Preimplantation mouse embryos and liver express the same type I keratin gene product

The cytoskeletal B protein isolated from extraembryonic endodermal cells (Endo B) is a 50-kDa subunit of intermediate filaments that is expressed in trophoblast and extraembryonic endoderm of early mouse embryos. Endo B was compared to cytokeratin D of adult mouse liver by immunoprecipitation, two-dimensional gel electrophoresis, and peptide mapping. The two proteins were indistinguishable. A cDNA probe for Endo B mRNA identified mRNA species of similar size in liver and endoderm, and primer extension analysis indicates that the Endo B mRNAs from the two cell types have similar 5' ends. An internal fragment of the Endo B cDNA was found to cross-hybridize with a conservative domain of a human type I keratin cDNA under low stringency conditions, demonstrating that Endo B is related to type I keratins. However, under stringent conditions necessary for genomic Southern analysis, mouse and human genomic fragments homologous to the Endo B cDNA were distinct from those defined by hybridization with the type I keratin cDNA. These results indicate that Endo B is related to the type I keratin family and expands the number of type I keratin genes identified in both the mouse and human genomes. It is likely that extraembryonic endoderm, one of the first differentiated cell types of the mammalian embryo, and adult liver express the same Endo B gene.     

Intermediate filaments of the midgestation rat trophoblast giant cell

Trophectoderm (TE) of the rodent blastocyst, the preimplantation precursor of the trophoblast giant cell (TGC), is the first embryonic cell to exhibit intermediate filaments (IF). The two IF proteins of TE (54K and 46K) have been variously described as trophectoderm specific, noncytokeratin, or cytokeratin and have been identified with Endo A and Endo B, IF proteins extracted from extraembryonic endodermal cells. IF proteins of midgestation rat TGC, the postimplantation descendant of TE, were compared to IF proteins of various rat simple epithelial cells by two-dimensional gel electrophoresis, partial proteolytic digest, antibody recognition on electrophoretic transfer, and antibody recognition by indirect immunofluorescence. The two TE IF proteins at 54K and 46K were identified in TGC IF and recognized by anti-Endo A, anti-Endo B, respectively, and anticytokeratins. TGC were found to possess additional cytokeratins at 52K, 45K, 43K, and 40K. The profile of TGC cytokeratins was qualitatively identical to that of various rat simple epithelial cells. The results suggest that (a) TE and TGC IF proteins are cytokeratins, (b) TE and TGC cytokeratins are characteristic of a simple epithelial cell, and (c) the morphologic and functional differentiation of TE to TGC is accompanied by elaboration of the cytokeratin profile.     

Murine endodermal cytokeratins Endo A and Endo B are localized in the same intermediate filament

The intracellular distribution of extra-embryonic endodermal, cytoskeletal proteins A (Endo A) and B (Endo B) was investigated by double-label immunofluorescent microscopy and double-label immunoelectron microscopy. In parietal endodermal cells, the immunofluorescent distribution of Endo B was always coincident with that of Endo A and could be distinguished from vimentin, particularly at the periphery of the cell. At the electron microscopic level, antibodies against both Endo A and Endo B recognized both bundles and individual intermediate filaments. Double-label immunoelectron microscopy was achieved by use of two sizes of colloidal gold particles (5 nm and 20 nm) that were stabilized with secondary antibodies. These results show that Endo A and B are found in the same intermediate filament and probably co-polymerize to form such structures.     

Morphological and radiochemical evidence for the metabolism of exogenous proteins by the preimplantation sheep blastocyst.

The ability of the trophoblast of the ovine preimplantation blastocyst to take up and metabolise proteins has been investigated using two experimental approaches, microscopical and radiochemical. The ultrastructure of the expanded blastocyst obtained from 14 and 17 day pregnant ewes was examined. The morphology of tissues maintained in culture for 24 h has been compared with that of fresh tissues. After culture, the cellular morphology of the explants was well preserved. Fresh and 24 h cultured tissues were incubated with horse-radish peroxidase and ferritin and these proteins subsequently were found to be localized in coated pits, caveolae and secondary lysosomes of the trophoblast. Comparison of the uptake of [3H]dextran and of 125I-labelled bovine serum albumin indicated that proteins could be taken up by cultured tissue by mechanisms in addition to simple fluid phase endocytosis. During culture of explants of blastocyst with 125I-labelled bovine serum albumin, a large fraction of the radioactivity taken up by the tissue appeared in the TCA-soluble fraction of the culture medium indicating that cultured trophoblast hydrolysed proteins. That amino acids released from captured protein could be used for protein synthesis by the trophoblast was indicated by the labelling of tissue and medium proteins after culturing explants with beta-lactamase labelled with [14C]leucine. A major product (Mr approximately 17 x 10(3) present in the medium was likely to have been ovine trophoblast protein-1. It is concluded that, during the expansion of the ovine blastocyst, the trophoblast has the ability to take up proteins, transport them to lysosomes and degrade them to amino acids which are used for protein synthesis. Thus proteins, as well as free amino acids, present in the histotrophe may be an important source of nitrogen for the sheep conceptus in the critical period just prior to implantation.     

Protein secretion by the mouse trophoblast during attachment and outgrowth in vitro

Protein secretion from mouse blastocysts undergoing attachment and trophoblast outgrowth in vitro was assessed. When Day 5 blastocysts were cultured in serum-containing medium, secretion of several 'attachment-associated' proteins (PAS) was initiated within 24 h, coincident with attachment and outgrowth. Those proteins characteristic of the pre-attachment blastocyst disappeared or made-up only a small portion of the secretions once attachment began. The major secreted protein from attached embryos, PA1, is a 35,000-45,000 Mr acidic glycoprotein with multiple isoelectric forms. PA2, a group of basic 40,000 Mr proteins and PA3 a group of 72,000 Mr proteins were also produced during outgrowth. PAS were secreted during outgrowth on fibronectin-coated plastic in serum-free medium, but not by blastocysts held in a non-attachment state during culture in serum-free medium on uncoated plastic. In pre-attachment blastocysts, secreted proteins were produced by trophoblast vesicles, but not by isolated inner cell masses. Both trophoblast vesicles growing out in vitro and surgically isolated trophoblast from spreading blastocysts had secreted protein patterns qualitatively similar to those of intact blastocyst outgrowths. The results indicate that development of trophoblast protein secretion continues through the period of outgrowth and giant cell transformation. These changes are apparently dependent on attachment of the blastocyst to a suitable substrate, but not dependent on any other serum influence.     

DNA synthesis in the mouse blastocyst during the beginning of delayed implantation

The spatiotemporal pattern of DNA synthesis in the mouse embryo at the beginning of metabolic dormancy was examined. Embryos were recovered from females at intervals following ovariectomy at 1100 hours on day 4 of pregnancy, incubated in vitro for 1 h in the presence of [3H]thymidine, and prepared for light microscopic autoradiography. The proportion of labeled cells in the embryo remained high (40-60%) for 18 h after ovariectomy and then declined gradually to 12% by 96 h. However, analysis of individual cell subpopulations showed that the decline was not uniform in all regions of the blastocyst. Labeling was high over the inner cell mass (ICM) during all time intervals in the study, while labeling over the mural trophoblast cells declined sharply by 24 h after ovariectomy. Labeling over the polar trophoblast also declined but had values that were intermediate between the ICM and mural trophoblast regions of the blastocyst. These regional differences in DNA synthesis during the arrest of development suggest that intermediate steps are involved in control of DNA synthesis in the embryo and that the ICM may play a role in the different responses of the trophoblast cell populations.     

Influx of bivalent cations can be independent of receptor stimulation in human endothelial cells.

The major active forms of cathepsins B and L were identified in Kirsten-virus-transformed mouse fibroblasts by the use of a specific radiolabelled inhibitor, benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2. No other proteins were labelled, demonstrating the specificity of this inhibitor for cysteine proteinases. Cathepsins B and L were distinguished by the use of specific antibodies. One active form of cathepsin B, Mr 33,000-35,000, and two active forms of cathepsin L, Mr 30,000 and 23,000, were identified. The intracellular precursors of these proteins had higher Mr values of 39,000 and 36,000 for cathepsins B and L respectively, as shown by pulse-chase experiments with [35S]methionine-labelled proteins. These did not react with the inhibitor under our culture conditions. The precursor of cathepsin L was secreted whereas the precursor of cathepsin B was not, demonstrating that secretions of the two enzymes are regulated differently. In contrast with results found previously for the purified protein [Mason, Gal & Gottesman (1987) Biochem. J. 248, 449-454], the secreted precursor form of cathepsin L did not react with the inhibitor either, indicating that it is not active and therefore, as such, cannot be directly involved in tumour invasion. The secreted protein did react with the inhibitor when incubated at pH 3.0, showing that the protein can be activated, although this did not occur under our culture conditions.     

Discrimination between the nuclear lamin and intermediate filament (cytokeratin/vimentin) proteins of rat hepatic tumor cells by differential solubility and electrophoretic criteria

1. The major proteins which comprise the high salt/detergent-insoluble cytoskeletal matrix of rat hepatic tumor cells containing abnormal (Mallory body-like) aggregates of intermediate filaments were distinguished on the basis of electrophoretic mobility and differential solubility. 2. Gel electrophoresis of the intermediate filament-enriched cytoskeletal fraction of Mallory body hepatic tumor cells revealed the presence of: (a) intermediate filament proteins typical of cultured liver epithelial cells (cytokeratins A and D, vimentin), (b) some residual actin and, (c) two peptides of Mr = 68,000-72,000. 3. Analysis of the products of filament disassembly/reassembly mixtures indicated that the two Mr = 68,000-72,000 peptide species had the solubility characteristics of nuclear lamins. 4. The presence of nuclear lamin proteins in the high salt/detergent-resistant fraction of cultured liver cells was consistent with the resolution of residual nuclear-like structures in extracted cell monolayers. 5. Thus, while cytokeratin/vimentin-class intermediate filament proteins and nuclear lamins co-isolate from rat liver cells under conditions of high salt/detergent extraction, these two types of cytoskeletal proteins could be distinguished on the basis of their differential solubility and molecular weight.     

Spectrin synthesis in the preimplantation mouse embryo

The preimplantation mouse embryo expresses two polypeptides, Mr 240,000 and Mr 235,000, that are immunologically cross-reactive with antibody to the alpha and beta subunits of mouse brain spectrin. We investigated the synthesis of the spectrin subunits in the Triton-soluble and Triton-insoluble fractions of fertilized eggs, two-cell embryos, compacted morulae, and blastocysts labeled with L-[35S]methionine. Synthesis of embryonic spectrin began in the Triton-soluble fraction with significant levels of alpha-spectrin synthesis first detected in the morula stage and significant levels of beta-spectrin synthesis detected in the blastocyst stage. Incorporation of newly synthesized alpha- and beta-spectrin into the cytoskeletal fraction took place in the blastocyst when equal amounts of both subunits were assembled. Previous studies have shown Triton-insoluble spectrin to be concentrated in regions of cell-cell contact in the embryo (J. S. Sobel and M. A. Alliegro, 1985, J. Cell Biol. 100, 333-336). The temporal and spatial correlation between the assembly of newly synthesized spectrin and its concentration in regions of cell apposition is consistent with the hypothesis that cell contact may influence the assembly of embryonic spectrin.     

Maternal recognition of pregnancy in the goat: effects of conceptus removal on interestrus intervals and characterization of conceptus protein production during early pregnancy

Goat conceptuses were surgically removed from the uterus at different days during early pregnancy and cultured for 24-30 h in the presence of L-[3H]leucine to determine the effects of embryo removal on the interestrus interval and to characterize in vitro synthesis and release of conceptus proteins. Normal cyclic and animals (controls) exhibited interestrus intervals of 20.44 +/- 0.89 days. Removal of conceptuses on Days 13 and 15 did not alter interestrus intervals compared to cyclic animals. Removal of conceptuses on Day 17 and times thereafter resulted in significant (p less than 0.05) prolongation of interestrus intervals. These results demonstrate that maternal recognition of pregnancy in the goat occurs between Days 15 and 17. Proteins synthesized and released into the medium by conceptuses were first detectable at Day 16 by the analytical method employed (two-dimensional polyacrylamide gel electrophoresis followed by fluorography). The major protein synthesized at this time was acidic (pI = 5.2-5.7) and consisted of two isotypes with molecular weights of about 17,000. Although patterns of protein production became more complex with conceptus development, this protein remained as a major product through Day 21 but not afterwards. This protein, as well as two other low molecular weight acidic proteins (Mr approximately equal to 21,000, 23,000; pI = 5.7-6.0) were shown by immunoprecipitation to react with anti-ovine trophoblast protein-1 (oTP-1) serum. Hence, these products may comprise a caprine trophoblast protein-1 (cTP-1) complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of the restriction endonuclease BglII from Bacillus globigii

The restriction endonuclease BglII from Bacillus globigii has been purified to homogeneity. The enzyme is a dimer of two subunits of Mr = 27000. The reaction mechanism does not involve the accumulation of a DNA intermediate nicked in one strand and the enzyme is not affected by superhelical twists in the substrate DNA, indicating that DNA binding does not involve either winding or unwinding of the double helix. Antibodies were prepared against BglII. These antibodies did not cross react with any other restriction endonucleases tested, including other enzymes from B. globigii or from closely related strains. It is thus unlikely that type II restriction enzymes represent a closely related group of proteins.     

Expression of new proteins of the intermediate filament protein family in differentiating F9 embryonal carcinoma cell cytoskeleton

Differentiation of F9 embryonal carcinoma cells by retinoic acid treatment results in extraembryonic endoderm-like cells. The effects of this process on the protein composition of the intermediate filaments were studied by two-dimensional gel electrophoresis and by immunoblotting. By this approach, two new proteins induced in differentiating cells, p57 and p54, were identified in cytoskeletal preparations enriched in intermediate filaments. The 57-kDa protein could be resolved into at least three components (pI 5.6-5.9), and the 54-kDa protein into at least two components (pI approximately 5.6). Both proteins reacted with a monoclonal antibody which recognizes an antigenic determinant common to all intermediate filaments. Based on these results, the two proteins were identified as members of the intermediate filament protein family. Partial digestion with V8 protease showed that p57 was different from vimentin, another intermediate filament protein present in these cells. p57 and p54 were also immunodetected by a polyclonal anti-keratin anti-serum, which suggests that these proteins share some homology with the keratins. These two proteins are different from the endodermal cytoskeletal protein A and B (endo A and endo B) keratins, which are known to be present in extraembryonic endoderm-like cells. They were also more abundant than endo A and endo B in differentiating F9 embryonal carcinoma cells, but almost undetectable in terminally differentiated extraembryonic endoderm-like cells, where endo A and endo B are readily detectable. This suggests that p57 and p54 have a different pattern of expression than endo A and endo B.     

Clonal tumorigenic endodermal cell lines producing basement membrane components

Tumors were developed from the mouse teratocarcinoma-derived endodermal cell line PF HR-9 by subcutaneous injections in syngeneic mice of large numbers of cells previously cultured for several years at high cell density. Cell cultures were established from the tumors and the cells were cloned. The cloned sublines were highly malignant in vivo and tumor metastases were occasionally observed. The tumors contained abundant extracellular material, which was distinctly laminated and contained type IV collagen, laminin, and heparan sulfate proteoglycan. The tumorigenic sublines were also shown to have retained markers of the original cells, such as the cytoskeletal proteins Endo A and B. These cell lines should be useful for biosynthetic studies on basement membrane and cytoskeletal components and the tumors for isolation of these macromolecules and their mRNAs.     

Analysis of the carbohydrate composition of the pancreatic plasmalemmal glycoprotein affinity labeled by short probes for the cholecystokinin receptor

Affinity labeling of the rat pancreatic cholecystokinin (CCK) receptor with decapeptide probes has identified an Mr = 85,000-95,000 protein, distinct from the Mr = 80,000 component previously labeled with 125I-Bolton Hunter-CCK-33. We have characterized the carbohydrate composition of this novel protein labeled with 125I-D-Tyr-Gly-[(Nle28,31)-CCK-26-33] and disuccinimidyl suberate by using chemical and enzymatic deglycosylation and lectin chromatography. The Mr = 85,000-95,000 component was demonstrated to be an N-linked sialoglycoprotein based on neuraminidase digestion to Mr = 75,000-85,000 and endo-beta-N-acetylglucosaminidase F (Endo F) digestion to Mr = 42,000. This was distinct from the Mr = 65,000 product of Endo F digestion of the protein labeled with 125I-Bolton Hunter-CCK-33. Lack of an effect of endo-beta-N-acetylglucosaminidase H demonstrated the absence of N-linked simple oligosaccharides, while products of chemical deglycosylation with hydrogen fluoride and endo-alpha-N-acetylgalactosaminidase supported the absence of O-linked carbohydrate. The presence of at least four oligosaccharide chains on the core protein was suggested by Endo F digestion of the Mr = 85,000-95,000 protein using limiting enzyme conditions. This glycoprotein was retained on wheat germ agglutininagarose and eluted by N,N',N"-triacetylchitotriose. Identification of the Mr = 85,000-95,000 component on the ectodomain of the plasmalemma of intact pancreatic acini confirmed this to be the fully processed form of the CCK-binding protein.     

Purification, characterization, and immunocytochemical localization of the major basic protein of pig blastocysts

The major basic protein (BP) synthesized and secreted by elongating pig blastocysts was purified from medium of Day 14-17 conceptus cultures. Sequential ion-exchange and gel-filtration chromatographies resulted in isolation of BP as a single polypeptide of Mr = 43,100 or 42,800 under denaturing or native conditions, respectively. BP was found to be a glycoprotein by incorporation of [3H] glucosamine and susceptibility to N-glycopeptidase F. Two BP polypeptides were produced by N-glycopeptidase F (Mr = 39,800 and 36,300). Antiserum to BP immunoprecipitated radiolabeled BP from blastocyst culture medium. BP was not detected in medium from 1-2 mm diameter spherical (Day 10) blastocysts but was found in medium from 3-5 mm spherical (Day 10) and filamentous (less than 50 cm, Day 12) conceptuses, suggesting that BP synthesis and secretion began at the initiation of trophoblast expansion. With immunocytochemical procedures, BP was located in the apical cytoplasm of trophectoderm cells of Day 11 expanding (5-7 and 10-20 mm) blastocysts. These results suggest that trophoblast epithelium secrete BP apically toward the uterine lumen and that BP may play a role in maternal-fetal interactions during the peri-implantation period.     

Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos

A cDNA clone of a keratin-related, intermediate filament protein, designated Endo B, was constructed from size-fractionated parietal endodermal mRNA and characterized. The 1466-nucleotide cDNA insert contains an open reading frame of 1272 nucleotides that would result in 5' and 3' noncoding sequences of 54 and 60 nucleotides, respectively. The predicted amino acid composition, molecular weight (47,400), and peptide pattern correlate well with data obtained on the isolated protein. The predicted amino acid sequence fits easily into the general domain structure suggested for all intermediate filament proteins with a unique amino-terminal head domain, a large conserved central domain of predominantly alpha-helical structure, and a relatively unique carboxyl-terminal or tail domain. Over the entire molecule, Endo B is 43% identical with human 52-kDa epidermal type I keratin. However, over two of the three regions contained in the central domain that are predicted to form coiled-coil structures, the Endo B is 54-68% identical with other type I keratin sequences. This homology, along with the presence of the completely conserved sequence DNARLAADDFR-KYE, which is found in all type I keratins, permits the unambiguous identification of Endo B as a type I keratin. Comparison of the Endo B sequence to other intermediate filament proteins reveals 22 residues which are identical in all intermediate filament proteins regardless of whether filament formation requires only one type of protein subunit (vimentin, desmin, glial fibrillar acidic protein, or a neurofilament protein) or two dissimilar types (type I and type II keratins). Endo B mRNA was detectable in RNA isolated from F9 cells treated with retinoic acid for 48 h. Approximately three to five genes homologous to Endo B were detected in the mouse genome.     

Calcium-dependant binding proteins associated with human placental syncytiotrophoblast microvillous cytoskeleton

Isolated human placental syncytiotrophoblast microvillous plasma membrane vesicles were extracted with Triton X-100 to yield a detergent-insoluble residue. The residue contained approx. 50% of the total membrane protein and was qualitatively different from untreated trophoblast on SDS-polyacrylamide gel electrophoresis, Western blots and dot-immunobinding assay. Three major proteins, with molecular weights of 68, 36 and 34 kDa, dissociated from this non-ionic detergent-insoluble submembranous cytoskeletal fraction in the presence of calcium chelators. They were immunologically related to human lymphocyte cytoskeletal calcium-binding proteins, and the 36 kDa component reacted with antisera to the phospholipase A2 inhibitor, lipocortin II. Anti-lipocortin I sera did not recognise the 34 kDa protein, but did react with a series of trophoblast cytoskeletal proteins in the 34-37 kDa region. Incubation of epidermal growth factor with isolated trophoblast membrane vesicles stimulated the phosphorylation of a 36 kDa protein on tyrosine residues. Immunoprecipitation studies further showed there was no phosphorylation of the 34 kDa protein, but the 68 kDa protein was a major phosphorylated component of isolated syncytiotrophoblast membranes. p68 was principally phosphorylated on serine with slight tyrosine phosphorylation which showed an apparent increase after epidermal growth factor treatment. These results indicate a family of calcium-dependant binding proteins, some of which are phosphorylated, associated with the submembranous cytoskeleton of syncytiotrophoblast microvilli.     

Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

Cholera toxin catalyzes transfer of radiolabel from [32P]NAD+ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of Mr = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (Mr = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and [32P]NAD+ caused radiolabeling of purified microtubule and intermediate filament proteins.     

Expression of the actin stress fiber-associated protein CLP36 in the human placenta

Differentiation processes in the trophoblast comprise polarization, cell fusion and migration. All these processes involve dramatic reorganizations of cytoskeletal proteins such as intermediate filaments or actin. Due to very restricted knowledge on cytoskeletal changes in trophoblast, we analyzed the protein expression of an actin stress fiber-associated protein, the carboxy-terminal LIM domain protein (CLP36). CLP36 belongs to the enigma family of proteins, binds to α-actinin and is involved in the cytoskeletal reorganization and signal transduction of a variety of cells. CLP36 protein was found to be exclusively expressed in the cytotrophoblast layer. Colocalization of CLP36 with Mib-1 revealed that CLP36 protein expression is restricted to proliferative and early post-proliferative trophoblast cells. Blockage of syncytial fusion by culture of villous explants in the presence of caspase 8 inhibitors further supported this notion since CLP36 was only found in the basal and proliferative layer of the multilayered cytotrophoblast. We present evidence for the exclusive protein expression of CLP36 in proliferative and early post-proliferative trophoblast cells. Pathological pregnancy syndromes such as preeclampsia are driven by alterations of trophoblast differentiation and turnover, where it needs to be elucidated whether CLP36 is involved in these alterations.