Altered cohesin gene dosage affects Mammalian meiotic chromosome structure and behavior

Abstract

Based on studies in mice and humans, cohesin loss from chromosomes during the period of protracted meiotic arrest appears to play a major role in chromosome segregation errors during female meiosis. In mice, mutations in meiosis-specific cohesin genes cause meiotic disturbances and infertility. However, the more clinically relevant situation, heterozygosity for mutations in these genes, has not been evaluated. We report here evidence from the mouse that partial loss of gene function for either Smc1b or Rec8 causes perturbations in the formation of the synaptonemal complex (SC) and affects both synapsis and recombination between homologs during meiotic prophase. Importantly, these defects increase the frequency of chromosomally abnormal eggs in the adult female. These findings have important implications for humans: they suggest that women who carry mutations or variants that affect cohesin function have an elevated risk of aneuploid pregnancies and may even be at increased risk of transmitting structural chromosome abnormalities.

Figure 1. Synaptic errors are increased in cohesin heterozygotes.

(A–D) Proportion of pachytene stage cells exhibiting minor and major synaptic defects. SCs were visualized using an antibody against SYCP3 to detect the axial/lateral elements of the synaptonemal complex. For Smc1b, n = 350 for heterozygous and n = 300 for wild-type siblings; for Rec8, n = 450 for heterozygous and n = 250 for wild-type siblings. (A) Proportion of cells (± SE) exhibiting minor defects. (B) Representative images of SCs with minor defects; SC with a fork (top), SC with an internal bubble (bottom). (C) Proportion of cells (± SE) exhibiting major synaptic defects. (D) Representative images of SCs with major defects; partial asynapsis (top), complete asynapsis (bottom).

Figure 2. Recombination levels are reduced in cohesion heterozygotes.

(A) The number of MLH1 foci in pachytene cells from Smc1b heterozygotes was significantly decreased by comparison with wild-type controls (mean MLH1 foci/cell ± SE = 25.0±0.25 and 27.9±0.25, respectively; t = 8.2; p<0.0001). Data represent 126 cells from 5 heterozygous females (grey bars) and 113 cells from 5 wild-type siblings (black bars). (B) A similar reduction was evident in Rec8 heterozygotes (mean MLH1 foci/cell ± SE = 26.6±0.26 and 28.7±0.28, respectively; t = 5.3; p<0.0001). Data represent 181 cells from 7 heterozygotes (grey bars) and 124 cells from 5 sibling controls (black bars). (C, D) SC length was also significantly decreased for both (C) Smc1b (mean ± SE = 186.7±1.18 µm for 126 cells from 5 heterozygotes (grey bars) and 211.7±1.05 µm for 113 cells from 5 controls (black bars); t = 7.2; p<0.0001) and (D) Rec8 heterozygotes (181.1±0.96 µm for 179 cells from 7 heterozygotes (grey bars) and 194.3±1.14 µm for 123 cells from 5 controls (black bars); t = 3.1; p<0.001).

Figure 3. Synaptonemal complex formation is disturbed in cohesin heterozygotes.

(A, B) Representative images of pachytene stage oocytes from control and Rec8 heterozygous females. SCs were visualized using antibodies against SYCP1, to detect the transverse filament of the central element (green), and SYCP3, to detect the axial/lateral elements (red). (A) Pachytene cell from wild-type female; arrow denotes single SC shown in enlarged images below showing SYCP3 signal (left panel), SYCP1 signal (middle panel) and merged signals (right panel). (B) Pachytene cell from a Rec8 heterozygote showing non-uniform SC staining, with red staining at the ends of most SCs; arrow denotes single SC shown in enlarged images below showing SYCP3 (left panel), SYCP1 (middle panel) and merged (right panel) signals.

Figure 4. An Smc1b hypomorphic mutation affects synapsis and recombination.

(A) Proportion of cells (± SE) with apparently normal synapsis, minor, or major defects. Although levels of synaptic defects were similar in wild-type and heterozygous females (+/hy), a marked increase in major defects was evident in homozygotes for the Smc1b hypomorphic allele (hy/hy); this produced a highly significant among-group difference in the frequency of synaptic defects (χ² _{2 df} = 35.7; p<0.0001). (B) Similarly, mean genome-wide MLH1 values ± SE were markedly decreased in homozygotes (20.7.9±0.21) by comparison with controls and heterozygotes (27.3±0.32 and 26.5±0.33, respectively) (F = 118.2; p<0.0001). These data represent 101 cells from 4 hypomorphic females (hashed bars), 100 cells from 5 hypomorphic heterozygotes (grey bars) and 100 cells from 4 wild-type siblings (black bars).

Figure 5. Chromosome abnormalities are increased in cohesin heterozygotes.

Representative images of numerical and structural defects in cohesin heterozygotes. (A) Premature sister chromatid separation in MII eggs; arrows denote the unattached single chromatids. (B) Anaphase bridges between MII eggs and first polar bodies. (C) Chromatid defects in MII eggs; arrows denote abnormal chromatid. (Left) acentric fragment; (middle) deletion of almost an entire chromatid; (right) proximal chromatid break; note that acentric fragment remains attached to telomere of intact sister chromatid. (D) Defects in diakinesis stage oocytes: (left) bivalent with a chromatid break in one chromosome; (middle) nonhomologous end-to-end chromosome fusion that appears to involve both chromatids of two bivalents; (right) proximal break in a single chromatid.

Figure 6. The effect of variable levels of SMC1β-LAP expression during meiosis.