Fig. 6

Generation of the Sirt1^-/- genotype, embryonic development and transcriptomic analysis of Sirt1^null embryos. (A) Number of pups per litter after mating with a Sirt1^+/- male. The dots represent individual litters, and the lines represent the medians; significance was tested via the Mann‒Whitney U test (**, P < 0.01). Ratio of offspring genotypes following mating with Sirt1^+/- males. Stacked columns show the cumulative proportion of recorded genotypes. A one sample t test was used to compare the recorded genotype ratio with a hypothetical value (i.e., 0.5). (B) Live-cell imaging and time analysis of early embryonic development of the Sirt1^+/+ and Sirt1^-/- genotypes following the activation of wt and Sirt1^null oocytes, respectively (red: H2B‐tdTomato). Assessment of cleavage (24 h) and blastocyst rates (96 h). The columns represent the means ± SEMs of three independent IVF assays, and significance was evaluated via paired t tests (**, P < 0.01). (C) Zygote development and viability. Stacked columns show the cumulative proportions of recorded phenotypes of zygotes; significance was evaluated via the chi-square test (***, P < 0.001). (D) Design of two schemes of nascent mRNA analysis using 5-ethynyl uridine (5-EU) treatment. (E) Representative images and quantification of 5-EU incorporated into nascent mRNA in parthenogenetically activated wt and Sirt1^null oocytes. (F) Immunocytochemical staining and analysis of H4K16ac in two-cell parthenote embryos. Dot plots show individual values (i.e., embryos) of integrated density (IntDen), and the lines represent the median; significance was evaluated via the Mann‒Whitney U test. (G) Heatmap of transcripts expressed in bulk samples of wt and Sirt1^null parthenote embryos. (H) Qualitative RNA analysis and volcano plot of the RNA-seq data. FDR = false discovery rate; |LFC|= absolute value of log₂ fold change. Scale bar: 50 µm.