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R M Roberts

Publications and source records attributed to R M Roberts.

At least 217 records · Page 12Linked to original sources
PubMed1983

A function of the allantoic sac in the metabolism of uteroferrin and maternal iron by the fetal pig.

The possible roles of the allantoic sac in metabolism of uteroferrin, the iron-containing, purple phosphatase, were examined. This protein, which originates in the maternal endometrium, was measured in allantoic fluid by a specific double-antibody radioimmunoassay and by its beta-mercaptoethanol-activated acid phosphatase activity. Total uteroferrin reaches a maximum between days 60 and 75 of pregnancy, when it constitutes up to one-third of the total protein, and then declines towards term. Fluid volume, total protein and total iron also reach a maximum around day 60. Two-dimensional polyacrylamide-gel electrophoresis was employed to identify proteins in allantoic fluid. Of the proteins detected, at least three basic and two acidic polypeptides are also characteristic of maternal uterine secretions. The remaining proteins appeared identical to those found in fetal serum and included transferrin, albumin and alpha-fetoprotein. Although uteroferrin acid phosphatase activity is stable when incubated in buffer, the enzyme loses activity in sterile allantoic fluid collected at all stages of pregnancy. Experiments with 59Fe-uteroferrin have shown that allantoic fluid promotes iron loss from uteroferrin and that the metal appears in transferrin. Thus the allantoic sac may serve not only as a depot for uteroferrin accumulation but as a site of active iron metabolism.

Acid Phosphatase↗
PubMed1982

Purification and properties of a progesterone-induced plasmin/trypsin inhibitor from uterine secretions of pigs and its immunocytochemical localization in the pregnant uterus.

The porcine uterus secretes a group of basic, low molecular weight protease inhibitors under the influence of progesterone, but not estrogen. One of these inhibitors (Mr approximately 14,500) which inhibits trypsin, plasmin, and chymotrypsin, but not other proteases tested, has been purified 10- to 15-fold from uterine secretions of pseudopregnant pigs using Sephadex G-100 chromatography, CM-cellulose ion exchange chromatography, and Sephadex G-50 or Bio-Gel P-10 chromatography. The inhibitor which is relatively heat- and pH-stable forms a 1:1 molar complex with trypsin which is not dissociated in sodium dodecyl sulfate except by boiling. Chymotrypsin appears to bind at the same site on the inhibitor as trypsin. The inhibitor is high in half-cysteine residues and basic amino acids, and appears not to be a glycoprotein. Antiserum has been raised against the purified inhibitor in rabbits and used to test its distribution in pigs using the immunoperoxidase-staining technique on tissue sections. The inhibitor is associated only with the glandular and surface epithelium of the uterus. Endometrial explants from pseudopregnant animals, cultured in presence of L-[3H]leucine, release the inhibitor in radioactive form indicating that it is a uterine product. The antiserum against the inhibitor cross-reacts with at least three other, basic, low molecular weights plasmin/trypsin inhibitors in porcine uterine secretions, suggesting that a family of isoinhibitors exists which may constitute up to 15% of the protein in porcine uterine secretions. The inhibitor(s) appears to coat and to be taken up by the trophoectoderm cells of the elongating blastocyst during pregnancy. It is suggested that the inhibitors may serve to protect the uterus from proteases released by the porcine trophoblast or to prevent degradation of essential macromolecules, such as uteroferrin, which have to be taken up by the conceptus.

Animals↗
PubMed1982

Iron transfer between the purple phosphatase uteroferrin and transferrin and its possible role in iron metabolism of the fetal pig.

Uteroferrin, a purple-colored, iron-containing phosphatase which is induced by progesterone in the porcine uterus, has been proposed to be an intermediary in iron transfer between the mother and conceptus in the pig. Along with a number of other uterine proteins of maternal origin, it accumulates in the allantoic fluid during mid-pregnancy. When [59Fe]uteroferrin was introduced into the allantoic sacs of five fetuses at Day 60 of pregnancy, its iron was transferred to another protein, identified as transferrin. The half-life of iron loss from uteroferrin was approximately 24 h and the kinetics suggested an approximately second order process. 59Fe was also distributed to the fetus and was recovered at high specific activity in the fetal red blood cells (as hemoglobin), liver, and spleen. Introduction of [59Fe] transferrin to allantoic fluid resulted in an almost identical distribution of isotope in the fetus as was observed with [59Fe]uteroferrin. Whereas [125I]transferrin has been shown to be capable of leaving the allantoic sac in intact form, [125I]uteroferrin either has only a limited capacity to exit or else is almost immediately degraded upon entering the fetal circulation. Following loss of its iron to transferrin in allantoic fluid, uteroferrin rapidly loses immunological reactivity and is cleaved proteolytically into small peptides. The transfer of iron between uteroferrin and transferrin has also been followed in vitro. Iron on uteroferrin is relatively stable to pH and is not readily lost to transferrin at around neutral pH unless low molecular iron chelators such as citrate, pyrophosphate, ATP, or ascorbate are present. Iron transfer was promoted at ascorbate concentrations (approximately 50 microM) and at pH values (approximately 6.8) approximating those found in allantoic fluid. Results are consistent with a model in which maternal uteroferrin can transfer its iron to fetal transferrin via a low molecular weight intermediary in the allantoic sac. Transferrin is then responsible for iron distribution to the fetus.

Acid Phosphatase↗
PubMed1982

Iron content and molecular weight of uteroferrin and a comparison of its iron and copper forms.

The molecular weight of the purple, iron-containing glycoprotein uteroferrin has been determined to be 35140 +/- 1610 by sedimentation equilibrium centrifugation. This is consistent with measurements carried out by other procedures. The dry weight of a solution of uteroferrin with an absorption of 1.00 cm -1 at 280 nm was 0.87 mg/ml. Highly purified uteroferrin has a ratio of absorbance at 280 nm to 545 nm of about 13.2 and a revised extinction coefficient at 545 nm of 3.1 . 10(3) M -1 is presented. The iron content of uteroferrin has been determined both colorimetrically and by atomic absorption spectroscopy. One iron atom is present per polypeptide chain. Reduction with dithionite leads to loss of iron, allowing the apoprotein to be prepared. Enzymatic activity can be restored to the apoprotein with Fe(III) or with Cu(II). The copper enzyme has no visible color and has a higher pH optimum than the Fe-uteroferrin. Whereas the phosphatase activity of the latter is increased several-fold by beta-mercaptoethanol, reduction inactivates Cu-uteroferrin. Both forms of uteroferrin have similar Km values for p--nitrophenyl phosphate and are inhibited by molybdate but not by tartrate. Excess Cu(II) and Fe(III) strongly inhibit uteroferrin phosphatase activity and these results may explain the failure of other to restore activity to the apoprotein using Cu(II) and Fe(III) salts.

Acid Phosphatase↗
PubMed1982

Comparison of glucose, fructose, ascorbic acid and glucosephosphate isomerase enzymatic activity in uterine flushings from nonpregnant and pregnant gilts and pony mares.

In Experiment 1, 40 gilts and 30 pony mares were used to characterize changes in glucose, fructose, ascorbic acid and glucosephosphate isomerase (GPI) enzymatic activity in uterine flushings collected either during the estrous cycle or early pregnancy. Total recoverable glucose was greater (P less than 0.01) in uterine flushings from pregnant gilts, but pregnancy status had no effect on total recoverable glucose in pony mare uterine flushings. Fructose was undetectable in uterine flushings from nonpregnant gilts and pony mares and pregnant gilts and pony mares prior to Day 14, but occurred in increasing amounts between Days 14 and 18 or 20 of pregnancy. In Experiment 2, it was demonstrated that the porcine conceptus is the primary source, if not the sole source of fructose. Total recoverable ascorbic acid in uterine flushings was not affected by pregnancy in gilts, but was greater (P less than 0.01) in pregnant versus nonpregnant pony mares. In both species, total recoverable ascorbic acid was affected (P less than 0.01) by day of the estrous cycle and pregnancy. The GPI enzyme allows for the interconversion of glucose-6-PO4 and fructose-6-PO4. GPI total and specific activities were greater (P less than 0.01) for pregnant than nonpregnant gilts and pony mares. The periods of greatest GPI activity were temporally associated with elevated estrogens of either ovarian or blastocyst origin. Results from Experiment 3 indicated a marked increase (P less than 0.01) in GPI activity in uterine flushings from gilts treated with estradiol valerate. Results of this study indicate that glucose (gilt only), fructose, ascorbic acid and GPI activity are increased in uterine flushings of gilts and pony mares during early pregnancy. The increase in these constituents may reflect increased carbohydrate metabolism in ways which are uniquely beneficial to conceptus development in ungulates.

Animals↗
PubMed1982

Placental transport and distribution of uteroferrin in the fetal pig.

Placental transport of uteroferrin (Uf), the progesterone-induced iron transport glycoprotein, and its distribution within the fetus were investigated by the peroxidase-antiperoxidase bridge (PAP) technique. In Experiment 1, Uf was localized in endometrial and placental tissues taken from gilts on Days 60, 75, 90 and 105 of pregnancy. Uteroferrin was observed within cells of the endometrial glands and surface epithelium adjacent to placental areolae but not in endometrial surface epithelium between areolae. Heavy staining for Uf was observed in cells of the areolae and was associated with both supra- and infranuclear cytoplasmic vesicles. Vesicles located within the infranuclear cytoplasm were occasionally observed to be releasing their contents into capillaries surrounding the areolae. In Experiment 2, Uf was measured by radioimmunoassay in blood samples taken from the umbilical vein and artery of fetuses on Day 75 of pregnancy. Uteroferrin concentrations were greater (P less than 0.07) in umbilical vein blood (79.8 +/- 13.1 ng/ml) than in umbilical artery blood (43.9 +/- 13.1 ng/ml). Uteroferrin binding by Day 75 fetal liver membranes was examined in Experiment 3. Binding of 125I-Uf increased linearly with increasing quantities of membrane protein and binding of 125I-Uf was competitively inhibited by adding unlabeled Uf to the assay. In Experiment 4, urine samples were taken from Day 75 fetuses and assayed for beta-mercaptoethanol activated acid phosphatase activity which is indicative of Uf. In addition, urine proteins were analyzed for Uf by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Ouchterlony double immunodiffusion (ID). Acid phosphatase specific activity was 3.37 +/- 1.83 mumol substrate hydrolyzed/10 min per mg protein. Uteroferrin was detected in two of three urine samples by 2D-PAGE and in three of eight samples by ID. In Experiment 5, tissue distribution of Uf was determined by the PAP technique in liver and kidney tissue taken from fetuses on Day 75 of pregnancy. Staining for Uf was observed in collecting ducts and proximal tubules of kidney tissue, but staining was not observed in liver tissue. These results indicate that Uf is transported by the areolae into the chorioallantoic capillaries and to the fetus by the umbilical vein. Within the fetus Uf is either bound by the liver, probably to supply iron for hematopoiesis, or cleared by the kidney and transported within the urine to the allantoic sac to serve as a temporary iron storage reservoir.

Acid Phosphatase↗
PubMed1982

Identification of stage-specific and hormonally induced polypeptides in the uterine protein secretions of the mare during the oestrous cycle and pregnancy.

Uterine secretions were obtained on Days 4, 8, 12, 14, 16, 18 and 20 of the oestrous cycle and early pregnancy. Acid phosphatase activity was significantly affected by day of the cycle, reaching a maximum at days 12-14 during the luteal phase and then declining to almost undetectable levels, by Day 20. In pregnant animals, activity continued to increase beyond Day 14. Two-dimensional polyacrylamide gel electrophoresis showed that albumin was a major component. However, a number of unique proteins of non-serum origin appeared in mid-cycle but had disappeared by Day 20. One of these was a basic protein indistinguishable in electrophoretic properties from the uterine acid phosphatase of the pig, uteroferrin, which is believed to be involved in iron transport from the uterine endometrial epithelium to the conceptus. These same polypeptides, including the putative uteroferrin, were also present in uterine flushings from pregnant animals until Day 20, and in flushings from ovariectomized mares treated with progesterone but not in those given only oestradiol-17 beta. Flushings from all ovariectomized animals contained a non-serum, acidic polypeptide (pI 5.3) of molecular weight 70 000. One basic polypeptide (molecular weight approximately 17 000) appeared by Day 4 of the oestrous cycle and disappeared by Day 16 but was maintained during pregnancy until Day 20. It was absent, however, in flushings from a Day 45 pseudopregnant mare. Like the sow, therefore, the mare possesses a number of proteins associated with cyclic changes in steroid hormones during the oestrous cycle and early pregnancy.

Acid Phosphatase↗
PubMed1982

Purification and properties of a major, low molecular weight protein released by the trophoblast of sheep blastocysts at day 13-21.

Sheep blastocysts (Day 13-21) incubated in a modified Minimum Essential Medium released proteins into the medium at an approximately linear rate over a 24-h period. Single Day-16 blastocysts converted 2-8% of the radioactivity supplied (100 muCi L-[3H]leucine) into non-dialysable macromolecules which were released into the medium. Two-dimensional polyacrylamide gel electrophoresis revealed that at Day 13 there was only one major product (Protein X), consisting of three closely similar isoelectric species of (pI of denatured polypeptides about 5.5), each with molecular weights of 17 000. Between Days 14 and 21 additional proteins were detected. One of these was of high molecular weight (greater than 660 000) and did not appear on the two-dimensional gels, but Protein X continued to predominate until Day 23 when it could not be detected. Explants of chorion from Day 30 of pregnancy failed to secrete Protein X. Protein X was released in significant quantities (50-100 micrograms per 24 h) by the trophoblast but not the yolk sac of Day-14 and Day-16 conceptuses, but was present in very low amounts in the tissues. Protein X from the incubation medium of Day-14 and Day-16 conceptuses was purified by successive DEAE ion exchange and Sephacryl S-200 gel chromatography. Because Protein X and some of the other proteins are produced transiently between Days 13 and 21, it is possible that they may play a role in maternal recognition of pregnancy in sheep.

Animals↗
PubMed1982

High molecular weight glycoproteins released by expanding, pre-attachment sheep, pig and cow blastocysts in culture.

Blastocysts isolated from sheep (Day 14--16), pigs (Day 16) and cows (Day 19) during the pre-attachment elongation phase were cultured for up to 30 h in a modified MEM medium in the presence of radioactive amino acids (L-[14C]leucine or L-[35S]methionine) to label protein and D-[3H]glucosamine to label complex saccharides. All the blastocysts released considerable quantities of non-dialysable radioactive material into the medium at an approximately linear rate over the course of the incubation. Ion-exchange chromatography on DEAE cellulose at pH 8.2 revealed that the major glucosamine-labelled product in the medium was a non-sulphated glycoprotein which eluted early in the salt gradient. None of the blastocysts produced any detectable glycosaminoglycan-like materials such as hyaluronic acid. The glycoprotein was purified by ion-exchange and gel filtration chromatography and had a molecular weight of greater than 660 000. Up to 100 micrograms of this material could be isolated from incubations of 2 sheep conceptuses. It was relatively resistant to protease hydrolysis and consisted of approximately 50% carbohydrate and 50% protein. The main monosaccharide constituents, as revealed by gas-liquid chromatography, were galactose and N-acetylglucosamine plus some mannose and fucose. No sialic acid was present. The linkages between the carbohydrate chains and the peptide appeared to be resistant to alkaline borohydride cleavage and were probably, therefore, N-glycosidic.

Animals↗