Aghigh, A. | bioRxiv Science

bioRxiv2026

Late gestational exposure to flutamide alters stromal composition and immune landscape in the rat mammary gland during pre-puberty, peri-puberty, and adulthood

Perinatal development of the mammary gland is regulated by hormonal signals that influence cell proliferation, extracellular matrix remodeling, immune cell recruitment, and intracellular signaling. While the role of estrogen in mammary gland development is well established, the impact of androgens remains less understood. To address this gap, we inhibited androgen signaling in utero using the anti-androgen flutamide (FLU) and investigated the effects on mammary gland development in rats. Using an integrative strategy combining histology, transcriptomics, lipidomics, cytokine profiling, and high-resolution imaging, mammary tissue were analyzed at pre-puberty (postnatal days (PND) 21), peri-puberty (PND46), and adulthood (PND9O). FLU exposure induced subtle, yet significant, alterations in mammary morphology and molecular signatures. At PND2l, the FLU exposed group exhibited an increased number of adipocytes with reduced size. Transcriptomic analysis revealed differentially expressed genes at PND2l and enrichment in pathways related to androgen response and immune signaling, but minimal changes at later developmental stages. Lipidomic profiling showed transient disruption in long-chain fatty acid composition at early developmental stages. Cytokine profiling revealed a reduced adaptive immune response at PND46 and PND9O, and second harmonic generation imaging demonstrated changes in collagen fiber orientation and density across all developmental stages. These data indicate that prenatal androgen signaling is essential for proper stromal development and the establishment of early transcriptional networks in the mammary gland, with only minor long-term effects on glandular architecture in adult nulliparous females.

pharmacology and toxicology↗

bioRxiv2025

An in utero exposure to the synthetic estrogen diethylstilbestrol affects the fat pad composition in post-natal mammary glands

In utero exposure to the synthetic estrogen diethylstilbestrol (DES) has been linked to developmental abnormalities and elevated breast cancer risk in adulthood in human and rodent models. While the impact of DES on the mammary epithelium has been thoroughly investigated, its effect on the other cell types of the mammary gland remains understudied. Here, given that the mammary gland development is strongly associated with its microenvironment, we aimed to investigate how in utero DES exposure alters the mammary glands stromal and immune function across key developmental stages. To achieve this aim, timed-pregnant rats were gavaged daily with DES or vehicle from gestation days 16-21, and female offspring mammary glands were analyzed at pre-puberty (postnatal day 21 (PDN21)), puberty (PND46), and adulthood (PND90). We assessed morphological and extracellular matrix changes, performed transcriptomic cell-type enrichment analysis, measured cytokine expression, and quantified immune cell populations. DES-exposed mammary glands exhibited pronounced stromal remodeling, including increased collagen deposition and orientation by adulthood. Gene expression profiling indicated DES-induced stage-specific immune alterations: immune cell signatures were enriched at PND21 and PND90 but diminished at PND46. Correspondingly, DES increased macrophage populations at PND21 while reducing T-lymphocyte numbers at PND46 and PND90. DES exposure also dysregulated inflammatory cytokine/chemokine expression in adult glands, suggesting a persistent inflammatory environment. In conclusion, in utero exposure to an estrogenic compound can reprogram mammary development, inducing long-term changes in the extracellular matrix and immune landscape. These disruptions to stromal-immune homeostasis may impair normal mammary morphogenesis and increase susceptibility to breast pathologies later in life.

pharmacology and toxicology↗