Purification of a candidate gonadotrophin surge attenuating factor from human follicular fluid

Gonadotrophin surge attenuating factor (GnSAF) is a new non-steroidal ovari an substance, different from inhibin, which attenuates the pre-ovulatory lu teinizing hormone (LH) surge in superovulated women. Human follicular fluid (FF) was used as a source for the isolation of GnSAF, the activity of whic h was monitored in an in-vitro pituitary bioassay. Primary rat pituitary ce lls were incubated with test substances for 48 h and subsequently washed an d incubated with 0.1 mu mol/l gonadotrophin releasing hormone (GnRH) plus t est substances for 4 h. GnSAF activity was expressed as the reduction of Gn RH-induced LH secretion in the 4 h incubation. GnSAF was purified from 250 mi of FF which was heat-treated at 80 degrees C for 5 min. Heparin-sepharos e chromatography, Con-A sepharose chromatography, reversed-phase high-perfo rmance liquid chromatography (HPLC) and preparative native gel electrophore sis were used for GnSAF fractionation. Using these purification steps, we h ave obtained an apparently homogeneous preparation that stains as a single band on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, G nSAF has an apparent molecular weight of 12.5 kDa and was identified by ami no acid sequence (mass spectrometry) to be the C-terminal fragment of human serum albumin.