The cytochemistry of glycoconjugates in the zona pellucida of murine ovarian oocytes and two-cell embryos

Summary Changes in glycoconjugates of the zona pellucida induced by maturation, ovulation and fertilization of mouse oocytes have been studied by means of light microscopic methods of cytochemistry. These methods consisted of periodic acid-Schiff (PAS), Alcian Blue pH 1.0 and pH 2.5, and peroxidase-labelled lectin diaminobenzidine (PO—LT—DAB) procedures in combination with the digestion technique with neuraminidase. According to the results obtained, glycoconjugates of the zona pellucida of fertilized eggs contained a smaller amount of sulphate groupings than that in ovarian oocytes, whereas their reactions for sialic acid and fucose residues were significantly stronger in intensity in the former, as compared with those in the latter. The cytophysiological significance of such cytochemical changes in glycoconjugates of the zona pellucida has been discussed with special reference to its functional alterations following maturation, ovulation and fertilization.     

Cytochalasin B induces changes in cytoplasmic Na+/K+ balance of two-cell mouse embryos

Changes in intracellular elemental (Na, K) concentrations caused by cytochalasin B were measured by electron probe microanalysis. Cytochalasin B is applied to transfer somatic cell nuclei into early embryo cells. This chemical causes a cytoskeleton rearrangement that may activate potassium channels, which, in turn, results in a cytoplasmic Na⁺/K⁺ imbalance. Our study showed that cytochalasin B reduced the intracellular sodium concentration. After the exposure of the mouse embryo with Dulbecco’s solution free from chemical, the Na⁺/K⁺ balance in cytoplasm reached the initial level. Possible mechanisms of registered changes in intracellular Na⁺ concentration are discussed.     

Heterogenous nuclear and cytoplasmic RNA in Li-treated sea urchin embryos

Ara-CTP differentially inhibits two types of DNA synthetic activity occurring in isolated hepatocyte nuclei in vitro. Ara-CTP inhibits type A synthesis (replication) with a K₁ of 5 × 10^?7 M, whereas type C synthesis (presumed repair) is much less sensitive, the K₁ being 5 × 10^?4 M. Significant inhibition of type C synthesis does not occur until type A synthesis is suppressed by more than 50%.     

Factors affecting the cryosurvival of mouse two-cell embryos

A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycoproteins of murine zona pellucida from unfertilized eggs and two-cell embryos: comparison of the reactivity to lectins

Glycoproteins of zona pellucida were fractionated by SDS-polyacrylamide gel electrophoresis, and their lectin binding was examined after blotting onto nitrocellulose. The specificity of lectin binding to the major zona glycoproteins was the same for zonae isolated from eggs or from two-cell embryos; RCA, WGA, SBA and DBA reacted with all of the glycoproteins, PNA with ZP-1 and ZP-3, and Con A with ZP-2. However, the reactivity of ZP-3 to SBA and DBA was decreased in zonae from two-cell embryos. In addition, two-cell embryo zona contained a band which had different binding specificity to lectins from the major glycoproteins.     

Viability of nuclei of two-cell mouse embryos stored at 4 degrees C and fused with blastomeres of fresh two-cell embryos.

This study compares the resistance of the nuclei and the cytoplasm of two-cell mouse embryos to short-term storage at low temperature above 0 degrees C. Two-cell embryos were stored at 4 degrees C for 24-96 h in PB1 containing 0.25, 0.5, 0.75, and 1.0 M sucrose. The development to blastocysts in culture was highest in the presence of 0.5 M sucrose. However, only 3% of the embryos developed into blastocysts after 96 h of storage. On the other hand, the viability of the nuclei of two-cell embryos stored at 4 degrees C was significantly prolonged when they were transplanted into a blastomere of enucleated fresh F1 (C57BL/6JXCBA) two-cell embryos. The proportions of chimeric embryos that developed to blastocysts were 88, 67, 76, 71, 64, 45, 32, and 20% following storage for 0, 48, 72, 96, 120, 144, 168, and 192 h, respectively. In addition, there was no difference in the coat color of the young derived from nuclei stored at 4 degrees C or fresh nuclei, although the proportions of chimeric embryos that developed into live young after transfer tended to decrease with increased storage time. Moreover, the viability of nuclei stored at 4 degrees C for 192 h was confirmed in the germ cell population of chimeric mice mated with albino mice. These results demonstrated that the nuclei in the two-cell mouse embryos were more resistant to storage at low temperature than the cytoplasm.     

On the size relationship between nuclear and cytoplasmic RNA in sea urchin embryos

The approximate sizes of heterogeneous nuclear (HnRNA) and cytoplasmic RNA of sea urchin embryos were determined by DMSO density gradient centrifugation and acrylamide-formamide gel electrophoresis. The data suggest that the sizes of these molecules are smaller than those estimated under nondenaturing conditions. The size of most of the nuclear RNA ranges from 0.5 to 3 × 10⁶ daltons, while that of the cytoplasmic RNA ranges from 0.1 to 2 × 10⁶ daltons. Both nuclear and cytoplasmic RNA of sea urchin embryos may have a minor fraction (5–10%) of very large species with molecular weights up to 4 to 5 × 10⁶ daltons.The idea that the size of HnRNA may be larger in organisms higher on the evolutionary scale is discussed.     

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.     

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.     

Fourier analysis of nuclear and cytoplasmic shape of blood lymphoid cells from healthy donors and chronic lymphocytic leukemia patients

The folding rates of the contours of nuclei and entire lymphoid cells were analyzed by Fourier analysis of the shapes. Smears of peripheral blood from healthy subjects and from patients with chronic lymphocytic leukemia (CLL: type B, stage zero) were routinely prepared and stained. The shapes of lymphoid cells from CLL patients revealed a higher folding rate (from fifth to tenth harmonics) than did those of lymphocytes from healthy subjects. Accordingly, the roughness coefficient (describing the folding rate of the surface) for CLL cells was 0.036, as compared to 0.028 for the cells of healthy subjects. The shapes of nuclei of CLL lymphoid cells also had a higher folding rate than did those of lymphocytes from healthy subjects, but a significant difference was found only for the highest harmonic calculated (the tenth harmonic); the respective roughness coefficients for nuclei were 0.037 and 0.033.     

Extremely rapid and reversible optogenetic perturbation of nuclear proteins in living embryos

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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.     

Polarized distribution of membrane components on two-cell mouse embryos

Summary In two-cell mouse embryos, membrane components detected by a variety of antisera, lectins and lipid analogues and covalent labeling were found to be localized in the poles of the two blastomeres opposite the cleavage furrow. The proportion of polarized blastomeres increased rapidly during the first 4–5 h following first cleavage and then diminished approximately two-fold over the remaining period before second cleavage. Concurrent with this decrease in percent polarization, observed poles were found to be less spatially restricted. This polarization is not the result of a ligand induced capping or a manifestation of differences in surface topography. In light of recent measurements of lateral diffusion, the polarization of membrane components may be significant for the formation of morphogenetic gradients during cleavage.     

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.