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R B Heap

Publications and source records attributed to R B Heap.

At least 19 recordsLinked to original sources
PubMed1995

Specific interactions of progestins and anti-progestins with progesterone antibodies, plasma binding proteins and the human recombinant receptor.

This structure-activity study compares the affinity of a series of progestins, progesterone metabolites and anti-progestins for a panel of monoclonal antibodies to progesterone, coypu (Myocastor coypus) or guinea pig plasma progesterone-binding proteins (PPBPs) and the human recombinant progesterone receptor A form (PR-A). The compounds tested were progesterone, Promegestone (R5020), Mifepristone (RU486), ZK98,734, Onapristone (ZK98,299), 11 alpha-hydroxyprogesterone, 11 alpha-progesterone hemisuccinate, androsterone, etiocholanolone, 5 alpha- and 5 beta-pregnane-3,20-diones, and 20 alpha- and 20 beta-hydroxyprogesterones. The Ki values for these ligands were determined by competitive binding assays using radiolabelled progesterone as the binding site ligand. For anti-progesterone antibodies (e.g. DB3 and 11/32), only progesterone (3.6-8.8 nM), the 11 alpha-derivatives (1.0-5.5 nM) used to prepare the immunogen and the two 5-pregnanediones (20.9-45.1 nM) were bound with high affinity. For PR-A, high affinity binding was found with receptor agonists (Ki = 1.1-6.2 nM), both 5- and 20-reduced metabolites, and antagonists (0.6-28.0 nM), but not with the 11 alpha-derivatives (950 nM-1.0 microM). In contrast, the PPBPs displayed high affinity interactions with progesterone (3.5-4.2 nM) and both 5 alpha- and 20 alpha-reduced metabolites (2.4-3.4 nM). Binding with the beta-isomers and R5020 was less pronounced (22-170 nM) and there was no evidence of high affinity binding with PR antagonists (> 1.0 microM). Analogs with the 17-keto group did not bind to any of the binders studied. Thus, commonalities among the three types of protein binders were their comparable binding affinities for progesterone (3.5-8.8 nM) and 5-pregnanedione isomers (2.4-330 nM), and a lack of binding for two C17-keto steroids (androsterone and etiocholanolone). The results imply that the tertiary features of the binding domain of these three types of proteins are sufficiently different to result in unique binding structures.

Animals↗
PubMed1995

Anti-progesterone antibody administration and the impairment of postpartum maternal care in mice.

Passive transfer of a monoclonal antibody against progesterone produces a high incidence of maternal rejection in mice after recovery from antibody-induced infertility. To investigate the mechanisms involved in this reduction of maternal care, we have examined whether the effect is due to long-term exposure to antibody. Antibody was administered i.p. either on day 2 or day 17 of pregnancy. When a low dose (1.0 nmol) was given on day 2, pregnancy proceeded normally but 44.8% pups delivered at term were rejected compared with 12.7% in the control group. When a higher dose (4.5 nmol) of antibody was given on day 17, pregnancy continued normally to term and the rejection rate was 48.8% (control: 11.1%). When the same amount of antibody was injected after delivery (day 1 of lactation), no detrimental effect was found on subsequent maternal care to the young, the rejection rate being comparable between antibody-treated and control groups (5.3% vs 4.6%). To determine if the presence of antibody interfered with lactation or suckling, a bolus injection of 10 microCi [3H]H2O was given to mice treated at day 17 with antibody or saline. The levels of radioactivity present in both mothers and pups and the first 5-day pup growth curves showed identical patterns, indicating that milk availability and the suckling process were not affected. Crossfostering studies revealed that antibody-treated mothers rejected 25.5% of fostered pups compared with 8.5% found in the control females when antibody was administered on day 17 of pregnancy and the entire litters were crossfostered between the two groups immediately after delivery.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Aberrant maternal behaviour in mice treated with a progesterone receptor antagonist during pregnancy.

The rapid onset of normal maternal behaviour after parturition in mice, consisting of cleaning, warming, feeding and protection of offspring, is primed by oestrogen, progesterone and oxytocin. Previous studies showed that passive transfer of monoclonal antibodies against progesterone significantly increases the incidence of maternal rejection of pups. To test the hypothesis that aberrant maternal behaviour is due to partial progesterone withdrawal leading to hormonal imbalance during late pregnancy, maternal rejection was assessed following treatment with a progesterone receptor antagonist. Mifepristone (RU486) was given subcutaneously on either day 2 (100 micrograms) or day 17 (50 micrograms) of pregnancy, or on the first day of lactation (100 micrograms). Maternal behaviour was monitored twice daily for the first 6 days of lactation and pup rejection recorded for a further 15 days. Maternal rejection was significantly greater after mifepristone administration on either day 2 or day 17 (28.6% and 38.3%) compared with controls (11.1% and 5.2% respectively). Rejection was negligible in both treated and control groups if mifepristone was given after parturition. When mothers were treated at day 17, the length of the latent period before pups were retrieved and returned to the nest was markedly increased in mifepristone-treated mothers (46.3 s) compared with controls (4.4 s) though the effect was transient. The results indicate that mifepristone interferes with the hormonal priming mechanism(s) necessary for the onset of normal maternal behaviour by a receptor-mediated effect. The similarity of the present results and those obtained with anti-progesterone antibodies implies that receptor antagonism or antibody scavenging of progesterone influence a common central nervous mechanism that is essential for the normal priming process.

Animals↗
PubMed1995

The local differential effect of prostacyclin, prostaglandin E2 and prostaglandin F2 alpha on mammary blood flow of lactating goats.

Mammary blood flow (MBF) and milk yield are closely related in dairy ruminants, but little is known about the regulation of MBF in vivo. The local effects on MBF of injections or continuous infusions into the mammary artery of prostaglandins (PG) or indomethacin (an inhibitor of prostaglandins) respectively, were investigated in surgically prepared conscious goats. Prostacyclin (PGI2) was found to be a potent stimulator of MBF which increased linearly over the dose range 50-1000 ng. PGE2 was almost as potent as PGI2 at low doses, but tachyphylaxis occurred at doses at and above 100 ng. The response to repeated injections of PGE2 quickly declined depending on the dose. PGF2 alpha had no effect on MBF. During infusion of indomethacin into the mammary artery MBF was reduced markedly, showing that endogenous mammary prostaglandins are involved in the regulation of vasodilatation. The results indicate that PGI2 (and to a lesser extent PGE2) has an important role in the local regulation of vascular tone in the mammary gland.

Animals↗
PubMed1995

Anti-progesterone effects on maternal recognition and behaviour imprinted during first pregnancy in mice.

Anti-progesterone treatment using specific anti-progesterone antibodies or a progesterone receptor (PR) antagonist during first pregnancy impairs postpartum maternal behaviour in mice. This effect is demonstrable only if the treatment is given during pregnancy but not immediately after parturition. The purpose of the present studies was to investigate if maternal behaviour is also impaired by anti-progesterone treatment in subsequent pregnancies. Studies with a monoclonal antibody to progesterone (DB3; 4.5 nmol/mouse) showed that injection of females on day 17 of second pregnancy did not cause maternal rejection but the latency of pup retrieval was prolonged especially during the first 3 days of lactation. This phenomenon was not observed in animals that had previous experience of full length lactation. Experiments were carried out with mifepristone (RU486; 10 micrograms/mouse) injected at day 17 of first, second or third pregnancies. Pup rejection (22.5% vs 12.3%) and prolongation of the retrieval latency (62.3 +/- 13.3 vs 19.7 +/- 6.5 s; P < 0.02) were observed following the first pregnancy. No abnormal behavioural effects were found in mothers treated in second or third pregnancy who had prior full length lactation experience. Control females subjected to only one pup retrieval test after first delivery rejected their pups if treated in their second pregnancy (27.3% vs 4.4%; P < 0.001) and displayed a marginal prolongation of the retrieval latency period (20.9 +/- 7.0 vs 7.4 +/- 2.6 s). Anti-progesterone treatment had no negative influence when administered during third pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Proto-oncogene erbA expression and increased abundance of progesterone receptors in the mouse uterus after passive immunisation against progesterone before implantation.

Passive immunisation with a monoclonal anti-progesterone antibody (DB3) prevents pregnancy in the mouse, and antibody is localised in the endometrium before the onset of implantation. BALB/c female mice were injected intraperitoneally with 9 nmol of DB3 (a dose known to cause 100% infertility) 32 h post coitum, and the uterus was removed at various times after injection. Using a monoclonal anti-progesterone receptor antibody (PR6), expression of progesterone receptors was found to be abundant in uterine tissue of DB3-treated mice; this was associated with substantial progesterone receptor mRNA levels and with maximum localisation of DB3 antibody as detected by anti-idiotype antibody. Control animals treated with an equal amount of the mouse myeloma protein P3 showed very low levels of progesterone receptor in the uterus. DB3 treatment also affected uterine expression of the proto-oncogene erbA product (which shows primary sequence homology with the progesterone receptor) as revealed by specific antiserum to the ERBA protein and by in situ hybridisation with a cDNA probe to v-erbA. Time-course studies indicated that the erbA gene was expressed at a high level before progesterone receptor expression increased, that its expression was dependent on the presence of the embryo and that erbA expression persisted longer in DB3-treated females. The observations suggest that anti-progesterone immunisation has a direct effect within the uterus, involving persistence of proto-oncogene erbA expression (which itself may represent an early maternal response to pregnancy) and increased progesterone receptor levels resulting from an unopposed oestrogen effect derived from local ligand withdrawal.

Amino Acid Sequence↗
PubMed1992

Biotinylated anti-progesterone monoclonal antibodies specifically target the uterine epithelium and block implantation in the mouse.

Post-coital administration of a mouse monoclonal antibody (mAb) against progesterone (DB3; IgG1) prevents pregnancy in several species. Our previous studies in mice have shown that passively transferred DB3 specifically targets the uterus before the expected time of implantation, probably through progesterone-binding sites. DB3 and two other anti-progesterone mAbs, i.e. 11/34 (IgG1) and 11/64 (IgM), were biotinylated and 9 nmol of each (a dose known to reduce pregnancy rate by greater than 80% with unconjugated mAbs) was injected into BALB/c female mice 32 h post coitum (p.c.). The biotin-mAb complexes were highly effective in blocking pregnancy (83-100%) compared with control animals that had received the biotinylated MOPC21 myeloma protein P3 (IgG1). Using the streptavidin-FITC (fluorescein isothiocyanate) reporter complex or second-stage anti-biotin-FITC antibodies, biotinylated conjugates of DB3, 11/34 and 11/64 were specifically localized on the uterine luminal epithelium at 68 h p.c. (i.e. 36 h after i.p. injection). Neither P3-biotin-treated pregnant mice nor biotinylated anti-progesterone mAb-treated pseudopregnant females showed a positive reaction. In addition, the localization of biotin-conjugated anti-progesterone mAbs could be blocked by absorption of the antibodies with free progesterone or progesterone conjugates prior to injection. These results show that localization to the uterine epithelium occurs with different anti-progesterone mAbs, and that this phenomenon is probably associated with progesterone-binding sites on the luminal epithelium.

Animals↗
PubMed1992

Antibodies, implantation and embryo survival.

The diverse strategies adopted among species for the maintenance of luteal function converge to meet the indispensable requirement for this hormone particularly at the time of onset of implantation. What has emerged from immunization studies is the difference that exists between various species in the effects of progesterone depletion. The findings affirm the uterus as a primary site of progesterone action in the preimplantation period of gestation in the mouse, whereas in the hamster the positive feedback by progesterone on pituitary luteotrophin secretion is important in maintaining luteal function and hormone secretion at a level necessary for uterine preparation. The impact of progesterone in the rat seems to be established within 48 h after fertilization, whereas in the ferret the hormone's action ensures a suitable uterine environment in which the early embryo can flourish. Its effects in early pregnancy in the marmoset are ambiguous from the present studies unless it emerges that target organ responses are tuned to low concentrations of active steroid. However, the discoveries in mice of a conserved family of immunoglobulin genes used exclusively by immunogenic forms of progesterone conjugated to proteins to stimulate antibody production, and of antibody binding to the uterine epithelium, reveal systems potentially inimical for embryo survival. The endometrial expression of the proto-oncogene erb-A at a time when the embryo has not yet arrived in the uterus, and of antibody-binding to the uterine epithelium, are early maternal responses whose significance require closer examination. The present findings support the hypothesis that the primary site of action of progesterone during the preimplantation period differs between species, and that it is not only the mother that recognizes, but the embryo that initiates early changes before the onset of implantation 'in the bed of soil that nourishes it', a symbiosis that may yet prove to be a common feature of the adoption of viviparity as a preferred mode of reproduction.

Animals↗
PubMed1992

Regulation of steroid synthesis and metabolism in isolated binucleate cells of the placenta in sheep and goats.

Binucleate cells of sheep and goat fetal placentae comprise about one-fifth of the trophectodermal layer at the feto-maternal interface. When isolated and incubated in vitro they produce the steroids that are synthesized by the placenta in vivo (progesterone in sheep, 5 beta-pregnane-3 alpha,20 alpha diol in goats). This study demonstrates that progesterone synthesis in binucleate cell preparations in sheep was increased by prostaglandin (PG) E-2, nordihydroguaiaracetic acid (NDGA) and methylisobutylxanthine, but reduced by indomethacin, whereas in goats only NDGA produced any effect (an increase). None of the other compounds tested (luteinizing hormone, follicle stimulating hormone, prolactin, dibutyryl cAMP, A23187 or phorbolmyristic acetate) had any effect. Sheep binucleate cells also produced PGE-2 from arachidonic acid. These results suggest that, in sheep, products of both the cyclooxygenase (producing PGE-2) and lipoxygenase (inhibited by NDGA) pathways of arachidonic acid metabolism have regulatory roles in placental steroid synthesis, but only the lipoxygenase pathway is relevant in goats.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Abnormal maternal behaviour in mice previously immunized against progesterone.

Anti-progesterone immunization leads to reversible infertility in mice; this can be achieved by passive immunization with a monoclonal antibody to progesterone (DB3), or by active immunization with either a progesterone-protein (bovine serum albumin; BSA) conjugate or anti-idiotype directed against DB3. Recovery of fertility in treated females varied from 39.5 to 75.5 median days after passive or active (progesterone-BSA) immunization respectively. Litter size after the first pregnancy also differed from 8.6 +/- 0.8 to 5.0 +/- 0.6 (mean +/- S.E.M.) per mother after passive or active immunization respectively. When litter size was standardized to a maximum of four pups per litter, aberrant maternal responses were observed in the first 5 days after delivery in 40-70% of the nursing mothers. These responses took the forms of cannibalism and failure to retrieve or to nurse pups and resulted in a high incidence of pup rejection (up to 40%), compared with no rejection in control mothers. When mothers were allowed to keep entire litters, an even higher incidence of pup rejection occurred (51% compared with 8% in controls). There was an apparent relation between the degree of negative maternal behaviour and the progesterone antibody concentration in the circulation during the infertile period. Whereas aberrant behaviour occurred mainly within the first 5 days of lactation, it was significantly reduced thereafter. Aberrant behaviour of the mother towards pups may be a consequence of the presence of residual progesterone antibodies in the circulation which affects the process of progesterone withdrawal at parturition that is essential for the establishment of normal maternal responses to the neonate.

Animals↗
PubMed1991

Oestrogen production by the preimplantation donkey conceptus compared with that of the horse and the effect of between-species embryo transfer.

Aromatase distribution in membranes of preimplantation horse and donkey conceptuses was compared by measuring the incorporation of [3H]androstenedione into oestrone and oestradiol-17 beta. In the donkey conceptus, aromatase activity was similar in all the tissues examined (yolk sac, chorionic girdle and allantochorion), whereas in the horse it was generally lower and showed the relationship chorionic girdle greater than yolk sac greater than allantochorion. A higher proportion of labelled precursor was incorporated into oestradiol-17 beta by extra-embryonic tissues of the donkey compared with those of the horse. In contrast to previous results, aromatase in the chorionic girdle did not decline progressively before its migration into the endometrium on Day 36 to form the endometrial cups. The chorionic girdle of a donkey conceptus carried in the uterus of a mare failed to invade the surrogate horse endometrium and aromatase activity was still high in this tissue at Day 42. Aromatase distribution in 2 transferred donkey-in-horse conceptuses resembled that of the fetal, rather than the maternal, genotype indicating a lack of effect of the maternal environment.

Amnion↗
PubMed1991

Progesterone and 5 beta-pregnanediol production by isolated fetal placental binucleate cells from sheep and goats.

Enzymic dispersion and density gradient separation were used for the isolation of enriched populations (60-90%) of cells from the corpus luteum, placenta and peripheral blood of pregnant sheep and goats. Analysis of the steroids produced from radioactive pregnenolone demonstrated that placental binucleate cells can produce progesterone and 5 beta-pregnanediol whereas white blood cells were relatively inactive. Thus, sheep binucleate cells converted pregnenolone predominantly to progesterone as did sheep luteal cells. However, goat binucleate cells produced 5 beta-pregnanediol as the major metabolite, which is consistent with its production in vivo during pregnancy. Production of progesterone (sheep) or 5 beta-pregnanediol (goat) by binucleate cells was shown to be proportional to the number and viability of the cells. In contrast with the binucleate cells there was no evidence that trophectodermal uninucleate cells play a significant role in placental progesterone or 5 beta-pregnanediol synthesis in either species.

Animals↗
PubMed1991

Mechanism of secretion of plasma insulin-like growth factor-I into milk of lactating goats.

125I-Labelled insulin-like growth factor-I (IGF-I) was infused as the free form directly into the pudic artery supplying one gland of lactating goats (n = 6). The infusion was for 60 min and 0.4 +/- 0.09% (S.E.M.) of the infusate was secreted into milk from the infused gland during its first passage through that gland. A large proportion of the 125I-labelled IGF-I escaped into the systematic circulation and was secreted into milk of both glands. A total of 5.2 +/- 0.4% of infused radioactivity was recovered in milk from both glands from 0 to 720 min. Radioactivity consisted of trichloroacetic acid (TCA)-precipitable and -soluble counts which were shown by gel filtration to be authentic IGF-I and degraded products of the peptide. The amount and time course of TCA-soluble radioactivity in milk from both glands was similar, suggesting degradation of 125I-labelled IGF-I at extramammary sites. Maximum specific activity for 125I-labelled IGF-I in milk from the infused gland was reached 80-120 min after the start of infusion and was 2.5-fold greater than milk from the non-infused gland. The time course of appearance of 125I-labelled IGF-I in milk suggests that transfer was via the transcellular pathway and this was further supported by comparing the pattern of transfer of [14C]sucrose and [14C]amino acids. When excess unlabelled IGF-I was included in the infusate, specific activity in milk from the infused gland was reduced to that of the non-infused gland, indicating a competitive and saturable mechanism of secretion for 125I-labelled IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Production of 5 alpha-dihydroprogesterone during late pregnancy in the mare.

Changes in the progesterone metabolite 5 alpha-dihydroprogesterone (5 alpha-DHP) in maternal plasma in late gestation, and possible sites of production of this steroid were studied in pony and Thoroughbred mares by an enzyme-linked immunosorbant assay for 5 alpha-DHP. In Thoroughbred mares, plasma 5 alpha-DHP increased from 63.7 +/- 10.5 ng/ml (27 days pre-partum) to 161.7 +/- 30.8 ng/ml (1 day pre-partum) falling to 90.2 +/- 16.1 ng/ml on the day of parturition. In pony mares, values rose from 30.8 +/- 8.1 ng/ml (27 days pre-partum) to 79.1 +/- 30.8 ng/ml (3 days pre-partum) and then decreased to 28.2 +/- 7.1 ng/ml on the day of parturition. Concentrations of 5 alpha-DHP were greater in the umbilical and uterine veins than in the corresponding umbilical and uterine artery samples. Tissues incubated with isotopically-labelled substrates showed that the fetal placenta converted pregnenolone to progesterone (47.5 +/- 9.0%). Endometrium metabolised [3H]pregnenolone to progesterone and 5 alpha-DHP (11.0 +/- 3.0% and 7.8 +/- 2.4%, respectively), and [14C]progesterone to 5 alpha-DHP (9.3 +/- 2.5%). Deuterium-labelled substrates were used to confirm the identity of these products by gas chromatography-mass spectrometry. Negligible amounts of 5 alpha-DHP were formed by other fetal tissues (adrenal, liver, kidney and testis). The results show that 5 alpha-DHP production in late pregnancy is derived primarily from endometrial metabolism of pregnenolone and/or progesterone.

5-alpha-Dihydroprogesterone↗
PubMed1990

Purification and characterization of heparin-binding growth factors from porcine uterus.

Heparin-binding growth factors present in pig uterine tissue were purified by approx. 50,000-fold using a combination of ammonium sulphate precipitation, ion-exchange chromatography and heparin-affinity chromatography. Purification of the uterus-derived growth factors (UDGFs) was monitored by the stimulation of [3H]thymidine incorporation into Swiss 3T3 cells and by a radioreceptor assay using 125I-labelled epidermal growth factor (EGF) as the ligand. The latter was shown to be a novel, rapid and reliable assay for heparin-binding growth factors which utilizes their trans-modulation of EGF receptor affinity. UDGFs exhibit strong affinity for immobilized heparin and two forms, named alpha UDGF and beta UDGF, were distinguished by salt gradient elution from heparin-agarose affinity columns. beta UDGF activity was eluted from heparin-agarose between 1.5 M- and 1.8 M-NaCl, and was correlated with the elution of a protein doublet of 17.2 kDa and 17.7 kDa. Immunoblotting of heparin-purified beta UDGF indicated that the beta UDGF doublet is immunologically related to the 146-amino-acid form of bovine basic fibroblast growth factor (bFGF), and that the 17.2 kDa component is an N-terminally truncated form of the 17.7 kDa component. After purification by C4 reversed-phase h.p.l.c., this doublet was biologically active and greater than 95% pure as assessed by silver-stained SDS/PAGE. Amino acid composition and sequence analysis confirmed that these beta UDGF polypeptides were microheterogeneous forms of bFGF. Fractions containing alpha UDGF activity were eluted from heparin-agarose in 1.3 M-NaCl. These fractions contained a 16.5 kDa protein which co-migrated on SDS/polyacrylamide gels with recombinant human acidic FGF (aFGF) and which which cross-reacted with an antiserum raised against aFGF. The identification of heparin-binding growth factors in porcine uterus at the time of implantation raises the possibility that they function in the reproductive tract during early pregnancy.

Amino Acid Sequence↗