Fig 1: Depletion of Snf2h leads to dysregulation of genes related to MPF activation. (A) Schematic illustration of oocytes collection for RNA-seq. Oocytes were collected based on the age of the mice and the size of the oocytes. Twenty oocytes were used for each RNA-seq library and two RNA-seq replicates were performed for WT and CKO groups at each stage. (B) Scatter plots comparing the gene expression profiles of WT and CKO fully grown GV oocytes. Two replicates for WT and CKO FGO oocytes were used for differential gene expression analyses (FC > 1.5, P-value <0.05, RPKM > 1). (C) Gene ontology analysis of the differentially expressed genes in CKO FGO oocytes. (D) Functional classification of differentially expressed genes associated with cAMP signaling, oocyte meiosis, meiotic M phase, abnormal female meiosis, and other reported factors involved in oogenesis. Up-regulated and down-regulated genes are colored with red and blue, respectively. (E) Heat map showing the log2 (fold change) of genes listed in D for GO1, GO2, and FGO oocytes.
Fig 2: Oocyte-specific depletion of Snf2h blocks meiotic resumption. (A) GVBD ratio of WT and Snf2h CKO oocytes after in vitro maturation (IVM) for 2 h. The left panel shows the representative bright field images. Arrowheads point to germinal vesicles. Scale bar, 50 µm. The right panel shows the quantification of GVBD ratio for WT and CKO oocytes. One-hundred-forty-four WT and 155 CKO GV oocytes from adult ovaries (6–8 wk) were used for the analysis. N = 6 independent biological replicates. (B) Oocyte maturation ratio of WT and CKO oocytes after IVM for 16 h. Left panel shows the representative bright field images. Arrows point to first polar bodies and arrowheads point to germinal vesicles. Scale bar, 50 µm. Right panel shows the quantification of maturation ratio for GV oocytes. Sixty-six WT and 61 CKO GV oocytes from adult ovaries (6–8 wk) were used for the analysis. N = 3 independent biological replicates. (C) Immunostaining showing the maturation process of WT and CKO oocytes. Stages are determined based on DAPI and α-Tubulin staining. The numbers represent the combined number of oocytes exhibiting the staining pattern and the total number of oocytes analyzed in three independent biological replicates. Scale bar, 20 µm. (D) Immunostaining showing the level of γH2AX in WT and CKO oocytes during IVM. The numbers represent the combined number of oocytes exhibiting the staining pattern and the total number of oocytes analyzed in three independent biological replicates. Scale bar, 20 µm.
Fig 3: Snf2h is essential for female fertility in mouse. (A) Immunostaining of Snf2h in oocytes at germinal vesicle (GV), germinal vesicle breakdown (GVBD), and meiosis metaphase II (MII) stages. Oocytes were collected from adult ovaries (6–8 wk). Scale bar, 20 µm. (B) Western blotting of Snf2h at GV, GVBD, and MII stages. a-Tubulin was used as a loading control. (C) Immunostaining of Snf2h in WT and CKO GV oocytes. Oocytes were collected from adult WT and CKO ovaries (6–8 wk). The numbers represent the number of oocytes showing the staining pattern and the total number of oocytes analyzed, respectively. Scale bar, 20 µm. (D) Fertility test of WT and CKO female mice. The WT (n = 6) and CKO (n = 6) female mice were co-caged with WT fertile male mice for 6 mo and the total numbers of pups per female are shown. (E) The numbers of MII oocytes per adult female mice (6–8 wk) after superovulation. The WT (n = 10 from three independent biological replicates) and CKO (n = 10 from three independent biological replicates) female mice were used for the analysis. Data are presented as mean ± SEM. Each dot represents a single female mouse analyzed. (****) P < 0.0001 by two-tailed Student's t-tests. (F) Quantification of oocyte size by measuring the diameters of GV oocytes (n = 40 from WT and n = 68 from CKO adult female mice, N = 3 independent biological replicates). Each dot represents a single oocyte analyzed. Data are presented as mean ± SEM. (****) P < 0.0001 by two-tailed Student's t-tests.
Fig 4: Meiotic resumption defects caused by Snf2h deficiency can be rescued by restoring MPF activity. (A) Schematic illustration of the cAMP-PKA-MPF pathway. Red words with yellow background positively regulate cAMP-PKA activity, which leads to meiotic arrest at prophase I stage. Black words with gray background negatively regulate cAMP-PKA activity, which activates MPF and promotes meiotic resumption. Red words with no background are the strategies used for the rescue experiments. (B–F) GVBD ratio at different time points during IVM for oocytes injected with Prkar2b mRNA (B), Prkaca siRNA (C), Cdk1AF mRNA (D), Ccnb2 mRNA (E), or Ccnb1 mRNA (F). N = 3 independent biological replicates for each rescue experiment and n > 10 oocytes were used for each replicate. Data are presented as mean ± SEM.
Fig 5: Snf2h regulates transcription by altering chromatin accessibility. (A) Metaplot showing the ATAC-seq signals at transcription start sites (TSSs) in WT and CKO growing oocytes (GO1) and fully grown GV oocytes (FGO). (B) Heat map showing the ATAC peaks classified according to their changes in CKO versus WT oocytes. Each row represents a locus (ATAC-seq peak center ± 5 kb), and the green gradient color indicates the ATAC-seq signal intensity. (C) Metaplot showing the ATAC-seq signals at TSS of down-regulated genes in CKO FGO. (D) Genome browser views showing the ATAC-seq and RNA-seq signals at Prkar2b, Ccnb2, and Ndc80 loci. The pink boxes indicate the ATAC peaks at the promoter regions, and the green boxes indicate the RNA-seq signals.
Supplier Page from Abcam for Anti-SNF2H antibody