ABSTRACT
A hierarchically organized lymphatic vascular system extends throughout the vertebrate body for tissue fluid homeostasis, immune trafficking, and the absorption of dietary fats. Intralymphatic dye injection and serial sectioning have been the main tools for visualizing lymphatic vessels. Specific markers for identifying the lymphatic vasculature in zebrafish and medaka have appeared as new tools that enable the study of lymphangiogenesis using genetic and experimental manipulation. Transgenic fishes have become excellent organisms for visualizing the lymphatic vasculature in living embryos, but this method has limited usefulness, especially in later developmental stages. The functional lymphatic endothelium predominantly takes up foreign particles in zebrafish and medaka. We utilized this physiological activity and lymph flow to label lymphatic vessels. Intraperitoneal injection of trypan blue is useful for temporal observations of the lymphatic ducts, which are essential for large-scale genetic screening, while cinnabar (HgS) injection allows identification of the lymphatic endothelium under electron microscopy, avoiding artefactual damage. This injection method, which is not high in cost and does not require high skill or special devices, is applicable to any live fish with functioning lymphatic vessels, even mutants, with high reproducibility for visualizing the entire lymphatic vascular system.
Subject(s)
Lymphatic Vessels , Oryzias , Animals , Injections, Intraperitoneal , Lymphangiogenesis , Reproducibility of Results , Zebrafish/geneticsABSTRACT
Macrophage colony-stimulating factor receptor (M-CSFR), a member of the group of type III protein tyrosine kinase receptors, is expressed primarily by monocyte/macrophage lineage cells. In order to describe the distribution of macrophages at the maternal-fetal interface in Neoditrema ransonnetii, a viviparous fish species, M-CSFR cDNA was sequenced. Two sequences were obtained: NrM-CSFR1 (4381 bp, encoding 980 amino acids), and NrM-CSFR2 (3573 bp, encoding 1016 amino acids). Both the genes were expressed in the ovary of pregnant females. In situ hybridization revealed that a number of cells that were positive for NrM-CSFR1 and/or NrM-CSFR2 populated the ovigerous lamellae of the ovary during pregnancy. Following parturition, M-CSFR-positive cells disappeared from the subepithelial region of ovigerous lamellae, and were localized in perivascular tissues. These results suggest the role of M-CSFR-positive cells, which appear to be macrophages, in N. ransonnetii during pregnancy.
Subject(s)
Fish Proteins/genetics , Macrophages/metabolism , Ovary/metabolism , Receptor, Macrophage Colony-Stimulating Factor/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/metabolism , Female , Fish Proteins/chemistry , Fish Proteins/metabolism , Perciformes , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Macrophage Colony-Stimulating Factor/chemistry , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Sequence Alignment/veterinary , Viviparity, NonmammalianABSTRACT
As the fetus expresses paternal major histocompatibility complex molecules, viviparous vertebrates require sophisticated mechanisms to modulate maternal immunology to ensure successful pregnancy. We anticipated that ovarian cavity fluid (OCF) is likely to feature significantly in the modulation of ovarian cavity immunology. Consequently, we examined the effects of OCF upon leukocyte function in Neoditrema ransonnetii. OCF did not affect phagocytosis or superoxide production by phagocytes. However, OCF suppressed lymphocyte proliferation induced by ConA almost completely. As OCF contained PGE(2) at high levels during late pregnancy, we also investigated the effect of PGE(2) upon lymphocyte expansion. PGE(2) exhibited negative effects upon lymphocyte mitogenesis in a dose-dependent manner (10-1000 ng/ml). PGE(2) significantly suppressed lymphocyte proliferation when present at levels equivalent to that seen in OCF (30.2 +/- 16.1 approximately 185.4 +/- 107.4 ng/ml). Data indicate that PGE(2) is one of the key modulatory molecules of the maternal immune system ensuring successful pregnancy in this viviparous species.
Subject(s)
Lymphocytes/physiology , Ovary/physiology , Perciformes/physiology , Viviparity, Nonmammalian/physiology , Animals , Cell Proliferation , FemaleABSTRACT
The C1q family is a growing group of proteins with a globular C1q domain in the C-terminal region. We purified a new member of this family with L-fucose-binding activity from the plasma of surfperch, Neoditrema ransonnetii through L-fucose-affinity chromatography and anion-exchange chromatography. N-terminal amino acid sequencing followed by cDNA sequencing revealed that the protein was composed of 212 amino acids including a signal peptide of 20 amino acids. The gene expression analysis by RT-PCR showed that the gene was transcribed in the liver, stomach and intestine. The hepatic gene expression was up-regulated within 3 h of an intraperitoneal injection of formalin-killed Edwardsiella tarda. A phylogenetic analysis of gC1q domains placed the 23 kDa protein in the same cluster as other fish non-complement C1q-like proteins including a precerebellin-like protein of rainbow trout and ovary-specific protein of crucian carp. Interestingly, sialic acid-binding lectins of mollusca were located on the neighboring branch. Though the lectin activity has yet to be ascribed to the gC1q domain, these findings, together with former findings on lectin activity of lamprey and human C1q, indicate that sugar-binding activity is relatively common among the C1q family.
Subject(s)
Complement C1q/isolation & purification , Fucose/metabolism , Perches/metabolism , Amino Acid Sequence , Animals , Base Sequence , Chromatography, Affinity/veterinary , Complement C1q/genetics , Complement C1q/metabolism , Female , Gene Expression Profiling , Male , Molecular Sequence Data , Perches/genetics , Phylogeny , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Sequence Alignment , Sequence Analysis, DNAABSTRACT
Developing fetuses of surfperch (Neoditrema ransonnetii, Perciformes; Embiotocidae) are retained in the ovarian cavity until birth, where they are surrounded by ovarian cavity fluid (OCF). Expecting the OCF to have key roles in maintaining pregnancy, we purified and characterized a major glycoprotein of 51 kDa in the OCF of surfperch. On the basis of the N-terminal amino acid sequence, we cloned and sequenced a full-length cDNA. The deduced sequence comprises 214 amino acids (aa) including a signal peptide of 20 aa and a mature protein of 194 aa. This protein had an extremely low pI (below 2.8) and extraordinarily high glycosylation rate (more than 50%), characteristics being shared with alpha-1-acid glycoprotein (AGP), a member of the lipocalin superfamily. A homology search and phylogenetic analysis indicated that the 51 kDa protein and tributyltin-binding protein found in Japanese flounder are the closest known relatives of AGP. We therefore named the protein nrF-AGP. Messenger RNA of nrF-AGP was expressed intensively in the liver, but not at all in the ovarian tissue. Because nrF-AGP is the most salient component in OCF but not in plasma, we reasoned that it was selectively sequestered from blood to the ovarian cavity in pregnant females, and consequently, plays crucial roles in pregnancy.
Subject(s)
Body Fluids/metabolism , Orosomucoid/isolation & purification , Ovary/metabolism , Perciformes/physiology , Viviparity, Nonmammalian/physiology , Amino Acid Sequence , Animals , Electrophoresis, Gel, Two-Dimensional , Female , Molecular Sequence Data , Orosomucoid/metabolism , Phylogeny , RNA, Messenger/genetics , Sequence Alignment , Sequence Analysis, Protein , Species SpecificityABSTRACT
Cell accumulation technique is an extracellular matrix (ECM) nanofilm-based tissue-constructing method that enables formation of multilayered hybrid culture tissues. In this method, ECM-nanofilm is constructed using layer-by-layer assembly of fibronectin and gelatin on culture cells. The ECM-nanofilm promotes cell-to-cell interaction; then the three-dimensional (3D) multilayered tissue can be constructed with morphological change of the cells mimicking living tissues. By using this method, we have successfully produced tubular networks of human umbilical venous endothelial cells (HUVECs) and human dermal lymphatic endothelial cells (HDLECs) in 3D multilayered normal human dermal fibroblasts (NHDFs). This study demonstrated morphological characteristics of the vascular networks in the engineered tissues by using light and electron microscopy. In light microscopy, HUVECs and HDLECs formed luminal structures such as native blood and lymphatic capillaries, respectively. Electron microscopy showed distinct ultrastructural aspects of the vasculature of HUVECs or HDLECs with intermediated NHDFs and abundant ECM. The vasculature constructed by HUVECs exhibited structures similar to native blood capillaries, involving overlapping endothelial connections with adherens junctions, abundant vesicles in the endothelial cells and basement membrane-like structure. The detection of laminin around HUVEC-constructed vessels supported the presence of perivascular basal lamina. The vasculature constructed by HDLECs showed some ultrastructural characteristics similar to those of native lymphatic capillaries such as irregular vascular shape, loose adhesive connection and gap formation between endothelial cells. In conclusion, our novel vascular network models fabricated by the cell accumulation technique provide highly organized blood and lymphatic capillary networks mimicking the vasculatures in vivo.