A new embryonic antigen,p67, present during mouse preimplantation development

An antibody prepared against nullipotential teratocarcinoma stem cells (A-N₁) detects cell surface antigens expressed by early mouse embryos and inhibits in vitro development of embryos in the absence of complement [Calarco and Banka, 1979]. Here we report the immunoprecipitation and electrophoretic characterization of A-N₁-detected antigens from preimplantation mouse embryos. Predominant antibody activity is directed against a 67,000-dalton glycoprotein (p67) with a mean pI of 5.3, which has not been previously described. This protein is not detected, at least as p67, after culture of embryos in tunicamycin. The p67 antigen is also expressed by pluripotential PSA1 teratocarcinoma cells but not by several different differentiated mouse cell types.     

Immunological characterization of embryonic cell surface antigens recognized by antiblastocyst serum

Stage-specific cell surface antigens expressed during mouse preimplantation development and detected by a rabbit antiserum prepared against mouse blastocysts (A-BL₂) have been characterized by serological and biochemical techniques. Immunofluorescence, immunoradiolabeling, and complement-mediated cytotoxicity assays reveal the expression of A-BL₂ surface antigens beginning at the 4-cell stage and reaching a maximum at the 8-cell to morula stages. At earlier times in development A-BL₂ antigens are not detectable, and there is a decline in expression at the blastocyst stage. No antibody reactivity is detected against adult mouse tissues or teratocarcinoma cell lines. The presence of A-BL₂ antibodies during in vitro embryo culture interferes with normal development. Treatment of embryos with β-N-acetylglucosaminidase, but not other glycosidases, proteases, or lipases, results in a quantitative decrease in the binding of A-BL₂ antibodies to surface antigens. Immunoprecipitation and electrophoretic analyses of A-BL₂ antigens demonstrate specific antibody activity against a pair of embryonic glycoproteins of 65,000 to 70,000 daltons which can be metabolically labeled with ³⁵S-methionine and ³H-glucosamine. Tunicamycin treatment alters the form of the A-BL₂ immunoprecipitate to a single 60,000-dalton protein.     

Metabolic and physiologic studies of nonimmune lymphoid cells cytotoxic for fibroblastic cells in vitro

An in vitro reaction between mouse lymphoid cells and target fibroblastic cells in wells of microtest plates, which appears to simulate the in vivo rejection of hemopoietic allografts, has been analyzed for metabolic and physiologic requirements. Protein synthesis was required for only the first few hours of culture. Inhibition of RNA synthesis and alteration of cell surface charge with various agents were without obvious effects. Metabolic slowing at 4 °C or deviation of the pH of the culture medium suppressed the reaction. Thymus cells, which are not cytotoxic in this system, significantly but not completely inhibited the cytotoxicity of lymph node cells. Antiserum directed against target cells specifically protected them from the cytotoxic lymphoid cells in the absence of complement.Precursors of cytotoxic lymphoid cells were radiosensitive, unlike the cytotoxic cells themselves. BALB/c anti-C57BL/6 spleen cell serum and ⁸⁹Sr both are able to prevent rejection of marrow allografts in vitro. Lymphoid cells incubated with this antiserum plus complement lost much of their cytotoxicity but were still effective at high ratios of aggressor to target cells. Lymphoid cells of mice treated with ⁸⁹Sr were effectively cytotoxic but lost practically all of their cytotoxicity afer incubation with the antiserum plus complement. Thus, it appears that this reaction detects two different cytotoxic lymphoid cells, either of which can function in vitro. Both cell types may need to cooperate in vivo during marrow allograft rejections.     

Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct.

Populations of fibroblast-like cells from 14 day embryonic chick cornea, heart, and skin were grown in vitro as primary cultures and found to be antigenically distinct from one another. Corneal fibroblasts were obtained by dissection, whereas heart and skin fibroblast-like cells were separated from nonfibroblastic cell types by their rapid adhesion to substrata. Cultured cells were used as antigens in rabbits. Antisera were first absorbed against homogenates of embryonic chicks from which the homologous tissue was removed. Each such 1° absorbed antiserum then was absorbed against homogenates of the two respective heterologous fibroblast-like cell populations (2° and 3° absorptions). Resulting 3° absorbed antisera were tested for specificity by immunodiffusion, immune agglutination, immune cytotoxicity (trypan blue uptake and ⁵¹Cr release), and indirect immunofluorescence. Each 3° antiserum was judged tissue specific when it reacted only with the fibroblast-like cells of its own tissue, i.e., the homologous population. Unabsorbed antisera reacted with both homologous and heterologous fibroblast-like cells, as did 1° absorbed antisera. Absorption of 1° antisera with homogenates of the two heterologous fibroblast-like populations removed antibodies against the heterologous populations without significantly reducing the 3° antiserum titer against the homologous fibroblast cell type. Moreover, absorption of 1° antisera with each of the two heterologous fibroblast-like populations removed antibodies not removed by the other. Thus, the fibroblast-like cells from cornea, heart, and skin are antigenically different from one another in vitro. The stable antigenic differences detected may have arisen during the differentiation of these cells in vivo. Some of the tissue-specific antigens detected must occur on the cell surface.     

Differences in Gene Expression between Mouse and Human for Dynamically Regulated Genes in Early Embryo

Infertility is a worldwide concern that can be treated with in vitro fertilization (IVF). Improvements in IVF and infertility treatment depend largely on better understanding of the molecular mechanisms for human preimplantation development. Several large-scale studies have been conducted to identify gene expression patterns for the first five days of human development, and many functional studies utilize mouse as a model system. We have identified genes of possible importance for this time period by analyzing human microarray data and available data from online databases. We selected 70 candidate genes for human preimplantation development and investigated their expression in the early mouse development from oocyte to the 8-cell stage. Maternally loaded genes expectedly decreased in expression during development both in human and mouse. We discovered that 25 significantly upregulated genes after fertilization in human included 13 genes whose orthologs in mouse behaved differently and mimicked the expression profile of maternally expressed genes. Our findings highlight many significant differences in gene expression patterns during mouse and human preimplantation development. We also describe four cancer-testis antigen families that are also highly expressed in human embryos: PRAME, SSX, GAGE and MAGEA.     

Laser Fusion of Mouse Embryonic Cells and Intra-Embryonic Fusion of Blastomeres without Affecting the Embryo Integrity

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.     

Characterization of antisera against mouse teratocarcinoma OTT6050: Molecular species recognized on embryoid bodies,preimplantation embryos,and sperm

Antisera raised in rabbits by hyperimmunization with small embryoid bodies of the transplantable teratocarcinoma OTT6050 recognize several distinct antigenic protein species on the surfaces of cells of the immunogen. Some of these antigens were found on the cells of preimplantation mouse embryos, on cells from parietal yolk sac carcinoma, and on mouse sperm. These antigens have been distinguished by polyacrylamide electrophoresis of the immune precipitates from detergent extracts of lactoperoxidase-iodinated cells. The intact embryoid bodies from the ascitic form of the OTT6050 teratocarcinoma exhibited five major protein bands (approximate MW 150K, 115K, 82K, 48K, and 12K), one band that ran at the dye front of the gels (R_f ≥1) and one minor band (approximate MW 22K). Two different rabbit antisera recognized an essentially identical pattern of antigens which, however, varied on the different cell types tested. Antisera were also elicited in syngeneic male mice using glutaraldehyde-fixed or irradiated OTT6050 embryoid bodies. The isoantisera had very poor titers in comparison to the absorbed xenoantisera, as assessed by complement-mediated cytotoxic activity against the immunizing cell types. Complement-mediated cytotoxicity could also be demonstrated using parietal yolk sac carcinoma cells, preimplantation mouse embryos from all cleavage stages, blastocysts, and immunosurgically isolated inner cell masses, as targets. The complexity of the antisera generated by intact embryoid bodies described here indicates that these structures bear multiple antigenic specificities not present on adult somatic cells, some of which are stage-specific embryonic polypeptides.     

Trypanosoma vivax: Absence of host protein on the surface coat

The possible presence of host serum proteins on the surface of Trypanosoma vivax stock Zaria Y486 was studied. Intact washed bloodstream forms from mice were not lysed or neutralized by antisera against mouse serum proteins. Serum against T. vivax prepared in rabbits against an antigen which was a water-soluble trypanosome extract, failed to cross-react with mouse serum when tested by immunoelectrophoresis and immunodiffusion. The T. vivax antigen failed to cross-react with three different anti-mouse sera when tested by the same techniques.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of ¹²⁵I-surface-labeled parasites showed the presence of a cluster of proteins ranging in molecular weights between 57,000 and 45,000 daltons. None of these proteins was precipitated by anti-mouse serum protein sera. The serum against T. vivax precipitated a protein of 50,000 daltons molecular weight.     

Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos

This study was undertaken to obtain specific information on the characteristics of spontaneous and induced apoptosis during preimplantation development of rabbit in vivo and in vitro developed embryos and mouse in vitro embryos. After reaching appropriate developmental stages, embryos were transferred into culture media with or without apoptotic inductor (actinomycin D 500 ng/mL) and cultured for 10 h. The identification of apoptotic cells was based on morphological assessment of nuclei and on detection of specific DNA degradation, phosphatidylserine redistribution and active caspase-3 under fluorescence microscope.Our experiments proved that apoptosis is a frequent physiological event occurring during normal preimplantation development. A high number of untreated rabbit and mouse blastocysts contained at least one apoptotic cell. Rabbit embryos showed a lower incidence of spontaneous apoptosis. Treated blastocysts of both species responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and significant increase in the incidence of apoptotic cell death. The occurrence of spontaneous apoptosis during earlier preimplantation development was sporadic and its presence was observed only at stages following embryonic genome activation (at 4-cell stage and later in mouse, at 16-cell and morula stage in rabbit). The susceptibility of embryos at early stages to the apoptotic inductor was much lower. The presence of actinomycin D did not increase the incidence of apoptotic embryos or apoptotic cells. Nevertheless, it slowed down embryo growth and triggered earlier appearance of some apoptotic features (at the 6-cell stage in rabbit). The results show that the occurrence of both spontaneous and induced apoptosis in preimplantation embryos is stage- and species-specific.     

Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro

Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro.     

Models of normal and transformed cell adhesion,and capping and locomotion in vitro

It is proposed that patching, capping and endocytosis, and cell locomotion are manifestations of a single process whereby the cell discards foreign materials. Capping results from the binding to the cell surface of particulate (or molecular) objects which cannot function as immovable substratum. This might be described as unsuccessful or abortive cell adhesion in that the particles adhere to the cell rather than the cell adhering to the substratum. Lateral particle movements on the cell surface membrane are effected by the submembranous microfilament-microtubule system, resulting in capping without displacement of the cell. Successful adhesion of the cell to a substratum renders capping and endocytosis impossible and the cell attempts to discard the substratum by mechanisms analogous to capping. The cell achieves this by lateral movement and detachment of the trailing edge.The concept of abortive adhesion leading to capping has been amplified by the development of molecular models of normal and neoplastic cell adhesion in vitro in the presence and absence of serum. In these models, the normal cells have molecule A (adhesion sites) on their surface; they can spread on the substratum in the absence of serum. In the presence of serum, the A molecules on the normal cell surface bind with B molecules in serum, which may be substratum-bound or free in suspension. Binding of free B molecules with cell surface A molecules results in blockage of adhesion sites; these are cleared via capping. New adhesion sites (A molecules) are produced at the active edges of the cell. Binding of cell surface A molecules with the substratum bound B molecules results in cell adhesion. Transformed cells do not have A molecules on their surface; they cannot spread in the absence of serum. The transformed cells may recruit A molecules from the serum to attain deformability and spreading.These models also relate to capping of gold or resin particles, cell locomotion and regulation of cell division, and lectin-induced agglutination of transformed cells.     

Inhibition of the lytic phase of murine T-cell-mediated alloimmune cytotoxicity by a rat antiactivated T-cell antiserum

Antisera produced in rats by immunization with alloimmune murine C57Bl/6 anti-P815 splenic lymphocytes or purified T cells activated in vitro by coculture with phytohemagglutinincoated L-929 cells were found to inhibit the in vitro cytolytic action of in vivo and in vitro alloimmune C57Bl/6 anti-P815 cytotoxic T cells in a 4-hr chromium-51 release assay. The rat anti-murine-activated lymphocyte (anti-MAL) or antiactivated T-cell (anti-ATC) serum inhibited lysis in the absence of exogenously added complement activity and were not directly cytotoxic to CTL. Absorption of anti-MAL with target cells P815, L-929, EL-4, and normal C57Bl/6 lymphocytes removed a limited amount of the CTL-inhibitory activity. In contrast, lectin-activated alloimmune lymphocytes fully absorbed the inhibitory activity indicating these antisera preferentially recognize unique antigenic determinants associated with the activated CTL cell surface. The anti-ATC was found to block alloimmune lysis by CTL from several inbred mouse strains suggesting these antisera recognized antigenic determinants of a common lytic mechanism. A kinetic analysis of the inhibitory activity of the anti-MAL on the CTL reaction scheme revealed this antiserum inhibited lysis at a post-Ca²⁺-dependent step, presumably during the target cell lytic phase. This result suggests the rat antiserum can neutralize the CTL lytic mechanism.     

Ability of outside cells from preimplantation mouse embryos to form inner cell mass derivatives

Previous studies have shown that inside cells in the preimplantation mouse embryo do not become committed to the formation of inner cell mass until after blastocyst formation. However, it is not yet clear whether outside cells are also labile late in preimplantation development or whether they become restricted to trophectoderm development at an earlier stage. The present study investigates the potency of outside cells isolated from late morulae just prior to blastocyst formation and shows that some, if not all, outside cells retain the potential to form inner cell mass derivatives in vitro and in vivo. This suggests that trophectoderm cells are not restricted in potential earlier than ICM cells and that all cells of the early embryo may be labile at least until blastulation.     

Effects of oxygen tension and humidity on the preimplantation development of mouse embryos produced by in vitro fertilization: analysis using a non-humidifying incubator with time-lapse cinematography

To examine the effects of oxygen tension and humidity on early embryonic development, the preimplantation development of mouse embryos produced by in vitro fertilization was assessed by time-lapse cinematography to evaluate morphokinetic development with higher precision. Zygotes were produced from spermatozoa and oocytes from ICR mice and cultured in KSOM under low or high oxygen tension in a non-humidified incubator with time-lapse cinematography (CCM-iBIS). The developmental rates of embryos to the 4-cell and blastocyst stages under lower oxygen tension in CCM-iBIS were significantly higher than those under higher oxygen tension in CCM-iBIS. Ninety-six hours after insemination, a large number of embryos cultured under low oxygen tension developed to the hatching blastocyst stage. Embryonic development was more synchronized under lower oxygen tension. Non-humidified cultures did not affect embryonic development. On average, mouse embryos cultured at lower oxygen tension reached 2-cell at 18 h, 3-cell at 39 h, 4-cell at 40 h, initiation of compaction at 58 h, morula at 69 h, and blastocyst at 82 h after insemination. In conclusion, lower oxygen tension better supports preimplantation development of mouse embryos fertilized in vitro, and non-humidified culture conditions do not influence the embryonic development in vitro.     

The human LT system. V. A comparison of the relative lytic effectiveness of various MW human LT classes on 51Cr-labeled allogeneic target cells in vitro: enhanced lysis by LT complexes associated with Ig-like receptor(s).

The present studies examine the in vitro cell-lytic capacity of various molecular weight (MW) human lymphotoxin (LT) classes obtained from lectin-activated normal or immune lymphocytes on allogeneic target cells. The findings reveal that the high-MW complex class of LT is up to 100 times more effective than the smaller MW LT forms (α, β, and γ) in causing lysis of various allogeneic cell types including lymphoid cells in vitro. Moreover, the data suggest that lectin-stimulated alloimmune cells (MLC sensitized) release complex LT forms in association with a specific antigen-binding receptor(s), and that these complexes are from 3 to 10 times more effective on the sensitizing target cell than complexes obtained from lectin-stimulated nonimmune cells. Positive evidence that complex-induced lysis involved LT was indicated by the finding that lysis was completely neutralized by incubation with heterologous antisera directed against a refined human α₂-LT subclass (anti-α₂) and partially neutralized with anti-human Fab₂′ serum. These findings support the concept that LT molecules may represent a system of related cell-lytic molecules. While the smaller MW forms are only weakly lytic by themselves, they can be assembled into highly lytic complexes which may be focused or directed by an antigen-binding receptor(s).     

X-chromosome inactivation in monkey embryos and pluripotent stem cells

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.     

Effect of Anti-immunoglobulin Antisera on Homograft Rejection in Mice

HETEROLOGOUS antisera against immunoglobulins or their component protein chains have been shown to inhibit the immune response in a variety of systems. Antibodies against mouse immunoglobulins, for example, inhibit the response of mouse spleen cells cultured in vitro^1–3. Antibodies against the heavy chains of chicken IgM (anti-μ), administered during embryonation and again at hatching, have produced agamma-globulinaemia in bursectomized chickens⁴, apparently by plasma cell line elimination⁵. Graft-versus-host (GVH) and delayed hypersensitivity reactions have been suppressed in neonatal mice by in vitro pretreatment of injected lymphoid cells with antiserum against light chains⁶. Similar pretreatment with univalent fragments (Fab) of anti-immunoglobulin antibodies has diminished the GVH reaction in adult mice⁷.     

Betaine Homocysteine Methyltransferase Is Active in the Mouse Blastocyst and Promotes Inner Cell Mass Development

Methyltransferases are an important group of enzymes with diverse roles that include epigenetic gene regulation. The universal donor of methyl groups for methyltransferases is S-adenosylmethionine (AdoMet), which in most cells is synthesized using methyl groups carried by a derivative of folic acid. Another mechanism for AdoMet synthesis uses betaine as the methyl donor via the enzyme betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5), but it has been considered to be significant only in liver. Here, we show that mouse preimplantation embryos contain endogenous betaine; Bhmt mRNA is first expressed at the morula stage; BHMT is abundant at the blastocyst stage but not other preimplantation stages, and BHMT activity is similarly detectable in blastocyst homogenates but not those of two-cell or morula stage embryos. Knockdown of BHMT protein levels and reduction of enzyme activity using Bhmt-specific antisense morpholinos or a selective BHMT inhibitor resulted in decreased development of embryos to the blastocyst stage in vitro and a reduction in inner cell mass cell number in blastocysts. The detrimental effects of BHMT knockdown were fully rescued by the immediate methyl-carrying product of BHMT, methionine. A physiological role for betaine and BHMT in blastocyst viability was further indicated by increased fetal resorption following embryo transfer of BHMT knockdown blastocysts versus control. Thus, mouse blastocysts are unusual in being able to generate AdoMet not only by the ubiquitous folate-dependent mechanism but also from betaine metabolized by BHMT, likely a significant pool of methyl groups in blastocysts.