Fig. 4.

Quantitative analysis of abnormal chromocenter condensation in mice with perturbed PAR metabolism. a. Three-dimensional reconstructions of laser scan microscopy z-stack images of individual sperm were used to quantify the percentage of nuclear volume occupied by intensely stained major satellite DNA, i.e. the highly condensed portion of the heterochromatin in the chromocenter that yielded signals above the imaging threshold, as well as the individual number of intense major satellite signals per sperm nucleus. b. Statistical analysis of quantification results demonstrated that sperm from Parp1^−/− and Parg(110)^−/− animals had a significantly lower degree of condensation of their pericentromeric heterochromatin (n= 252 (wild type), 284 (Parg(110)^−/−), 154 (Parp1^−/−) and 230 (Parp1^−/−Parg(110)^−/− double knock out)) individual sperm were analyzed in 3 independent hybridization experiments, significance was determined on the means obtained in 3 individual hybridization experiments, and calculated by 1-way ANOVA with Bonferroni correction for multiple measurements; mean values are indicated in red). c. Graphs on the left side of the panel show frequency distributions of nuclear volumes occupied by intense major satellite signals, i.e. pericentromeric heterochromatin that is condensed above signal detection limit (bin width = 2, position and value of median binned value is indicated by red arrows). The right hand side of the panel illustrates the corresponding frequency distribution of numbers of individual Major Satellite FISH signal per sperm nucleus (n= 52–99 sperm per genotype; the analysis was repeated in three independent experiments; one-way ANOVA followed by Dunnett’s Multiple Comparison Test showed significant differences between results from wild type compared to Parp1^−/− or Parg(110)^−/−, but not to Parp1^−/− / Parg(110)^−/− double knock out