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

Paul Alfred Francois Fowler, Tarja Kristina Sorsa-Leslie, Phillip Cash, Bryan Dunbar, William Thomas Melvin, Y. Wilson, H. D. Mason, William Joseph Harris

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)823-832
Number of pages9
JournalMolecular Human Reproduction
Volume8
Issue number9
DOIs
Publication statusPublished - 2002

Keywords

  • FSH
  • GnRH
  • granulosa cells
  • LH
  • pituitary
  • follicle-stimulating-hormone
  • inhibiting factor
  • superovulated women
  • factor GNSAF
  • gel-electrophoresis
  • in-vitro
  • fluid
  • purification
  • identification
  • resolution

Cite this

A 60-66 kDa protein with gonadotrophin surge attenuating factor bioactivity is produced by human ovarian granulosa cells. / Fowler, Paul Alfred Francois; Sorsa-Leslie, Tarja Kristina; Cash, Phillip; Dunbar, Bryan; Melvin, William Thomas; Wilson, Y.; Mason, H. D.; Harris, William Joseph.

In: Molecular Human Reproduction, Vol. 8, No. 9, 2002, p. 823-832.

Research output: Contribution to journalArticle

Fowler, Paul Alfred Francois ; Sorsa-Leslie, Tarja Kristina ; Cash, Phillip ; Dunbar, Bryan ; Melvin, William Thomas ; Wilson, Y. ; Mason, H. D. ; Harris, William Joseph. / A 60-66 kDa protein with gonadotrophin surge attenuating factor bioactivity is produced by human ovarian granulosa cells. In: Molecular Human Reproduction. 2002 ; Vol. 8, No. 9. pp. 823-832.
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abstract = "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.",
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T1 - A 60-66 kDa protein with gonadotrophin surge attenuating factor bioactivity is produced by human ovarian granulosa cells

AU - Fowler, Paul Alfred Francois

AU - Sorsa-Leslie, Tarja Kristina

AU - Cash, Phillip

AU - Dunbar, Bryan

AU - Melvin, William Thomas

AU - Wilson, Y.

AU - Mason, H. D.

AU - Harris, William Joseph

PY - 2002

Y1 - 2002

N2 - 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.

AB - 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.

KW - FSH

KW - GnRH

KW - granulosa cells

KW - LH

KW - pituitary

KW - follicle-stimulating-hormone

KW - inhibiting factor

KW - superovulated women

KW - factor GNSAF

KW - gel-electrophoresis

KW - in-vitro

KW - fluid

KW - purification

KW - identification

KW - resolution

U2 - 10.1093/molehr/8.9.823

DO - 10.1093/molehr/8.9.823

M3 - Article

VL - 8

SP - 823

EP - 832

JO - Molecular Human Reproduction

JF - Molecular Human Reproduction

SN - 1360-9947

IS - 9

ER -