RESUMEN
The rF1+rV candidate sub-unit vaccine for plague, formulated by adsorption to alhydrogel, has been demonstrated to be immunogenic in the cynomolgus macaque in a clinically relevant dose-range (5-40 microg of each sub-unit) and regimen. Following two doses of vaccine, a specific IgG titre developed in a dose-related manner with predominance of the IgG1/IgG2 isotypes. Groups of macaques receiving only a single dose of vaccine at the 40 microg dose-level had a significantly reduced peak IgG response and faster decline to baseline. Serum collected at week 5 from 19 immunised animals competed with and displaced murine Mab7.3 from binding to the V antigen in vitro. By week 53 of the schedule, although absolute IgG titres had declined, 17/19 macaque sera tested contained competing antibody, indicating the durability of a functional immune response to rF1+rV in this species. Thirteen of these week 53 sera were passively transferred into groups of naive mice, and all conferred full or partial protection against subsequent challenge of the mice with plague. Generally, those sera which were most competitive with Mab 7.3 for binding to V antigen were fully protective by passive transfer, although one week-53 serum sample was fully protective by passive transfer but not active by competitive ELISA. The early development of protective immunity in macaques was also indicated from the protection conferred on naive mice by the passive transfer of immune macaque serum collected at 2-10 weeks of the immunisation schedule. Serum samples from representative macaques within this time period also inhibited the Yersinia-mediated cytotoxicity of J774 macrophages in a qualitative in vitro assay of type three secretion.
Asunto(s)
Inmunización Pasiva , Vacuna contra la Peste/inmunología , Peste/inmunología , Yersinia pestis/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Pruebas Inmunológicas de Citotoxicidad , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Esquemas de Inmunización , Inmunoglobulina G/inmunología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Peste/prevención & control , Proteínas Citotóxicas Formadoras de Poros/inmunología , Proteínas Recombinantes/inmunología , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Immunization with a recombinant form of the protective antigen (rPA) from Bacillus anthracis has been carried out with rhesus macaques. Rhesus macaques immunized with 25 mug or more of B. subtilis-expressed rPA bound to alhydrogel had a significantly increased immunoglobulin G (IgG) response to rPA compared with macaques receiving the existing licensed vaccine from the United Kingdom (anthrax vaccine precipitated [AVP]), although the isotype profile was unchanged, with bias towards the IgG1 and IgG2 subclasses. Immune macaque sera from all immunized groups contained toxin-neutralizing antibody and recognized all the domains of PA. While the recognition of the N terminus of PA (domains 1 to 3) was predominant in macaques immunized with the existing vaccines (AVP and the U.S. vaccine anthrax vaccine adsorbed), macaques immunized with rPA recognized the N- and C-terminal domains of PA. Antiserum derived from immunized macaques protected macrophages in vitro against the cytotoxic effects of lethal toxin. Passive transfer of IgG purified from immune macaque serum into naive A/J mice conferred protection against challenge with B. anthracis in a dose-related manner. The protection conferred by passive transfer of 500 mug macaque IgG correlated significantly (P = 0.003; r = 0.4) with the titers of neutralizing antibody in donor macaques. Subsequently, a separate group of rhesus macaques immunized with 50 mug of Escherichia coli-derived rPA adsorbed to alhydrogel was fully protected against a target dose of 200 50% lethal doses of aerosolized B. anthracis. These data provide some preliminary evidence for the existence of immune correlates of protection against anthrax infection in rhesus macaques immunized with rPA.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/inmunología , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Proteínas Recombinantes/inmunología , Administración Intranasal , Aerosoles , Animales , Vacunas contra el Carbunco/administración & dosificación , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/genética , Bacillus anthracis/genética , Bacillus subtilis/genética , Bacillus subtilis/inmunología , Escherichia coli/genética , Escherichia coli/inmunología , Inmunización Secundaria , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Macaca mulatta , Ratones , Ratones Endogámicos A , Estructura Terciaria de Proteína , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genéticaRESUMEN
The human immune response to a new recombinant plague vaccine, comprising recombinant F1 (rF1) and rV antigens, has been assessed during a phase 1 safety and immunogenicity trial in healthy volunteers. All the subjects produced specific immunoglobulin G (IgG) in serum after the priming dose, which peaked in value after the booster dose (day 21), with the exception of one individual in the lowest dose level group, who responded to rF1 only. Three subjects, found to have an anti-rV titer at screening, were excluded from the overall analysis. Human antibody functionality has been assessed by quantification of antibody competing for binding to rV in vitro and also by the transfer of protective immunity in human serum into the naive mouse. Human and macaque IgG competed for binding to rV in vitro with a mouse monoclonal antibody, previously shown to protect mice against challenge with plague, suggesting that this protective B-cell epitope on rV is conserved between these three species. Total IgG to rV in individuals and the titer of IgG competing for binding to rV correlated significantly at days 21 (r = 0.72; P < 0.001) and 28 (r = 0.82; P < 0.001). Passive transfer of protective immunity into mice also correlated significantly with total IgG titer to rF1 plus rV at days 21 (r(2) = 98.6%; P < 0.001) and 28 (r(2) = 76.8%; P < 0.03). However, no significant vaccination-related change in activation of peripheral blood mononuclear cells was detected at any time. Potential serological immune correlates of protection have been investigated, but no trends specific to vaccination could be detected in cellular markers.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Vacuna contra la Peste/inmunología , Vacunas Sintéticas/inmunología , Adulto , Anticuerpos Antibacterianos/sangre , Método Doble Ciego , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Recuento de Leucocitos , Masculino , Proteínas Citotóxicas Formadoras de PorosRESUMEN
In order to evaluate the immunogenicity and protective efficacy of anthrax vaccine candidates a suitable small animal model is required. The inbred A/J strain of mouse has been selected as a potential model, and its immune response to immunisation with recombinant protective antigen (rPA) vaccine characterised, by assessment of rPA specific antibody production, and protection against injected challenge, with the unencapsulated STI strain of Bacillus anthracis. Studies were conducted to determine the time required post immunisation to develop a protective immune response, to define the minimum protective dose of vaccine required and to assess the long-term immune response to immunisation. From the results of these studies it was possible to establish that the A/J mouse is a consistent and robust small animal model for rPA vaccine testing. A comparison of the immune response to rPA vaccine immunisation in the Turner Outbred (TO) mouse strain was also conducted. Both inbred and outbred mouse strains displayed a predominantly Th2 biased immune response and showed a comparable antibody response to rPA immunisation. An assessment of protection in the TO mouse against aerosol challenge with the fully virulent strain of B. anthracis, Ames, was also made.
Asunto(s)
Vacunas contra el Carbunco , Carbunco/inmunología , Carbunco/prevención & control , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Animales , Vacunas contra el Carbunco/administración & dosificación , Vacunas contra el Carbunco/inmunología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta Inmunológica , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos A , Análisis de Supervivencia , Factores de Tiempo , Vacunación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
Insulin-like growth factor I (IGF-I) is essential for normal growth and development, regulating cell proliferation, differentiation, and survival. Little IGF-I exists in the free form; rather, it is bound to one of a family of six specific IGF-binding proteins (IGFBPs). Usually, IGFBPs have a high affinity for IGF-I and inhibit its activity. Intriguingly, some IGFBPs also potentiate IGF-I action; the precise mechanism of this is unclear, but it is thought to include modification of the IGFBP to lower its affinity for IGF-I. We have previously generated a novel antihuman (h) IGF-I antiserum that, instead of inhibiting IGF-I activity, enhances it in vivo. As the enhancing anti-IGF-I antiserum and potentiating IGFBPs share several properties with regard to IGF action, the antibody may provide a model for examining the actions of enhancing IGFBPs. In this study we demonstrate that the antiserum can also enhance IGF-I activity in vitro, assessed as cell number of a bovine fibroblast cell line, suggesting that its actions might not merely be confined to changing the kinetics of IGF-I clearance or degradation. Epitope scanning using overlapping octamer and hexamer peptides spanning the entire sequence of IGF-I indicates that the enhancing antiserum recognizes a specific linear region spanning the C-terminal region of the C domain and the proximal A domain (residues Ser33 to Cys47), and that this recognition is not present in nonenhancing antisera. Further, this region is located on the opposite surface of IGF-I from putative type 1 receptor-binding residues, allowing the possibility that the antiserum might be able to modulate IGF-I receptor binding. Antibodies raised against a synthetic peptide corresponding to Ser33 to Cys47 of IGF-I also potentiated IGF-I activity in vivo. As IGF-I may be beneficial in various clinical conditions associated with catabolism or cell repair, we suggest that this potentiating anti-IGF-I antiserum has favorable properties that could form a basis for therapeutic strategy.
Asunto(s)
Sueros Inmunes/farmacología , Factor I del Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/farmacología , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Cisteína , Enanismo/fisiopatología , Epítopos/análisis , Fibroblastos , Humanos , Insulina/química , Factor I del Crecimiento Similar a la Insulina/fisiología , Factor II del Crecimiento Similar a la Insulina/química , Ratones , Ratones Mutantes , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina , Vertebrados , Aumento de Peso/efectos de los fármacosRESUMEN
The aims of this investigation were (1) to examine IGF-binding protein-3 (IGFBP-3) mRNA levels in candidate tissues which might be important sources for blood IGFBP-3 (liver and skin) and in a target tissue for IGF-I action (skeletal muscle), and (2) to examine the effects of a single dose (500 micrograms) of GH or IGF-I on IGFBP-3 message levels in these tissues since temporal responses (4, 8 and 24 h after the single subcutaneous dose of peptide to GH-deficient dwarf rats) would indicate which peptide is the primary modulator of IGFBP-3 synthesis. Circulating IGF-I and IGFBP-3 concentrations were significantly increased (P < 0.05) by IGF-I and GH. GH treatment increased liver IGFBP-3 mRNA levels by 4 h (P < 0.001 over the 24 h) whereas IGF-I had no effect. Similarly, GH, but not IGF-I, increased muscle IGFBP-3 mRNA levels (P < 0.001 for the 24 h study period). However, both IGF-I and GH induced increases in skin IGFBP-3 mRNA abundance throughout the 24 h period (P < 0.001 and P < 0.01 respectively) and skin IGFBP-3 message abundance was greater that in the liver. Liver IGF-I mRNA levels were, as expected, increased after GH and tended to decrease after IGF-I treatment; muscle IGF-I mRNA was increased by GH (P < 0.001) and, interestingly, progressively increased by IGF-I (P < 0.05 for the 24 h period); skin IGF-I mRNA levels were unchanged by both peptides. The IGF-I induced increase in serum IGFBP-3 concentrations in the absence of an increase in hepatic IGFBP-3 mRNA levels and a paucity of liver IGF-I type 1 receptor mRNA imply that other sources of IGFBP-3 protein or synthesis must exist. The response of skin IGFBP-3 mRNA levels to both GH and IGF-I suggests that other cell types, such as fibroblast-derived cells, could be more important than the liver in the regulation of circulating reservoir IGFBP-3 in certain circumstances. In contrast to some current suggestions, the rapid and consistent GH-induced increase in IGFBP-3 message levels in all tissues studied implies that GH might have a direct function in the regulation of IGFBP-3 synthesis.
Asunto(s)
Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Hígado/metabolismo , Músculo Esquelético/metabolismo , Neuropéptidos/farmacología , ARN Mensajero/metabolismo , Piel/metabolismo , Animales , Autorradiografía , Western Blotting , Hormona del Crecimiento/deficiencia , Hormona del Crecimiento/farmacología , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Masculino , Modelos Biológicos , Sondas ARN , ARN sin Sentido , ARN Mensajero/análisis , Ratas , Ratas Mutantes , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismoRESUMEN
We have previously demonstrated that a specific anti-IGF-I antibody will enhance the growth-promoting activity of IGF-I in vivo (Stewart et al. 1993). Since the antibody had a modest affinity for IGF-I we suggested that the antiserum might protect IGF-I from degradation whilst maintaining it in a bioavailable form. The aim of this investigation was to test that hypothesis by determining the plasma clearance and tissue distribution of tracer IGF-I in the presence of the enhancing anti-IGF-I immunoglobulin (anti-IGF-I Ig) or non-immune immunoglobulin (NI Ig). Dwarf rats were treated with saline, NI Ig or anti-IGF-I Ig for 4 days. On day 4, 125I-IGF-I (1.6 x 10(7) c.p.m.) was injected into the jugular vein and blood sampled over the next 330 min when the rats were killed: samples of liver, kidney and skeletal muscle were quickly dissected out. Mean plasma trichloroacetic acid (TCA)-precipitable 125I-IGF-I was always significantly greater (P < 0.001 for each time point) from anti-IGF-I Ig rats versus the NI Ig or saline groups (which exhibited practically identical decay curves), implying increased binding capacity for IGF-I in the anti-IGF-I Ig rats. Pharmacokinetic parameters were calculated by resolution of the decay curves using a two-phase model. The total clearance rate of 125I-IGF-I was significantly decreased (P < 0.001) by almost twofold in the anti-IGF-I versus the two control groups, consistent with the increased binding capacity in the anti-IGF Ig rats. The half-lives of the faster-decaying phase were not significantly different between treatment groups but, surprisingly, that for the slower-decaying phase was significantly decreased (P < 0.001) in the anti-IGF-I Ig rats versus the two control groups; this may reflect the low affinity of the anti-IGF-I Ig for IGF-I and its enhancing properties. The degradation of 125I-IGF-I was significantly decreased in animals receiving the anti-IGF-I Ig. In support of this, kidney TCA-precipitable radioactivity (c.p.m.) was seven-fold less (P < 0.001) in the anti-IGF-I Ig groups versus the controls, indicative of reduced excretion. Liver TCA-precipitable radioactivity was increased (P < 0.001) in the anti-IGF-I Ig rats, probably due to reticuloendothelial clearance of non-self antibodies: skeletal muscle TCA-precipitable radioactivity tended to increase in the anti-IGF-I Ig group versus the controls which might indicate increased targeting of IGF-I to muscle. Size exclusion chromatography of plasma 15 and 120 min after administration of 125I-IGF-I demonstrated a broad peak of radioactivity with a molecular mass of 150-300 kDa in the anti-IGF-I Ig-treated rats, which was responsible for more than 90% of the eluted radioactivity. This suggests that: (1) 125I-IGF-I was bound to the anti-IGF-I Ig and might also be able to associate with IGFBPs or (2) the polyclonal antibody might recognise more than one antigenic site on IGF-I. These data indicate that the anti-IGF-I Ig was protecting IGF-I from degradation, leading to a large plasma pool of IGF-I but that IGF-I could be transferred readily from the plasma pool to tissues. We suggest that administration of IGF-I in conjunction with a binding molecule similar to the antibody described here could provide the basis for effective IGF-I treatment strategy.
Asunto(s)
Sueros Inmunes/farmacología , Factor I del Crecimiento Similar a la Insulina/inmunología , Factor I del Crecimiento Similar a la Insulina/farmacocinética , Animales , Disponibilidad Biológica , Cromatografía , Femenino , Inmunoglobulinas/farmacología , Radioisótopos de Yodo/farmacocinética , Tasa de Depuración Metabólica , Ratas , Ratas Mutantes , Distribución TisularRESUMEN
Oxytocin-induced prostaglandin F2 alpha (PGF2 alpha) responses were measured in explants of uterus from ovariectomized ewes on the day of tissue collection or after culture for 72 h in the presence or absence of oestradiol-17 beta (100 nmol/l). Oxytocin receptor binding activity was 210 +/- 47 fmol [3H]oxytocin bound per mg protein in fresh tissue and 89 +/- 24 and 90 +/- 17 fmol/mg in tissue cultured with control medium or with oestradiol respectively (means +/- S.E.M.). PGF2 alpha production during the hour following oxytocin administration to freshly collected tissue was 272 +/- 77 ng/g/h compared with 193 +/- 35 ng/g/h in the absence of oxytocin. These rates were 2789 +/- 1085 and 353 +/- 135 ng/g/h after culture for 72 h in control medium and 2022 +/- 496 and 342 +/- 134 ng/g/h after culture with oestradiol. Thus oestradiol had no effect on the culture-induced maturation of the PGF2 alpha response. Short-term exposure to arachidonic acid (66 mumol/l) did not increase PGF2 alpha production in fresh tissue but significantly increased basal but not oxytocin-induced PGF2 alpha production after 72 h in culture (P < 0.05). There was an absence of oxytocin-induced inositol phosphate turnover in fresh tissue but after culture concentrations of inositol mono-, bis- and trisphosphates were all significantly increased by oxytocin (P < 0.005). Antisera directed against G-protein alpha sub-units alpha i3, alpha o, alpha q, alpha 11 and the common beta subunit, and prostaglandin H-synthase-1 detected proteins that were present before and after culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Dinoprost/metabolismo , Oxitocina/farmacología , Ovinos/fisiología , Útero/metabolismo , Animales , Ácido Araquidónico/farmacología , Técnicas de Cultivo , Electroforesis en Gel de Poliacrilamida , Femenino , Proteínas de Unión al GTP/metabolismo , Immunoblotting , Fosfatos de Inositol/metabolismo , Ovariectomía , Fosfatidilinositoles/metabolismo , Receptores de Oxitocina/metabolismo , Útero/efectos de los fármacosRESUMEN
We have previously shown that a polyclonal anti-IGF-I antiserum administered together with IGF-I potentiates IGF-I activity in vivo. The anti-IGF-I antiserum has a modest affinity for IGF-I, similar to that for enhancing IGFBPs, and treated animals have significantly higher circulating IGF-I concentrations than their controls. Our recent findings have demonstrated that the anti-IGF-I activity decreases the clearance of IGF-I by at least 2-fold and that it abolishes the acute hypoglycaemic action of a single subcutaneous dose of IGF-I. Interestingly, we have been unable to demonstrate potentiation of the growth-promoting activity of the potent non-IGFBP binding IGF-I analogue LR3IGF-I, even though the analogue binds to the antiserum in vitro; rather native IGF-I/antibody complexes perform even better than LR3IGF-I. In IGF-I/antibody-treated dwarf rats, most IGF-I may be found in an uncharacterised high molecular weight antibody complex which is probably responsible for improved IGF-I performance. Thus, the anti-IGF-I antibody may be behaving in a similar manner to a high molecular weight IGFBP and is effective in potentiating IGF-I action in vivo.
Asunto(s)
Anticuerpos Bloqueadores/fisiología , Hipoglucemia/inducido químicamente , Factor I del Crecimiento Similar a la Insulina/análogos & derivados , Factor I del Crecimiento Similar a la Insulina/inmunología , Animales , Formación de Anticuerpos , Peso Corporal , Ensayo de Inmunoadsorción Enzimática , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Tasa de Depuración Metabólica , Ratas , Distribución TisularRESUMEN
PhosphoIipid/Ca(2+) -dependent protein kinase C (PKC) and oxytocin receptor were measured in sheep endometrial explants after culture for up to 96 h. Oxytocin receptor binding and PKC activity were reduced by up to 90% in explants exposed to recombinant ovine trophoblast interferon (rolFN-τ), recombinant bovine IFN-α(1) or ovine conceptus secretory proteins (a source of IFN-τ). Inhibition occurred in both caruncular and intercaruncular endometrium taken between days 7 and 10 of the oestrous cycle and in intercaruncular (but not caruncular) endometrium on day 6. Down-regulation of PKC by continued exposure of expiants to 4ß-phorbol myristate acetate, or treatment with PKC inhibitors reduced both oxytocin receptor binding and PKC activity by up to 70%. Tyrosine kinase inhibitors were ineffective. Addition of oxytocin or progesterone, which reduce oxytocin receptor bindingin vivo, also lowered oxytocin receptor bindingin vitro in the absence of any effect on PKC. The data indicate that IFN-τ inhibits oxytocin receptor synthesis by a mechanism involving PKC inhibition, but that a non-PKC pathway also operates to control oxytocin receptor binding in non-pregnant animals. These conclusions were supported by measuring PKC activity and oxytocin receptor binding in endometrium without culture. Prolonged exposure of the endometrium to IFN-τin vivo may lead to PKC down regulation by a mechanism analogous to that involved in the action of continuous activation by agonist, and this may represent one function of the prolonged secretion of IFN-τ over a 10-day period in early pregnancy.
RESUMEN
Oxytocin receptor binding activity in explants of caruncular and intercaruncular endometrium collected from luteal phase ewes increased during culture. An initial rise in binding activity occurred during the first 24 h of culture in both tissues; binding activity in intercaruncular endometrium continued to increase until day 6, remained unchanged on day 8 and had decreased by day 10 of culture. The maximum concentration of receptors in caruncular endometrium was significantly lower than that in intercaruncular endometrium (P < 0.001) and did not change significantly between days 4 and 10 of culture. Apparent dissociation constants and maximal binding of oxytocin receptor in cultured caruncular and intercaruncular endometrium were 3.09 and 2.72 nmol l-1 and 249 and 459 fmol [3H]oxytocin bound mg-1 protein, respectively. Concentrations of oxytocin receptor remained constant in myometrium during 96 h of culture. The rise in endometrial oxytocin receptor concentration did not result from exposure to fetal calf serum, phenol red or insulin in the culture medium. Substituting fetal calf serum with sheep serum or BSA did not block the rise in receptor binding activity. Actinomycin D and cycloheximide inhibited the rise in receptor concentration in both tissues. Co-culture of lung or kidney with endometrium had no effect on binding activity, whereas co-culture with luteal tissue effectively reduced the rise in oxytocin receptor concentration. To establish whether synthesis of functional oxytocin receptors occurred during culture, the effect of oxytocin on prostaglandin F2 alpha (PGF2 alpha) production was assessed in fresh tissue and after 48 h in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Endometrio/metabolismo , Oxitocina/metabolismo , Receptores de Oxitocina/metabolismo , Ovinos/metabolismo , Animales , Cuerpo Lúteo/metabolismo , Medios de Cultivo , Técnicas de Cultivo , Cicloheximida/farmacología , Dactinomicina/farmacología , Dinoprost/biosíntesis , Femenino , Fase Luteínica/fisiología , Oxitocina/farmacologíaRESUMEN
The effects of oestradiol, progesterone, oxytocin and combinations of these hormones on oxytocin receptor binding in explants of uteri from ovariectomized ewes were determined. Receptor binding remained unchanged after 96 h in culture in control medium. Oestradiol at concentrations of 1 pmol-10 mumol l-1 did not alter receptor binding activity in tissue cultured for 96 h, but at 100 mumol l-1 oestradiol significantly reduced (P < 0.01) receptor binding activity. Progesterone and oxytocin significantly reduced receptor binding activity in explants cultured for 96 h (P < 0.05). Explants cultured in medium containing progesterone and oestradiol or oxytocin and oestradiol showed receptor binding characteristics similar to those found in tissue cultured with progesterone or oxytocin alone. When explants were cultured for 72 h in medium containing oestradiol followed by 24 h in medium containing oestradiol alone, oestradiol with oxytocin, oestradiol with progesterone, oxytocin alone, progesterone alone, or in medium with no added hormones, receptor binding activity was always reduced in the presence of progesterone and oxytocin whether or not oestradiol was present in the medium. Receptor binding activity in explants cultured for the final 24 h in medium containing oestradiol or no added hormones were similar to those in tissue cultured in control medium for a total of 96 h. These data show that progesterone and oxytocin reduce oxytocin receptor binding activity in cultured uterine tissue and, in contrast to its effect on the rat uterus, that oestradiol is not a potent stimulator of oxytocin receptor synthesis in uterine tissue of ovariectomized ewes in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)