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Condensin dysfunction is a reproductive isolating barrier in mice

Nature volume 623, pages 347–355 (2023)Cite this article

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Abstract

Reproductive isolation occurs when the genomes of two populations accumulate genetic incompatibilities that prevent interbreeding1,2. Understanding of hybrid incompatibility at the cell biology level is limited, particularly in the case of hybrid female sterility3. Here we find that species divergence in condensin regulation and centromere organization between two mouse species, Mus musculus domesticus and Mus spretus, drives chromosome decondensation and mis-segregation in their F1 hybrid oocytes, reducing female fertility. The decondensation in hybrid oocytes was especially prominent at pericentromeric major satellites, which are highly abundant at M. m. domesticus centromeres4,5,6, leading to species-specific chromosome mis-segregation and egg aneuploidy. Consistent with the condensation defects, a chromosome structure protein complex, condensin II7,8, was reduced on hybrid oocyte chromosomes. We find that the condensin II subunit NCAPG2 was specifically reduced in the nucleus in prophase and that overexpressing NCAPG2 rescued both the decondensation and egg aneuploidy phenotypes. In addition to the overall reduction in condensin II on chromosomes, major satellites further reduced condensin II levels locally, explaining why this region is particularly prone to decondensation. Together, this study provides cell biological insights into hybrid incompatibility in female meiosis and demonstrates that condensin misregulation and pericentromeric satellite expansion can establish a reproductive isolating barrier in mammals.

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Fig. 1: Species-specific chromosome mis-segregation driven by domesticus centromere stretching.
Fig. 2: Condensin II dysfunction in hybrid oocytes causes egg aneuploidy.
Fig. 3: Species divergence in condensin regulation and centromere organization creates a reproductive isolating barrier.
Fig. 4: Major satellites reduce condensin II further through TOP2A.
Fig. 5: Condensin II regulation across the Mus genus.

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Data availability

The datasets analysed during the current study are available at Figshare (https://doi.org/10.25444/nhlbi.23671194). Source data are provided with this paper.

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Acknowledgements

We thank A. E. Kelly, N. M. Rusan and N. Phadnis for comments on the manuscript; M. A. Lampson, B. E. Black and the members of the Akera laboratory for discussions; T. Hirano for the NCAPD3 and NCAPG antibodies; and M. E. Torres-Padilla and Y. Miyanari for the TALE constructs. Schematics in the figures were created using BioRender. This work was funded by Division of Intramural Research at the National Institutes of Health/National Heart, Lung and Blood Institute (1ZIAHL006249 to T.A.).

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  1. Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Warif El Yakoubi & Takashi Akera

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  1. Warif El Yakoubi
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  2. Takashi Akera
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Contributions

Conceptualization: T.A. Methodology: W.E.Y. and T.A. Investigation: W.E.Y. and T.A. Funding acquisition: T.A. Supervision: T.A. Writing—original draft: W.E.Y. Writing—review and editing: W.E.Y. and T.A.

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Correspondence to Takashi Akera.

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Extended data figures and tables

Extended Data Fig. 1 Morphologically distinct chromosomes in hybrid and spretus oocytes.

a, domesticus, hybrid and spretus oocytes were imaged live in anaphase I in the presence of sirDNA to visualize chromosomes. PB, polar body; dashed lines, oocyte cortex. See Fig. 1b for the quantification of the lagging chromosome rate. b,c, The chromosome length (b) and width (c) were quantified. Each dot in the graph represents a single oocyte (b, n = 26, 21 and 21 oocytes for domesticus, hybrid and spretus, respectively; c, n = 31, 25 and 19 oocytes for domesticus, hybrid and spretus, respectively); unpaired t-test (two-sided) was used for statistical analysis; **P = 0.0042, ***P < 0.0007, ****P < 0.0001. d, The graph shows line scans of the DNA intensity across the chromosome arm region to quantify the individualization of sister chromatids. domesticus chromosomes tend to be more individualized, indicated by the dip in the DNA intensity in the middle of the line scan (i.e., inter-sister chromatid space), compared to hybrid and spretus chromosomes. The line scans are averaged over n = 188, 121 and 154 chromosomes for domesticus, hybrid and spretus, respectively. e, hybrid oocytes were analysed for cold-stable microtubules at metaphase I. Enlarged images are optical slices showing individual bivalents with the spretus centromere correctly attached to the spindle (i.e., end-on attachment), whereas the domesticus centromere has lateral/merotelic attachment. We speculate that major satellite stretching would reduce the abundance of the error-correction machinery (i.e., Aurora B kinase and MCAK) localized at the pericentromere, leaving mis-attachments at domesticus centromeres uncorrected. Consistent with this idea, knocking down condensin in mitosis reduces Aurora B kinase at pericentromeres60. Furthermore, we previously reported that domesticus centromeres have less MCAK compared to spretus centromeres51. The uncorrected mis-attachments would eventually cause lagging chromosomes in anaphase as observed in a. The graph shows the percentage of centromeres in each attachment category; each dot represents an independent experiment (n = 63, 62 and 66 bivalents analysed in each experiment); unpaired t-test (two-sided) was used for statistical analysis; NS = 0.7457, **P = 0.0014 (lateral/merotelic), **P = 0.0015 (end-on); red line, mean; scale bar: 5 µm.

Source Data

Extended Data Fig. 2 Reduced condensin II abundance leads to centromere stretching in hybrid oocytes.

a, domesticus, hybrid and spretus oocytes were fixed at metaphase I and stained for NCAPD3, using a different antibody (Bethyl, A300-604A) from Fig. 2b where the NCAPD3 antibody from the Hirano lab was used. NCAPD3 intensities on the chromosome were quantified, showing a similar trend with Fig. 2b (n = 22, 22 and 23 oocytes for domesticus, hybrid and spretus, respectively); each dot in the graph represents a single oocyte; unpaired t-test (two-sided) was used for statistical analysis; ***P = 0.0002, ****P < 0.0001; red line, mean. b, domesticus, hybrid and spretus oocytes were fixed at metaphase I and stained for NCAPD3 and ACA (kinetochores) (dataset from Fig. 2b). NCAPD3 intensities at major satellites (white arrowheads in the images, top graph, n = 16 and 15 oocytes for domesticus and hybrid, respectively) and kinetochores (bottom graph, n = 32, 26 and 21 oocytes for domesticus, hybrid and spretus, respectively) were quantified; each dot in the graph represents a single centromere; unpaired t-test (two-sided) was used for statistical analysis; ****P < 0.0001; red line, mean. NCAPD3 levels at major satellites were reduced in hybrid oocytes compared to domesticus oocytes, consistent with the idea that reduced condensin II at major satellites drives centromere stretching in hybrid oocytes. Kinetochore NCAPD3 intensities were similar among domesticus, hybrid and spretus oocytes, indicating that the kinetochore condensin II pool is regulated differently from the rest of the chromosome, similar to fission yeast condensin59. This analysis suggests that the spretus genetic background reduces the condensin II abundance on the chromosome axis and centromeric satellites but not as much at the kinetochore. c, Hybrid oocytes were fixed at metaphase I and stained for NCAPD3 (dataset from Fig. 2b plus one additional independent experiment). Chromosomal NCAPD3 intensities relative to the number of stretched centromeres per oocyte were plotted in the graph (n = 31 oocytes); white arrowheads, stretched centromeres; red line, a simple linear regression between chromosomal NCAPD3 intensities and the number of stretched centromeres (R2 = 0.2595, P < 0.0034). d, To partially deplete NCAPH2 by the TrimAway method61, hybrid oocytes expressing mCherry-Trim21 with the control IgG or the anti-NCAPH2 antibody microinjection were fixed at prometaphase I and stained for TOP2A (a major satellite marker, Extended Data Fig. 8b). The number of stretched centromeres per oocyte (top graph; n = 13 and 13 cells for + IgG and + anti-NCAPH2 antibody, respectively) and the major satellite length was quantified (bottom graph; n = 277 and 259 centromeres for + IgG and + anti-NCAPH2 antibody, respectively); unpaired t-test (two-sided) was used for statistical analysis; *P = 0.015, ****P < 0.0001. Images are maximum intensity z projections to show all chromosomes or optical slices magnified to show individual chromosomes; red line, mean; scale bars: 5 µm.

Source Data

Extended Data Fig. 3 Localization of condensin I and II in hybrid oocytes and somatic cells.

a, Schematic of the condensin I complex. domesticus, hybrid and spretus oocytes were fixed at metaphase I and stained for NCAPG (condensin I). NCAPG intensities on the chromosome were quantified, showing a similar trend with condensin II (n = 44, 24 and 25 oocytes for domesticus, hybrid and spretus, respectively; each dot in the graph represents a single oocyte; unpaired t-test (two-sided) was used for statistical analysis; ****P < 0.0001; red line, mean. Condensin I levels were variable among hybrid oocytes similar to condensin II (Extended Data Fig. 2c) and more often than not were lower compared to domesticus oocytes (P = 0.0539). b, Chromosomes were spread using bone marrow cells isolated from domesticus and the hybrid and stained for NCAPD3 and NCAPG. The graph shows line scans of the NCAPD3 and NCAPG intensity across the chromosome arm region to quantify the chromosome axis enrichment of condensin II and I, respectively. Condensin II levels were similar between domesticus and the hybrid, whereas condensin I levels were slightly higher in the hybrid. The NCAPD3 line scans were averaged over n = 100 and 100 chromosomes for domesticus and hybrid, respectively, and the NCAPG line scans were averaged over n = 100 and 100 chromosomes for domesticus and hybrid, respectively; error bars, standard deviation; scale bars: 5 µm. The schematic in a and b was created using BioRender.

Source Data

Extended Data Fig. 4 Overexpressing NCAPD3 or NCAPH2 did not rescue centromere stretching.

Hybrid oocytes expressing eGFP-NCAPD3 (a) or NCAPH2-eGFP (b) derived from domesticus or spretus were fixed at prometaphase I and stained for TOP2A (a major satellite marker, see Extended Data Fig. 8b) and eGFP. The length of major satellites was quantified; each dot in the graph represents a single centromere (a, n = 752, 683 and 743 centromeres for control, + spretus eGFP-NCAPD3 and + domesticus eGFP-NCAPD3, respectively; b, n = 464, 294 and 451 centromeres for control, + spretus NCAPH2-eGFP and + domesticus NCAPH2-eGFP, respectively). NCAPH2-eGFP was not able to localize on the chromosome probably because of the competition with endogenous NCAPH229. The enhanced centromere stretching upon overexpressing NCAPH2-eGFP implies that NCAPH2-eGFP sequesters other condensin II subunits in the cytoplasm, reducing the functional condensin II pool. Images are maximum intensity z projections to show all chromosomes or optical slices to show individual chromosomes; red line, mean; scale bars: 5 µm.

Source Data

Extended Data Fig. 5 NCAPG2 amino acid sequences are mostly conserved between domesticus and spretus.

a, The entire amino acid sequences of NCAPG2 from H. sapiens, M. musculus domesticus (musculus), and M. spretus were aligned. Residues diverged between domesticus and spretus were highlighted in yellow. Although there are several residues diverged between domesticus and spretus NCAPG2, these differences are probably not part of the hybrid incompatibility, because both of them showed similar localization pattern and efficiently rescued centromere stretching when overexpressed in hybrid oocytes (Fig. 2c and Extended Data Fig. 7b). NCAPG2 antibodies used in this study (Bioss bs-7721R and Bethyl A300-605A) were raised against human NCAPG2 fragments. Antigen sequences used for the antibody productions were highlighted in light green for the Bioss antibody and light blue for the Bethyl antibody. b,c, Part of the amino acid sequences of human NCAPD3 (b) and NCAPG2 (c) that were used as the antigens (highlighted in grey) to produce the Bethyl A300-604A and A300-605A antibodies were aligned with NCAPD3 and NCAPG2 sequences from M. musculus domesticus, M. spretus and M. spicilegus. A single residue diverged among mouse species were highlighted in yellow. Since the sequences are basically conserved across the mouse species for the region corresponding to the antigen sequences (a-c), it is unlikely that the distinct NCAPG2 and NCAPD3 staining patterns among these mouse species are due to the differential efficiency in antibody recognition (Figs. 2b, 3a, 5b, d and Extended Data Fig. 2a, 6b, d).

Extended Data Fig. 6 Nuclear enrichment of condensin II subunits in late prophase I.

a, domesticus and hybrid oocytes were fixed at prophase I and stained for SMC2 and SMC4. The graph shows the quantification of SMC2 and SMC4 intensities in the nucleus; each dot in the graph represents a single oocyte (SMC2: n = 26 and 21 oocytes for domesticus and hybrid, respectively; SMC4: n = 24 and 27 oocytes for domesticus and hybrid, respectively). b, domesticus, hybrid and spretus oocytes were fixed at prophase I and stained for NCAPG2 (Bethyl A300-605A). The graph shows the quantification of NCAPG2 intensities in the nucleus, which shows a similar trend with Fig. 3a where a different NCAPG2 antibody (Bioss bs-7721R) was used; each dot in the graph represents a single oocyte (n = 27, 19 and 24 oocytes for domesticus, hybrid and spretus, respectively); unpaired t-test (two-sided) was used for statistical analysis; *P = 0.015. c, Western blot of NCAPG2 using thymus tissues and oocyte samples. As a loading control, stain-free gel image was shown to indicate the total protein amount loaded to each lane. Oocyte collection and western blot were repeated four times to quantify the total NCAPG2 protein level in each genotype. Total NCAPG2 protein levels were equivalent among domesticus, spretus and hybrid oocytes. For gel source data, see Supplementary Fig. 1. d, domesticus, musculus, spretus and spicilegus oocytes were fixed at prophase I and stained for NCAPG2. NCAPG2 intensities in the nucleus were quantified; each dot in the graph represents a single oocyte (n = 18, 24, 21 and 33 oocytes for domesticus, musculus, spretus and spicilegus, respectively); unpaired t-test (two-sided) was used for statistical analysis; ****P < 0.0001. Images are optical slices to show the nucleus. e, Chromosome spreads were performed at metaphase I using domesticus, spicilegus and domesticus × spicilegus hybrid oocytes expressing MajSat and counterstained with DAPI. Centromeric MajSat intensities were quantified in domesticus and spicilegus oocytes (n = 488 and 354 centromeres for domesticus and spicilegus, respectively); unpaired t-test (two-sided) was used for statistical analysis; ****P < 0.0001. Centromere MajSat signal ratios in domesticus × spicilegus hybrid oocytes were calculated as the domesticus centromere divided by the spicilegus centromere signal for each bivalent (n = 183 chromosomes); each dot in the graph represents a single bivalent chromosome. The quantification shows that spicilegus centromeres have less major satellites compared to domesticus ones, consistent with a previous study using dot-plot hybridization58. Images are maximum intensity z projections to show all chromosomes or optical slices to show individual chromosomes; red line, mean; scale bars: 5 µm.

Source Data

Extended Data Fig. 7 NCAPG2 nuclear enrichment is critical for timely chromosome condensation in mouse oocytes.

a, Hybrid oocytes expressing spretus NCAPG2-eGFP were fixed at metaphase I and stained for NCAPD3 and eGFP. NCAPD3 intensities on chromosomes were quantified (n = 61 and 48 oocytes for control and spretus NCAPG2-eGFP, respectively); unpaired t-test (two-sided) was used for statistical analysis; *P = 0.0423. b, Hybrid oocytes expressing domesticus NCAPG2-eGFP, spretus NCAPG2-eGFP or spretus NCAPG2-NES-eGFP were fixed at prophase I and stained for eGFP. Nuclear/cytoplasmic ratios of eGFP intensities were quantified; each dot in the graph represents a single oocyte (n = 37, 31, 40 and 29 oocytes for control, domesticus NCAPG2-eGFP, spretus NCAPG2-eGFP and spretus NCAPG2-NES-eGFP, respectively); unpaired t-test (two-sided) was used for statistical analysis; ****P < 0.0001. c, Hybrid oocytes expressing spretus NCAPG2-eGFP or spretus NCAPG2-NES-eGFP were fixed at prophase I and stained for NCAPG2 and eGFP. NCAPG2 intensities in the nucleus were quantified; each dot in the graph represents a single oocyte (n = 16, 25 and 30 oocytes for control, spretus NCAPG2-eGFP and spretus NCAPG2-NES-eGFP, respectively); unpaired t-test (two-sided) was used for statistical analysis; *P = 0.0165, ****P < 0.0001. Specifically, the spretus NCAPG2-eGFP overexpression increased the total NCAPG2 levels in the nucleus. d, Hybrid oocytes expressing spretus NCAPG2-eGFP or spretus NCAPG2-NES-eGFP were fixed either at prometaphase I or metaphase I and stained for eGFP and TOP2A. White arrowheads indicate stretched centromeres. eGFP intensities on chromosomes (prometaphase I: n = 37, 37 and 25 oocytes for control, spretus NCAPG2-eGFP and spretus NCAPG2-NES-eGFP, respectively; metaphase I: n = 61, 48 and 43 oocytes for control, spretus NCAPG2-eGFP and spretus NCAPG2-NES-eGFP, respectively; each dot represents a single oocyte) and the major satellite length in prometaphase I were quantified (n = 488, 549 and 368 chromosomes for control, spretus NCAPG2-eGFP and spretus NCAPG2-NES-eGFP, respectively; each dot represents a single centromere); unpaired t-test (two-sided) was used for statistical analysis; ****P < 0.0001. Images are maximum intensity z projections to show all chromosomes or optical slices to show individual chromosomes; red line, mean; scale bars: 5 µm.

Source Data

Extended Data Fig. 8 High TOP2A and low condensin II abundance at major satellites.

a, domesticus oocytes expressing MajSat were fixed at metaphase I and stained for NCAPD3 (antibody from the Hirano lab). The graph is line scans of MajSat (green), centromere (magenta), and NCAPD3 (black) intensities across the chromosome length. Major satellites had reduced levels of condensin II compared to the rest of the chromosome in both domesticus and hybrid (Fig. 4a) oocytes, indicating that condensin II has an intrinsic property of loading less on major satellites. This experiment was repeated independently two times with similar results. b, Chromosome spreads were performed at metaphase I using hybrid oocytes expressing MajSat and stained for TOP2A. “D” and “S” indicate domesticus and spretus centromeres, respectively. domesticus centromeres with major satellites highly enrich TOP2A compared to spretus centromeres. This experiment was repeated independently two times with similar results. c, Images taken for Fig. 4b were re-analysed to plot the centromeric TOP2A intensities relative to the length of major satellites on each chromosome (n = 159 chromosomes); red line, a simple linear regression between the centromeric TOP2A intensities and the length of major satellites (R2 = 0.1784, P < 0.001). d, Hybrid oocytes expressing eGFP-TOP2A were fixed at prometaphase I and stained for TOP2A and eGFP. The major satellite length was quantified (n = 543 and 653 centromeres for control and eGFP-TOP2A, respectively); unpaired t-test (two-sided) was used for statistical analysis; ****P < 0.0001. Overexpressing TOP2A enhanced the centromere stretching phenotype rather than rescuing it. Images are maximum intensity z projections to show all chromosomes; red line, mean; scale bars: 5 µm.

Source Data

Extended Data Fig. 9 TOP2A’s catalytic activity and its guiding function to localize to the chromosome axis are critical to reduce condensin II abundance at centromeres.

a, Hybrid oocytes expressing MinSat or MinSat-TOP2A were fixed at prometaphase I and stained for TOP2A. TOP2A levels recruited to spretus centromeres by this targeting strategy was equivalent to that on major satellites. This experiment was repeated independently two times with similar results. b,c, Hybrid oocytes expressing MinSat-TOP2AY804F, MinSat-TOP2A∆CTD or MinSat-TOP2ACTD were fixed either at metaphase I and stained for NCAPD3 (b) or fixed at prometaphase I and counterstained with DAPI (c). These experiments were repeated independently two to four times with similar results. MinSat-TOP2AY804F, MinSat-TOP2A∆CTD and MinSat-TOP2ACTD were less efficient in reducing condensin II abundance and inducing centromere stretching compared to MinSat-TOP2A (wild-type), indicating that both TOP2A’s catalytic activity and its guiding function to localize to the chromosome axis are important to reduce condensin II abundance at centromeres62. “D” and “S” indicate domesticus and spretus centromeres, respectively. Images are maximum intensity z projections to show all chromosomes (left) or optical slices magnified to show individual chromosomes (right). Scale bar: 5 µm. d, Schematic showing two pathways that regulate condensin II levels. Nuclear NCAPG2 levels dictate the condensin II abundance on the overall chromosome, while major satellites locally reduce condensin II abundance via TOP2A. spretus pericentromeres have very little major satellites, maintaining their centromeres compact despite they have lower basal condensin II levels on the chromosome.

Extended Data Fig. 10 Major satellite-specific TOP2A reduction increases condensin II at major satellites and rescues centromere stretching.

a, Hybrid oocytes over-expressing MajSat were fixed at prometaphase I and stained for TOP2A. Centromeric TOP2A levels (top graph; n = 528 and 361 centromeres for control and MajSat o.e., respectively; each dot represents a single centromere), chromosome axis TOP2A levels (middle graph; n = 124 and 133 chromosomes for control and MajSat o.e., respectively; each dot represents a single chromosome), and the number of stretched centromere per oocyte (bottom graph; n = 36 and 27 oocytes for control and MajSat o.e., respectively; each dot represents a single oocyte) were quantified; unpaired t-test (two-sided) was used for statistical analysis; ***P = 0.0004, ****P < 0.0001. b, domesticus oocytes over-expressing MajSat were fixed at metaphase I and stained for NCAPD3. Graphs show major satellite enrichment of NCAPD3, calculated as the signal at major satellite divided by the chromosome arm signal for each half-bivalent (top graph; n = 210 and 262 for control and MajSat o.e., respectively) or individualization of sister chromatids at major satellites based on the DAPI staining (bottom graph; n = 23 and 37 for control and MajSat o.e., respectively); unpaired t-test (two-sided) was used for statistical analysis; ****P < 0.0001. Images are maximum intensity z projection to show all chromosomes or optical slices magnified to show individual chromosomes; red line, mean, scale bar: 5 µm.

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Supplementary Information

Supplementary Fig. 1 and Supplementary Table 1. Supplementary Fig. 1 contains the uncropped western blot and gel images shown in this study, including the western blot of NCAPG2 using thymus and oocyte samples in Extended Data Fig. 6c. The stain-free gel image serves as a loading control. After acquiring the stain-free gel image, proteins were transferred to the PDVF membrane to perform western blotting. Supplementary Table 1 contains a list of the total numbers of events analysed and the numbers of repeats of each experiment.

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El Yakoubi, W., Akera, T. Condensin dysfunction is a reproductive isolating barrier in mice. Nature 623, 347–355 (2023). https://doi.org/10.1038/s41586-023-06700-6

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