Fig. 5.

Perinatal androgen exposure programs bone marrow MC progenitors for reduced MC granule mediator concentrations in adulthood. (A) Schematic depicting derivation of BMMCs from femoral bone marrow of 7- to 8-wk-old GDX CM, CF, and AF mice. Created with BioRender.com. (B) Total histamine concentration was reduced in perinatally AF BMMCs compared to CF BMMCs. (C) Hdc gene expression in BMMCs. One-way ANOVA with Tukey’s multiple comparisons test. (D) BMMCs were sensitized with monoclonal anti-DNP IgE (0.5 µg/mL) overnight and later stimulated with 0, 15, 31, and 62 ng/mL DNP-HSA for 1 h. Supernatants from AF BMMCs had reduced concentrations of histamine after FcεRI stimulus compared with CF BMMCs, and had similar responses to CM BMMCs, with the exception of 15 ng/mL DNP stimulus. Two-way ANOVA with Tukey’s multiple comparisons test. (E) BMMCs were stained with fluorescent-conjugated c-kit (Pacific Blue) and FcεRI (PE) antibodies, and representative images of two-color flow cytometric analysis of FcεRI (horizontal axis) and c-kit (vertical axis) receptor expression from CM, CF, and AF adult BMMCs are shown. Flow cytometry geometric means (bar graphs) for c-kit, FcεRI, and c-kit/FcεRI double positive cell percentages showed no difference between BMMC groups. One-way ANOVA with Tukey’s multiple comparisons test. (F) Androgenized BMMCS exhibited reduced levels of MC proteases MCPT4 and CPA (Western blot analysis), and 5HT (ELISA) in BMMC pellets. (G) Relative mRNA expression for Mcpt4, Cpa3, and Tph in BMMC pellets was not different across experimental groups. One-way ANOVA with Tukey’s multiple comparisons test. Data are representative of experiments from five or six independent BMMC cultures in each experimental group to establish reproducibility. #P < 0.10, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.