Meiotic cohesin subunits RAD21L and REC8 are positioned at distinct regions between lateral elements and transverse filaments in the synaptonemal complex of mouse spermatocytes

Abstract

Cohesins containing a meiosis-specific α-kleisin subunit, RAD21L or REC8, play roles in diverse aspects of meiotic chromosome dynamics including formation of axial elements (AEs), assembly of the synaptonemal complex (SC), recombination of homologous chromosomes (homologs), and cohesion of sister chromatids. However, the exact functions of individual α-kleisins remain to be elucidated. Here, we examined the localization of RAD21L and REC8 within the SC by super-resolution microscopy, 3D-SIM. We found that both RAD21L and REC8 were localized at the connection sites between lateral elements (LEs) and transverse filaments (TFs) of pachynema with RAD21L locating interior to REC8 sites. RAD21L and REC8 were not symmetrical in terms of synaptic homologs, suggesting that the arrangement of different cohesins is not strictly fixed along all chromosome axes. Intriguingly, some RAD21L signals, but not REC8 signals, were observed between unsynapsed regions of AEs of zygonema as if they formed a bridge between homologs. Furthermore, the signals of recombination intermediates overlapped with those of RAD21L to a greater degree than with those of REC8. These results highlight the different properties of two meiotic α-kleisins, and strongly support the previous proposition that RAD21L is an atypical cohesin that establishes the association between homologs rather than sister chromatids.

Fig. 1.

Comparison of the resolution power between conventional confocal microscopy and 3D-SIM. (A–D) Mouse spermatocytes at the pachytene stage were immunofluorescently labeled with anti-SYCP3 (A, B) or anti-SYCP1 antibodies (C, D). Subsequently, the signals were observed either by conventional confocal microscopy (LSM) (A, C) or by 3D-SIM (B, D). The nuclei in A and B were from different samples. A single optical section is shown. Scale bars: 1 µm. (E–G). The 3D image of the SC in an entire nucleus of a pachytene spermatocyte was visualized using the “Volume Viewer” function of Priism suite. In the merged image, SYCP3 and SYCP1 signals appear in green and purple, respectively. Scale bar: 1 µm. (H) Linearized SC images from three-dimensional z-stacks obtained by 3D-SIM. The top, middle, and bottom panels show SYCP3, SYCP1, and the merged image (SYCP3 in green and SYCP1 in purple), respectively. Scale bar: 0.5 µm. (I) The average spacing of SYCP3 or SYCP1 (n = 98 homologs, * P < 0.01 by t-test). (J, K) In a similar manner as shown in H and I, the experiments were repeated, exchanging the fluorescent dyes that were conjugated to secondary antibodies. In the merged image, SYCP3 and SYCP1 signals appear in purple and green, respectively. (n = 72 homologs, * P < 0.01 by t-test).

Fig. 2.

RAD21L is localized to a more interior position than REC8 in the synaptonemal complex. (A–H) Mouse spermatocytes at the pachytene stage were immunofluorescently labeled with anti-SYCP3 (B and F) and either anti-RAD21L (C) or anti-REC8 (G) antibodies. (A and E) DNA was counterstained with DAPI. (D) SYCP3 (green) and RAD21L (purple) staining in a merged image is shown. Scale bars: 1 μm. (I) Linearized SC images from three-dimensional z-stacks as in Fig. 1H. The top, middle, and bottom panels show signals of SYCP3, RAD21L, and the merged image (SYCP3 in green and RAD21L in purple), respectively. Scale bar: 0.5 μm. (J) The average spacing of SYCP3 or RAD21L (n = 87 homologs, * P < 0.01 by t-test). (K) Linearized SC images from three-dimensional z-stacks as described above. The top, middle, and bottom panels show signals of SYCP3, REC8, and the merged image (SYCP3 in green and REC8 in purple), respectively. Scale bar: 0.5 μm. (L) The average spacing of SYCP3 or REC8 (n = 239 homologs, * P < 0.01). (M–O) Mouse spermatocytes at the pachytene stage were immunofluorescently labeled with anti-RAD21L (M) and anti-REC8 (N) antibodies. (O) The merged image is shown (RAD21L in green and REC8 in purple). RAD21L and REC8 show both asymmetric (white arrow in O) and symmetric localization (yellow arrow in O) between homologous axes. Scale bars: 1 μm.

Fig. 3.

The chromosomal localization of RAD21L and REC8 in zygotene spermatocytes. (A–I) Mouse spermatocytes at the zygotene stage were immunofluorescently labeled with either anti-SYCP3 and anti-RAD21L antibodies (A–C), with anti-SYCP3 and anti-REC8 antibodies (D–F), or with anti-RAD21L and anti-REC8 antibodies (G–I). The merged images are shown (C, F, and I). Also shown at the bottom are magnified images of unsynapsed chromosomes co-labeled with the above antibodies. (C’, F’, I’) RAD21L forms bridges between two linear axes (white arrows). Scale bars: 1 μm for main panels and 0.5 μm for magnified images.

Fig. 4.

RAD21L signals overlap with recombination intermediates to a greater extent than REC8 signals. (A–D) The volume viewers of mouse spermatocytes at the zygotene stage were immunofluorescently labeled with both anti-RAD21L and anti-MSH4 antibodies (A), with both anti-REC8 and anti-MSH4 antibodies (B), with both anti-RAD21L and anti-RAD51 antibodies (C), or with both anti-REC8 and anti-RAD51 antibodies (D). (A–D) White arrows indicate the signals of MSH4 or RAD51 at the synaptic regions of the SC. Scale bars: 1 μm. (E) The ratios of overlapping areas of RAD21L-MSH4 signals and REC8-MSH4 signals relative to MSH4 signals were calculated and compared (n = 60 homologs, * P < 0.01 by t-test). (F) The ratios of overlapping areas of RAD21L-RAD51 signals and REC8-RAD51 signals relative to RAD51 signals were calculated and compared (n = 80 homologs).

Fig. 5.

The precise positioning of meiotic cohesins in the synaptonemal complex and the mechanism of synapsis and recombination during prophase I in mammals.