Establishing a 3d mouse primary cell culture model to study the direct effects of environmental estrogens on the functional differentiation of the mammary gland

RODRIGUEZ HA; KASS L; GOMEZ AL; MUÑOZ-DE-TORO M; ALTAMIRANO GA; LUQUE EH

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedades de Biociencias

There are several human-made chemical compoundswith hormonal action. Among them, Bisphenol A (BPA) and benzophenone-3 (BP3)have been shown to have estrogenic activities. Previously, we had demonstratedthat the development and differentiation of the mammary gland are affected byperinatal exposure to xenoestrogens in vivo.In the present study, our aim was to develop and establish an in vitro culture model to study thedirect effects of environmental estrogens during the functional differentiationof the mammary gland. Mammary organoids were obtained and isolated from 8weeks-old C57BL/6 female mice using enzymatic and mechanical digestion. Theorganoids were cultured on top of Matrigel with growth medium for 24 hs at37°C, 5% CO2. The next day, the medium was changed to adifferentiation one containing lactogenic hormones and exposed to: vehicle (1%ethanol), 1 µM of BPA and 1 µM or 1 ηM of BP3 for 72 hs. The mRNA expression ofestrogen alpha (ERS1), progesterone (PR) and prolactin receptors (PRLR) and themRNA levels of the milk protein beta-casein (CSN2) were analyzed by real timeRT-PCR. Also, CSN2 and alpha-lactoalbumin (LALBA) protein expression wasquantified in histological sections by an immunofluorescence assay. After 72 hsof exposure with 1 µM BPA, ESR1 and PR mRNA levels were like controls, but β-CasmRNA and protein expression was increased, indicating that this 3D culturemodel responded to BPA treatment during differentiation. Furthermore, theexposure to both doses of BP3 induced an increase of ERS1, PR and PRLRexpression; and 1 µM BP3 augmented CSN2 and 1 ηM BP3 increased LALBA proteinexpression compared to controls. In conclusion, our results show that a 3Dmouse primary cell culture model could be an appropriate tool to study thedirect effects of environmental estrogens, and demonstrate that direct exposureto low doses of BPA and BP3 induce alterations on mammary gland functionaldifferentiation and milk protein synthesis.