Response to epidermal growth factor of skin fibroblasts from donors of varying age is modulated by the extracellular matrix.

The present study was undertaken to investigate the effect of epidermal growth factor (EGF) on the biosynthetic activity of skin fibroblasts from donors of varying age and the modulation of their response to this growth factor by culture in a three-dimensional extracellular matrix. When cultured in monolayer on plastic or at the surface of a collagen gel, EGF specifically inhibited collagen synthesis whatever the age of the donor (from 17 to 84 years, n = 11). This inhibition was paralleled by a significant decrease in the steady-state level of procollagen type I mRNAs. When embedded in a three-dimensional floating collagen lattice, EGF stimulated the non-collagen protein (NCP) synthesis in fibroblasts from younger donors (5 out of 6) while fibroblasts from the older ones were not affected. Collagen production by fibroblasts from younger donors was not inhibited as in monolayer (some being even stimulated) while that of the older donors was inhibited as observed in monolayer. The steady-state level of procollagen type I mRNA was not modified by EGF in the three-dimensional culture. No significant difference was observed in the affinity and the number of EGF receptors of the fibroblasts on plastic or embedded in a collagen lattice between young and aged donors. Our results suggest that the environment of the cells can modulate the reactivity to EGF and reveal differences related to in vivo aging.     

Ligand-induced protein tyrosine kinase activity in living cells coexpressing intact EGF receptors and receptors with an extensive cytosolic deletion.

A population of stable NIH 3T3 transfectants with two molecular weight classes of membrane-bound EGF receptors encoded by a human EGF receptor cDNA has been identified and characterized. In addition to intact EGF receptors, these cells also express a molecule with an extensive cytosolic deletion. This deletion includes the ligand-activated intrinsic protein tyrosine kinase catalytic domain. Treatment with EGF caused dimerization of intact and truncated receptors, allowing us to assess protein tyrosine kinase activity in the heterodimer isolated from living cells. In contrast to homodimeric complexes with intact EGF receptor only, heterodimers were deficient in protein tyrosine kinase activity. Moreover, physical association between intact and truncated molecules suppressed receptor auto-phosphorylation by EGF receptor protein tyrosine kinase activated by antibody binding in vitro. Evidence presented here supports the idea that protein tyrosine kinase activation is facilitated by interaction between adjacent receptor molecules with intact catalytic domains. Furthermore, molecules with cytoplasmic deletions that are physically associated with kinase-active EGF receptors appear to behave as dominant negative mutations. The HerC cl cells used in this study were selected with methotrexate to amplify the EGF receptor cDNA, and in that sense may resemble certain tumor-derived cells characterized by overexpressed and rearranged EGF receptor genes.     

The effect of age on the antigen-presenting mechanism in limiting dilution precursor cell frequency analysis

We have utilized limiting dilution analysis (LDA)2 to compare the intrinsic precursor cytotoxic T lymphocyte (pCTL) frequency for influenza-plus-self in young and old C57BL/6 mice. Under conditions of excess interleukin 2 (IL-2) and antigen presenting cells (APC) derived from spleens of mice matched in age to those being tested, we found more than a twofold difference in pCTL frequency between young and old animals. However, there was no difference in pCTL frequency between the two age groups if antigen was presented to the old responder cells on spleen cells derived from young mice. The apparent decrease in pCTL frequency in old mice by standard LDA may in fact be due to a defect in the antigen processing and/or presentation mechanism of old spleen cells. We conclude that the age-associated defective CTL activity previously reported by us and by others may be due at least in part to a defect in the antigen presentation mechanism of aging mice.     

人肝癌细胞表皮生长因子受体以及佛波酯对它的调度

Using radioligand binding assay, the presence of epidermal growth factor (EGF) receptors in cells of two human liver cancer cell lines, BEL-7402 and SMMC-7721, was demonstrated. The ligand binding data were analyzed by a computer program. The dissociation constants (KD) of the ligand-receptor binding complex at equilibrium for 7402 and 7721 cells were 1.2 nM and 0.8 nM respectively, and their number of EGF receptors per cell were 6.2 x 10(4) and 2.5 x 10(4) respectively. After the treatment of cells with phorbol 12-myristate 13-acetate (PMA), no change either in the affinity or in the number of EGF receptors was found in 7721 cells. However, in the case of 7402 cells, while the number of receptors, like 7721 cells, remained unchanged, the affinity of EGF receptors displayed a time dependent modulation after PMA treatment. It dropped within the first hour to a KD value of 3.0 nM and then gradually returned to the normal control value at 48 hours or even slightly higher than normal (0.95 nM) at 96 hours of treatment. The modulation or down-regulation of EGF receptors by PMA in 7402 cells was paralleled by the simultaneous inhibition of DNA synthesis in these cells as evidenced from their reduction of 3H-TdR uptake. It is not clear what is the basis for the differences found between 7402 cells and 7721 cells in their number of EGF receptors per cell and their responsiveness to PMA treatment. It might be related to their difference in autocrine secretion of alpha-transforming growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of epidermal growth factor receptors and influence of epidermal growth factor on proliferation and aging in cultured smooth muscle cells.

Epidermal growth factor (EGF) at nanomolar concentrations stimulated DNA synthesis in confluent, serum-starved cultures of calf aorta and human uterine smooth muscle cells. Stimulation of DNA synthesis in lens epithelial cells was studied for comparison. L and D-ascorbic acid potentiated the effect of serum and EGF on DNA synthesis in calf aorta cells. In contrast L-ascorbic acid had minimal potentiating effect with serum and no effect with EGF present along with serum on DNA synthesis in human uterine smooth muscle and rabbit lens epithelial cells. EGF and ascorbic acid increased cell number when added to stationary phase cultures. Specific binding of 125I-labelled EGF to smooth muscle cells was demonstrated. Receptor concentration in calf-aorta smooth muscle cells was higher in dense cultures compared to sparse cultures. The time course of binding and dissociation of 125I-labelled EGF was similar in "dense" and "sparse" cultures. Human uterine smooth muscle cells in culture exhibited a finite lifespan. There was no stimulation of DNA synthesis in response to serum and EGF in cells of high population doubling level (PDL); although 125I-labeled EGF binding was higher in old cells (high PDL) compared to young cells (low PDL). This increase in binding was shown to be due to changes in the concentration of receptors without changes in their affinity for EGF.     

Human epidermal growth factor (EGF) receptor sequence recognized by EGF competitive monoclonal antibodies. Evidence for the localization of the EGF-binding site

Epitopes recognized by three epidermal growth factor (EGF) competitive monoclonal antibodies, LA22, LA58, and LA90, have been localized to a 14-amino acid region in the extracellular domain of the human EGF receptor. The binding of each of these mutually competitive antibodies to A431 epidermoid carcinoma cells was inhibited up to 87% by EGF. Furthermore, binding to A431 cells was inhibited 100% by the EGF competitive monoclonal antibody 528 IgG. The EGF receptor monoclonal antibody 455 IgG, which recognizes a blood group A-related carbohydrate modification of A431 receptors and does not inhibit EGF binding, did not inhibit the binding of these three antibodies to A431 cells. Antibodies LA22, LA58, and LA90 were unusual in that they bound to recognized denatured and endoglycosidase F-treated antigenic determinants in Western blots. This suggested that the antibodies recognized continuous peptide epitopes. The epitopes for these antibodies were first localized in cyanogen bromide- and V8 protease-generated fragments of a truncated form of the EGF receptor secreted by A431 cells. In experiments with synthetic peptides, all three antibodies were found to bind to the 14 amino acids from Ala-351 to Asp-364 of the mature human EGF receptor. These amino acids are located between the two Cys-rich regions of the extracellular domain of the receptor, and they include an Arg-Gly-Asp-Ser recognition site for adhesion molecule receptors. The homologous sequence in the chicken EGF receptor, which binds mouse EGF with a 100-fold lower affinity than the human EGF receptor, contains four amino acid differences including two in the Arg-Gly-Asp-Ser tetramer. The mutually competitive binding of EGF and antibodies LA22, LA58, and LA90 implied that the amino acids between Ala-351 and Asp-364 participated in the formation of the EGF-binding site of the human EGF receptor.     

Biosynthesis of the epidermal growth factor receptor in human squamous cell carcinoma lines: secretion of the truncated receptor is not common to epidermal growth factor receptor-hyperproducing cells

The biosynthesis of the EGF receptor was examined in the epidermoid carcinoma cell line A431 and five novel cell lines from human squamous cell carcinomas possessing high numbers of EGF receptors. Newly synthesized EGF receptors were visualized by labeling with [35S]methionine and immunoprecipitation with a monoclonal anti-EGF receptor antibody. In addition, the processing of the EGF receptor and its intracellular transport was analyzed by distinguishing cell surface receptors from intracellular receptors and by treating cells with inhibitors such as tunicamycin, monensin and brefeldin A. These analyses revealed that in all the tumor cell lines the EGF receptor is synthesized as a glycosylated protein of Mr 160,000 which is converted to the receptor of Mr 170,000 through posttranslational glycosylation. The receptors of Mr 160,000 and 170,000 appeared to possess high mannose type oligosaccharide chains because endoglycosidase H treatment reduced their molecular weights by approximately 30,000. A431 was the only tumor cell line studied that secreted the truncated EGF receptor of Mr 110,000. In A431 cells, the truncated EGF receptor was generated from a protein of Mr 60,000 through tunicamycin- and monensin-sensitive glycosylation. A431 cells treated with monensin secreted the truncated receptor as a Mr 95,000 form.     

Heterodimerization of the erbB-1 and erbB-2 receptors in human breast carcinoma cells: a mechanism for receptor transregulation

The erbB-1 and erbB-2 protooncogenes encode homologous membrane receptors that respectively bind epidermal growth factor (EGF) and a still incompletely characterized ligand. Binding of EGF to its receptor is known to increase tyrosine phosphorylation of the erbB-2/neu receptor in tumor cells. To investigate the mechanism of this transregulatory pathway, we analyzed the interactions between the two receptors in SKBR-3 human breast carcinoma cells. Chemical cross-linking of 125I-labeled EGF revealed that the radiolabeled EGF receptor coimmunoprecipitates with the erbB-2/neu receptor. In addition a cross-linked species of 360-kdalton molecular mass is also coimmunoprecipitated. The formation of the latter species is absolutely dependent on the presence of EGF receptor and thus appears to represent a heterodimer of the erbB-1 and erbB-2 receptors. In vitro kinase reaction assays revealed that receptor heterodimerization is induced by EGF binding and leads to a dramatic increase in the self-phosphorylation capacity of the dimerized receptors. Moreover, analysis of living SKBR-3 cells suggested that most of the EGF-induced transregulation of the erbB-2/neu receptor is due to receptor heterodimerization. In conclusion, heterodimers of erbB-1 and erbB-2 receptors may provide a mechanism for dual transductory functions of growth factors of breast tumor cells.     

Age related induction of platelet-derived growth factor A-chain mRNA in normal human fibroblasts

We have previously found that stimulation of normal neonatal fibroblasts with PDGF or EGF leads to a transient induction of PDGF A-chain mRNA and the synthesis of PDGF-AA proteins. This finding may imply the existence of an autocrine feedback mechanism to amplify the mitogenic signal under certain conditions. We have now studied the PDGF-BB mediated PDGF A-chain induction in a set of fibroblasts from young and old donors to clarify if the levels of induction are correlated to the donor age and the replicative capacity of the cells. The PDGF A-chain induction was found to be reduced in cells from old donors compared with cells from embryonic and neonatal donors. The different cell strains were also characterized further with respect to PDGF receptor expression and PDGF binding properties. PDGF β-receptors were found to be enhanced in old donor cells strains, whereas the PDGF α-receptors showed more variability in expression between the strains. The PDGF A-chain mRNA induction was also decreased or absent in late passage human fibroblasts (senescent cells) when compared with early passage cells. These data suggest that the PDGF A-chain mRNA induction is regulated by an age related mechanism in human fibroblasts. © 1994 Wiley-Liss, Inc.     

Membrane vesicles of A431 cells contain one class of epidermal growth factor binding sites

Epidermoid carcinoma A431 cells exhibit two classes of epidermal growth factor (EGF) receptors as deduced from Scatchard analysis. Steady-state binding of EGF to isolated A431 membranes indicated, however, the presence of only one class of EGF binding sites. The apparent dissociation constant (Kd) of these sites was approx. 0.45 nM which is similar to that of the high-affinity receptor of intact A431 cells. These results suggest that the vesicle receptor population consists only of high-affinity receptors. However, further studies indicated that the binding sites were similar to the low-affinity class, since binding of EGF could be blocked entirely by 2E9, a monoclonal anti-EGF receptor antibody which is able to inhibit specifically EGF binding to low-affinity receptors in A431 cells. The difference in affinity of the receptors in membrane vesicles as compared to intact cells may be explained by differences in biophysical parameters such as diffusion-limited EGF binding and receptor distribution. Based upon these considerations, it is concluded that membrane vesicles of A431 cells contain one class of EGF receptors which are apparently identical to the low-affinity receptors of intact cells.     

Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.     

Up-regulation of caveolin attenuates epidermal growth factor signaling in senescent cells

Senescent human diploid fibroblasts do not respond to growth factors like epidermal growth factor (EGF), although they have a normal level of receptors and downstream signaling molecules. To examine the mechanism of signaling attenuation, we investigated Erk activation after EGF stimulation in senescent cells. Senescent cells did not phosphorylate Erk-1/2 after EGF stimulation, whereas young cells did. In those senescent cells, we found an increased level of caveolin proteins and strong interactions between caveolin-1 and EGF receptor. Electron microscopic analysis demonstrated an increased number of caveolae structures in senescent cells. More interestingly, brain, spleen, and lung from 26-month-old rats showed substantial increases of caveolin proteins. However, in the case of p53-induced senescence, caveolin-1 was not induced, and EGF stimulation phosphorylated Erk-1/2 as much as young control cells. Finally, we overexpressed caveolin-1 in young human diploid fibroblasts in which the activation of Erk-1/2 upon EGF stimulation was significantly suppressed. These results suggest that the unresponsiveness of senescent fibroblasts to EGF stimulation may be due to the overexpression of caveolins, which seems to be independent of growth arrest and other aging phenotypes.     

Decreased enzymic protection and increased sensitivity to oxidative damage in erythrocytes as a function of cell and donor aging.

Erythrocytes from young and old rats were separated into four age fractions by density-gradient centrifugation. The specific activities per cell were determined for glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glutathione peroxidase (EC 1.11.1.9), glutathione reductase (EC 1.6.4.2) and catalase (EC 1.11.1.6). Decreased specific activities were observed with increasing cell age for all four enzymes in both young and old animals. In addition, significant differences in the activities of these enzymes were observed between cells of the same age fraction from young and old donors. Susceptibility of fractionated erythrocytes to oxidative attack in vitro generated by incubation with xanthine/xanthine oxidase increased with both cell and animal age. The amount of membrane-lipid peroxidation also increased with cell and animal aging, as measured by both thiobarbituric acid and fluorescent chromolipid assays. Increases of 2-3-fold in the contents of lipid peroxides were observed between the youngest and oldest age fractions of young rats. Lipid peroxide contents in young cells of old animals were equal to those in old erythrocytes from young rats and increased by 30% with cell aging in the old donors. These results suggest that the extent of enzymic protection against oxidative and peroxidative damage decreases with erythrocyte aging. More importantly, enzymic protection in cells of old rats is considerably decreased already in the early stages of their lifespan.     

Receptor-mediated vectorial transcytosis of epidermal growth factor by Madin-Darby canine kidney cells

Transcellular transport of a variety of ligands may be an important mechanism by which regulatory substances reach their site of action. We have studied the transcellular transport of two 6,000-mol-wt proteins, epidermal growth factor (EGF) and insulin, across polarized Madin-Darby canine kidney (MDCK) cells grown on dual-sided chambers on a nitrocellulose filter substrate. When grown on these chambers, MDCK cells are polarized and express distinct basal and apical surfaces. MDCK cells are capable of unidirectional transport of EGF from the basal-to-apical direction, 50% of bound EGF transported in 2 h. Transport was inhibited by the addition of unlabeled EGF in a dose-dependent manner. Anti-EGF receptor Ab, which inhibited binding, also inhibited transport. No transport in the apical-to-basal direction is noted. Insulin transport is not observed in either direction. Transport correlates with the presence of ligand-specific receptors on the cell surface. Hence, EGF receptors (Ro = 48,000, Kd = 3.5 X 10(-10) M) are found only on the basal surface of the MDCK cells and neither surface expresses insulin receptors. Characterization of the EGF receptors on MDCK cells, as assessed by affinity, molecular mass, and anti-receptor antibody binding reveals that this receptor has similar characteristics to EGF receptors previously described on a variety of cells. Hence, the EGF receptor can function as a transporter of EGF across an epithelial cell barrier.     

青年猫与老年猫初级听皮层GABA_A受体免疫表达的比较研究

目的对5只老年猫(12岁,3-3.5kg)与5只青年猫(2岁,3-3.5kg)初级听皮层(AI)γ-氨基丁酸(gamma-aminobutyric acid,GABA)A受体神经元进行免疫表达比较研究,探索老年猫与青年猫初级听皮层(AI)GABAA受体年龄性变化及产生可影响的生理作用。方法运用免疫组织化学反应与免疫印迹相结合的方法对不同年龄组动物(AI)组织进行染色。光学显微镜下观察、拍照;免疫组织化学阳性反应示GABAA R-IR(GABAA receptor-immunoreaction)神经元形态、密度及分布;免疫印迹示GABAA受体蛋白含量变化。结果老年猫的AI区GABAA R-IR神经元密度比青年猫的GABAA R-IR下降了29.19%,阳性反应强度减弱了20.7%,老年猫阳性反应细胞占神经元总数百分比比青年猫的减少了5.32%;老年猫的GABAA受体蛋白表达量比青年猫的下降了23.16%。结论初级听皮层GABAA受体细胞及受体表达下调可能是老年个体听觉功能减退的重要原因。     

The control of DNA synthesis in primary cultures of hepatocytes from adult and young rats: interactions of extracellular matrix components, epidermal growth factor, and the cell cycle

Hepatocytes from adult and 4-week-old rats cultured on one of several extracellular matrix components were stimulated to replicate by epidermal growth factor (EGF). DNA synthesis was increased at 44-48 hr in adult hepatocytes and at 24, 48, and 72 hr in hepatocytes from young rats when EGF was added 2 hr after explantation. When EGF was added at 24 hr, maximal DNA synthesis of adult hepatocytes was observed at 48 hr, whereas that of 4-week-old hepatocytes was seen at 48 and 72 hr. Ten ng EGF per ml was the optimal concentration for maximal DNA synthesis in both adult and young cells. DNA synthesis decreased with increasing cell density, but this effect was less in hepatocytes from young than in those from adults. When hepatocytes were cultured on substrata consisting of individual extracellular matrix components, neither the time that adult cells needed to respond to EGF nor the time from stimulation by EGF to the peak of maximal DNA synthesis was altered in either adult or young cells. The optimal EGF concentration for maximal DNA synthesis and the cell density control of replication were also not altered by the substrata used. Substrata made from each of the extracellular matrix components studied enhanced DNA synthesis of adult and young hepatocytes stimulated by EGF in the following decreasing order: fibronectin, type IV collagen, type I collagen, and laminin. In both adult and young hepatocytes the enhancement of DNA synthesis was greatest when cultured on fibronectin. Thus the initiation and magnitude of DNA synthesis in primary cultures of rat hepatocytes were altered both by the age of the donor and the substratum on which the cells were explanted.     

Thermosensitivity of Nuclear RNA Polymerase during the in vitro Senescence of Mouse Fibroblasts

Embryonic mouse fibroblasts divide approximately twelve times in vitro prior to cessation of mitotic activity. During this period of cellular senescence the thermosensitivity of the RNA polymerase activity of isolated nuclei has been examined as a means of detecting the possible accumulation of defective enzyme molecules, as has been found by other workers for several cytoplasmic enzymes during the ageing of human fibroblasts in vitro.
The total RNA polymerase activity of nuclei isolated from old (10th generation) cells is more thermoresistant than that of young (2nd generation) cells. However, the net RNA polymerase activity of nuclei from non-dividing (confluent) cells is more thermoresistant than that of exponentially growing cells of the same age. When allowance is made for the state of growth of the cultures, little difference is seens in the thermosensitivity of the activities of nuclei from old and young cells. Neither is there any difference between the thermosensitivity of the net activity of an established line of murine fibroblasts (L-cells) and cells in primary culture.
Preheating nuclei increases the inhibition of their total RNA polymerase activity by or-α-amanitin, indicating that RNA polymerase II is the most heat resistance species present. There appears to be no difference between the thermosensitivity of the α-amanitin sensitive and resistance species of the enzyme in the nuclei of old and young cells.
It is concluded that old cells resemble non-dividing young cells in containing a higher proportion of RNA polymerase II in their nuclei, resulting in greater thermoresistance of the total RNA polymerase activity over that of exponentially growing cells. However, there appears to be no increase in thermosensitivity of the enzymes with age.     

Decreased response to epidermal growth factor during cellular senescence in cultured human microvascular endothelial cells.

We have previously demonstrated that epidermal growth factor (EGF) induces cell migration, tissue-type plasminogen activator synthesis, as well as tubular formation in microvascular endothelial cells from human omental tissue. In this study, we compared the responsiveness to EGF of late passaged (senescent) human omental microvascular endothelial (HOME) cells with that of early passaged (young) HOME cells. We have employed HOME cells derived from surgically resected omental samples from 14 patients. EGF-stimulated cell migration significantly more in the young cells than in the senescent cells during serial cultivation (aging) in vitro. Scatchard analysis demonstrated that the number for both high and low affinity receptors for EGF in HOME cells was decreased dramatically during serial cultivation. The expression of EGF receptor mRNA was also decreased in the senescent HOME cells. Treatment of HOME cells with EGF significantly increased cellular mRNA levels of tissue-type plasminogen activator, and two protooncogenes, c-fos and c-myc, in young HOME cells, but not in senescent HOME cells. Thus HOME cells aged in vitro show a decreased responsiveness to EGF, resulting in decreased migration of human endothelial cells. The serial cultivation of human endothelial cells in vitro may downregulate EGF receptor and decrease responsiveness to exogenous EGF, a potent angiogenic factor.     

Receptors for epidermal growth factor and insulin-like growth factor-I on preimplantation trophoderm of the pig.

125I-labelled epidermal growth factor (125I-EGF) and 125I-labelled insulin-like growth factor-I (125I-IGF-I) bound to trophoderm cells from pig blastocysts obtained on days 15-19 of pregnancy. Specific binding was detected on freshly isolated cell suspensions and on cells cultured for several days. The binding of 125I-EGF was inhibited by increasing concentrations of EGF, but not by various other growth factors and hormones. Chemical cross-linking of 125I-EGF to its receptors using disuccinimidyl suberate (DSS) revealed a radiolabelled band of relative molecular mass 160,000, similar to that identified as the EGF receptor in other cell types. The binding of 125I-IGF-I was inhibited by both IGF-I and insulin, indicating that the receptors were either type I IGF receptors or insulin receptors. Cross-linking of 125I-IGF-I to serum-free supernatants from trophoderm cultures showed that the cells secreted an IGF-binding protein, giving a complex of relative molecular mass about 45,000. The presence of receptors for EGF and IGF/insulin suggests that these factors could be involved in regulating the growth and development of the early blastocyst.