PubMed Health⌕ Search

Biomedical subjects

T A Bramley

Publications and source records attributed to T A Bramley.

At least 73 records · Page 4Linked to original sources
PubMed1985

Specific binding of gonadotrophin-releasing hormone and an agonist to human corpus luteum homogenates: characterization, properties, and luteal phase levels.

The binding of radiolabeled GnRH and GnRH agonist (GnRHA) was studied in homogenates of human luteal tissue using a polyethylene glycol precipitation technique. GnRHA binding to human luteal homogenates was dependent on pH, was inhibited by a number of divalent metal ions, and was of moderately high affinity (Ka = 3 X 10(7) M-1). Binding of both radiolabeled GnRH and GnRHA increased linearly with increasing homogenate concentration, and binding of either tracer was parallel with different corpus luteum (CL) homogenates. Moreover, similar concentrations of unlabeled GnRH or GnRHA blocked the binding of either tracer, indicating that both hormones bound to a common site. The rate of dissociation of bound ligand was very low at 0 C, but was extremely rapid at physiological temperatures (t1/2 = 2.5-3.5 min). Human luteal GnRHA-binding sites were partially solubilized by treatment with Triton X-100 and were sensitive to trypsin and pronase, but not hyaluronidase. GnRHA binding to homogenates of human CL obtained at each stage of the luteal phase varied markedly, with levels ranging from less than 20 to greater than 1600 pg/mg DNA for CL from the (midluteal) stage of the menstrual cycle. This variability could not be accounted for by assay irreproducibility, loss of binding activity during storage, or occupancy of binding sites by endogenous ligand. These observations may help to explain negative reports from other groups, which demonstrated little or no specific binding of GnRH to human luteal tissue and no effect of GnRH on steroidogenesis using dispersed human luteal cells in vitro. We conclude that human luteal homogenates possess specific, moderately high affinity binding sites for GnRH and its agonist analogs.

Adult↗
PubMed1983

Specific binding of luteinizing hormone releasing hormone to human luteal tissue.

Homogenates of human luteal tissue bound radioiodinated luteinizing hormone releasing hormone agonist. Specific binding was both time- and temperature-dependent. Native LHRH and two LHRH agonists competed for binding, whereas TRH, somatostatin and oxytocin did not, indicating that the binding sites were specific. The apparent Ka values were 2 X 10(7)M-1 for both LHRH agonists and 10(6)M-1 for native LHRH. This is the first demonstration of specific binding of LHRH to human ovarian tissue. No binding could be detected to ovarian tissue from postmenopausal women.

Binding, Competitive↗
PubMed1981

Inactivation of porcine corpus luteum lutropin receptors by a microsomal enzyme. Activation in ageing and regressing corpora lutea.

The binding of 125I-labelled human choriogonadotropin (hCG) to homogenates of porcine corpora lutea showed a marked departure from ideal behaviour, due to a time- and temperature-dependent inactivation of both free hormone and unoccupied receptors. Occupied receptors were not affected. Lutropin (LH)-receptor-inactivating activity was detected after preincubation of homogenates, particulate fractions and microsomes, but little activity could be demonstrated in cytosol fractions. Inactivation was dependent on the temperature and pH of preincubation, and on tissue concentration: LH-receptor inactivation was first-order with respect to preincubation time. Lutropin-receptor-inactivating activity was low in early-luteal and mid-luteal phase in pig corpora lutea, but was increased significantly in late-luteal and regressing corpora lutea.

Animals↗
PubMed1981

Properties of LH/hCG receptors in porcine corpus luteum homogenates and subcellular fractions, and factors influencing the recovery of membrane-bound hormone.

An evaluation of several techniques commonly used to separate bound from free hormone was made for the binding of radiolabelled human chorionic gonadotropin to mid-luteal pig corpus luteum homogenates and subcellular fractions. Compared to millipore filtration with 0.22 or 0.45-mu HAWP filters, centrifugation of homogenates at 2 500 x gav or 1 000 x gav for 15 min recovered only 50 and 33% respectively of the total bound hormone present. This discrepancy was accentuated further when hormone binding to post-nuclear subcellular fractions was studied. Centrifugation of a nuclear fraction at 2 500 x gav for 15 min recovered 70% of the membrane-bound receptors present (as measured by filtration), whereas centrifugation of mitochondrial, lysosomal and microsomal fractions under these conditions recovered only 46, 16 and 4% respectively. Divalent metal ions influenced hormone binding in 2 ways: (i) Specific bonding of hCG was greatest in the absence of metal ions and in the presence of low levels of chelating agents. Increasing concentrations of magnesium and calcium ions appeared to interfere with the interaction of hormone and receptor. (ii) Divalent metal ions reduced the recovery of particle-bound hormone by centrifugation, at both high and low speeds. The results illustrate the pitfalls of using low-speed centrifugation to recover membrane-bound hormone-receptor complexes when small membrane vesicles are present.

Animals↗
PubMed1981

Glutamic-oxalacetic transaminase leakage from human spermatozoa as an indicator of cryodamage.

The levels of extracellular glutamic-oxalacetic transaminase (GOT: EC 2.6.1.1) activity have been measured in cell-free supernatants of human semen and of semen + cryoprotectant mixtures both before freezing by either cryostorage or quench-freezing regimes, and after subsequent thawing. The mean level of GOT activity in seminal plasma was 293 (+/- 15 sem) IU/l at 37 degrees C, this being 15 times the level in normal blood plasma. Seminal GOT is positively correlated (p less than 0.001) with the sperm density. Increases in the extracellular GOT activity were measured after both cryostorage and quench-freezing, but the latter procedure, which destroys all sperm motility, caused an average of 2.8 times more enzyme release than cryostorage. Although the release of GOT from human spermatozoa during freezing and thawing does not correlate with the commonly used percent return of motility as an estimate of cryosurvival, possible explantations for this finding, and its importance, are discussed. Objective measurements such as GOT release may be developed to augment, if not replace, subjective estimations of sperm motility as indicators of cellular integrity in studies of sperm cryostorage.

Aspartate Aminotransferases↗
PubMed1980

Interactions of gonadotropins with corpus luteum membranes. VIII. The different properties of rat luteal cell light and heavy membranes cannot be explained by fractionation of inside-out and outside-out plasma-membrane vesicles.

Fractions enriched in luteal cell plasma-membranes were isolated from superovulated rat ovaries. Cell surface-membrane marker enzyme activities were measured in both light and heavy membrane fractions, with or without treatment with various detergents, to assess whether these fractions consisted of resealed vesicles with different orientations (i.e. inside-out or outside-out). Control and digitonin-treated membranes were also fractionated on sucrose-density gradients to monitor the buoyant density perturbation of each surface-membrane marker induced by this detergent. In another experiment, hCG-binding sites of superovulated rat ovaries were labelled in vivo by injecting [131I]hCG intravenously 2 h prior to fractionation and isolation of ovarian membranes. Membranes were then tested for the ability to bind [125I]hCG and [131I]hCG in vitro. These experiments indicated that light and heavy rat luteal cell membrane fractions had similar levels of inside-out membrane vesicles (ranging from 0 to 34%), and that the hCG-binding sites of the 2 fractions appeared to be equally freely accessible to hormone in vivo. It is concluded that differences in membrane orientation cannot account for the distinct properties and marker enzyme profiles of rat ovarian light and heavy membranes, providing further support that these membrane are derived from different regions of the luteal cell surface-membrane.

5'-Nucleotidase↗
PubMed1980

Changes in the binding of human chorionic gonadotrophin/luteinizing hormone, follicle-stimulating hormone and prolactin to human corpora lutea during the menstrual cycle and pregnancy.

The changes in the binding of human chorionic gonadotrophin/luteinizing hormone (HCG/LH), follicle-stimulating hormone (FSH) and prolactin to 44 corpora lutea have been assessed during the luteal phase of the human menstrual cycle and early pregnancy. All corpora lutea bound HCG but out of 32 only ten bound FSH and only seven bound prolactin specifically. While binding of HCG increased to maximal levels in the mid-luteal phase, binding of FSH and prolactin was most often found in the early luteal phase. Maximum binding of HCG was associated with maximum serum levels of progesterone. Luteal regression was associated with a decrease in the binding of HCG but a causal relationship could not be established. Very low binding of HCG was found to corpora lutea of pregnancy. These results show that (1) the changes in binding of HCG during the luteal phase of the human menstrual cycle are similar to those in other species and (2) there are specific binding sites for prolactin and FSH in the human corpus luteum.

Chorionic Gonadotropin↗
PubMed1979

Interactions of gonadotropins with corpus luteum surface membranes. V. Differential effects of digitonin on the buoyant densities of light and heavy rat ovarian membrane fractions.

Previous studies have indicated that rat luteal cells at certain stages of development can be fractionated so as to obtain two plasma membrane fractions with different densities and different profiles of marker enzymes. The light membrane fractions (density 1.13) contain the majority of hCG-binding sites and little or no cyclase enzyme, while the heavy membranes (density 1.17) contain the majority of cyclase enzyme and lesser quantities of hormone-binding sites. These membrane fractions were further compared with respect to their susceptibility to perturbation by digitonin. The buoyant density of luteal cell light membrane fractions, as marked by [125I]iodo-hCG binding, Mg2+-dependent ATPase, and 5'-nucleotidase, were highly perturbable by digotonin (delta density, greater than 0.05), while adenylate cyclase activity and phosphodiesterase activity associated with this fraction were only slightly perturbed (delta density, less than 0.02). The buoyant density of luteal cell heavy membrane fractions, as marked by adenylate cyclase, ATPase, and nucleotidase, was not significantly perturbed by digotonin. The hCG binding associated with the heavy membrane fraction was not perturbed by digitonin. From these studies, we conclude that the adenylate cyclase activity associated with light membrane fractions is due to contamination by heavy membranes, while the hCG-binding activity in heavy membrane fractions is intrinsic to that membrane. Except for the lysosomal marker (glucuronidase), which was solubilized by digitonin, the detergent had no significant effect on the density of mitochondrial, Golgi, GERL (Golgi, endoplasmic reticulum, and lysomal), or endoplasmic reticulum membranes. Plasma membranes from isolated granulosa cells and ovaries obtained 24 h after priming with PMS gonadotropin-hCG behaved as heavy membranes (density, 1.17) which contained hCG-binding sites, adenylate cyclase, nucleotidase, and Mg2+-dependent ATPase. These were not significantly perturbed by digitonin. The appearance of light membranes and the segregation of adenylate cyclase from the majority of hCG-binding sites is a development feature of the luteal cell.

Adenylyl Cyclases↗
PubMed1978

Interations of gonadotropins with corpus luteum membranes. VII. Association of hCG-binding and adenylate cyclase activities with rabbit corpus luteum plasma-membranes.

Previous studies with rat luteal cell plasma membranes indicated a dissociation of hCG binding sites and adenylate cyclase activity. The hormone binding sites were associated with a light membrane (d 1.13) fraction derived from the microvillous surface while the cyclase enzyme was associated with a heavy membrane (d 1.17) fraction derived from the basolateral surface of the cell. In this study of plasma membranes from 8-day pseudopregnant rabbit corpora lutea both hCG binding and adenylate cyclase activity were associated with a light membrane (d 1.13) fraction. No heavy membrane material was detected.

Adenylyl Cyclases↗
PubMed1978

Interactions of gonadotropins with corpus luteum membranes. I. Properties and distributions of some marker enzyme activities after subcellular fractionation of the superovulated rat ovary.

The properties of a number of enzyme activities of the superovulated rat ovary have been studied to establish optimal assay conditions and specific assay procedures for each activity. The activities were chosen on the basis of their extensive use in other tissues of the rat as marker enzymes for the major cell organelles. Homogenates of superovulated rat ovaries were subjected to fractionation by differential rate centrifugation, and sedimentation profiles were constructed for each marker enzyme activity. The various subcellular fractions were also monitored by electron microscopy. The enrichment of fractions with particular organelles by electron microscopy, and enrichment of the appropriate organelle marker enzyme activities correlated well. Sedimentation profiles of a number of plasma membrane marker enzymes demonstrated a marked discrepancy between hCG-binding activity, and 5'-nucleotidase-, alkaline phosphatase-, and Mg2+-dependent ATP-ase on the one hand, and basal, hCG-stimulated, and fluoride-stimulated adenylate cyclase activities on the other hand. Fractions enriched in hCG-binding and adenylate cyclase activities were subjected to further fractionation on discontinuous sucrose density gradients. The distributions of the various plasma membrane markers again indicated a partial dissociations between hCG-binding and adenylate cyclase activities of luteinized rat ovaries, suggesting the existence of two distinct major plasma membrane populations, with different buoyant densities, marker enzyme profiles and adenylate cyclase and hormone-binding levels.

Adenylyl Cyclases↗
PubMed1978

Interactions of gonadotropins with corpus luteum membranes. II. The identification of two distinct surface membrane fractions from superovulated rat ovaries.

Fractions enriched in hCG-binding activity were prepared by differential rate centrifugation of superovulated rat ovarian homogenates and were applied to continuous sucrose density gradients (20-55%). After centrifugation at 63,000 x gav for 3.5 h, fractions of each gradient were collected and assayed for a range of marker enzyme activities characteristic of surface membranes and subcellular organelles. Mitochondria, lysosomes, and rough and smooth endoplasmic reticulum membranes accumulated in the gradient between 38-41% sucrose (1.165-1.180 g/cm3). Nuclei passed through the gradient. However, the various surface membrane markers concentrated in two distinct regions of the gradient. Alkaline phosphatase, phosphodiesterase, (Na+ + K+)ATPase I, and hCG-binding activity concentrated at 29-32% sucrose (1.120-1.135 g/cm3), whereas 5'-nucleotidase, Mg2+-dependent ATPase, and adenylate cyclase activities (and minor peaks of hCG-binding and phosphodiesterase activities) were enriched at 36-38% sucrose (1.16-1.17 g/cm3). A second ATPase, [(Na+ + K+)ATPase II], was also observed in this region of the gradient, which could be distinguished from (Na+ + K+)ATPase I of the light membrane fraction by its sensitivity to the Ca2+-chelating agent, ethylene glycol bis-(aminoethyl)tetraacetic acid (EGTA). The kinetics of binding of radioiodinated hCG to the gonadotropin receptors of the light and heavy membrane fractions were very similar. It is suggested that fractionation of superovulated rat ovaries yields two distinct populations of surface membrane material which have distinct densities and marker enzyme profiles. Furthermore, in contrast to the heavy membrane fraction, light membranes seem to possess considerable amounts of hCG receptor activity but very little adenylate cyclase.

Animals↗
PubMed1976

Subcellular distribution of a gonadotropin-induced form of mouse ovarian alkaline phosphatase.

The subcellular distributions of alkaline phosphates I (the major activity of prepubertal mouse ovaries) and alkaline phosphatase Ib (a kinetically distinct isoenzyme induced in large amounts by injection of human chorionic gonadotropin or luteinizing hormone) were studied by differential rate centrifugation and discontinuous density gradient centrifugation of ovarian homogenates from control and gonadotropin-treated mice. The distributions of the two alkaline phosphatases were alike and were similar to those of nucleotidase, Mg2+ -dependent ATPase and Co2+ -stimulated naphthylamidase activities, suggesting that they were associated with plasma-membrane vesicles.

Alkaline Phosphatase↗
PubMed1976

Neonatal androgenization: effects of responsiveness of ovarian alkaline phosphatase to gonadotropins.

Five-day-old female mice were injected subcutaneously with 100 mug of testosterone benzoate in oil, or with oil only. At various ages thereafter, they received either 5 I.U. human chorionic gonadotropin (hCG) per 10 g body weight or saline only, and were killed 24 h later. Alkaline phosphatase activity was measured in whole ovarian homogenates. Neonatal androgenization failed to affect the early phases of ovarian responsiveness, but selectively abolished both the normal rise in alkaline phosphatase which precedes the onset of puberty and the responsiveness of the enzyme to hCG stimulation at this time.

Aging↗
PubMed1976

Mouse ovarian alkaline phosphatase activities that respond to gonadotropins: histochemical and biochemical studies.

Alkaline phosphatase activity was measured in whole ovarian homogenates from pre-pubertal mice of different ages, with and without prior injection of human chorionic gonadotropin. Alkaline phosphatase activity was also scored in the different cell types in sections of similar ovaries, using two distinct histochemical procedures. The results from those methods differed. Biochemical studies indicated the presence of three distinct alakaline phosphatase activities: I and Ib, both optimal at pH 10.4 and with similar substrate requirements and inhibitor sensitivities (phosphatase I being characteristic of unstimulated ovaries and Ib of ovaries stimulated with human luteinizing hormone or human chorionic gonadotropin), and phosphatase II, optimal at pH 9.4, with different substrate requirements and inhibitor sensitivities. The differences observed using the histochemical procedures can probably be accounted for by the effects of different incubation conditions on the activities of these three enzymes.

Aging↗