Initiation of angiogenesis by porcine follicular fluid

Angiogenesis was observed and measured after injection of porcine follicular fluid into rabbit corneas. A qualitative response (0 to 6+) and quantitative measurement (mm/day) were obtained 9 days after injection. Undiluted porcine follicular fluid stimulated angiogenesis with new blood vessels visible by the third day after injection, extending 2.0 to 3.0 mm into the site of injection from the corneal scleral limbus (1 to 4+) by day 9. Angiogenic activity was consistently found in fractions of porcine follicular fluid which precipitated in 20% to 40% saturated ammonium sulfate. Sephadex gel filtration of the 20% to 40% saturated ammonium sulfate fraction resulted in fractions with molecular weights of 45,000 to 60,000 and less than or equal to 1500 daltons which stimulated angiogenesis. Charcoal treatment of active fractions did not remove angiogenic activity. Angiogenic activity was retained after heating at 56 degrees C for 1 hour but was lost after boiling (20 minutes). Quantitative measurements of chemotaxis with use of Boyden chambers and mitogenesis by means of tritiated thymidine incorporation were performed. Follicular fluid from small follicles contained greater chemotactic activity than follicular fluid from medium or large follicles. The 20% to 40% saturated ammonium sulfate precipitate that eluted through Sephadex G-100 with a molecular weight of 45,000 to 60,000 daltons contained angiogenic, mitogenic, and chemotactic activity. In conclusion, porcine follicular fluid contains angiogenic factors that may be associated with perifollicular neovascularization during folliculogenesis.