Long-Term Retention of CENP-A Nucleosomes in Mammalian Oocytes Underpins Transgenerational Inheritance of Centromere Identity

Abstract

Centromeres control genetic inheritance by directing chromosome segregation but are not genetically encoded themselves. Rather, centromeres are defined by nucleosomes containing CENP-A, a histone H3 variant [1]. In cycling somatic cells, centromere identity is maintained by an established cell-cycle-coupled CENP-A chromatin assembly pathway, but how centromeres are inherited through the mammalian female germline is unclear because of the long (months to decades) prophase I arrest. Here we show that mouse oocytes retain the pool of CENP-A nucleosomes assembled before birth, and that this pool is sufficient for centromere function, fertility, and genome transmission to embryos. Indeed, oocytes lack any measurable CENP-A nucleosome assembly through the entire fertile lifespan of the female (>1 year). Thus, the remarkable stability of CENP-A nucleosomes confers transgenerational centromere identity in mammals.

Figure 1. Absence of CENP-A chromatin assembly in full grown oocytes or MII eggs

(A–C) Oocytes were injected with cRNA for CENP-A-GFP and H3.3-mCherry or GFP-CENP-C and H3.3-mCherry, and imaged live in germinal vesicle-intact (GV) or metaphase II (MII) stages as shown in the schematic (A). Representative images show CENP-A-GFP and H3.3-mCherry (B) or GFP-CENP-C and H3.3-mCherry (C) at GV or MII stages (n≥10 in each case). Images are maximal intensity projections of confocal z-series; insets show 1.6x magnification of centromeres labeled with GFP-CENP-C. White circle represents nuclear envelope. (B) 1-cell stage embryos were injected with cRNA for mCherry-CENP-C and CENP-A-GFP and imaged live at the 4-cell stage, as shown in the schematic (D). Representative images show an optical section of 3 blastomeres (n=27); insets show 3x magnification of CENP-A-GFP and mCherry-CENP-C localized at centromeres. Scale bars 5 µm. See also Figure S1.

Figure 2. CENP-A nucleosomes are stably retained at oocyte centromeres for >1 year

(A) Oocytes arrest in prophase I in resting primordial follicles, which are cyclically recruited starting five days after birth to begin growth towards a Graafian follicle, the final stage before ovulation. Primordial follicles can remain in the resting phase for a period lasting >1 year before they are recruited for maturation and ovulation. After fertilization, the maternal and paternal pronuclei enter G1-phase and begin mitotic cell cycles. Cre expression driven by the Gdf9 promoter excises Cenpa in resting primordial follicles two days after birth. (B) Three models for centromere inheritance in the mammalian oocyte. See Results & Discussion for details. (C) Schematic of Cenpa conditional knockout gene locus. The neomycin selection cassette used for selection of ES cells is flanked by FRT sites. Cenpa protein coding exons 2–5 are flanked by loxP sites. (D–F) Oocytes were collected from 11–14.5 month old WT and KO mice, or from young C57BL/6J (BL6) controls, and CENP-A levels were analyzed by immunofluorescence, with ACA to co-label centromeres (schematic, D). Images (E) of oocytes with intact germinal vesicles (GV) are maximal intensity projections of confocal z-series; scale bar 5 µm. Total centromeric CENP-A staining was quantified for each oocyte (n=64 oocytes from 4 WT mice; n=85 oocytes from 5 KO mice) and normalized to young C57BL/6J controls (n=155 oocytes, 8 mice) for each experiment. Normalized values were averaged over multiple experiments (F; error bars, s.d.). (G) The number of centromere clusters was counted in each oocyte and averaged over each group (error bars, s.d.). See also Figures S2, S3, S4, and Table S1.

Figure 3. CENP-A nucleosomes assembled early in prophase I support normal meiotic centromere function

(A–C) Oocytes were collected from 12 month old WT and KO females, or young C57BL/6J controls, matured in vitro to MII, and stained for β-tubulin, ACA to label centromeres, and DNA (schematic, A). Representative images (B) show MII eggs at 20X magnification (scale bar 10 µm), white circle represents zona pellucida; dashed squares are regions imaged at 100x (scale bar 5 µm). The percent in each group that remained arrested with an intact germinal vesicle (GV), arrested at metaphase I, or progressed normally to MII was quantified (C, n=27 oocytes from 3 WT mice, 23 oocytes from 3 KO mice, or 26 oocytes from 2 C57BL/6J (BL6) mice). The table summarizes how many MII eggs from WT and KO females had chromosomes aligned at metaphase.

Figure 4. CENP-A nucleosomes assembled in early prophase I support normal fertility and transgenerational centromere inheritance

(A) WT (n=5) and KO (n=6) females were mated with either C57BL/6J or B6SJLF1/J males to determine fertility. Average litter size (± s.d.), number of litters, and age of the females are reported. (B) Cenpafl/⁺;Ddx4-Cre/+ males (blue box) were mated to Cenpa^fl/fl;Gdf9-Cre/+ females (n=2 females, 3–6 months old). The red box represents offspring that are either predicted to die in utero (all Cenpa^−/−) or are predicted to be impossible to produce if there is full excision of the Cenpa^fl allele. The green box represents animals that are predicted to survive (all Cenpa^−/+) if all oocytes are indeed Cenpa^−/−. Surviving pups were genotyped and found to be exclusively Cenpa^−/+ with no intact Cenpa^fl allele. (C) Breeding scheme to generate F1 pups that are Cenpafl/⁺ or Cenpa^−/+. Green boxes indicate mice used in experiment. (D,E) Oocytes from F1 pups were fixed in metaphase I and stained for CENP-A and DNA. CENP-A intensity was measured at each centromere, and the ratio of brighter/dimmer intensity was calculated (E) for each bivalent (n≥238 bivalents for WT and KO). Error bars s.d. Scale bar 5 µm.