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F B Wooding

Publications and source records attributed to F B Wooding.

At least 37 records · Page 2Linked to original sources
PubMed1992

Active lymphocyte traffic induced in the periphery by cytokines and phytohemagglutinin: three different mechanisms?

Phytohemagglutinin (PHA) injection induces transient protease-sensitive traffic of lymphocytes in skin and other tissues in several species. Examination of the possible roles of cytokines in such reactions showed that recombinant bovine and human tumor necrosis factor (TNF)-alpha potently induce dose-dependent lymphocyte traffic in pig skin (and in other tissues including the draining lymph nodes) with early kinetics and a morphology of the inflammatory reaction similar to that of PHA (peaking 9-12 h). Recombinant human interleukin (IL)-1 alpha also induces dose-dependent lymphocyte traffic, but it peaks at 4 h. Entry of labeled lymphocytes into inflammatory sites induced by PHA, TNF-alpha and IL-1 alpha, but not into normal skin, is inhibited by approximately 80% by their pretreatment with trypsin, indicative of the induction of endothelial determinants recognized by protease-sensitive surface molecules on the lymphocytes. Even the minimal lymphocyte traffic induced by interferon-gamma and lipopolysaccharide was similarly protease sensitive. At the earliest stage (approximately 2 h) of significant induction of lymphocyte entry by TNF-alpha and IL-1 alpha the inductive signal for each appears easily saturated. Thus lymphocyte entry is little increased by increasing low cytokine doses over 100-fold: However, these reactions are additive, and this was used to confirm that they are distinct from each other and from PHA. A further distinction was revealed by the homing of lymphocytes pretreated with pertussis toxin: such lymphocytes were greater than 90% inhibited in their homing to tissues through constitutive high endothelial venules (HEV) and greater than 60% inhibited in homing to TNF-alpha and IL-1 alpha skin sites, but unaffected in homing to PHA skin sites (like most non-HEV-mediated traffic). Moreover, potent chicken anti-TNF-alpha, which prevented TNF-induced lymphocyte entry, did not affect PHA-induced traffic. Thus, these three agents which induce peripheral lymphocyte traffic appear to involve different mechanisms as shown by differences in (i) their kinetics; (ii) the effect of anti-TNF-alpha and (iii) the effect of pertussis toxin treatment of the lymphocytes and by the fact that their inductive mechanisms are additive in effect.

Animals↗
PubMed1992

Light and electron microscopic studies of cellular localization of oPL with monoclonal and polyclonal antibodies.

Accurate knowledge of placental lactogen localization is fundamental to any hypothesis of its synthesis and secretion. We used locally generated monoclonal and polyclonal antibodies from three separate sources to localize ovine placental lactogen immunoreactivity on light and electron microscope Lowicryl K4M sections of ovine placentomes of 97-145 days of gestation, using immunogold techniques. All antibodies demonstrated that immunoreactivity was exclusively localized in the trophoectoderm binucleate cell Golgi body and granules and in granules in the syncytium derived from binucleate cell migration. No evidence was found to support a recent claim that monoclonal antibodies to oPL that were produced in Canada indicated a predominant localization of ovine placental lactogen to uninucleate trophectodermal cells.

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↗
PubMed1991

Quantitative immunogold ultracryomicrotome studies of the distribution of periimplantation proteins in the sheep.

Several mammalian uterine and conceptus proteins are produced at specific stages of implantation. Ovine trophoblast protein-1 (OTP-1) is only synthesised in vitro by conceptus tissue from between 13 and 21 days of pregnancy (dpc). This immunogold ultracryosection study shows that, during this period, OTP-1 immunoreactivity is only found in the Golgi body of the trophectodermal cells. A second protein, of 14 kD molecular weight (14 K protein), has a more varied distribution being found in membrane-bounded crystals in uterine epithelium and trophectodermal cells, and distributed throughout the cytosol and nucleoplasm of the uterine epithelium. There are only trace amounts of the 14 K protein in the fetomaternal syncytium which replaces the uterine epithelium during implantation, and no crystals are found in the trophectoderm after cotyledonary villus formation is initiated at 24-25 dpc. The crystals containing 14 K protein persist throughout pregnancy in the intercotyledonary areas. The narrow time window of OTP-1 occurrence reinforces the suggestion that this represents an important developmental signal, whereas the distribution of the 14 K protein indicates a more general nutritive function.

Animals↗
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↗
PubMed1990

Characterization of the synthetic capacities of isolated placental binucleate cells from sheep and goats.

Sheep and goat binucleate cells (BNC) play a central role in placental growth and development. This study reports a simple method for isolating 60-70% pure populations of BNC of high viability. After incubation of the isolated BNC with a brief pulse of 14C-leucine or 3H-fucose or 3H-galactose, electron microscope autoradiography showed that label was eventually incorporated into the characteristic BNC granules via the Golgi body. Fucose and galactose initially showed a much higher Golgi body label than leucine, which was at first predominantly localised in the endoplasmic reticulum. 35S-methionine incorporation by BNC suspensions was extensive enough to allow an immunoprecipitation investigation which demonstrated that the protein hormone ovine placental lactogen and the SBU-3 antigen were synthesised de novo. Previous studies with isolated BNC have shown a remarkable range of substances to be released into the incubation medium but not necessarily synthesised during the incubation. The results demonstrate unequivocally that isolated BNC's are capable of total synthesis in vitro of two of the proteins that these same cells are known to secrete in vivo.

Animals↗
PubMed1990

Phytohemagglutinin induces major short-term protease-sensitive lymphocyte traffic involving high endothelium venule-like blood vessels in acute delayed-type hypersensitivity-like reactions in skin and other tissues.

Injection of phytohemagglutinin induces dose-dependent acute delayed-type hypersensitivity-like reactions in young pig skin which attract large numbers of labeled peripheral blood lymphocytes and lymphoblasts but not red cells. Similar reactions are induced in the gut wall, joints, and draining lymph nodes, and also in the skin of sheep and cattle, and by concanavalin A. Labeled peripheral blood lymphocyte entry starts within 2 h, is maximal at 6-18 h and is effectively over by 48 h and, both early and late, is markedly inhibited in a selective way by trypsinization, like high endothelium venule-mediated entry to lymphoid tissues. Electron microscopy showed development of high endothelium venule-like blood vessels with intramural lymphoid cells in mononuclear infiltrates. Inductive processes in this model short-term peripheral defense mechanism are being investigated.

Animals↗
PubMed1990

An immunogold, cryoultrastructural study of sites of synthesis and storage of chorionic gonadotropin and placental lactogen in human syncytiotrophoblast.

The sites of intracellular synthesis and storage of human placental lactogen (hPL) and human chorionic gonadotropin (hCG) are controversial. We have used one of the most sensitive methods, cryoultramicrotomy and immunogold labelling, to localise these hormones at the electron-microscopic level. In both 12-week and term placentas hCG and hPL are present throughout the rough endoplasmic reticulum cisternae, in the Golgi bodies, and in the infrequent small dense granules of the syncytiotrophoblast. Previous assays have shown that hCG is at a higher concentration in early pregnancy and hPL peaks in late pregnancy, and our results corroborate these findings. No significant localisation of either hormone was seen in the cytotrophoblast or villous stroma. The results suggest that both hCG and hPL are synthesised and packaged by the classical secretory pathway, although the level of hormone stored in granules at any one time is small.

Chorionic Gonadotropin↗
PubMed1990

A progesterone-modulated, low-molecular-weight protein from the uterus of the sheep is associated with crystalline inclusion bodies in uterine epithelium and embryonic trophectoderm.

A protein of Mr 14,000 (14K protein) has been identified in the medium after culturing sheep conceptuses in vitro. However, it was also a component of uterine flushes of nonpregnant ewes during the luteal phase of the estrous cycle. The 14K protein, which consists of several isoforms, was purified from conceptus culture medium by a simple three-step procedure, involving ammonium sulfate precipitation, high-performance anion-exchange chromatography, and gel filtration on Sephacryl S-200. Western blotting procedures with an anti-14K antiserum showed that endometrium from ovariectomized ewes began to release the 14K protein after 6 to 14 days of progesterone treatment. Immunostaining of tissue from Day 16 pregnant ewes and from ewes treated with progesterone for 14 or 30 days showed the protein to be confined to the surface and upper glandular epithelium of the endometrium and to be absent from the deep glands. It was also present in trophectoderm of Day 16 conceptuses. Immunogold labeling in conjunction with electron microscopy revealed that within trophectoderm the 14K protein was localized to large, membrane-bound rhomboidal or needle-shaped crystal structures, and it seems likely that the protein was accumulated by the conceptus as a result of uptake of uterine histotroph. In the uterine epithelium, immunogold label was again most strongly concentrated over crystal-like structures but was also uniformly present over the cytoplasm and nucleoplasm. It was absent over mitochondria, over lipid droplets, and over non-epithelial types of endometrial cell. Such a cellular distribution for a secretory protein is novel, and it remains unclear whether the protein is synthesized by these cells.

Animals↗
PubMed1990

The role of trophoblastic binucleate cells in implantation in the goat: a morphological study.

In the goat conceptus individual intra-epithelial trophectodermal binucleate cells first appear 18 days post coitum and their incidence rapidly increases where the trophectoderm is apposed to the caruncular and intercaruncular sites of initial attachment to the uterine epithelium. Special staining techniques reveal that these cells, when mature, contain prominent Golgi bodies and numerous characteristic granules. Our evidence shows that at 19 days post coitum the binucleate cells migrate to the microvillar junction and fuse with individual uterine epithelial cells to form hybrid feto-maternal trinucleate cells. It is proposed that subsequent continued binucleate cell migration and fusion with trinucleate cells produce the syncytial plaques typical of the remainder of pregnancy. It is further suggested that the fusion is important in facilitating the delivery of the characteristic granules to the base of the uterine epithelial layer with subsequent exocytosis of their contents into maternal tissue.

Animals↗
PubMed1989

An immunological cryo-ultrastructural study of a sequential appearance of proteins in placental binucleate cells in early pregnancy in the cow.

Using the most sensitive immunocytochemical method available, on ultrathin frozen sections, the results in this paper demonstrate that bovine placental lactogen (bPL) is present in the earliest fetal binucleate cells found at 21 days post coitum in the trophectoderm. A second protein, the SBU-3 antigen, which is absent in the early stages of pregnancy appears abruptly in the binucleate cell granules at 30 days post coitum coincident with the start of villus development. Subsequently, the granules contain both bPL and the SBU-3 antigen. This sequential production of unlike proteins indicates that the binucleate cell has different functions depending on the stage of pregnancy and has important roles to play both at implantation and in villus development.

Animals↗
PubMed1988

Morphological characterisation of lactotrophs separated from the bovine pituitary by a rapid enrichment technique.

The morphological characteristics of bovine pituitary cells separated by a rapid enrichment procedure are described. Single-cell suspensions were prepared from pituitary glands of steers by use of a collagenase technique and separated by discontinuous gradient centrifugation. The separation of prolactin- and growth hormone-containing cells was assessed by radioimmunoassay of hormone content and immunocytochemistry, and the distribution of fibroblasts assessed after establishing cell cultures. Morphometric analysis of the fine structure of two fractions respectively enriched and depleted in the proportion of immuno-cytochemically-identified lactotrophs was performed after labelling with anti-prolactin antiserum coupled to immunogold complex. Cells recovered from the higher-density fraction were more highly granulated, suggesting that this was a major characteristic determining separation. Cells labelled for prolactin could not be distinguished from unlabelled cells on the basis of their granule size range, but unlabelled cells had a significantly greater coefficient of variation. These data suggest that granule density and distribution, but not granule size per se, are useful characteristics for the identification of bovine lactotrophs.

Animals↗
PubMed1988

Endoplasmin is a reticuloplasmin.

The location of endoplasmin in the endoplasmic reticulum was investigated by biochemical and immunoelectron microscopic analyses. The protein could be obtained in a soluble form by procedures that do not involve the use of any detergents. The soluble protein has the amino- and carboxy-terminal sequences of the intact molecule, showing that it has not been proteolysed. Application of the Triton X-114 phase-separation test does not reveal significant hydrophobicity in the molecule. Immunogold labelling studies on cells with a dilated endoplasmic reticulum (ER) lumen show that endoplasmin is uniformly distributed throughout the lumen, with no evidence of a preferential association with the membrane. These studies clearly demonstrate that endoplasmin is a luminal protein of the ER, i.e. a reticuloplasmin, and not an integral membrane protein.

Animals↗
PubMed1988

Dissociation and re-assembly of the endoplasmic reticulum in live cells.

The endoplasmic reticulum (ER) of a typical interphase 3T3 fibroblast consists of a compact perinuclear arrangement of cisternae and lamellae which can be observed by immunofluorescence with anti-endoplasmin. During mitosis the reticulum dissociates into small fragments from which it appears to re-assemble in the daughter cells. When interphase 3T3 cells are exposed to calcium ionophores, but not other ionophores, there is a similar dissociation of the ER into small uniform fragments, which are dispersed throughout the cytoplasm. Electron microscopy shows that the fragments consist of small vesicular structures and that essentially all of the rough ER except the nuclear envelope is dissociated. The dissociation of the ER by calcium ionophore is a relatively specific process since other organelles and supramolecular assemblies remain unaffected. When cells with dissociated ER are returned to normal medium, there is a rapid reassembly of the fragments into the continuous reticulum. In a proportion of the cells it is possible to observe linear arrays of the fragments, which probably represent intermediates in the re-assembly process. These observations demonstrate that the ER in interphase 3T3 cells can be dissociated into, and re-assembled from, small fragments. Re-assembly of the ER from the fragments is dependent on the presence of millimolar levels of calcium in the external medium. In the presence of calcium, re-assembly is inhibited by the calcium channel blocker, verapamil. Thus calcium ions appear to play an important role in ER structure and assembly.

Animals↗
PubMed1988

Alterations in uterine epithelial tight junction structure during the oestrous cycle and implantation in the pig.

The structure of the pig uterine epithelium tight junction has been studied using freeze-fracture methods during oestrus and implantation. Compared with non-pregnant non-cyclic pigs, in both pregnancy and during the cycle the number of intersections per unit area of the tight junction ridges increases to a maximum 16 days after ovulation. There was no significant alteration in the depth or number of ridges in the tight junction band. The only significant difference between tight junctions in cyclic and pregnant pigs was a brief increase in the depth during the cycle (12-15 days after ovulation). The number of intersections is thought to be a far more important measure than depth in determining the permeability of the tight junction. It therefore seems unlikely that changes in tight junction structure play any direct role in the rescue of the corpus luteum or in establishing pregnancy but merely reflect the alterations in progesterone concentrations. However, the decrease in uterine permeability indicated by the increase in intersection frequency has been reported for other species and may reflect an important change in the uterine milieu in preparation for a possible pregnancy, apparently induced by alterations in progesterone concentration.

Animals↗
PubMed1987

Trinucleate cells and the ultrastructural localisation of bovine placental lactogen.

Bovine placental lactogen activity is shown by immunogold electron microscopy to be restricted to the granules and the Golgi body from which they form in the bovine fetal trophectodermal binucleate cell, and granules of similar size and staining reaction in trinucleate "giant" cells found in the maternal uterine epithelium throughout pregnancy. These results support the hypothesis that a fetal binucleate cell forms a maternal giant cell by migration to and fusion with a uterine epithelial cell.

Animals↗