Female mice with an ancestral history of Khdc3 mutation manifest hepatic transcriptional dysregulation that is independent of genotype.

(a) Gene ontology scatterplot for dysregulated genes in Khdc3-null oocytes, based on RNA-Seq. The y-axis represents −log FDR (false discovery rate) and the x-axis represents log2 enrichment ratio. Dysregulation of genes important in lipid and carbohydrate metabolism are highlighted. (b) Murine expression of Khdc3 in various tissues, detected by qPCR (N=3). (c) Relative mRNA expression of Cyp17a1 in the livers of WT, WT*, KOKO, and KO* mice measured by qPCR. Each dot represents an individual mouse. (d) Relative mRNA expression of Cyp17a1 in the livers of WT* female mice generated from various male and female Khdc3-null grandparents measured by qPCR. (e) Volcano plots depicting differentially expressed genes identified by RNA-seq in the livers of WT vs. WT*, KO* and KOKO mice, respectively (N=4-6). Red dots represent upregulated genes in WT*, KO* and KOKO mice while blue dots represent genes that were downregulated in WT*, KO* and KOKO mice. The x-axis shows log2 fold change values, and the y-axis denotes −log10 p-values. (f) Dot plots of common dysregulated liver genes amongst the WT*, KO*, and KOKO mice compared to WT mice; ***p-adjusted < 1 × 10−5. (g) Dot plot of the significantly dysregulated genes in KO* livers (x-axis) versus WT* livers (y-axis), based on adjusted p-value. Red dots reveal significantly dysregulated genes in KO* mice, blue dots reveal significantly dysregulated genes in WT* mice, and grey dots represent commonly significantly dysregulated genes in both KO* and WT* mice. (h) Gene ontology of the most common dysregulated genes identified in the livers of both WT* and KO* revealed abnormalities in pathways critical for lipid and glucose metabolism.

WT* defects persist over multiple generations and can be passed through the maternal and paternal ancestral lines.

(a) Schematic of experimental mating to form WT**(P) mice. Khdc3-null mice are represented by black filled shapes, WT mice represented by white filled shapes, and Khdc3-heterozygote mice represented by half-filled black shapes. Volcano plot displaying dysregulated genes in the livers of WT**(P) mice compared to WT mice (N=4). Red dots represent upregulated genes and blue dots represent downregulated genes in the WT**(P) mice. (b) Schematic of experimental mating to form WT**(M) mice. Volcano plot displaying dysregulated genes in the livers of WT**(M) mice compared to WT mice (N=4). Red dots represent upregulated genes and blue dots represent downregulated genes in the WT**(M) mice. (c) Venn diagram representing the number of significantly dysregulated liver genes of WT**(P) and WT**(M) female mice compared to WT mice. (d) Pedigree schematic representing mating of WT*(P) male and female mice over successive generations to form WT******(P) mice. Dot plots represent relative liver mRNA expression of Cyp17a1 and 2610507I01Rik of the 2nd through 7th generation WT female mice descended from male Khdc3-null mice as measured by qPCR. (e) Pedigree schematic representing creation of a F2 outcross WT female from the mating of a female Khdc3-null female and WT male and subsequent F1 generation Khdc3-heterozygote female with WT male. Venn diagram depicting the overlap of commonly dysregulated genes in the livers of F2 outcross WT and WT**(M) female mice compared to WT mice (N=4-7). (f) Dot plots representing liver gene dysregulation amongst WT, WT*(P), WT*(M), and F2 outcross WT female mice. ***p < 1 × 10−5.

Wild type mice with ancestral history of Khdc3 mutation have dysregulation of multiple hepatically-metabolized molecule in the serum.

(a) Litter sizes in wild type (WT x WT) and WT* (WT* x WT*) matings. (b) Weights of WT** pups at birth, 3 weeks, and 8 weeks. (c) Metabolic phenotype of WT****** females at 8 months of age, including weight, fat composition, food intake, and energy expenditure. (d-e) Dot plots revealing concentration of bile acids and metabolic cofactors in KOKO, WT**(P), and WT****(P) female mice compared to WT mice. (f) Dot plots revealing metabolic cofactors in WT****(P) female mice exposed to a HFD compared to WT**** mice fed a conventional diet and WT mice fed a HFD. (g) Dot plots revealing metabolites in WT****(P) female mice exposed to a HFD compared to WT**** mice fed a conventional diet and WT mice fed a HFD. *p < 0.05, **p < 0.005, ***p < 0.0005.

Oocytes of KOKO and WT**(P) mice have dysregulated expression of multiple miRNAs and tsRNAs.

(a) Dot plots representing dysfunctional expression of small RNA-processing genes of the livers of WT**(P) mice as measured in RNA-seq. (b) Volcano plots depicting differentially expressed miRNAs and tRNA fragments in KOKO oocytes compared to WT oocytes. Red dots represent upregulated small RNAs, and blue dots represent downregulated small RNAs (N=4). (c) Volcano plots depicting differentially expressed miRNAs and tRNA fragments in WT**(P) oocytes compared to WT oocytes. Red dots represent upregulated small RNAs, and blue dots represent downregulated small RNAs (N=4). (d) Scatterplot of the most significantly dysregulated small RNAs of WT**(P) oocytes (x-axis) versus KOKO oocytes (y-axis), based on p-value. Green dots reveal dysregulated miRNAs and tRNA fragments in WT**(P) oocytes, brown dots reveal dysregulated miRNAs and tRNA fragments in KOKO oocytes, and black dots represent commonly dysregulated small RNAs in both WT**(P) and KOKO oocytes.

Injection of serum from KO mice in WT females is sufficient to cause transcriptional dysregulation in WT offspring.

(a) Schematic of serum transfer experiment. (d) Volcano plot depicting differentially expressed genes in the wild type offspring born to mothers who received injection with either WT or KO serum. *p < 0.05, **p < 0.005, ***p < 0.0005.

Khdc3 expression in ovaries of WT, KO*, and WT* mice, from RNA-Seq.

Primer sequences used for qPCR