Table of Contents

Chromosome errors, or aneuploidy, affect an exceptionally high number of human conceptions, causing pregnancy loss and congenital disorders. Here, we have followed chromosome segregation in human oocytes from females aged 9 to 43 years and report that aneuploidy follows a U-curve. Specific segregation error types show different age dependencies, providing a quantitative explanation for the U-curve. Whole-chromosome nondisjunction events are preferentially associated with increased aneuploidy in young girls, whereas centromeric and more extensive cohesion loss limit fertility as women age. Our findings suggest that chromosomal errors originating in oocytes determine the curve of natural fertility in humans. [Abstract copyright: Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Cytological Studies of Human Meiosis: Sex-Specific Differences in Recombination Originate at, or Prior to, Establishment of Double-Strand Breaks

Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB) formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC) length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin difference

Cytological studies of human meiosis: sex-specific differences in recombination originate at, or prior to, establishment of double-strand breaks

Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB) formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC) length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway

Chromosome-specific MLH1 values in males and females.

<p>Ten representative large, medium and small chromosomes were identified by FISH and chromosome-specific MLH1 values determined. For all ten chromosomes, mean values were lower in males (white) than females (black), and for nine of the ten the differences were statistically significant: chromosome 1 (10 males, n of cells =139; 7 females, n=83; t=14.1; p<0.0001), 6 (5 males, n=174; 3 females, n=30; t=8.2; p<0.0001), 13 (4 males, n=139; 8 females, n=109; t=8.4; p<0.0001), 14 (4 males, n=142; 4 females, n=70; t=3.2; p<0.005), 15 (6 males, n=130; 3 females, n=45; t=3.3; p<0.005), 16 (12 males, n=204; 9 females, n=63; t=8.8; p<0.0001), 18 (2 males, n=187; 7 females, n=100; t=6.9; p<0.0001), 21 (10 males, n=302; 11 females, n=218; t=7.1; p<0.0001), and 22 (10 males, n=313; 11 females, n=161; t=4.4; p<0.0001). The difference did not reach significance for chromosome 9 (5 males, n=58; 2 females, n=34; t=1.9; p=0.064).</p

Chromosome-specific MLH1 localization patterns in males and females.

<p>The chromosomal locations of MLH1 foci were determined using the same cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085075#pone-0085075-g002" target="_blank">Figure 2</a> for ten representative large, medium and small chromosomes. Each chromosome arm was arbitrarily divided into five equal regions – centromeric, proximal, interstitial, distal, and telomeric – and the distribution of MLH1 foci recorded for both chromosome arms for metacentric and sub-metacentric chromosomes or for the q-arm only of acrocentric chromosomes. The distribution differed significantly between females (black) and males (white) for seven of the ten chromosomes: 1 (χ²=24.9; p<0.005), 6 (χ²=24.8; p<0.005), 13 (χ²=13.8; p=0.01), 16 (χ²=32.1; p<0.0001), 18 (χ²=47.7; p<0.0001), 21 (χ²=22.3; p<0.0001), and 22 (χ²=20.8; p<0.0001). However, sex-specific differences were not evident for chromosomes 9 (χ²=15.1; p=0.088), 14 (χ²=5.3; p=0.262), or 15 (χ²=7.8; p=0.101).</p

Comparison of SC length and DNA loop size in males and females.

<p>Male data are in white, female data in black. (A) Chromosome-specific SC lengths were determined for cells scored in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085075#pone-0085075-g002" target="_blank">Figure 2</a> and striking sex-specific differences were evident on all ten chromosomes analyzed: 1 (t=8.9; p<0.0001), 6 (t=23.1; p<0.0001), 9 (t=12.8; p<0.0001), 13 (t=26.7; p<0.0001), 14 (t=30.8; p<0.0001), 15 (t=24.1; p<0.0001), 16 (t=21.7; p<0.0001), 18 (t=28.6; p<0.0001), 21 (t=36.4; p<0.0001), and 22 (t=35.4; p<0.0001). (B, C) Three individual chromosomes (1, 16 and 21) were analyzed for DNA loop size, using the deflection of FISH paint probes from the SC as a surrogate for loop size. For chromosomes 1 and 16, we measured the width of the FISH signal at the centromere and three points on each chromosome arm, and averaged the seven values. For chromosome 21, loop size was taken as the average of three measurements, one at the centromere and two on the long arm. (B) Blow-up image of a portion of a representative pachytene stage oocyte, labeled with DAPI (blue) and a chromosome 1 paint probe (red). White bars represent the seven individual DNA loop measurements, three from each chromosome arm and one at the centromere. The centromere was identified using CREST prior to FISH. (C) DNA loop size means were significantly greater in males for each chromosome; i.e., for chromosome 1 (2 males, n of cells=24; 3 females, n=23; t=15.2; p<0.0001), for 16 (2 males, n=39; 3 females, n=32; t=20.8; p<0.0001) and for 21 (2 males, n=37; 2 females, n=43; t=16.0; p<0.0001).</p

Genome-wide mean MLH1 values in human males and females.

<p>MLH1 foci were used as a marker for meiotic recombination events. (A) shows a representative pachytene oocyte and (B) a representative pachytene spermatocyte, that were immunostained for SYCP3 (in red), a component of the synaptonemal complex; CREST (in blue), detecting centromeric regions; and MLH1 foci (in green), detecting crossovers. (C) In total 4660 spermatocytes from 56 males (white) and 2038 oocytes from 63 females (black) were examined. Mean MLH1 counts (± S.E.) were significantly lower in males than females (49.09 ± 0.07 vs. 69.25 ± 0.29; t=92.5, p<0.0001) and the range was narrower in males than females (30-66 vs. 27-119). (D) Mean number (± S.E.) of MLH1 foci per cell for individual male and female samples, demonstrating the lack of overlap between the sexes, and the increased variation in individual female cases by comparison with males.</p

Spacing between adjacent MLH1 foci in males and females.

<p>Inter-focal distances, calculated as the percent of the length of the synaptonemal complex between adjacent MLH1 foci, were determined using the same cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085075#pone-0085075-g002" target="_blank">Figure 2</a>; male data are depicted in white, female data in black. To obtain sufficient numbers of cells for direct male:female comparisons, we restricted our analysis to chromosomes having the same number of MLH1 foci in males and females; i.e., for chromosome 1 we analyzed cells in which the chromosome exhibited four MLH1 foci and for chromosomes 13, 14, 16, 18 and 22, cells in which the relevant chromosome exhibited two MLH1 foci. Thus, for chromosome 1, we made three measurements of inter-focal distances per cell, while for chromosomes 13, 14, 16, 18 and 22 we made a single measurement of inter-focal distance per cell. For chromosomes 6, 9, 15 and 21 we had a limited number of cells with the same number of MLH1 foci in both sexes; thus, these chromosomes were excluded from the analysis. For each chromosome, inter-focal distances were binned (by % value) into ten groups. The distribution of categories of inter-focal distances was significantly different between males and females for each of the six chromosomes: 1 (χ²=51.7; p<0.0001), 13 (χ²=26.7; p<0.0005), 14 (χ²=30.6; p<0.0001), 16 (χ²=31.9; p<0.0001), 18 (χ²=50.0; p<0.0001), and 22 (χ²=48.8; p<0.0001).</p

Correlations between Synaptic Initiation and Meiotic Recombination: A Study of Humans and Mice

Meiotic recombination is initiated by programmed double strand breaks (DSBs), only a small subset of which are resolved into crossovers (COs). The mechanism determining the location of these COs is not well understood. Studies in plants, fungi, and insects indicate that the same genomic regions are involved in synaptic initiation and COs, suggesting that early homolog alignment is correlated with the eventual resolution of DSBs as COs. It is generally assumed that this relationship extends to mammals, but little effort has been made to test this idea. Accordingly, we conducted an analysis of synaptic initiation sites (SISs) and COs in human and mouse spermatocytes and oocytes. In contrast to our expectation, we observed remarkable sex- and species-specific differences, including pronounced differences between human males and females in both the number and chromosomal location of SISs. Further, the combined data from our studies in mice and humans suggest that the relationship between SISs and COs in mammals is a complex one that is not dictated by the sites of synaptic initiation as reported in other organisms, although it is clearly influenced by them