Maternal CENP-C restores centromere symmetry in mammalian zygotes to ensure proper chromosome segregation

Abstract

Across metazoan species, the centromere-specific histone variant CENP-A is essential for accurate chromosome segregation, yet its regulation during the mammalian parental-to-zygote transition is poorly understood. To address this, we generated a CENP-A-mScarlet mouse model that revealed sex-specific dynamics: mature sperm retain 10% of the CENP-A levels present in MII oocytes. However, this difference is resolved in zygotes prior to the first mitosis, using maternally inherited cytoplasmic CENP-A. Notably, the increase in CENP-A at paternal centromeres is independent of sensing CENP-A asymmetry or the presence of maternal chromosomes. Instead, CENP-A equalization relies on the asymmetric recruitment of maternal CENP-C to paternal centromeres. Depletion of maternal CENP-A decreases total CENP-A in both pronuclei without disrupting equalization. In contrast, reducing maternal CENP-C or disruption of its dimerization function impairs CENP-A equalization and chromosome segregation. Therefore, maternal CENP-C acts as a key epigenetic regulator that resets centromeric symmetry at fertilization to preserve genome integrity.

Keywords: CENP-A; CENP-C; MIS18BP1; centromere; epigenetics; intergenerational; mouse; oocyte; sperm; zygote.

Figure 1:. Loss of CENP-A in male germ cells precedes histone-to-protamine exchange.

A) Schematic of the Cenpa-GS-mScarlet-i-V5 transgene (https://biorender.com/ewmqnxf). B) Immunoblot of H3 and kinetochore components in flow sorted spermatogonia (‘gonia), pachytene/diplotene spermatocytes (‘cytes), round spermatids (‘tids), and mature sperm from Cenpa^mScarlet/+ males. Alpha tubulin serves as a loading control. Blots were stripped and re-blotted for the indicated proteins in n=2 mice. C) Schematic overview of germ cell markers used to define germ cell stages: GFRα1 (undifferentiated spermatogonia), SOHLH1 (differentiating A/Intermediate), STRA8 (B spermatogonia/preleptotene), and γH2AX (mid–late pachytene) (https://biorender.com/ella0sc). D) Representative immunofluorescence images of whole-mount Cenpa^mScarlet/+ tubules with various germ cell markers (n=200 cells per cell type; n=3 mice; scale bar: 20 μm). E) Quantification of CENP-A-mScarlet fluorescence across germ cell stages, testicular somatic cells, and intestinal cells (n=200 germ cells and intestinal crypt cells; n=100 for Sertoli and Myoid cells; n=2 mice). Each dot is the sum of CENP-A-mScarlet intensity per cell. F) Immunoblots of CENP-B protein levels in flow sorted germ cells and mature sperm from C57Bl/6J males. Representative image from n=2 replicates from n=2 mice.

Figure 2:. CENP-A asymmetry between oocytes and sperm is conserved across flies, mice, and humans.

A) Immunoblot of CENP-A and CENP-C in n=450 GV or MII oocytes and increasing sperm inputs. Membranes were stripped and re-probed with the indicated antibodies. Representative of two blots using n=12 females and n=2 males. B) Quantification of band intensities from (A), data normalized for ploidy, cellular input, and to GV protein levels. C) Quantification of total CENP-A immunofluorescence in MII oocytes and mature sperm from C57BL/6J mice (n=32 oocytes from n=6 females and n=600 sperm from n=2 males). D) Representative images of CENP-A-mScarlet fluorescence inherited from Cenpa^mScarlet/+ males and females. Data is from n=23 zygotes, n=2 technical replicates. Scale bar: 20 μm, 10 μm in inset. E) Quantification of CENP-A-mScarlet fluorescence in the maternal and paternal pronuclei of zygotes shown in panel (D). F) Quantification of total CENP-A immunofluorescence in human oocytes and sperm; n=5 oocytes and n=49 sperm. Data are from n=3 female donors and n=3 male donors and are normalized for ploidy. G) Representative images of Drosophila gametes with endogenous CID-Dendra2 fluorescence (red) and Hoechst (green); scale bars: oocyte = 10 μm, sperm = 1 μm. H) Quantification of CID-Dendra2 fluorescence from Drosophila gametes in (G) (n=62 oocytes, 94 sperm). ****p < 0.0001.

Figure 3:. Maternal- and paternal-derived CENP-A nucleosomes are inherited intergenerationally.

A) Schematic of zygotic pronuclear (PN) stages. B) CENP-A-mScarlet fluorescence in IVF zygotes from C57Bl/6J females × Cenpa^mScarlet/+ males at indicated PN stages (https://biorender.com/a1ojl2y). Maternal and paternal pronuclei were distinguished by size and position relative to polar body (n=4 males; 4 IVF/immunostaining replicates). Scale bars: 20 μm (main) and 10 μm (insets). C) Maternal CENP-A-mScarlet fluorescence across PN stages (n=12 females; EdU marks S-phase entry). Scale bars: 50 μm (main) and 20 μm (insets). D) Percent of zygotes with maternal CENP-A-mScarlet in one or both pronuclei at each PN stage. E) Percent of EdU positive or negative zygotes at each PN stage. F) Total CENP-A immunofluorescence in maternal vs. paternal pronuclei at PN5 (n = 25 zygotes); each dot represents summed puncta, normalized to maternal pronuclei. G) Comparison of maternally vs. paternally derived CENP-A-mScarlet levels at 8 hours post-fertilization. Only zygotes with maternal CENP-A-mScarlet present in both pronuclei were included. Each dot represents the total CENP-A-mScarlet in a single pronucleus. n=119 for maternally inherited CENP-A-mScarlet in maternal pronucleus, n=103 for maternal CENP-A-mScarlet in the paternal pronucleus embryos, and n=35 for paternal CENP-A-mScarlet in the paternal pronucleus. *: p < 0.05, ****: p < 0.0001.

Figure 4:. CENP-A incorporation in the paternal pronucleus is autonomously regulated.

A) Schematic of androgenetic embryo generation (https://biorender.com/fjua7jj). B) Quantification of total CENP-A immunofluorescence 16 hours after ICSI in control and androgenetic embryos (n=2 ICSI experiments). Each dot represents summed puncta per pronucleus; for fused androgenetic pronuclei, values were halved to normalize per genome. Fluorescence was normalized to the mean control maternal pronucleus (n=24 control maternal, 24 control paternal, and 38 androgenetic genomes). ns, not significant.

Figure 5:. CENP-C and MIS18BP1 are asymmetrically recruited to paternal centromeres.

A) Representative immunofluorescence images of PN0–2 zygotes co-stained for CENP-C and maternal CENP-A-mScarlet (top) or total CENP-A and CENP-B-eGFP (bottom). Images are representative of n=2 IVF experiments using six Cenpa^mScarlet/+ or three CF-1 females and 1–2 B6D2F1/J male. Scale bars: 20 μm. B) Quantification of total CENP-C immunofluorescence or CENP-B-eGFP fluorescence in panel (A). Each dot is the sum of the total puncta in one pronucleus. Values are normalized to the maternal pronucleus (n=34 for CENP-C; n=8 for CENP-B-eGFP). C) Paternal/maternal ratios of CENP-C fluorescence in early (PN0–2, n=44) and late (PN3–5, n=90) zygotes. Each dot represents one zygote. The red dashed line represents a paternal/maternal ratio of 1. D) Representative images of MIS18BP1 localization across pronuclear stages (n=3 IVFs using n=7 females). Scale bars: 20 μm. E) Quantification of MIS18BP1 fluorescence in early (PN 1–3) and late (PN 4–5) zygotes from panel (D). Fluorescence was measured from a central z-slices and normalized to pronuclear area. Shown are paternal/maternal ratios; each dot is one zygote (n_early =10, n_late =10). Red dashed line = ratio of 1. p < 0.05; ns = not significant.

Figure 6:. CENP-A equalization and chromosome segregation rely on maternal, dimerized CENP-C.

A) RT-qPCR confirms efficient CENP-C knockdown in meiotic oocytes. Cenpc transcript levels were quantified from not injected, negative control siRNA (20–40 nM), and Cenpc siRNA (20–40 nM) groups (n= ~20 oocytes per replicate; n=2 biological replicates each with n=3 technical replicates. B) Paternal/maternal CENP-C fluorescence ratios in early zygotes (PN0–2) (n=3 replicates, using 3 CF-1 females and 1 B6D2F1/J each). The red dashed line denotes a ratio of 1. C) Representative PN4/5 zygotes from GV oocytes injected with Cenpc siRNA, control siRNA, or not injected controls stained for CENP-A (n=6 IVFs using n=3–4 CF-1 females and n=1 B6D2F1/J male each). Zygotes with paternal/maternal total CENP-A ratios above 5 were excluded from further analysis. Scale bar: 20 μm, 10 μm in inset. D) Quantification of paternal/maternal CENP-A ratios from (C). Each dot represents one zygote. Data: not injected (n=54), negative control (n=23), and siRNA-treated (n=35) zygotes from n=4 biological replicates. E-F) Scatterplots of maternal (x-axis) vs. paternal (y-axis) CENP-A fluorescence from (D) for not injected (E) and Cenpc siRNA-treated (F) zygotes. Linear regressions were performed with y-intercepts fixed at 0. Each dot = 1 zygote. G) Representative DAPI images of 2-cell embryos from conditions in (C), cultured for 24 hours post-fertilization. Scale bar: 20 μm. H) Proportion of embryos from (G) collected at each PN stage. “Stalled” indicates zygotes that failed to form a proper paternal pronucleus. Percentages were calculated by combining all embryos from all replicates. Data is representative of n=227 not injected, n=43 neg ctrl, and n=76 siRNA embryos, from at least n=3 replicates using n=5 CF-1 females and n=1 B6D2F1/J male each. I) Percentage of 2-cell embryos with at least one chromosome segregation defects from (G). Number of 2-cell embryos: not injected = 191, neg ctrl = 33, siRNA = 62. J) Rescue of CENP-A equalization by co-injection of siRNA-resistant Cenpc^WT RNA but not a dimerization-deficient mutant (Cenpc^Dimer). Paternal/maternal CENP-A ratios: not injected n=29, siRNA n=37, Cenpc^WT n=14, Cenpc^Dimer n=23. Data from n=3 replicates with n=5 CF-1 females and n=1 B6D2F1/J each. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Figure 7:. CENP-C compensates for acute CENP-A loss to preserve faithful chromosome segregation.

A) RT-qPCR quantification of Cenpa transcript levels in not injected, negative control siRNA (100–150 nM), and Cenpa siRNA–injected oocytes (100–150 nM) (n=6 biological replicates, n=10–20 oocytes each, 3 technical replicates). B) Representative PN3–PN5 zygotes (controls vs. Cenpa siRNA) stained for CENP-A (n=5 IVFs, 4–5 CF-1 females and n=1 B6D2F1/J male each). Scale bars: 20 μm. C) Quantification of total CENP-A immunofluorescence in maternal or paternal pronuclei from (B). Fluorescence measurements are normalized to the mean total maternal fluorescence in not injected oocytes. Shown above are the average paternal-to-maternal ratios for each sample. n=41 not injected zygotes, n=21 negative control zygotes, and n=26 siRNA zygotes. D) Quantification of total CENP-C in PN2/3 zygotes from Cenpa siRNA-treated zygotes generated as described for (C) from n=3 IVFs. n=54 not injected, n=23 negative control, and n=20 siRNA zygotes. E) Developmental outcomes at 24 hours post-fertilization for siRNA-treated embryos or embryos from Cenpa^+/– females. “Stalled” indicates zygotes that failed to form a paternal pronucleus. Data from all replicates consists of n=227 not injected, n=81 neg ctrl, and n=85 siRNA injected embryos (n=3 replicates with 5 CF-1 females and n=1 B6D2F1/J male each). Data also includes n=56 WT and n=56 Cenpa^+/− from n=2 replicates with 2–4 females for each genotype and n=2 B6D2F1/J males. Not injected embryos are the same as in Fig. 6H. F) Frequency of chromosome segregation defects in 2-cell embryos from (E): not injected n=191, negative control n=61, siRNA n=75, WT n=55, Cenpa^+/– n=56. G) Model for centromere remodeling and its impact on 2-cell embryo outcomes (https://biorender.com/lopqbuz). *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. ns indicates not significant.