Petr Dráber

Differentiation antigens of mouse teratocarcinoma stem cells defined by monoclonal antibodies

Three differentiation antigens of mouse teratocarcinoma stem cells are defined using a panel of ten IgM-class monoclonal antibodies raised against teratocarcinoma F9 cells. TEC-01 and four other antibodies define an antigen that corresponds to SSEA-1. TEC-02 antibody defines an antigen that is expressed on teratocarcinoma stem cells, parietal yolk sac cells PYS-2, unfertilized eggs including the zona pellucida and blastocysts. It is absent from all mouse adult tissues tested. Three other antibodies exhibit binding properties similar to TEC-02. TEC-03 antibody defines an antigen that is expressed on teratocarcinoma stem cells, PYS-2 cells and mouse blastocysts. It is absent from all mouse adult tissues except for lungs.

Inhibition of fertilization by a monoclonal antibody recognizing the oligosaccharide sequence Ga1NAcβ1→4Ga1β1→4 on the mouse zona pellucida

Abstract Mouse eggs and pre-implantation stage embryos express on their surfaces a carbohydrate epitope, TEC-2, defined by an IgM monoclonal antibody, TEC-02. The TEC-2 epitope involves the oligosaccharide sequence Ga1NAcβ1→4Ga1β1→4 that is expressed on the plasma membrane and zona pellucida of mouse eggs and on a very limited number of other cell types. In this study we addressed the question whether or not the binding of TEC-02 antibody to the mouse eggs would interfere with their fertilization. Our data showed that the TEC-2 epitope is carried by two zona pellucida glycoproteins, ZP2 and ZP3. Binding of TEC-02 antibody to mouse eggs inhibited specifically and in a dose-dependent manner their fertilization in vitro. The inhibitory effect of TEC-02 antibody was dependent on the presence of an intact zona pellucida. Direct radioantibody binding assays indicated that the TEC-02 antibody completely inhibited fertilization at a concentration at which one quarter of all available TEC-2 binding sites was occupied. Binding of TEC-02 antibody to an egg did not interfere with initial attachment of the sperm to the egg but inhibited maintenance of sperm binding to the zona pellucida, the secondary binding. The combined data indicate that TEC-2, which is a well-defined zona pellucida specific carbohydrate epitope, might be a part of the secondary sperm receptor.

The epitope of mouse embryonic antigen(s) recognized by monoclonal antibody TEC-02 is a carbohydrate carried by high-molecular-weight glycoconjugates.

The expression and properties of mouse embryonic antigens, recognized by monoclonal antibody TEC-02, were analyzed in teratocarcinoma-derived cell lines. TEC-2 antigens were found in the majority of the parietal endoderm cells PYS-2 and in a fraction of cultured embryonal carcinoma cells but not in other cell lines tested. During the course of retinoic acid-induced differentiation of embryonal carcinoma cells F9, the expression of TEC-2 was transiently increased. Immunolabeling of extracts from F9 and PYS-2 cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that TEC-2 antigens are polydisperse glycoconjugates of high molecular weight (mostly greater than 100,000). The TEC-2 epitope was shown to be carbohydrate which in F9 cells might be attached to the same carrier as another developmentally regulated carbohydrate epitope TEC-1. The TEC-2 antigens, isolated by indirect immunoprecipitation, were degraded by extensive pronase digestion or mild alkaline treatment to mostly large products. Immunostaining of glycolipid standards suggested that TEC-2 epitope involves the GalNAc beta 1----4Gal beta 1----4R sequence. Combined data indicate that TEC-2 is a new developmentally regulated carbohydrate epitope carried in embryonal carcinoma cells predominantly on glycoprotein-bound large carbohydrates.

Cytochemical localization of GalNAc and GalNAcβ1,4Galβ1,4 disaccharide in mouse zona pellucida

Abstract Carbohydrate residues contained in the zona pellucida play a key role in the process of sperm-egg interaction. In vitro fertilization experiments have shown that a specific monoclonal antibody against GalNAcβ1,4Galβ1,4 disaccharide inhibits fertilization in mice. In the present study, the ultrastructural cytochemical localization of GalNAc residues and the GalNAcβ1,4Galβ1,4 disaccharide was carried out in ovarian and postovulatory oocytes by using lectin-gold cytochemistry and immunocytochemistry. Plant lectins SBA and DBA showed an affinity for the entire zona pellucida matrix of ovarian oocytes throughout the follicular maturation; however, immunoreactivity for GalNAcβ1,4Galβ1,4 disaccharide was not detected in ovarian oocytes at the earliest stages of follicular development but was found to be associated with the inner region of the zona matrix at the trilaminar primary follicle stage. The Golgi apparatus, vesicular aggregates, and cortical granules of the oocyte were intensely labeled by SBA and DBA throughout follicular development. Immunoreactivity to GalNAcβ1,4Galβ1,4 disaccharide was first observed in the Golgi apparatus and vesicular aggregates in trilaminar primary follicles. No immunoreactivity was observed in the cortical granules. In postovulatory oocytes, results were similar to those observed in ovarian oocytes. Our results thus suggest that (1) GalNAcβ1,4Galβ1,4 disaccharide residues are present only in the inner region of the zona pellucida and, therefore, might be involved in sperm penetration through the zona pellucida, (2) the inner and outer regions of the zona pellucida contain different oligosaccharide chains, (3) the vesicular aggregates detected in the oocyte could represent an intermediate step in the secretory pathway of zona pellucida glycoproteins and might be involved in the formation of cortical granules.

Localization of mouse TEC-1,2,3 antigens in adult and fetal tissues in the rat and human

Cryostat sections of adult and fetal tissues of the rat and human were investigated by a double immunoperoxidase technique for the presence of three developmentally-regulated mouse antigens, TEC-1,2,3. In the adult rats the TEC-1 antigen was detected on the epithelium of the isthmic portion of the oviduct, in some kidney tubuli, and in the brain; the TEC-2 antigen was distributed on the epithelium of various tissues, but it was also present on cells that may correspond to phagocytizing macrophages; the TEC-3 antigen was detected on endothelia of some vessels or capillaries. In rat fetuses only the TEC-1 antigen was detected; it was found on digestive tract epithelium and some kidney tubuli. In adult human tissues the distribution of TEC-1 antigen resembled that known for SSEA-1; the TEC-2 antigen was detected in some kidney tubules. In human midpregnancy fetuses the TEC-1 antigen was found in the same tissues as in adults; TEC-2 was detected in the thyroid gland. There was no staining of adult or fetal human tissues with TEC-03 antibody. The data show different patterns of expression of TEC-1,2,3 antigens among the species studied.

Biological activity of hormonally active and non-active androgen derivatives

Androgen derivatives appeared to have different biological activities in vivo and in vitro. Testosterone-17-isobutyrate given in three doses of 50 or 200 microgram increased significantly the weight of seminal vesicles and reduced thymus weight in castrated males, whereas testosterone-17-hemisuccinate, testosterone-D-beta-glucoside and testosterone-3-(O-carboxymethyl)-oxime had no such effects. Similarly, ten 1-mg doses of testosterone-17-isobutyrate, unlike testosterone-17-hemisuccinate, resulted in a marked reduction of thymus weight in non-castrated males and in a significant inhibitory effect on the activity of spermatogenesis. On the other hand, all three androgen derivatives, which had appeared inactive in vivo, had similar effects in vitro (as had active testosterone) as demonstrated by inhibition of Concanavalin A-induced lymphocyte activation expressed by 14C thymidine incorporation and inhibition of cell agglutination. These results seem to suggest that as for the regulation of androgen-dependent organs and functions (such as the size of seminal vesicles, activity of spermatogenesis, thymus size), hormonally active androgens are also involved in certain immunosuppressive effects in vivo. On the other hand, in vitro immunological effects are produced by both hormonally active and non-active androgen derivatives as well as by other steroid hormones, the common denominator being the steroid structure.

Differential expression of mouse embryonic antigens TEC-1 and TEC-2 in the epididymis of four rodent species

The expression, properties and relationship of two mouse embryonic antigens (TEC-1 and TEC-2), which are defined by monoclonal antibodies, were investigated in the epididymis of four rodent species. Absorption analysis, indirect immunofluorescence microscopy and immunohistochemistry revealed that all the species studied contained in their epididymides, but not in testes, either TEC-1 (Chinese hamster), TEC-2 (guinea pigs, rats) or both TEC-1 and TEC-2 (mice) antigens. In an indirect immunofluorescence assay, the antigens were found on spermatozoa isolated from caudae epididymides of guinea pigs, rats and Chinese hamsters but not mice. On the other hand, the TEC-2 antigen, which is expressed on mouse eggs, was not detected on eggs from the other species studied. Immunolabeling of epididymal extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that both epididymal antigens have apparent molecular weights of greater than 200,000. In guinea pigs, rats and mice, the antigens were detected by a two-site sandwich radioantibody-binding assay in which the antigen is immobilized and detected with the same antibody; this indicates that several antigenic determinants were present on the same carrier. In mice, some carriers seem to express both TEC-1 and TEC-2 epitopes. In Chinese hamsters, TEC-1 antigen was only detected by the solid-phase assay, suggesting that in this species there are markedly fewer antigenic determinants per carrier molecule. Interspecies differences in the activities of epididymal glycosyltransferases and/or glycosidases appear to be the biochemical mechanism of the species-specific expression of these antigens.

Developmentally regulated surface structures of teratocarcinoma stem cells studied by mutant cell lines.

Monoclonal antibodies TEC-01, TEC-02, and TEC-03, which define three developmentally regulated antigens TEC-1 (SSEA-1-like), TEC-2, and TEC-3, have been used to isolate and characterize teratocarcinoma stem cell mutants with altered expression of surface glycoconjugates. Mutants lacking TEC-1 antigen have been isolated by exposing mutagenized P19S1801A1 cells to TEC-01 antibody, which was conjugated to the toxin from Ricinus communis. None of the mutants exhibits significant changes in the expression of TEC-3 antigen, but some are defective in the expression of TEC-2 antigen. Analysis of the expression of TEC-1,2,3 antigens in different lectin-resistant F9 and OTF9-63 cell lines has shown that all express TEC-1 antigen, but some lectin-resistant phenotypes exhibit reduction in the expression of TEC-2 and/or TEC-3 antigens. Mutational events in genes regulating the expression of specific glycosyltransferases or glycosidases appear to be the biochemical mechanism regulating the expression of TEC-1 and TEC-2 antigens.

Cytochalasin B-induced changes in concanavalin A-activated lymphocytes

SummaryCytochalasin B (CB) administered simultaneously with a mitogenic dose of concanavalin A (Con A) interferes with the activation process. This interference involves structural alterations of cellular membrane which do not include a reduced Con A-binding capacity. This conclusion is supported by the observation of deformities in both nuclear and cytoplasmic membranes in Con A-activated lymphocytes subsequently treated with CB. The high incidence of membrane blebs and pseudomyelin bodies in the cytoplasm points to a general effect of CB on the structural organization of membrane which may secondarily interfere with some specific event such as generation or transfer of signals for activation or cytokinesis.

Retinoic acid-induced changes in differentiation-defective embryonal carcinoma RAC65 cells

RAC65 is a mutant clone of mouse embryonal carcinoma cells, P19, which does not undergo terminal differentiation upon treatment with retinoic acid (RA), RAC65 cells express a truncated RA receptor α (RARα) which, however, does not fully explain their defect. Here we show that RAC65 cells exhibit an additional defect in RARα mRNA which may reflect a defect in RNA splicing. The parental and mutant cells also differ in their capacities to bind [3H]RA into nuclear fractions and in expression of cellular RA binding protein (CRABP) mRNA after treatment with RA. The combined data suggest that the deffect in RAC65 RARα results in reduced expression of the CRABP gene after RA treatment and, therefore, increased flow of RA into the nucleus.