Human Reproduction, Vol. 14, No. 6, 1449-1456,
June 1999
© 1999 European Society of Human Reproduction and Embryology
Purification of a candidate gonadotrophin surge attenuating factor from human follicular fluid
1 Department of Pharmacology and 2 Laboratory of Biological Chemistry, University of Ioannina Medical School, Greece, 3 Protein & Peptide Group, EMBL, Heidelberg, Germany and 4 Department of Obstetrics and Gynaecology, University of Thessalia Medical School, 22 Papakiriazi Str., 412 22 Larissa, Greece
Gonadotrophin surge attenuating factor (GnSAF) is a new non-steroidal ovarian substance, different from inhibin, which attenuates the pre-ovulatory luteinizing hormone (LH) surge in superovulated women. Human follicular fluid (FF) was used as a source for the isolation of GnSAF, the activity of which was monitored in an in-vitro pituitary bioassay. Primary rat pituitary cells were incubated with test substances for 48h and subsequently washed and incubated with 0.1µmol/l gonadotrophin releasing hormone (GnRH) plus test substances for 4 h. GnSAF activity was expressed as the reduction of GnRH-induced LH secretion in the 4 h incubation. GnSAF was purified from 250 ml of FF which was heat-treated at 80°C for 5 min. Heparin-sepharose chromatography, Con-A sepharose chromatography, reversed-phase high-performance liquid chromatography (HPLC) and preparative native gel electrophoresis were used for GnSAF fractionation. Using these purification steps, we have obtained an apparently homogeneous preparation that stains as a single band on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. GnSAF has an apparent molecular weight of 12.5 kDa and was identified by amino acid sequence (mass spectrometry) to be the C-terminal fragment of human serum albumin.
Key words: gonadotrophin surge attenuating factor/human follicular fluid/luteinizing hormone
5 Present address: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado, USA
6 To whom correspondence should be addressed
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