Figure 2. Estrous cycling affects obesogenic APC proliferation.

(A) Schematic of circulating estradiol (E2) levels in female mice during the 4 stages of the estrous cycle (adapted from McLean et al.⁷¹). Grouping of females based on transition into proestrus happening early (days 0 to 2) or late (days 3 to 7) of a 1-week-long HFD feeding.

(B) Representative images of vaginal smears stained with crystal violet from female mice during the 4 stages of the estrous cycle. Nucleated epithelial cells are highlighted by purple arrows, cornified squamous epithelial cells by black arrows, and leukocytes by green arrows. Scale bar is 100 μm, images taken at 20×.

(C) Representative flow cytometry dot plots to measure BrdU incorporation into APCs. Briefly, APCs are lineage negative (CD45−, CD31−) and positive for CD34, CD29, and Sca-1. BrdU incorporation is measured to assess proliferation.

(D) Representative BrdU histograms from APCs in the different groups including BrdU FMO.

(E) APC proliferation from females after 1 week of SD or HFD feeding.

(F) CD45+ cell proliferation from females after 1 week of SD or HFD feeding.

n = 7–11 mice per group. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s test for (E) and (F). Error bars represent mean ± SEM. ns, not significant, *p < 0.05, **p < 0.01, ****p < 0.0001. APCs, adipocyte precursor cells; VWAT, perigonadal fat; SWAT, inguinal subcutaneous fat; E2, 17β-estradiol; SD, standard diet; HFD, high-fat diet; BrdU, bromodeoxyuridine; FMO, fluorescence minus one.