Fig. 3: Changes in gene expression and reduced ovarian follicular fluid-meiosis-activating sterol (FF-MAS) in the ovary of infertile Abcg5/g8 knockout mice.

a RNA-seq analysis of ovaries collected from control diet (CD)-fed wild-type (WT) (n = 2), CD-fed Abcg5/g8 knockout (KO) (n = 3), and vegetable oil-free diet (VOFD)-fed KO mice (n = 2) were conducted. Venn diagrams show the number of genes whose expression levels were significantly lower than two-thirds (left diagrams) or higher than 1.5-fold (right diagrams) in the ovaries of CD-fed KO mice compared with the ovaries of CD-fed WT mice and VOFD-fed KO mice. b Phenotype enrichment analyses of the downregulated (top) and the upregulated (bottom) transcripts in CD-fed KO mice compared with CD-fed WT and VOFD-fed KO mice. The top five enriched phenotypes are listed. c Volcano plot of RNA-seq data showing log₂ (fold change, FC) against −log₁₀ (p value) of transcripts identified using RNA-seq analysis of ovaries from fertile mice [n = 4: CD-fed WT mice (n = 2) and VOFD-fed KO mice (n = 2)] vs. infertile CD-fed KO mice (n = 3). The red and blue dots represent upregulated and downregulated genes in infertile KO mice compared with the fertile mice group, respectively. The larger blue dots represent genes whose disruption impaired reproductive systems, based on the Mouse Genome Informatics database (https://www.informatcs.jax.org). d Gene ontology (GO) analysis of transcripts with reduced expression in the ovaries of infertile CD-fed KO mice compared with those of fertile CD-fed WT and VOFD-fed KO mice according to the values in the enrichment score under the theme of biological processes. The top five enriched processes are listed. e Selected list of differentially expressed transcripts involved in sterol biosynthetic processes in the ovaries of CD-fed WT and CD- or VOFD-fed KO mice. f Metabolic processes from squalene to FF-MAS and their associated enzymes. Representative photographs (g) and fold changes in size (h) of follicles (n = 40–50: integrated data from two independent experiments with 16–26 follicles per group) from baseline (Day 0) to 10 days (Day 10) after incubation in the absence (−) or presence (+) of the phytosterol mix (PS mix) and 1 µM FF-MAS [PS (−), FF-MAS (−) (n = 40); PS (+), FF-MAS (−) (n = 50); PS (+), FF-MAS (+) (n = 47)]. Close-up images of the representative follicle surrounded by squares are shown below each photograph in (g). The scale bar in g indicates 500 µm. Horizontal lines and error bars in h show the mean ± S.D. The dots represent individual data points. **p < 0.01, significantly different using Sidak’s multiple comparisons test compared with PS mix (−) and FF-MAS (−). N.S. not significantly different. Relative mRNA levels of Sqle, Lss, and Cyp51 analyzed using quantitative real-time PCR (i) and concentrations of FF-MAS (j) in the ovaries of CD-fed WT mice (n = 4 for analyzing Sqle, n = 8 for analyzing Lss and Cyp51, and n = 6 for analyzing FF-MAS), CD-fed KO mice (n = 4 for analyzing Sqle, n = 8 for analyzing Lss and Cyp51, and n = 6 for analyzing FF-MAS), and VOFD-fed KO mice (n = 4 for analyzing Sqle, n = 8 for analyzing Lss and Cypr51, and n = 5 for analyzing FF-MAS). Bar graphs represent mean ± S.D. The dots represent individual data points. *p < 0.05, **p < 0.01, significantly different using the Kruskal–Wallis test followed by Dunn’s post hoc test compared with CD-fed WT mice. N.S. not significantly different.