Production of identical twins by separating two-cell rat embryos

Rat identical twins were produced from two-cell embryos. In the presence of cytochalasin B, rat two-cell embryos could be separated efficiently into two blastomeres by micromanipulation. Isolated blastomeres, embedded in agar cylinders and cultivated in ligated rat oviducts for 3 days, developed to the morula or blastocyst stage. After removing the agar, pairs of developed one-half embryos were transferred into Day 1 oviducts or Day 4 uteri of pseudopregnant rats. The percentage of embryos, separated either in the presence or absence of cytochalasin B, that developed into live fetuses was higher in cases of uterine transfer than in cases of oviduct transfer (38% vs. 18%, 31% vs. 15%, respectively). Throughout the present experiment, nine pairs of identical twins were successfully produced. This is the first report of the production of identical rat twins by separating two-cell embryos.     

Co-culture of two-cell rat embryos on cell monolayers

Summary Monolayers of 2 different populations of uterine cells and of fetal fibroblasts were evaluated for the support of rat embryo development in vitro. Compared to controls, cultures performed in Earle's buffered saline solution (EBSS) alone, the cleavage rate of 2-cell embryos to the 4-cell stage was significantly increased when the embryos were cocultured for 24 h with mixed uterine stromal and myometrial cells (70.7 vs. 56.0%;P<0.01). Coculture of 2-cell embryos with either uterine epithelial-stromal or stromal-myometrial cells in medium TC 199 (M199) for 24 h significantly increased the cleavage rate to the 4-cell stage compared to controls in the same medium (respectively, 78.3 and 77.6 vs. 49.9%;P<0.01). The development was not improved when fibroblasts were used as feeder cells. After 48 h, the proportion of 4-cell embryos showing cellular fragmentation was significantly decreased in the presence of either epithelial-stromal or stroma-myometrial cells in M199 compared to controls (respectively, 18.4 and 20.0 vs. 43.8%;P<0.01). Coculture in EBSS or with fibroblasts failed to prevent embryo degeneration. In one coculture with stromal-epithelial cells in M199, 6/11 embryos proceeded beyond the 4-cell stage, two of them reaching the 8-cell stage. No embryo developed beyond that stage in our study. Although considerable efforts remain necessary to achieve further growth, these results suggest that coculture offers promise as a means of supporting the in vitro development of rat embryos.     

Niridazole metabolism by rat embryos in vitro

We report the results of studies on the reductive activation of the schistosomicidal agent, niridazole (NDZ). Intact rat embryos in vitro reduced this compound, generating a stable metabolite in the presence of 5% O2. By contrast, embryo and yolk sac homogenates or liver microsomes appeared to require anaerobiasis. Malformation incidence--specifically, axial asymmetry--showed a strong correlation with nitroreductase activity rates when the latter were modulated by oxygen tension. Data presented here suggest that when embryos are exposed to NDZ under conditions of low oxygen in vitro, redox cycling ensues with molecular oxygen serving to oxidize early reduction products. This process continues, regenerating the parent compound until oxygen is depleted locally. The basis of this localized depletion is unknown, but inability of the immature supply system to replete oxygen or demand by precociously aerobic tissues may be involved. Once local anaerobiasis is attained, further reduction could generate toxic metabolites capable of covalently binding cellular macromolecules. Localized hypoxia represents another potential mechanism of dysmorphogenesis.     

Production of reconstructed two-cell rat embryos after chemical inactivation of chromosomes in MII oocytes by etoposide

The application of etoposide for chemical enucleation of rat oocytes was tested. The reconstruction efficiency after chemical and mechanical enucleation was comparatively analyzed. The obtained data indicate similar viability of reconstructed rat embryos irrespective of the enucleation technique.     

In vitro formation of tetraploid rat blastocysts after fusion of two-cell embryos

Gene targeting technology is not available in the rat which is an animal model of major importance, e.g., in cardiovascular research. This is due to the fact that the rat embryonic stem cell (ESC)-like cells established by several groups do not form germ-line chimeras when injected into blastocysts. In the mouse, the aggregation of ESC with tetraploid embryos has allowed the generation of animals completely derived from these cells. However, aggregation of rat ESC-like cells with tetraploid rat embryos has not yet been attempted to evaluate their developmental capacity. Therefore, we established a method to produce tetraploid rat embryos by fusion at the two-cell stage. Chemical fusion by polyethylene glycol (PEG) was shown to be less efficient (56.3% fused embryos) than electrofusion (96.1% fused embryos). The rate of development of fused embryos to blastocysts was independent of the fusion method and similar to the rate of control embryos. However, this rate was lower when the embryos had been cultured from the zygote state before fusion (14-20%) compared to freshly isolated two-cell embryos (41-63%). Alike for the mouse, blastocysts derived from fused two-cell rat embryos contained about half the number of cells as control blastocysts and were homogeneously tetraploid with no evidence of mosaicism. This method may be useful for the establishment of gene-targeting technology in the rat.     

Full-term development of rat after transfer of nuclei from two-cell stage embryos

Cloning technology would allow targeted genetic alterations in the rat, a species which is yet unaccessible for such studies due to the lack of germline-competent embryonic stem cells. The present study was performed to examine the developmental ability of reconstructed rat embryos after transfer of nuclei from early preimplantation stages. We observed that single blastomeres from two-cell embryos and zygotes reconstructed by pronuclei exchange can develop in vitro until morula/blastocyst stage. When karyoplasts from blastomeres were used for the reconstruction of embryos, highest in vitro cleavage rates were obtained with nuclei in an early phase of the cell cycle transferred into enucleated preactivated oocytes or zygotes. However, further in vitro development of reconstructed embryos produced from blastomere nuclei was arrested at early cleavage stages under all conditions tested in this study. In contrast, immediate transfer to foster mothers of reconstructed embryos with nuclei from two-cell embryos at an early stage of the cell cycle in preactivated enucleated oocytes resulted in live newborn rats, with a general efficiency of 0.4%-2.2%. The genetic origin of the cloned offspring was verified by using donor nuclei from embryos of Black Hooded Wistar rats and transgenic rats carrying an ubiquitously expressed green fluorescent protein transgene. Thus, we report for the first time the production of live cloned rats using nuclei from two-cell embryos.     

Biosynthesis of platelet-activating factor by two-cell mouse embryos.

Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media. The demonstration that known precursors ([1-14C]hexadecanol, [1-3H]hexadecanol, 1-O-[alkyl-1'2'-3H]lyso-PAF, 1-O-[alkyl-1'2'-3H]acetyl-glycerol and [methyl-3H]choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells. The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as [1-3H]hexadecanol was incorporated into PAF. Incorporation of label from alkylacetyl-glycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF. Incubation of embryos with each of three [14C]carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors. Incorporation of label from [2-14C]pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development. L-[U-14C]Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less. The incorporation of D-[U-14C]glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.     

DNA synthesis in developing two-cell mouse embryos

Mated CF1 (Carworth) female mice were sacrificed at 2 hr intervals between 29 and 43 hr after human chorionic gonadotrophin (HCG) administration. One- and two-cell eggs were incubated in [³H]thymidine for 1 hr. Labeled two-cell embryos were first observed at 31 hr and reached a maximum number at 35 hr. The S period is approximately 6 hr in duration. Although both blastomeres were labeled in most cases, embryos with only one labeled blastomere were more numerous at later times. In vitro labeling was corroborated by injecting [³H]thymidine directly into the isthmic portion of the oviduct. Embryos usually complete the second cleavage division 18–20 hr after onset of DNA synthesis. The cell cycle at the two-cell stage is thus characterized by a G₁ of close to 1 hr, a 6 hr S, and a G₂ of about 12 hr.Embryos developing in vitro frequently fail to progress beyond the two-cell stage. The block is not due to absence of DNA synthesis since these embryos were found to incorporate [³H]thymidine.     

Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis.

Culture of hamster embryos with 0.35 mM inorganic phosphate results in developmental arrest at the 2-cell stage. These arrested 2-cell embryos were found to have significantly elevated levels of both intracellular pH and intracellular free calcium. Culture of 2-cell embryos with both glucose and phosphate did not further alter intracellular ionic homeostasis. Developmental arrest of 2-cell embryos was dependent on the concentration of phosphate used. Culture with 1.25 microM phosphate did not alter development, while concentrations of 2.5 microM and 5.0 microM resulted in a percentage of embryos arresting development at the 2-cell stage. Analysis of intracellular levels of pH and calcium after culture with different phosphate concentrations revealed a significant negative correlation between intracellular calcium levels and development beyond the 2-cell stage. There was no correlation between the increase in intracellular pH and embryo development in the presence of phosphate. The increase in intracellular calcium levels after culture with phosphate appears to be derived from intracellular pools, as preventing the influx of extracellular calcium did not alter development beyond the 2-cell stage. Therefore, it is apparent that a disruption in ionic homeostasis is associated with developmental arrest of hamster embryos cultured with phosphate.     

RNA synthesis in starfish embryos: developmental consequences of its inhibition by formycin

Embryos of the starfish Asterina pectinifera were examined with regard to their ability to undergo the early events of embryonic development in the presence of formycin, an analogue of adenosine and a reported inhibitor of RNA synthesis. It was shown that in normal embryos the pool of ribonucleoside 5'-triphosphates increased during the period of blastula formation. The increase of the UTP pool was blocked nearly completely by 25 micrograms/ml formycin, and that of the CTP pool was inhibited partially by the same concentration of the drug. On the other hand, the pools of ATP and GTP were the same for both control and formycin-treated embryos. The development of embryos cultured in the presence of 25 micrograms/ml formycin stopped at the early blastula stage. Addition of 100 micrograms/ml each of uridine and cytidine to cultures of embryos that had been placed in 25 micrograms/ml formycin at the onset of blastulation allowed gastrulation to occur, suggesting that the developmental arrest produced by formycin is due primarily to the inhibition of pyrimidine nucleotide biosynthesis.     

A study on cryoprotectant solution suitable for vitrification of rat two-cell stage embryos

The present study was performed to develop a suitable cryoprotectant solution for cryopreservation of rat two-cell stage embryos. First, we examined the cell permeability of several cryoprotectants; propylene glycol had the fastest permeability compared to dimethyl sulfoxide, ethylene glycol, and glycerol. Embryos were then exposed to a solution containing propylene glycol to evaluate its effects on fetal development. As the development was similar to that of fresh embryos, P10 (10% v/v propylene glycol in PB1) was used as a pretreatment solution. Next, the effects of the vitrification solution components (sucrose, propylene glycol, ethylene glycol, and Percoll) were examined by observing the vitrification status; 10% v/v propylene glycol, 30% v/v ethylene glycol, 0.3 mol sucrose, and 20% v/v Percoll in PB1 (PEPeS) was the minimum essential concentration for effective vitrification without the formation of ice crystals or freeze fractures.     

Actin localization in nuclei of two-cell mouse embryos

In this study, preimplantation mouse embryos were used as a new model for the study of actin distribution in nuclei and the identification of functional forms of nuclear actin. The combination of the direct detection of actin by fluorescent-conjugated phalloidin and DNase I with indirect immunofluorecence was applied as an integrated approach to studying the localization of actin in nuclei of two-cell mouse embryos. Aggregates of monomeric actin and two oligomeric forms of actin were revealed in nuclei. Each of these forms demonstrated their own pattern of distribution. Oligomeric actin recognized by antibodies to the actin C-terminal domain was associated with condensed chromatin, as well as with metaphase chromosomes and chromatin of the second polar body. Monomeric actin and another oligomeric form recognized by antibodies to actin N-terminal domain were revealed in the area of dispersed chromatin localization.     

Transitional changes in arachidonic acid metabolism by bovine embryos at different developmental stages

In order to determine the profile of arachidonic acid (AA) metabolites synthesized by bovine embryos during early developmental stages, embryos collected from superovulated beef cattle (days 6 through 17) were incubated with AA and its metabolites were analyzed by high performance liquid chromatography and radioimmunoassay (RIA). Embryos harvested and cultured before day 12 of the estrous cycle metabolized AA primarily to prostaglandin E2 (PGE2), whereas, those harvested on day 13 of the cycle metabolized AA to both PGE2 and PGF2 alpha. Furthermore, embryos collected after day 15 of the cycle metabolized AA to PGI2 in addition to PGE2 and PGF2 alpha. In view of the luteotropic properties that have been attributed to PGE2 and the vasodilatory effect of PGI2, this transitional change in prostaglandin synthesis during early stages of embryonic development may be a part of the mechanism by which the embryo exerts a luteotropic effect leading to maternal recognition of pregnancy and by which the conceptus begins preparing for subsequent implantation.     

Reorganization of microfilaments and microtubules by thermal stress in two-cell bovine embryos

Two-cell bovine embryos become arrested in development when exposed to a physiologically relevant heat shock. One of the major ultrastructural modifications caused by heat shock is translocation of organelles toward the center of the blastomere. The objective of the present study was to determine if heat- shock-induced movement of organelles is a result of cytoskeletal rearrangement. Two-cell bovine embryos were cultured at 38.5 degrees C (homeothermic temperature of the cow), 41.0 degrees C (physiologically relevant heat shock), or 43.0 degrees C (severe heat shock) for 6 h in the presence of either vehicle, latrunculin B (a microfilament depolymerizer), rhizoxin (a microtubule depolymerizer), or paclitaxel (a microtubule stabilizer). Heat shock caused a rearrangement of actin-containing filaments as detected by staining with phalloidin. Moreover, latrunculin B reduced the heat-shock-induced movement of organelles at 41.0 degrees C but not at 43.0 degrees C. In contrast, movement of organelles caused by heat shock was inhibited by rhizoxin at both temperatures. Furthermore, rhizoxin, but not latrunculin B, reduced the swelling of mitochondria caused by heat shock. Paclitaxel, while causing major changes in ultrastructure, did not prevent the movement of organelles or mitochondrial swelling. It is concluded that heat shock disrupts microtubule and microfilaments in the two-cell bovine embryo and that these changes are responsible for movement of organelles away from the periphery. In addition, intact microtubules are a requirement for heat-shock-induced swelling of mitochondria. Differences in response to rhizoxin and paclitaxel are interpreted to mean that deformation of microtubules can occur through a mechanism independent of microtubule depolymerization.