1 Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer
Institute at Frederick, 1050 Boyles Street, Frederick, MD 21702, USA
2 Department of Biochemistry and Cellular and Molecular Biology, University of
Tennessee, Knoxville, TN 37996, USA
3 Department of Epidemiology and Preventive Medicine, School of Medicine,
University of Maryland, Baltimore, MD 21201, USA
4 Department of Biochemistry and Molecular Biology, Division of Reproductive
Biology, Johns Hopkins University, Bloomberg School of Public Health,
Baltimore, MD 21205, USA
* Author for correspondence (e-mail: ssharan{at}mail.ncifcrf.gov)
Accepted 29 September 2003
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SUMMARY |
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Key words: BRCA2, Spermatogenesis, Oogenesis, Meiosis, DNA repair
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Introduction |
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The involvement of BRCA1 and BRCA2 mutations in human
ovarian cancer as well as the possible roles of the proteins in DNA dynamics
has suggested the possibility that breast cancer susceptibility genes may
function during normal gametogenesis. Until recently, evidence supporting this
idea has derived primarily from expression studies. In mice, both
Brca1 and Brca2 are expressed during spermatogenesis,
particularly during meiotic prophase
(Zabludoff et al., 1996;
Blackshear et al., 1998
;
Chen et al., 1998
).
Intriguingly, the BRCA2 protein is localized on meiotic chromosomes
(Chen et al., 1998
), suggesting
a role in the dynamics of meiotic recombination and/or chromosome pairing and
synapsis.
However, the role of BRCA2 in meiosis and gametogenesis has been difficult
to define because loss-of-function mutations in the mouse Brca2 gene
result in early embryonic lethality (Hakem
et al., 1998). We report a viable but infertile mouse model with
impaired BRCA2 expression. The mutant mice lack endogenous Brca2
function but carry a human BRCA2 gene present in a bacterial
artificial chromosome (BAC). The human transgene rescues the embryonic
lethality phenotype of the Brca2-null mice; however, there is poor
expression of the human BRCA2 transgene in the testes and ovaries,
with ensuing infertility. We use this mouse model to define stages of
gametogenesis impaired by deficient BRCA2 expression and find evidence for a
role in meiosis and gametogenic success in both males and females.
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Materials and methods |
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Generation of BAC transgenic mice
BAC DNA was extracted using standard alkaline lysis and purification on a
cesium chloride gradient. Supercoiled DNA was dialyzed overnight in 1xTE
(10mM Tris pH 7.6, 1mM EDTA) and diluted to 0.5-1.0 ng/µl for pronuclear
microinjection. Pronuclear microinjection was performed as described by Hogan
et al. (Hogan et al.,
1994).
Genotyping mice by Southern analysis and genetic crosses
Transgenic mice were identified by Southern analysis of BamHI
digested tail DNA using the chloramphenicol resistance gene present in the BAC
vector as a probe to detect a 8.0 kb fragment. Brca2 heterozygous and
homozygous mutant mice were genotyped by hybridizing EcoRV digested
genomic DNA on a Southern blot with a 1.5 kb fragment from exon 11 of murine
Brca2 gene nucleotides 5208-6710 of NM 009765. The probe detects a 5
kb wild-type fragment and a 6.5 kb fragment corresponding to the mutant
allele. BAC transgenic founders were crossed with Brca2 Ko/+ mice to
obtain Brca2 Ko/+;Tg/+ F1 progeny. The Brca2 Ko/+;Tg/+ F1
mice were crossed to Brca2 Ko/+ mice to obtain Brca2
Ko/Ko;Tg/+ mice.
Expression analysis by in situ hybridization
E9.5 and E11.5 embryos, ovaries and testes were fixed in freshly prepared
4% paraformaldehyde and embedded in paraffin wax for sectioning. In situ
hybridization was performed as described by Tessarollo and Parada
(Tessarollo and Parada,
1995).
Probes
PCR fragments containing regions of mouse Brca2 (nucleotides
9500-10129, NM 009765) and human BRCA2 (nucleotides 10141-10770, NM
000059) cDNA were cloned into the pGEM-T vector (Promega) and
sequenced to determine their orientation. 35S-labeled-antisense
human BRCA2 and mouse Brca2 probes were generated by in
vitro transcription using T7 RNA polymerase and NotI linearized
template. The corresponding sense probes were generated using SP6 RNA
polymerase and NcoI (mouse Brca2) or SacII (human
BRCA2) linearized templates. In situ hybridization was performed in
triplicate for each sample and the experiments were repeated at least once to
evaluate the consistency of the results.
Histology
E9.5 and E11.5 embryos, testes and ovaries were fixed in Bouin's solution.
Samples were dehydrated through an ethanol series, embedded in paraffin wax,
serially sectioned and stained with Hematoxylin and Eosin. Histological
sections of testes were stained with periodic acid Schiff (PAS)-Hematoxylin
and the ovaries from postnatal day 2 pups were stained with Weigert's
Hematoxylin-picric acid Methyl Blue. Slides were examined using brightfield
microscopy. To examine the consistency of the phenotype, samples from at least
two different mice were examined with the exception of the testis from a
7-month-old mouse where a single male was examined.
Cell death assay
A TUNEL assay (In Situ Cell Death detection kit, Roche Molecular
Biochemicals) was performed according to the manufacturer's instructions on
testes of 3-week-old mice. Testes were fixed in 4% paraformaldehyde and
processed as described by Tessarollo and Parada
(Tessarollo and Parada, 1995).
Anti-fluorescein antibody conjugated with horse-radish peroxidase (POD) was
used to detect fluorescein labeled dUTP. DAB substrate (Roche Molecular
Biochemicals) was used to detect POD and visualized by light microscopy.
Fixation and immunofluorescent labeling of isolated germ cells
Cell preparations enriched in germ cells were prepared as previously
described (Cobb et al., 1999).
Briefly, testes were detunicated and digested in 0.5 mg/ml collagenase (Sigma)
in Krebs-Ringer buffer for 20 minutes at 32°C and then in 0.5 mg/ml
trypsin (Sigma) for 13 minutes, followed by filtering through 80 µm Nitex
mesh and washing in buffer. Surface-spread preparations were used to visualize
nuclei as previously described (Cobb et
al., 1997
). Cell preparations from at least two rescued mice were
used for each antibody staining.
Antisera used were polyclonal human BRCA2 antibody, Ab-2 (Oncogene
Research, 1:50), anti-mouse BRCA2 (Pep-3, polyclonal antisera against mouse
BRCA2 amino acids 2330-2345, 1:50), anti-SYCP3 (1:500)
(Eaker et al., 2001),
anti-SPO11 (Trevigen, 1:25), anti-DMC1
(Masson et al., 1999
) (a
generous gift from Madalena Tarsounas and Stephen West, Cancer Research UK),
anti-RAD51 (Oncogene 1:100), anti-H1t (polyclonal antisera raised against
full-length H1t cDNA, 1:1000), anti-
H2AX (Upstate; Lake Placid, NY,
1:500) and anti-RPA (Oncogene 1:25). After overnight incubation in primary
antibody, slides were incubated with rhodamine- or fluorescein-conjugated
secondary antibodies (Pierce, 1:500), followed by mounting with Prolong
Antifade (Molecular Probes) containing DAPI (Molecular Probes) to stain DNA.
Control slides were stained with either secondary antibodies only, or
pre-immune sera to replace primary antibody. Localization was observed with an
Olympus epifluorescence microscope and images were captured and transferred to
Adobe PhotoShop with a Hamamatsu color 3CCD camera.
Follicular count
Quantitative analysis of ovarian follicles was completed by marking every
tenth section and counting the total number of primordial, primary, preantral
and antral follicles present in each of these marked sections. Follicles were
counted as primordial if they contained an intact oocyte and were surrounded
by a single layer of flattened granulosa cells. Follicles were counted as
primary if they contained an intact oocyte and were surrounded by a single
layer of cuboidal shaped granulosa cells; as preantral if they contained an
intact oocyte, more than one layer of granulose cells and lacked antral
spaces; and as antral if they contained an intact oocyte, more than one layer
of granulosa cells and antral spaces. To avoid double-counting follicles, only
follicles containing an oocyte with a visible nucleus were counted. The number
of follicles was multiplied by 10 to account for the fact that every tenth
section was used in the analysis (Smith et
al., 1991). To avoid bias, all ovaries were analyzed without
knowledge of genotype or age.
Superovulation
Females were superovulated and oocytes were collected as described by Hogan
et al. (Hogan et al., 1994).
Briefly, 0.1 ml of PMS (5 IU) was injected into the intraperitoneal cavity of
6-week-old females. After 46 hours, 0.1 ml of hCG (5 IU) was injected into
each female. After 18 hours the females were sacrificed and the oviducts were
dissected out. The oviducts were opened with fine forceps and the eggs were
collected. Cumulus cells were dissociated in presence of 700 units/ml of
hyaluronidase.
In vitro oocyte maturation
Forty-eight hours after PMSG injection, germinal vesicle (GV)-intact
oocytes were collected from ovarian follicles by piercing the ovaries of two
control and two rescue mice (7 and 9 weeks of age) with 27.5 gauge syringe
needles in Whitten's medium containing 0.05% polyvinyl alcohol (Sigma, St.
Louis, MO) and 0.25 mM dibutyryl cAMP (dbcAMP, Sigma)
(Cho et al., 1974). All oocytes
collected, those enclosed in cumulus cells and those not enclosed in cumulus
cells, were used in studies of in vitro maturation. For those oocytes that
emerged from follicles enclosed in cumulus cells, the cumulus cells were
removed by pipetting the oocyte-cumulus cell complexes through a thin-bore
pipet. GV-intact oocytes were matured in vitro by washing the oocytes through
six 100 µl drops of Whitten's medium lacking dbcAMP, and then cultured
overnight at 37°C in 100 µl drops Whitten's medium (10-25 oocytes per
drop) in 5% CO2 in air. At 16 hour post-dbcAMP removal, the cells
were viewed by dissecting microscope to assess what percentage of the oocytes
had emitted the first polar body (PB1), undergone GV breakdown (GVBD) but not
emitted PB1, were still GV intact, or had other phenotypes. Less than 50% of
the control oocytes had emitted the first polar body by this time. As oocytes
of some strains of mice undergo meiotic maturation more slowly than others
(Polanski et al., 1998
), we
cultured these oocytes for additional 3 hours to 19 hours post-dbcAMP removal.
Some oocytes remained GV intact after these 19 hours [27% (22/81), control;
14% (4/29)]; this might be attributable to the use of all oocytes collected
(i.e. potentially including some meiotically incompetent oocytes) in these
maturation studies. At 19 hour post-dbcAMP removal, the cells were prepared
for staining as follows. The zonae pellucidae were removed by a brief
incubation in acidic medium-compatible buffer (10 mM HEPES, 1 mM
NaH2PO4, 0.8 mM MgSO4, 5.4 mM KCl, 116.4 mM
NaCl, final pH 1.5), and then the cells were fixed in freshly prepared 3.7%
paraformaldehyde in PBS for 1-2 hours. Fixation and all subsequent steps were
performed at room temperature in a humidified chamber as described previously
(Evans et al., 2000
). The
fixed cells were washed in PBS, then permeabilized in PBS containing 0.1%
Triton X-100 for 15 minutes, and then incubated in blocking solution (PBS
containing 0.1% BSA and 0.01% Tween-20) for 60 minutes. Cells were stained
with 50 ng/ml of TRITC-conjugated phalloidin (Sigma) for 45 minutes to label
F-actin. Cells were then washed three times (in blocking solution, 10-20
minutes each), and then mounted in VectaShield mounting medium (Vector Labs;
Burlingame, CA) containing 1.5 µg/ml 4',6-diamidino-2-phenylindole
(DAPI; Sigma). Cells were viewed on a Nikon Eclipse fluorescent microscope,
and digital images were captured with a Princeton 5 MHz cooled interlined CCD
camera (Princeton Instruments, Trenton, NJ) using IP Labs software
(Scanalytics, Fairfax, VA). All images were collected with similar exposure
times and were not further manipulated, except for cropping in Photoshop 6.0
(Adobe systems Incorporated, San Jose, CA) for figure preparation. Throughout
this work, the term `oocyte' refers to GV-intact oocytes at prophase I; the
term `egg' refers to a metaphase II egg.
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Results |
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Expression of the endogenous Brca2 gene and the human BRCA2 transgene
To investigate the rescue of embryonic lethality by the human
BRCA2-containing BAC, the expression of the human transgene was
examined by RT-PCR. Although the level of expression of the human gene
appeared lower than that of the endogenous Brca2 gene, the two
exhibited qualitatively identical expression patterns revealed by RT-PCR. Both
were expressed in brain, heart, liver, lung, kidney, spleen, ovary, testis,
thymus and the mammary gland of pregnant and lactating females (data not
shown). Interestingly, neither the mouse nor the human gene was expressed in
the mammary gland of virgin females. These RT-PCR results suggest that the
tissue-specific regulation of the human gene under the control of its own
promoter is similar to the endogenous mouse Brca2 gene.
Expression was further examined in embryos, where expression of endogenous
Brca2 is first detected at E7.5
(Sharan et al., 1997). As
development progresses, Brca2 expression appears to be upregulated in
cells that are rapidly dividing while reduced in cells undergoing
differentiation. We examined the expression of the BRCA2 transgene
and compared it with the endogenous Brca2 expression pattern in
embryos at E9.5 by in situ hybridization. The human BRCA2 gene was
expressed in the developing embryo and the pattern of expression was identical
to that of the endogenous Brca2 gene
(Fig. 2B-D). However,
expression of the human BRCA2 gene was reduced compared with the
mouse gene. At E11.5 expression of both the human transgene and the mouse gene
was found to be restricted to regions of rapid cell division (data not shown).
From the expression pattern seen in the brain, both the mouse and the human
genes were expressed in the neuroepithelium of the ventricular layer. Similar
to E9.5 embryos, expression of the human BRCA2 gene in E11.5 embryos
was considerably lower than that of the endogenous gene. The qualitatively
identical expression pattern of the mouse and human genes during embryogenesis
provides insight into the viability of the Brca2 homozygous mutant
mice. Although the level of transcript expression is reduced, it must provide
an amount of BRCA2 protein that is above the threshold level required for
normal function during embryogenesis.
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We conclude from these expression analyses that the infertility of the rescued animals is due to poor expression or function of the human BRCA2 protein in germ cells. Thus, the Brca2 Ko/Ko; Tg mice, in effect, are equivalent to a conditional knockout with respect to the gonads and can be used to study the function of BRCA2 in gametogenesis.
Male infertility phenotype
None of the four Brca2 Ko/Ko; Tg/+ males produced any offspring
after repeated mating for 2-6 months with either rescued or wild-type females,
while their Brca2 Ko/+; Tg/+ littermates sired multiple litters
suggesting that the rescued males were infertile. To define further the
infertility phenotype, the histology of the testes from males of different
ages was examined. Testes of the rescued mutant males were small compared with
controls. Histological examination of 3-week-old Brca2 Ko/+; Tg/+
males revealed seminiferous tubules that were filled with spermatocytes
(Fig. 3A). The rescued male
testes also showed the presence of spermatocytes in seminiferous tubules but
their number appeared reduced and some seminiferous tubules were completely
devoid of germ cells (Fig. 3B).
The seminiferous tubules of 2-month-old Brca2 Ko/Ko; Tg/+ males
appeared smaller compared with their normal littermate controls and showed
interstitial cell hyperplasia (Fig.
3C,D). The Leydig cells appeared to be functionally normal as the
weight of the seminal vesicles was unaffected in the mutant mice (data not
shown). No germ cells beyond early meiotic prophase were present in the
seminiferous tubules of the rescued males. Several tubules were completely
devoid of germ cells and contained only Sertoli cells, suggesting that the
germ cells were undergoing rapid degeneration. With germ cell deficiency,
there was an increase in the relative number of Sertoli cells per seminiferous
tubule in the mutants compared with the control tubules. The degeneration was
more severe in 7-month-old rescued males, where very few seminiferous tubules
had spermatocytes and majority of the tubules were filled with vacuolated
Sertoli cells (Fig. 3E). The
nuclei of these remaining spermatocytes appeared condensed and darkly
stained.
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Analysis of meiotic progress in mutant males
As spermatocytes in testes of Brca2 Ko/Ko; Tg/+ rescued animals
appeared to be blocked during prophase I of meiosis, spermatocytes were
examined by immunofluorescence with various antibodies to determine more
precisely the timing of the block in prophase I. First, the localization of
the endogenous mouse BRCA2 protein during normal meiosis was confirmed. As
seen in Fig. 4A, signal for
BRCA2 protein in zygotene spermatocytes is diffuse throughout the nucleus. As
synapsis occurs in normal spermatocytes, BRCA2 staining begins to disappear
and is no longer detected by the end of pachynema (data not shown) in
accordance with previous findings (Chen et
al., 1998). Mouse BRCA2 protein was not detected in Brca2
Ko/Ko; Tg/+ spermatocytes (Fig.
4B). Similarly, no human BRCA2 protein was detected in either the
heterozygous or homozygous mutant transgenic spermatocytes, confirming the
negligible expression of the human protein in spermatocytes of transgenic mice
(Fig. 4C,D).
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Events surrounding the initiation of meiotic recombination in the
BRCA2-deficient spermatocytes were examined by determining the localization of
two proteins known to be associated with the formation of
recombination-related DNA double-strand breaks. First, we assessed the
presence of SPO11, an evolutionarily conserved protein required for normal
formation of meiosis-specific double strand breaks
(Keeney et al., 1997).
Positive staining for SPO11 protein was found in both Brca2 Ko/Ko;
Tg/+ and Brca2 Ko/+; Tg/+ control spermatocytes
(Fig. 4G,H). Second, we
determined the presence of the phosphorylated form of histone H2AX
(
-H2AX), a marker for chromatin containing DNA double-strand breaks.
Brca2 Ko/Ko; Tg/+ spermatocytes show positive
-H2AX staining,
again suggesting that the chromatin in BRCA2-deficient spermatocytes contains
DNA with double-strand breaks. Normally,
-H2AX staining disappears in
pachynema, except for its localization with the sex chromosomes
(Mahadevaiah et al., 2001
).
However, mutant spermatocytes do not exhibit either loss or re-localization of
staining with anti-
-H2AX, providing further evidence that they are
blocked at a stage prior to pachynema.
As BRCA2 interacts with RAD51 protein
(Patel et al., 1998) and
co-localizes with RAD51 in human spermatocytes
(Chen et al., 1998
), we
examined localization of RAD51 protein and its meiosis-specific variant, DMC1,
in BRCA2-deficient spermatocytes. Western blot analysis of the testes of
Brca2 Ko/Ko; Tg/+ mice showed presence of RAD51 and DMC1 proteins at
levels similar to the control testes (data not shown). Interestingly, the
number of RAD51 foci per nucleus was reduced in Brca2 Ko/Ko; Tg/+
spermatocytes compared with heterozygous transgenic control spermatocytes
(Fig. 5A-C). Analysis of RAD51
foci in mutant and control nuclei revealed that 98% of mutant spermatocytes in
the leptotene and zygotene stages had reduced numbers of RAD51 foci (58% with
no foci and majority of nuclei in 1-100 group had less than 10 foci and only a
few had 20-30), while greater than 98% of control spermatocytes had 100-250
RAD51 foci per nuclei (Fig.
5C). Additionally, although many discrete DMC1 foci were visible
over zygotene-stage spermatocytes in normal animals, few to no foci were
detected in BRCA2-deficient spermatocytes, similar to the reduction seen in
RAD51 foci (Fig. 5D,E). Finally, we examined the expression of replication protein A (RPA), a
single-stranded DNA-binding protein involved in DNA replication and repair,
which is associated with synapsed regions of the chromosomes during the
zygotene and pachytene stages (Plug et
al., 1997
; Plug et al.,
1998
), and has been shown to interact with RAD51 (Golub, 1998).
Surprisingly, despite reduced number of RAD51 foci and lack of extensive
synapsis, we found abundant RPA foci present in mutant spermatocytes
(Fig. 5F,G).
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Analysis of meiotic progress in mutant females
Because meiotic progress is arrested in mutant males, we hypothesized that
there could be some abnormalities in meiosis in females as well. However, the
presence of oocytes in young animals suggested that impairment to meiotic
progress could be at a stage after the late prophase, or dictyate, arrest that
normally occurs during female gametogenesis. Successful oocyte `meiotic
maturation' or progress through meiosis from prophase I to metaphase II is
crucial for successful fertilization and embryo development.
To address our hypothesis, oocytes from control and rescued females were
examined for their abilities to undergo meiotic maturation in vitro. Ninety
GV-intact oocytes were collected from two control females (55 from a
9-week-old and 35 from a 7-week-old) and 31 oocytes were collected from two
rescued females (21 from a 9-week-old and 10 from a 7-week-old), demonstrating
that the rescued females had fewer germinal vesicle (GV)-intact oocytes than
did the controls, as was anticipated from the ovarian histology. Additionally,
the oocytes from rescued females showed a reduced ability to complete oocyte
maturation successfully when compared with controls. GV-intact oocytes from
control and rescued females were cultured for 16 hours in medium lacking
dbcAMP to allow progression into meiosis from prophase I to metaphase II
(Cho et al., 1974). Entry into
meiosis is indicated by the breakdown of the nuclear envelope of the GV, known
as GV breakdown (GVBD). Completion of the first stage of female meiosis
(progression to metaphase II) is indicated by the emission of the first polar
body (PB1), the product of asymmetric cytokinesis. As shown in
Fig. 7, oocytes from the
rescued females appeared capable of initiating meiotic maturation, as only 14%
(4/29) remained arrested at GV stage after 16 hours of culture. However,
abnormalities of meiotic maturation were observed; most notably, 21% (6/29) of
the polar bodies appeared to be abnormally large, and a low number (9/29, 31%)
had normal first polar bodies. It should be noted that the PB1 emission in the
controls by 16 hours was relatively low (44%). Because oocytes of some strains
of mice undergo meiotic maturation more slowly
(Polanski, 1986
), we cultured
the oocytes that had begun maturation (59 controls, 25 rescued) for 3
additional hours, to 19 hours post-dbcAMP removal. In this time,
50% of
the GVBD oocytes in both the control and rescue groups progressed to emit the
first polar body (Fig. 7B). To
asses meiotic progress further, cells then were fixed and stained to label the
F-actin and DNA (four out of the 59 control oocytes were lost in this process,
and therefore data are reported for 55 oocytes.) Normal metaphase II-arrested
eggs show first polar body (indicative of completion of meiosis I), chromatin
arranged on the metaphase II plate and an actin-rich cap over the meiotic
spindle (Fig. 8A). Although
nearly 50% (12/25) of the rescued oocytes had this phenotype, abnormalities
were observed in the others. The most common anomaly was an oversized polar
body-like structure (7/25, 28%; Fig.
8B,C). While eggs with large polar bodies have been observed in
some instances of female meiosis abnormalities (e.g. the Mos-deficient and
endothelial nitric oxide synthase-deficient mice
(Choi et al., 1996
;
Jablonka-Shariff and Olson,
1998
), the eggs with oversized polar body-like structures observed
here were different. In the majority of these eggs (6/7), all of the DNA was
in the PB-like structures and no DNA present in the oocyte
(Fig. 8B,C). Other
abnormalities in the oocytes from rescued females included apparent impairment
of the first meiotic division, including abnormal chromatin condensation
(Fig. 8D), chromatin
condensation but little organization of the metaphase I spindle
(Fig. 8E), formation of a
metaphase I spindle but failure to emit PB1
(Fig. 8F), or failure to
progress to metaphase II and instead of having an abnormal, tightly condensed
chromatin `wad' in an egg that had emitted PB1
(Fig. 8G). By contrast, 85%
(47/55) of the control oocytes emitted the first polar body and 71% (39/55)
showed proper metaphase II arrest. The other eight control cells with PB1s had
slight metaphase II abnormalities, namely incomplete chromosome congression to
the metaphase II spindle (Fig.
8H). Of the eight control oocytes that had undergone GVBD but not
emitted PB1, seven of these appeared to be normal metaphase I cells, with the
meiotic spindle positioned at the periphery of the cell.
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Discussion |
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Previously, the expression of BRCA2 protein in adult testes and ovaries and
its colocalization with RAD51 on synapsed chromosomes in spermatocytes had
suggested a role for this protein in meiosis
(Chen et al., 1998), but this
was difficult to test experimentally because of the lethality of
Brca2-null embryos and the early germ-cell loss in mice homozygous
for either of the two hypomorphic alleles of Brca2
(Connor et al., 1998
;
Friedman et al., 1998
). Here,
we have circumvented these problems by showing that Brca2-null mice
rescued by a human BRCA2 transgene survive to adulthood, but with
impaired germ-cell development. As the human BRCA2 transgene is
poorly expressed in the gonads and human protein could not be detected in
ovaries or testes, we presume that infertility results from BRCA2 deficiency
in the gonads, especially in the germ cells.
The phenotype of spermatocytes in Brca2 Ko/Ko;
Tg/+ males reveals a requirement for adequate levels of
BRCA2 protein for normal meiotic prophase progression, including assembly of
protein complexes onto chromosomal axes. Although synapsis is initiated in
BRCA2-deficient spermatocytes, it is never completed and mutant spermatocytes
do not reach the pachytene stage of meiotic prophase. The presence of both of
SPO11 and -H2AX proteins on chromatin of mutant spermatocytes suggests
that early recombination-related DNA double-strand break formation probably
occurs in BRCA2-deficient spermatocytes. However, recombination complexes of
RAD51 and DMC1 are not appropriately assembled onto meiotic chromosomal axes
in the absence of BRCA2 protein. Thus, the role of BRCA2 in meiotic
recombination could be similar to that hypothesized for double-strand break
repair in mitotic cells, where it is thought that BRCA2 recruits RAD51 to
sites of DNA damage, thus initiating repair
(Davies et al., 2001
). The
BRCA2 protein may also be directly or indirectly responsible for recruiting
DMC1 protein to meiotic chromosomal axes at the site of RAD51 foci, as DMC1
localization was dramatically diminished in mutant spermatocytes. In normal
spermatocytes, RAD51 and DMC1 colocalize and interact with SYCP3
(Tarsounas et al., 1999
),
suggesting functional interaction of these proteins in recombination events.
The presence of RAD51 foci in DMC1-deficient spermatocytes
(Pittman et al., 1998
;
Yoshida et al., 1998
) implies
that DMC1 is not required for localization of RAD51 onto chromosomal axes.
However, the role of RAD51 in DMC1 recruitment to chromatin is not known, as
there is early embryonic lethality of Rad51-null mice
(Lim and Hasty, 1996
;
Tsuzuki et al., 1996
). Hence,
the reduced number of DMC1 foci in BRCA2-deficient spermatocytes could be
because of lack of BRCA2 or because of failure of RAD51 to load onto
chromatin. In spite of greatly diminished RAD51 and DMC1 localization in
BRCA2-deficient spermatocytes, numerous foci of RPA are found on chromosomal
axes. RPA and RAD51 do not always colocalize in spermatocytes
(Tarsounas and Moens, 2001
)
and the pattern of RPA localization in BRCA2-deficient spermatocytes suggests
that neither BRCA2 nor RAD51 may be required for RPA localization on
chromosomal axes, but that BRCA2- or RAD51-mediated processes could be
required for removal of RPA from chromosomal axes. Taken together, these
observations of BRCA2-deficient spermatocytes suggest a pivotal role for BRCA2
protein in organizing the complex of proteins that assemble onto chromosomal
axes during early meiotic prophase. Additionally, the failure of spermatocytes
to progress in their developmental program suggests a strong dependency upon
completion of early events of recombination.
Although it is not known if absence of BRCA2 protein can cause some prophase arrest of oocytes, it is clear that some oocytes survive and progress to the end of meiotic prophase, where they become normally arrested. Thus, there is sexual dimorphism between spermatocytes and oocytes in their dependency on BRCA2-mediated meiotic events. However, the fact that we do detect the human BRCA2 transcript by RT-PCR in the gonads, the possibility that some human protein may be present cannot be ruled out. This level may be beyond the level of detection by immunohistochemistry but may be sufficient to partially rescue the phenotype. Therefore, the possibility that a low level of BRCA2 protein in the ovary might be sufficient for meiotic maturation in females but not in males cannot be ruled out. Nonetheless, oocyte survival, the progress of oogenesis and acquisition of meiotic competence is indeed compromised by the absence of BRCA2; there is a marked reduction in the number of oocytes in the rescued females. Even though the rescued females appear to make some normal GV-intact, diplotene oocytes, multiple abnormalities are seen in oocytes undergoing maturation in vitro. Additionally, after gonadotropin treatment, there were 2.5-fold fewer metaphase II eggs from the BRCA2-deficient females compared with the controls, and 10-fold fewer E12.5 embryos in rescued females compared with the controls. These analyses of both in vitro and in vivo maturation, fertilization and embryogenesis suggest that the oocytes from rescued BRCA2-deficient females have reduced competence to undergo maturation and fertilization and to support embryonic development. Taken together, these abnormalities reveal a complex role for BRCA2 in oogenesis, and raise the possibility that all oocytes might be abnormal, but some escape arrest of development.
Thus, aspects of the meiotic phenotype of BRCA2 deficiency appear sexually
dimorphic: spermatocytes arrest in early meiotic prophase, while at least some
oocytes survive to the end of meiotic prophase, but many exhibit abnormalities
of subsequent maturation and developmental competence. Similar sexually
dimorphic phenotypes have been observed for many meiotic mutations
(Hunt and Hassold, 2002).
Interestingly, these include many of those mutations where the male phenotype
is meiotic arrest at or around the transition from zygonema to pachynema:
Brca2 (this report), Msh4
(Kneitz et al., 2000
),
Scp3 (Yuan et al.,
2000
; Yuan et al.,
2002
) and Mei1 (Libby
et al., 2002
); in these mutant female mice, oocytes can progress
through meiotic prophase. Taken together, these phenotypes reveal profound
differences between male and female meiosis in mice. At the moment, it is not
possible to discriminate between two alternative explanations: either the
mechanics of meiosis differ between sexes in such a way that the relevant
proteins are not required for recombination and meiotic progress in oocytes or
meiotic checkpoints present in spermatocytes are lacking or inefficient in
oocytes.
The phenotype of the rescued Brca2 mutant mice suggests that human
BRCA2-mutation carriers could also be predisposed to fertility
problems. In one recent study (Vachon et
al., 2002), individuals with a family history of breast and
ovarian cancer were found to be at risk for nulliparity. Thus, given the
proposed role of BRCA2 in DNA repair in somatic as well as germ cells, cancer
predisposition and infertility may represent two different tissue-specific
phenotypes of mutation in a single gene, more fully revealing its
function.
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ACKNOWLEDGMENTS |
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