Interplay between synaptonemal complex, homologous recombination, and centromeres during mammalian meiosis

Abstract

The intimate synapsis of homologous chromosome pairs (homologs) by synaptonemal complexes (SCs) is an essential feature of meiosis. In many organisms, synapsis and homologous recombination are interdependent: recombination promotes SC formation and SCs are required for crossing-over. Moreover, several studies indicate that initiation of SC assembly occurs at sites where crossovers will subsequently form. However, recent analyses in budding yeast and fruit fly imply a special role for centromeres in the initiation of SC formation. In addition, in budding yeast, persistent SC-dependent centromere-association facilitates the disjunction of chromosomes that have failed to become connected by crossovers. Here, we examine the interplay between SCs, recombination, and centromeres in a mammal. In mouse spermatocytes, centromeres do not serve as SC initiation sites and are invariably the last regions to synapse. However, centromeres are refractory to de-synapsis during diplonema and remain associated by short SC fragments. Since SC-dependent centromere association is lost before diakinesis, a direct role in homolog segregation seems unlikely. However, post-SC disassembly, we find evidence of inter-centromeric connections that could play a more direct role in promoting homolog biorientation and disjunction. A second class of persistent SC fragments is shown to be crossover-dependent. Super-resolution structured-illumination microscopy (SIM) reveals that these structures initially connect separate homolog axes and progressively diminish as chiasmata form. Thus, DNA crossing-over (which occurs during pachynema) and axis remodeling appear to be temporally distinct aspects of chiasma formation. SIM analysis of the synapsis and crossover-defective mutant Sycp1⁻/⁻ implies that SCs prevent unregulated fusion of homolog axes. We propose that SC fragments retained during diplonema stabilize nascent bivalents and help orchestrate local chromosome reorganization that promotes centromere and chiasma function.

Figure 1. Centromere association and synapsis during meiotic prophase I.

(A) Criteria for assigning “associated” and “synapsed” centromeres. Magnified images from nuclei stained with CREST (white) and SYCP3 (red) are shown. (B and C) Surface spread pre-leptotene spermatocyte nucleus immunolabeled for RAD21L (green), γH2AX (red), and CREST (white). (D–O) Representative prophase spermatocyte nuclei immunolabled for SYCP3 (green), SYCP1 (red), and CREST (white). To highlight the association status of centromeres, CREST and SYCP1 channels are shown separately. Nuclei stages are as follows: (D and E) leptonema; (F and G) early zygonema; (H and I) late zygonema; (J and K) pachynema; (L and M) diplonema; (N and O) diakinesis/metaphase. (P and Q) Levels of centromere association and centromere synapsis in individual nuclei at the various prophase stages (note that “associated centromeres” includes “synaspsed centromeres”). P values for comparisons of associated and synapsed groups (Mann-Whitney tests) for the various stages are as follows: leptotene, 0.0009; early zygotene, 0.078, mid/late zygotene, 0.52; pachytene, 0.99; diplotene, 0.52. (R and S) Correlations between centromere association and centromere synapsis. For the graph in (R), individual nuclei were ranked according to their level of centromere association. The graph in (S) shows the correlation for zygotene nuclei (R-squared = 0.97). The X intercept of 4.9% confirms the existence of a low level of associated, but not synapsed centromeres. Scale bar = 10 µm.

Figure 2. Localization of synaptonemal complex central element components during diplonema.

Diplotene stage spermatocyte nuclei, immunolabeled for SYCP3 (red), CREST (white) and various SC central-element proteins (green). (M–P) Magnifications of bivalent chromosomes (indicated by arrows in panels I–L), highlighting the localization of SC central element proteins to centromeres and nascent chiasmata. Scale bars = 10 µm for panels A–L; 1 µm for panels M–P.

Figure 3. Centromere association in the absence of crossing-over.

Representative diplotene-stage spermatocytes from wild-type (A and B) and Rnf212^−/− (C and D) mice, immunostained for SYCP3 (green), SYCP1 (red) and CREST (white). Selected homolog pairs are magnified in panels B and D. Note the absence of chiasmata in D, with homologs remaining associated solely via their centromeres. (E) Levels of associated centromeres in diplotene spermatocytes from wild-type and Rnf212^−/− mice. The two distributions are not statistically different (P = 0.35, Mann-Whitney test). Scale bars = 10 µm for panels A and C; 1 µm for B and D.

Figure 4. Axis remodeling revealed by structured illumination microscopy of diplotene-stage spermatocytes.

All panels show chromosomes from diplotene-stage nuclei immunolabled for SYCP3 (green) and SYCP1 (red). (A–C) A representative diplotene-stage nucleus. The arrow highlights the X-Y chromosome pair. The chromosome highlighted by the white box is magnified in (D). Note the two foci of SYCP1-staining SC central-element localized between the SCYP3-staining homolog axes. Terminal accumulation of SYCP3 indicates the position of the centromeres. (E–G) Selected examples of nascent chiasmata showing various patterns of axis fusion and associated SC central element. Panels in E show the SYCP3 and SYCP1 channels merged; F shows the SYCP1 channel only; G shows the SYCP3 channel only. Scale bars = 5 µm for panels A–C; 1 µm for D–G.

Figure 5. Chiasma-like structures and terminal fusions in the absence of synapsis and crossing-over.

(A and B) Representative diplotene-like nuclei from the Sycp1^−/− knock-out immunostained for SYCP3. (C) Gallery of selected examples of axial association sites showing a variety of axis morphology: clearly separated, touching, converging and fused. (D) Fusion of non-centromeric termini into terminal loops. Scale bars = 5 µm for panels A and B; 1 µm for C and D.

Figure 6. Centromere-associated SC fragments during diplonema.

(A–C) A representative diplotene-stage nucleus immunostained for SYCP3 (green) and SYCP1 (red). Arrowheads indicate paddle-like terminal structures with and without axis splitting. The arrow in C highlights the X-Y chromosome pair. (D) Selected examples of associated centromeres highlighting individual SYCP3-staining homolog axes connected by SYCP1-staining SC central element. (E) Selected examples of associated centromeres showing varying degrees of axis splitting. (F) Selected examples of separated centromeres showing retention of SYCP1. (G) Example of an ostensibly achiasmate homolog pair connected solely by SC-associated centromeres. Scale bars = 5 µm for panels A–C; 1 µm for D–G.

Figure 7. Centromere association and morphology of centromere regions in the absence of synapsis.

Spread spermatocyte nuclei from the Sycp1^−/− knock-out immunostained for SYCP3 (red) and CREST (white). (A and B) Pachytene-like nucleus showing extensive coalignment or pseudo-synapsis of homologous chromosomes and a significant fraction of associated centromeres. The chromosome pair highlighted by a white box is magnified in panel (C). Note that the centromeres are associated and the centromeric termini are clearly fused into a terminal loop. (D) Levels of centromere association in pachytene-like Sycp1^−/− nuclei plotted as a function of the level of pseudo-synapsis. (E and F) Representative diplotene-like nucleus in which homologs remain connected at one or more sites but the centromeres are clearly separated. The homologs highlighted by a white box are magnified in panel G. Note the thickened SYCP3 centromeric termini typical of diplotene nuclei. (H) Structured illumination microscopy images of selected centromeric termini from diplotene-like Sycp1^−/− nuclei showing duality, splitting and fracturing (fractured and split axes are highlighted by arrows). Scale bars = 10 µm for panels A, B, E and F; 1 µm for C, G and H.

Figure 8. Identification of post-synapsis inter-centromeric CREST-staining bridges.

(A) Selected image of a late diplotene/early diakinesis spermatocyte from an Rnf212^−/− mouse, immunostained for SYCP3 (red) and CREST (green). The arrow in panel A highlights the chromosomes magnified in panels B and C. Additional examples of CREST-staining bridges are shown in D–G. (H–K) Examples of CREST-staining bridges from wild-type spermatocytes. Scale bars = 10 µm for panel A; 1 µm for B and C; 5 µm for D–K.

Figure 9. Identification of inter-centromeric SYCP3-staining bridges in diakinesis/metaphase I spermatocytes.

(A–C) Selected diakinesis/metaphase-I stage spermatoytes from wild-type and Rnf212^−/− mice, immunostained for SYCP3 (red), SYCP1 (green), and CREST (white). Circles highlight pairs of CREST foci associated with interconnected bi-lobed SYCP3 structures. The white rectangle in A indicates the SYCP3 structure magnified in panel C. Note the absence of SYCP1 staining at this stage. (D–L) Selected SIM images of diakinesis/metaphase-I stage spermatocytes from wild-type mice, immunostained for SYCP3 (red), SYCP1 (white) and CREST (green) (note that only SYCP3 and CREST channels were imaged by SIM; SYCP1 was imaged in the same microscope by conventional epifluorescence to confirm the absence of SYCP1 staining at this stage). Panels G–L show a gallery of selected pairs of CREST structures interconnected by contiguous SYCP3-staining structures. Note the duality of most CREST-staining structures, consistent with the individualization of sister-kinetochores. Scale bars = 10 µm for panels A and B; 1 µm for C; 5 µm for D–F; 1 µm for G–L.