BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 232, 340–344 (1997) ARTICLE NO. RC976283 Estrogen Affects Development of Alveolar Structures in Whole-Organ Culture of Mouse Mammary Glands John Barlow,* Theresa Casey,* Jen-Fu Chiu,† and Karen Plaut* ,1 *Department of Animal and Food Sciences and †Department of Biochemistry, University of Vermont, Burlington, Vermont 05405 Received January 13, 1997 with the former being an early stage in alveolar growth In order to determine if estrogen affects lobulo-alveo- and a precursor to the latter. Lobulo-alveoli develop after lar development, and to determine if whole organ culture 5 days in culture, and elimination of estrogen and proges- of mouse mammary glands is an appropriate model of terone from the medium does not affect growth (5,6). It estrogen effects on mammary development, mouse mam- is possible that estrogen and progesterone are only re- mary glands were cultured in the presence or absence quired in mouse mammary development during prepu- of phenol red and estrogen. Lobulo-alveolar development bertal stages of ductal elongation. This is consistent with did not occur in vitro when phenol red was eliminated the present descriptions of hormone requirements for lo- from culture medium. Lobulo-alveolar development was bulo-alveolar development in the organ culture model, stimulated by 17b-estradiol in a dose dependent manner but not with results of in vivo studies (1,3,7). Another over a physiological range. These results support the hy- possibility is that a component of the mouse mammary pothesis that estrogen plays a critical role in alveolar gland culture system mimics or replaces the requirement development in the mammary gland. In this report an of estrogen or progesterone during lobulo-alveolar devel- in vitro model of mammary development is identified in opment. We hypothesized that the apparent differences which the effects of estrogen are morphologically de- between hormone requirements in whole organ culture scribed and quantified.  1997 Academic Press and in vivo may be due to estrogenic activity of phenol red, a pH indicator dye commonly added to tissue culture media (8,9). Estrogen and progesterone are required for normal In order to determine whether phenol red or estrogen mammary gland development (1,2). While estrogen is affects development of mouse mammary glands in organ necessary for ductal growth and branching in vivo (3), culture, we quantified extent of alveolar development there is no effect of this hormone on proliferation of after 5 days of culture in a lactogenic medium with and mammary epithelial cells in primary culture (2,4). without phenol red, and in a phenol red free medium Therefore, it has been hypothesized that the growth containing various concentrations of estradiol-17b. promoting effects of systemic estrogen are indirectly controlled by local factors within the mammary gland. MATERIALS AND METHODS The role of estrogen in alveolar development is not well described, although in mature mice ovarian steroids Reagents. Waymouth’s Medium 752/1, 17b-estradiol, progester- are required for maintenance and development of ducts one, hydrocortisone (H), aldosterone (A), and insulin (Ins) were pur- and lobulo-alveoli (1,3). chased from Sigma Chemical Company (St. Louis, MO). Waymouth’s Postpubertal stages of mammogenesis can be repro- Medium without phenol red (Formula No. 95-0060DJ), was pur- chased from Gibco/BRL (Frederick, MD). Human recombinant epi- duced in vitro using a mouse mammary gland whole or- dermal growth factor (EGF) was obtained from Collaborative Bio- gan culture system provided prepubertal mice are first medical Products, Becton Dickinson (Bedford, MA). Ovine prolactin primed with ovarian steroids (5). When exposed to appro- (Prl; lot# AFP10677C) was provided by National Hormone and Pitu- priate hormones and growth factors, in a serum-free me- itary Program of the National Institute of Diabetes, Digestive and dium, alveolar structures proliferate from the ductal Kidney Diseases. framework within a cultured gland. Two types of alveolar Whole-organ culture. Female 21 to 23 day old BALB/c mice were structures are formed, alveolar buds and lobulo-alveoli, obtained from Charles River Breeding Labs (Montreal, Quebec or Kingston, NY). Animals were maintained in accordance with the NIH Guide for Care and Use of Laboratory Animals. At 25-28 days of age, mice were primed by implantation with a subcutaneous pellet 1 To whom correspondence should be addressed. Fax: (802) 656- 8196. E-mail: kplaut@zoo.uvm.edu. of estrogen and progesterone. After nine days of priming, mice were 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 340