Molecular Human Reproduction, Vol. 9, No. 5, 291-300,
May 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Human placental GnRH-like factors: II. Inhibition of enzymatic degradation of GnRH-II and [D-Trp6]GnRH ethylamide tracers by human term placental cytosol fractions reveals the presence of GnRH-binding protein(s)
Submitted on November 28, 2002; accepted on January 29, 2003
1 Division of Reproductive & Developmental Sciences and 2 The University of Edinburgh Medical School, The Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK
3 To whom correspondence should be addressed. e-mail: tbramley{at}staffmail.ed.ac.uk
We describe the preliminary characterization of GnRH-binding protein(s) in human placental cytosol. Samples were analysed by chromatography on Sephadex G25. Radiolabelled GnRH and its analogues elute significantly later than the total column volume (Vt) on Sephadex G25 column chromatography. However, incubation of GnRH II or GnRH agonist tracers with human placental cytosol reduced the intact tracer peak, with the concomitant appearance of a new peak eluting in the total column volume (Vt). This peak increased with increasing cytosol concentration and duration of incubation, and probably represented degraded GnRH tracer, since (i) degradation-resistant GnRH agonist tracer, [D-Trp6]GnRH EtA, was inactivated more slowly than GnRH II, (ii) boiling of cytosol fractions abolished formation of this peak and (iii) peptidase inhibitors blocked its formation. A second new tracer peak eluted in the column void volume (Vo) and was largely unaffected by peptidase inhibitor concentrations that blocked tracer degradation. The magnitude of this high molecular weight peak depended on the GnRH tracer employed, cytosol concentration, and the pH, duration and temperature of incubation. Tracer associated with this third peak appeared similar to intact GnRH tracer by TLC. Unlabelled GnRH analogues and isoforms decreased both tracer degradation and formation of the Vo peak, but their specificity and affinity for the two processes differed. Ligand blots identified several bands that were abolished by inclusion of unlabelled agonist during incubation. Our data indicate the presence of specific GnRH binding protein(s) and GnRH peptidases that may modulate local actions of GnRH in the human placenta.
Key words: GnRH analogue/GnRH-binding protein/GnRH II/GnRH peptidase/trophoblast