Role of polycomb group protein cbx2/m33 in meiosis onset and maintenance of chromosome stability in the Mammalian germline

Abstract

Polycomb group proteins (PcG) are major epigenetic regulators, essential for establishing heritable expression patterns of developmental control genes. The mouse PcG family member M33/Cbx2 (Chromobox homolog protein 2) is a component of the Polycomb-Repressive Complex 1 (PRC1). Targeted deletion of Cbx2/M33 in mice results in homeotic transformations of the axial skeleton, growth retardation and male-to-female sex reversal. In this study, we tested whether Cbx2 is involved in the control of chromatin remodeling processes during meiosis. Our analysis revealed sex reversal in 28.6% of XY(-/-) embryos, in which a hypoplastic testis and a contralateral ovary were observed in close proximity to the kidney, while the remaining male mutant fetuses exhibited bilateral testicular hypoplasia. Notably, germ cells recovered from Cbx2((XY-/-)) testes on day 18.5 of fetal development exhibited premature meiosis onset with synaptonemal complex formation suggesting a role for Cbx2 in the control of meiotic entry in male germ cells. Mutant females exhibited small ovaries with significant germ cell loss and a high proportion of oocytes with abnormal synapsis and non-homologous interactions at the pachytene stage as well as formation of univalents at diplotene. These defects were associated with failure to resolve DNA double strand breaks marked by persistent γH2AX and Rad51 foci at the late pachytene stage. Importantly, two factors required for meiotic silencing of asynapsed chromatin, ubiquitinated histone H2A (ubH2A) and the chromatin remodeling protein BRCA1, co-localized with fully synapsed chromosome axes in the majority of Cbx2((-/-)) oocytes. These results provide novel evidence that Cbx2 plays a critical and previously unrecognized role in germ cell viability, meiosis onset and homologous chromosome synapsis in the mammalian germline.

Figure 1

Targeted deletion of Cbx2 leads to sex reversal and gonadal hypoplasia. (A) Correlation between genotype, phenotype and chromosomal sex in fetal offspring of heterozygous (Cbx2^(+/−) intercrosses; (B) Gonadal differentiation in a female wild type fetus on day 18.5 pc; (C) Stage-matched Cbx2^(XX−/−) mutant fetus showing ovarian hypoplasia; (D) Anatomic disposition and testicular morphology in a male Cbx2^(XY+/+) control fetus with prominent testis cord development; (E) Sex reversal in a Cbx2^(XY−/−) male containing both a small ovary as well as a contralateral small testis; (F) Wild type fetuses exhibit well-developed testicular cords. In contrast, mutant Cbx2^(XY−/−) fetuses with bilateral testicular hypoplasia as well as intersex embryos show poor definition of tubular structures; (G) Ovarian histology in control ovaries reveals numerous oocytes in meiosis (arrows). In contrast, mutant gonads show reduced meiotic germ cell numbers and hypoplasia.

Figure 2

Precocious meiosis onset in Cbx2^(XY−/−) fetal gonads. (A) Cbx2^(XY−/−) oocyte recovered from an ovary of a unilaterally sex reversed embryo showing completely asynapsed sex chromosomes and persistent γH2AX staining associated with the X chromosome; (B) The position of the X (green, arrow) and the Y (red, arrowhead) chromosome is shown; (C, D) Partial synapsis of the sex chromosomes associated with abundant γH2AX signals (green) at sex chromatin and autosomes in a Cbx2^(XY−/−) oocyte at the late zygotene-early pachytene stage; (E, F) Evidence for precocious onset of meiotic prophase I in fetal testicular germ cells obtained from a Cbx2^(XY−/−) embryo with bilateral testes. Synaptonemal complexes at meiotic chromosome cores are labeled with SYCP3 (red) and γH2AX staining (green). DNA is shown in blue. Scale bar = 10 μm.

Figure 3

Abnormal synapsis and non-homologous chromosome interactions in Cbx2^(XX−/−) mutant oocytes. (A) Chromosome synapsis (arrow) in control oocytes at the pachytene stage showing complete co-localization of lateral (SYCP3, red) and central (SYCP1, green) elements of the synaptonemal complex; (B) Stage-matched Cbx2^(XX−/−) oocyte presenting abnormal synapsis (arrowhead), non-homologous chromosome interactions between two partially synapsed bivalents (arrows) as well as structural damage in the form of prominent chromosome breaks (asterisk). DNA is shown in blue; (C) Proportions of pachytene stage oocytes obtained from control and mutant fetuses on day 18.5 pc showing incomplete chromosome synapsis and non-homologous interactions. Scale bar = 10 μm.

Figure 4

Persistence of H2AX phosphorylation and presence of univalent chromosomes in Cbx2^(XX−/−) oocytes. (A) Late pachytene oocyte obtained from a control Cbx2^(XX+/+) ovary on day 18.5 pc exhibiting complete chromosome synapsis (20 bivalents). Note the absence of γH2AX staining; (B) The position of the X chromosome bivalent (white) is indicated; (C, D) Mutant oocyte exhibiting a chromosome bivalent with axial gaps in the synaptonemal complex decorated by γH2AX staining (green) suggesting the presence of structural chromosome damage (arrow); (E, F) Persistence of H2AX phosphorylation (green) in Cbx2^(XX−/−) oocytes showing failure to resolve DSBs at X chromosome univalents (arrows); (G, H) Cbx2^(XX−/−) oocyte at diplotene stage exhibiting γH2AX staining at asynapsed chromosomes (arrow). Note the presence of X chromosome univalents and non-homologous associations lacking γH2AX foci (arrowhead). Synaptonemal complexes are labeled with SYCP3 (red) and DNA is shown in blue. The position of the X chromosomes is marked with (X); (I) Proportion of mutant oocytes exhibiting abnormal γH2AX patterns. Scale bars = 10 μm.

Figure 5

Rad51 foci associate with asynapsed chromosomes and chromosome segments in Cbx2 mutant oocytes. Complete meiotic chromosome synapsis in control oocytes is associated with the timely resolution of DSBs and lack of Rad51 foci at the pachytene stage (upper panel). In Cbx2^(XX−/−) mutant oocytes non-homologous and incomplete chromosome synapsis leads to persistence of Rad51 foci (green, arrow, thin arrow) on meiotic chromosome axes (lower panel). Moreover, Rad51 foci are also retained at chromosome cores that appear fully synapsed but exhibit axial gaps on the synaptonemal complex (arrowhead). SYCP3 is shown in red and DNA is shown in blue. Scale bar = 10 μm.

Figure 6

Activation of meiotic silencing of unsynapsed chromatin (MSUC) in Cbx2^(XX−/−) oocytes. (A) Compared to oocytes isolated from control ovaries (upper panel), Cbx2^(XX−/−) oocytes show prominent BRCA1 labeling (green) at a subset of synapsed chromosome cores (red, arrow), while the majority of incompletely synapsed bivalents show BRCA1 with a focal distribution (arrowheads); (B) The histone modification ubH2A (red) is not detectable in control oocytes at the pachytene (upper panel) or the diplotene (lower panel) stage of meiosis; (C) In Cbx2^(XX−/−) mutant oocytes, ubH2A associates with the chromosome cores (green) of asynapsed meiotic bivalents at the pachytene stage (arrows, upper panel) and persists at these chromosomal domains until the diplotene stage (arrow, lower panel). DNA is shown in blue. Scale bars = 10 μm.

Figure 7

Crossover formation in mutant Cbx2^(XX−/−) oocytes. Control oocytes (upper panel) as well as oocytes recovered from mutant Cbx2^(XX−/−) fetuses (lower panel) at 18.5 dpc exhibit 1-2 Mlh1 foci (red) per fully synapsed chromosome bivalent (arrows). In contrast, asynapsed meiotic chromosomes in pachytene stage Cbx2^(XX−/−) oocytes lack Mlh1 foci due to failure to establish homologous recombination sites (arrowhead). DNA is shown in blue. Scale bars = 10 μm.

Supplemental Figure S1

Co-localization of ubH2A (red, arrow) with fully synapsed chromosome bivalents (SYCP1, green) in Cbx2^(XX−/−) oocytes at the diplotene stage. Arrowhead points to faintly labeled desynapsed bivalent. Scale bar = 10μm.

Supplemental Figure S2

Attenuation of the MSUC response (BRCA1, green) in Cbx2^(XX−/−) oocytes exhibiting excessive asynapsis (SYCP3, red). Scale bar = 10μm.