Department of Biochemistry and Cellular and Molecular Biology, University
of Tennessee, Knoxville, TN, USA
*
Author for correspondence (e-mail:
mahandel{at}utk.edu
)
Accepted May 11, 2001
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SUMMARY |
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Key words: Meiosis, Aneuploidy, Spermatogenesis, Spindle, Checkpoint, Apoptosis
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INTRODUCTION |
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Mitotic checkpoints govern functional assembly of the mitotic spindle
apparatus and bipolar attachment of chromosomes (Burke,
2000; Gardner and Burke,
2000
). These mechanisms
respond to malattachment of chromosomes by delaying exit from mitosis. Tension
developed as chromosomes achieve bipolar attachment appears to be crucial to
the mechanism (Li and Nicklas,
1995
; Nicklas et al.,
1995
). Tension can be assessed
by changes in phosphorylation of kinetochore proteins (Gorbsky et al.,
1999
; Li and Nicklas,
1997
; Waters et al.,
1999
), and these proteins
appear to monitor both spindle assembly and chromosome attachment, signaling
the onset (or delay) of anaphase. For example, centromeric protein CENP-E is a
kinesin-like motor protein whose activity at kinetochores is thought to be
monitored by the spindle assembly checkpoint (Yen et al.,
1992
). CENP-E is essential for
microtubule/kinetochore attachment, as chromosomes from HeLa cells that lack
the CENP-E gene or are injected with antibodies against the CENP-E protein do
not align properly on the metaphase plate, and subsequently do not proceed
through division (Schaar et al.,
1997
). Human CENP-E is thought
to be involved in checkpoint signaling as it associates with the checkpoint
protein hBUBR1 (Chan et al.,
1999
). The association of the
checkpoint protein MAD2 with the kinetochore has recently been shown to be
dependent on the presence of CENP-E, thus linking CENP-E to another component
of the spindle assembly checkpoint mechanism (Abrieu et al.,
2000
).
Little is known about the checkpoints that govern the meiotic divisions or
their similarity to mechanisms that act in mitosis. The first meiotic division
differs markedly from mitosis. In the first meiotic division, homologous
chromosome pairs are separated from each other in a reductional division,
while in the second mitotic-like equational division, sister chromatids are
separated from each other. The information governing the mode of meiotic
anaphase separation is apparently contained within the chromosome and is not a
property of the spindle (Paliulis and Nicklas,
2000). Checkpoint mechanisms
signaling error in the two distinct division processes might also be intrinsic
to the meiotic chromosomes. Investigations using model organisms have
confirmed localization of checkpoint protein BUB1 in Drosophila
spermatocytes (Basu et al.,
1998
), and MAD2 in maize
gametocytes (Yu et al., 1999
)
and mouse spermatocytes (Kallio et al.,
2000
). CENP-E is localized on
the kinetochores in metaphase mouse spermatocytes (Kallio et al.,
1998
) and in pig oocytes
during both meiotic divisions (Lee et al.,
2000
), and has been implicated
as essential for MII arrest of mouse oocytes (Duesbery et al.,
1997
). Whether these proteins
act singly or in concert as a spindle checkpoint mechanism during meiotic
divisions is not known, nor is it known what the consequences of the
checkpoint might be. One likely consequence of checkpoint-detected error might
be apoptosis, which plays an important role in male germ cell development and
regulation (Print and Loveland,
2000
; Sinha Hikim and
Swerdloff, 1999
).
Interestingly, evidence suggests that mammalian female meiosis lacks
stringent checkpoint control, which could explain high rates of aneuploidy,
particularly in humans (Hunt and LeMaire-Adkins,
1998; LeMaire-Adkins et al.,
1997
). It has generally been
assumed that there is more effective quality control during male meiosis, but
this assumption has not been experimentally tested. Identifying division-phase
mechanisms that might detect chromosomal abnormalities and eliminate defective
gametes is not easy in normal males, where the number of abnormal cells is
small by comparison with the vast numbers of normal gametes.
Checkpoint mechanisms might more readily be revealed in males where the
potential for chromosomal error in alignment and segregation is elevated. The
model used here is male mice heterozygous for Robertsonian (Rb)
translocations. Rb chromosomes are metacentric, or nearly metacentric, and are
formed by the centric fusion of two acrocentric chromosomes (Robertson,
1916). During the first
meiotic prophase in individuals heterozygous for Rb chromosomes, the Rb
participates in a trivalent with the two homologous acrocentric chromosomes
(Fig. 1). Pairing defects in
this unusual configuration could give rise to the potential for error in
either chromosome alignment at metaphase I (MI) or unbalanced segregation at
anaphase I (Fig. 1). We have
used mice simultaneously heterozygous (Rb/+) for four different Rb
chromosomes, Rb(5.15)3Bnr, Rb(11.13)4Bnr, Rb(16.17)7Bnr and Rb(2,8)2Lub,
produced by mating individuals quadruply homozygous (RBJ/Dn) to chromosomally
normal individuals. Two lines of previous evidence had suggested that these
heterozygous mice could provide a model to test for meiotic checkpoints
responding to chromosomal misalignment and malsegregation. First,
heterozygotes for the single translocations Rb(5.15)3Bnr, Rb(11.13)4Bnr and
Rb(16.17)7Bnr have each been shown to be prone to nondisjunction of the
involved chromosomes, both by assessment of metaphase II (MII) spermatocytes
and by zygotic loss (Cattanach and Moseley,
1973
; Nijhoff and de Boer,
1979
). Elevation of sperm
aneuploidy has been documented by fluorescence in situ hybridization (FISH)
analysis of sperm from male mice carrying Rb(8.14), a Rb chromosome not
present in the RBJ/Dn stock used in this study (Lowe et al.,
1996
). Second, heterozygosity
for some of these Rb translocations is associated with abnormalities in
pairing and recombination suppression (Cattanach and Moseley,
1973
; Davisson and Akeson,
1993
), both of which could
lead to delays in synapsis and abnormalities during segregation (Koehler et
al., 1996
). However, extent of
error appears to be chromosome-specific (Davisson and Akeson,
1993
; Winking et al.,
2000
). Spindle abnormalities
and lagging chromosomes have also been observed in oocytes of Rb-heterozygous
mice (Eichenlaub-Ritter and Winking,
1990
).
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We provide new data on meiotic pairing abnormalities and nondisjunction that leads to apoptosis and gametic aneuploidy in male Rb heterozygotes. Our observations indicate that the unusual chromosome constitution in Rb-heterozygous males leads to abnormalities during meiotic division, with concomitant cell death consistent with checkpoint surveillance of chromosome alignment on the spindle. Nonetheless, aneuploid gametes are produced, suggesting that checkpoint mechanisms do not reliably eliminate all aneuploid germ cells.
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MATERIALS AND METHODS |
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Three-color fluorescence in situ hybridization (FISH)
For sperm FISH analysis, four Rb/+ and six B6 mice were killed by cervical
dislocation and sperm from epididymides were collected in 2.2% citrate. Sperm
were spread onto a slide and dried. The slides were soaked in DTT on ice for
30 minutes and placed immediately into LIS (diiodosalicyclic acid) for 1 hour.
The slides were air dried and dehydrated in ethanol. Slides and probes were
denatured at 78°C in formamide, then dehydrated and air-dried. The probes
were specific for chromosomes 8, X and Y (gifts from Terry Hassold, Case
Western University, Cleveland, OH). Biotin was used to label 1 µg of the Y
probe pERS-532 (Eicher et al.,
1991); the chromosome 8 probe,
which was a mixture (2 µg total) of four subclones (Boyle and Ward,
1992
), was labeled with
digoxigenin; and 1 µg of the X chromosome-specific probe DXWas (Disteche et
al., 1987
) was labeled with
both biotin and digoxigenin separately. Probes were labeled with digoxigenin
and biotin using a nick translation kit (Roche Pharmaceutical), and purified
over a Sephadex- G50 column. The probe mix was added to each slide and allowed
to incubate at 37°C overnight. The next day, the slides were washed in 50%
formamide/2xSSC, in 2xSSC and then in PN Buffer (0.1M
NaH2PO4, 0.1M Na2HPO4, 0.05%
NP-40, pH 8). Slides were incubated in a BSA-blocking buffer and the
appropriate fluorochrome-conjugated detector, also in BSA, at 37°C for 30
minutes, then washed in PN buffer twice. After adding DAPI/Antifade (Molecular
Probes), slides were viewed with an epifluorescent microscope using a
100x lens. Estimates of sperm aneuploidy were deliberately conservative.
Only hyperhaploidy, and not hypohaploidy, was scored; the aneuploidy frequency
represents twice the hyperhaploidy frequency. Additionally, sperm were deemed
suitable for scoring only when the following criteria were met: fluorescent
signals were clearly within and not on the edge of the sperm nucleus,
fluorescent signals were all in the same plane of focus, and any two signals
scored as separate were separated by a distance equal or greater than one
signal domain.
Chromosome painting was performed on testicular cells fixed in 3:1
ethanol:acetic acid and then air-dried onto slides (Evans et al.,
1964). The slides were
air-dried overnight and dehydrated in a 70%, 90%, 90% and 100% ethanol series,
then air-dried again. The DNA of the cells was denatured by incubation in 70%
formamide/2xSSC at 65°C for 2 minutes. The slides were then quenched
in the ethanol series above and air-dried again. Chromosome paint probes, for
chromosome 2 and chromosome 8 (Cambio, Cambridge, UK), were warmed to
37°C, and denatured at 65°C for 10 minutes, then at 37°C for 60-90
minutes. Subsequently, 15 µl of each chromosome paint probe was added to
each slide and the cells were coverslipped, sealed and incubated overnight at
37°C in a humidified chamber. The slides were washed twice for 5 minutes
at 45°C in 50% formamide/2xSSC and then twice in 0.1xSSC.
Detection reagents 1 and 2, provided by the manufacturer (Cambio) for the
chromosome 2 probe, which required amplification, were made in a 3%
BSA/4xSSC blocking solution and slides were incubated with the
appropriate detection reagent for 40 minutes in a humidified chamber at
37°C. The slides were then processed for visualization as above.
Testis fixation and in situ apoptosis detection
Mice were killed by cervical dislocation, testes removed, and fixed either
in 4% paraformaldehyde overnight at 4°C or in Bouin's solution overnight
at room temperature. Testes from three Rb/+ mice and three B6 mice at 14, 18
and 23 days old, as well as adult, were fixed in this manner. The testes were
dehydrated through an ethanol series and toluene, then embedded in paraffin.
The tissue was sectioned at 3-6 µm, and the sections placed on slides to
dry. After deparaffination in xylene and rehydration in a decreasing ethanol
series, the slides were subjected to the TUNEL reaction for assessment of
apoptosis (see below). For staging of tubule sections, Periodic Acid-Schiff
(PAS) staining was performed following the manufacturer's (Sigma) protocol
with some modifications. After the TUNEL reaction, slides were rinsed in
phosphate-buffered saline (PBS), then placed in 0.5% periodic acid for 10
minutes. After a 10 minute wash in dH2O, followed by incubation for
1 hour in Schiff reagent in the dark, the slides were placed in 1% potassium
metabisulfite for 2 minutes. The slides were then washed in dH2O,
stained with Hematoxylin for 2 minutes, rinsed with tap water, and placed in
lithium carbonate (1.38 g/100 ml dH2O saturated) for 3 seconds.
After washes in an increasing ethanol series and xylene, the slides were
mounted with Permount (Fisher).
Apoptosis assays were performed using the In Situ Cell Death Detection Kit (Roche/Boehringer Mannheim), employing the TUNEL reaction following the manufacturer's protocol, with the exception that the enzyme incubation was for 15 minutes. Scoring of apoptosis frequency was performed by counting alkaline phosphatase-positive (brown) cells in tubule sections. Tubule cross-sections were scored as apoptotic when three or more apoptotic meiotic cells were observed per tubule cross-section.
Fixation and immunofluorescent labeling of tubule segments and
isolated germ cells
To obtain cytological preparations enriched in meiotically dividing
spermatocytes (stage XII of the mouse seminiferous epithelium), a variation of
the transillumination procedure (Parvinen et al.,
1993) was used. Testes from
adult mice (three B6 males and three Rb/+ males) were detunicated, then
digested with collagenase for 8 minutes at 33°C in Krebs-Ringer
bicarbonate (KRB)-buffered media. Transillumination patterns were observed
using a dissecting microscope and the desired stage XII segments (visualized
as 3 mm beyond the site of transition from optically dense to light) were
excised and transferred onto a microscope slide in KRB. For fixation, a
coverslip was placed on top of the segment, then the entire slide was frozen
in liquid N2 for 30 seconds. The coverslip was removed, and the
slide was fixed in 3:1 ethanol/acetic acid. Before incubation with antibody,
the slide was placed in PBS/0.2% Triton X-100 (Sigma) for 5 minutes, then
placed in blocking solution (PBS/10% goat serum) for 30 minutes.
Cell preparations enriched in germ cells were prepared as previously
described (Cobb et al., 1999a).
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 mesh
and washing in buffer. To make surface-spread preparations for visualization
of nuclei, cells were fixed in 2% paraformaldehyde with 0.03% SDS (Cobb et
al., 1999a
). Spermatocytes from
germ cell preparations were also embedded in a fibrin clot using modifications
to a previously published protocol (LeMaire-Adkins et al.,
1997
). Germ cells were
isolated as mentioned above, and brought to a concentration of
25x106 cells/ml. Onto a slide, 3 µl of fibrinogen
(Calbiochem, 10mg/ml fresh) and 1.5 µl of the cell suspension were mixed.
Then 2.5 µl of thrombin (Sigma, 250 units) was added, and allowed to clot
for 5 minutes. The slide was fixed in 4% paraformaldehyde, washed in 0.2%
Triton X-100, then processed for immunofluorescence.
The antisera used were polyclonal anti-SYCP3, anti-ß-tubulin
(Amersham), anti-phosphorylated histone H3-Ser10 (Upstate Biotech),
anti-CENP-E (Schaar et al.,
1997), anti-CENP-F (Liao et
al., 1995
; generously provided
by T. Yen) and anti-MPM-2 (Upstate Biotech). The polyclonal antibody
recognizing SYCP3 was prepared by Covance Research Products (Richmond, CA)
against recombinant his-tagged protein expressed in Escherichia coli.
The Sycp3 cDNA was synthesized by RT-PCR from testicular RNA, cloned
into the pPROExHta expression vector (GibcoBRL) and the sequence verified by
direct sequencing. Rats were injected intramuscularly with 0.5 mg of purified
SYCP3 protein in 6 M urea followed by booster injections of 0.25 mg protein at
3-week intervals. Serum was collected at 3-week intervals beginning one month
after the initial injection. All sera collected after the injections contained
specific antibodies that recognized the SYCP3 protein. The specificity of the
antiserum was determined by immunoblotting using extracts from pachytene
spermatocytes, known to contain SYCP3 protein. Preimmune serum did not
recognize any proteins in extracts from pachytene spermatocytes and did not
stain cells. Serum collected after antigen injection recognized only protein
of the appropriate molecular weight and stained axial elements and
synaptonemal complexes in spermatocytes.
After overnight incubation in primary antibody, slides were incubated with rhodamine- or fluorescein-conjugated secondary antibodies (Pierce), 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 as a primary antibody. Staining was observed with an Olympus epifluorescence microscope and images were captured and transferred to Adobe PhotoShop with a Hamamatsu color 3CCD camera. Confocal imaging was performed using a Leica TC SP2 laser-scanning confocal microscope.
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RESULTS |
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Spermatocytes from Rb-heterozygous mice exhibit meiotic pairing
abnormalities and chromosome misalignment
As failure to maintain normal bivalent chromosomes could cause the observed
gamete aneuploidy, metaphase chromosome pairing was examined from air-dried
chromosome preparations. FISH with chromosome-specific paint probes was used
to examine MI pairing configurations of the Rb(2.8)Lub and its homologs, the
acrocentric chromosomes 2 and 8. Fig.
3A shows among spermatocytes from B6 control males, the signals
for chromosomes 2 and 8 are combined, suggesting maintenance of homologous
pairing at MI. By contrast, typical images of spermatocyte nuclei from the
Rb/+ males revealed two types of signal configurations: those suggesting
apparent homologous pairing (Fig.
3B) and those with apparent pairing disruption, where signals for
homologous chromosomes are separated and sometimes one of the painted
chromosomes (either chr. 2 or chr. 8) is juxtaposed with an unpainted
DAPI-stained chromosome (Fig.
3C). Among the five individual males scored, the overall frequency
of spermatocytes with apparently unpaired chromosomes 2 and/or 8 was 28.18%
compared with 0% for B6 control spermatocytes
(Table 2).
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In addition to the univalence and pairing abnormalities illustrated in Fig. 3, earlier prophase pairing abnormalities were seen in surface-spread spermatocyte nuclei stained with antiserum against mouse SYCP3 for visualization of the synaptonemal complex. These pairing abnormalities consisted primarily of incompletely paired regions, sometimes seen as pairing `protrusions' at the centromeric regions (Fig. 4).
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Additionally, unaligned chromosomes were seen on meiotic MI spindles, which
could be a consequence of the observed pairing abnormalities. In order to
retain the three-dimensional configuration of division-phase spermatocytes,
germ cells were fixed and embedded in a fibrin clot and visualized by confocal
microscopy. When scoring these cells, prometaphase cells were identified as
having condensed chromosomes, loss of nuclear envelope and a unipolar spindle.
Metaphase cells exhibit bipolar spindles and aligned chromosomes. Metaphase I
cells retain SYCP3 epitopes, while MII cells are spatially close to their
sister cell and do not retain SYCP3 epitopes. These criteria allowed us to
determine that cells with unaligned chromosomes were in metaphase (not
prometaphase). Additionally, all frequencies of abnormalities were compared
with control B6 spermatocytes. Spermatocytes from B6 mice consistently
exhibited a `compact' MI configuration of chromosomes, with all chromosomes
congressed to the meiotic spindle equator
(Fig. 5A), revealed by
immunofluorescence with antibodies against the phosphorylated form of histone
H3-Ser10 and ß-tubulin. Phosphorylation of histone H3 on Ser10 is
correlated with chromosome condensation at G2/M in spermatocytes (Cobb et al.,
1999b) and thus antibody
staining provides a marker for cells in the division phase. In contrast to
chromosomally normal spermatocytes, MI spermatocytes from Rb/+ mice frequently
exhibited chromosomes that were unaligned or malattached at a distance from
the metaphase equator (Fig.
5B). This pattern of misalignment was seen after establishment of
the bipolar and elongated spindle, which in rodents occurs during prometaphase
(Kallio et al., 1998
). This
configuration was sometimes accompanied by abnormalities in spindle structure;
for example, in Fig. 5C, note
that one spindle pole is not developed, while the microtubule arrays radiate
away from the metaphase plate. Such spindle abnormalities may be an early step
in apoptosis (see below). Although spindle abnormalities were less frequent,
nonaligned chromosomes were found in 23% of the 500 phospho-histone
H3-positive MI spermatocytes scored in each of 3 Rb/+ mice, whereas unaligned
chromosomes were seen in only 4.8% of 500 MI spermatocytes from each of three
B6 males. In addition to the MI abnormalities, some MII spermatocytes from
Rb/+ mice also exhibited aberrant chromosome configurations; for example,
chromosomes that are positioned behind rather than between the spindle poles
(Fig. 5D).
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The frequency of spermatogenic cell stages was determined to test the hypothesis that these pairing and metaphase alignment abnormalities could cause a loss of cells and/or delay in the normal progression of spermatogenesis. Germ cells were isolated and the frequency of cell types was obtained from nuclei spreads (Fig. 6). The frequency of postmeiotic round spermatids, relative to the frequency of leptotene/zygotene spermatocytes, was decreased in the germ cell population from Rb/+ compared with that from B6 mice, and a concomitant increased frequency of pachytene spermatocytes, but not of leptotene/zygotene spermatocytes, was found among germ cells from Rb/+ mice compared with germ cells from the control B6 mice (Fig. 6). Additionally, when sectioned material was analyzed, the frequencies of stage XII, and VII-IX, tubule sections in Rb/+ testes were found to be greater than those in control B6 testes, while the frequency of the other stages did not differ statistically between the two (Fig. 7). Taken together, these data suggest loss of cells and possible delay in progress of spermatogenesis in Rb/+ mice.
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Spermatocytes from Rb-heterozygous males with misaligned chromosomes
exhibit elevated frequency of apoptosis in meiotic division phase
To test the hypothesis that chromosome abnormalities might activate a
meiotic checkpoint leading to apoptosis, apoptotic cells in tissue sections
were identified and enumerated using the TUNEL reaction and Periodic
Acid-Schiff reagent to stage tubule sections. The criterion for identifying
individual cross-sections as apoptotic was the presence of three or more
apoptotic cells per tubule section. Relatively few cells were found to be
apoptotic in testes of control B6 males (Figs
7 and
8). However, in testes of Rb/+
males, apoptosis was found to be elevated among MI spermatocytes in
seminiferous epithelium stage XII. In a developmental analysis of the onset of
apoptosis, testes from three Rb/+ and three control B6 mice were examined on
days 14, 18 and 23 after birth. These time points were chosen to precede (days
14 and 18) and coincide with (day 23) appearance of significant numbers of MI
cells. An elevated number of apoptotic meiotic germ cells were found in testes
from Rb/+ mice only after 23 days of age
(Fig. 8), with a frequency of
15.8±2.8 apoptotic cells/stage XII tubule cross-section (data not
shown).
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In order to determine if the apoptotic cells seen in stage XII cross sections were due to MI spermatocytes with unaligned chromosomes, microdissected stage XII segments were analyzed for both apoptosis by the TUNEL reaction, for DNA by DAPI stain and by immunofluorescence with antibody against phosphorylated histone H3 to visualize chromosome alignment at MI. This analysis revealed that a significant proportion of the apoptotic MI spermatocytes exhibited a misaligned chromosome (Fig. 8C). A total of 1000 apoptotic MI spermatocytes were scored in tubules from three Rb/+ males and 79.8% contained chromosomes not properly aligned on the metaphase plate (Table 3). This observation directly links apoptosis to spermatocytes with unaligned chromosomes. Interestingly, it was observed that many of the apoptotic MI spermatocytes did not stain with the antibody against phosphohistone H3, suggesting loss of phosphorylation on Ser10 as part of the apoptotic process. This observation suggests that the previous estimate that 23% of MI spermatocytes in Rb/+ testes have misaligned chromosomes (above and Fig. 5) is low, as this was derived only from MI spermatocytes that stained positively for phospho-histone H3.
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Taken together, these analyses show increased germ-cell apoptosis in testes of Rb/+ mice, provide evidence that the susceptible meiotic stage encompasses the division phases, and suggest that it is cells with chromosomal abnormalities that are undergoing apoptosis.
Meiotic spermatocytes from Rb-heterozygous mice exhibit features of
normal G2/M but also abnormalities in behavior of putative checkpoint
proteins
In spite of chromosome pairing abnormalities and apparent meiotic delay,
many features of the meiotic prophase-metaphase (G2/M) transition were normal
in spermatocytes from Rb/+ mice compared with those from B6 controls. In
surface-spread spermatocytes from both control and Rb/+ spermatocytes, we
observed orderly disassembly of the synaptonemal complex, chromatin
condensation and individualization, and appearance at MI of newly
phosphorylated epitopes, detected by phospho-histone H3-Ser10 antibody (a
marker for chromosome condensation) and MPM antibody (a marker for epitopes
phosphorylated at division phase; data not shown).
Because of evidence for spindle abnormalities in Rb/+ spermatocytes and for
elimination of spermatocytes by apoptosis, attention was given to localization
of proteins that might act directly or indirectly in checkpoint mechanisms. In
mitotic HeLa cells, kinetochores on lagging chromosomes stain more intensely
with antibodies against hBUBR1 and CENP-E, known spindle assembly checkpoint
proteins, than do kinetochores on chromosomes that are properly aligned (Chan
et al., 1999). Consequently,
the pattern of localization and intensity of signal of CENP-E was monitored at
kinetochores of chromosomes associated with spindles in Rb/+spermatocytes,
especially at the kinetochores of improperly attached or lagging chromosomes
(as in Figs 5,
8). In
Fig. 9, the normal prometaphase
(Fig. 9A,B) and metaphase
(Fig. 9C,D) patterns of CENP-E
staining of B6 spermatocytes from microdissected stage XII tubule sections is
shown. This pattern of staining was also the predominant one in Rb/+
spermatocytes, with the important exception of kinetochores of chromosomes not
aligned at the metaphase spindle equator (arrows in
Fig. 9E-G), where staining was
more intense. Kinetochores on all of the malattached chromosomes stained more
intensely with antibodies against CENP-E. A similar staining pattern was seen
with antibodies against CENP-F (Fig.
10). The increase in fluorescence intensity in detection of these
proteins may be due to either an increase in the amount of protein at the
kinetochore on unaligned chromosomes, or an increased accessibility of the
epitope to the antibody, possibly due to a conformational alteration.
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Antibodies against the polo-like kinase PLK1 protein, MAD2 and SYCP3, as
well as CREST antisera, were used to assess the possibility of an elevation in
staining intensity of other proteins in centromeric regions, as well as to
detect multiple kinetochores (data not shown). Staining with these antibodies
revealed similar intensity on kinetochores of both properly aligned
chromosomes and those that were not in Rb/+ spermatocytes. Although a role for
PLK1 protein in both DNA repair and centrosome maturation has been suggested,
it is not thought to be involved in a spindle checkpoint mechanism. While MAD2
is thought to play a role, its function during mammalian meiosis has not been
established and awaits further experimental evidence. We observed staining
patterns similar to those found previously (Kallio et al.,
2000), but also found
considerable inconsistency in staining patterns between cells.
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DISCUSSION |
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Meiotic division of spermatocytes from Rb-heterozygous mice is
error-prone
There are three lines of evidence from this work suggesting that gametic
aneuploidy is a characteristic of Rb/+ mice. The first, and most direct,
derives from assessment of sperm aneuploidy by FISH. Sperm aneuploidy for
chromosome 8, participating in a Rb chromosome, was 8.8%, compared with 0.15%
for control sperm. Similarly increased aneuploidy has been observed
previously; specifically, a tenfold increase in the sperm aneuploidy frequency
was found for males heterozygous for the Rb(8.14)16Rma translocation, although
hyperhaploidy in the sex chromosomes did not differ from control values (Lowe
et al., 1996). It is highly
likely that most of the sperm aneuploidy we have observed derives from
unbalanced segregation of metacentric Rb chromosomes and their acrocentric
homologs at anaphase I. As chromosome 8 is involved in only one of the four Rb
chromosomes (Rb(2,8)2Lub), 9% is likely to be a minimum estimate of the total
sperm aneuploidy. The overall aneuploidy frequency could be as high as 72% if
sperm FISH probes for all the other seven chromosomes involved in Rb
translocations (chromosomes 2, 5, 11, 13, 15, 16 and 17) had been used.
However, aneuploidy frequencies are most probably chromosome specific (Winking
et al., 2000
), and thus the
multiplicative estimate of 72% could be inaccurate. Previous observations also
suggest that heterozygotes for the single translocations Rb(5.15)3Bnr,
Rb(11.13)4Bnr and Rb(16.17)7Bnr are prone to nondisjunction, as assessed both
by MII chromosome analysis and by zygotic loss (Cattanach and Moseley,
1973
). Additionally, other data
derived from scoring chromosome arms in MII spermatocytes suggest
malsegregation of chromosomes in Rb(11.13)4Bnr heterozygotes (Everett et al.,
1996
). Thus, Rb/+ mice are a
model for production of aneuploid sperm, and, furthermore, the aneuploidy
appears to be restricted to the chromosomes involved in the Rb
translocations.
The second line of evidence provides clues to what could be an origin of
meiotic error in spermatocytes of Rb/+ mice. Spermatocytes scored at MI with
whole-chromosome paint probes for chromosomes 2 and 8 (forming Rb(2,8)2Lub)
displayed an abnormally high level (28%) of apparent univalence or
nonhomologous pairings for these two chromosomes. This observation suggests
that homolog pairing is diminished or that chiasmata are lacking or are
prematurely resolved. Reduced chiasmata formation is also suggested by
observations of pachytene spermatocytes that show abnormalities of chromosome
pairing. Other studies have also shown that mispairing and recombination
suppression occurs in Rb/+ spermatocytes (Davisson and Akeson,
1993; Everett et al.,
1996
). However, this is the
first study where mispaired chromosomes have been positively identified at MI
by the use of chromosome-specific paint probes.
The third line of evidence also provides insight to a possible mechanism of
aneuploidy. Spermatocytes scored at MI for misaligned or malattached
chromosomes, or for failure in congression showed an abnormally high frequency
(23%) of these errors. The lagging chromosomes were seen after establishment
and elongation of the bipolar spindle in prometaphase (Kallio et al.,
1998). Careful comparison was
made with the frequency of lagging chromosomes in control (B6) spermatocytes
with elongated spindles to ensure that we were observing metaphase and not a
stage in prometaphase congression. Although scoring was based on a sensitive
immunofluorescence detection of MI chromosomes with phosphorylated histone H3,
specific chromosomes could not be identified, as the preparative techniques
for visualization of spindles were not compatible with chromosome FISH.
Furthermore, the estimate of mislaigned chromosomes derived by using antibody
to phosphorylated histone H3 may be low, because further analysis showed that
many MI spermatocytes with misaligned chromosomes were apoptotic and did not
stain with antibody against phospho-histone H3
(Fig. 8C;
Table 3). These observations
are consistent with a previous finding of lagging chromosomes in anaphase
mouse oocytes containing Rb translocations (Eichenlaub-Ritter and Winking,
1990
). We assume, but do not
know, that univalent or mispaired Rb trivalents contributed to the majority of
misaligned chromosomes detected at MI. Taken together, these observations
imply that there was premature separation of chromosome homologs and that
univalent or non-homologously paired chromosomes were delayed in spindle
attachment and/or congression.
Taken together, these three lines of evidence lend support to the hypothesis that meiosis in Rb/+ mice is fraught with an increased level of error. The common effect is malsegregation leading to gametic aneuploidy, but the causes can lie in diminished chromosome pairing that leads to univalence or misaligned chromosomes, as well as unbalanced segregation of paired trivalents involving Rb chromosomes. These errors undoubtedly contribute to germ cell aneuploidy and embryo death. As roughly 9% of sperm from these quadruple Rb/+ mice were aneuploid for chromosome 8, we estimated (above) that the total of the four translocations involving eight chromosomes events could produce an aneuploidy frequency as high as 72%. Nonetheless, somewhat amazingly, male mice heterozygous for four different Rb chromosomes are fertile, in spite of seemingly great potential for chromosomal disaster.
Apoptosis may serve as an elimination mechanism for abnormal germ
cells
Evidence for a testicular mechanism for elimination of chromosomally
aberrant germ cells was found in the elevated frequency of stage-specific
apoptosis observed in testes of Rb/+ mice compared with both chromosomally
normal B6 mice and Rb homozygotes. Apoptosis is a known mechanism for control
of germ-cell number and elimination of abnormal and/or damaged germ cells in
the testis (Print and Loveland,
2000); however, background
levels of apoptosis are normally low. For example, previous observations
documented a mean value of 1.9±0.2 apoptotic cells per tubule in testes
of B6 mice (Kon et al., 1999
),
which this is consistent with our values for apoptosis frequency in control B6
mice (Figs 7,
8). In contrast, germ cell
apoptosis was elevated in Rb/+ mice. Most apoptosis was seen in spermatocytes
of stage XII tubules where spermatocytes undergo meiotic divisions. Moreover,
apoptosis was not detected in Rb/+ mice until 23 days after birth, a time
point that coincided with an increase in MI spermatocytes. Most significantly,
apoptosis was found predominantly in MI spermatocytes that exhibited
misaligned chromosomes (Fig. 8;
Table 3). Previously,
correlations have been made between induced chromosome damage or genetic
abnormalities and increased apoptosis. Now, these data directly link apoptosis
to the presence of misaligned chromosomes at MI, thereby suggesting that the
presence of a misaligned chromosome triggers a spindle checkpoint mechanism
leading to cell death. Analysis of apoptosis in testes of mice homozygous for
the Rb chromosomes revealed more frequent cell death in stage XII tubules than
was detected in B6 mice, but the frequency was not as high as found in the
testes of Rb/+ mice (data not shown). Importantly, apoptosis was not detected
during pachynema, even though Rb/+ mice exhibited an elevated frequency of
pachytene spermatocytes compared with B6 mice
(Fig. 6). Thus, there was no
evidence for a `pachytene checkpoint', one that might monitor success in
pairing of homologous chromosomes. Such a checkpoint might be expected to lead
to apoptotic cells at a stage earlier than stage XII, although the more
convoluted scenario of detection of error in pachytene leading to elimination
at MI cannot be excluded.
The fact that Rb/+ testes contain an increased frequency of stage XII
sections compared with B6 testes (Fig.
8), in spite of the fact that there was no significant variation
in frequency for any other stage between the two strains, is also suggestive
of an arrest, or delay, in meiosis. This was also observed in various other
Rb/+ strains (Hansmann et al.,
1988) and suggests that some
consequence of heterozygosity for Rb chromosomes, most likely a
checkpoint-mediated mechanism that detects misaligned chromosomes, activates
developmental arrest and elimination by apoptosis. Elimination of
division-phase spermatocytes was also reflected a reduced number of round
spermatids among germ cells from testes of Rb/+ mice compared with B6 controls
(Fig. 6), in spite of the fact
that no differences were ascertained in frequencies of early prophase,
leptotene and zygotene, spermatocytes. The elevated frequency of pachytene
spermatocytes and reduced frequency of round spermatids in Rb heterozygotes
compared with controls (Fig. 6)
suggests that there was a delay in entry into division phase. Similar
conclusions were reached from different kinds of analyses of mice carrying
fewer and different Rb translocations (Nijhoff and de Boer,
1979
; Speed and de Boer,
1983
).
Surprisingly, the concurrent analysis of TUNEL reaction and phosphorylated
histone H3 (Fig. 8C) revealed
that most apoptotic MI spermatocytes do not react with the antibody to
phosphorylated histone H3, suggesting that the epitope may be dephosphorylated
or no longer accessible. Antibody to another protein, SYCP3, also did not
react with many apoptotic cells, suggesting changes in either antibody
penetration or accessibility of epitopes in apoptotic cells. Although it has
previously been determined that phosphorylation of histone H3 is not involved
in apoptosisinduced condensation of interphase chromatin (Hendzel et al.,
1998), this is, to our
knowledge, the first suggestion that phosphorylated histone H3 could be
dephosphorylated as part of apoptosis.
Taken together, these data suggest a delay in completion of MI and elimination of spermatocytes by apoptosis in Rb/+ mice. If this is checkpoint mediated, the important biological problem is to determine the checkpoint signal. The main events that culminate in the first metaphase are chromosome condensation, spindle morphogenesis and alignment of chromosomes onto the spindle at the equator. In our observations, no differences were detected between Rb/+ and B6 mice with respect to timing of chromosome condensation and spindle formation. Thus, if a checkpoint is present, we hypothesized that the signal is improper alignment of chromosomes at metaphase.
Altered staining intensity of CENP-E and CENP-F proteins on
improperly aligned kinetochores may reveal an element of a meiotic spindle
checkpoint mechanism
Evidence for a spindle checkpoint mechanism responding to improperly
attached chromosomes in Rb/+ spermatocytes stems from differences between
properly attached and malaligned chromosomes in the staining intensity of
proteins known to localize to kinetochores and to be components of a spindle
assembly checkpoint mechanism. Among metaphase spermatocytes identified by
anti-phospho-histone H3 staining from Rb/+ mice, 23% contain unaligned
chromosomes (Fig. 5B). All of
the unaligned kinetochores assessed stained more intensely with antibodies
against CENP-E and CENP-F than did kinetochores of chromosomes that were
properly positioned on the spindle. However, antibodies against proteins that
are unrelated to the spindle assembly checkpoint (PLK1, CREST and SYCP3)
yielded equal staining signal on aligned compared with unaligned chromosomes
in Rb/+ metaphase spermatocytes. This observation suggests that the increased
signal of CENP-E and CENP-F on unaligned chromosomes is specific and signals
the state of chromosome alignment or attachment on the spindle.
Similar observations have been made of mitotic cells, where it was shown
that kinetochores on lagging chromosomes stained more intensely with
antibodies against CENP-E than did chromosomes aligned on the metaphase plate
(Chan et al., 1999). Dynein has
been shown to relocate onto kinetochores of chromosomes that are mechanically
detached from spindle microtubules in grasshopper spermatocytes (King et al.,
2000
), where the relocation of
dynein is a transient interaction and not caused by structural alterations of
the dynein protein itself that affect antibody binding. Comparable results
have been obtained for Drosophila mitotic and meiotic cells (Basu et
al., 1998
) using antibodies
recognizing the BUB1 spindle checkpoint protein. CENP-E is a kinesin-like
motor protein whose function in kinetochore-microtubule attachments has been
proposed to be monitored by the hBUBR1 checkpoint kinase (Chan et al.,
1999
). During mitosis, this
mechanosensor complex relays signals from the kinetochore to inhibit the
anaphase-promoting complex (APC) from ubiquitinating proteins whose
destruction is required for entry into anaphase. We hypothesize that the
increase in staining intensity for CENP-E and CENP-F on malattached meiotic
chromosomes in Rb/+ spermatocytes may initiate a signal either to correct the
attachment problem, or, if the error cannot be corrected, to initiate
apoptotic elimination of a spermatocyte that is likely to give rise to
aneuploid gametes.
Abnormalities of meiotic chromosome behavior may activate a
checkpoint leading to elimination of aberrant germ cells
Good gamete quality in males with increased potential for gametic
aneuploidy could be maintained by the operation of checkpoint mechanisms.
Indeed, this study provided data consistent with the hypothesis that
chromosomal abnormalities, specifically misalignment, are detected in the
meiotic division phase and lead to elimination of aberrant germ cells by
apoptosis. These data suggest that mechanisms ensure the elimination of germ
cells with abnormal chromosomal configurations or behavior. Similar mechanisms
have been implicated by the MI arrest of male mice with a single sex
chromosome, the XOSxr male (Kot and Handel,
1990; Sutcliffe et al.,
1991
) and the improvement of
gametogenic progress resulting from providing a partner for the XSxr
chromosome (Burgoyne et al.,
1992
). These examples are in
contrast to the situation of mammalian female meiosis, where data suggest that
checkpoint mechanisms may be inefficient or absent. For example, a single
unpaired sex chromosome (in the XO female) does not trigger a meiotic arrest
(LeMaire-Adkins et al., 1997
),
suggesting lack of apparent checkpoint control. Arrest in the female does
occur when the oocyte is faced with massive chromosome univalency, as in the
Mlh1-null female. Here, spindle assembly fails, suggesting a role for
the chromosomes in the morphogenesis of the MI spindle of the oocyte (Woods et
al., 1999
).
Surprisingly, however, it is not clear how much improvement in gamete quality is brought about by elimination of aberrant MI germ cells. From FISH analysis of MI spermatocytes, it can be extrapolated that the frequency in the sperm population of sperm disomic or nullisomic for chromosome 8 would be 10-11% if there were no elimination of chromosomally aberrant germ cells. This frequency is not greatly different from the observed frequency of 9%. However, the potential frequency of aneuploidy deriving from adjacent segregation of the trivalent is not known, and it might increase the predicted aneuploidy for chromosome 8. Thus, at this point, it is not known if the frequency of 9% sperm scored as aneuploid for chromosome 8 represents a reduction from the expected frequency.
Taken together, the results from this study provide evidence for a meiotic spindle checkpoint mechanism in male gametogenic cells, but one that may not be totally efficient in eliminating germ cells destined to form aneuploid gametes. First, data on meiotic pairing abnormalities and nondisjunction leading to gametic aneuploidy in Rb/+ spermatocytes validate Rb/+ mice as a model for error-prone meiotic chromosome segregation. Second, abnormalities of chromosome attachment to and alignment on the meiotic spindle were prevalent in Rb/+ spermatocytes. Third, staining patterns for candidate checkpoint proteins differed between properly attached and malaligned chromosomes in meiotic metaphase spermatocytes. Fourth, an increased frequency of post-prophase meiotic germ cell death was seen in testes of Rb/+ mice, as well as developmental delays consistent with checkpoint surveillance. Most significantly, the data show that cells with misaligned chromosomes account for the apoptotic cells, providing a direct link between chromosome error and elimination by apoptosis. However, when aneuploidy for chromosome 8 was considered, the frequency of chromosomally unbalanced sperm was not substantially less than the frequency estimated from observed meiotic abnormalities. Thus, considered together, these observations provide indirect but compelling evidence for detection of meiotic chromosome error leading to subsequent elimination of spermatocytes. What is not yet known is how effective the checkpoint is. Clearly, aneuploid sperm are produced and this can undoubtedly lead to reduction of reproductive efficiency. Further insight into the role and efficacy of the spindle checkpoint mechanism in male gametes is sorely needed, and will derive, in part, from mutation of putative checkpoint genes and analysis of the phenotypic effects in models for meiotic error, such as Rb/+ mice.
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ACKNOWLEDGMENTS |
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