Expression of gamma-glutamyl transpeptidase in midgestation mouse yolk sac and mouse visceral yolk sac carcinoma cells.

gamma-Glutamyl transpeptidase (gamma GT) is a crucial enzyme for the metabolism of xenobiotics and endogenous mediators of biological functions (leukotrienes, prostaglandins, and hepoxillins). Yet little is known about its potential role during development. It is a single copy gene expressed from at least seven promoters. Using histochemistry and immunohistochemistry we demonstrate that gamma GT first appears in the midgestational yolk sacs of mouse embryos. Established cell lines with phenotypic features of yolk sac endoderm (JC-44) or embryonic stem cells were also assayed for the expression of gamma GT. Significant levels were detected in JC-44 cells and higher levels were found in JC-44-derived embryoid bodies. Because this cell line appears to be a good in vitro counterpart of yolk sac differentiation, we characterized the gamma GT mRNA types expressed in JC-44 cells. By ribonuclease protection analysis, gamma GT RNA types IV and VI represent about 80% of the total gamma GT RNA in JC-44 embryoid bodies. Reverse transcription-mediated polymerase chain reaction detected low amounts of gamma GT RNA types I, III, and V. Expression of gamma GT in yolk sac follows a pattern seen in many tissues in which one or two gamma GT RNA types dominate the expression profile; however, the reason for this tissue specificity is unknown.

Globo-series carbohydrate antigens are expressed in different forms on human and murine teratocarcinoma-derived cells.

The glycolipids of human teratocarcinoma-derived cell line NCCIT were compared with those of 5 murine teratocarcinoma-derived cell lines. Glycolipid antigens were identified by cell surface immunofluorescence and high-performance thin-layer chromatography (HPTLC) immunostaining with a panel of monoclonal anti-carbohydrate antibodies. Human NCCIT embryonal carcinoma (EC) cells contained extended globo-series glycolipids Gb5 (galactosyl globoside) and GL7 (sialyl galactosyl globoside) recognized by antibodies to stage-specific embryonic antigens 3 and 4 (SSEA-3 and -4). SSEA-4 was not detected by immunofluorescence on the surface of any of the 5 murine teratocarcinoma-derived cell lines examined; however, SSEA-3 was detected on the surface of murine cell lines resembling primitive endoderm (JC44, NF-PE) and trophectoderm (E6496D). HPTLC analysis revealed a large amount of globoside (Gb4) in these differentiated cells, which may account for their labeling with anti-SSEA-3 antibody. Globo-series glycolipids were also detected in murine EC cells; however, differences were noted between the 2 cell lines examined. F9 cells contained primarily Gb4 and Forssman glycolipid, whereas NF-1 cells contained only minor amounts of Gb4 and lacked Forssman glycolipid entirely. Our results, coupled with the known distribution of Forssman antigen in the egg cylinder-stage mouse embryo, suggest that F9 and NF-1 murine EC cells are replicas of cells at different stages of development of the embryonic ectoderm. Glycolipids of normal mouse embryos were examined for comparison. Gb4 and Forssman glycolipid were presents in both embryonic and extra-embryonic tissues, whereas Gb5 and GL7 were restricted to visceral yolk sac and placenta. Our results demonstrate that human and murine teratocarcinoma-derived cells both synthesize extended globo-series glycolipids; however, oligosaccharide chain elongation takes different pathways in the 2 species. These differences reflect species-related and cell type-specific patterns of glycosylation.

Molecular cloning and characterization of murine cyclin E.

Using a monoclonal antibody developed to a mouse trophectodermal carcinoma stem cell line E6496D lysate, we have identified the gene that encodes murine cyclin E. The cDNA contains a consensus cyclin box and a long 3' untranslated region and shares over 75% homology with human cyclin E cDNA. We show that the gene is expressed in fetal tissues, embryonal carcinoma F9 cells and yolk sac carcinoma PYS-2 cells, but is not expressed in preimplantation stages of development. In adult tissues, cycE mRNA is detectable in the spleen and to a lesser extent in the testis and brain. These data show that cycE is developmentally regulated and unevenly expressed in fetal and adult mouse tissues.

Isolation of serine protease from granulated metrial gland cells of mice and rats with lectin from Dolichos biflorus.

Granulated metrial gland cells were the only cells in the endometria of pregnant mice and rats that reacted histochemically with fluoresceinated lectin (DBA) from Dolichos biflorus. Cell extracts of uteri of pregnant animals, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analysed by lectin overlay blotting, contained DBA-reactive, 40-50 kDa, doublet glycoprotein bands. This glycoprotein was purified on a DBA agarose affinity column. It was identified by amino acid sequencing as a serine protease closely related to granzymes of T lymphocytes. We conclude that this granzyme accounts for the selective reactivity of granulated metrial gland cells with fluoresceinated DBA in histological sections of uteri of pregnant rodents and show that DBA affinity columns can be used for purification of granzyme derived from granulated metrial gland cells.

Technetium-99m-labeled proteins for imaging inflammatory foci.

Polyclonal human IgG (IgG), antinuclear antibody (TNT-1), and human serum albumin (HSA), were labeled with 99mTc by a method recently developed in our laboratory, and administered i.v., each to a separate group of five mice, bearing inflammatory foci induced by an i.m. injection of 40 microL turpentine or 5 x 10(8) E. coli and 5 x 10(8) Entercocci. TNT-1 labeled with 125I served as a control and 67Ga-citrate as a "gold standard". At 4 or 24 h post injection, animals were imaged and sacrificed for tissue distribution studies. At 4 h in the turpentine group, the abscess-to-muscle ratios were: 67Ga, 4.8 +/- 2.1, 125I-TNT-1, 4.3 +/- 1; 99mTc-TNT-1, 3.5 +/- 1.8; 99mTc-IgG, 3.9 +/- 0.6; and 99mTc-HSA, 4.3 +/- 1. In the microorganism group, these ratios were 2.6 +/- 0.6, 3.3 +/- 0.5, 3.4 +/- 0.08, 3 +/- 1.1 and 4.1 +/- 0.6, respectively. Autoradiographic examination of infected tissues indicated that leakage of labeled proteins into interstitial space due to increased capillary permeability may be one of the major mechanisms of uptake.

Basement membrane components secreted by mouse yolk sac carcinoma cell lines.

Three new cell lines (NE, ME, LRD) were cloned from mouse-embryo-derived teratocarcinomas and characterized on the basis of developmental, ultrastructural, and cytochemical criteria as nullipotent embryonal carcinoma (EC), pure parietal yolk sac (PYS) carcinoma and mixed parieto-visceral yolk sac carcinoma respectively. Cell lines NE and ME were composed of a monomorphous cell population; however, the morphology of ME was growth-medium-dependent. LRD was composed of a heterogeneous cell population and formed embryoid bodies. NE secreted soluble laminin, osteonectin, entactin and fibronectin but did not form visible pericellular matrix. ME formed pericellular matrix which was composed of laminin and entactin, but did not contain fibronectin. The LRD cells formed pericellular matrix which was composed of laminin, entactin and fibronectin. Whereas laminin from ME and LRD reacted with polyclonal antibodies and a monoclonal antibody to parietal yolk sac laminin, the laminin from NE cells was unreactive with the monoclonal antibody. Osteonectin was found in the supernatant of LRD and ME, but could not be demonstrated immunohistochemically in the extracellular matrix. We conclude that some extracellular matrix components, such as laminin and fibronectin, are produced not only by yolk sac carcinoma cells but by nullipotent EC as well, although the latter do not assemble them into extracellular matrix. Laminin produced by EC is immunochemically different from laminin secreted by yolk sac carcinoma. The extracellular matrix produced by mixed parieto-visceral yolk sac carcinoma is different from the matrix laid down by the pure PYS in that the latter does not contain fibronectin. The lack of osteonectin in the extracellular matrix of yolk sac carcinoma cells indicates that not all polypeptides secreted by these cell lines are incorporated into the extracellular matrix. The new cell lines described in this paper differ with regard to their capacity to form extracellular matrix and secrete its various components. Hence they could be used for further studies of basement membrane assembly in vitro.

Preparation and evaluation of new bifunctional chelating agents: a preliminary report.

A scheme has been designed to synthesize a homologous series of new bifunctional chelating agents (BFCAs), which may increase the thermodynamic stability of metal chelates and conjugate at the specific sites on the monoclonal antibody molecule (MoAb) permitting us to analyze the structure-activity relationships of the series of compounds. Four such compounds have been prepared and characterized by FT-i.r. and NMR spectroscopy. One of them has been used to label an antibody with 111In, the stability and distribution of which has been examined in tumor-bearing mice and compared to that of the 111In-MoAb prepared using cyclic anhydride of DTPA. Enhanced tumor/blood ratios (9 vs 6.5), tumour to muscle ratios (7 vs 3), and decreased liver uptake (4 vs 12%) have been obtained.

Distinct antigenic characteristics of murine parietal yolk sac laminin.

Two monoclonal antibodies (LAM-A and LAM-B) specific for laminin from normal and neoplastic parietal yolk sac (PYS) cells were produced in rats immunized with a mouse yolk sac carcinoma cell line. Both antibodies immunoprecipitated the 400,000- and 200,000-Da chains of laminin and reacted with purified PYS laminin in ELISA. LAM-A reacted with mouse and rat PYS laminin, whereas LAM-B reacted only with mouse PYS laminin. Formaldehyde- and methanol-fixed adult and fetal somatic tissues were immunohistochemically unreactive with either of the two antibodies. In acetone-fixed tissue sections, both antibodies reacted weakly with some medullary tubules of the kidney, the follicular basement membrane of the ovaries, and the seminiferous tubules. The antibodies appear to react with the polypeptide determinants residing on the terminal portion of the long arm of laminin. It is concluded that laminin derived from normal or malignant PYS cells has distinct antigenic sites that are immunochemically not apparent in most other basement membranes.

Developmentally regulated expression of the cell-cell adhesion glycoprotein cell-CAM 120/80 in peri-implantation mouse embryos and extraembryonic membranes.

Peri-implantation mouse embryos and extraembryonic membranes were examined immunohistochemically for the expression of the cell-cell adhesion molecule (cell-CAM) 120/80. Cell-CAM 120/80 was seen along the lateral borders of all cells in the blastocyst but became undetectable on trophoblastic giant cells, some mononuclear trophoblastic cells and parietal yolk sac cells when blastocysts were cultured in vitro. In postimplantation embryos in vivo, all parts of the early egg-cylinder reacted with the antibody to cell-CAM 120/80 except for the cells of the parietal endoderm and the primary trophoblastic giant cells. In the late stage egg-cylinder, no cell-CAM 120/80 was seen on the cells of the primitive mesoderm or on the primordial germ cells. The germ cells in genital ridges and fetal gonads remained cell-CAM 120/80-negative throughout the fetal stages of development. In the extraembryonic membranes, the visceral yolk sac, amnion, and the cells of the placental labyrinth were cell-CAM 120/80-positive, whereas, the parietal yolk sac cells and the spongiotrophoblast cells were negative. These data show that cell-CAM 120/80 is found on cells arranged into epithelial layers in the early embryo and extraembryonic tissues, but is not expressed in the dissociated cells differentiating from these epithelia. Thus, the expression of cell-CAM 120/80 appears to be developmentally regulated.

Spectrin in mouse gametogenesis and embryogenesis.

Antibodies to nonerythroid alpha spectrin (p 230) were used to study the distribution of this polypeptide in mouse germ cells, zygote, and early embryonic cells. In the primordial germ cells, fetal oocytes, and spermatogonia, spectrin was found predominantly in the form of a narrow condensed subplasmalemmal band, as in all other somatic cells. During spermatogenesis, spectrin is condensed into the supraacrosomal cytoplasm and is lost during the reduction of the cytoplasm of the maturing spermatozoa. The postnatal growth of the oocyte is accompanied by a loss of the dense cortical band of spectrin and its redistribution in the cytoplasm. Zygotes also contain granular dispersed spectrin. Cortical condensation of spectrin filaments gradually reappears in the blastomeres at the two-cell stage and in the secondary polar body. Cortically condensed filaments represent thereafter the predominant form of spectrin in all preimplantation stage embryonic cells. Trophoblastic cells spreading out from explanted blastocysts are devoid of the cortically condensed spectrin and contain, instead, spectrin arrays in the cytoplasm. Trophoblastic cells, which surround the implanted embryo in vivo, also show diffuse cytoplasmic spectrin which subsequently undergoes subplasmalemmal condensation. These data show that spectrin is present in all stages of gametogenesis and embryogenesis, except in mature spermatozoa; and that it undergoes cytoplasmic redistribution during morphogenesis.

Mouse endometrial stromal cells produce basement-membrane components.

During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations. The decidualization of stromal cells is characterized by the accumulation of pericellular basement-membrane material. Decidualized stromal cells of pregnancy differ in this respect from stromal cells obtained from nonpregnant uteri, which are not associated with any significant amounts of basement-membrane components. Mouse decidual cells isolated from 6- to 7-day pregnant uteri explanted in vitro continue to synthesize basement-membrane-like extracellular matrix. Using immunohistochemistry and metabolic labeling followed by immunoprecipitation, SDS-PAGE, and fluorography, it was shown that the decidual cells synthesize laminin, entactin, fibronectin, type-IV collagen, and heparan sulfate proteoglycan. Stromal cells isolated from nonpregnant uteri and explanted in vitro produced the same basement-membrane components. Apparently, these cells were stimulated by the procedure used during isolation and explanation to undergo pseudodecidualization. We thus showed that stromal cells from pregnant and nonpregnant mouse uteri synthesize significant amounts of basement-membrane components in vitro, and hence could serve as a good model for the study of normal basement-membrane components.

Trophectodermal carcinoma: mouse teratocarcinoma-derived tumour stem cells differentiating into trophoblastic and yolk sac elements.

The transplantable tumour line derived from a spontaneous ovarian murine teratocarcinoma (Fekete & Ferigno, 1952) was cloned and characterized using light and electron microscopic and immunohistochemical techniques. Grown in ascites, the tumour consisted predominantly of stem cells and a small number of differentiated derivatives. The stem cells expressed surface reactivity with antibody to SSEA-3 and Forssman antigen, alkaline phosphatase, focal cytoplasmic reactivity with antibody to SSEA-1, and varying amounts of cytoplasmic glycogen and 3 beta-hydroxysteroid dehydrogenase. Their cytoskeleton reacted with antibodies to keratin and vimentin. The differentiated derivatives formed approximately 5-15% of the total cell population in ascites and appeared either as giant cells or were characterized by their reactivity with antibodies to H-2 or alpha-foetoprotein or intracellular and pericellular laminin or high levels of 3 beta-hydroxysteroid dehydrogenase activity. Solid tumours produced from subcutaneously injected cells had a variegated appearance suggesting, that like the limited differentiation in the ascites, the stem cells can give rise to trophoblastic, as well as parietal and visceral yolk sac elements. On the basis of the presented data the tumour stem cells were considered as representing malignant equivalents of the common precursor of trophoblastic, visceral and parietal yolk sac cells most likely corresponding to trophectoderm. Accordingly, the tumour was designated as trophectodermal carcinoma.