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Tumor Necrosis Factor Receptors in Microvascular Endothelial Cells from Bovine Corpus Luteum¹

^a Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan ^b Institute of Physiology, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany

There is sufficient evidence to prove that tumor necrosis factor (TNF) modulates bovine corpus luteum (CL) function. Our previous study demonstrated that functional TNF receptors are present on luteal cells in bovine CL throughout the estrous cycle. The purpose of the present study was to identify the presence of functional TNF receptors on the microvascular endothelial cells derived from developing bovine CL. TNF receptors were analyzed by a radioreceptor assay using ¹²⁵I-labeled TNF on two types of cultured endothelial cells. One has a cobblestone appearance (CS cells), and the other has a tube-like structure (TS cells). ¹²⁵I-Labeled TNF binding was maximal after incubation for 30 h at 37°C, and the specificity of binding was confirmed. A Scatchard analysis showed the presence of two binding sites (high- and low-affinity) for TNF receptors on both CS and TS cells. The dissociation constant (K_d) values and concentrations of the high-affinity binding sites for TNF receptors were similar for CS and TS cells. However, K_d values and concentrations of the low-affinity binding sites in CS cells were significantly higher than those in TS cells (P < 0.05 or lower). The expression of TNF receptor type 1 (TNF-RI) mRNA was determined in both cell types. Furthermore, TNF significantly stimulated prostaglandin E₂ and endothelin-1 secretion by both CS and TS cells (P < 0.05 or lower). These results indicate the presence of two types of TNF receptors and the expression of TNF-RI mRNA in the endothelial cells derived from bovine CL, and suggest that TNF plays two or more roles in regulating the secretory function of the endothelial cells.

¹ This research was supported by a Grant-in-Aid for Scientific Research (No. 11460129) from the Ministry of Education, Science, Sports and Culture of Japan; the Japanese-German Cooperative Science Promotion Program of the Japanese Society for the Promotion of Science (JSPS); and the German Research Foundation (Scha 257/14-1). R.S. (No. 8763) and Y.U. (No. 6032) are Research Fellows of JSPS. A.M. is supported by Alexander von Humbolt Stiftung.

² Correspondence. FAX: 81 86 251 8388; kokuda{at}cc.okayama-u.ac.jp

³ Current address: Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan.

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