Homeostatic control of recombination is implemented progressively in mouse meiosis

Abstract

Humans suffer from high rates of fetal aneuploidy, often arising from the absence of meiotic crossover recombination between homologous chromosomes. Meiotic recombination is initiated by double-strand breaks (DSBs) generated by the SPO11 transesterase. In yeast and worms, at least one buffering mechanism, crossover homeostasis, maintains crossover numbers despite variation in DSB numbers. We show here that mammals exhibit progressive homeostatic control of recombination. In wild-type mouse spermatocytes, focus numbers for early recombination proteins (RAD51, DMC1) were highly variable from cell to cell, whereas foci of the crossover marker MLH1 showed little variability. Furthermore, mice with greater or fewer copies of the Spo11 gene--with correspondingly greater or fewer numbers of early recombination foci--exhibited relatively invariant crossover numbers. Homeostatic control is enforced during at least two stages, after the formation of early recombination intermediates and later while these intermediates mature towards crossovers. Thus, variability within the mammalian meiotic program is robustly managed by homeostatic mechanisms to control crossover formation, probably to suppress aneuploidy. Meiotic recombination exemplifies how order can be progressively implemented in a self-organizing system despite natural cell-to-cell disparities in the underlying biochemical processes.

Figure 1. Cell-to-cell variability in numbers of recombination intermediates decreases as meiotic prophase progresses

a) Representative spermatocyte chromosome spreads from various meiosis prophase I stages, stained for the indicated proteins. Scale bar, 5 µm. b) Total foci per nucleus in wild-type spermatocytes. Each dot is the count from a single nucleus. Bars, mean ± standard deviation (SD). RAD51 and DMC1 at early zygonema (219.2 ± 69.8 and 185.8 ± 67.8, respectively; mean ± SD) and MSH4 at late zygonema and early pachynema (144.9 ± 30.9) are plotted on the left axis; MLH1 at mid-pachynema (23.6 ± 2.7) is plotted on the right axis (presented with the mean focus count at the same height as for MSH4, to facilitate comparison). Total mice analyzed: RAD51, n = 8; DMC1, n = 5; MSH4, n = 4; MLH1, n = 9. c) Coefficients of variation (CV, black line) with 95% confidence intervals (blue line) estimated by bootstrapping (see Methods).

Figure 2. Spo11 locus number modulates early recombination indicators but not crossover numbers

Spo11^het and Spo11^wt+tg mice are fertile and show no obvious meiotic defects, including no difference in the fraction of cells at various stages of meiotic prophase (data not shown). a) Immunoprecipitation followed by western blotting for SPO11 protein. Spo11 locus composition: Spo11^null (null), Spo11^het (het), Spo11^wt (wt), or Spo11^wt+tg (wt+tg). The two major isoforms of SPO11 (α and β) are indicated on the left of the western blots, and the position of a molecular weight marker (kDa) is indicated in the center. Coomassie-stained gels were used to verify equivalent amounts of whole cell extracts from wt and het for immunoprecipitation (see Fig. S3a). The transgene expresses only SPO11β (which is known to be proficient for autosomal DSB formation) such that SPO11α serves as standard for comparison. Representative blots from multiple experiments (n ≥ 4) are shown. b) RAD51 and DMC1 foci in early meiotic prophase (leptonema and early zygonema, total early in Table 1) per nucleus in spermatocytes with the indicated genotypes. Each circle represents the focus count of a single nucleus. Black bars, means; brackets, p values for the indicated comparisons (Mann-Whitney, one-tailed). Mice analyzed: RAD51: het, n = 5; wt, n = 8; wt+tg, n = 3; DMC1: het, n = 2; wt, n = 5; wt+tg, n = 3. c) Left, whole cell extracts from juvenile testes 12 days post partum (dpp) immunoblotted for γH2AX protein. Spo11 locus composition and the positions of molecular weight markers (kDa) as in (a). Right, quantification of γH2AX intensity relative to wild type in juvenile testis extracts (mean ± SD). Subtracting SPO11-independent γH2AX determined from null mice, hets have on average 55% the level of gH2AX (range ~30–70%) as wt, while wt+tg have twice the level (~180–240%). Number of independent mice analyzed and ages: null and het vs. wt, n = 5 (11 to 14 dpp); wt vs. wt+tg, n = 3 (12 dpp). Because SPO11-independent γH2AX accompanies formation of the sex body later in meiosis, we analyzed juvenile males whose testes contain leptotene and zygotene spermatocytes of the first semi-synchronous meiotic wave. d) Autosomal MLH1 focus counts in mid-pachytene spermatocytes. Black bars show the means, which were not statistically significantly different (Mann-Whitney, one-tailed; het vs. wt, p>0.07; wt vs. wt+tg, p>0.3) Total mice analyzed: het, n = 7; wt, n = 9; wt+tg, n = 3. CV, coefficient of variation; CI, 95% confidence interval for the CV.

Figure 3. Changes in numbers of recombination-associated foci as meiosis progresses

a) RAD51 foci at the indicated stages. Black bars, means; brackets, statistically significant p values (Mann-Whitney, one-tailed). Mice analyzed: Spo11^het, n = 5; Spo11^wt, n = 8; Spo11^wt+tg, n = 3. b) DMC1 foci. Mice analyzed: Spo11^het, n = 2; Spo11^wt, n = 5; Spo11^wt+tg, n = 3. c) MSH4 foci. Mice analyzed: Spo11^het, n = 3; Spo11^wt, n = 5; Spo11^wt+tg, n = 3. n.d., not determined; n.a., not applicable.

Figure 4. Cytological interference is unchanged despite decreased or increased early recombination intermediates

a) Cytological interference in Spo11^het vs. Spo11^wt spermatocytes. Distances between MLH1 foci were measured on autosomal bivalents containing ≥two foci. The cumulative fraction of the inter-focus distances measured as a percentage of synaptonemal complex length or µm (inset) is shown. Number of bivalents analyzed: Spo11^het, n = 206; Spo11^wt, n = 248. The ^Spo11het data was previously published. b) Cytological interference in Spo11^wt vs. Spo11^wt+tg spermatocytes. Number of bivalents analyzed: Spo11^wt, n = 154; Spo11^wt+tg, n = 192. c) Homeostatic regulation of recombination occurs at two stages. Phases of meiotic prophase are indicated. Black line, wild-type mean focus counts for RAD51 (early recombination intermediate), MSH4 (transition recombination intermediate) and MLH1 (crossovers); dotted line, the maximal (max) and minimal (min) focus count for each marker. A model for progressive homeostatic implementation is depicted below. NCO, noncrossovers.