Interactions of glycoconjugates with endogenous galectins, have been long proposed to participate in several reproductive processes including implantation. In human placenta gal-1, gal-3, gal-8, and ...gal-13 proteins are known to be present. Each of them has been proposed to play multiple functions, but so far no clear picture has emerged. We hypothesized that gal-1 participates in trophoblast invasion, and conducted Matrigel invasion assay using isolated cytotrophoblast from first trimester placenta and HTR-8/SVneo cell line to test it.
Function blocking anti-gal-1 antibody was employed to assess participation of endogenous gal-1 in cell adhesion, cell invasion of HTR-8/SVneo cells. When gal-1 was blocked in isolated trophoblast cell invasion was reduced to 75% of control (SEM ± 6.3, P<0.001) and to 66% of control (SEM ± 1.7, P<0.001) in HTR-8/SVneo cell line. Increased availability of gal-1, as two molecular forms of recombinant human gal-1 (CS-gal-1 and Ox-gal-1), resulted in increased cell invasion by cytotrophoblast to 151% (SEM ± 16, P<0.01) with 1 ng/ml of CS-gal-1, and to 192% (SEM ± 51, P<0.05) with 1 µg/ml of Ox-gal-1. Stimulation was also observed in HTR-8/SVneo cells, to 317% (SEM ± 58, P<0.001) by CS-gal-1, and to 200% (SEM ± 24, P<0.001) by Ox-gal-1 at 1 µg/ml. Both sets of results confirmed involvement of gal-1 in trophoblast invasion. Galectin profile of isolated cytotrophoblast and HTR-8/SVneo cells was established using RT-PCR and real-time PCR and found to consist of gal-1, gal-3 and gal-8 for both cell types. Only gal-1 was located at the trophoblast cell membrane, as determined by FACS analysis, which is consistent with the results of the functional tests.
These findings qualify gal-1 as a member of human trophoblast cell invasion machinery.
Galectin-1 (gal-1) is the best studied member of the galectin family of the
human placenta assumed to play important roles in pregnancy. Standard
isolation of gal-1 from human placenta using lactose ...extraction and affinity
chromatography in the presence of a reducing agent, produced several known
forms of gal-1, which were compared to the recombinant human gal-1 (rhgal-1)
and oxidized recombinant human gal-1 (Ox-gal-1). Isolated placental gal-1
retained lectin binding activity, evidenced by hemagglutination and dot blot
lectin assays. Characterization of the forms present by surface-enhanced
laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS),
based on hydrophilic interactions or immunorecognition, provided a sensitive
tool for detection of the fine differences among the diverse molecular
forms. The forms detected included previously established biologically
active oxidized gal-1 and reduced gal-1, as well as some other currently
uncharacterized (less investigate forms.
Our previous findings showed that galectin-1 (LGALS1) plays an important role in the in vitro invasion of normal human trophoblast cells. In the present study, choriocarcinoma JAr cells were found to ...express LGALS1, -2, -3, -8, -10, and -13 mRNA and at least LGALS1, -3, and -8 protein, as determined by reverse-transcriptase PCR and Western blot, respectively. The galectin mRNA signature of JAr cells thus differed from that of normal first-trimester extravillous trophoblasts. A Matrigel migration assay was also used to investigate and confirm the relevance and effect of LGALS1 on the invasive potential of JAr cells, as observed in other trophoblast models. This modulation in behavior was achieved by specific lectin-glycan binding.
The aim of this work was to study the involvement of IGFBP-3/Tf complexes in the pathology of colorectal carcinoma (CRC), quantify them, investigate their relation to iron concentration and binding ...to transferrin receptor (TfR) in colon tissue (non-cancer and cancer), and to assess the priority of this pathway for internalization of IGFBP-3.
The presence of IGFBP-3/Tf complexes was analyzed in sera from healthy persons and patients with CRC, and in colon tissue by immunoblotting. Complexes were immunoprecipitated, quantified by immunoassay and structurally characterized by immunoblotting, lectin blotting and mass spectrometry. Complexes which interacted with colon cells were immunoprecipitated with anti-TfR1 antibody and studied. Colon tissue slides were subjected to immunohistochemical analysis.
The concentration of IGFBP-3/Tf complexes was three times lower in patients with CRC. They were increasingly carbonylated, sialylated, contained more Galβ4GlcNAc units, expressed altered charge density and increased affinity for metal ions. Immunoprecipitation experiments revealed more TfR1 on membranes than in cytosol of colon cells, also more in cancer than non-cancer tissue. TfR1 on membranes were less occupied with IGFBP-3/Tf complexes than in cytosol. Immunofluorescent staining indicated a remarkable degree of co-localization of IGFBP-3 and TfR1, evenly distributed in non-cancer tissue and both evenly and cell surface concentrated in cancer tissue.
Increased expression of TfR1 on colon cell membranes in patients with CRC compensates for the reduced extracellular availability of IGFBP-3/Tf and TfR1 is the principal binding partner of extracellular IGFBP-3. IGFBP-3/Tf complexes in patients with CRC exhibit increased affinity for iron ions.
Integrin β1 is bound to galectin-1 in human trophoblast Bojic-Trbojevic, Žanka; Jovanovic Krivokuca, Milica; Stefanoska, Ivana ...
Journal of biochemistry (Tokyo),
2018-Jan-01, 2018-01-01, 20180101, Letnik:
163, Številka:
1
Journal Article
Recenzirano
Interaction of sugar binding proteins-galectins, with glycoconjugates is considered relevant for various reproductive processes. Galectin-1 (gal-1) is a molecule involved in trophoblast cell ...invasion, which is accomplished through interaction with cell surface and/or extracellular matrix glycoproteins. A possibility of interaction of endogenous gal-1 and trophoblast β1 integrins, both previously shown relevant for trophoblast invasion, was investigated. Confocal microscopy showed overlap in gal-1 and β1 integrin localization at the plasma membrane of isolated cytotrophoblast, HTR-8/SVneo extravillous trophoblast cell line and JAr choriocarcinoma cells. Immunoprecipitation confirmed an interaction of gal-1 with integrin β1, but not with α1 or α5 integrin subunits. Nondenaturing electrophoresis and subcellular fractionation suggested association of gal-1 with β1 integrin in intracellular and plasma membrane compartments of HTR-8/SVneo cells. Gal-1/β1 integrin complex was sensitive to chemical and enzyme treatments, indicating carbohydrate dependent interaction. Down-regulation of gal-1 by siRNA, however, had no effect on level or distribution of β1 integrin, as determined by qPCR and flow cytometry. These results suggest complex lectin type interaction of gal-1 with β1 integrin at the trophoblast cell membrane, which could influence trophoblast cell adhesion, migration and invasion.
In the course of embryo implantation extensive interaction of the trophoblast with uterine tissue is crucial for adequate trophoblast invasion. This interaction is highly controlled, and it has been ...pointed out that a specific glycocode and changes in glycosylation may be important for successful implantation and maintenance of pregnancy. Both uterine and trophoblast cells have been shown to express cell surface glycoconjugates and sugar binding proteins, such as mucins (MUC) and galectins (gals). An increasing number of studies have investigated potential candidates interacting in this process. However, knowledge about the biochemical nature of the interactions and their importance for trophoblast cell function, and, consequently, for pregnancy outcome are still lacking. This review is aimed at deliberating the possibility that mucins, as heavily glycosylated proteins, might be among the functionally relevant galectin ligands in human trophoblast, based on both published data and our original research.
Galectin-1 binds mucin in human trophoblast Bojic-trbojevic, Anka; Jovanovic Krivokuca, Milica; Kolundzic, Nikola ...
Histochemistry and cell biology,
11/2014, Letnik:
142, Številka:
5
Journal Article
Recenzirano
Mucins are multifunctional highly glycosylated proteins expressed by the female reproductive tract. Differential expression of MUC1 and MUC15 has been shown in trophoblast. This study was undertaken ...to establish the distribution of mucin(s) in cytotrophoblast cell cultures using anti-bovine submaxillary mucin (BSM) and to investigate the possibility of MUC1/mucin(s) being a binding partner of trophoblast galectin-1. MUC1 is demonstrated here using immunocytochemistry on isolated cytotrophoblast and the HTR-8/SVneo extravillous trophoblast cell line but detection of additional trophoblast mucins cannot be excluded. Western blot analysis showed similar bands ranging from 30 to >200 kDa with anti-BSM and the well-known mucin antibodies HMFG1 and B72.3. Immunocytochemistry and cell-based ELISA data were found to support that all of the antibodies used are reactive with BSM, suggesting the presence of shared epitopes between BSM and trophoblast mucin(s). Binding of galectin-1 to trophoblast MUC1/mucin(s) was analyzed using a solid-phase assay and co-immunoprecipitation. Recombinant galectin-1 binding to isolated trophoblast mucin in solid-phase assay was sensitive to lactose, a carbohydrate inhibitor of galectin binding. In whole HTR-8/SVneo lysates, ~200 kDa mucin was detected in galectin-1 immunoprecipitates, while endogenous galectin-1 was present in BSM-immunoprecipitates. Furthermore, double fluorescence immunocytochemistry showed overlap of galectin-1 and trophoblast mucins at the plasma membrane of HTR-8/SVneo cells. These results suggest that trophoblast mucin(s) could act as binding partners of galectin-1, in a carbohydrate-dependent manner.
Summary
Stem cells are unique cell populations able to copy themselves exactly as well as specialize into new cell types. Stem cells isolated from early stages of embryo development are pluripotent, ...i.e., can be differentiated into multiple different cell types. In addition, scientists have found a way of reverting specialized cells from an adult into an embryonic‐like state. These cells, that are as effective as cells isolated from early embryos, are termed induced pluripotent stem cells (iPSCs). The potency of iPSC technology is recently being employed by researchers aimed at helping wildlife and environmental conservation efforts. Ambitious attempts using iPSCs are being made to preserve endangered animals as well as reanimate extinct species, merging science fiction with reality. Other research to sustain natural resources and promote animal welfare are exploring iPSCs for laboratory grown animal products without harm to animals offering unorthodox options for creating meat, leather, and fur. There is great potential in iPSC technology and what can be achieved in consumerism, animal welfare, and environmental protection and conservation. Here, we discuss current research in the field of iPSCs and how these research groups are attempting to achieve their goals. Stem Cells Translational Medicine 2019;8:7–13
Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Although it has been demonstrated that thyroid hormone supplementation improves bovine ...embryo development in vitro, the cellular mechanisms underlying these effects are so far unknown. In this study, we investigated the role of thyroid hormone in development of human preimplantation embryos. Embryos were cultured in the presence or absence of 10−7 M triiodothyronine (T3) till blastocyst stage. Inner cell mass (ICM) and trophectoderm (TE) were separated mechanically and subjected to RNAseq or quantification of mitochondrial DNA copy number. Analyses were performed using DESeq (v1.16.0 on R v3.1.3), MeV4.9 and MitoMiner 4.0v2018 JUN platforms. We found that the exposure of human preimplantation embryos to T3 had a profound impact on nuclear gene transcription only in the cells of ICM (1178 regulated genes—10.5% of 11 196 expressed genes) and almost no effect on cells of TE (38 regulated genes—0.3% of expressed genes). The analyses suggest that T3 induces in ICM a shift in ribosome and oxidative phosphorylation activity, as the upregulated genes are contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Our findings might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Our data also raise a possibility that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization.
Embryos cultured in the presence or absence of triiodothyronine (T3) till blastocyst stage upregulate genes contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression.
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