Figure 1.
Tissue changes in the FRT during the estrous cycle of the mouse.
Vaginal smears were taken from naïve virgin C57BL/6 female mice (8–12 weeks old) over the course of one estrous cycle and used for determination of cycle stage. Mice were killed at PE (A), E (B), ME (C) and DE (D) and tissue sections of vagina were cut. Vaginal smears (top panel) and tissue sections (bottom panel) were stained with H&E. The insets in the top panel show typical cell types found in vaginal smears of each cycle stage. The inset in the bottom panel shows neutrophils present in vaginal epithelium. Representative images from conventional cycle stage assessments are shown. Top panel, 100× magnification; bottom panel, 400× magnification.
Figure 2.
Influence of the estrous cycle on cervico-vaginal IgG levels.
Vaginal washings were taken from naïve adult female C57BL/6 mice (8–12 weeks old) throughout the course of the estrous cycle. Samples were analyzed by mouse IgG-specific ELISA. Each graphic symbol represents one experimental animal. Data are derived from three independent experiments (n = 4) and were analyzed using one-way ANOVA with post-test (Tukey's). The mean±SEM is shown (*, p≤0.05; **, p≤0.01).
Figure 3.
Detection and quantification of neutrophils and monocytes in the total lower FRT during the estrous cycle by flow cytometry.
Lower FRT was isolated from naïve adult virgin female C57BL/6 mice and prepared for flow cytometry. Cells were gated as CD45.2+CD11b+CD11clo (as shown in Figure S1) prior to further analysis. (A) Percentage of CD45.2+CD11b+CD11cloLy6C+Ly6G+ cells (neutrophils) and CD45.2+CD11b+CD11cloLy6C+Ly6G− (monocytes) at each cycle stage (estrus,E; proestrus, PE: metestrus, M; diestrus, DE). (B–D) Frequency (B), absolute number (C) and morphology (D) of neutrophils at each cycle stage. (E–G) Frequency (E), absolute number (F) and morphology (G) of monocytes at each cycle stage. Samples in (D) and (G) were Giemsa stained cytospins after sorting. Representative images shown in (D) and (G) were taken at 400× magnification. Data derived from three independent flow cytometry experiments (each run with tissue pooled from four mice per time point) were quantified and analyzed using one-way ANOVA with Tukey's post-test. The mean±SEM is shown (*, p≤0.05; **, p≤0.01; ***, p≤0.001).
Figure 4.
Detection and quantification of Gr-1+ neutrophils/monocytes in the vagina during the estrous cycle by immunofluorescence.
Tissue was taken from naïve adult virgin female C57BL/6 mice and vaginal tissue sections were stained with DAPI (blue) and mAb RB6-8C5 (Gr-1; white). Representative images from one experiment are shown. The scale bar represents 50 µm. *, lumen; **, lamina propria; thick line, basal membrane; thin line, epithelial cell-lumen border. Data represent a single optical slice (A). Images (n = 12) were quantified with the Image J software by pixel count per unit area. Analyses were performed on lamina propria (B) and epithelial compartment (C) using one-way ANOVA with Tukey's post test. The mean±SEM is shown (**, p≤0.01; ***, p≤0.001; n.d., not detected).
Figure 5.
Effects of Ly6G depletion on cervico-vaginal IgG.
Naïve virgin C57BL/6 mice were treated with the Ly6G-depleting antibody 1A8 or the isotype control 2A3 at PE (A), E (B), ME (C) or DE (D). Cervico-vaginal washings were taken 24 and 48h after mAb administration according to the described protocol. Data are presented as ratios of genital IgG levels post-injection, measured by ELISA, relative to genital IgG levels pre-injection (baseline). Data are derived from one experiment (n≥4 mice) and were analyzed using unpaired t test. The mean±SEM is shown (n.s., non-significant).
Figure 6.
Estrous cycle progression in Ly6G-depleted mice.
Four groups of naïve virgin C57BL/6 mice were injected i.p. with 200 µg of mAB 1A8 (A) or control mAB 2A3 (B). Each group was injected at a different time point during the estrous cycle (group 1 = PE, group 2 = E, group 3 = ME, group 4 = DE). Vaginal smears were taken each day of the experiment and stained with H&E. The smears were used for determination of cycle stage and cycle progression. Representative images from five mice in each experimental group are shown. Images were taken with 100× magnification.
Figure 7.
Vaginal epithelial shedding in Ly6G-depleted mice.
Four experimental groups of naïve virgin C57BL/6 female mice were injected i.p. with mAb 1A8 (upper panel) or control mAb 2A3 (lower panel). Each mouse group was injected at a different cycle stage. FRT tissue sections were cut and stained with H&E. The tissue sections were used to assess vaginal epithelial shedding in Ly6G-depleted mice. Representative images are shown from five mice in each group. Images were taken at 400× magnification.