Epithelial-stromal cell interactions are important for normal development and function of the mouse mammary gland. The steroid hormone estrogen is required for epithelial cell proliferation and ductal development in vivo. Recent studies of estrogen receptor alpha knockout mice indicate that estrogen-induced proliferation is dependent upon the presence of estrogen receptor in mammary stromal cells, but not in epithelial cells. The purpose of the present study was to identify the underlying mechanism of estrogen-dependent stroma-derived effects on mammary epithelium. We have developed a minimally supplemented serum-free medium, collagen gel primary mammary coculture system to address the issue of stroma-derived, estrogen-dependent effects on epithelial cell proliferation. Conditioned medium from mammary fibroblasts or coculture with mammary fibroblasts caused increased epithelial cell proliferation and produced tubular/ductal morphology. Hepatocyte growth factor (HGF) was identified as the mediator of this effect, as the proliferative activity in fibroblast-conditioned medium was completely abolished by neutralizing antibody to HGF, whereas neutralizing antibodies to either epidermal growth factor or IGF-I had no effect. Treatment of mammary fibroblasts with estrogen increased the production of HGF. From these results we conclude that estrogen may indirectly mediate mammary epithelial cell proliferation via the regulation of HGF in mammary stromal cells and that HGF plays a crucial role in estrogen-induced proliferation in vivo.

The steroid hormones, estrogen and progesterone, are required for mammary epithelial cell proliferation and alveolar morphogenesis in vivo. We have developed a minimally supplemented, serum-free medium, collagen gel primary mammary culture system to determine the mechanism of progestin-induced proliferation and alveolar morphogenesis. In epithelial cells cultured alone, treatment with progestin (R5020) alone produced a lumen within the epithelial organoids, but did not stimulate epithelial cell proliferation. The formation of lumens was associated with increased apoptosis, targeted within the organoids. We have previously reported that in our culture system hepatocyte growth factor (HGF) increases epithelial cell proliferation and induces a tubulo-ductal morphological response. In the present report we show that treatment with HGF and progestin (R5020) further increases epithelial proliferation above that with HGF alone and also produces an alveolar-like morphology similar to that observed in vivo in response to progestin treatment. To the best of our knowledge this is the first in vitro demonstration of both progestin-induced proliferation and alveolar-like morphogenesis of normal nonpregnant mouse mammary epithelial cells in vitro. These results suggest that HGF may play a crucial role in progestin-induced proliferation and morphogenesis in vivo.

The steroid hormones, estrogen and progesterone, are required for mammary epithelial cell proliferation and alveolar morphogenesis in vivo. We have developed a minimally supplemented, serum-free medium, collagen gel primary mammary culture system to determine the mechanism of progestin-induced proliferation and alveolar morphogenesis. In epithelial cells cultured alone, treatment with progestin (R5020) alone produced a lumen within the epithelial organoids, but did not stimulate epithelial cell proliferation. The formation of lumens was associated with increased apoptosis, targeted within the organoids. We have previously reported that in our culture system hepatocyte growth factor (HGF) increases epithelial cell proliferation and induces a tubulo-ductal morphological response. In the present report we show that treatment with HGF and progestin (R5020) further increases epithelial proliferation above that with HGF alone and also produces an alveolar-like morphology similar to that observed in vivo in response to progestin treatment. To the best of our knowledge this is the first in vitro demonstration of both progestin-induced proliferation and alveolar-like morphogenesis of normal nonpregnant mouse mammary epithelial cells in vitro. These results suggest that HGF may play a crucial role in progestin-induced proliferation and morphogenesis in vivo.

The influence of oestradiol and progesterone either singly or in combination with each other on the levels of hyaluronic acid, heparan sulphate, chondroitin sulphate, and on the activity of hyaluronidase and chondroitinase were investigated in the mammary gland of ovary-intact and in ovariectomized rats, administered oestradiol and/or progesterone. Administration of oestradiol to ovary-intact rats elevated the levels of hyaluronic acid and decreased the levels of heparan sulphate while progesterone, when administered alone, could elevate only chondroitin sulphate when compared with controls. The steroids when administered in combination, however, increased the levels of all glycosaminoglycans studied. Ovariectomized animals showed a decrease in heparan sulphate alone as compared with controls while administration of oestradiol to these rats elevated the levels of both heparan sulphate and chondroitin sulphate as compared with ovariectomized rats. Also the administration of progesterone either singly or in combination increased the levels of heparan sulphate and also decreased the levels of hyaluronic acid with no impact on the levels of chondoritin sulphate. In ovary-intact animals administration of oestradiol alone had no effect on hyaluronidase activity. Progesterone either singly or in combination with oestradiol reduced the activity of hyaluronidase, whereas it had no influence on the activity of chondroitinase. The activities of both the enzymes were decreased in ovariectomized animals and administration of oestradiol and/or progesterone to the above groups resulted in an increase. This study demonstrates that oestradiol anzd progesterone play an important role in modulating glycosaminoglycans and their depolymerizing enzymes, thereby influencing the activities of the mammary epithelium.

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