IDENTIFICATION OF GONADOTROPIN SURGE-INHIBITING FACTOR (GNSIF) ATTENUIN IN HUMAN FOLLICULAR-FLUID/

Evidence from several laboratories suggests that the ovaries of many s pecies produce a non-steroidal factor called gonadotrophin surge-inhib iting or attenuating factor (GnSIF) which may regulate the response of the pituitary to gonadotrophin-releasing hormone (GnRH) and as such, may modulate the timing and/or amplitude of the luteinizing hormone (L H) surge. We have recently isolated a candidate GnSIF from porcine fol licular fluid (PFF). Porcine GnSIF is a 69 kDa protein which has undet ectable inhibin and follistatin immunological and biological activity. The present study was designed to purify and identify GnSIF from huma n follicular fluid, GnSIF activity was measured as suppression of GnRH -stimulated LH secretion from rat pituitary cells in primary culture. Human follicular fluid (similar to 500 ml) was recovered from patients undergoing in-vitro fertilization (IVF). GnSIF was purified by hepari n-sepharose, Q-sepharose, S-sepharose, and hydrophobic interaction chr omatography followed by isoelectric focusing. Gel electrophoresis and Western blot were used to identify human GnSIF and compare it with por cine GnSIF. Using these steps, we obtained a highly-purified preparati on of GnSIF that manifests in-vitro bioactivity and chromatography cha racteristics similar to those observed for porcine GnSIF and that hybr idizes with a porcine GnSIF antibody. Following treatment with human c horionic gonadotrophin/human menopausal gonadotrophin (HMG/HCG), human follicular fluid contained roughly 25% of the GnSIF (per mg protein) present in porcine follicular fluid. We conclude that GnSIF is present in human follicular fluid and may participate in the regulation of go nadotrophin secretion in this species.