Mice heterozygous for Robertsonian centric fusion chromosomal translocations frequently produce aneuploid sperm. In this study RBJ/Dn× C57BL/6J F1 males, heterozygous for four Robertsonian translocations (2N=36), were analyzed to determine effects on germ cells of error during meiosis. Analysis of sperm by three color fluorescence in situ hybridization revealed significantly elevated aneuploidy, thus validating Robertsonian heterozygous mice as a model for production of chromosomally abnormal gametes. Primary spermatocytes from heterozygous males exhibited abnormalities of chromosome pairing in meiotic prophase and metaphase. In spite of prophase abnormalities, the prophase/metaphase transition occurred. However, an increased frequency of cells with misaligned condensed chromosomes was observed. Cytological analysis of both young and adult heterozygous mice revealed increased apoptosis in spermatocytes during meiotic metaphase I. Metaphase spermatocytes with misaligned chromosomes accounted for a significant proportion of the apoptotic spermatocytes, suggesting that a checkpoint process identifies aberrant meioses. Immunofluorescence staining revealed that kinetochores of chromosomes that failed to align on the spindle stained more intensely for kinetochore antigens CENP-E and CENP-F than did aligned chromosomes. Taken together, these observations are consistent with detection of malattached chromosomes by a meiotic spindle checkpoint mechanism that monitors attachment and/or congression of homologous chromosome pairs. However, the relatively high frequency of gametic aneuploidy suggests that the checkpoint mechanism does not efficiently eliminate all germ cells with chromosomal abnormalities.
Accurate meiotic segregation of chromosomes is essential for normal reproduction and a major determinant of gamete quality. Errors in chromosome segregation in either of the two meiotic divisions can lead to gametic loss,reduced fertility or aneuploidy in offspring. Understanding the mechanisms that ensure normal chromosome segregation and of checkpoints that come into play in cases of chromosomal meiotic abnormalities could provide insights into the origin of aneuploidy, such as trisomy 21 (Down syndrome), in our own species. However, little is known of the mechanisms that determine gamete genetic quality.
Mitotic checkpoints govern functional assembly of the mitotic spindle apparatus and bipolar attachment of chromosomes (Burke,2000EF5; Gardner and Burke,2000EF17). 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,1995EF30; Nicklas et al.,1995EF34). Tension can be assessed by changes in phosphorylation of kinetochore proteins (Gorbsky et al.,1999EF18; Li and Nicklas,1997EF31; Waters et al.,1999EF44), 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.,1992EF47). 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.,1997EF40). Human CENP-E is thought to be involved in checkpoint signaling as it associates with the checkpoint protein hBUBR1 (Chan et al.,1999EF7). 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.,2000EF1).
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,2000EF36). 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 Drosophilaspermatocytes (Basu et al.,1998EF2), and MAD2 in maize gametocytes (Yu et al., 1999EF48)and mouse spermatocytes (Kallio et al.,2000EF23). CENP-E is localized on the kinetochores in metaphase mouse spermatocytes (Kallio et al.,1998EF22) and in pig oocytes during both meiotic divisions (Lee et al.,2000EF28), and has been implicated as essential for MII arrest of mouse oocytes (Duesbery et al.,1997EF12). 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,2000EF38; Sinha Hikim and Swerdloff, 1999EF41).
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,1998EF21; LeMaire-Adkins et al.,1997EF29). 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,1916EF39). 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,1973EF6; Nijhoff and de Boer,1979EF35). 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.,1996EF33). Second, heterozygosity for some of these Rb translocations is associated with abnormalities in pairing and recombination suppression (Cattanach and Moseley,1973EF6; Davisson and Akeson,1993EF10), both of which could lead to delays in synapsis and abnormalities during segregation (Koehler et al., 1996EF25). However, extent of error appears to be chromosome-specific (Davisson and Akeson,1993EF10; Winking et al.,2000EF45). Spindle abnormalities and lagging chromosomes have also been observed in oocytes of Rb-heterozygous mice (Eichenlaub-Ritter and Winking,1990EF13).
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.
MATERIALS AND METHODS
RBJ/Dn mice (The Jackson Laboratory, Bar Harbor, ME) were crossed with C57BL/6J (B6) mice (The Jackson Laboratory, Bar Harbor, ME) to produce F1 individuals heterozygous (Rb/+) for each of the four Rb chromosomes present in the Rb-homozygous parent (Rb(5.15)3Bnr, Rb(11.13)4Bnr,Rb(16.17)7Bnr and Rb(2,8)2Lub).
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.,1991EF14); the chromosome 8 probe,which was a mixture (2 μg total) of four subclones (Boyle and Ward,1992EF3), was labeled with digoxigenin; and 1 μg of the X chromosome-specific probe DXWas (Disteche et al., 1987EF11) 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/2×SSC, in 2×SSC 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 100× 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.,1964EF15). 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/2×SSC 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/2×SSC and then twice in 0.1×SSC. Detection reagents 1 and 2, provided by the manufacturer (Cambio) for the chromosome 2 probe, which required amplification, were made in a 3%BSA/4×SSC 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.,1993EF37) 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 25×106 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-β-tubulin(Amersham), anti-phosphorylated histone H3-Ser10 (Upstate Biotech),anti-CENP-E (Schaar et al.,1997EF40), anti-CENP-F (Liao et al., 1995EF32; 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.
Rb-heterozygous mice exhibit elevated levels of sperm aneuploidy
Analysis of sperm by three-color fluorescence in situ hybridization (FISH)was used to seek evidence that Rb/+ mice are models for error-prone meiosis. This analysis revealed elevated levels of sperm aneuploidy in Rb/+ males by comparison with age-matched control males(Table 1). Disomy for chromosomes 8, X and Y was determined using probes specific for each chromosome, and sperm from 6 Rb/+ males were scored. The combined hyperhaploidy frequency for chromosomes 8, X and Y was 4.57% in sperm from Rb/+ males compared with 0.25% in sperm from B6 males(Fig. 2; Table 1). The hyperhapoidy frequency for chromosome 8 was 4.38% in sperm from Rb/+ males compared with 0.075% in sperm from B6 males. As nullisomic sperm (those lacking signals)were not scored, the estimated overall aneuploidy frequency for chromosome 8 is twice the disomy frequency, or approximately 9%. Moreover, sperm from Rb/+males were characterized by a significantly elevated level of chromosome 8 aneuploidy, compared with sex-chromosome aneuploidy. This is an important point, as chromosome 8 is involved in Rb(2.8)Lub in the Rb/+ males, while chromosomes X and Y are not a member of any of the translocation chromosomes. As strict criteria were used for scoring sperm aneuploidy (see Materials and Methods), this estimate is a conservative one. Although probes were not used to detect aneuploidy of the chromosomes involved in the other Rb translocations in this RBJ/Dn stock, it can be assumed that a frequency of 9%sperm aneuploidy is a minimal estimate of the overall frequency and that Rb/+mice are a model for meiosis with errors in chromosome segregation.
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).
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).
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 β-tubulin. Phosphorylation of histone H3 on Ser10 is correlated with chromosome condensation at G2/M in spermatocytes (Cobb et al.,1999bEF9) 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., 1998EF22). 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).
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.
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).
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.
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., 1999EF7). 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.
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.,2000EF23), but also found considerable inconsistency in staining patterns between cells.
This study was conducted to seek evidence for how male germ cells cope with error in meiotic division. Mice heterozygous for Rb chromosome translocations have previously been shown to produce aneuploid gametes, and it is not clear if there are any correction mechanisms that might diminish the overall level of gamete aneuploidy. The results show that mice heterozygous for four different Rb chromosomes derived from RBJ/Dn are a good model in that they produce sperm characterized by a higher than normal frequency of aneuploidy. Abnormalities of chromosome pairing at metaphase of the first meiotic division were demonstrated by use of whole-chromosome FISH paint probes. Additionally,MI spermatocytes from Rb heterozygotes were characterized by an elevated frequency of chromosomes misaligned and failing to congress on the spindle. Evidence that there is ensuing cell death, delay and arrest, which could be mediated by a checkpoint mechanism, includes detection of increased apoptosis of meiotic division-phase spermatocytes, predominantly those with misaligned chromosomes, as well as changes in the kinetics of spermatogenesis and presence of putative checkpoint signals on misaligned chromosomes at metaphase. Nonetheless, the relatively high frequency of gametic aneuploidy suggests that the checkpoint mechanism might not be an efficient inhibitor of meiotic progress in cells faced with multiple chromosomal abnormalities.
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., 1996EF33). 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., 2000EF45), 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,1973EF6). Additionally, other data derived from scoring chromosome arms in MII spermatocytes suggest malsegregation of chromosomes in Rb(11.13)4Bnr heterozygotes (Everett et al.,1996EF16). 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,1993EF10; Everett et al.,1996EF16). 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.,1998EF22). 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,1990EF13). 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,2000EF38); 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., 1999EF26),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.,1988EF19) 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,1979EF35; Speed and de Boer,1983EF42).
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.,1998EF20), 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., 1999EF7). Dynein has been shown to relocate onto kinetochores of chromosomes that are mechanically detached from spindle microtubules in grasshopper spermatocytes (King et al.,2000EF24), 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., 1998EF2) 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.,1999EF7). 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,1990EF27; Sutcliffe et al.,1991EF43) and the improvement of gametogenic progress resulting from providing a partner for the XSxrchromosome (Burgoyne et al.,1992EF4). 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., 1997EF29),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., 1999EF46).
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.
This work was supported by a grant from the NIH, HD33816 to M. A.H. We are grateful to Debby Andreadis, Sally Fridge and Trisha Smith for maintenance of mice, to Dr John Dunlap for his generosity in assistance with confocal imaging, to Dr Terry Hassold for initial instruction in procedures for FISH and for providing FISH probes, to Dr Tim Yen for generously providing antibodies recognizing CENP-E and CENP-F, and to Dr Marko Kallio for advice on microdissection by transillumination. We are indebted to Drs John Eppig, Bruce McKee and Tim Yen, members of the Handel laboratory and two anonymous reviewers for critical comments on the manuscript and discussions.