Molecular Human Reproduction, Vol. 5, No. 8, 777-783,
August 1999
© 1999 European Society of Human Reproduction and Embryology
Effects of alcohol on the human placental GnRH receptor system
Department of Obstetrics and Gynaecology, Division of Reproductive Medicine and Development, 37 Chalmers Street, Edinburgh EH3 9EW, Scotland, UK
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Isolation of human term placental membranes in the presence or absence of protease inhibitors indicated that protease inhibitors significantly reduced the amounts of [125I]-labelled gonadotrophin-releasing hormone (GnRH) binding to membrane GnRH-receptors in vitro by ~20%. This decrease was largely due to the ethanol used to dissolve the serine protease inhibitor, phenylmethylsulphonylfluoride (PMSF). Ethanol alone decreased the specific binding of [125I]-labelled GnRH isoform (IC50, 7.9 ± 0.8 mg/ml; n = 6) or agonist tracers (IC50, 10.0 ± 1.4 mg/ml; n = 6) to human placental membranes in a dose-dependent manner. Other alcohols also interfered with [125I]-GnRH isoform or agonist binding: inhibition increased with increasing carbon chain length and was dependent on the isomeric position of the hydroxyl group. Fractionation of term placental cytosol by gel chromatography demonstrated the presence of a high molecular weight fraction (~60–70 kDa) which inhibited [125I]-GnRH binding to human placental membranes. However, placental cytosol fractions did not cross-react significantly with a specific anti-GnRH antibody. Surprisingly, re-assay of cytosol fractions in the presence of a cocktail of protease inhibitors generated a factor (molecular weight ~40–50 kDa) which did cross-react strongly with the GnRH antibody. The generation of this factor was due to the ethanol solvent rather than to the protease inhibitors per se, as treatment of pooled `latent' cytosol fractions with ethanol alone generated GnRH-like immunoactivity (irGnRH) which competed in parallel with GnRH standard. The amount of irGnRH generated depended on the concentration of ethanol added to the `latent' cytosol fractions. However, ethanol had no effect on the assay in the absence of cytosol fraction, or with inactive cytosol fractions. Thus, ethanol can perturb the human placental GnRH/GnRH-receptor system in vitro in two distinct ways: by inhibition of GnRH binding to receptor, and by dissociation of complexed endogenous GnRH-like factor(s) from a GnRH-binding protein. It is postulated that high alcohol consumption in vivo may interfere with placental GnRH secretion/action and affect placental secretion of factors important to the establishment and maintenance of pregnancy.
fetal alcohol syndrome (FAS)/GnRH-binding protein/pregnancy/trophoblast
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Introduction |
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Gonadotrophin-releasing hormone (GnRH) plays an important role in a local, closed-loop feedback system in the human placenta involving the GnRH-receptor, human chorionic gonadotrophin (HCG), inhibin, activin, and pro-inflammatory cytokines (Petraglia et al., 1987
, 1989, 1994; Zhuang et al., 1991; Keelan et al., 1998) which operates in a paracrine/autocrine manner to control placental hormone secretion (Siler-Khodr et al., 1991; Currie and Leung, 1993; Keelan et al., 1994, 1998; Wolfahrt et al., 1998).
Ethanol is a teratogenic agent (Kaufman, 1997) which freely passes through the placenta and enters fetal tissues. In rodents, ethanol ingestion affects cell function in a variety of organs, including lung, liver, pituitary, nervous system and brain, and affects blastocyst outgrowth and trophoblast formation (Stachecki et al., 1994), giving rise in rats to defects in placental histology (Padmanabhan, 1985; Eguchi et al., 1989; Seyoum and Persaud, 1995). Increased infertility and spontaneous abortion rates have been observed in women who drink heavily (Bradley et al., 1998), and chronic alcohol consumption prior to conception and during pregnancy can lead to abnormal fetal growth and may give rise in ~10% of women who drink heavily during pregnancy to fetal alcohol syndrome (FAS). FAS is a leading cause of mental retardation (Abel, 1990; Shibley and Pennington, 1997), affecting ~1% of newborns (Sampson et al., 1997).
Although placental weight is unaffected in women who are alcohol abusers, birth weights and amniotic fluid steroid concentrations are decreased (Westney et al., 1991). Alcohol is known to suppress proliferation of trophoblast cells whilst stimulating their differentiation (Karl et al., 1996), and acute exposure of placental cells to moderate ethanol concentrations in vitro affects the placental secretion of steroids, prostaglandins, cytokines and HCG (Ahluwalia et al., 1992; Wimalasena, 1994; Randall and Saulnier, 1995; Wimalasena et al., 1994; Karl et al., 1998; Svinarich et al., 1998). Altered cytokine expression is known to be an important factor in some forms of spontaneous abortion (Shaarawy and Nagui, 1997).
Whilst studying the effects of protease inhibitors on human placental GnRH receptors, a decrease was observed in GnRH binding associated with the inclusion of ethanol in isolation media. This led to a study of the effects of alcohol on the human placental GnRH system in greater detail. Some novel and potentially important effects of alcohol(s) on the human placental GnRH system are reported.
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Materials and methods |
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All fine chemicals, enzyme inhibitors and reagents were from either Sigma (UK) (Poole, Dorset, UK) or BDH (Poole, Dorset, UK). Radiolabelled sodium iodide (Na125I) and [125I]-labelled and unlabelled recombinant human epidermal growth factor (rhEGF) were from Amersham International plc, (Amersham, Bucks, UK). Chicken GnRH II (cGnRH II) was purchased from Peninsula Laboratories (Belmont, CA, USA). The GnRH agonist buserelin ([D-Ser (tBu)6] 1–9 GnRH ethylamide) was the kind gift of Dr J.Sandow, Hoescht AG, Frankfurt, Germany. All other GnRH analogues were purchased from Sigma. Anti GnRH antibody (Ab 10.2; Sharp et al., 1987
) was the generous gift of Professor Peter Sharp, Roslin Institute (Midlothian, Scotland, UK).
Preparation and measurement of specific activities of [125I]-labelled GnRH tracers
GnRH peptides were radioiodinated by a glucose oxidase/lactoperoxidase method, and purified on Sephadex G25 columns as described previously (Bramley et al., 1992). Specific activities of radiolabelled GnRH isoform preparations were estimated by self-displacement assay of binding to cGnRH II antibody (85–1100 Ci/g; n = 9 separate cGnRH II isoform preparations), whilst specific activities of GnRH agonist tracers were estimated by self-displacement assay of binding to homogenates or membrane fractions of immature female rat pituitary glands (350–1105 Ci/g, n = 12 preparations).
Placentae
Human placentae were obtained by spontaneous vaginal delivery or by elective Caesarian section at term from normal women. Placental villous tissue was dissected free of other tissues, and washed extensively in ice-cold isotonic phosphate-buffered saline (PBS; Flow Laboratories, Irvine, Scotland) to reduce blood contamination. Villi were minced, weighed and homogenized (Polytron homogenizer with two 10 s bursts at full speed, separated by a 1 min cooling period in ice) in 5 ml/g of either ice-cold 0.3 mol/l sucrose–10 mmol/l Tris–HCl–1 mmol/l EDTA, pH 7.4 (SET buffer alone; Protocol 1) or SET containing 5 mmol/l EDTA–1 mmol/l N-ethylmaleimide (N-EM)–1 mmol/l phenylmethylsulfonylfluoride (PMSF)–10 µg/ml pepstatin A (Protocol 2). PMSF stock solutions were freshly prepared as 100x stock solutions in ethanol, and added immediately before homogenization. After filtration through four layers of cheesecloth, homogenates were centrifuged at 1000 g for 10 min (4°C) to remove nuclei and cell debris. Supernatants were re-centrifuged at 100 000 g for 60 min in a Sorvall OTD-50 refrigerated ultracentrifuge (4°C), and microsomal pellets obtained were gently rehomogenized (5–10 strokes in a loose Dounce homogenizer) in the appropriate SET medium with or without protease inhibitors. After recentrifugation at 100 000 g, microsomes were resuspended in SET without protease inhibitors and stored in 2 ml aliquots at –70°C or in liquid nitrogen until required. Cytosol fractions were stored in 5 ml aliquots at –20°C.
Assays
Protein was measured by the Lowry method using crystalline bovine serum albumin (BSA) as a standard (Lowry et al., 1951) .
Receptor binding
Specific binding of radiolabelled GnRH agonist and GnRH isoforms to human placental membranes was measured by incubation of triplicate aliquots (0.5–1.0 mg protein/tube) at 20°C for 1 h in a 0.5 ml incubation system containing 40 mmol/l Tris–HCl, pH 7.4, 0.5% BSA and 100 000 cpm of [125I]-labelled GnRH tracer. Non-specific binding was measured in duplicate in the presence of 10 µg of unlabelled buserelin. Bound hormone was recovered by polyethyleneglycol (PEG) precipitation (Bramley et al., 1992), and pellets counted for [125I] in a Packard `CobraTM II' 
-counter at an efficiency of 75%. The difference between binding in the presence and absence of unlabelled GnRH agonist represented specific binding (normally 15–35% of total counts added). Controls without tissue, with and without unlabelled GnRH agonist, were included to correct for displacement of tracer from assay tubes by cold analogue (usually 0.5–1% of total counts added). Specific GnRH binding was corrected for protein content of the placental membrane fraction used, and is reported as pg GnRH bound/mg membrane protein.
[125I]-labelled rhEGF binding
Specific binding of [125I]-labelled rhEGF to placental microsomes was measured as described previously (Bramley and Menzies, 1992).
Statistical analysis
Statistical significance was estimated by Student's t-test (paired or unpaired) with Bessel's correction for small numbers, or by Wilcoxon's rank order test. P < 0.05 was considered to be statistically significant.
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Results |
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Effects of preparation protocol on [125I]-GnRH binding to placental membranes
Overall, villous membranes prepared from human term placentae in the absence of a protease inhibitor cocktail (Protocol 1) tended to have higher specific binding of [125I]-GnRH agonist (239 ± 29 pg/mg membrane protein; n = 27) than membranes prepared in the presence of inhibitors (Protocol 2; 187 ± 24 pg/mg membrane protein; n = 32). Because of the variability in GnRH binding values between different term placental membrane preparations, this difference did not reach statistical significance (P < 0.2). However, preparation of villous membranes from six term placentae by both protocols in parallel demonstrated that inclusion of inhibitors (Protocol 2) significantly reduced [125I]-GnRH binding values by ~20% (mean binding, 218 versus 186 pg/mg membrane protein; P < 0.05 by Student's paired t-test).
To establish which protease inhibitor(s) was responsible for the reduction in binding, placental membranes were incubated with each inhibitor, alone or together, at the final concentrations used in Protocol 2. No significant inhibition was observed with EDTA (metalloprotease inhibitor) or pepstatin A (carboxypeptidase inhibitor; Table I). N-ethyl maleimide (N-EM; thiol protease inhibitor) decreased GnRH binding, but this effect failed to reach significance (P > 0.05). PMSF (a serine protease inhibitor) decreased GnRH-binding to the same value as all four inhibitors together (Table I). However, PMSF was added as an ethanolic solution immediately prior to incubation. Surprisingly, ethanol controls decreased placental GnRH binding to the same extent as PMSF (Table I).
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Effects of other alcohols on GnRH binding
Ethanol inhibited specific binding of radiolabelled cGnRH II (IC50, 7.9 ± 0.8 mg/ml; n = 6) and agonist tracers (IC50, 10.0 ± 1.4 mg/ml; n = 6) to term placental microsomes in a dose-dependent manner (Figure 1
). Other alcohols also decreased the specific binding of GnRH isoform (Figure 2A) or agonist tracers in a dose-dependent manner (Figure 2B). Inhibitory activity was related to both carbon chain length and to the position of the hydroxyl group (Table II). These alcohols also inhibited the specific binding of [125I]-rhEGF to placental membranes: however, compared with their effects on GnRH binding, higher alcohol concentrations were required to inhibit EGF binding, and the different isomers of butanol had similar potencies for placental EGF receptors, in contrast to their effects on GnRH binding (Table II).
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) or buserelin (•) in the absence, or in the presence of increasing concentrations of ethanol. Non-specific binding was measured in duplicate at each ethanol concentration by the inclusion of excess unlabelled GnRH agonist (10 µg buserelin/tube). After 2 h at 20°C, bound hormone was precipitated by ice-cold immunoglobulin G (IgG)/polyethyleneglycol, and specific binding calculated and expressed as percentage of controls with no ethanol. Values are given as mean ± SEM for 4–6 separate experiments in triplicate.
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= methanol; • = ethanol; 
= n-butanol; and 
= n-penterol. Non-specific binding was measured in duplicate at each ethanol concentration by the inclusion of excess unlabelled GnRH agonist (10 µg/tube). After 2 h at 20°C, bound hormone was precipitated by ice-cold immunoglobulin G/polyethyleneglycol, specific binding was calculated and expressed as percentage of controls with no ethanol. Values are given as mean ± SEM for 2–5 separate experiments in triplicate.
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Effects of ethanol on GnRH-like activity of placental cytosol
Gel exclusion chromatography of a human placental cytosol fraction, followed by assay of each fraction for inhibition of [125I]-GnRH binding to placental membranes and GnRH immunoactivity, indicated a broad peak of receptor-active material (molecular weight, ~60–70 kDa; Figure 3A
), but no GnRH-like immunoactive material was observed (Figure 3B; open circles). However, radioimmunoassay of these same cytosol fractions in the presence of the cocktail of the protease inhibitors used in Protocol 2 and Table I
generated a clear peak of GnRH-like activity (molecular weight, ~40–50 kDa; Figure 3B; solid symbols). Once again, this effect was not due to the protease inhibitors themselves (Table III), but to the ethanol solvent in which PMSF was dissolved. Treatment of `active' pooled cytosol fractions (Fractions 25–32; Figure 3B) with increasing ethanol concentrations generated increasing levels of an immunoreactive GnRH-like factor (irGnRH; Figure 4). The irGnRH formed by treatment of pooled `latent' fractions (fractions 25–32) with ethanol competed for binding of GnRH tracer to anti-GnRH antibody in a dose-dependent fashion, and in parallel to GnRH standard (Figure 5). Ethanol had no effect on the radioimmunoassay standard curve. Moreover, `inactive' fractions (fractions 45–50; Figure 3B) failed to generate irGnRH when treated with ethanol (Figure 5).
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Discussion |
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This study showed that treatment of human term placental membranes with alcohols in vitro significantly affected the placental GnRH system. Firstly, addition of protease inhibitors to membrane isolation media significantly decreased amounts of GnRH receptors in human term placental microsomal fractions. Investigation of the effects of each inhibitor alone on GnRH binding in vitro indicated that the effect was due to the ethanol solvent used to dissolve the serine protease inhibitor, PMSF. Ethanol decreased binding of radiolabelled cGnRH II and GnRH agonist to human placental villous membranes in vitro in a dose-dependent manner, and the concentrations required to give 50% inhibition of GnRH binding (IC50, ~10 mg/ml) were similar to those shown to affect placental function in other studies (Schenker et al., 1989
; Karl and Fisher, 1993; Wimalasena, 1994; Wimalasena et al., 1994; Svinarich et al., 1998). Significant effects were observed at or below the UK legal limits for blood alcohol concentrations (Figure 1). Other alcohols also inhibited [125I]-labelled GnRH binding in a dose-dependent manner, and their inhibitory effects increased with increasing chain length, suggesting that inhibition depended on hydrophobicity. These alcohols also inhibited EGF binding to placental membranes, though (despite the much lower placental membrane concentrations used in the EGF receptor assay) higher alcohol concentrations were required for inhibition (Table II). Moreover, inhibition of radiolabelled GnRH (but not EGF) binding was affected by the position of the hydroxyl group. These different effects of alcohols on GnRH and EGF binding suggest that inhibition was unlikely to be due to a general perturbation of membrane structure.
Secondly, ethanol acts in a dose-dependent manner on partially-purified human placental cytosol fractions to generate a high molecular weight factor irGnRH-like activity. Although a high molecular weight (60–70 kDa) GnRH-receptor binding inhibitor was observed in untreated cytosol fractions, placental cytosol fractions had no GnRH-like immunoactivity until activated by ethanol treatment (Figure 3B). Fractions eluting on either side of this `latent' GnRH-like peak had no immunoactivity even in the presence of ethanol (Figure 3B, Figure 5 and unpublished data). Furthermore, radioimmunoassay standard curves were unaffected by the addition of ethanol, emphasizing that ethanol itself did not interfere in the GnRH radioimmunoassay.
One interpretation of this data is that the `latent' peak contains an endogenous GnRH-like factor(s) complexed with a GnRH-binding protein(s). Ethanol treatment appears to disrupt this binding, releasing the bound irGnRH-like factor, which can then interact with the antibody. Indeed, an earlier study (Flanaghan et al., 1996) characterized a high molecular weight (67 kDa) GnRH-binding protein in extracts of some extrahypothalamic tissues, whilst other authors (Siler-Khodr et al., 1997) described a GnRH-binding protein, present in the serum of a minority (three out of 39) of pregnant women, with characteristics resembling those of an antibody to GnRH, and which was precipitable by ethanol. We have other data also indicating the presence of a GnRH-binding protein in some placental extracts (Mullen and T.A.Bramley, unpublished data).
If an endogenous GnRH-like factor(s) is normally associated with a GnRH-binding protein in the blood of pregnant women, the procedures used to extract GnRH from plasma or serum may affect the subsequent measurement of GnRH immunoreactivity (Kawamura et al., 1985; Petraglia et al., 1994; Sorem et al., 1996), and may account for some of the differences between these studies. It will be of interest to examine the presence of the immunoactive GnRH-like factor(s) in placental extracts from normal and abnormal pregnancies at different gestations, using different extraction, processing and GnRH assay procedures.
The current studies have examined the effects of ethanol only in vitro. However, the fact that changes were observed at ethanol concentrations similar to legal UK driving limits suggest they may be physiologically relevant. GnRH is an important part of the autocrine/paracrine regulation of placental secretion of hormones and cytokines (Siler-Khodr et al., 1986a,b,c; Currie and Leung, 1993; Keelan et al., 1994; Svinarich et al., 1998; Wolfahrt et al., 1998). Given the importance of HCG in the rescue of the corpus luteum of pregnancy in the human, and the known interference of alcohol with cytokine networks (Deaciuc, 1997), we speculate that disruption of GnRH/GnRH-receptor interactions by ethanol, either directly, by inhibition of the GnRH receptor (Figure 1), or indirectly, by disrupting binding of endogenous (ir)GnRH to binding protein (Figure 3B and Figure 5) could interfere with placental function, affecting the processes of implantation, luteal rescue and/or maternal recognition of pregnancy, and leading eventually to pregnancy loss or fetal damage. It will now be important to examine amounts of placental GnRH receptors in women with different levels of alcohol consumption during pregnancy, and to correlate placental GnRH binding with exposure in vivo to different patterns of alcohol intake and with pregnancy outcome. Furthermore, since extrapituitary GnRH receptors have been described in several other human tissues and cancers (Qayum et al., 1991; Kakar and Jennes, 1995; Emons et al., 1998), heavy drinking may also disturb GnRH autocrine/paracrine systems in other tissues and contribute to the development of other pathological processes.
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Acknowledgments |
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We are most grateful to our clinical colleagues in the Simpson Memorial Maternity Pavilion for the collection of term placentae, to Dr Peter Sharp (Roslin Institute, Roslin, Scotland) for the generous gift of anti-GnRH antisera, Dr J.Sandow (Frankfurt, Germany) for the GnRH agonist, buserelin, and to T.Pinner and T.McFetters for their photographic expertise.
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Notes |
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1 To whom correspondence should be addressed
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Submitted on March 3, 1999; accepted on May 7, 1999.
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