Corpora lutea induced by gonadotrophin-releasing hormone treatment of anoestrous Welsh Mountain ewes: reduced sensitivity to luteinizing hormone in vivo and to chorionic gonadotrophin in vitro.

Seasonally anoestrous Welsh Mountain ewes received 250 ng gonadotrophin-releasing hormone (GnRH) every 2 h, with (Group 1; n=13) or without (Group 2; n=14) progesterone priming for 48 h. Fourteen control ewes (Group 3) were studied during the luteal phase in the breeding season. Animals in Group 4 (n=12) received progesterone priming followed by 250 ng GnRH at increasing frequency for 72 h, while ewes in Group 5 (n=13) were given three bolus injections of 30 microg GnRH at 90-min intervals. All treatment regimens induced ovulation. However, only corpora lutea (CL) from ewes in Group 3 (breeding season) or Group 4 exhibited normal luteal function. Luteal luteinizing hormone (LH) receptor levels were significantly higher on day 12 than day 4, and CL from groups with adequate CL (3 and 4) had significantly higher (125)I-human chorionic gonadotrophin (hCG)-binding levels than the three groups with inadequate CL on day 12. LH-binding affinity was unchanged. Exogenous ovine LH (10 microg) in vivo on days 3 or 11 after ovulation induced a pulse of progesterone in ewes with adequate CL: however, ewes in Groups 1, 2 and 5 showed no significant response. Basal progesterone secretion in vitro was significantly greater on day 4 than on day 12. Maximal steroidogenic responses of adequate and inadequate CL to hCG and to dibutyryl cyclic-3',5'-AMP were similar at both stages of the luteal phase. However, the EC50 for hCG on days 4 and 12 was 10-fold lower for groups with an adequate CL (0.1 IU hCG/ml) than for inadequate-CL groups (1 IU hCG/ml; P <0.05). Thus, in addition to the well-characterized premature sensitivity of GnRH-induced inadequate CL to endometrial luteolysin, we have shown (1) a marked decrease in total number of cells in the CL, a profound reduction in vascular surface area, and a decrease in mean large luteal cell volume (with no change in large luteal cell numbers), (2) decreased luteal LH receptor and progesterone content compared with adequate CL and (3) that CL that were becoming, or were destined to become, inadequate failed to respond to ovine LH in vivo and were 10-fold less sensitive to hCG in terms of luteal progesterone secretion in vitro.

Reduced LH sensitivity in vivo and in vitro of corpora lutea induced during anoestrus by GnRH, and during the late breeding season, in Scottish Blackface ewes.

Scottish Blackface ewes were synchronised in mid-breeding (November; group 1; n=12 ewes) or late-breeding season (March; group 2; n=16). Anoestrous ewes (May) were treated with progestagen sponges for 7 days and then given 250 ng GnRH 3-hourly for 24 h, 2-hourly for 24 h and hourly for a further 24 h (group 3; n=12). A second group of anoestrous ewes (group 4, n=19) received three bolus injections (30 microg) of GnRH at 90-min intervals without progestagen pretreatment. After ovulation, ewes were bled twice daily until slaughter (day 4 or day 12: oestrus=day 0). Mid-breeding season (group 1) and anoestrous ewes in group 3 formed 'adequate' corpora lutea (CL) with high plasma progesterone levels (3-4 ng/ml) maintained for at least 12 days, and responded in vivo to ovine LH (oLH) (10 microg) with a rise in plasma progesterone on day 11 (group 3, but not group 1, ewes also responded on day 3). CL minces from these ewes responded to human chorionic gonadotrophin (hCG) in vitro with a dose-dependent increase in progesterone secretion. Ewes in group 4 had a foreshortened luteal phase (8-10 days) and low plasma progesterone levels (approximately 1 ng/ml), consistent with formation of inadequate CL. LH injection failed to induce a significant plasma progesterone increase. Furthermore, although progesterone secretion in vitro in response to maximally stimulating doses of hCG or dibutyryl cAMP (dbcAMP) was similar to that in adequate CL, the sensitivity of these CL to hCG (EC (effective concentration)50, 1 IU hCG/ml) was reduced 10-fold compared with adequate CL (EC50, 0.1 IU hCG/ml; P<0.01). Ewes that ovulated in the late breeding season (group 2) had high plasma progesterone, although levels began to decrease after day 10. Injection of oLH in vivo increased plasma progesterone. However, sensitivity to hCG in vitro (EC50, 0.5 IU hCG/ml) was intermediate between that of adequate luteal tissue (groups 1 and 3; EC50, 0.1 IU/ml) and that of group 4 ewes (EC50, 1 IU hCG/ml). Our data demonstrate a markedly reduced luteal sensitivity to LH in vivo and hCG in vitro in Scottish Blackface ewes with inadequate CL, and suggest that a similar loss of sensitivity to LH may occur in the late breeding season.

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).

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 (V(t)) 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 (V(t)). 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-Trp(6)]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 (V(o)) 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 V(o) 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.

Non-genomic steroid receptors in the bovine ovary.

Over the last few years, rapid and physiologically important non-genomic actions of all classes of steroid hormones have been described in many cell types. A putative non-genomic membrane progesterone receptor (NGPR) was the first, and so far the only, non-genomic steroid receptor cloned. Two homologous NGPR proteins have been identified in the human, and a similar protein in the bovine and rat. Various detection methods have been used to identify putative NGPRs in a range of tissues: however, different methods often yield quite different molecular weights, and probably detect distinct moieties. We describe some properties of the specific cell-surface membrane binding sites for [3H]-progesterone in enriched cell membrane preparations of bovine luteal and follicular cells. Similar binding sites were also detected in cell-membranes of some (but not all) bovine tissues. Western blots of detergent extracts of bovine luteal membranes identified a protein (85kDa) that reacted with an antiserum to the N-terminal peptide of porcine NGPR. Activity was low in native non-denatured extracts, but increased dramatically in a dose-dependent manner following pretreatment with the cholesterol-complexing agent, digitonin. This protein was co-precipitated by antisera to caveolin. In contrast, a specific monoclonal antibody to the ligand binding domain of the genomic progesterone receptor (Mab C262) detected two proteins (M(r), 55 and 60kDa) in luteal membrane detergent extracts. Immunostaining of these proteins by Mab C262 was abolished by digitonin concentration-dependent manner in non-denatured extracts. However, both proteins were unaffected by digitonin in fully denatured detergent extracts, suggesting that digitonin induced a conformational change in the native protein that prevented binding of Mab C262 to its epitope. Our data suggest the presence of a complex of two or more distinct membrane-associated progesterone-binding proteins in bovine luteal membranes. Moreover, their conformations are specifically affected by removal of bound cholesterol.

Human placental gonadotrophin-releasing hormone-like factors: an artefact of human placental peptidases?

Non-denatured human placental cytosol fractions displaced tracer binding in parallel with gonadotrophin-releasing hormone (GnRH) isoform and agonist peptides in GnRH-specific radioimmunoassays and radioreceptor assays. However, placental immuno- and receptor binding-GnRH-like activity was highly correlated with inactivation of GnRH tracers, suggesting that placental GnRH-like factors may be an artefact of ligand degradation during assay. The properties and inhibitor sensitivities of the major (125)I-labelled GnRH-degrading enzymes of term placental cytosol were studied using a dextran-coated charcoal (DCC) adsorption assay as a rapid screen for GnRH tracer inactivation. Three different activities were demonstrable: (i) a cathepsin D-like enzyme (M(r) 55 kDa), active against all radiolabelled GnRH isoforms and agonists tested, optimal at acid pH, and inhibited specifically by pepstatin; (ii) a metallo-thiol endopeptidase activity (M(r) 70 kDa) optimal at alkaline pH (7-9) which degraded GnRH isoforms to a greater extent than GnRH analogues, inhibited dose-dependently by low concentrations of thiol reagents (N-ethylmaleimide, thimerosal), chelating agents (o-phenanthroline, EDTA), and by tosyl-phenylalanyl-chloromethyl ketone but not by other serine protease inhibitors; and (iii) a bacitracin-sensitive enzyme optimal at physiological pH. These observations permitted the development of a robust radioreceptor assay which minimized GnRH tracer degradation. Under these assay conditions, the GnRH-like radioreceptor assay activity of human placental cytosol fractions was markedly reduced.

Human placental GnRH-like factors: parallel displacement in GnRH immuno- and receptor-binding assays can be caused by degradation of radiolabelled GnRH tracers.

Human term placental cytosol fractions decreased the specific binding of gonadotrophin-releasing hormone (GnRH) isoform tracers to placental membranes (and to rat pituitary GnRH receptors and anti-GnRH antibodies) in a dose-dependent manner, and in parallel to GnRH standard curves. However, cytosol fractions had little or no effect on the binding of two GnRH superagonist tracers. The specificity of placental binding sites for a range of GnRH-like and unrelated peptides was shown to be similar with GnRH isoforms or GnRH agonists as binding ligands, suggesting that isoforms and agonists did not bind to different forms of the GnRH-receptor. Inclusion of a cocktail of protease inhibitors during the preparation of placental cytosol significantly reduced immuno- and receptor-binding activity. Moreover, incubation of radiolabelled chicken GnRH II with placental cytosol led to marked inactivation of tracer, as assessed by radioreceptor and radioimmunoassays for GnRH, high resolution liquid chromatography, thin layer chromatography and adsorption to dextran-coated charcoal and other matrices. There was a good negative correlation between tracer degradation and apparent GnRH immuno- and receptor-binding activities. These results emphasize the important effects which proteases in un-denatured tissue extracts can have on radioreceptor and radioimmunoassays due to inactivation of peptide tracers, and suggest that previous measurements of receptor- and immuno-active GnRH-like factors may have been over-estimated due to peptidase action during the GnRH assay.

Stimulation of specific binding of [3H]-progesterone to bovine luteal cell-surface membranes: specificity of digitonin.

Non-genomic actions of progesterone have been described in the ovary, and luteal membranes of several species have been shown to possess specific binding sites for [3H]-progesterone. However, binding of radiolabelled progesterone to luteal membranes was demonstrable only in the presence of digitonin. Digitonin is a non-ionic detergent which is thought to act by forming one-to-one complexes with certain sterols. It is also a cardiotonic agent, inhibiting (Na+-K+) ATPase activity by interaction with the extracellular (ouabain/K+) binding site. We therefore investigated which properties of digitonin were responsible for its stimulatory actions on progesterone binding to bovine luteal membranes. A range of compounds with detergent, cardiotonic and or cholesterol-complexing activities were tested for their effects on [3H]-progesterone binding to bovine luteal membrane fractions, and on haemolysis of rat erythrocytes. Stimulation of progesterone binding to luteal membranes was highly specific for digitonin, and a number of ionic and non-ionic detergents, cardenolides, saponins and cholesterol-complexing reagents tested failed either to stimulate [3H]-progesterone binding to bovine luteal membranes in the absence of digitonin, or to inhibit binding specifically in the presence of digitonin. When digitonin was first reacted with excess cholesterol or pregnenolone to form the respective digitonides, stimulatory activity was greatly reduced, suggesting that the ability of digitonin to interact with (an) endogenous steroid(s) may be important in its action. High performance liquid chromatography (HPLC)-mass spectrometry of commercially available digitonin preparations indicated the presence of numerous minor impurities in most commercial digitonin preparations. Three major UV-absorbing peaks were isolated and characterised by mass spectrometry: all stimulated progesterone binding to bovine luteal membrane receptors in a dose-dependent manner, though to differing extents. Our data suggest that the unique action of digitonin on luteal membrane progesterone receptors is not related to its detergent or cardiotonic properties, but appears to be related to its ability to complex with membrane sterols.

Effects of alcohol on the human placental GnRH receptor system.

Isolation of human term placental membranes in the presence or absence of protease inhibitors indicated that protease inhibitors significantly reduced the amounts of [(125)I]-labelled gonadotrophin-releasing hormone (GnRH) binding to membrane GnRH-receptors in vitro by approximately 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 [(125)I]-labelled GnRH isoform (IC(50), 7.9 +/- 0.8 mg/ml; n = 6) or agonist tracers (IC(50), 10.0 +/- 1.4 mg/ml; n = 6) to human placental membranes in a dose-dependent manner. Other alcohols also interfered with [(125)I]-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 ( approximately 60-70 kDa) which inhibited [(125)I]-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 approximately 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.

Bovine ovarian non-genomic progesterone binding sites: presence in follicular and luteal cell membranes.

We have shown recently that the bovine corpus luteum (CL) possesses specific luteal cell surface membrane binding sites for progesterone. We have now confirmed and extended these observations to compare the subcellular distribution of these binding sites in developing, mature and regressing CL. The median buoyant densities of luteal progesterone binding sites from early-, mid- and late-luteal phase CL were similar (though three of five density profiles for late-luteal phase CL showed association of steroid binding with a fraction with a lower density), and clearly resolved from nuclear, mitochondrial, lysosomal, peroxisomal, Golgi-endoplasmic reticulum-lysosomal and smooth endoplasmic reticulum markers. Specific binding of [3H]progesterone overlapped with the distributions of 5'-nucleotidase and luteinizing hormone receptor (luteal cell surface membrane markers) in both control and digitonin-treated gradients at all stages of the luteal phase. Since steroidogenic 'large luteal' and 'small luteal' cells of the CL are derived from the granulosa cells (GC) and theca of the preovulatory follicle, we also investigated whether similar receptors were present in the follicle, and describe for the first time specific membrane binding sites for progesterone in purified GC and thecal membranes from healthy bovine follicles of different sizes. Specific binding increased linearly with GC and thecal membrane protein concentration; however, it was detectable only when digitonin was included in the binding incubation. Binding sites were specific for progesterone; unlabelled progesterone competed for [3H]progesterone binding at low concentrations (IC50, 35 and 31 nmol/l) compared with testosterone (IC50, 905 and 870 nmol/l) and delta4-androstenedione (IC50, 1050 and 660 nmol/l) for GC and thecal receptors respectively. In contrast, oestradiol, oestrone, pregnenolone, cortisol, cholesterol, and a genomic progesterone receptor antagonist, RU486, competed poorly. Steroid binding was present in GC and thecal membranes of follicles of all sizes, but [3H]progesterone binding to GC membranes decreased significantly with increasing follicle size (P<0.02), perhaps indicating developmental regulation of GC membrane non-genomic progesterone receptors in the preovulatory bovine follicle. We suggest that these membrane steroid receptors may be involved in the autocrine/paracrine regulation of follicular function by progesterone.

Specific non-genomic, membrane-localized binding sites for progesterone in the bovine corpus luteum.

Fractionation of bovine corpus luteum (CL) homogenates on continuous sucrose density gradients with and without preincubation with 3H-progesterone demonstrated high levels of tracer binding and high content of endogenous progesterone associated with particulate membrane fractions. Analysis of gradient fractions for a range of luteal plasma membrane and intracellular organelle marker enzyme activities indicated that endogenous progesterone content and 3H-progesterone-binding activity were associated with fractions enriched in luteal plasma membrane markers. This was confirmed by pretreatment of homogenates with the saponin, digitonin, prior to fractionation. Digitonin perturbed the buoyant density of luteal surface membrane markers and 3H-progesterone binding to a similar extent, but did not perturb the buoyant densities of other intracellular markers to the same degree. Interestingly, digitonin pretreatment also increased the proportion of progesterone tracer that entered the gradients. We consistently failed to demonstrate significant binding of 3H-progesterone to membrane fractions incubated with progesterone tracer in vitro. However, when digitonin was included in the in vitro binding assay, we observed a dramatic, dose-dependent stimulation of 3H-progesterone binding by digitonin. Other radiolabeled steroids tested (3H-cortisol, 3H-testosterone) bound poorly in the presence or absence of digitonin. 3H-Progesterone binding in the presence of optimal digitonin concentrations increased linearly with increasing luteal membrane concentration; was dependent on the pH, duration, and temperature of incubation; and low levels of progesterone (68 nM) competed for tracer binding. A range of other steroids tested (androgens, estrogens, corticosteroids, steroid precursors) competed at higher concentrations (10- to 100-fold) or did not compete at all for 3H-progesterone binding. There was no correlation between the hydrophobicity of various steroids and their ability to compete for binding. Moreover, a number of agonists and antagonists specific for the genomic progesterone receptor, agonists of peripheral benzodiazepine receptors, and inhibitors of a range of steroidogenic enzymes did not compete for 3H-progesterone binding. Western blots confirmed that detergent-solubilized progesterone-binding sites could be resolved from cytochrome P450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase. Moreover, extraction of bound steroid from the binding site and HPLC demonstrated identity to progesterone, suggesting that no metabolism of the progesterone tracer had occurred during incubation. Progesterone binding to membranes of large luteal cells was higher compared with binding to small luteal cells, and levels were similar in membranes prepared from CL at all stages of the luteal phase. We suggest that bovine luteal progesterone-binding sites may play a role either in sequestration of newly synthesized progesterone or in the mediation of autocrine and/or paracrine actions of progesterone in the CL.

Co-purification of a ribonuclease and human chorionic gonadotrophin beta-core protein from human urine and displacement of 125I-human luteinizing hormone from Candida albicans binding sites by ribonucleases.

An 18 kDa pregnancy urine protein preparation, purified to apparent electrophoretic homogeneity as judged by silver-staining of polyacrylamide gels, inhibited binding of 125I-hLH (human luteinizing hormone) to Candida albicans microsomes, reacted with monoclonal and polyclonal antibodies raised against human chorionic gonadotrophin (hCG) beta-core protein and exhibited ribonuclease (RNase) activity. Eleven of the 12 amino acids at the N-terminus of a protein in this preparation were identical to those of the N-terminus of human non-secretory ribonuclease. These results indicate co-purification of hCG beta-core with a RNase. An 18 kDa RNase was also purified from a commercial hCG preparation (Chorulon). However, no RNase activity was detected in a highly purified commercial preparation (Profasi). Three commercial RNase preparations displaced 125I-hLH from C. albicans binders at extremely low concentrations (< 0.001 microg/ml RNase) whereas only slight displacement of 125I-hLH from sheep luteal binding sites was observed with very high concentrations of the RNases (100 microg/ml RNase). The co-purification of hCG beta-core and RNase from pregnancy urine and the displacement of 125I-hLH from C. albicans binding sites by RNases may be related to the close relationship that has been identified between mammalian RNase inhibitors and the extracellular domain of gonadotrophin receptors. The presence of RNase in commercial preparations of gonadotrophins should be borne in mind during any investigations that involve impure preparations of these hormones.

Measurement of luteal and placental gonadotrophin-releasing hormone (GnRH) binding sites: role of inactivation of GnRH tracer.

The ability of radiolabelled gonadotrophin-releasing hormone (GnRH) and GnRH analogues to bind to homogenates and membranes of rat, rabbit and sheep pituitary and to luteal homogenates and membranes from a number of species was measured. In addition, inactivation of tracer during binding incubation was estimated by measurement of the ability of the unbound tracer fraction to bind to fresh GnRH-receptor or anti-GnRH antibody. Following incubation of rat, sheep and rabbit pituitary gland with a radiolabelled GnRH agonist tracer, appreciable amounts of specific binding of GnRH agonist to fresh rat pituitary or human placental GnRH receptors could still be demonstrated. In contrast, no specific binding of [125I]-labelled GnRH analogues were measured following incubation of the tracer with homogenates or membranes of bovine, sheep and porcine luteal tissue, nor with rabbit and rat placental homogenates. However, during incubation of GnRH tracers with these tissues, almost complete inactivation of GnRH tracers occurred. There was an inverse relationship between binding and inactivation of [125I]-labelled GnRH for a number of human corpora lutea. We conclude that degradation of GnRH tracers may prevent the measurement of specific GnRH binding sites in some tissues.

Human placental gonadotrophin-releasing hormone (GnRH) binding sites: III. Changes in GnRH binding levels with stage of gestation.

We have measured the levels of gonadotrophin-releasing hormone (GnRH) binding sites in human placental villous membrane fractions obtained at different stages of gestation. There was a marked decrease in the specific activity of 125I-labelled GnRH binding to membrane fractions obtained between 10-20 weeks gestation, but there was no change in either affinity or ligand specificity of these binding sites. The observed decrease in binding was not due to contamination of placental villous membranes by membranes from other tissues, since there was no gestation-dependent decrease in the specific activity of epidermal growth factor receptor or alkaline phosphatase activity in villous membrane fractions between 10-20 weeks of gestation. Furthermore, incubation of GnRH tracer with membranes from different stages of gestation, followed by re-incubation of the unbound tracer fraction with fresh membranes, demonstrated unequivocally that decreased GnRH binding to 10-20 week membranes was not due to increased degradation of GnRH tracer. We conclude that the observed changes in GnRH receptor levels between 10-20 weeks gestation must reflect either decreased expression/synthesis (or increased catabolism) of placental GnRH receptors, or increased occupancy (or down-regulation) of placental GnRH receptors by an endogenous GnRH-like ligand.

Isolation of cell populations from the mare corpus luteum: comparison of mechanical and collagenase dissociation.

Corpora lutea were obtained from mares at days 3, 10 and 14 after ovulation, and examined histologically. The morphology of isolated luteal cells obtained by either mechanical or collagenase dissociation of the tissue was examined and the cells stained to detect the steroidogenic enzyme delta 5, beta-hydroxysteroid dehydrogenase. The ratio of large:small cells was significantly higher for cells obtained from mechanically dissociated luteal tissue than for cells obtained by collagenase dissociation (P < 0.01). Cells obtained by both mechanical and collagenase dissociation secreted progesterone, although neither cell population responded to exogenous gonadotrophin with an increase in progesterone secretion. Homogenates of equine luteal tissue bound 125I-labelled human LH with high affinity and specificity, and the specific activity and binding affinity of luteal LH receptors did not change significantly from day 3, to days 10 and 14 after ovulation. However, mechanically dissociated cells on days 10 and 14 bound significantly more LH than did collagenase-dissociated cells on these days (P < 0.05). These results indicate that (i) collagenase dissociation of mare luteal tissue yields a population of cells that is unrepresentative of the corpus luteum, and (ii) the mare corpus luteum is not responsive to LH in vitro at the stages examined.

Specific binding sites for progesterone in subcellular fractions of the porcine corpus luteum.

Subcellular fractionation of porcine corpus luteum (CL) homogenates on continuous sucrose gradients has previously demonstrated that most of the endogenous progesterone of the CL was associated with a unique particulate fraction. Exogenous radiolabelled steroids were also sequestered with some specificity by this fraction. We now report that this particulate fraction is capable of binding high levels of exogenous 3H-labelled progesterone (and pregnenolone) in vitro, but only in the presence of the saponin, digitonin. Binding was dependent on the pH, temperature and duration of incubation, and showed specificity and high affinity for progesterone (Kd, 79 nM). Androgens, oestrogens and pregnenolone competed for porcine luteal [3H]progesterone binding sites, but only at much higher concentrations, whereas cholesterol, a number of progesterone receptor agonist and antagonist analogues and inhibitors of 3 beta-hydroxysteroid dehydrogenase and C17-hydroxylase/C17,20-lyase did not compete. Analysis of profiles for a number of luteal cell-surface membrane and intracellular organelle markers confirmed previous studies showing the association of an NADH-cytochrome C reductase with this fraction. Moreover, the content of endogenous progesterone associated with particulate subcellular fractions isolated from porcine granulosa cell (GC) and CL homogenates at different stages of the luteal phase and early pregnancy waxed and waned with the stage of the luteal phase (and the secretory activity of the CL). Binding of [3H]progesterone in vitro equilibrated at the same buoyant density as endogenous progesterone: levels of both were highest during the mid-luteal phase and during early pregnancy, lower in early and late luteal CL, and undetectable in corpora albicantia. In contrast, relaxin secretory granules were readily resolved from progesterone binding sites. We propose that these particulate progesterone binding sites may be involved in the sequestration and/or packaging of newly-synthesized steroid for secretion by the luteal cell, or may mediate actions of progesterone within the luteal cell.

Particulate binding sites for steroid hormones in subcellular fractions of the ovine corpus luteum: properties and hormone specificity.

We have described previously the presence of binding sites in particulate fractions of the porcine corpus luteum (CL) which were specific for progesterone. We now demonstrate the presence of similar progesterone-specific binding sites in particulate fractions of the ovine CL. Preincubation of ovine luteal membranes with radiolabelled steroids demonstrated binding of progesterone and pregnenolone to a low-density particulate fraction (1.07-1.09 g/cm3). Preincubation with digitonin perturbed the buoyant density of this fraction (to 1.10-1.14 g/cm3) without causing release of steroid. Androgens and oestrogens did not bind appreciably to control luteal membranes, but were bound when preincubated with digitonin. In contrast, steroid conjugates (oestrone sulfate, pregnanediol glucuronide), cortisol, fatty acids (arachidonic acid, prostaglandin F2 alpha) and cholesterol ester failed to bind to ovine luteal membranes, with or without digitonin pretreatment. The effects of digitonin on steroid binding led us to examine its effects on steroid binding to ovine luteal membrane fractions in vitro. Specific progesterone binding was absent in the absence of digitonin, even at very high membrane concentrations. However, binding of 3H-labelled progesterone was stimulated 5-15-fold in a dose-dependent fashion by increasing digitonin concentrations, reaching a plateau at about 100 microM. In the presence of digitonin, [3H]progesterone binding increased linearly with luteal membrane concentration. Other detergents, saponins and cardiotonic steroids tested did not stimulate progesterone binding to ovine luteal membranes. [3H]Progesterone binding was dependent on the pH, duration and temperature of incubation. Unlabelled progesterone decreased binding of [3H]progesterone (half-maximal displacement of specific binding (IC50) at about 60 nM) whereas androgens were less potent (IC50, 500-3300 nM), whilst a wide range of other steroids and inhibitors of steroidogenic enzymes were ineffective, except at very high concentrations. Similarly, a number of progesterone receptor agonist and antagonist analogues failed to compete for progesterone binding to luteal membranes, suggesting that these binding sites were unrelated to progesterone receptors. Modifications to the 3, 4, 5 and 11 positions of progesterone, removal of the steroid side-chain or aromatization of the A-ring decreased binding potency dramatically, whereas changes to the 17 or 20 positions had relatively minor effects. Our results indicate the presence of a low density particulate fraction in ovine corpora lutea which contains specific binding sites for endogenous and exogenous progesterone.

Specificity studies of particulate binding sites for steroid hormones in subcellular fractions of the porcine corpus luteum.

We have studied the binding of a number of radiolabelled steroids and lipophilic substances to porcine corpus luteum (CL) particulate fractions. Following preincubation of CL homogenates with radiolabelled progesterone or pregnenolone prior to fractionation on continuous sucrose density gradients, a broad peak of binding was observed associated with a particulate fraction of buoyant density 1.05-1.10 g/cm3. Progesterone content also peaked at a similar buoyant density (1.06-1.12 g/cm3). Pretreatment of luteal homogenates with digitonin perturbed the buoyant density of the progesterone-binding particulate fraction to 1.10-1.14 g/cm3 and sharpened the binding peak. Progesterone content was also perturbed to a similar extent by digitonin pretreatment, without release of the steroid. Oestrogens were also sequestered by this fraction, but steroid precursors (cholesterol, cholesterol ester), corticosteroids (cortisol, corticosterone), sterol conjugates (oestrone sulphate, pregnanediol glucuronide) and other lipophilic substances (arachidonic acid, phospholipid, prostaglandins E1, E2 and F2 alpha) were not bound. Androgens were bound weakly by fractions from control gradients but, in the presence of digitonin, significant binding could be demonstrated. Radiolabelled steroids were shown to interact directly with luteal membrane fractions, rather than interacting first with cytosolic steroid receptors which then bound to membranes. Furthermore, [3H]progesterone was not bound by porcine granulosa cell particulate fractions. These observations suggest that this fraction may be involved in sequestration or packaging of progesterone for secretion by the luteal cell.

Human placental gonadotrophin-releasing hormone (GnRH) binding sites: I. Characterization, properties and ligand specificity.

Radioiodinated gonadotrophin-releasing hormone tracers were prepared from mammalian (m GnRH), salmon (s GnRH), lamprey (l GnRH) and the two forms of chicken GnRH (ch GnRH I and ch GnRH II), and also from the GnRH agonist (GnRHA) analogues, Buserelin ([D-Ser(tBu)6] 1-9 GnRH ethylamide) and Tryptorelin ([D-Trp6 GnRH] 1-9 ethylamide) and a GnRH antagonist (GnRHANT; [Ac 3,4-dehydro-Pro1, D-p-F-Phe2, D-Trp3,6] LHRH). Specific binding of hormone tracers was compared in homogenates and membrane fractions from human placenta and rat pituitary. GnRH agonist tracers bound readily to pituitary and placental binding sites. Binding of m GnRH to rat pituitary membranes was low compared to agonist binding, whereas other GnRH iso-forms were not bound. Binding of 125I-labelled m GnRH to human placental membranes was also low compared to that of Buserelin, and l GnRH and ch GnRH I tracers bound poorly. However, human placental membranes bound s GnRH and ch GnRH II to the same extent as GnRHA. Studies of the inactivation of GnRH tracers following incubation with rat pituitary and human placental membranes demonstrated that, although GnRH isoforms were degraded at different rates in these tissues, the differential ability of GnRH isoforms to bind to pituitary or placental binding sites was not related to differences in degradation of tracers, but rather to differences in ligand specificity. Specific binding of 125I-labelled GnRH agonists (GnRHA) and mammalian GnRH (m GnRH), s GnRH and ch GnRH II tracers to human placental membrane fractions increased linearly with increasing membrane protein at low concentrations. Binding was dependent on both the duration and temperature of incubation, and pH profiles of 125I-labelled GnRHA, s GnRh and ch GnRH II binding to placental membranes were similar. Once bound s GnRH formed a tighter complex with placental receptors than GnRHA, though 125I-labelled s GnRH was inactivated more rapidly than agonist tracer during incubation with placental membranes. Binding of GnRH tracers was specific for molecules with the GnRH structure. Deletions of amino acid residues at positions 1-3 and/or deamidation at Gly10 reduced binding potencies for both human placental and rat pituitary binding sites, indicating that both ends of the GnRH molecule were required for optimal binding. Modifications which conferred increased agonist activity led to markedly increased receptor binding potency in the rat pituitary, but only slightly increased potency for placental receptors. In contrast, GnRH antagonist analogues had increased potency towards pituitary receptors, but much reduced potency towards human placental binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Human placental gonadotrophin-releasing hormone (GnRH) binding sites: II. Comparison of binding and inactivation of 125I-labelled GnRH agonist to subcellular fractions following density gradient centrifugation.

Human placental homogenates and membrane fractions were centrifuged on continuous sucrose density gradients, with or without buoyant density perturbation of plasma-membranes by digitonin, and aliquots of each gradient fraction were assayed for a range of plasma-membrane and intracellular organelle markers, and for specific binding and inactivation of radiolabelled GnRH agonist (GnRHA), Buserelin ([D-Ser(tBu)6] GnRH 1-9 ethylamide). GnRH agonist (GnRHA) binding equilibrated in the same regions of control gradients as the plasma-membrane markers, EGF-receptor and alkaline phosphatase. Moreover, binding of 125I-labelled GnRHA and 125I-labelled chicken GnRH II (ch GnRH II) was enriched in the same regions of the gradients, indicating that both bound to the same membrane fractions. Digitonin pretreatment increased the buoyant density of all three placental plasma-membrane markers to a similar degree. Intracellular organelle markers (and hCG content) equilibrated in different regions of the gradient to placental surface-membranes, and were not perturbed appreciably by digitonin. In contrast, inactivation of 125I-labelled GnRHA was associated largely with the soluble (cytosol) fraction which failed to enter the gradient, and little tracer inactivation was observed in fractions enriched in GnRHA binding activity. Similar results were obtained with fractionated rat pituitary membranes. We conclude that: (a) placental GnRH binding sites do not represent binding of radiolabelled ligand to GnRH-degrading enzymes, (b) degradation of radiolabelled ligand is associated largely with placental cytosol fractions, and (c) GnRH binding activity appears to be associated largely with placental plasma-membranes.

Specific binding sites for 125I-labelled epidermal growth factor in the ovine corpus luteum and human placenta.

Specific, high-affinity binding sites for radiolabelled mouse epidermal growth factor (mEGF) were demonstrated in homogenates and membranes of ovine corpora lutea. Subcellular fractionation on sucrose density gradients demonstrated that luteal EGF receptors were associated with fractions enriched in cell surface-membrane markers. Binding of 125I-labelled mEGF to ovine luteal EGF receptors was dependent on the pH, temperature and duration of incubation, and on the concentration of metal ions present in the incubation medium. Unlabelled mEGF and human EGF (urogastrone) competed for the binding of radiolabelled mEGF to ovine luteal homogenates at low doses (half-maximal inhibition of binding (IC50) at 2-3 nmol/l). Transforming growth factor-alpha also competed for mEGF-binding sites (IC50, 4-10 nmol/l), but a range of peptides, growth factors and protein hormones were ineffective at much higher concentrations. Concave Scatchard plots for 125I-labelled EGF binding and Hill coefficients of < 1 for displacement radiolabelled EGF suggested negative co-operativity of binding sites, and dilution at equilibrium accelerated the rate of dissociation of 125I-labelled EGF from human placental (but not from ovine luteal) receptors. Specific EGF-binding sites were also demonstrated in rat and rabbit placental homogenates, and in luteal homogenates of the pig. Luteal concentrations of EGF receptors appeared to be reduced significantly during early pregnancy in both the pig and sheep.