Identification of activins and follistatin proteins in human follicular fluid and placenta

Abstract

Follistatin, an activin-binding protein, is able to neutralize the various activities of activin by forming an inactive complex with it. The widespread tissue localization of follistatin is very similar to that of activin, which suggests that it plays a local modulatory role in the various paracrine/autocrine actions of activin. We detected significant activin-binding activities in human follicular fluid and placental homogenates, although they were much lower than those in porcine and bovine follicular fluids, which raised the possibility that follistatin is present in human follicular fluid and placenta. Therefore, we attempted to identify the protein molecules responsible for this activin-binding activity in human follicular fluid and placental homogenates. Human follicular fluid, collected from in vitro fertilization patients, was processed by affinity chromatography successive steps on sulfated gel matrices and reverse phase high performance liquid chromatography (HPLC). The final HPLC yielded abundant follistatin and almost equimolar amounts of activin-A, -AB, and -B. The follistatin protein showed characteristic multiple bands when analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ability of each band to bind activin specifically was determined by activin binding assay and ligand blotting analysis. Several pieces of evidence, including the immunoblotting analysis and functional assay results, demonstrated the presence of three activin isoforms, A, AB, and B, in the follicular fluid. In contrast, human placental homogenates were found to contain follistatin and activin-A proteins only. Activin-AB and -B were not detected in any HPLC fraction, indicating that activin-A is the major form of activin in the human placenta. The present data indicate that the three activin isoforms and multiple forms of follistatin exist in human follicular fluid, and the activin-A isoform and follistatin exist in human placenta. They suggest that the physiological functions of the activin isoforms during embryonic development differ and that follistatin plays a functional role in the local control system(s) that regulates human reproduction.