RESEARCH ARTICLE 2417 Coordinating the segregation of sister chromatids during the first meiotic division: evidence for sexual dimorphism

Craig A. Hodges, Renée LeMaire-Adkins and Patricia A. Hunt* Department of Genetics and Center for Human Genetics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106-4955, USA *Author for correspondence (e-mail: [email protected])

Accepted 5 April 2001 Journal of Cell Science 114, 2417-2426 © The Company of Biologists Ltd

SUMMARY

Errors during the first meiotic division are common in our chromosome at prophase to determine if this factor species, but virtually all occur during female meiosis. The influenced the propensity of the chromosome for self- reason why oogenesis is more error prone than synapsis. We were unable to directly correlate synaptic spermatogenesis remains unknown. Normal segregation of differences with subsequent segregation behavior. homologous chromosomes at the first meiotic division (MI) However, unexpectedly, we uncovered a sexual dimorphism requires coordinated behavior of the sister chromatids that may partially explain sex-specific differences in the of each homolog. Failure of sister kinetochores to act fidelity of meiotic chromosome segregation. Specifically, in cooperatively at MI, or precocious sister chromatid the male remnants of the synaptonemal complex remain segregation (PSCS), has been postulated to be a major associated with the centromeres until anaphase of the contributor to human nondisjunction. To investigate the second meiotic division (MII), whereas in the female, all factors that influence PSCS we utilized the XO mouse, since traces of synaptonemal complex (SC) protein components the chromatids of the single X chromosome frequently are lost from the chromosomes before the onset of the first segregate at MI, and the propensity for PSCS is influenced meiotic division. This finding suggests a sex-specific by genetic background. Our studies demonstrate that the difference in the components used to correctly segregate strain-specific differences in PSCS are due to the actions chromosomes during meiosis, and may provide a reason for of an autosomal trans-acting factor or factors. Since the high error frequency during female meiosis. components of the synaptonemal complex are thought to play a role in centromere cohesion and kinetochore Key words: Meiosis, Nondisjunction, Sister chromatid cohesion, orientation, we evaluated the behavior of the X Synaptonemal complex, XO mouse

INTRODUCTION form attachments to the same rather than opposing spindle poles. The second meiotic division (MII) is similar to a mitotic Proper chromosome segregation during mitotic cell division cell division, involving the segregation of sister chromatids. requires at least two chromosome-associated protein However, because MI and MII occur without an intervening S complexes. At the centromere, functional kinetochores must be phase, successful chromosome segregation at MII requires formed on individual sister chromatids to facilitate the a mechanism whereby cohesion is released along the attachment of the chromatids to opposite spindle poles. In chromosome arms at anaphase I but maintained between sister addition, cohesion must be established between the centromeres until anaphase II. centromeres and along the arms of the sister chromatids and It is commonly assumed that the specialized meiotic maintained until anaphase to prevent their precocious cohesion requirements depend upon the unique events of separation. meiotic prophase, e.g. synapsis and recombination. The role of By comparison with mitotic cell division, the segregation recombination in the disjunction of homologous chromosomes behavior of chromosomes during meiotic cell division is at MI is well established (e.g. Carpenter, 1994; reviewed in complex, necessitating modification of both the kinetochore Hassold et al., 2000; Hawley, 1988; Koehler et al., 1996; Lamb and cohesion complexes. Our understanding of these meiotic et al., 1996). The role of synapsis remains less well modifications remains rudimentary. The first meiotic division characterized, but the inferential evidence is compelling: both (MI) involves the segregation of homologs rather than defects in homolog synapsis and the absence of a homologous sister chromatids (Fig. 1A). Thus, successful chromosome partner are associated with an increased frequency of segregation at MI requires specialized cohesion mechanisms premature separation of sister chromatids at MI in a variety of that provide physical connections between homologs (rather species (reviewed in Moore and Orr-Weaver, 1998; Wolf, than sister chromatids), and impose constraint on the 1993). Moreover studies in corn, yeast and mammals provide centromeres of sister chromatids so that their kinetochores evidence for the involvement of components of the 2418 JOURNAL OF CELL SCIENCE 114 (13) synaptonemal complex (SC), the proteinaceous structure influence the behavior of sister kinetochores at the first meiotic involved in homolog synapsis, in MI segregation. In maize, the division. We report here the results of detailed meiotic studies analysis of a variety of mutants and structural rearrangements that exclude X-chromosome specific differences and suggest provides compelling evidence for a relationship between the that segregation is influenced by the action of an autosomal gene SC and sister chromatid cohesion and suggests a direct or genes. We hypothesized that this trans-acting factor(s) would correlation between synaptic failure in the centromeric region influence the synaptic behavior of the X chromosome, and that and precocious sister chromatid segregation (PSCS) (reviewed failure to undergo self-synapsis involving the centromere of the in Maguire, 1995). In yeast, mutations in the meiosis-specific chromosome would result in the premature separation of X component of the cohesion complex, Rec8, show an increase chromatids at MI. Our observations did not fit this expectation, in PSCS at MI (Watanabe and Nurse, 1999). Finally, in but our studies provide new insight to the complexity of the mammals, two pieces of data implicate the involvement of SC synaptic process and suggest that inferences about subsequent proteins in meiotic sister chromatid cohesion. First, remnants meiotic events based on pachytene analysis may be misleading. of the lateral element persist at the centromere until anaphase More importantly, however, our efforts to correlate segregation II, as revealed by immunolocalization studies using antibodies behavior with the retention of synaptonemal complex proteins to two protein components, SCP3 and SCP2 (Dobson et al., revealed a surprising difference in centromere-associated 1994; Offenberg et al., 1998). Second, studies of the recently proteins between oogenesis and spermatogenesis. Differences identified cohesin components, SMC1 and SMC3, provide in the protein components of the meiotic chromosome cohesion evidence of interactions with SCP3 and SCP2 (Eijpe et al., complex may influence the fidelity of meiotic chromosome 2000). Thus, there is considerable indirect evidence for the segregation, thus this intriguing sexual dimorphism may involvement of SC proteins in the unique behavior of sister provide a partial explanation for the high chromosome error rate kinetochores at MI, in the maintenance of connections between during human female meiosis. homologs, and in the maintenance of cohesion between sister centromeres until anaphase II. However, the mechanistic details remain unclear. MATERIALS AND METHODS These processes are not only of academic interest to students of meiotic cell division but rather have a dramatic impact on Production of XO female mice human life: the meiotic divisions in the human female are XO female mice and XX sibling controls were produced on two extraordinarily error prone. Estimates of the error frequency different inbred strain backgrounds using previously described mating range from one in twenty to one in three human oocytes, schemes involving males prone to meiotic sex chromosome depending upon the age of the woman (reviewed in Hassold et nondisjunction. Specifically, C57BL/6 females were mated to al., 1996). Despite intensive investigation, the reason for the C57BL/6 males carrying the Y* chromosome (Eicher et al., 1982) and high error rate in our species remains unknown. It is clear, C3H females were mated to C3H males carrying the X-linked Paf however, that the majority of errors occur during the first mutation (Lane and Davisson, 1990). Both crosses produce meiotic division, that the propensity for segregation errors is approximately 20% XO females. chromosome-specific, and that the fidelity of chromosome Intercrosses of the two inbred strains were made to generate F1 hybrid females that differed only in the origin of their X chromosome: segregation is influenced by the number and placement of C57BL/6 females were mated to C3H males carrying the Paf mutation recombination events along the chromosome arms (reviewed to produce XO females with a C57BL/6 X chromosome and C3H in Hassold et al., 2000). It has been postulated that PSCS is a females were mated to C57BL/6 males carrying the Y* mutation to major mechanism responsible for the age-related increase in produce XO females with a C3H X chromosome. segregation errors in our species (Angell, 1997; Angell et al., 1994; Wolstenholme and Angell, 2000), and the small amount Oocyte collection, culture and fixation of data available from direct studies of human oocytes are To assess the segregation of the X chromosome at the first meiotic consistent with this suggestion (Angell, 1997; Angell et al., division, oocytes arrested at metaphase of MII were obtained as 1994; Mahmood et al., 2000; Volarcik et al., 1998). follows: meiotically arrested oocytes at the germinal vesicle stage Studies of human nondisjunction have been limited both by were collected from the ovaries of 3.5-week-old females by piercing the follicles with 26-gauge needles. Oocytes were placed in 10 µl the difficulty of obtaining human oocytes and the lack of a drops of Waymouth’s MB752/1medium (Gibco BRL) supplemented suitable animal model for experimental studies aimed at with 10% fetal bovine serum and 0.23 mM sodium pyruvate, overlaid understanding the age effect in our species. However, the high with mineral oil, and incubated overnight at 37°C in an atmosphere degree of conservation among species in proteins that mediate of 5% CO2 in air. After 14-16 hours in culture oocytes were scored some of the specialized meiotic chromosome behaviors for polar body extrusion, indicating completion of the first meiotic suggests that the mouse may provide valuable mechanistic division and arrest at MII. Oocytes exhibiting polar body formation insight to human aneuploidy. Accordingly, we have utilized the were embedded in a fibrin clot attached to a microscope slide as female XO mouse to study the factors that influence PSCS at previously described (Hunt et al., 1995), fixed in 2% formaldehyde, 1% Triton X-100, 0.1 mM Pipes, 5 mM MgCl2, and 2.5 mM EGTA MI. As in other organisms (reviewed in Wolf, 1993), the sister ° chromatids of the univalent X segregate at anaphase of MI in a for 30 minutes at 37 C, washed in 0.1% normal goat serum (NGS) in PBS for 15 minutes at 37°C, and blocked in 10% NGS containing proportion of cells (Hunt et al., 1995); however, the frequency 0.1% Triton X-100 for 1 hour at 37°C. Fixed oocytes were stored in of PSCS versus ‘intact’ segregation (i.e. in which the sister 10% NGS at 4°C until FISH analysis was performed as described chromatids of the univalent remain attached) is influenced by below. genetic background (LeMaire-Adkins and Hunt, 2000). This difference in MI segregation on two inbred backgrounds Synaptonemal complex studies provides a genetic approach to understanding the factors that To assess meiotic pairing we used combined immunofluorescence Coordinated segregation of sister chromatids 2419 staining (to visualize the SC) and FISH (to identify the X chromosome and the telomeres). Preparations of oocytes at the pachytene stage were made from ovaries from 16-21 days post coitus (d.p.c.) fetuses and newborns according to the method described by Peters et al. (Peters et al., 1997), and stored at −20°C. To visualize both the SC and the centromeres, indirect immunofluorescence staining was performed with combinations of the following antibodies: (1) SCP2 or SCP3, which recognize two different protein components of the lateral element of the SC, (2) SCP1, which recognizes a protein component of the central element of the SC and (3) CREST serum, which recognizes centromere-associated proteins. For immunostaining, slides were washed in 1% donkey serum in PBS for 1 hour at room temperature, incubated with primary antibody diluted in 1% donkey serum for 2 hours at 37°C, and washed in 1% donkey serum for 1 hour at room temperature. The primary antibodies were detected with an FITC-conjugated donkey anti-goat IgG (Jackson Immunoresearch) for SCP3, Rhodamine-conjugated donkey anti- rabbit IgG (Jackson Immunoresearch) for SCP1 and SCP2, and Rhodamine-conjugated donkey anti-human IgG (Jackson Immunoresearch) for CREST in PBS for 1 hour at 37°C and washed in 1% donkey serum for 1 hour at room temperature. Slides were stored at 4°C in 1% donkey serum until FISH was performed as described below. Fluorescence in situ hybridization (FISH) To evaluate X chromosome segregation at the first meiotic division, Fig. 1. Segregation of X chromosome homologs and univalents at MI. MII arrested cells were hybridized with the X-linked probe, Left: Cartoons depict MI segregation of (A) X homologs to the oocyte DXWas70, which recognizes repetitive sequences near the centromere and polar body in control XX females, (B) intact segregation and (C) of the mouse X chromosome. The slides were washed in 2× SSC for precocious segregation of sister chromatids (PSCS) of the univalent X 5 minutes at room temperature, blotted and covered with 30 µl of chromosome in oocytes from XO females (Note that the termed Hybrisol VII (Oncor) containing 30 ng of digoxigenin-labeled ‘precocious’ refers to the fact that sister segregation occurs at MI DXWas70 probe. A coverslip was applied and sealed with rubber rather than MII; a physical association is maintained between the cement, the slides were denatured at 85°C for 10 minutes, and sister chromatids until anaphase I). Right: Actual images of oocyte hybridized overnight at 37°C in a humid chamber. Following and polar body chromosomes in MII arrested oocytes from XX and hybridization, slides were washed in 50% formamide/2× SSC at 37°C XO females, hybridized with an X-chromosome specific probe. In all for 10 minutes followed by a wash of 2× SSC at 37°C for 5 minutes. images, the group of chromosomes (red) segregated to the polar body Hybridized slides were washed in PN buffer for 2 minutes, blocked at MI is on the right and the group remaining in the oocyte is on the in PN buffer containing 5% non-fat dry milk and 0.02% sodium azide left. Each FISH signal (yellow) represents a single X chromatid. for 5 minutes at room temperature, detected with an antidigoxigenin- (A) Normal MI segregation of homologs results in two X signals (or conjugated fluorochrome for 1 hour at 37°C, and washed for 1 hour one doublet) in both the oocyte and polar body; (B) intact segregation in PN buffer. Prior to analysis oocytes were counterstained with 100 of the univalent X is evident either as two signals in the egg or in the ng/ml propidium iodide and mounted with 50% glycerol/PBS polar body (not shown); (C) PSCS of the univalent X chromosome is containing 0.1 µg/ml ρ-phenylenediamine and a coverslip. Oocytes evident as a single signal in both the oocyte and the polar body. were visualized on a BioRad MRC600 confocal system. Hybridization of pachytene preparations was essentially the same as MII arrested oocytes with the following exceptions: (1) in addition were denatured for only 5 minutes and (3) an avidin-conjugated to the DXWas70 probe, 10 µl of a biotin-labeled human pan-telomeric fluorochrome was added at the detection step to detect the telomere probe that recognizes mouse telomeres was applied, (2) slides probe.

Table 1. X chromosome segregation at MI in oocytes from XO females* Type of segregation n Intact PSCS Significance Inbred strains C57BL/6 369 286 (76%) 83 (24%) χ2=85.06, P<0.001 C3H 427 194 (45%) 233 (55%)

F1 hybrids XB6O 209 102 (49%) 107 (51%) χ2=0.63, P>0.1 XC3HO 232 122 (53%) 110 (47%)

*For inbred strains, data from the present analysis (representing 47 and 205 oocytes from XO females produced on the C57BL/6 and C3H backgrounds, respectively) were combined with previously reported data (LeMaire-Adkins and Hunt, 2000). All F1 hybrid data comes from the present study. n, number of oocytes studied. 2420 JOURNAL OF CELL SCIENCE 114 (13)

Fig. 2. Analysis of X chromosome synapsis in pachytene stage oocytes. (A) Pachytene stage oocyte from an XO female mouse immunostained with an antibody to SCP3 (green) to visualize the lateral elements of the SC, and hybridized with X-chromosome specific (purple) and telomere-specific (red) probes. The dispersed X chromosome signal (arrow) is due to the nature of the probe (DXWAS70 detects a multicopy sequence close to the centromere of the X chromosome) and the decondensed state of the chromatin at this stage. (B-F) Synaptic configurations of the univalent X chromosome during pachytene, (B) ‘asynapsed’ X chromosome showing a lateral element with telomeres present at both ends, (C) ‘fully self-synapsed’ X chromosome forming a hairpin structure with overlapping telomeres that appear as a single signal, (D) ‘partial self-synapsis including the centromere’ showing a portion of the lateral element synapsed with both telomeres overlapping and appearing as one, (E) ‘partial self-synapsis excluding the centromere’ showing a portion of the lateral element synapsed with two telomeres distinguishable, (F) ‘X/autosome association’ showing a triradial structure with three telomeres representing the X and an autosomal bivalent.

Chromosome preparations of spermatocytes and oocytes difference in X chromosome segregation between the two at diakinesis/MI inbred strains (χ2=85.06, P<0.001), with the incidence of Chromosome preparations of spermatocytes at diakinesis/MI were PSCS significantly higher on the C3H inbred background prepared from testes from 6-week-old males as previously described (Table 1). (Peters et al., 1997). To obtain comparable preparations in the female, oocytes from 3.5-week-old females were collected and cultured as Differences in X chromosome segregation do not described above. After 4 hours in culture the zona pellucida was reflect X-chromosome specific differences removed by brief incubation in 2% pronase (Calbiochem) in Waymouth’s MB752/1. Zona-free oocytes were placed on a slide To determine if the observed segregation differences were due dipped in 1% paraformaldehyde (pH to 9.2) and dried in a humid to X-chromosome specific differences, we crossed the two chamber overnight. inbred strains to generate F1 hybrid females that were identical genetically, except that one type of female carried a single X chromosome of C57BL/6 origin (XB6O females) and the other RESULTS an X of C3H (XC3HO females) origin. X chromosome segregation in the two types of F1 females was virtually At MI, the single X chromosome in XO females can either identical (Table 1) and resembled one of the progenitor strains. segregate intact to one spindle pole or undergo PSCS at That is, segregation in both F1 females was similar to that anaphase I, segregating one chromatid to each pole (Fig. 1 and observed for the C3H parental strain, but significantly different B6 Hunt et al., 1995). However, previous studies in our laboratory from the C57BL/6 strain (e.g. X O F1 females versus suggested that the segregation frequencies were markedly C57BL/6 females, χ2=49.82, P<0.001). The altered different in XO females produced on C57BL/6 and C3H inbred segregation pattern of the C57BL/6-derived X chromosome on strain backgrounds (LeMaire-Adkins and Hunt, 2000). To an F1 background excludes an X-chromosome specific effect, confirm this observation, we conducted a new analysis. The and demonstrates that genetic differences in segregation of the results were not significantly different from the original study, univalent X chromosome are mediated by an autosomal gene and the combined data demonstrate a highly significant or genes.

Fig. 3. Colocalization of SCP1 and SCP3 on the univalent X chromosome. (A-C) Combined SCP3 and SCP1 staining (green and red respectively, with yellow colocalization) and FISH with X-chromosome specific (purple) and telomeric (purple) probes. (D-F) SCP1 staining alone. (A,D) ‘Fully self-synapsed’ X chromosome showing colocalization of SCP3 and SCP1 (yellow) along the length of the SC. (B,E) ‘Asynapsed’ X chromosome showing no evidence of SCP1 staining. (C,F) ‘Asynapsed’ X chromosome showing SCP1 staining along the entire length of the SC. Note: the magnification is the same in all images; however, SC length is dependent upon the stage of the cell (e.g. B,E represent early pachytene) and the synaptic behavior of the X (e.g. the fully self-synapsed X, as in A,D, is half the length of the asynapsed X, as in C,F). Coordinated segregation of sister chromatids 2421

Frequency of oocytes with asynapsed and distinguish the centromeric and telomeric ends of the chromosomes chromosome. Fig. 2 shows an example of this methodology and of the different types of X chromosome synaptic 100% configurations observed. These configurations are similar to those reported in previous EM studies of oocytes from XO 90% female mice (Speed, 1986). 80% To compare synapsis on the two inbred backgrounds, configurations were categorized as detailed in Fig. 2. Data from 70% the study of 678 pachytene cells from 17-20 d.p.c. C57BL/6 60% Swiss Albino fetuses and 623 cells from 17-19 d.p.c. C3H fetuses are shown C57Bl/6 50% in Table 2. In addition, a total of 361 control oocytes from XX C3H females produced on either the C57BL/6 or C3H strain were 40% scored, and no synaptic defects involving the X bivalent were

percent of oocytes 30% observed (data not shown). Inspection of the data from the two

20% types of XO females revealed no striking difference in the synaptic behavior of the univalent X chromosome on the two 10% genetic backgrounds (Table 2). However, to specifically assess 0% self-synapsis involving the centromeric region of the 1 6 1 7 1 8 1 9 2 0 chromosome (e.g. to test the hypothesis that PSCS is a Days post coitus consequence of failure of the X to undergo self-synapsis involving the centromeric region of the chromosome), we Fig. 4. Percentage of pachytene oocytes with an asynapsed X compared the proportion of cells in which synapsis included chromosome on successive days of gestation. The two strains used in the centromere (e.g. ‘fully self-synapsed’ and ‘partially self- this study, C57Bl/6 (squares) and C3H (triangles), exhibit an initial decline in such cells followed by a slight increase while the Swiss synapsed including the centromere’; Fig. 2C,D). Contrary to Albino strain (diamonds) (used by Speed et al., 1986), exhibits a expectation, the background showing the highest incidence of steady decline. centromeric self-synapsis (C3H) was also the background with the highest incidence of PSCS. Thus, the hypothesis that nonhomologous synapsis involving the centromeric region of Does synaptonemal complex formation during the X chromosome in some way prevents PSCS at MI was not prophase influence segregation? supported by the synaptic profiles of the two different inbred Coordinated behavior of the centromeres of sister chromatids strains. at MI (Fig. 1A) is thought to result from events unique to Because SCP3 only recognizes one component of the SC meiotic prophase (Maguire, 1995). Thus, we hypothesized that (the lateral element), we could not conclude that all cases strain-specific segregation differences might reflect differences scored as self-synapsed actually involved the formation of a in the synaptic behavior of the X chromosome that influence fully mature tripartite SC. To examine this further, we repeated the deposition or retention of cohesion proteins between sister the analysis using the SCP3 antibody in combination with centromeres. SCP1, an antibody that recognizes a component of the central The absence of a homolog prevents the univalent X from undergoing normal synapsis during meiotic prophase but, in the mouse, the single X chromosome has been reported to exhibit frequent non-homologous self- synapsis (Speed, 1986). To determine if differences in synaptic behavior were correlated with subsequent segregation events, we compared X chromosome synaptic configurations on the two inbred backgrounds. Pachytene cells were examined by combining immunofluorescence to visualize the SC and two-color FISH to identify the X chromosome

Fig. 5. SCP2/ SCP3 localization at diakinesis/metaphase I in spermatocytes and oocytes. (A,C) Chromosomes (blue) immunostained with CREST antiserum, which localizes to the centromeres (green). (B,D) Chromosomes (blue) and SCP3 localization (red). (A,B) Two spermatocytes at diakinesis/MI exhibiting SCP3 localization to all centromeres. (C,D) A comparably fixed oocyte showing CREST staining but no SCP3 localization. (Note: the localization pattern of SCP2 is identical.) 2422 JOURNAL OF CELL SCIENCE 114 (13)

Fig. 6. SCP3 localization during the transition from pachytene to dictyate arrest. (A,D,G) CREST localization at the centromeres (red), (B,E,H) SCP3 (green) and (C,F,I) merged images. (A- C) Pachytene stage oocyte with SCP3 localization along the length of the SC. (D-F) Diplotene/early dictyate oocyte showing clustering of centromeres in regions of the nucleus and SCP3 localization at most but not all centromeres. (G-I) Late diplotene/dictyate cell exhibiting clustering of centromeres but no SCP3 staining above background levels. (Note: the temporal localization pattern of SCP2 is similar; see text for details.) element of the fully formed SC. SCP1 staining was evident on all self- synapsed X chromosomes (Fig. 3D). However, unexpectedly, a significant proportion of cells with an asynapsed X chromosome exhibited SCP1 staining along the length of the X chromosome lateral element (Fig. 3F). Is there a pachytene checkpoint difference between backgrounds? It is generally thought that a checkpoint mechanism operates to cull cells with synaptic abnormalities (Odorisio et al., 1998). Perinatal germ cell loss is dramatically cells (e.g. combined tunel assay and immunostaining with increased in XO females by comparison with XX siblings antibodies to SC proteins). Thus, to test the hypothesis that the (Burgoyne and Baker, 1985), suggesting that aberrations in X survival of cells with an asynapsed X differed on the two chromosome synapsis increase germ cell loss at this stage. genetic backgrounds, we compared the proportion of cells with Because self-synapsis of the X chromosome has been an asynapsed X among late pachytene cells. For this analysis, suggested to be necessary for progression beyond pachytene we scored pachytene cells in the perinatal ovary at a time at (Speed, 1986), we reasoned that differences in the stringency which, for both strains, greater than 60% of prophase cells had of the checkpoint mechanism on the two genetic backgrounds progressed beyond pachytene (e.g. 20 d.p.c. for C57BL/6 and might account for the observed segregation differences. That 19 d.p.c. for C3H). Our expectation was that the accumulation is, the increased incidence of PSCS in the C3H strain might of pachytene cells with an asynapsed X chromosome (e.g. reflect a strain-specific relaxation in cell cycle control that pachytene arrest followed by apoptosis) or their rapid allowed a greater number of cells with an asynapsed X elimination would skew the distribution of synaptic chromosome to bypass the pachytene checkpoint mechanism. configurations. However, although temporal fluctuation in the The rapidity of germ cell loss in the perinatal ovary number of cells with an asynapsed X chromosome was precludes the analysis of synaptic behavior among apoptotic observed on both backgrounds (see Fig. 4, and Discussion

Table 2. Frequency of different X chromosome synaptic configurations among pachytene cells from XO females produced on two different inbred backgrounds Partially self-synapsed Fully including excluding Fetus d.p.c. n Asynapsed self-synapsed centromere centromere X/autosome C57BL/6 17 161 107 (66%) 14 (9%) 7 (4%) 19 (12%) 14 (9%) 18 223 97 (43%) 66 (30%) 23 (10%) 23 (10%) 14 (6%) 19 126 60 (48%) 34 (27%) 8 (6%) 18 (14%) 6 (5%) 20 168 87 (52%) 35 (21%) 21 (13%) 20 (12%) 5 (8%) C3H 17 233 126 (54%) 49 (21%) 10 (4%) 23 (10%) 25 (11%) 18 182 78 (43%) 58 (32%) 7 (4%) 26 (14%) 13 (7%) 19 208 94 (45%) 81 (39%) 14 (7%) 9 (4%) 10 (5%) Coordinated segregation of sister chromatids 2423 below), no obvious stain-specific differences were observed; an chromosome-associated proteins. Because the fixation asynapsed X chromosome was observed in 87/168 (52%) cells methodology is essentially the same as that used for pachytene from C57BL/6 females and 94/208 (45%) of cells from C3H cells, we were confident that any chromosome-associated females (Table 2). Thus, our results provide no evidence SCP3 protein would be visible. To our surprise, we could not for a strain-specific difference in the synaptic checkpoint detect either SCP2 or SCP3 in oocytes from either XO or mechanism. control females, although the same fixation procedure yielded The high incidence of cells with an asynapsed X among late strong centromeric staining in the male (e.g. Fig. 5). Similarly, pachytene cells was unexpected, since these cells should be by western analysis SCP3 was not detectable in samples of 500 strongly selected against by the pachytene checkpoint control diakinesis stage oocytes; further, although a band mechanism. As shown in Fig. 4, both XO females produced on corresponding to SCP3 was detected in ovaries from newborn the C57BL/6 and C3H background as well as the previously females, signal intensity diminished with age, becoming studied XO females produced on the Swiss Albino background undetectable during the second week post partum (data not by Speed (Speed, 1986) show an initial decline in the number shown). of pachytene cells with an asynapsed X chromosome. To determine the timing of the disappearance of SCP2 and However, this decline is followed by a slight increase on both SCP3 from the chromosomes, we analyzed oocytes from 3-, 5- backgrounds in this study (Fig. 4). These data, coupled with and 7-day-old females. This allowed us to assess the the fact that our analysis of SCP1 stained preparations revealed localization of these proteins during the transition from positive SCP1 staining on a proportion of asynapsed X diplotene to the dictyate arrest stage. To specifically assess chromosomes, led us to suspect that the synaptic configuration localization at the centromere, preparations were double- of the X chromosome was changing over time. That is, we labeled with either the SCP2 or SCP3 antibody and CREST could not rule out the possibility that cells that escaped the serum and counterstained with DAPI. As is true at the earlier pachytene checkpoint mechanism via self-synapsis might stages of meiotic prophase, diplotene and dictyate stage subsequently ‘open-out’, appearing asynapsed at late oocytes could be distinguished from somatic cells on the basis pachytene. Indeed, a comparison of SCP1 staining between of size, the dispersed nature of the chromatin and the fact that oocytes at early and late pachytene (e.g. from 17 and 19 d.p.c. all centromeres were replicated. During the course of the fetuses, respectively) on the C3H background revealed a analysis, it became evident that oocytes at the diplotene stage significant increase in the number of apparently asynapsed X show a characteristic congregation of centromeres at several chromosomes exhibiting SCP1 staining among late pachytene distinct locations within the nucleus, which becomes more cells (Table 3; χ2=13.65, P<0.001). However, there was no pronounced as the cells enter dictyate arrest (e.g. Fig. 6D,G). significant difference between backgrounds with respect to A minimum of 50 cells were scored at each developmental such cells (Table 3; χ2=0.28, P>0.5). time point (i.e. 3, 5 and 7 days). A progressive decline in the number of oocytes exhibiting staining was observed for SCP3, Disappearance of lateral element proteins at the with nearly 90% of cells from 3-day-old females but only 16% dictyate stage of cells from 7-day-old females showing discrete SCP3 foci Previous immunolocalization studies in mouse spermatocytes (χ2=70.72, P<0.001). The disappearance of SCP2 appeared to using antibodies directed against the lateral element proteins be even more rapid, as none of the cells from 7-day-old females SCP2 and SCP3 have demonstrated that remnants of the SC exhibited staining. Moreover, as shown in Fig. 6, although the persist at the centromere until anaphase II (Dobson et al., 1994; latest persisting foci of staining tended to be found at or near Offenberg et al., 1998). On the basis of these observations, the centromeres, among dictyate cells (i.e. cells exhibiting tight SCP3 has been postulated to facilitate sister chromatid and clustering of centromeres), there was no evidence that either sister kinetochore cohesion (Dobson et al., 1994). Thus, we protein was retained in the centromeric region. Thus, our reasoned that PSCS might be explained by the premature analysis suggests that, in contrast to spermatogenesis, neither disappearance of one or both of these SC proteins from the X SCP2 or SCP3 is present at the centromere during the first centromere. Accordingly, we conducted immunolocalization meiotic division in females. studies of cells at prometaphase of the first division. Our initial attempts to visualize SCP3 were unsuccessful, and we assumed that this was a technical artifact resulting from the fixation of DISCUSSION intact oocytes. To overcome this difficulty, we devised a fixation protocol for fixing and spreading diakinesis stage Under normal meiotic conditions, where bivalents rather than oocytes onto a microscope slide while maintaining the univalents are segregated at MI, premature separation of sister

Table 3. SCP1 staining on asynapsed X chromosomes SCP1 staining Background Stage (d.p.c.) n Present Absent* Significance C3H Early pachytene (17) 46 5 (11%) 41 (89%) χ2=13.65, P<0.001 Late pachytene (19) 41 19 (46%) 22 (54%) χ2=0.28, P>0.5 C57BL/6 Late pachytene 30 12 (40%) 18 (60%)

*Includes oocytes with no or only very weak SCP1 staining. 2424 JOURNAL OF CELL SCIENCE 114 (13) chromatids results in the production of aneuploid gametes. mutations in the SC components of S. cerevisiae (e.g. red1, Indeed, our interest in the factors that predispose to this type zip1 and hop1), which exhibit a slight increase in PSCS at MI of segregation error is motivated by the fact that PSCS has been (Hollingsworth and Byers, 1989; Smith and Roeder, 1997; postulated to be the major mechanism of age-related Sym and Roeder, 1994), (3) immunolocalization studies in nondisjunction in our species (Angell, 1997; Angell et al., male mammals, which demonstrate that remnants of the lateral 1994; Wolstenholme and Angell, 2000). This aberrant MI element of the SC are retained at the centromere until anaphase segregation behavior requires two events that normally do not II (Dobson et al., 1994; Offenberg et al., 1998) and (4) recent occur until MII: (1) the differentiation of functionally distinct observations suggesting that the SC components SCP2 and kinetochores at the centromeres of sister chromatids and (2) SCP3 interact with the cohesion proteins SMC1 and SMC3 the premature release of cohesion between sister centromeres (Eijpe et al., 2000). at anaphase. The coordinated segregation of sister chromatids We hypothesized that genetic differences in MI segregation during mammalian female meiosis remains poorly understood. behavior of the univalent X chromosome might reflect Hence differences in the propensity for premature segregation differences in the synaptic behavior of the X chromosome that of X chromatids in oocytes from XO females produced on two influence the deposition or retention of cohesion proteins at the different inbred strains provide a genetic tool for understanding centromere. The behavior of the univalent X during prophase the factors that influence sister chromatid behavior at MI. is intriguing; although the X has no homolog, previous studies have demonstrated that the univalent chromosome frequently An autosomal, trans-acting factor influences sister exhibits a fold-back, self-synaptic behavior at pachytene chromatid segregation at MI (Speed, 1986). Indeed, self-synapsis has been suggested to be Based on meiotic studies in lower eukaryotes, it seemed likely essential for germ cell survival, with the high incidence of cells that X chromosome segregation differences would reflect with an asynapsed X providing an explanation for the increased subtle differences between the X chromosomes on the two perinatal germ cell loss observed in the XO mouse (Burgoyne inbred strains. That is, detailed studies of univalent and Baker, 1985; Speed, 1986). chromosomes in the grasshopper suggest that the patterns of Detailed analysis of pachytene stage oocytes revealed no meiotic behavior and segregation of univalent chromosomes striking difference in the self-synaptic behavior of the X reflect chromosome-specific features (Rebollo et al., 1998). chromosome on the two genetic backgrounds. Our expectation Similarly, in yeast, mutants with a single division meiotic was that the strain with the highest incidence of PSCS would phenotype exhibit a mixed segregation pattern, with some have more cells in which self-synapsis excluded the chromosomes exhibiting a proclivity for intact and others for centromeric region of the chromosome. Although we did equational segregation. Experiments in which centromeric observe a small difference between the two genetic sequences were transferred between chromosomes with backgrounds, it was the opposite of our prediction; the C3H different segregation patterns demonstrated that the background, which showed a higher frequency of PSCS, had a segregation phenotype is a property of the centromere slightly increased frequency of cells in which the X (reviewed in Simchen and Hugerat, 1993). chromosome centromeric region was self-synapsed. To test for X-chromosome specific differences, we generated The analysis of X chromosome synapsis is complicated by F1 hybrid females carrying either a single X chromosome the fact that cell selection is occurring, and the assumption derived from the parental C57BL/6 or the C3H inbred strain. that all pachytene configurations are equally likely to survive With the exception of the X chromosome, the two types of F1 is almost certainly not valid. Although the exact timing of the females were genetically identical. Meiotic studies revealed no so called ‘pachytene’ checkpoint control mechanism is not difference in the segregation pattern of the univalent X known, a previous study of XO mice by Speed suggested a chromosome between the F1 females. Thus, contrary to decline in cells with an asynapsed X with advancing expectation, X-chromosome specific differences are not a developmental age (Speed, 1986). Thus, we attempted to plausible explanation for the observed segregation differences. enrich for late pachytene cells to test the hypothesis that We conclude that the actions of an autosomal gene or genes selection against cells with an asynapsed X chromosome influence X chromosome segregation. Further, since the might differ on the two backgrounds, and that enhanced segregation phenotypes of both F1 females and one of the survival of such cells might be associated with an increased parental strains were identical, this trans-acting genetic effect frequency of PSCS. We observed a slight but non-significant appears to be dominant. difference in the frequency of pachytene cells with an asynapsed X chromosome; however, the background with the Synaptic behavior at pachytene is not a reliable higher level of PSCS (C3H) had the lowest frequency of such predictor of segregation cells. Thus, our studies provided no evidence of relaxed cell Based on studies of maize mutants, Maguire postulated that the selection. proteins of the synaptonemal complex confer the specialized The observation by Speed of a decline in cells with an meiotic centromere cohesion requirements necessary for asynapsed X chromosome with advancing developmental age proper chromosome segregation (reviewed in Maguire, 1995). (Speed, 1986), is consistent with selection against this category Support for this hypothesis can be drawn from a variety of of cells. In contrast, we observed an initial decline followed by sources, including (1) mutations with precocious separation of a slight increase at the most advanced developmental ages (Fig. sister chromatids indicative of complete loss of sister 4). Although the difference between Speed’s study and our own chromatid cohesion (e.g. in Drosophila, S. pombe and Sordaria may be due to strain differences, our subsequent studies macrospora), which also exhibit defects in synapsis and/or suggested that the apparent increase we observed reflected a recombination (reviewed in Moore and Orr-Weaver, 1998), (2) change over time in the conformation of the X chromosome. Coordinated segregation of sister chromatids 2425 That is, in immunolocalization studies using the SCP1 have gone unnoticed, as persistence of SCP3 at the centromere antibody to assess formation of mature SC, we observed SCP1 throughout MI is frequently mentioned as though it is a staining on asynapsed X chromosomes in some cells. This may universal feature of mammalian meiosis. The disappearance of simply reflect a pattern of binding similar to that observed on the two lateral element components, SCP2 and SCP3, the unsynapsed portion of the X as occurs during male meiosis coincident with entry into meiotic arrest suggests that, at least (P. Moens, personal communication). However, a comparison in the female, these proteins are not essential components of of early and late pachytene cells demonstrated a significant the cohesion complex that mediates the specialized MI increase in such cells with advancing developmental age. behavior of sister centromeres. This difference between male Hence, we suggest that the presence of SCP1 is a remnant of and female meiosis may have functional consequences, e.g. the self-synapsis, and that the apparent asynapsed configuration is disappearance of these proteins prior to the first division may analogous to the synaptic adjustment described in tandem contribute to the vulnerability of the female meiotic process by duplications (Moses and Poorman, 1981), or the residual SCP1 increasing the likelihood of PSCS. observed on separated cores of autosomal chromosomes at Interestingly, the phenotype of the recently reported SCP3 diplotene (Moens and Spyropoulos, 1995). However, there was knockout mouse suggests that sex-specific differences in the no difference between strains in the frequency of such cells, meiotic role of this protein may not be limited to the meiotic hence it does not provide insight to the genetic background divisions: the SCP3 null mutant male is sterile, exhibiting effect on segregation. synaptic failure and meiotic arrest at the zygotene stage (Yuan et al., 2000). In contrast, the SCP3 null female is apparently Evidence that meiotic centromeric cohesion is fertile. Detailed meiotic studies of the mutant female have yet sexually dimorphic to be conducted. It will not only be interesting to learn how Given the complications of cell selection and the apparent homolog synapsis and recombination proceed in the absence changes in the synaptic configuration of the X over time, of this protein, but also whether chromosome segregation is further studies of meiotic prophase seemed unlikely to provide affected. Thus, the SCP3 protein and other SC components insight to the segregation behavior of the X chromosome. may provide long-sought insight to sex-specific differences in However, previous immunolocalization studies in the male meiotic cell division in mammals. demonstrated persistence of the lateral element proteins SCP2 and SCP3 at the centromere until anaphase II (Dobson et al., We are grateful to T. Hassold and H. F. Willard for helpful 1994; Offenberg et al., 1998). Thus we reasoned that PSCS discussions and comments on the manuscript. In addition, we thank segregation might be explained by the premature T. Ashley, C. Heyting, R. Jessberger and P. Moens for generous gifts disappearance of these proteins from the X chromosome of the synaptonemal complex antibodies used in these studies. This work was supported by National Institutes of Health grant R01 centromere. 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