Microsome fractions prepared from the mammary glands of non-pregnant, pregnant and lactating sheep have been used to study binding of 125I-labelled transforming growth factor-α (TGF-α). 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-α 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 (Kd) was similar in all physiological states (2·43±0·27 mol/l × 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-a (maximum response at 10 μg/l; 1·8 nmol/l) but not by EGF. Cells derived from 15- to 20-week pregnant sheep responded significantly to TGF-α 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-α and IGF-I produced an additive effect on DNA synthesis. Oestradiol (10−12 to 10−9 m), a potential stimulator of the TGF-α 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.
Journal of Endocrinology (1995) 144, 165–171
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