Production of mitogenic factor(s) by ovine corpora lutea throughout the estrous cycle.

Abstract

To evaluate secretion of mitogenic factors by ovine corpora lutea (CL) at several stages of development, luteal explants from days 5 (n = 12 ewes), 10 (n = 6 ewes), and 15 (n = 6 ewes) of the estrous cycle were incubated in serum-free medium for 24 h. Luteal-conditioned media (LCM) were evaluated for their ability to stimulate proliferation of endothelial, BALB/3T3, and ovarian granulosa cells. After mitogenic activity of LCM from individual animals was evaluated, pools of LCM from each day of the estrous cycle were subjected to anion-exchange, cation-exchange, and heparin-affinity chromatography to characterize mitogenic activity. Pools of LCM also were utilized for ultrafiltration, heat-treatment, trypsin-treatment, and immunoneutralization studies. Results demonstrated that ovine CL secrete mitogenic activity that stimulates (P less than 0.01) proliferation of endothelial (135.7 +/- 5.3% of control) and granulosa cells (188.9 +/- 2.9%) but not 3T3 (103.2 +/- 2.5%) cells. Differences between stages of luteal development were not observed. The mitogenic activity bound to diethylaminoethyl-Sephacel and heparin-agarose, but not to carboxymethyl-Sepharose, indicating that ovine luteal mitogenic factor(s) is anionic and may belong to the heparin-binding growth factor (HBGF) family. In addition, the mitogenic activity was heat-labile, trypsin-sensitive, and appeared to have a M(r) greater than 100,000. Mitogenic activity for endothelial cells was partially neutralized with a specific antibody against HBGF-1 and was completely abolished with a specific antibody against HBGF-2. Moreover, HBGF-1 and HBGF-2 were immunolocalized in histological sections of CL from days 5 (n = 5 ewes), 10 (n = 5 ewes), and 15 (n = 5 ewes) after estrus. These findings are the first report of a major mitogenic factor(s) produced by cyclic ovine CL and indicate this factor is an HBGF-2-like molecule.