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Biomedical subjects

L Myatt

Publications and source records attributed to L Myatt.

At least 91 records · Page 5Linked to original sources
PubMed1984

A novel oestrogen-binding species in rat granulosa cells.

The dissociation constants (Kd) and steroid specificities of oestrogen-binding species in rat granulosa cell cytosol and nuclei have been studied. Preliminary work, where diethylstilboestrol was employed as competitor in binding assays, identified the oestrogen receptor in whole ovarian tissue nuclei (Kd 0.35 +/- 0.09 nmol/l) and cytosol (Kd 0.39 +/- 0.03 nmol/l). Isolation of granulosa cells revealed that the majority of this receptor (75-96%) was present in these cells. Specificity studies on the binding of [3H]oestradiol in granulosa cell cytosol indicated the presence of an additional class of oestrogen-binding sites which were, however, not present in nuclei. Saturation analysis over an extended range of [3H]oestradiol concentrations and using unlabelled oestradiol as competitor revealed a binding species of Kd 45.8 +/- 6.9 nmol/l (capacity 16.7 pmol/mg cytosol protein) for oestradiol in addition to the cytosol oestrogen receptor of Kd 0.58 +/- 0.22 nmol/l (capacity 2.8 pmol/mg cytosol protein). The low affinity of this novel species implies that the dextran-coated charcoal techniques used in previous studies on ovarian oestrogen-binding species would cause dissociation of ligand and not allow it to be measured. The second oestrogen-binding species displayed affinity for oestradiol-17 beta, oestriol, oestrone, testosterone, 5 alpha-dihydrotestosterone, methyltrienolone, progesterone and the antioestrogens tamoxifen, nafoxidine and clomiphene citrate. The species, however, did not bind diethylstilboestrol, a characteristic shared with other low affinity cytosol oestrogen-binding species which have been reported in dog prostate, chick oviduct and male rat liver but not shared with uterine type II oestrogen receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Further characterization of the second oestrogen-binding species of the rat granulosa cell.

Rat granulosa cell cytosol contains a second oestrogen-binding species (SOB) distinguished from the classical oestrogen receptor by its lower dissociation constant (approx. 45 nmol/l) and the ability to bind oestrogens, antioestrogens, androgens and progesterone but not diethylstilboestrol. The SOB and the oestrogen receptor can be further distinguished by their differential adsorption to spheroidal hydroxylapatite and Concanavalin A-Sepharose. Addition of chaotropic salts or molybdate to granulosa cell cytosol did not alter the concentration of SOB or oestrogen receptor measured, indicating that there are no 'masked' binding sites in the two species caused by aggregation phenomena. The association rate of oestradiol with SOB at 4 degrees C (1.72 +/- 0.27(S.E.M.) X 10(8) mol/h) and 25 degrees C (4.50 +/- 0.36 X 10(8) mol/h) was faster than with the oestrogen receptor (7.20 +/- 0.15 X 10(7) mol/h and 1.23 +/- 0.15 X 10(8) mol/h respectively). The biphasic dissociation kinetics of [3H]oestradiol from the oestrogen receptor at 25 degrees C (rate constants k-1 = 0.30 +/- 0.07/min and k-2 = 3.73 +/- 0.57 X 10(-3)/min) were similar to those reported in other target tissues but the dissociation of [3H]oestradiol from SOB appeared to be much more rapid and could not be measured by the Sephadex LH-20 separation method employed for determining receptor kinetics. Using sucrose density-gradient (SDG) analysis and Sephacryl S-200 gel chromatography the oestrogen receptor fractionated in an aggregated form (10.3S, Stokes radius greater than 5.2 nm) in low ionic strength buffers and as a small species (4.4S, Stokes radius 3.5 nm) in buffers containing 0.4 M-KCl. However, the SOB fractionated as 2-3S, Stokes radius 3.7-4.0 nm at low ionic strength and as 5.8S, Stokes radius 3.5 nm in 0.4 M-KCl. In contrast to the receptor from other target tissues the granulosa cell oestrogen receptor did not bind to the artificial acceptor matrix oligo(dT)-cellulose and heat activation did not promote a 4S to 5S conversion when analysed on SDG. The salt-extracted form of nuclear receptor sedimented at 4.6S, mol. wt 69-72000 on SDG.

Animals↗
PubMed1983

Decreased prostacyclin production by placental cells in culture from pregnancies complicated by fetal growth retardation.

Production of prostacyclin (PGI2) in vitro by human placental cells from pregnancies complicated by fetal growth retardation was significantly reduced compared with that in placental cells from normal pregnancies of either matched gestation or at term. This appeared to be due to a reduction of synthesis of PGI2 rather than to any alteration in the rate of its enzymic metabolism. Addition of oestradiol and progesterone increased PGI2 production by cells from pregnancies with fetal growth retardation in a similar manner to that by cells from first trimester pregnancies, implying that the placental cells are not irreversibly damaged by ischaemia. The decreased PGI2 production by cells of trophoblastic origin may be an aetiological factor in the thrombotic occlusion of the uteroplacental circulation which impairs fetal growth.

Cells, Cultured↗
PubMed1982

The binding of rat uterine cytosol oestrogen receptors to oligodeoxythymidylate--cellulose. Its relationship to a stable form of receptor complex with separate ligand- and oligonucleotide-binding sites.

The interaction of rat uterine cytosol oestrogen-receptor complexes with the synthetic acceptor oligo(dT)--cellulose was studied. Differences in the stability of receptor complexes and their ability to bind to oligo(dT)--cellulose on storage at 4 degrees C or when exposed to increased temperatures indicated heterogeneity of steroid- and oligonucleotide-binding sites. Dilution, dialysis and (NH4)2SO4 precipitation increased the interaction of receptor complexes with oligo(dT)--cellulose (a step termed activation). This increase may be the result of the removal of low-molecular-weight cytosol components which inhibit receptor activation, dimerization to the 5 S form, which binds to oligo(dT)--cellulose, or interaction of 5 S receptor with the oligonucleotide. Cytosol oestradiol--receptor complexes exhibited biphasic dissociation kinetics. All these manipulations resulted in an increase in the proportion of the slow-dissociating component equivalent to the increase in receptor binding to oligo(dT)--cellulose. In contrast, addition of 10mM-sodium molybdate to cytosol decreased both oligo(dT)--cellulose binding and the proportion of receptor with slow dissociation kinetics. The inclusion of proteinase inhibitors did not affect interactions of receptor with oligo(dT)--cellulose nor the dissociation kinetics. These results suggest that oligo(dT)--cellulose binding may serve to quantify the proportion of cytosol receptor in an active form capable of nuclear interaction and to help to ascertain whether a receptor system is fully functional. This binding procedure could prove useful in the evaluation of oestrogen responsivity under normal and pathological conditions.

Animals↗
PubMed1982

Peripheral plasma immunoreactive 6-oxo-prostaglandin F1 alpha and gynaecological tumours.

Peripheral plasma levels of immunoreactive 6-oxo-PGF1 alpha, the stable hydrolysis product of prostacyclin, were significantly higher in female patients with tumours of the genital tract than in normal controls. In the groups with malignant tumours, these high levels declined after operation and/or radiotherapy if the tumour responded to treatment. In patients who did not respond to treatment or with tumour recurrence, levels of plasma 6-oxo-PGF1 alpha remained high or even rose further. Benign gynaecological tumours were also associated with significantly raised plasma 6-oxo-PGF1 alpha levels, and these fell to normal levels immediately on surgical removal of the tumour. Possible reasons for these alterations are described. Further investigations are warranted to see whether serial measurements of plasma 6-oxo-PGF1 alpha could be used as a prognostic index for the clinical status of patients with gynaecological tumours.

6-Ketoprostaglandin F1 alpha↗
PubMed1981

A comparison of the quantitative analysis of 6-oxo-PGF1 alpha in biological fluids by gas chromatography mass spectrometry and radioimmunoassay.

Two sensitive and selective quantitative methods for 6-oxo-PGF1 alpha, the stable hydrolysis product of prostacyclin are described and compared. Prostaglandins were extracted from biological fluids with organic solvents. Samples for gas chromatographic mass spectrometric analysis required additional thin-layer chromatographic separation prior to conversion to the O-methyloxime, methyl ester, tri-trimethylsilyl ether. Ion fragments at m/z 418 and 508 (protium) and m/z 422 and 512 (deuterium) were monitored to prepare 6-oxo-PGF1 alpha standard curves. The gas chromatographic mass spectrometric detection limit was 500 pg injected on column with a method coefficient of variation of 11.6% at this level. At 590 pg ml-1 the coefficient of variation for reproducibility of measurement was 1.77%. Antisera raised against a 6-oxo-PGF1 alpha-bovine serum albumin conjugate in sheep had a higher titre and greater selectivity than those raised in rabbits. Cross-reaction of sheep antisera with all prostaglandins and fatty acids tested was less than 0.5%. The radioimmunoassay limit of detection was 60 pg ml-1 (6 pg per tube) with a coefficient of variation of 10.4% (intra-assay) and 10.75% (inter-assay). The double blind comparison of gas chromatographic mass spectrometric and radioimmunoassay quantitation of the same samples gave a correlation coefficient of 0.97. Both methods offer sensitivity, selectivity and reproducibility. Gas chromatography mass spectrometry is necessary to validate the radioimmunoassay method which offers advantages of time, sample capacity and volume, expenditure and sensitivity.

6-Ketoprostaglandin F1 alpha↗
PubMed1980

Effect of an intra-uterine device on intracellular relationships of the uterine oestrogen receptor, particularly during pregnancy.

The presence of an intra-uterine device in the rat results in a lower nuclear concentration of the oestrogen receptor in the treated horn at pro-oestrus when it is compared with the contralateral control horn. This effect was also seen after the administration of hyperphysiological doses of oestradiol and when the horn was exposed in vitro to high concentrations of oestradiol. The cyclic changes during the oestrous cycle in the activity of the oestrogen-induced enzyme peroxidase were similar in the treated and control horns. These observations have discounted the possibility that the relatively lower nuclear receptor content in the treated horn at pro-oestrus was due to a decreased exposure to oestrogen. A significantly lower nuclear content was also observed in the treated horn on days 4 and 5 of pregnancy. This was not associated with a deficiency in cytosol receptor content which increased concurrently with that of the control horn in the 6 days of pregnancy that were studied. The proportional content of the putative cytosol factor implicated in receptor translocation was similar in both horns, increasing on days 4 and 6 in concert with reported changes in 'induced protein' synthesis. There appeared to be reduced levels of nuclear receptor at a time when blastocyst implantation normally occurs.

Animals↗
PubMed1980

Alterations in progesterone receptors in the rat uterus bearing an intra-uterine device during the oestrous cycle and early pregnancy.

The binding characteristics, content and intracellular distribution of cytosolic and nuclear progesterone receptors have been investigated, using [3H]progesterone as ligand, in the rat uterus bearing a unilateral intra-uterine device (IUD) during the oestrous cycle and from days 3 to 6 of pregnancy. The dissociation constants of nuclear and cytosolic progesterone-receptor complexes for IUD-containing and control uterine horns were similar. Cytosolic receptor concentrations in the IUD-containing uterus were always lower but changed in a manner similar to the control during the periods studied. Nuclear receptor concentrations in the control horn reflected changes in hormone levels during the oestrous cycle although concentrations measured were greater than previously reported. However, in IUD-containing uteri the pattern was completely reversed with minimal levels at pro-oestrus. Nuclear receptor concentrations were little different in both horns during early pregnancy. Total progesterone receptor synthesis determined between metoestrus and pro-oestrus in IUD-containing horns was significantly less than that of control horns. This correlated with the attenuated rise of nuclear oestrogen receptor levels previously observed between these times in IUD-containing uterine horns.

Animals↗
PubMed1978

The oestrogen receptor in the rat uterus in relation to intra-uterine devices and the oestrous cycle.

We investigated the binding characteristics, content and intracellular distribution of nuclear and cytosolic oestrogen receptors in the uteri of rats bearing a unilateral intrauterine device, fitted 14--18 days earlier, at four phases of a 5-day oestrous cycle. The patterns of changes in wet weight and content of cytosolic and nuclear receptor that normally occur during the oestrous cycle were not altered by the presence of the device. At all stages of the cycle the intra-uterine-device-containing horn had a greater wet weight and a correspondingly higher content of cytosolic receptor than its contralateral control horn, the cellular concentration of cytosolic receptor being apparently maintained. However, the intra-uterine-device-containing horn had significantly lower cellular concentrations (i.e. per mg of DNA) of nuclear receptor, particularly at late dioestrus and pro-oestrus. Thus the treated horn showed a decreased translocation of receptor in response to increases in circulating oestrogens. Both horns contained equivalent amounts of an activating factor implicated in translocation and measured in vitro by binding of cytosol receptor to oligo(dT)-cellulose. The presence of an intra-uterine device neither altered the dissociation constants (Kd) of the nuclear and cytosolic oestrogen-receptor complexes nor the stability of the nuclear receptor complex in vitro. The decreased translocation cannot thus be directly attributed to changes in the physical properties of the receptor. This decrease may be responsible for the anti-fertility effect of the intra-uterine device (which affects only the treated horn of the bicornuate rat uterus), since implantation of the blastocyst requires correct concentrations of nuclear oestrogen receptor.

Animals↗
PubMed1978

The effect of norgestrel and clogestone on the spontaneous motility of the human fallopian tube.

Spontaneous isometric contractions in vitro of both isthmic and ampullary parts of the human fallopian tube were studied. The tubes were obtained during the early luteal phase of the menstrual cycle from patients undergoing abdominal hysterectomy and who had ingested clogestone acetate or norgestrel, either 1 or 12 hours prior to the experiment. A dose of 150 microgram dl-norgestrel given orally 12 hours preoperatively caused a significant increase in the frequency of spontaneous isometric contractions together with a significant reduction in the area under the curve for each contraction. The possible effect of this altered pattern of motility is discussed.

Fallopian Tubes↗