Characterization of effects of estrogen, progesterone and the endocrine-disrupting chemicals on mouse mammary gland in 4-vinylcyclohexene diepoxide-induced menopause model via single-cell RNA-sequencing

Mammary gland is a dynamic organ which undergoes most of its structural development after birth under cyclic control of ovarian hormones such as estrogen and progesterone. Using 4-vinylcyclohexene diepoxide (VCD) menopause model, we investigated the effect of ovarian hormones on mouse mammary glands. In particular, we focused on mouse mammary gland fibroblasts because they are one of the known crucial players but yet to be characterized well. With integrated analysis including six other publicly available datasets as well as mammary epithelium atlas data, we comprehensively described the characters of mouse mammary gland (e.g. potential commitment to mammary gland development, response to estrogens, developmental relationship, and crosstalk with mammary epithelium) in a population specific manner. Furthermore, we investigated the effect of endocrine disrupting chemical named polybrominated diphenyl ethers (PBDEs) in either of absence or presence of ovarian hormones on whole cellular components of mouse mammary gland including epithelial cells, fibroblasts, and immune cells at a single cell level. Overall design: Female 9-weeks-old C57BL/6 mice were administered with VCD for 15 days. At 100 days after starting the VCD administration, when menopause is accomplished by complete ovarian failure, the mice were treated with 11 different conditions; #1) Vehicle_NormEnv (with potential exposure for environmental PBDEs derived from plastics in cages), #2) Vehicle (no exposure to PBDEs and ovarian hormones), #3) 17ß-estradiol (E2), #4) E2 and progesterone (P4), #5) E2 and ICI 182, 780 (ER antagonist; ICI), #6) low dose of PBDEs for 20 weeks (Low_PBDE), #7) Low_PBDE and E2, #8) Low_PBDE, E2, and P4, #9) high dose of PBDEs (20 times of Low_PBDE) for 1 week (High_PBDE), #10) High_PBDE and E2, #11) High_PBDE, E2, and P4. Ovarian hormones and ICI were administered for 1 week and once, respectively, at the final week of PBDEs treatment. A sample from age-matched intact mice were also included in the experimental groups. The 4th mammary glands were harvested at the end point and single-cell RNA sequencing was performed.