INITIAL CHARACTERIZATION OF MITOGENIC ACTIVITY OF OVINE CORPORA-LUTEAFROM EARLY-PREGNANCY

To characterize angiogenic factors produced by ovine corpora lutea (CL ) during early pregnancy, two experiments were performed. In Experimen t 1, luteal explants from days 12, 18, 24, and 30 (n = 4 ewes/day) aft er mating were incubated in serum-free medium for 6 h. Luteal-conditio ned media (LCM) were evaluated for their ability to stimulate prolifer ation of endothelial and 3T3 cells, as well as migration of endothelia l cells. Pools of the LCM (one pool/day) then were characterized bioch emically. In Experiment 2, two pools of LCM from day 24 of pregnancy w ere evaluated for their effects on endothelial cell, 3T3 cell, and ovi ne luteal cell proliferation. These pools of LCM then were concentrate d by ultrafiltration and subjected to heparin-agarose affinity chromat ography with salt gradient (0-4 M NaCl in buffer) elution, and fractio ns were evaluated for mitogenic activity for endothelial and 3T3 cells . The resulting five peaks of mitogenic activity from heparin-agarose chromatography were characterized biochemically. The five peaks of mit ogenic activity were further purified by using chromatography, then we re concentrated and subjected to SDS-PAGE and Western analysis for FGF -2. Ovine CL from each day of early pregnancy secreted mitogens (P<0.0 5) for endothelial (285+/-8% of unconditioned media controls) and 3T3 (142+/-7%) cells as well as factors which stimulated migration of endo thelial cell (153+/-8% of controls). LCM pool from day 24 of pregnancy also stimulated (P<0.05) proliferation of ovine luteal cells in a dos e-dependent manner. In Experiment 1, mitogenic activity for endothelia l cells was greater than 100 kDa, heat-labile, trypsin-sensitive and b ound to DEAE-Sephacel and heparin-agarose columns, but not to a CM-Sep harose column. Antibody against FGF-1 did not affect mitogenic activit y of LCM for endothelial and 3T3 cells, whereas treatment with FGF-2 a ntibody decreased (P<0.05) mitogenic activity of LCM for both endothel ial and 3T3 cells. In Experiment 2, heparin-agarose affinity chromatog raphy resolved five peaks of mitogenic activity: a non-heparin-binding peak that was specific for 3T3 cells, three heparin-binding peaks tha t were specific for endothelial cells, and one heparin-binding peak th at was specific for 3T3 cells. In Experiment 2, heparin-, heat-, or tr ypsin-treatment and immunoneutralization with FGF-1 or FGF-2 antibodie s influenced mitogenic activity of all of the peaks. Whereas SDS-PAGE demonstrated several bands of protein within each peak, Western analys is was unable to detect the presence of FGF-2 in any of the heparin-bi nding peaks. These data demonstrate that ovine CL from early pregnancy produce mitogenic factors that can be resolved into 5 separate peaks of activity with differing affinities for heparin. These data also ind icate that the endothelial mitogens produced by CL of early pregnancy are immunologically related to, but biochemically distinct from FGF-2. Mitogens for endothelial and other cells likely play a role in regula tion of luteal function during early pregnancy in sheep.