TRANSFORMING GROWTH-FACTOR-ALPHA - RECEPTOR-BINDING AND ACTION ON DNA-SYNTHESIS IN THE SHEEP MAMMARY-GLAND

Microsome fractions prepared from the mammary glands of non-pregnant, pregnant and lactating sheep have been used to study binding of I-125-Iabelled transforming growth factor-alpha (TGF-alpha). Binding was dependent on microsomal protein concentration, time and temperature. It showed the characteristics of an epidermal growth factor (EGF) receptor, being displaced by TGF-alpha and EGF, but not by insulin or IGF-I. The non-linear curve fitting program LIGAND was used to determine affinity and number of binding sites. A single class of high-affinity binding sites was found. The apparent dissociation constant (K-d) was similar in all physiological states (2.43+/-0.27 mol/l x 10(-10), n=23). Numbers of binding sites were lower in late-pregnant (20 weeks) and lactating sheep (14.07+/- 2.45 fmol/mg protein, n=10) than in non-pregnant, 10- or 15-week pregnant sheep (43.04+/-5.93 fmol/mg protein, n=13). DNA synthesis by mammary alveolar epithelial cells cultured on collagen gels was increased twofold by TGF-alpha (maximum response at 10 mu g/l; 1.8 nmol/l) but not by EGF. Cells derived from 15- to 20-week pregnant sheep responded significantly to TGF-alpha on day 3 of culture, but the response was delayed to day 4-5 of culture in cells from other physiological states. Dose-response was not significantly affected. TGF-alpha and IGF-I produced an additive effect on DNA synthesis. Oestradiol (10(-12) to 10(-9) M), a potential stimulator of the TGF-alpha gene, did not stimulate DNA synthesis alone, or in combination with IGF-I. It is concluded that growth factors acting via the EGF receptor play a role in ruminant mammary development, but whether they mediate oestradiol effects remains unresolved.