Defective chorioallantoic fusion in mid-gestation lethality of parthenogenone<-->tetraploid chimeras.

Previous studies of parthenogenetic embryos revealed severe perturbations of both embryonic and extraembryonic tissue lineages during postimplantation development. The majority of pure parthenogenetic concepti have no recognizable axis and exhibit preferential terminal differentiation of their trophectoderm and primitive endoderm. To further define the role of the extraembryonic lineages in parthenogenetic development, we provided them with zygote-derived extraembryonic tissues by aggregating them with fertilized tetraploid embryos. On Day 12 of combined in vitro and in vivo development, most of the embryos proper in these chimeras were entirely derived parthenogenetically, whereas their trophectoderm and primitive endoderm tissues were derived from the tetraploid component. No Igf2 expression was detected in the parthenogenetic embryo proper, indicating that imprinting was manifested in such chimeras. Typical development of the parthenogenetic embryo proper was markedly improved in comparison with pure parthenogenetic concepti, with such chimeras attaining an average of 23 somites (range, 10 to 35). However, most of the chimeras died abruptly at Day 13, and all were being resorbed at Day 14 of development. The gross normality of axial structures and organ development suggests that a major cause of failure of these chimeric parthenogenones to survive beyond mid-gestation was due to defective chorioallantoic fusion. Our results indicate that the severe perturbation of axial development seen in most pure parthenogenetic concepti is a secondary consequence of the effects of parthenogenesis on the trophectoderm and primitive endoderm lineages. Moreover, the mid-gestation death of parthenogenetic embryos proper despite the presence of zygote-derived tetraploid tissues implicates extraembryonic mesoderm in manifesting the effects of genomic imprinting.

Beneficial effects of taurine on mouse zygotes developing in protein-free culture medium.

The objectives of this study were to determine if mouse zygotes from outbred mice can develop in simple culture medium in the absence of bovine serum albumin (BSA), and if taurine can be used as a medium supplement to improve development. Zygotes from 2 stocks of outbred mice (CD-1 and CF-1) were cultured in simple embryo culture medium (TE medium) lacking BSA and with or without taurine (24 mM), or in regular TE medium with BSA. The presence of BSA had little or no effect on development, but development to post-blastocyst endpoints was enhanced when CD-1 zygotes were cultured in medium containing taurine. In addition, when CD-1 blastocysts were transferred to pseudopregnant animals, embryos cultured in the presence of taurine developed into fetuses more often than those cultured in medium without taurine, and their weights were higher than those of embryos cultured in regular TE medium with BSA. These beneficial effects of taurine do not appear to be the nonspecific effects of a fixed nitrogen source, because the addition of glycine to BSA-free TE medium did not have similar beneficial effects. It was concluded that mouse zygotes from outbred mice do not require BSA for their preimplantation development in culture and that the presence of taurine in preimplantation culture medium is beneficial not only for preimplantation development of the zygotes, but also for their post-blastocyst development.

In vitro development of one-cell embryos from outbred mice: influence of culture medium composition.

One-cell embryos from outbred mice (CF1, CD-1, and Dub:ICR) were cultured in various modifications of egg culture medium (ECM). The best development was observed in medium in which inorganic salts of modified T6 medium (mT6) replaced those of ECM. In this modification (TE), 66% of one-cell CF1 embryos developed into blastocysts, compared to 46 and 43% for ECM and mT6, respectively. Moreover, the cell numbers of blastocysts developing in TE (7.49 +/- 3.3) were higher than the cell numbers of those developing in ECM (55.1 +/- 2.4). The culture requirements of embryos varied between different stocks of mice: Fewer CF1 embryos developed to the blastocyst stage than either Dub:ICR embryos (90%) or CD-1 embryos (84%). Lowering the osmolarity of the medium from 300 to 280 mOSM, increasing the concentration of KC1 from 1.42 to 25 mM, or omitting lactate from the medium during Day 1 of culture did not further improve development of embryos, in contrast to previous reports. However, the time at which embryos were transferred to outgrowth medium influenced their postblastocyst development. The best development was observed when embryos were transferred on Day 4 of culture at the late morula-early blastocyst stage.

Stage-specific response of preimplantation mouse embryos to W-7, a calmodulin antagonist.

Involvement of calmodulin-dependent processes in preimplantation development of mouse embryos was studied with the use of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific antagonist of calmodulin. At 25 microM, W-7 interfered with compaction of eight-cell embryos, caused decompaction of compacted eight-cell embryos, inhibited cavitation of late morulae, and caused collapse and degeneration of blastocysts. These effects of W-7 appear to be due to specific inhibition of calmodulin-dependent processes, because W-5, a less active analogue of W-7, was less effective in interfering with development; at 25 microM, W-5 had only a slight effect on compaction and had no effect on blastocyst formation, maintenance of blastocoels, or post-blastocyst development. In addition to the developmental effects just described, W-7 inhibited cell proliferation in four-cell embryos and reduced cell numbers of morulae after treatment at the two- to eight-cell stages. There was a marked increase in embryos' sensitivity to W-7 at the late morula stage, and the sensitivity increased further as embryos developed into blastocysts; the effects of W-7 were largely reversible after treatment at the two-cell through the compacted eight-cell stages, but not after treatment at the late morula or blastocyst stage. At the blastocyst stage, inner cell mass cells appeared to be slightly more resistant to W-7 than trophectoderm cells. This differential sensitivity became more pronounced at the late blastocyst stage: after 3.5-4-h exposure of late blastocysts to 25 microM W-7, all trophectoderm cells degenerated but most of the inner cell masses survived. From these results it appears that calmodulin-dependent processes are involved in development of mouse embryos at all of the preimplantation stages examined.

Midgestational exposure of pregnant BALB/c mice to magnetic resonance imaging conditions.

The potential for producing reproductive toxicity or teratogenesis in mice by exposure to magnetic resonance imaging (MRI) conditions was evaluated by means of reproduction studies and the homeotic shift test. Embryos from pregnant BALB/c mice were exposed in vivo for 16 hours beginning on gestation day 8.75 to MRI conditions of modest field strength (static field, 0.35 tesla (T); pulsed gradients, 2.3 X 10(-4) T/cm for 2.5 to 10 msec; and radio frequency, 15 MHz at an average of 61.2 mW). Unexposed, sham-exposed (both MRI and X-ray) and X-irradiated (0.5 Gy) animals were the control groups. Neither placental resorptions nor stillbirths were increased by MRI. Fetal weight at birth and crown-rump length were proportional; however, crown-rump length was significantly less (p less than 0.001) in the MRI-exposed fetuses (respective mean values for MRI-exposed fetuses were 21.8 +/- 0.2 mm compared to 22.4 +/- 0.1 for sham-exposed fetuses). Both crown-rump length and fetal weight were significantly reduced after X-irradiation. The percentage of homeotic skeletal shifts was scored for each of eight anatomic sites. Only X-radiation produced significant increases in skeletal shifts. Prolonged midgestational exposure of mice to MRI conditions currently used for human clinical imaging, therefore, failed to reveal overt embryotoxicity (resorptions, stillbirths) or teratogenicity (homeotic shifts), consistent with the non-ionizing properties of MR. However, the slight but significant reduction in fetal crown-rump length after prolonged exposure justifies further study of higher MRI energy levels and consideration of other endpoints for establishing with greater confidence the safety of MRI during pregnancy.

Enhancement of methylmercury toxicity by L-cystine in cultured mouse blastocysts.

Expanded mouse blastocysts incubated with 1 to 2 microM methylmercury (MeHg) in modified Eagle's basal medium (BME + AA), which contains amino acids, collapsed and degenerated within 24 h. In contrast, blastocysts incubated with the same concentration of MeHg in egg culture medium (ECM), which does not contain amino acids, survived and remained expanded as control embryos did. By systematically omitting each BME amino acid from BME + AA and adding each BME amino acid to egg culture medium, we determined that L-cystine (0.5 mM in BME + AA) was the component of BME + AA that was responsible for the enhancement of the toxicity of MeHg. The shortest incubation time during which the cystine-enhanced MeHg toxicity became irreversible was 2 h, and the addition of any of the neutral BME amino acids (except threonine) or non-BME neutral amino acids (alanine, glycine, or serine) during the 2 h incubation eliminated or reduced the cystine-enhanced MeHg toxicity. Basic amino acids (except histidine) were less effective in protecting embryos: Glutamine and lysine reduced the toxic effect only slightly, and arginine had no effect. DL-buthionine sulfoximine (7.5 mM), a specific inhibitor of glutathione, also reduced cystine-enhanced MeHg toxicity. It therefore appears that cystine enhances MeHg toxicity indirectly, at least in part, by stimulating the synthesis of cellular glutathione, which may in turn enhance MeHg transport. In the absence of cystine, 10 microM MeHg (2 h incubation) was necessary to cause the collapse and degeneration of all blastocysts treated.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of sister chromatid exchange in preimplantation mouse embryos in vitro by 3H-thymidine or ultraviolet light in combination with caffeine.

Preimplantation mouse embryos were exposed in vitro to 3H-thymidine (25, 100, or 250 Bq/ml) or ultraviolet (UV) light (1.35 or 4.05 J/m2), either alone or in combination with caffeine (1 mM with 3H-thymidine and 0.5 mM with UV light). Exposure to 3H-thymidine lasted for 2 days, from the two-cell stage to the late morula/early blastocyst stage, and UV radiation was applied acutely at the late morula/early blastocyst stage. The effects were quantified by the sister chromatid exchange (SCE) assay. All three agents induced SCEs when used singly. 3H-thymidine was effective in inducing SCEs only at 250 Bq/ml, whereas UV light was effective at both fluences. Although caffeine did not induce SCEs when it was added before exposure to bromodeoxyuridine (BrdUrd), which is used to visualize SCEs, it did induce SCEs when present during the entire culture period (3H-thymidine experiments) or during incubation in BrdUrd (UV experiments). Caffeine markedly enhanced the SCE-inducing effect of UV light but did not influence the effect of 3H-thymidine.

Developmental and cytogenetic effects of caffeine on mouse blastocysts, alone or in combination with benzo(a)pyrene.

Mouse blastocysts were treated with caffeine and/or benzo(a)pyrene (BP), and the effects on development and on induction of sister chromatid exchanges (SCEs) were examined. Caffeine interfered with blastocyst development in a dose-related manner. At 4 mM, the highest concentration tested, caffeine interfered with development of blastocysts to all four endpoints: hatching, trophoblast outgrowth, inner cell mass (ICM) growth, and two-layer (primary endoderm and ectoderm) differentiation of ICMs. At 2 mM, caffeine reduced the incidence of both ICM growth and differentiation but did not affect hatching or formation of trophoblast outgrowths. At 1 mM, caffeine interfered only with ICM differentiation. Cell proliferation was least sensitive to caffeine and was reduced at concentrations of greater than or equal to 2 mM. Induction of SCEs was most sensitive to caffeine exposure; an increase in SCE frequency was observed at 0.1 and 0.5 mM. When caffeine was added to cultures with BP (1 microM, a concentration that was not embryotoxic and did not induce SCEs), both embryotoxic effects and SCE frequency were increased. The enhancing effect on SCE induction was particularly marked; as little as 0.1 mM caffeine was sufficient to cause doubling of induced SCE frequencies when added to cultures with BP.

Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin.

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.

Cytochrome P-450 metabolic activity in embryonic and extraembryonic tissue lineages of mouse embryos.

Mouse morulae, blastocysts, and embryonic and extraembryonic tissue layers were examined for benzo[a]-pyrene metabolism by cytochrome P-450, using the sister chromatid exchange assay. Benzo[a]pyrene exposure in vitro increased sister chromatid exchanges in blastocysts of all genetically responsive mice examined [BALB/cDub, C3H/AnfCum, and outbred Dub:(ICR) strains] but not blastocysts of the nonresponsive AKR/J strain. Benzo[a]pyrene treatment of responsive 7 1/2- and 8 1/2-day (postimplantation-stage) embryos, either intact or as separate tissue layers, increased sister chromatid exchanges in tissues of both embryonic and extraembryonic lineages--i.e., in the embryo proper, in isolated embryonic ectoderm, and in yolk sac, chorion, extraembryonic ectoderm, and extraembryonic endoderm layers. These results indicate that cytochrome P-450 is active in most or all tissues of the early mammalian embryo. It could metabolize xenobiotic molecules reaching the conceptus near the onset of morphogenesis and organogenesis, or it could have another as yet undefined role in normal development.

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation.

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17beta-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or epsilon-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0

Chlamydia trachomatis infection of mouse trophoblasts.

Chlamydia trachomatis (strains L2 and DE) were found to infect fertilized mouse ova in vitro. Inclusions were found in the trophoblast, but not in the embryo.

Plasminogen activator activity in mouse embryos cultured on decidual cell monolayers.

The nature of proteolytic activity in the mouse blastocyst was investigated in vitro using cell monolayers as a substrate. Blastocysts obtained from mice on day 4 of gestation were placed directly on various types of cell monolayers. The embryos had hatched at 34 hrs, attached at 45 hrs, and at 56 hrs of culture they formed trophoblast outgrowths by displacing the underlying monolayer cells. Because blastocysts developed equally well on all types of cell monolayers tested, a decidual cell monolayer was used in all the following experiments. The plasminogen activator activity in blastocysts was examined using the fibrin-agar overlay assay. There was a strong relationship between the levels of activity and the extent of trophoblast outgrowth. At the hatching stage, 20% of the embryos showed fibrinolysis, whereas 86% showed clear fibrinolysis at the trophoblast outgrowth stage. After the loss of invasiveness, only 32% of them showed fibrinolysis. Lysed zones surrounding trophoblast always extended beyond the area which displaced the previously occupied cell monolayer. We never observed a clear zone (halo) between the trophoblast and monolayer cells nor cytolytic activity inside the fibrinolytic zone until the embryos had lost their invasive nature.

Inhibition of mouse blastocyst attachment and outgrowth by protease inhibitors.

Effects of protease inhibitors on development of mouse blastocysts and fibrinolytic activity of trophoblast were examined by growing embryos on monolayers of decidual cells in the presence of inhibitors. Nitrophenol-p-guanidino benzoate (NPGB) was the most effective inhibitor; 10(-4) M NPGB inhibited attachment and trophoblast outgrowth by 24% and 66%, respectively, and inhibited the fibrinolytic activity of trophoblast by 86%. The effects of NPGB were reversible, as demonstrated by the embryos' ability to attach and resume normal development when transferred to NPGB-free medium. Soybean trypsin inhibitor and epsilon-aminocaproic acid were less effective than NPGB in inhibiting blastocyst development. When 10(-4) M NPGB and 350 microgram/ml of soybean trypsin inhibitor were added together, blastocyst development and fibrinolytic activity were inhibited more severely than when either inhibitor was added alone. We suggest that at least two types of proteolytic activities in mouse blastocysts are involved in implantation, attachment requiring trypsin-like activity, and trophoblast outgrowth requiring both plasminogen activator and trypsin-like activity.

An improved culture medium for mouse blastocysts.

Eagle's basal medium, modified to contain essential amino acids at the concentrations optimal for mouse blastocyst hatching, attachment, and outgrowth, supported in vitro development of the mouse blastocyst better than other tissue culture media tested. This medium was improved for growth and differentiation of the inner cell mass by doubling the concentration of amino acids and glucose and by adding uridine (10(-5) M) and beta-mercaptoethanol (10(-5) M). In this improved medium nearly all blastocysts grown from the two-cell stage hatched and formed trophoblast outgrowths, and 62% developed into two-layer egg cylinders.