Barbara Spyropoulos

RAD51 IMMUNOCYTOLOGY IN RAT AND MOUSE SPERMATOCYTES AND OOCYTES

Abstract.On the assumption that Rad51 protein plays a role in early meiotic chromosomal events, we examine the location and time of appearance of immuno-reactive Rad51 protein in meiotic prophase chromosomes. The Rad51 foci in mouse spermatocytes appear after the emergence of, and attached to, short chromosomal core segments that we visualize with Cor1-specific antibody. These foci increase in number to about 250 per nucleus at the time when core formation is extensive. The numbers are higher in mouse oocytes and lower in rat spermatocytes, possibly correlating with recombination rates in those cases. In the male mouse, foci decrease in number to approximately 100 while chromosome synapsis is in progress. When synapsis is completed, the numbers of autosomal foci decline to near 0 while the X chromosome retains about 15 foci throughout this time. This stage coincides with the appearance of testis-specific histone H1t at mid- to late pachytene. Electron microscopy reveals that at first Rad51 immunogold-labeled 100 nm nodules are associated with single cores, and that they come to lie between the chromosome cores during synapsis. It appears that these nodules may be the homologs of the Rad51-positive early nodules that are well documented in plants. The reciprocal recombination-correlated late nodules appear after the Rad51 foci are no longer detectable. The absence of Rad51 foci in the chromatin loops suggests that in wild-type mice Rad51/DNA filaments are restricted to DNA at the cores/synaptonemal complexes. The expected association of Rad51 protein with Rad52 could not be verified immunocytologically.

Immunocytology of chiasmata and chromosomal disjunction at mouse meiosis

Immunocytological and in situ hybridization evidence supports the hypothesis that at meiosis of chiasmate organisms, chromosomal disjunction and reductional segregation of sister centromeres are integrated with synaptonemal complex functions. The Mr 125,000 synaptic protein, Syn1, present between cores of paired homologous chromosomes during pachytene of meiotic prophase, is lost from synaptonemal complexes coordinately with homolog separation at diplotene. Separation is constrained by exchanges between non-sister chromatids, the chiasmata. We show that the Mr 30,000 chromosomal core protein, Cor1, associated with sister chromatid pairs, remains an axial component of post-pachytene chromosomes until metaphase I. We demonstrate that at this time the chromatin loops are still attached to their cores. A reciprocal exchange event between two homologous non-sister chromatids is therefore immobilized by anchorage of sister chromatids to their respective cores. Cores thus contribute to the sister chromatid cohesiveness required for maintenance of chiasmata and proper chromosomal disjunction. Cor1 protein accumulates in juxtaposition to pairs of sister centromeres during metaphase I. Presumably, independent movement of sister centromeres at anaphase I is restricted by Cor1 anchorage. That reductional separation of sister centromeres is mediated by Cor1, is supported by the dissociation of Cor1 from separating sister centromeres at anaphase II and by its absence from mitotic anaphases.

Initiation and resolution of interhomolog connections: crossover and non-crossover sites along mouse synaptonemal complexes.

Programmed double-strand breaks at prophase of meiosis acquire immunologically detectable RAD51-DMC1 foci or early nodules (ENs) that are associated with developing chromosome core segments; each focus is surrounded by a γH2AX-modified chromosome domain. The 250-300 ENs per nucleus decline in numbers during the development of full-length cores and the remaining foci are relatively evenly distributed along the mature cores (gamma distribution of ν=2.97). The ENs become transformed nodules (TNs) by the acquisition of RPA, BLM, MSH4 and topoisomerases that function in repair and Holliday junction resolution. At the leptotene-zygotene transition, TNs orient to positions between the aligned cores where they initiate structural interhomolog contacts prior to synaptonemal complex (SC) formation, possibly future crossover sites. Subsequently, TNs are associated with SC extension at the synaptic forks. Dephosphorylation of TN-associated histone γH2AX chromatin suggests annealing of single strands or repair of double-strand breaks DSBs at this time. Some 200 TNs per pachytene nucleus are distributed proportional to SC length and are evenly distributed along the SCs (ν=∼4). At this stage, γH2AX-modified chromatin domains are associated with transcriptionally silenced sex chromosomes and autosomal sites. Immunogold electron microscope evidence shows that one or two TNs of the 10-15 TNs per SC acquire MLH1 protein, the hallmark of reciprocal recombination, whereas the TNs that do not acquire MLH1 protein relocate from their positions along the midline of the SCs to the periphery of the SCs. Relocation of TNs may be associated with the conversion of potential crossovers into non-crossovers.

Mutant meiotic chromosome core components in mice can cause apparent sexual dimorphic endpoints at prophase or X–Y defective male-specific sterility

Genetic modifications causing germ cell death during meiotic prophase in the mouse frequently have sexually dimorphic phenotypes where oocytes reach more advanced stages than spermatocytes. To determine to what extent these dimorphisms are due to differences in male versus female meiotic prophase development, we compared meiotic chromosome events in the two sexes in both wild-type and mutant mice. We report the abundance and time course of appearance of structural and recombination-related proteins of fetal oocyte nuclei. Oocytes at successive days post coitus show rapid, synchronous meiotic prophase development compared with the continuous spermatocyte development in adult testis. Consequently, a genetic defect requiring 2–3 days from the onset of prophase to reach arrest registers pachytene as the developmental endpoint in oocytes. Pachytene spermatocytes, on the other hand, which normally accumulate during days 4–10 after the onset of prophase, will be rare, giving the appearance of an earlier endpoint than in oocytes. We conclude that these different logistics create apparent sexually dimorphic endpoints. For more pronounced sexual dimorphisms, we examined meiotic prophase of mice with genetic modifications of meiotic chromosome core components that cause male but not female sterility. The correlations between male sterility and alterations in the organization of the sex chromosome cores and X–Y chromatin may indicate that impaired signals from the XY domain (XY chromosome cores, chromatin, dense body and sex body) may interfere with the progression of the spermatocyte through prophase. Oocytes, in the absence of the X–Y pair, do not suffer such defects.

The synaptonemal complex karyotypes of palearctic hamsters, Phodopus roborovskii satunin and P. sungorus pallas

Synaptonemal complex (SC) karyotyping is used to analyse the relationship of two Palearctic hamsters. Male Phodopus sungorus have 13 SCs plus an X-Y pair while P. roborovskii has 16 SCs and an X-Y pair. P. sungorus has four nucleolar organizers as opposed to two in P. roborovskii. Centromeres of the X and Y are juxtaposed in the pairing region of the latter but not in the former. SC lengths and relative arm ratios are also discussed. Comparisons with karyotypes of other hamster genera indicate large intergeneric karyotypic differences and expectedly much smaller variation within each genus. Data on the two Phodopus species suggest that their presently accepted congeneric status warrants further examination.

Recycling cells in FISH and immunocytology studies

▼We have previously published protocols for combined fluorescent in situ hybridization (FISH) and immunocytology of meiotic chromosomes (Ref.1). Here we highlight two aspects of our procedures that are beneficial to other similar systems: the use of commercially available glass multiwell slides and an effective antifading mounting agent. With multiwell slides, material can be re-stained with different probes and/or antibodies. The 15 µm plastic coating around each well provides uniform support, protects cells from damage during removal of coverslips and precludes the need for wax or nail polish barriers. No sealants are necessary, discoloured mounting medium can be refreshed and the three-dimensional structure of the cell may be preserved for confocal microscopy, if desired. One can determine the most effective combination of experimental parameters by testing concentration gradients, comparing different antibodies, or screening combinations of fluorochromes. Testing requires minimal material − 5−10 µ lp er well− and the slides are less likely to break than coverslips, the alternative approach to minimizing reagent volumes. Multiwell slides may be recycled by washing in detergent, sonicating in a bleach/detergent solution and rinsing in water. The slides should be washed with a commercial glass cleaner (Windex) and rinsed in water just before use to ensure adhesion of tissue. The older and more used the slide, the better the adhesion of new material. An example of sequential staining is shown (Fig. 1). In this meiotic prophase nucleus, where homologous chromosomes form proteinaceous cores (synaptonemal complexes) and undergo pairing and recombination, the centromeres are stained with anti-centromere serum from a CREST (calcinosis, Raynaud’s phenomenon, oesophageal dysmotility, telangiectasia syndrome) patient. Centromeres are visualized yellow−orange with Cy3-conjugated antihuman secondary antibody through a fluorescein isothiocyanate (FITC) filter. The pairing region of the ho