INITIAL CHARACTERIZATION OF ENDOTHELIAL MITOGENS PRODUCED BY BOVINE CORPORA-LUTEA FROM THE ESTROUS-CYCLE

To further characterize mitogenic factor(s) present in luteal extracts or luteal explant conditioned media (LCM), bovine corpora lutea (CL) were homogenized or incubated in explant culture, respectively. After evaluation of luteal extracts and LCM by using an endothelial cell pro liferation bioassay, mitogenic activity was characterized by immunoneu tralization with antibodies against heparin-binding (fibroblast) growt h factor (HBGF) 1 or 2. LCM also were subjected to ultrafiltration, as well as anion-exchange, cation-exchange, and heparin-affinity chromat ography. The presence of HBGF-2 in LCM also was evaluated by using a d ot immunoblot assay. Extracts of luteal tissues and LCM stimulated (P < 0.05) proliferation of endothelial cells in a dose-dependent manner. Mitogenic activity of luteal extracts and LCM was decreased (P < 0.05 ) by treatment with specific antibodies against HBGF-2 or HBGF-1. LCM also contained immunoreactive HBGF-2. The mitogenic activity bound to anion exchangers, phenyl-Sepharose, and heparin-agarose, but not to ca tion exchangers, indicating that endothelial mitogenic activity is ani onic at neutral pH, has some hydrophobic characteristics, and belongs to the HBGF family of proteins. Following ultrafiltration, endothelial mitogenic activity was retained by membranes having a 30 000 or 100 0 00 molecular weight cutoff. In addition, LCM was resolved into four pe aks of heparin-binding endothelial mitogenic activity, each with a dif ferent affinity for heparin. These data demonstrate that bovine CL con tain and produce endothelial mitogens of large molecular size, which m ay be important regulators of luteal function. These endothelial mitog ens are heparin-binding and anionic at neutral pH. In addition, a larg e portion of the endothelial mitogenic activity produced by bovine CL appears to be immunologically related to HBGF-2 (basic fibroblast grow th factor).