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
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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
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GnRH stimulates the release of hCG from the human placenta in vivo (Iwashita et al., 1993) and in vitro (Li et al., 1994; Leung and Peng, 1996; Islami et al., 2001), forming an important component of a complex regulatory system involving activin and inhibin (Petraglia et al., 1996; 1998), interleukins (Nishino et al., 1990), prostaglandins (Kang et al., 1991) and steroids (Petraglia et al., 1995; Chen et al., 1998). The type 1 GnRH receptor gene is expressed in human placenta (Lin et al., 1995; Wolfahrt et al., 1998; 2001; Cheng et al., 2000), and it appears to be identical to that expressed in the human pituitary gland (Kakar and Jennes, 1995; Leung and Peng, 1996; Boyle et al., 1998). However, the apparent affinity of placental extra-pituitary receptors is too low to respond to the low concentrations of hypothalamic GnRH present in the circulation (20–200 pmol/l; Sorem et al., 1996), suggesting that placental GnRH receptors most probably respond in an autocrine/paracrine manner to locally produced GnRH. Indeed, the human placenta secretes GnRH (reportedly in a pulsatile manner in vivo (Petraglia et al., 1994), and in vitro (Barnea and Kaplan, 1989; Barnea et al., 1991; Islami et al., 2001), and expresses the gene for GnRH (Seeburg and Adelman, 1984; Radovick et al., 1990; Duello et al., 1993; Wolfahrt et al., 1998; 2001). There are, however, reported differences in the upstream start site employed compared to that utilized in the hypothalamus (Seeburg et al., 1987; Dong et al., 1993). In addition to messenger RNA for GnRH and the type 1 GnRH receptor, GnRH II is also expressed in human placenta (Islami et al., 2001) and granulosa–lutein cells (Kang et al., 2001). Moreover, different isoforms of GnRH (GnRH, GnRH II and salmon GnRH representing the three main phylogenic branches of GnRH; Fernald and White, 1999) not only bind to human placental membranes (Bramley et al., 1992; Siler-Khodr and Grayson, 2001b), but activate various signalling pathways in human placental cells (Keun Kang et al., 2000; Kleine et al., 2000; Siler-Khodr and Grayson, 2001a; Wolfahrt et al., 2001) and modulate human placental function (Islami et al., 2001; Siler-Khodr and Grayson, 2001a), as do post-translationally modified forms of GnRH (Gautron et al., 1992; Currie et al., 1993). Furthermore, sub-types of the GnRH receptor have been described in mammalian species including primates (Troskie et al., 1998; Santra et al., 2000; Millar et al., 2001; Neill, 2002), and GnRH-receptor splice variants are capable of modulating GnRH-receptor signalling in extra-pituitary tissues (Grosse et al., 1997; Kottler et al., 1999). The unusual specificity of placental GnRH receptor binding (Bramley et al., 1992) may indicate expression of other GnRH receptor types in placenta that might respond differently to the type 1 receptor with other GnRH isoforms.
Whilst investigating the presence of other forms of GnRH in placental extracts, we observed that incubation of GnRH isoform and agonist tracers with human term placental cytosol fractions led to their rapid and extensive degradation (Bramley and Menzies, 2000; Bramley et al., 1999). We also observed the formation of a high molecular weight peak of bound tracer. GnRH-binding proteins have been described in the serum of some fish (Huang et al., 1991; Lovejoy et al., 1993; D’Antonio et al., 1995), but also in the circulation of a few pregnant (Siler-Khodr et al., 1997) or post-menopausal women (Davis et al., 1987). We now describe the assay and preliminary characterization of GnRH-binding protein(s) in human placental cytosol.
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Materials
All fine chemicals, enzyme inhibitors, peptides, anti-GnRH antibodies and other reagents were obtained from sources described previously (Bramley et al., 1999). GnRH II was purchased from Peninsula Laboratories, USA and [D-Trp6]GnRH ethylamide ([D-Trp6]GnRH EtA) and the GnRH antagonist ([Ac-3-4 dehydro Pro1-D-pF-Phe2-D-Trp3,6]GnRH) were obtained from Sigma–Aldrich, UK. Buserelin ([D-Ser (tBu)6]GnRH ethylamide) was from Dr J.Sandow, Hoescht AG, Germany. Other GnRH peptides were the generous gift of Dr Judy King, University of Cape Town, South Africa.
GnRH peptides were iodinated using a glucose oxidase/lactoperoxidase method, purified by chromatography on Sephadex G25 columns (Bramley et al., 1992) and checked for their ability to bind to rat pituitary and human placenta microsomes. Tracers with low binding activities were discarded. Specific activities of GnRH agonist tracers were measured by self-displacement assay of binding to rat pituitary receptors, as described previously (Bramley and Menzies, 2000). Specific activities of radiolabelled GnRH isoform preparations were measured by self-displacement assay of binding to either a conformational anti-GnRH antibody (EL14; final dilution, 1:30 000) or an anti-cGnRH II antiserum (Antibody 11.3; final dilution, 1:20 000–1:50 000; Sharp et al., 1987; 1989), as described previously (Bramley et al., 1992).
Tissues
Rat pituitaries were obtained from adult female Sprague–Dawley rats humanely euthanized with CO2 vapour. All animal handling procedures conformed to UK Home Office guidelines. Pituitary glands were quickly dissected and homogenized in ice-cold 0.3 mol/l sucrose–10 mmol/l Tris–1 mmol/l EDTA, pH 7.4 (SET medium; 2 glands/ml) using a loose-fitting all-glass Dounce homogenizer. Aliquots (2 ml) were snap-frozen in solid CO2, and stored at –20°C until required.
Human placentae were obtained from normal women giving birth by spontaneous vaginal delivery at full term (n = 4). Approval for these studies was obtained from the Human Reproduction Ethical Committee of the Lothian Health Board Hospitals Trust. Placental microsomal membranes and cytosol fractions were prepared as described previously (Bramley and Menzies, 2000) except that the final centrifugation step was performed at 100 000 g for 1 h. Aliquots of cytosol (2 ml) were stored at –80°C or in liquid nitrogen until required.
Assays
Protein was measured by the method of Lowry et al. (1951) with crystalline bovine serum albumin as a standard.
GnRH binding to placental and pituitary membranes
Specific binding of GnRH agonist and isoform tracers to human placental membranes was measured in triplicate following incubation at 20°C for 1 h. Bound hormone was recovered by polyethylene glycol precipitation (Bramley et al., 1992). Non-specific binding was measured in duplicate in the presence of 10 µg of unlabelled buserelin. Specific binding of radiolabelled GnRH analogues to rat pituitary receptors was measured as described above, except that incubation was for 4 h at 4°C.
GnRH binding and tracer inactivation by placental cytosol
Aliquots of placental cytosol were incubated in a 0.5 ml system containing 40 mmol/l Tris–HCl, pH 7.4, 1% (w/v) bovine serum albumin (BSA) or 1% BSA–0.2 mol/l citrate buffer, pH 7.4, and 100 000 cpm of GnRH isoform or analogue tracer, as indicated. Tube contents were transferred immediately to a pre-calibrated 18x0.8 cm column of Sephadex G25 (fine) swollen in 1% BSA–Tris or BSA–citrate, pH 7.4. The void volume (Dextran Blue; Vo) and total volume (Na[125I]; Vt) of each column were measured before and after chromatography of GnRH tracers. Fractions (16 or 20 drops) were eluted with ice-cold BSA–Tris or BSA–citrate buffer and counted for 125I in a Packard ‘Crystal’ gamma counter at an efficiency of 75%. Controls always included chromatography of GnRH tracer alone, and a zero-time control (ice-cold cytosol to which tracer was added and then applied immediately to the G25 column). Radioactivity recovered in peaks 2 and 3 in zero-time and ‘minus cytosol’ controls was subtracted from that recovered in these peaks following incubation with cytosol, and corrected for total tracer added to the incubation.
In some experiments, incubates were treated with ice-cold dextran-coated charcoal (DCC), centrifuged (5000 g for 10 min at 0°C) before fractionation on Sephadex G25 chromatography or thin layer chromatography (TLC).
Thin layer chromatography
Aliquots (5–20 µl) of incubates or column fractions from the different radioactivity peaks were spotted onto polyethyleneimine cellulose TLC plates, dried under a stream of cold air, and developed in 0.1 mol/l ammonium bicarbonate, pH 7.8. TLC plates were dried, wrapped in plastic film and placed in a Molecular Dynamics (Symantech, USA) phosphor-imager cassette for 3–10 days as described previously (Bramley et al., 1999).
Statistics
The significance of differences between means was calculated by Student’s t-test, with the Bessel correction for small samples. P < 0.05 was considered to be significant.
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Chromatography of GnRH tracers
Chromatography of [D-Trp6]GnRH ethylamide ([D-Trp6]GnRH EtA) tracer on Sephadex G25 demonstrated a major peak (peak 1) that was eluted significantly later than the total volume of the column (Figure 1A). Only small amounts of radioactivity (<2% of total counts) were eluted in either the void volume (Vo) or the total volume (Vt) of the column (Figure 1A; arrows). However, following incubation of tracers at 20°C with non-denatured human term placental cytosol fraction, two new major tracer peaks appeared. One eluted in the total column volume (peak 2) and a second eluted in the void volume (peak 3; Figure 1A). If [D-Trp6]GnRH EtA tracer was incubated with boiled human term placental cytosol (Figure 1A; triangles), or if incubation with cytosol took place on ice (data not shown), elution profiles were indistinguishable from profiles of tracer alone, or zero-time controls (where tracer was added to placental cytosol and fractionated immediately). Essentially identical results were obtained when cytosol was incubated with GnRH II tracer (Figure 1B: Note that different fraction sizes were collected for GnRH II and [D-Trp6]GnRH EtA tracer in this experiment—see legend. All experiments thereafter routinely collected 16 drop (1 ml) fractions to allow optimal resolution of peaks).
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) human placental cytosol (10 µl). Fractions (20 drops for A; 16 drops for B) were eluted with ice-cold Tris–BSA buffer, and counted for 125I. Triangles indicate elution profiles following incubation of tracers with 10 µl of human placental cytosol that had been boiled for 10 min. Radioactivity peaks are as described in the text. Arrows indicate Vt = column total volume; Vo = column void volume.Identity of tracer in peaks 2 and 3
We have shown previously that the peak of GnRH II tracer that eluted in peak 2 did not bind either to a conformational or to a GnRH II-specific antibody (Bramley et al., 1999). It was incapable of binding to rat pituitary or human placental GnRH receptors (T.A.Bramley and G.S.Menzies, unpublished data), and behaved on high-power liquid chromatography (HPLC) or TLC quite differently to intact GnRH tracer (Bramley et al., 1999). To establish whether the material bound in the high molecular weight peak (peak 3) was intact or degraded GnRH, we subjected all three tracer peaks to TLC (Figure 2). Intact [D-Trp6]GnRH EtA (Figure 2A) and GnRH II tracers (Figure 2B) remained close to the origin (Tc; Rf < 0.05). Incubation of tracers with cytosol (20°C for 1 h) led to the appearance of a second spot with mobility (GnRH II; Rf = 0.45 ± 0.05; [D-Trp6]GnRH EtA, Rf = 0.10 ± 0.06) that was distinct from that of the intact hormone (+). Tracer eluted in peak 1 had an identical chromatographic mobility to intact tracer (Figure 2A, B) as expected. The mobility of tracer eluting in peak 2 was similar to that of the major novel products generated following incubation with cytosol (+; Figure 2A, B). However, tracer eluting in the void volume (peak 3) appeared to have a mobility that was similar to that of both peak 1 and intact unincubated tracer (Figure 2A, B), and (unlike tracer from peak 2) was capable of binding to anti-GnRH II antibody and to human placental receptors (Bramley et al., 1999; and unpublished data).
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Degradation of GnRH II and [D-Trp6]GnRH EtA tracers
[D-Trp6]GnRH EtA tracer was incubated with increasing concentrations of the human placental cytosol fraction and the proportions of intact and degraded tracer calculated after chromatography on Sephadex G25 (Figure 3A). The percentage of intact hormone decreased with increasing concentrations of placental cytosol, whilst the fraction of degraded product increased proportionately (Figure 3A). Similarly, when GnRH II tracer was incubated with two different human placental microsomal fractions (Figures 3B), and intact (Rf < 0.05) and degraded tracer (Rf = 0.45) quantified, loss of intact tracer was paralleled by the appearance of the degraded form.
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Intact (but not degraded) cGnRH II and [D-Trp6]GnRH EtA tracers are preferentially adsorbed by DCC (Bramley et al., 1999). Incubation of [D-Trp6]GnRH EtA tracer with placental cytosol, followed by treatment with ice-cold dextran-coated charcoal (DCC) prior to chromatography on Sephadex G25 greatly reduced the magnitude of peak 1 (Figure 4A, B) in a dose-dependent manner, but had little effect on the magnitude of peaks 2 and 3. DCC also reduced radioactivity recovered in peak 1 with GnRH II tracers (Figure 4C) in a dose-dependent manner, but had little effect on the magnitude of peaks 2 and 3.
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), peak 2 (
) or peak 3 (shaded columns) following post-incubation treatment with 0, 10, 30 or 100 µl of DCC was calculated relative to controls with no added DCC (means ± SEM for three separate experiments). The proportions of GnRH II tracer recovered in peak 1 (Effects of protease inhibitors on degradation of GnRH II and [D-Trp6]GnRH EtA tracers
Incubation of GnRH II and [D-Trp6]GnRH EtA tracers with placental cytosol in the presence of increasing concentrations of metallo- and/or thiol-peptidase inhibitors (EDTA, N-ethylmaleimide, thimerosal) or bacitracin strongly inhibited degradation of GnRH II and GnRH agonist tracers (Bramley and Menzies, 2000). We confirmed and extended our previous data, demonstrating that the loss of peak 1 and the formation of peak 2 following incubation of placental cytosol with [D-Trp6]GnRH EtA tracer was inhibited in a dose-dependent and reciprocal manner with increasing concentrations of N-ethylmaleimide (N-EM; Figure 5A, B) or thimerosal and bacitracin (data not shown). In contrast, inhibitors of serine proteases (benzamidine, trypsin inhibitor, antipain, leupeptin, phenylmethylsulphonyl fluoride [PMSF]) or carboxypeptidase (pepstatin A) had little or no effect on the magnitude of peak 2 (data not shown). N-EM (and thimerosal) also reduced binding of [D-Trp6] GnRH EtA tracer (peak 3) somewhat, but only at concentrations that abolished degradation (Figure 5B).
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), 5 mmol/l; (
), 3 mmol/l. (B) The magnitude of bound (N-EM also inhibited degradation of GnRH II tracer (measured as formation of peak 2) and increased the amount of intact tracer measured either by TLC (Figure 5C) or Sephadex G25 chromatography (Figure 5D). However, in contrast to its inhibitory effects on formation of peak 3 from [D-Trp6]GnRH EtA (Figure 5B), N-EM enhanced the formation of peak 3 from GnRH II tracer in a dose-dependent manner (Figure 5D).
Formation of peak 3 from GnRH II was also strongly stimulated in a dose-dependent manner by inclusion of citrate (an inhibitor of a GnRH II-degrading placental peptidase; Bramley et al., 1999) in the incubation medium (Figure 6A). Moreover, in the presence of citrate, the divalent metal ion chelating agent EDTA further increased formation of peak 3 from GnRH II tracer (Figure 6B). All further studies with GnRH II tracer therefore included citrate as the incubation buffer.
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Incubation characteristics
Degradation (peak 2; Figure 7A, C, E) and binding (peak 3; Figure 7B, D, F) of both GnRH II and [D-Trp6]GnRH EtA tracers by placental cytosol increased with duration of incubation (Figure 7A, B) and increasing placental cytosol concentration (Figure 7C, D), and was dependent on incubation pH (Figure 7E, F). However, formation of peak 2 (tracer degradation; Figure 7A, C, E) was significantly lower with [D-Trp6]GnRH EtA than with GnRH II tracers. Levels of binding (peak 3) achieved were similar with the two tracers (Figure 7B, F), though binding of GnRH II increased more rapidly (Figure 7B) and reached higher levels than with [D-Trp6]GnRH EtA tracer at each cytosol concentration tested (Figure 7D).
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Hormone specificity
Formation of peaks 2 and 3 was inhibited by a number of GnRH-related peptides. Their effects on binding and degradation, however, did not necessarily parallel one another (Figure 8). Thus, dogfish GnRH (Figure 8B) and the GnRH agonist nafarelin (Figure 8C) reduced D-Trp6]GnRH EtA tracer binding by 30 and 50% respectively, but inhibited degradation by 90% (Table I). A Scatchard plot derived from binding curves with increasing GnRH II tracer indicated a single class of binding sites with a Ka of
300 nmol/l (Figure 8D; R2 = 0.8).
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Both binding (Figure 9A) and degradation (Figure 9B) of [D-Trp6]GnRH EtA tracer were reduced dose-dependently by unlabelled [D-Trp6]GnRH EtA or a GnRH antagonist.
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Ligand binding of [D-Trp6]GnRH EtA tracer to cytosol proteins
Human placental cytosol proteins were precipitated by increasing concentrations of ammonium sulphate (0–80% saturation) and subjected to non-denaturing sodium dodecyl sulphate–polyacrylamide gel electrophoreseis. Proteins were transferred to nitrocellulose membranes and incubated with [D-Trp6]GnRH EtA tracer for 1 h at 20°C. After washing to adsorb unbound tracer, membranes were dried and radioactivity located by phosphor-imaging (Figure 0
). A strong band was observed at 80–90 kDa in unfractionated cytosol (lane 1), and in material precipitated by ammonium sulphate concentrations of >40%. Fainter bands were also apparent at 200, 60 and 30–40 kDa (Figure 0). Incubation with 10 µg/ml of unlabelled [D-Trp6]GnRH EtA abolished these bands (data not shown).
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Discussion |
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Chromatography on Sephadex G25 resolves (highly retarded) mono-iodinated GnRH isoform and analogue tracers from their non-iodinated forms (Bramley et al., 1992) and from degraded GnRH tracers (Bramley et al., 1999). Adsorption to Sephadex G25 varies for different GnRH analogues and isoforms (Bramley et al., 1992), and may be due to hydrophobic and/or ionic interactions with the gel. Using this simple technique, we have shown previously that GnRH-like immuno- and receptor-binding activity in non-denatured human placental extracts could be accounted for largely by tracer degradation by placental peptidases (Bramley et al., 1999; Bramley and Menzies, 2000). Other GnRH-degrading activities have been described in the human placenta, and proteolysis of GnRH may play an important role in regulation of hCG secretion (Siler Khodr et al., 1989; Kang and Siler-Khodr, 1992; Kikkawa et al., 2002). The present report extends our previous studies of GnRH tracer degradation by human placental cytosol. In particular, both GnRH II and GnRH agonist tracers were degraded rapidly in a time-, temperature- and pH-dependent fashion by human placental cytosol. Degrading activity was heat-labile, and was abolished by inhibitors of placental proteases in a concentration-dependent manner. Unlabelled GnRH analogues and isoforms decreased tracer degradation in a dose-dependent manner, presumably by competing for the substrate-binding site(s) of degrading peptidase(s).
However, during the course of these studies we observed that incubation of GnRH tracers with placental cytosol gave rise to a high molecular weight peak on gel exclusion chromatography. Contamination of cytosol fractions by human placental microsomes is unlikely in view of the high centrifugal force employed in preparing cytosol fractions (100 000 g for 1 h). Levels of human placental surface-membrane markers (epidermal growth factor receptor, alkaline phosphatase, GnRH agonist binding, etc.) in cytosol fractions were extremely low or undetectable (Menzies and Bramley, 1992). Moreover, using polyethyleneglycol precipitation (which efficiently recovers microsomal-sized placental membrane vesicles), we failed to demonstrate binding of GnRH tracers to placental cytosol fractions (Menzies and Bramley, 1992; Bramley et al., 1999). Furthermore, whereas placental membrane receptors showed a rank order of binding potency of GnRH agonist = GnRH 
GnRH II > cGnRH I >> GnRH antagonists (Bramley et al., 1992), GnRH antagonist had a similar potency to that of GnRH, GnRH II and GnRH agonists for cytosolic GnRH-binding sites (Table I). Hence, we believe that cytosolic GnRH-binding sites cannot be accounted for by contamination by GnRH receptor-bearing placental membrane vesicles.
Tracer associated with peak 3 appeared to be intact GnRH tracer by TLC (Figure 2) [and by HPLC and the ability to bind to receptor and/or anti-GnRH antibody (T.A.Bramley and G.S.Menzies, data not shown)]. Without chemical analysis, it is not possible to state unequivocally that tracer associated with peak 3 is bound intact GnRH tracer, nor to rule out that it has been modified in ways that do not affect its antigenicity, receptor binding or behaviour on TLC. However, if binding of GnRH tracers to peak 3 did require prior cleavage, binding should be inhibited under conditions where degradation was blocked. This was not so. Indeed, under conditions where degradation of [D-Trp6]GnRH EtA tracer was markedly inhibited by N-EM, the magnitude of peak 3 was largely unaffected (Figure 5B). On the contrary, GnRH II binding was dramatically increased in the presence of levels of inhibitors (N-EM, Figure 5D; citrate, Figure 6A; EDTA, Figure 6B) that strongly inhibited degradation. Furthermore, the increase in GnRH II binding to peak 3 with increasing N-EM concentrations closely paralleled the fraction of intact GnRH II rather than degraded GnRH II tracer (compare Figure 5C, D).
As expected, [D-Trp6]GnRH EtA tracers were degraded more slowly than GnRH II (Figure 7A, C, E). However, at equivalent cytosol concentrations, GnRH II tracer bound more rapidly (Figure 7B), and reached higher steady-state binding levels (Figure 7D) than [D-Trp6]GnRH EtA, suggesting that once bound, GnRH II dissociated only slowly and was protected from further proteolytic attack. This could also be inferred by the lack of effect of DCC on the magnitude of peak 3, in the face of extensive depletion of unbound tracer (Figure 4A–C).
Binding and degradation of both GnRH tracers were affected by a range of GnRH-like peptides (Figures 8, 9; Table I) in a dose-dependent manner. However, there was no significant correlation between their effects on binding and degradation (r2 = 0.027). Whilst lamprey GnRH 1 (Tyr3, Leu5, Glu6, Trp7, Lys8 GnRH) appeared to have little effect on tracer degradation (Table I), dogfish GnRH (His5, Trp7, Leu8 GnRH) (and nafarelin and GnRH antagonist) greatly reduced degradation. Both lamprey 1 and dogfish (as well as salmon GnRH) possess a Trp7 group, suggesting that this residue is not a major determinant for inhibition of degradation. It will be of great interest to extend these results to other GnRH peptides, and in particular, to examine the effects of step-wise replacement of the residues in lamprey GnRH 1 by the appropriate residues of dogfish GnRH. These studies may enable the identification of analogues with increased binding affinity and/or enhanced resistance to degradation by placental peptidases. Such analogues may be useful in defining the role of GnRH-binding proteins and peptidases in the human placenta.
Radioligand blots of undenatured placental cytosol [and proteins partially purified by precipitation with >40% (NH4)2SO4] clearly demonstrated a high molecular weight band (Mr, 90 kDa), with fainter bands at 200, 60 and 30–40 kDa (Figure 0
B). All these bands were abolished by the inclusion of GnRH agonist (Figure 0A). Specific GnRH-binding proteins have been described in the serum of some fish (Huang et al., 1991; Lovejoy et al., 1993; D’Antonio et al., 1995) and in the circulation of some pregnant (Siler-Khodr et al., 1997) or post-menopausal women (Davis et al., 1987) that have a similar binding affinity to the cytosolic factor described herein. Future work will establish the cellular localization of these proteins in the placenta. Their further purification and isolation will extend these preliminary studies of ligand specificity.
Degrading enzymes that rapidly inactivate GnRH (Figure 7A) and binding protein(s) that can complex GnRH peptides (for transport?) and/or protect them from inactivation add a further layer of complexity to the role of GnRH peptides and receptors in the human placenta, and suggest that there is still much to be learned concerning the role of GnRH in the regulation of human placental function.
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Acknowledgements |
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We should like to thank Dr J.King, Dr H.Urbanski and Prof. P.Sharp for their generous gifts of anti-GnRH antibodies, Drs J.Sandow and J.King for GnRH analogues and peptides, and the staff of the Department of Obstetrics & Gynaecology and the Labour Ward of the Simpson Memorial Maternity Pavilion, Edinburgh for the provision of human placental tissue.
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REFERENCES |
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