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W D Sierralta

Publications and source records attributed to W D Sierralta.

29 records · Page 2Linked to original sources
PubMed1992

Estradiol-promoted accumulation of receptor in nuclei of porcine endometrium cells. Comparison of the retention of receptor in nuclei during subcellular fractionation of untreated and hormone-treated cells.

Nuclei were isolated from porcine endometrium of castrated pigs either unexposed or exposed to estradiol in vivo by two techniques, one of which included a hypotonic step. Aliquots were analyzed for estradiol content. Receptor was extracted from buffered, Surfynol-stabilized suspensions by either (a) KCl alone, (b) in combination with dithiothreitol, or (c) by dithiothreitol with polypentosanesulfate and addition of KCl. The yields rose from a-->c. The same proportional gains with increasing extractant efficacies were obtained from nuclei of unstimulated and estradiol-treated cells. Receptor recovery with extractant "c" rose linearly over the range of 9-80 x 10(6) nuclei/mL and was independent of the technique used for isolation. Nuclear fractions isolated using steroid-free solutions contained more estrogen receptor than estradiol; the numerical excess in control nuclei persisted in the nuclei of stimulated cells featuring a stoichiometric rise of ligand and receptor contents. The increase of receptor contents in nuclei isolated from hormone-stimulated cells coincided with a decline in the cytoplasmic fractions. An excess of hormone over receptor was seen only when nuclei were isolated from untreated cells with media containing 10 nM estradiol. Our data strengthen earlier notions of an estradiol-promoted receptor translocation into the nucleus and are not compatible with the ligand-filling hypothesis of preexisting nuclear binding sites.

Animals↗
PubMed1992

Estradiol-promoted accumulation of receptor in nuclei of porcine endometrium cells. Immunogold electron microscopy of resting and estradiol-stimulated cells.

Endometrium was collected by curettage from castrated pigs, either untreated or exposed to estradiol in vivo by intrauterine injection, and processed for electron microscopy. The resin LR Gold was used for embedding, and sections were floated on droplets of 10 nm diameter gold particles, coated with the immunoglobulin-G1 (IgG1) fraction or its Fab2 fragment of a monospecific polyclonal antiserum raised in goats against the C-terminal half of the estradiol receptor. On average, only one gold particle per microns 2 became attached in the cytoplasmic area of untreated cells, whereas four were found over the nuclear area. These figures rose to 2-3/microns 2 and 15-26/microns 2, respectively, within 10 min after exposure to estradiol. The labeling intensities of nuclei in cell clusters and of coprocessed nuclei released from cells ruptured during curettage were identical in all situations. Nuclear pores were frequently tagged after estradiol treatment. The proportions of tagging densities in nuclei of untreated and estradiol-exposed cells corresponded to those of receptor contents measured in extracts of isolated nuclei by ligand binding. This correlation was not seen for the cytoplasmic compartment of untreated cells, the scarce tagging of which is interpreted by hidden antigenic determinants. Our morphological analyses support the conclusions drawn from biochemical data (Sierralta et al., 1992) of an estradiol-promoted translocation of receptor from the cytoplasm into the nucleus.

Animals↗
PubMed1991

[Transcervical injection into the uterine lumen of castrated swine as a cell and organ biological research design. The Mariensee model].

The technique and the equipment necessary for the transcervical intrauterine injection in trained, ovariectomized pigs is detailed. Experimental groups of 14-16 week-old animals are recruited from German landrace litters with large numbers (4-9) of females. A silastic tubing containing a crystalline suspension of estradiol in propylene glycol is subcutaneously implanted behind the right ear. Ovariectomy and the detachment of the left uterine horn from the cervix are performed 7-10 days later. The animals are then kept for 6-7 weeks in ventilated stables, heated to 20 degrees C in wintertime, with 12 hr light dark cycle, free access to water and semi-ad libitum supplies of pelleted standard feed. The silastic implant gives rise to plasma levels of 8-12 pg estradiol/ml and still contains crystalline sediments when removed before the experiment. The manipulations for the transcervical, intrauterine instillation of solutions, imitating the reproductive action of the boar, are practiced daily for at least one week. The animals learn quickly to enter a restraining box, which is facilitated by the simultaneous offering of pellets drenched with root beer. The intrauterine injections proceed thereafter without any signs of stress from the animals. Stress is also avoided during slaughter by unexpected electric stunning on leaving the box, followed by exsanguination. Uteri are quickly excised and chilled in crushed ice until processed. One to two grams of endometrium cells can be harvested by curettage from each, treated and untreated (detached) horn. The Mariensee model allows for defined kinetic analyses of treated cells and provides nontreated controls from the same animal. It combines the advantages of in-vivo and in-vitro experiments.

Animals↗
PubMed1989

Assignment of estradiol-17 beta dehydrogenase and of estrone reductase to cytoplasmic structures of porcine endometrium cells.

Homogenates of porcine endometrium contain substantial activity for the dehydrogenation of estradiol-17 beta but little for estrone reduction. Both activities are associated with cytoplasmic structures. The dehydrogenase is characterized by a pH 7.7 optimum, Km 2.2 x 10(-7) mol/l for estradiol and Km 4.4 x 10(-5) mol/l for the cosubstrate NAD+. The corresponding figures for the reductase are pH 6.6, Km 1.1 x 10(-6) mol/l for estrone and Km 2.1 x 10(-5) mol/l for the cosubstrate NADPH. The (mitochondrial/lysosomal) 17,000 x g particulate fraction contains a 52-fold higher dehydrogenase than reductase activity. The (microsomal) 200,000 x g particulate fraction is only 16-fold richer in dehydrogenase. Isopycnic centrifugations of the two fractions in Percoll gradients reveal that estrone reductase and the coequilibrating marker enzyme cytochrome c reductase occur in constant proportions, whereas the dehydrogenase/cytochrome c reductase ratios are different. Both, the kinetic data and the structural assignments speak in favour of individual enzymes catalyzing the dehydrogenation of estradiol and the reduction of estrone. All gradient fractions exhibiting dehydrogenase activity feature small, electrodense vesicles of 0.15-0.20 microns in diameter as a common structural element which might harbour the dehydrogenase.

17-Hydroxysteroid Dehydrogenases↗
PubMed1988

Harvesting and separation of two populations of lysosomes from porcine endometrium.

The lysosomes present in homogenates of porcine endometrium epithelium equilibrate in two density regions of Percoll gradients. Patterns with varying proportions between high and low density peaks are observed, when aliquots of a tissue sample are processed with different all-glass Potter-Elvejhem homogenizers. The described constant-tolerance shearing device (CTSD), in contrast, provides homogenate fractions with higher latencies and steady distribution patterns. They are characteristic for each of the six lysosomal markers and the six other structure-bound enzymes measured in gradient fractions of the particulate matter harvested between 600g and 17,000g. The 17,000g sediments of CTSD homogenates contain more than 40% of the total lysosomal enzymatic activities. Recoveries from Percoll gradients are between 93 and 101%. Enrichments in the high density region range from 35-fold (beta-glucosidase) to 82-fold (acid ribonuclease). Both lysosomal populations exhibit latencies between 89 and 94%. Our results indicate that light lysosomes can be artificially generated by inappropriate homogenization, which should be considered in experiments on the formation and maturation of lysosomes.

Animals↗
PubMed1987

Biosynthesis and posttranslational finishing of the estradiol receptor.

The time course of subcellular receptor distribution in porcine endometrial epithelium was studied after intrauterine administration of estradiol alone or in combination with puromycin. In untreated cells, the major proportion of receptor is associated with cytoplasmic membranes. The solubilization of receptor from isolated nuclei is independent of their estradiol content. Smooth cytoplasmic membranes are the site of origin of receptor which is swiftly translocated into the nucleus in a 1:1 ratio with the hormone after exposure of the cells to estradiol. Simultaneously administered puromycin delays receptor synthesis and reveals that the nuclear passage of receptor is terminated by receptor degradation. The synthesis of receptor proceeds in rough endoplasmic membranes. A subsequent finishing and deposition in smooth membranes depends on intact protein synthesis.

Animals↗
PubMed1983

Characterization of microsomal subfractions from porcine endometrium cells.

Crude microsomes from porcine endometrium and three subfractions obtained by a modification of Rothschild's technique were characterized by RNA/protein ratio, marker enzyme activities and morphological appearance. The microsomes were devoid of glucose-6-phosphatase activity. They contained approximately 10% of arylesterase-, approximately 30% of both NADPH-cytochrome reductase- and UDPgalactose-N-acetyl-glucosamine beta-D-galactosyltransferase- and approximately 60% of 5'-nucleotidase activities present in the homogenates. Subfraction I (smooth membranes) had twice the galactosyltransferase activity of Subfraction II (smooth and rough membranes + free ribosomes); both subfractions were rich in 5'-nucleotidase and cytochrome reductase activities. Subfraction III (rough membranes) had very low marker activities but exhibited the highest RNA/protein ratio, which was lowest in I.

Animals↗
PubMed1983

Structural assignment and extractability of microsomal estradiol receptors.

Two electrophoretically different forms of estradiol receptor can be extracted from crude porcine endometrium microsomes with low ionic strength buffers. Better yields (approximately 50%) of both forms are obtained in the presence of Surfynol 485. Dithiothreitol boosts the solubilization of basic receptor. Together, Surfynol and dithiothreitol have a more than additive effect, amounting to 3-4 times the quantities of receptor extracted with plain buffer. Trypsin more than triples the yields obtained with Surfynol/dithiothreitol, while degrading both receptor forms to a characteristic fragment. Hyaluronoglucosaminidase is somewhat less effective than trypsin. It changes acidic receptor to basic. The proportions of acidic/basic receptor in microsomal subfractions are different. Rough endoplasmic reticulum contains almost exclusively basic receptor. Smooth membranes are rich in acidic receptor. The efficacy of both enzymes is closely related to the proportion of acidic receptor found in Surfynol/dithiothreitol extracts.

Animals↗
PubMed1983

Origin and quantification of cytoplasmic estradiol receptor in resting target cells.

The exhaustive extraction of microsomal estradiol receptor by Surfynol/dithiothreitol/trypsin in low ionic strength buffer was employed for distribution studies on non-stimulated porcine endometrium. It was found that more than half of the cytoplasmic receptor contents were of microsomal origin. "Empty" structures did not interfere with receptor analysis by agargel electrophoresis. The combined yields from homogenate fractions corresponded to those obtained from unfractionated homogenates. Freeze-fracturing of endometrium had a moderate receptor-solubilizing effect.

Alkynes↗
PubMed1983

Subcellular distribution, properties and interrelationship of oestrogen receptors in endometrium and other target tissues.

More than half of the extranuclear receptor content of resting cells is associated with cytoplasmic structures. Subfractionation of microsomes reveals a preponderance of "basic" (low electrophoretic mobility) receptor in rough endoplasmic reticulum. Surfynol-dithiothreitol extracts of smooth membranes are rich in "acidic" (high electrophoretic mobility) receptor. Trypsin increases the yields up to seven-fold, and this increase is correlated (r = 0.993) with the acidic receptor content and 5'-nucleotidase activity of these microsomal preparations. Acidic microsomal "cytosolic" and nuclear receptor can be degraded to the "basic" variety of streptomyces hyaluronidase. All forms give rise to a tryptic fragment with unchanged affinity for oestradiol and dimerization ability. Basic receptor isolated after enzymatic conversion of acidic receptor is microheterogenous on isoelectric focusing, but gives rise to only one precipitation arc versus the IgG fraction of a polyclonal antiserum. The precipitation arc can be recharged with labelled oestradiol and autoradiographed. Non-immune IgG's from (unspecific) soluble complexes with oestrogen receptors, but not with their tryptic fragments. The polyclonal antioestrogen receptor IgG fraction precipitates the oestradiol-tagged tryptic receptor fragment from all subcellular sources of all homologous (porcine) and heterologous (human, ovine, bovine, goat, rabbit, guinea pig, rat) target tissues tested. Organ specificity can therefore be excluded and a high degree of phylogenetic conservation of the oestrogen receptor's protein moiety is implied. The presence, in the immune IgG fraction, of steroid releasing antibodies, which apparently distort the binding site, should spur the search for monoclonal antibodies with similar properties.

Animals↗