surprising given that mitotic cells fully degrade this cyclin in
prometaphase. In light of these findings, we wanted to examine
whether the degradation of cyclin A2 was impaired in Cdc202/H
oocytes. As for securin-EYFP, cyclin A2-EGFP inhibited PBE in
Cdc20+/+ oocytes when expressed at high levels (data not shown),
but again we were able to control this problem by injecting low
amounts of transcript. Consistent with the earlier data [17],
Cdc20+/+ primary oocytes rapidly destroyed cyclin A2-EGFP in
metaphase I (Figure 9). In contrast, both the onset and the rate of
cyclin A2-EGFP were substantially reduced in Cdc202/H oocytes.
Strikingly, Cdc202/H oocytes again entered anaphase I with higher
substrate levels than Cdc20+/+ oocytes. Taken together, the above
data demonstrate that multiple APC/C substrates are inefficiently
degraded when Cdc20 levels are low, raising the possibility that
persistent cyclin-CDK activity in metaphase I might underlie, at
least in part, the chromosome missegregation phenotype of
Cdc202/H oocytes.
It is conceivable that delayed cyclin and securin degradation
impairs separase activation, and therefore proper cleavage of
cohesin along chromosome arms of bivalents prior to anaphase
onset. To test for this possibility, we collected Cdc202/H and
Cdc20+/+ primary oocytes, cultured them in vitro until they
arrested in metaphase II and then stained chromosomes for the
presence of Rec8, a meiosis specific component of the cohesin
complex [25,26]. While Rec8 staining was readily detectable along
chromosome arms of metaphase I chromosomes, no such staining
was detectable in metaphase II oocytes, irrespective of Cdc20
genotype (Figure S1), implying that Cdc202/H oocytes generated
sufficient separase activity for complete cleavage of Rec8.
Furthermore, core mitotic checkpoint proteins that are involved
in kinetochore assembly, kinetochore-microtubule and/or spindle
assembly checkpoint activation, such as Bub1, BubR1, and Mad2,
were normally localized at kinetochores of Cdc202/H primary
oocytes (Figure S2).
Cdc20 Hypomorphism Prolongs Metaphase I
Orderly progression of oocytes through meiosis I is controlled
by the APC/C, which prompted us to examine whether timing of
meiosis I is deregulated at low Cdc20 levels. Cdc20+/+ and Cdc202/
H
oocytes were injected with H2B-mRFP mRNA and observed by
time-lapse microscopy while executing meiosis I. We found that
the time from germinal vesicle breakdown (GVBD) to metaphase
was similar in Cdc20+/+ and Cdc202/H oocytes (Figure 6A and 6B),
which is consistent with the notion that Cdh1 functions as the
primary ACP/C activator during the early stages of meiosis I [12].
However, the average time from metaphase entry to anaphase
onset was about two times longer in Cdc202/H oocytes than in
Cdc20+/+ oocytes (Figure 6A and 6B). This delay was unlikely to be
due to chromosome segregation errors as oocytes with misaligned
or lagging chromosomes were excluded from the analysis.
Consistent with delayed metaphase progression, PBE extrusion
was markedly delayed in Cdc202/H oocytes (Figure 6C). Taken
together, these data indicate that the timing of metaphase I is
subject to deregulation when the amount of Cdc20 protein is
limited.
Low Cdc20 Impairs Degradation of Mitotic Cyclins and
Securin in Metaphase I
To explore the mechanism underlying the chromosome
missegregation phenotype of Cdc202/H primary oocytes, we
measured the rate of degradation of two key APC/CCdc20
substrates, cyclin B1 and securin [12]. In the first set of
experiments, we injected Cdc202/H and Cdc20+/+ primary oocytes
with mRNA encoding cyclin B1-EGFP and monitored the
degradation of fluorescent protein by live-cell imaging. Oocytes
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Figure 5. Cdc202/H oocytes show increased chromosome missegregation in meiosis I. (A) Schematic overview of the experimental
procedure. A small amount of H2B-mRFP mRNA was injected into GV-positive Cdc20+/+ and Cdc202/H primary oocytes. After short recovery, oocytes
were released from prophase I arrest by removal of dbcAMP. About 1 h after GVBD, we started to monitor chromosome movements by live cell
imaging. We note that GVBD itself was not affected by Cdc20 hypomorphism. (B) Percentage Cdc20+/+ and Cdc202/H primary oocytes with the
indicated chromosome segregation errors. (C) Examples of Cdc202/H oocytes undergoing normal or aberrant anaphase I. Arrowheads highlight
misaligned and lagging chromosomes. We note that most oocytes with lagging chromosomes were able to complete meiosis I. Bar is 10 mm.
doi:10.1371/journal.pgen.1001147.g005
(Figure 10A), secondary spermatocytes of Cdc202/H males had much
lower aneuploidy rates than secondary oocytes of Cdc202/H females
(19% versus 90%). Chromosome counts on primary spermatocytes
revealed a 4-fold increase in aneuploidy due to Cdc20 hypomorphism, with 12% of spreads showing abnormal numbers of bivalents
(Figure 10B), suggesting that the mitotic divisions that spermatogonia
have to undergo to produce primary spermatocytes are error prone
at low Cdc20 levels. The rather modest increase in aneuploidy from
Aneuploidy Rates during Male Meiosis I Are Relatively
Low
Cdc202/H males appeared to have normal fertility, predicting that
male meiosis I is much less sensitive to Cdc20 hypomorphism. To
verify this, we prepared chromosome spreads of testicular cell
suspensions from Cdc20+/+ and Cdc202/H mice and performed
chromosome counts on secondary spermatocytes. Although aneuploidy was 5-fold higher at low than at normal Cdc20 levels
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The Female Germline Is Sensitive to Cdc20 Loss
Figure 6. Metaphase I is retarded in Cdc202/H oocytes. (A) The progression through meiosis I of Cdc20+/+ and Cdc202/H oocytes expressing
H2B-mRFP was monitored by live-cell microscopy and typical examples of image sequences are shown. Time after GVBD is indicated in each image
(h:min). Abbreviations: PM, prometaphase; M, metaphase; A, anaphase; and PBE, polar body extrusion. Scale bar is 10 mm. (B) Measurement of the
timing of meiosis I of H2B-mRFP-expressing Cdc20+/+ and Cdc202/H oocytes by live-cell imaging. Oocytes with congression defects were excluded
from the experiment. Data shown are mean 6 SEM. *p,0.05 (student t-test). (C) Polar body extrusion rates of cultured Cdc20+/+ and Cdc202/H
primary oocytes as assessed by time-lapse microscopy (DIC imaging). The time at which 50% of oocytes had completed PBE was 9.5 h for Cdc20+/+
oocytes and 13.6 h for Cdc202/H oocytes.
doi:10.1371/journal.pgen.1001147.g006
Because aneuploidy has been associated with reduced cell
growth and survival [27,28], one might have predicted that
oogenesis would be severely disrupted in Cdc20 hypomorphic
mice. Surprisingly, however, we did not observe significant
alterations in the number and morphology of follicles and corpora
lutea in these mice. These findings suggest that cellular pathways
that might inhibit cell proliferation or induce cell death in response
to chromosome missegregation are either not active in female
germ cells or require a higher threshold for activation than in
somatic cells [29]. Our finding that folliculogenesis was unperturbed was also unexpected in light of studies showing that
depletion of Cdc20 from primary oocytes by a morpholino causes
metaphase I arrest [12]. For somatic cells it has been estimated
that metaphase arrest requires a 20-fold or higher reduction in
cellular Cdc20 levels [30]. We suspect that morpholino treatment
reaches this level of reduction, whereas Cdc20 hypomorphism
does not.
In systematically karyotyping primary and secondary oocytes
and fertilized eggs, we discovered that Cdc20 hypomorphism
promotes aneuploidization at different stages of oogenesis,
12% to 19% as primary spermatocytes develop into secondary
spermatocytes underscores that the fidelity of male meiosis I remains
quite high at low Cdc20 levels. Furthermore, histology and apoptosis
rates were normal in testis of Cdc202/H males, as judged by
hematoxylin and eosin (H/E) and TUNEL staining of testis sections,
respectively (Figure 10C–10E).
Discussion
By generating a series of mice with graded reduction in Cdc20
levels, we discovered a remarkably sharp threshold for Cdc20
expression in female germ cells below which chromosome
segregation errors occur at high frequency, leading to production
of aneuploid eggs that are fertilization competent but fail to
progress beyond the first few embryonic divisions. On the other
hand, low Cdc20 levels are well tolerated by somatic tissues and
have no overt impact on the overall health and life expectancy of
mice. These findings raise the intriguing possibility that hypomorphic Cdc20 alleles may be responsible for unexplained fertility
problems in otherwise healthy women.
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The Female Germline Is Sensitive to Cdc20 Loss
Figure 7. Cyclin B1 degradation is delayed during metaphase I if Cdc20 are low. (A,B) Kinetics of cyclin B1-EGFP degradation during
meiosis I. Cdc20+/+ and Cdc202/H primary oocytes were collected and injected with transcripts encoding cyclin B1-EGFP and H2B-mRFP prior to GVBD.
Cyclin B1-EGFP degradation was monitored by time lapse microscopy as oocytes progressed through meiosis I. (A) Still images illustrating that cyclin
B1 degradation is delayed in Cdc202/H primary oocytes. Time after GVBD (h:min) is indicated for each image. Scale bar is 10 mm. (B) Graph showing
the mean cyclin B1-EGFP fluorescence intensities of the indicated numbers of Cdc20+/+ and Cdc202/H oocytes. For each oocyte, the fluorescence
intensity was normalized to the intensity recorded 1 h after GVBD. Abbreviations in (A,B) are as in Figure 6A. Error bars represent SEM. (C) Metaphase I
oocytes of the indicated genotypes stained for cyclin B1, p-(Ser) Cdk substrates, and DNA (Hoechst). Bar = 10 mm. Note that signals of both cyclin B1
and p-(Ser) Cdk substrates are increased in the Cdc202/H oocyte. (D) Quantification of cyclin B1 and p-(Ser) Cdk substrate signals. *p = 0.001 versus
Cdc202/H metaphase (unpaired t test). **p = 0.033 versus Cdc202/H metaphase (unpaired t test). Error bars represent SEM.
doi:10.1371/journal.pgen.1001147.g007
involving both mitotic and meiotic divisions. The highest increase
in aneuploidy, however, occurred in the first meiotic division. The
most prominent segregation errors that we observed during
meiosis I are chromosome misalignment and chromosome lagging.
Previous studies in HeLa and Ptk1 cells uncovered that cyclin A2
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overexpression causes chromosome misalignment [31], suggesting
that alignment defects in Cdc20 hypomorphic oocytes might be
related to their inability to destroy cyclin A2 in a timely fashion.
Resolution of chiasmata requires removal of cohesin from
chromosome arms, which involves cleavage of the cohesin subunit
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The Female Germline Is Sensitive to Cdc20 Loss
Figure 8. Cdc202/H oocytes have impaired securin destruction in metaphase I. Rates of securin-EYFP degradation during meiosis I. Cdc20+/+
and Cdc202/H primary oocytes were injected with transcripts encoding H2B-mRFP and securin-YFP. After induction of GVBD, degradation of securinEYFP was followed by time-lapse microscopy. (A) Time-lapse microscopy images illustrating that securin-EYFP degradation is delayed in Cdc202/H
primary oocytes. The time after GVBD (h:min) is indicated. Scale bar represents 10 mm. (B) Graph showing the mean securin-EYFP fluorescence
intensities for Cdc20+/+ and Cdc202/H primary oocytes. The fluorescence intensity for each oocyte was normalized to the intensity recorded 2 h after
GVBD. Abbreviations in (A,B) are as in Figure 6A. Error bars represent SEM.
doi:10.1371/journal.pgen.1001147.g008
Rec8 by separase [32]. In turn, activation of separase requires
APC/C-mediated degradation of securin and cyclin B, both of
which are delayed in Cdc20 hypomorphic primary oocytes. Thus,
PLoS Genetics | www.plosgenetics.org
it is possible that Cdc20 hypomorphic oocytes do not have enough
APC/C activity to fully activate separase and properly resolve
chiasmata, thereby prompting chromosome lagging and aneuploi11
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The Female Germline Is Sensitive to Cdc20 Loss
Figure 9. Cdc202/H oocytes show inefficient cyclin A2 destruction in metaphase I. (A) Still images illustrating that cyclin A2-EGFP
degradation is impaired in Cdc202/H primary oocytes (the time after GVBD is indicated). Scale bar represents 10 mm. (B) Graph showing the mean
cyclin A2-EYFP fluorescence intensities for Cdc20+/+ and Cdc202/H primary oocytes. The fluorescence intensity for each oocyte was normalized to the
intensity recorded 1 h after GVBD. Error bars represent SEM.
doi:10.1371/journal.pgen.1001147.g009
dization. Arguing against this explanation is the fact that
chromosome spreads of Cdc20 hypomorphic metaphase II oocytes
did not contain any bivalents or chromosomes that stained positive
for Rec8 along chromosome arms. Alternatively, chromosome
lagging in Cdc20 hypomorphic oocytes might be caused by
PLoS Genetics | www.plosgenetics.org
microtubule-kinetochore attachment defects [33,34]. For instance,
delayed degradation of cyclin B1 (or other APC/C substrates)
might promote such defects by disrupting key components of the
mechanisms that establish syntelic attachment or that correct
merotelic or amphitelic attachments. We found that two mitotic
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The Female Germline Is Sensitive to Cdc20 Loss
be emphasized that microtubule-kinetochore attachment is a
complex process requiring many different proteins, any of which
could be deregulated in our mutant oocytes. Interestingly, in
somatic cells, a small fraction of Cdc20 accumulates at
kinetochores during mitosis [35], which raises the possibility that
Cdc20 might have a more direct role in establishing proper
microtubule-kinetochore attachments.
Our finding that a significant percentage of Cdc202/H primary
oocytes were already aneuploid before resuming meiosis I suggests
that the mitotic divisions by which primordial germ cells develop
into primary oocytes are prone to chromosome missegregation
when Cdc20 levels are below a certain threshold. Due to technical
limitations, it was not possible to verify this experimentally. The
precise impact of Cdc20 hypomorphism on meiosis II is difficult to
decipher, largely because nearly all oocytes are aneuploid after
meiosis I. However, the presence of a sizeable amount of near
triploid fertilized eggs strongly suggests that Cdc20 insufficiency
can cause failure of maternal sister chromatids to separate during
meiosis II, although we note that it cannot be excluded that the
preexisting aneuploidy rather than the low Cdc20 levels drive the
separation defect. It could be argued that embryos from Cdc202/H
females bred to Cdc20+/+ males might fail to thrive due to a
potential lack of Cdh1 expression in the early embryos, rendering
embryonic mitotic divisions particularly dependent on Cdc20.
However, this explanation seems unlikely because Cdh1 has been
shown to be expressed in two-cell stage mouse embryos [23].
Furthermore, it should be considered that the embryos from
Cdc202/H females bred to Cdc20+/+ males that fail to thrive had
either Cdc20+/2 or Cdc20+/H genotypes. Embryos of both these
genotypes show normal survival rates when derived from Cdc20+/2
and Cdc20+/H females crossed with Cdc20+/+ males (Figure 2B and
2C), further supporting the idea that aneuploidy acquired during
oogenesis is largely responsible for the early death of embryos from
Cdc202/H females.
An intriguing finding was that the fertility problems of
Cdc202/H mice are restricted to females, even though our analysis
of aneuploidy in primary and secondary spermatocytes demonstrated that mitotic and meiotic divisions of male germ cells are
prone to aneuploidy. However, the key difference between males
and females that probably accounts for their distinct fertilities is
that the rate of aneuploidization during meiosis I is substantially
higher in females than in males. Why might female meiosis I be
more sensitive to Cdc20 hypomorphism? A recent study of mouse
oocytes suggests that mammals have a unique mechanism for
control of meiosis I in that they require APC/CCdh1 activity for
progression through prometaphase I [12]. Cdc20 is targeted for
destruction by this early APC/C activity and needs to be resynthesized during metaphase I to enable anaphase onset. It is
possible that Cdc20 destruction in prometaphase I only occurs in
females, perhaps creating a higher degree of Cdc20 insufficiency in
oocytes than in spermatocytes.
Figure 10. Spermatogenesis is aneuploidy prone at low Cdc20
levels. (A) Cdc202/H males are much less prone to aneuploidy in
meiosis I than Cdc202/H females. (B) Mitotic divisions that amplify
spermatogonia appear to be prone to aneuploidy when Cdc20 levels
are low. (C) H/E-stained testis sections from Cdc20+/+ and Cdc202/H
males. Paraffin sections (5 mm) were from 5 month-old mice. Bar in top
and bottom images represent 200 mm, and 25 mm, respectively. (D)
Representative images of TUNEL-stained testis sections from Cdc20+/+
and Cdc202/H males. TUNEL-positive cells are green. Cell nuclei were
visualized by Hoechst staining. Scale bar represents 100 mm. (E)
Quantitation of apoptosis in testes from Cdc20+/+ and Cdc202/H males.
The number of TUNEL-positive cells was counted in 50 tubules. Only
tubules that were cross-sectioned were considered. There is no
statistical difference between both groups (student t-test).
doi:10.1371/journal.pgen.1001147.g010
Materials and Methods
Generation of Cdc20 Mutant Mice
The gene targeting procedure used to produce the hypomorphic
Cdc20 allele (H) was as previously described [22]. To generate the
targeting construct, Cdc20 gene fragments of 3.9 kb (spanning
exons 1–3) and 4.7 kb (spanning exons 4–10) were PCR amplified
from 129Sv/E genomic DNA and cloned into HindIII-XbaI and
SalI-NotI sites of pNTKV1901 (Stratagene). The targeting
construct was linearized with NotI and electroporated into TL1
129Sv/E ES cells. Transfectants were selected in 350 mg/ml G418
and 0.2 mM FIAU, and expanded for Southern blot analysis using
checkpoint proteins required for proper microtubule-chromosome
attachment, Bub1 and BubR1, were normally localized at
kinetochores of Cdc20 hypomorphic oocytes. However, it should
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The Female Germline Is Sensitive to Cdc20 Loss
a 710 bp 39 external probe on EcoR1-digested genomic ES cell
DNA. This probe was amplified by PCR from 129Sv/E genomic
DNA using the following primers: 59-CATGGCTGGTTTGGGAGAGAATGC TG-39 and 59-CACAACACAGTTCATCTTCCCAGTG-39. Chimeric mice were produced by microinjection of targeted ES cell clones with 40 chromosomes into C57BL/
6 blastocysts. Chimeric males were mated with C57BL/6 females
and germline transmission of the Cdc20H allele was verified by
PCR analysis of tail DNA from pups with a agouti coat color. The
Cdc202 allele used in our studies was derived from gene trap ES
clone XE368 (purchased from BayGenomics). The following
primer combinations were used for PCR genotyping of mice used
in our studies: primers a (59-CAGAAAGCCTGGTCTCTCAACCTG-39) and b (59-CACAGTAGTCATTCCGGATT
TCGGG-39) for Cdc20+; primers b and c (59-TCCATTGCTCAGCGGTGCTG -39) for Cdc20H; and primers d (59-GTATCCAACCATGGCCAAGGTGGCTGAG-39) and e (59-TATACGAAGTTATCGATCTGCGATCTGC-39) for Cdc202. All
mouse experiments were conducted after approval of the Mayo
Clinic Committee on Animal Care and Use. All mice in the study
were of a 129Sv/E x C57BL/6 mixed genetic background.
anti-centromere antibody (Antibodies Inc, 15-235-0001), Bub1(25165) [28], Mad2 (polyclonal anti-mouse full-length Mad2
antibodies generated in a rabbit), and Rec8 (kindly provided by
Dr. J. Lee [25]).
Isolation and Culture of Oocytes and Fertilized Eggs
Primary oocytes were isolated from ovaries of 3- to 4-week-old
Cdc20+/+ and Cdc202/H mice as described [40], and cultured in
micro-drops of G-1 v5 plus medium (Vitrolife) under embryotested paraffin oil (Vitrolife). In case primary oocytes were used in
mRNA microinjection experiments, 50 mg/ml dibutyryl cyclic
AMP (dbcAMP) was added to the G-1 v5 plus medium to inhibit
GVBD. To obtain secondary oocytes, 3- to 4-week-old Cdc20+/+
and Cdc202/H females were injected with pregnant mare serum
gonadotropin (PMSG; 5 IU/mouse, Sigma G4527) and 46 h later
with human chorionic gonadotropin (hCG; 5 IU/mouse, Sigma
C0684). Eighteen h after the hCG injection, ovaries were collected
and secondary oocytes harvested from oviducts. Metaphase IIarrested oocytes for Rec8 immunostaining experiments were
prepared by culturing primary oocytes from ovaries of 3- to 4week-old Cdc20+/+ and Cdc202/H mice in G-1 v5 plus medium
until they arrested in metaphase. Fertilized eggs were produced by
mating 6- to 12 week-old Cdc20+/+ and Cdc202/H females with
Cdc20+/+ males. The next morning, one-cell stage embryos were
harvested from oviducts and freed of cumulus cells as described
[41]. Embryo culturing was done in micro-drops of G-1 v5 plus
medium as described [42]. Embryos were photographed daily
from day E0.5 to E4.5.
Fertility Analysis and Histology
Female fertility was measured by breeding 2-month-old females
of various Cdc20 genotypes to 2- to 3-month-old wild-type males
for a 3-month period. During this period, we recorded, for each
female, the number of vaginal plugs (to determine whether females
showed normal mating behavior), the number of litters produced,
and the amount of pups delivered. Histological evaluation of testes
and ovaries were as previously described [11]. Follicles and
corpora lutea were counted in five ovary sections of each mouse.
Follicle classification was according to Pedersen and Peters [36].
TUNEL staining was done on 5 mm testis sections using an in situ
cell death detection kit from Roche.
Chromosome Counts on Oocytes, One-Cell Stage
Embryos, and Spermatocytes
For chromosome counts on oocytes and one-cell stage embryos,
the procedure of Tarkowski [43] was followed. Briefly, freshly
harvested secondary oocytes and fertilized eggs were cultured for
20 h at 37uC in medium containing 0.5 mg/ml colcemid,
incubated in 1% sodium citrate for 20 min at RT and transferred
to glass slides. Ethyl alcohol and glacial acetic fixative (3:1) was
dropped on the zygotes and secondary oocytes three times. Airdried slides were Giemsa stained and chromosomes counted using
a light microscope with a 1006 objective. Primary oocytes were
collected and cultured in micro-drop cultures of G-1 v5 plus
medium. Upon GVBD, primary oocytes were harvested and
chromosome spreads prepared. For chromosome counts on
spermatocytes, testes were collected and minced between two
microscope slides. Released cells were suspended in 5 ml PBS,
centrifuged at 1,000 rpm for 5 min, resuspended 5 ml 0.075 M
KCl, and incubated at RT for 30 min. Cells were fixed in
Carnoy’s solution, washed, and finally resuspended in 0.5 ml
fixative. Twenty-five ml aliquots were dropped onto pre-wetted
microscope slides and chromosomes were stained with Giemsa.
Western Blot Analysis and Indirect Immunofluorescence
Western blot analysis was performed as described earlier [37].
Extracts of MEFs, splenocytes, and bone marrow were prepared in
PBS containing 0.1% NP40, 10% glycerol and complete protease
inhibitor cocktail (Roche). Extracts were centrifuged at 20,000 g
for 15 min (4uC), and supernatants collected for electrophoresis.
Quantitation of relative Cdc20 protein levels in Cdc20+/H,
Cdc20+/2, Cdc20H/H and Cdc202/H testis and ovary, and
Cdc202/H MEFs, spleen, and bone marrow was done as previously
described [38]. Briefly, Cdc20 western blot signals obtained with
rabbit Cdc20 antibody from Santa Cruz (SC-8358), were
quantified using ImageJ software (http://rsbweb.nih.gov) and
normalized to background and b-actin (Sigma A5441) or a-tubulin
(Sigma, T-9026) signals. Values obtained were normalized to those
of corresponding wild-type tissues and MEFs, where wild-type
signals were set at 100. Normalized signal values were converted to
percent protein using the graph of Figure S3. Relative Cdc20
protein amounts represent the average of at least two independent
samples.
Indirect immunofluorescence was performed as previously
described [37,39]. Immunofluorescence images were captured
using a Carl Zeiss LSM 510 laser-scanning microscope with a cApochromat 1006 oil immersion objective. Fluorescent signals
from cyclin B1 and P-(Ser) CDKs substrate labelings were
quantitated using ImageJ software. The mean fluorescence
intensity was determined after background subtraction of images
transformed to 8 bits grayscale. The following primary antibodies
were used: cyclin B1 (Calbiochem, PC-133), P-(Ser) CDKs
substrate (Cell Signaling, #2324), BubR1(1-350) [11], human
PLoS Genetics | www.plosgenetics.org
Live-Cell Imaging of Cultured Primary Oocytes
To measure the accuracy of chromosome segregation during
meiosis I, chromosome movements of primary oocytes were
followed by time-lapse microscopy. To this end, H2B-mRFP
mRNA was produced by in vitro transcription using the T3
mMESSAGE mMACHINE kit (Ambion Inc). Using a Femtojet
microinjector (Eppendorf), GV-stage primary oocytes were
microinjected with 5–10 picoliter of mRNA solution containing
0.5 mg/ml H2B-mRFP mRNA [44]. Injected oocytes were
allowed to recover for 30 min in micro-drops of M2 medium
containing 50 mg/ml dbcAMP and then transferred to 35 mm
glass-bottomed culture dishes (MatTek Corporation) containing
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The Female Germline Is Sensitive to Cdc20 Loss
G-1 v5 plus medium without dbcAMP to induce GVBD.
Chromosome movements were followed using a Zeiss Axio
Observer Z1 system with CO2 Module S, TempModule S,
Heating Unit XL, Pln 40x/0.6 Ph2 DICIII objective, AxioCam
MRm camera, and AxioVision 4.6 software [45]. The temperature of the imaging medium was kept at 37uC. Images were
collected at interframe intervals of 20 min.
To analyze timing of meiosis I, the time intervals from GVBD
to prometaphase, prometaphase to metaphase, and metaphase to
anaphase were measured. Importantly, only H2B-mRFP mRNAinjected Cdc20+/+ and Cdc202/H oocytes progressing through
meiosis I without any chromosome segregation errors were
included in our timing analysis.
To determine polar body extrusion rates, Cdc20+/+ and Cdc202/
H
oocytes were collected and monitored by differential interference
contrast (DIC) time-lapse microscopy as they progressed through
meiosis I.
To analyze the degradation kinetics of mitotic cyclins and
securin, coding sequences for cyclin B1-EGFP, securin-EYFP and
cyclin A2-EGFP were cloned into pBluescript RN3 or pMDL2
[46], and mRNAs were produced by in vitro transcription as
described above. GV-stage primary oocytes were microinjected
with 5–10 picoliter of mRNA solutions containing 0.5 mg/ml
H2B-mRFP +0.5 mg/ml cyclin B1-EGFP, 0.1 mg/ml H2B-mRFP
+0.1 mg/ml securin-YFP, or 0.1 mg/ml H2B-mRFP +0.1 mg/ml
cyclin A2-EGFP. Injected oocytes were allowed to recover for
30 min in micro-drops of M2 medium containing 50 mg/ml
dbcAMP and then transferred to 35 mm glass-bottomed culture
dishes. Time-lapse microscopy was initiated 1 or 2 h after GVBD
to allow for expression of fluorescent protein-tagged APC/C
substrates. Images were collected at interframe intervals of 20 min.
Quantification of fluorescence levels was as follows. For each
oocyte and for each time point, images detecting mRFP, EGFP/
EYFP, and DIC were acquired. Time-lapse images were then
exported as grayscale ‘‘avi’’ uncompressed files. Videos were
opened using ImageJ using avi reader plugin. DIC images were
used to highlight the area occupied by the oocyte using the
freehand tool in ImageJ. The highlighted area was moved to the
corresponding EGFP/EYFP image and the mean fluorescence
intensity within this area measured after background subtraction.
Mean fluorescence intensities were expressed in arbitrary units.
The value of time zero (the fluorescence intensity for the first
image acquired) was considered 100% and the subsequent timelapse intensities were normalized against it. Excel T-TEST
software was used for statistical analyses.
(Hoechst). Note that Rec8 signals are localized along chromosome
arms of bivalents. (B) Cdc20+/+ and Cdc202/H primary oocytes
were cultured until metaphase II arrest, collected and stained for
Rec8, centromeres and DNA. Note that chromosome arms in
metaphase II are negative for Rec8 irrespective of genotype,
indicating the lack of bivalents. Scale bar represents 10 mm.
Found at: doi:10.1371/journal.pgen.1001147.s001 (0.61 MB TIF)
Figure S2 Mitotic checkpoint proteins properly localize to
kinetochores of Cdc202/H primary oocytes. Cdc20+/+ and
Cdc202/H primary oocytes were harvested from ovaries and
cultured until they had progressed to metaphase I (,7 h after
GVBD). Oocytes were fixed and immunostained for centromeres
(ACA antibody) and either Bub1, BubR1 or Mad2. DNA was
visualized by Hoechst staining. Scale bar represents 10 mm.
Found at: doi:10.1371/journal.pgen.1001147.s002 (1.22 MB TIF)
Figure S3 Percent Cdc20 protein plotted versus the average
band intensity on western blots.
Found at: doi:10.1371/journal.pgen.1001147.s003 (0.14 MB TIF)
Example of a Cdc202/H oocyte undergoing chromosome missegregation during meiosis I. Chromosomes (red) were
marked by injection of H2B-mRFP prior to GVBD. Note the
presence of lagging chromosomes as sister chromatids move to
opposite poles in anaphase.
Found at: doi:10.1371/journal.pgen.1001147.s004 (2.82 MB
MOV)
Video S1
Second example of a Cdc202/H oocyte undergoing
chromosome missegregation during meiosis I. Chromosomes (red)
were marked by injection of H2B-mRFP prior to GVBD. Note the
presence of lagging chromosomes as sister chromatids move to
opposite poles in anaphase.
Found at: doi:10.1371/journal.pgen.1001147.s005 (2.65 MB
MOV)
Video S2
Acknowledgments
We would like to thank Darren Baker, Ming Li, and Dave Norris for
assistance; Paul Galardy and Robin Ricke for comments on the
manuscript; and members of the van Deursen laboratory for helpful
discussions. We thank Nigel Garrett (The Wellcome Trust/Cancer
Research Gurdon Institute) for providing pRN3 plasmid, Mark Levasseur
(University of Newcastle) for providing pMDL2 plasmid, Rob Wolthuis
(Netherlands Cancer Institute) for providing pCDNA3-HsCyclin A2cerulean plasmid, and Jibak Lee (Chromosome Dynamics Laboratory,
RIKEN Advanced Science Institute) for providing Rec8 antibody.
Supporting Information
Author Contributions
Chromosome missegregation in Cdc20-/H primary
oocytes does not seem to involve non-disjunction of bivalents. (A)
A Cdc202/H primary oocyte in metaphase I stained for the meiotic
cohesin component Rec8 [46], centromeres (ACA) and DNA
Figure S1
Conceived and designed the experiments: FJ MH LM KBJ WZ JMvD.
Performed the experiments: FJ MH LM KBJ WZ JMvD. Analyzed the
data: FJ MH LM KBJ WZ JMvD. Contributed reagents/materials/
analysis tools: DEM. Wrote the paper: FJ LM JMvD.
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September 2010 | Volume 6 | Issue 9 | e1001147