Mouse oocytes carrying metacentric Robertsonian chromosomes have fewer crossover sites and higher aneuploidy rates than oocytes carrying acrocentric chromosomes alone

Abstract

Meiotic homologous recombination during fetal development dictates proper chromosome segregation in adult mammalian oocytes. Successful homologous synapsis and recombination during Meiotic Prophase I (MPI) depends on telomere-led chromosome movement along the nuclear envelope. In mice, all chromosomes are acrocentric, while other mammalian species carry a mixture of acrocentric and metacentric chromosomes. Such differences in telomeric structures may explain the exceptionally low aneuploidy rates in mice. Here, we tested whether the presence of metacentric chromosomes carrying Robertsonian translocations (RbT) affects the rate of homologous recombination or aneuploidy. We found a delay in MPI progression in RbT-carrier vs. wild-type (WT) fetal ovaries. Furthermore, resolution of distal telomere clusters, associated with synapsis initiation, was delayed and centromeric telomere clusters persisted until later MPI substages in RbT-carrier oocytes compared to WT oocytes. When chromosomes fully synapsed, higher percentages of RbT-carrier oocytes harbored at least one chromosome pair lacking MLH1 foci, which indicate crossover sites, compared to WT oocytes. Aneuploidy rates in ovulated eggs were also higher in RbT-carrier females than in WT females. In conclusion, the presence of metacentric chromosomes among acrocentric chromosomes in mouse oocytes delays MPI progression and reduces the efficiency of homologous crossover, resulting in a higher frequency of aneuploidy.

Figure 1

MPI progression in the oocytes of RbT-carrier and WT mouse ovaries at 15.5–17.5 dpc. (a) MPI substages identified by SYCP3 immunofluorescence (IF) staining. In Rb5 and RBF oocytes, RbT-centromeres (indicated by arrowheads) were identified by IF-staining of centromeres with CREST antibody (red) without adjacent telomeres with anti-TRF1 antibody (green). C- and D-telomeres were also distinguished by IF-staining of TRF1 with and without adjacent CREST staining, respectively. Scale bar 10 μm. (b) Percentages of oocytes at progressive MPI substages in the ovaries of each genotype. The number of examined oocytes and females (in parentheses) are given on the top of each column. *Significant difference between WT and Rb5 or RBF ovaries at P < 0.05 by χ²-test.

Figure 2

C- and D-telomere clustering and resolution in Rb5, RBF, and WT BALB/c mouse oocytes. (a) Representative images showing four types of telomere clustering in RBF oocytes. Telomere clusters are indicated by white broken line circles. RbT-centromeres are marked with arrowheads. Scale bar 10 μm. (b) Percentages of oocytes having each type of telomere clusters at progressive MPI substages. The total number of examined oocytes is given on the top of each column. Four females were examined in each strain. *Significant difference between RBF and WT or Rb5 oocytes at P < 0.05 by χ²-test.

Figure 3

Initiation of chromosome synapsis in Rb5, RBF, and WT BALB/c oocytes at the early zygotene stage. (a) Rb5 oocyte with six short SYCP1 stretches (1–4 µm). C-telomeres, D-telomeres and interstitial regions are distinguished with IF-staining with anti-TRF1 and CREST antibodies. The merged image is followed by SYCP1, TRF1, and CREST staining alone. The circle with broken line indicates RbT-centromeres. (b) Percentages of short SYCP1 stretches at three chromosomal regions from the oocytes of three genotypes. The number of short stretches per oocyte is given on the X axis. The total number of examined oocytes is given on the top of each column. The results are combined as total at the right end. Four females were examined in each strain. *Significance in RBF oocytes compared to WT oocytes at P < 0.05 by χ²-test.

Figure 4

Crossover sites in Rb5, RBF, and WT BALB/c oocytes. (a) Crossover sites marked with MLH1 IF-staining in oocytes of each genotype. Arrowheads indicate RbT chromosomes. White circle with broken line indicates a chromosome lacking MLH1 foci in an RBF oocyte. (b) Histogram of the number of either acrocentric (blue) or metacentric (red) chromosome arms with 0–4 MLH1 foci in the oocytes of each genotype. (c) Percentage of either acrocentric or metacentric chromosome arms lacking MLH1 foci in each oocyte. The combined results are shown in the “All” column. The total number of examined chromosome arms is given on the top of each column. (d) Percentages of oocytes with at least one chromosome pair lacking MLH1 foci. The total number of examined oocytes and females (in parentheses) are given on the top of each column. *Significant difference at P < 0.05 by χ²-test.

Figure 5

Aneuploidy in MII-oocytes ovulated by RbT-carrier and WT females. (a) The average number of ovulated oocytes by WT, Rb5, and RBF young (3–4 months) or old (6–9 months) females. The total number of examined mice is given on the top of each column. *Significant difference at P < 0.05 by Mann–Whitney test. (b) Representative images of nondisjunction (NDJ) and precocious separation of sister chromatids (PSSC) aneuploidy. White circle with broken line indicates a single chromatid (Cht) as the PSSC type aneuploidy. Arrowhead indicates an RbT chromosome. Scale bar 10 µm. (c) The rate of NDJ and PSSC aneuploidy in the oocytes from young vs. old females as shown in (a). The total number of examined oocytes is given on the top of each column. *Significant difference at P < 0.05 by χ²-test.