Journal Article

A 60–66 kDa protein with gonadotrophin surge attenuating factor bioactivity is produced by human ovarian granulosa cells

Paul A. Fowler, Tarja Sorsa-Leslie, Phillip Cash, Bryan Dunbar, William Melvin, Yvonne Wilson, Helen D. Mason and William Harris

in MHR: Basic science of reproductive medicine

Published on behalf of European Society of Human Reproduction and Embryology

Volume 8, issue 9, pages 823-832
Published in print September 2002 | ISSN: 1360-9947
Published online September 2002 | e-ISSN: 1460-2407 | DOI: http://dx.doi.org/10.1093/molehr/8.9.823
A 60–66 kDa protein with gonadotrophin surge attenuating factor bioactivity is produced by human ovarian granulosa cells

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We aimed to confirm the ovarian site of gonadotrophin surge-attenuating factor (GnSAF) production and produce granulosa/luteal cell-conditioned medium (G/LCM) containing GnSAF for purification studies. Blue dye affinity chromatography followed by pseudochromatofocusing of G/LCM yielded bioactive fractions at pH 5.74 and 5.77. The former had a major 60–66 kDa band with an internal amino acid sequence of EPQVYVHAP following tryptic digestion. A rat polyclonal antiserum (rPAb) raised against this band completely blocked in-vitro GnSAF bioactivity in human follicular fluid, serum and G/LCM. GnSAF bioactivity was localized to a 64 kDa band of serum-free G/LCM and following 2D gel electrophoresis, one of the spots recognized by Western blotting with the GnSAF rPAb had an N-terminal amino acid sequence of NH-XVPQGNAXXN. Neither amino acid sequence had significant homology with proteins in the human genome database. When ovarian tissues from spontaneously cycling women were cultured under serum-free conditions, neither theca- nor stroma-conditioned media contained GnSAF bioactivity. However, granulosa cell-conditioned medium significantly reduced GnRH-induced LH secretion, an effect that was reversed by incubation with the GnSAF rPAb. In conclusion, we have confirmed that human granulosa cells produce GnSAF within the ovary and have two candidate amino acid sequences for GnSAF. We have also demonstrated that serum-free granulosa cell culture constitutes the method of choice for the characterization of GnSAF since recovery of bioactivity is superior in the presence of fewer serum proteins.

Keywords: FSH; GnRH; granulosa cells; LH; pituitary

Journal Article.  7685 words.  Illustrated.

Subjects: Reproductive Medicine

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