Early Postimplantation Mouse Embryos Research Articles

Teratogenicity of benzoic acid derivatives of retinoic acid in cultured mouse embryos

Isotretinoin (13- cis-RA) is a human teratogen and mouse embryos exposed to 13- cis-RA in vivo exhibit many of the same defects as humans. Early postimplantation mouse embryos exposed to 13- cis-RA in culture exhibit developmental alterations of the visceral arches, similar to those seen after in vivo exposure. Certain benzoic acid derivatives of retinoic acid have been shown to possess activity equal to or greater than retinoic acid in several in vitro systems. This study examines the teratogenic effects of some of these retinoids on mouse embryos in vitro. Day 8 CD-1 mouse embryos were cultured for 48 hours in the presence of these benzoic acid derivatives. With the exception of Ro-15-0778, all compounds produced visceral arch malformations similar to those seen in embryos exposed to 13- cis-RA, but at dramatically different effective concentrations. Extremely low concentrations of the retinoic acid-related compounds tested appear to have detrimental effects on embryonic development and these compounds may be poor candidates for therapeutic use.

Effects of cadmium on preimplantation and early postimplantation mouse embryos in vitro with special reference to their trophoblastic invasiveness.

Cadmium chloride, at concentrations of 0.5 or 1 microgram/ml medium, did not affect the trophoblastic invasiveness of mouse embryos treated for 24 hours at 4-cell and morula stages. At higher concentrations of 5 or 10 micrograms/ml medium, most treated embryos in vitro underwent degeneration while a few survivors formed trophoblastic outgrowths with variable areas. Cadmium chloride, at a low concentration of 0.5 microgram/ml medium presented continuously to blastocysts after attachment in vitro, has significantly retarded the trophoblastic outgrowth areas and reduced the number of trophoblastic giant-cell nuclei, though the spreading blastocysts appeared morphologically normal. At higher concentrations of 1 or 5 micrograms/ml medium, cytoplasmic disintegration and detachment of trophoblasts were observed. It is suggested that cadmium may interfere with the cell division and/or the transformation of trophectoderm cells into giant cells, resulting in the retardation of the trophoblastic outgrowths.

Differentiation and grafting of haemopoietic stem cells from early postimplantation mouse embryos.

Haemopoietic stem cells evidently arise in early post-implantation mouse embryos at day 6 of gestation, a day earlier than previously thought (Moore & Metcalf, 1970). Disaggregated embryonic cells were injected into mice given a lethal dose of X-irradiation. The presence of donor haemoglobin (Whitney, 1978) and donor lymphocytic glucose phosphate isomerase (GPI) (Siciliano & Shaw, 1976) to detect donor erythrocytes and lymphocytes, respectively, were monitored by starch gel electrophoresis. The presence of donor cells was also assessed by using donor embryos carrying the T6 marker chromosomes. Decidual cells dissected free of embryos did not colonize any recipients. Disaggregated cells from early mouse embryos first colonized the liver and then repopulated the haemopoietic systems of recipients, producing adult donor haemoglobin within 2-3 days and donor GPI within 3-5 days. 80% of grafted X-irradiated recipients survived and donor markers were found in each of them. All nongrafted controls died within 14 days of X-irradiation and none of them showed donor markers. Disaggregated embryonic cells could be grafted across major histocompatibility barriers unlike adult bone marrow. Haemopoietic stem cells could not be identified in disaggregated cells from embryos aged less than 6 days gestation.

Lectin receptors on the peri- and early post-implantation mouse embryo.

Mouse embryos at the blastocyst, blastocyst outgrowth, and primitive streak (day 7.5) stages of development were analysed for expression of lectin receptors using a panel of six FITC-conjugated lectins with affinities for five distinct saccharides (BSL, ConA, DBA, LTL, UEA and WGA). Blastocyst trophoblast expressed receptors for all the lectins but later tissues of the trophectoderm lineage lost receptors for distinct but overlapping subsets of the lectin panel. The inner cell mass (ICM) of the early blastocyst lacked receptors only for UEA. Differentiation of primary endoderm was accompanied by the aquisition of UEA receptors but subsequent differentiation into visceral and parietal endoderm involved the loss of receptors for both fucose binding lectins (UEA and LTL). Embryonic ectoderm in the day 7.5 egg cylinder retained receptors only for ConA and WGA. Thus, in general, differentiation during the peri- and early post-implantation period was associated with a differential loss of lectin receptors in all cell lineages of the mouse conceptus.

Enhancement of radiation effects by mercury in early postimplantation mouse embryos in vitro.

Two-cell mouse embryos were X-irradiated (1 Gy) and immediately thereafter exposed to mercuric chloride (3 microM) up to the blastocyst stage. When combined treatment started shortly (about 1 to 2 h) before mitosis to the four-cell stage, blastocyst formation, hatching of blastocysts, trophoblast outgrowth and ICM formation were impaired stronger than expected from the addition of the single effects. The enhancement of risk was maximal for hatching of blastocysts and no further increase was observed for trophoblast outgrowth and ICM formation. When exposure of embryos to X-rays and mercury began about 5 to 6 h before mitosis to the four-cell stage, only additive effects were obtained for the endpoints mentioned above.

Developmentally regulated expression of insulin-like growth factors by differentiated murine teratocarcinomas and extraembryonic mesoderm

The expression of plasma membrane receptors for insulin-like growth factors (IGFs) by PC13 embryonal carcinoma (EC) cells, and their immediate differentiated progeny PC13END was examined by binding radiolabelled IGF-I to cell monolayers. Both cell types express high-affinity IGF receptors, but the apparent number of unoccupied receptors sites falls by about 60% upon differentiation. Crosslinking studies reveal that both type 1 and type 2 IGF receptors are expressed by PC13EC cells. PC13END-cell-conditioned medium contains developmentally regulated, separable activities, one of which reacts directly with IGF-II, and the other with IGF for plasma membrane receptors. The former activity represents a soluble secreted IGF-binding protein. The latter activity is structurally and functionally similar to rat IGF-II. Polyclonal antibodies raised against purified rat IGF-II specifically recognize multiple forms of IGF in radiolabelled culture supernatants and material which closely resembles the soluble IGF-binding protein. Immunoprecipitation of radiolabelled culture supernatants with anti-rat IGF-II reveals that the differentiation of PC13EC cells is accompanied by the coexpression of IGF-like molecules and the soluble binding protein, and that IGF-like molecules are expressed by extraembryonic tissues of mesodermal origin in the early postimplantation mouse embryo. These findings show that IGF-like molecules are expressed in early mammalian development and may act in an autocrine fashion in vivo.

The development of 12th to 14th day foetuses following reimplantation of pre- and early-primitivestreak-stage mouse embryos

This paper describes a technique for transplanting early postimplantation mouse embryos from their own implantation site to a decidua in another pregnant mouse. Evidence is provided that this procedure is compatible with their continued development. At a low frequency both 6th and 7th day embryos can re-establish themselves and continue apparently normal development and placentation for at least 6-8 days.

Growth factors in mammalian embryogenesis.

Malignant murine embryonal carcinoma (EC) cells have been studied as a means of characterizing the identity and action of growth regulatory molecules in the early postimplantation mouse embryo. The differentiation of EC cells in vitro is accompanied by significant changes in the control of cell proliferation, including the acquisition of dependence on specific exogenous growth factors for cell multiplication. This is at least partly controlled by the developmentally regulated expression of specific growth factor receptors and their intracellular response systems. The development of defined media has allowed the identification of the principal factors required for EC cell proliferation in vitro. These factors are synthesized in vivo by the extra-embryonic tissues of the developing embryo and by the differentiated progeny of EC cells in vitro. EC cells secrete a potent growth factor (embryonal carcinoma-derived growth factor, ECDGF) which has been purified and partly characterized. ECDGF induces proliferation of the differentiated progeny of EC cells and specific normal embryonic cell types in vitro, suggesting that ECDGF may act as an embryonic growth factor in vivo. Together these findings suggest that proliferation in the embryo may be controlled by reciprocal interaction between primitive ectoderm cells and their differentiated derivatives, mediated by the developmentally regulated expression of specific soluble growth factors.

Interferon synthesis in the early post-implantation mouse embryo

A qualitative bioassay was adapted and used to determine the ability of the early post-implantation mouse embryo to synthesise interferon. Interferon production was not seen in any embryo tissue in the absence of an inducer and could only be detected in virus-induced tissue from the early 7th day of development. This induced interferon synthesis was initially confined to the trophoblast of the early 7th day embryo. It was then found in tissues of both trophoblast and inner cell mass origin in the early 8th day, and subsequently, in derivatives of the embryonic ectoderm in the 13th-day embryo.

The formation of the embryonic mesoderm in the early post-implantation mouse embryo

The formation of the mesoderm in early post-implantation mouse embryos is described and analysed. The outgrowth of the mesoderm was found to depend on the changes in the shape of the embryonic ectoderm, which lead to a relative displacement of the primitive streak in the caudal direction. The primitive streak deposits its cells laterally in the case of the lateral mesoderm, and medially in the case of the headprocess. In doing so, the primitive streak leaves a trail of mesoderm cells. This means that mesoderm cells do not migrate actively from the caudally located primitive streak towards more frontal positions in the embryo. This is confirmed by the results of scanning electron microscopy, which revealed that mesoderm cells show no polarity at all in the caudofrontal direction. In may therefore be concluded that these cells probably do not migrate. Studies on the cell-cycle parameters of the embryonic ectoderm, showed that mesoderm cells-to-be are probably recruited not only from the proliferation zone, but also from the lateral ectoderm. It is postulated that the lateral ectoderm gives rise, via the largest part of the primitive streak, to most of the mesoderm cells, whereas the proliferation zone gives rise to the head-process mesoderm, via the anterior part of the primitive streak.

Embryo‐derived teratocarcinoma II. Teratocarcinogenesis depends on the type of embryonic graft

Early post-implantation mouse embryos or their embryonic and extraembryonic portions were transplanted under the kidney capsule of adult isogenic recipients in order to test the tumor-producing potential of various grafts and to establish the optimal conditions for teratocarcinoma production. The highest yield of malignant tumors was obtained from 7-day embryos transplanted in toto. Severing the extraembryonic portion of 7-day egg cylinders prior to transplantation of the embryonic part reduced the number of teratocarcinomas to values comparable with those obtained from transplanted 6-day embryos. The extraembryonic portion of 7-day embryos produced no tumors. These data suggest that extraembryonic portions of the 7-day egg cylinder do not have tumorigenic potential alone, but may increase the yield of teratocarcinomas from the embryonic portion of the egg cylinder.

Differential mitosis and degeneration patterns in relation to the alterations in the shape of the embryonic ectoderm of early post-implantation mouse embryos

The shape of the embryonic ectoderm of early post-implantation mouse embryos changes greatly in the period of 6.2-7.3 days post coitum. The subcellular morphology of the embryonic ectoderm remains unchanged, except in the primitive-streak region. Cell kinetics differ between ectodermal regions. These differences may be related to the changes in the shape of the ectoderm. The increase in cell number in the lateral ectoderm (the prospective surface ectoderm) exceeds that in the frontal ectoderm (the future neurectoderm). This is not due to differences in the duration of the cell cycle. It can be explained, however, by the occurence of different relative numbers of dividing and non-dividing cells. These numbers vary between the two regions. The percentage of non-dividing cells in the frontal ectoderm may reach 45, whereas in the lateral ectoderm this percentage is not higher than 15. Autoradiography in tritiated thymidine-treated embryos combined with the mitotic indices gave us all of the parameters necessary to present a model capable of clarifying the growth of the ectoderm during gastrulation, as well as the changes in the shape of the ectoderm.

The localization and synthesis of some collagen types in developing mouse embryos

The location of type IV (basement membrane) collagen in early post-implantation mouse embryos was examined by immunoperoxidase reactions using a specific immunoglobulin raised against mouse lens capsule collagen. Reaction was positive in the earliest embryos studied—on the fifth day of gestation (the day of detection of the copulation plug is the first day). It was found only in the primitive endoderm adjacent to the blastocoelic cavity. Subsequently in development, strong staining reactions were found in the parietal endoderm, Reichert's membrane and an acellular layer which separates the visceral endoderm of the egg cylinder from the ectoderm. In tenth to eighteenth day visceral yolk sacs, the mesodermal portion was stained, which is consistent with the presence of basement membranes around blood vessels. The endodermal portion of the visceral yolk sac did not react, while small amounts were found in the amnion. By incubation of various embryonic tissues with tritiated amino acids, purification of the biosynthesized secreted collagens and their partial characterization, the differential expression of several collagen genes was detected. Identification of collagen types was made by: reaction with specific antibodies to type I and IV collagens; electrophoretic mobility; sensitivity to reduction and to collagenase; analysis of the proportions of 3-hydroxyproline, 4-hydroxyproline and hydroxylysine; and CNBr peptides. In agreement with the data of Minor et al. 1976a for the rat, mouse parietal endoderm synthesizes large amounts of type IV collagen. In contrast to their findings, however, the 165,000 molecular weight polypeptide is not converted to one of 100,000 after reduction, alkylation and repepsinization ( Dehm and Kefalides, 1978). The endoderm of the visceral yolk sac was shown to be synthesizing primarily type I collagen, while the mesoderm layer of this membrane synthesized both type I and IV collagens. Little or no type IV collagen synthesis was detected in the endoderm of the visceral yolk sac. If it is correct that the visceral endoderm of the early embryo makes a major contribution to the formation of the endoderm portion of the visceral yolk sac, then it is clear that a switch in collagen gene expression must occur as it does so.

Influence of copper on the early post-implantation mouse embryo: An in vivo and in vitro study.

Female mice were injected intravenously with copper sulphate on either the 7th day (early egg cylinder stage of development), the 8th day (late egg cylinder stage), or the 9th day (early somite stage of development), and examined on the 10th day of gestation. Injection on the 7th day was found to be embryo-lethal; when females were injected on the 8th day, the majority of the surviving embryos exhibited anomalies of the neural tube and/or the heart, while injection on the 9th day resulted in a very low incidence of anomalies. The most common malformations seen on the 10th day involved failure of closure of the neural tube in the head region of the embryo, and various types of anomalies of cardiac rotation and shape. When additional females injected on the 8th day were examined on the 12th day, a high proportion of the fetuses examined had developed exencephaly.A further group of embryos from untreated females were explanted on the 9th day and cultured in vitro in various concentrations of copper sulphate. The lowest levels tested had little obvious effect on neural tube closure. Intermediate doses resulted in, retarded and anomalous embryonic development, while the highest levels employed resulted in neural tube and cardiac anomalies similar to those produced in vivo.The results demonstrate both the direct toxic effect of copper on embryonic development and that the stage of embryonic development at the time of exposure determines both the nature and the extent of the effect.

Modulation of alphafetoprotein synthesis in the early postimplantation mouse embryo

The visceral endoderm of mouse egg cylinders on the 7th and 8th days of gestation is divided into the visceral embryonic (VE) endoderm cell population which synthesizes alphafetoprotein (AFP), and the visceral extra-embryonic (VEX) endoderm population which does not synthesize AFP. Embryonic (E) and extra-embryonic (EX) ectoderm and visceral endoderm tissues were enzymically separated, reassociated in different combinations, and cultured in vitro for 48 h. The immunoperoxidase reaction on sections of cultured tissues showed that both VE and VEX endoderm cells synthesize high levels of AFP when cultured in isolation or in association with E ectoderm, but do not synthesize AFP when in close association with EX ectoderm. Both 7th and 8th day VEX endoderm cells synthesize detectable levels of AFP 12 h after isolation, and contain high levels by 24 h. It is concluded that both VE and VEX endoderm cells have the ability to synthesize AFP, but modulation of expression occurs through an inhibitory influence of the EX ectoderm.

Localization of trophoblast-defined surface antigens during early mouse embryogenesis

The binding pattern of a rabbit antiserum raised against mouse-ectoplacental-cone trophoblast on component cell populations in the pre-implantation and early post-implantation mouse embryo has been examined at the electron-microscope level using an immunoperoxidase-labelling technique. Binding was not detectable on the 1-cell stage, appeared at low levels at the 8-cell stage and was heavy on the trophectoderm and its trophoblast giant cell and extra-embryonic ectoderm descendants in the post-implantation embryo. In contrast, immunosurgically isolated 3 1/2-day inner cell masses (ICM) showed only slight labelling, whilst ICM derivatives in the 7 1/2-day embryo were unlabelled. The results indicate that the antiserum may be identifying a trophoblast-specific surface determinant(s), which appears with the differentiation of the trohectoderm and is maintained on some of the cell populations derived from this tissue at least until the early post-implantation stages.