PMC

In vitro models with G. vaginalis clinical isolate. Three G. vaginalis clinical isolates (GV 1, GV 2 and GV 3) were obtained from vaginal swabs of subjects enrolled in this study and their ability to form biofilms was evaluated. Biofilm biomass was quantified using the crystal violet (CV) staining method after 48 h of incubation. The optical density (OD) was measured at 590 nm using a 96-well microplates reader. Data represent the mean ± SEM of duplicate samples from three different experiments. The statistical significance of differences between the groups was determined by ANOVA + Bonferroni as a post-hoc comparison. *P = 0.0111 GV 3 vs. GV 1; ^#P = 0.0077 GV 3 vs. GV 2; P > 0.9999 GV 1 vs. GV 2 (a). Vaginal ECs from healthy donors (n = 9) were washed three times, counted, adjusted to 2×10⁵/ml in RPMI 1640 supplemented with 10% FCS. Cells were then cultured for 18 h at 37 °C under 5% CO₂ in 96-well microtiter plates (100 µl/well) with or without cycloheximide (CHX) 100 μg/ml, GV 1, GV 2 and GV 3 at 2×10⁵/ml, 100 µl/well (1:1) or GV 1, GV 2 and GV 3 at 2×10⁶/ml, 100 µl/well (10:1). Immediately (t = 0) and after incubation, ECs were centrifuged, suspended in hypotonic propidium iodide solution and kept for 1 h in the dark at room temperature. Data are expressed as the percentage of vaginal ECs undergoing apoptosis. Data represent the mean ± SEM of duplicate samples from 9 different women for GV3 and 5 different women for GV 1 and GV 2. The statistical significance of differences between the groups was determined by Kruskal-Wallis + Dunn’s as a post-hoc comparison. *P = 0.0025 CHX vs. ECs from healthy donors (t = 18 h); *P = 0.0119 GV 1 (10:1) vs. ECs from healthy donors (t = 18 h); *P = 0.0175 GV 2 (10:1) vs. ECs from healthy donors (t = 18 h); *P = 0.0012 GV 3 (1:1) vs. ECs from healthy donors (t = 18 h); *P = 0.0002 GV 3 (10:1) vs. ECs from healthy donors (t = 18 h); P > 0.9999 ECs from healthy donors (t = 0) vs. ECs from healthy donors (t = 18 h); P = 0.1779 GV 1 (1:1) vs. ECs from healthy donors (t = 18 h); P = 0.1692 GV 2 (1:1) vs. ECs from healthy donors (t = 18 h) (b).

(A) Genome-wide distribution of ATAC-seq THSSs from GV and MII oocytes.
(B) Average profiles of ATAC-seq THSSs and nucleosome signals from GV and MII oocytes at TSSs.
(C) Pearson’s correlation of ATAC-seq THSSs with RNA-seq and H3K4me3 ChIP-seq at promoters in GV and MII oocytes.
(D) Heatmaps showing ATAC-seq THSSs and nucleosome signal at distal non-TSS peaks. Clusters derived by k-means clustering of ATAC-seq THSS reads. Cluster 1, GV-specific; Cluster 2, common between GV and MII; Cluster 3, MII-specific.
(E) Average profiles of ATAC-seq THSS and nucleosome signals from GV and MII oocytes at non-TSS peaks corresponding to clusters shown in Figure 4D.
(F) Venn diagram showing overlap of distal ATAC-seq THSS non TSS peaks between GV and MII oocytes. The lower part of the panel shows the number of peaks containing the CTCF motif.
(G) Metaplots of GV and MII oocyte Hi-C significant interactions between anchors occupied by ATAC-seq THSSs (cluster 2 in Figure 4D) containing the CTCF motif.
(H) Metaplots of GV and MII oocyte Hi-C significant interactions between anchors occupied by all distal ATAC-seq THSSs present in GV oocytes.
(I) Boxplot comparing ATAC-seq THSS reads on the loop anchors shown in Figure 4H between GV and MII oocytes. RPKM; Reads Per Kilobase Million
(J) Heatmaps showing RNA-seq, H3K4me3, and H3K27me3 ChIP-seq from GV and MII oocytes at gene promoters interacting with common THSSs (cluster 2 in Figure 4D) analyzed in Figure 4K.
(K) Metaplots of GV and MII oocyte Hi-C significant interactions between enhancers defined by the presence of THSSs and H3K27ac, and active promoter containing H3K4me3.
See also

(A1) Zona pellucida of mouse oocytes was cut with a sharp glass needle. (A2) Mouse GV was aspirated by a blunt-tip micropipette with an inner diameter of about 20 μm. (A3) Pig GV was transferred to the perivitelline space of enucleated mouse oocyte by a blunt-tip micropipette with an inner diameter of about 25 μm. (A4) Pig GV was fused to the mouse cytoplast by electric fusion. (B1) Zona pellucida of mouse oocyte was cut by using pressure with a sharp glass needle. (B2) Pig GV was squeezed out by pressing the zona pellucida. (B3) Mouse GV was directly injected into pig cytoplast by a blunt-tip micropipette with an inner diameter of about 20 μm with a piezo unit. (B4) A pCy-mGV oocyte was reconstructed with pig cytoplast and mouse GV. (C1) Pig GV was transferred to the micro-manipulation droplet with enucleated mouse oocyte. (C2) Mouse GV was transferred to the micro-manipulation droplet with enucleated pig oocyte.

Single-cell PAIso−seq for GV oocytes. a Global poly(A) tail length distributions of all detected CCS reads (Pass ≥10) in single GV oocyte C1 (cell 1), C2, C4, C7, C15, and GV rep.2, and single GV oocyte-combined (SCGV com.) datasets. The median length of each poly(A) tail of a transcript is shown as the red dot and the number below the violin plot. b The frequency of non-A residues in single GV oocytes C1, C2, C4, C7, C15, GV rep.2, and SCGV com. datasets. c The Spearman’s correlation of gene poly(A) tail length between single GV oocyte C4 and GV rep.2 (top panel, n = 928), single GV oocyte C15 and GV rep.2 (middle panel, n = 566), single GV oocyte C15 and C4 datasets (bottom panel, n = 597) datasets. The blue line represents linear regression line. The light-gray area represents confidence interval of the regression. Genes with at least four transcripts in each of the datasets are included in the analysis.