Polyethylene glycol-induced fusion of heat-inactivated and living protoplasts of Bacillus megaterium.

Protoplasts of Bacillus megaterium, incubated at 50 degrees C for 120 min, lost the ability to revert to bacillary form. Such heat-inactivated protoplasts, however, produced recombinants when fused by polyethylene glycol treatment with normal protoplasts. Although this differential inactivation effect is not yet fully reproducible, reciprocal inactivations of the parental protoplasts in genetic crosses have clearly shown that for protoplast fusion (i) either of the parents may serve as the viable recipient for markers coming from the heated parental protoplasts, and (ii) either of the parents may be rendered nonviable and yet, when fused with a viable partner, contribute to formation of a recombinant. Heat inactivation seems to provide a way to counterselect when few markers are available and one of the parents is prototrophic.     

Autocrine activation of ion currents in the two-cell mouse embryo

The actions of autocrine ligands are required for the normal development of the preimplantation embryo in vitro. These ligands act as survival factors for the preimplantation stage embryo. One autocrine ligand, paf (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine), induced a dihydropyridine-sensitive calcium transient in the zygote and two-cell embryo, and these transients were required for the normal preimplantation stage survival. Paf induces an influx of external calcium through a dihydropyridine-sensitive channel. Dihydropyridine-sensitive currents are voltage-regulated, yet to date there is no evidence of membrane voltage depolarization in the two-cell embryo. To define the paf-induced calcium influx we have examined the response of the membrane potential and ion currents to paf in two-cell embryos. An initial response to paf challenge was the expression of an ion current (-15.6+/-1.6 pA) that was dependent upon extracellular calcium, was not voltage-gated but was dihydropyridine (nifedipine)-sensitive. This calcium current was followed (91+/-6 s after paf) by a net outward current (284+/-59 pA) that was composed of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate-sensitive (anion channel blocker) and tetraethylammonium chloride-sensitive (K(+) channel blocker) currents. This current corresponded temporally with a marked paf-induced transient hyperpolarization of the membrane potential (-8.4+/-1.2 mV) that was dependent upon the generation of the calcium transient. The results directly demonstrate the activation of a voltage-independent calcium current in response to paf and show for the first time the expression of an afterhyperpolarization that occurs as a response to the calcium transient.     

F-actin is involved in the polyethylene glycol-induced fusion of chick embryo fibroblasts

Cytochalasin B inhibits the polyethylene glycol (PEG)-induced fusion of chick embryo fibroblasts. Induction of fusion of these cells by PEG is associated with transient changes in the pattern of F-actin organization within the cell. The changes include the disappearance of stress fibres and accumulation of F-actin under the plasma membrane. These results suggest an involvement of F-actin in PEG-induced cell fusion.     

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.     

Three dimensional reconstruction of two-cell mouse embryo by laser scanning microscopy

The direct method of volumetric parameters’ measurement of early mammal embryo was developed. The sample preparation was based on embryo cryofixation followed by laser scanning microscopy. Digital image processing and three dimensional reconstruction of embryos was performed with standard graphic software. The availability of a developed technique for the analysis of cell physiology at cultivation conditions was demonstrated on isolated two-cell mouse embryo.     

Atypical GATA protein TRPS1 plays indispensable roles in mouse two-cell embryo

Zygotic genome activation (ZGA) is one of the most critical events at the beginning of mammalian preimplantation embryo development (PED). The mechanisms underlying mouse ZGA remain unclear although it has been widely studied. In the present study, we identified that tricho-rhino-phalangeal syndrome 1 (TRPS1), an atypical GATA family member, is an important factor for ZGA in mouse PED. We found that the Trps1 mRNA level peaked at the one-cell stage while TRPS1 protein did so at the two/four-cell stage. Knockdown of Trps1 by the microinjection of Trps1 siRNA reduced the developmental rate of mouse preimplantation embryos by approximately 30%, and increased the expression of ZGA marker genes MuERV-L and Zscan4d via suppressing the expression of major histone markers H3K4me3 and H3K27me3. Furthermore, Trps1 knockdown decreased the expression of Sox2 but increased Oct4 expression. We conclude that TRPS1 may be indispensable for zygotic genome activation during mouse PED.     

Electrofusion of mouse blastomeres

Fusion of blastomeres of 2-cell mouse embryos with an intact zona pellucida can be induced with electric pulses. Fusion was most frequent with the field strength of 1 kV/cm and direct current pulses of 100-250 microsec duration. An electrolyte solution (PBS) can be used instead of a non-electrolyte solution (0.3 M mannitol). The viability of blastomeres fused in these two types of solution is similar. Fused 2-cell blastomeres develop into tetraploid blastocysts but die after implantation. Embryos in which blastomeres failed to fuse despite the treatment (diploid controls) can develop till term. The technique can also be applied to 3- and 4-cell embryos and to zona-free oocytes and blastomeres.     

Apparent absence of Na+/H+ antiport activity in the two-cell mouse embryo

We have used the pH-sensitive dye BCECF to investigate the regulation of intracellular pH (pHi) by two-cell stage mouse embryos in bicarbonate-free medium. There is no indication of a Na+/H+ antiport active in regulating pHi, as recovery from acid-loading was insensitive to amiloride, ethylisopropylamiloride, or the absence of extracellular Na+. Instead, protons appear to be in equilibrium across the plasma membrane, as indicated by the response of pHi to changes in external K+. The embryos have an intracellular buffering power in the normal range (25.3 mM/pH); their apparent permeability to protons is, however, very high (0.22 cm/sec).     

Mitotic spindles and cleavage planes are oriented randomly in the two-cell mouse embryo

Most experimental embryological studies performed on the early mouse embryo have led to the conclusion that there are no mosaically distributed developmental determinants in the zygote and early embryo (for example see [1-6]). It has been suggested recently that "the cleavage pattern of the early mouse embryo is not random and that the three-dimensional body plan is pre-patterned in the egg" (in [7] for review see [8-10]). Two major spatial cues influencing the pattern of cleavage divisions have been proposed: the site of the second meiotic division [11, 12] and the sperm entry point [13-14], although the latter is controversial [15-17]. An implication of this hypothesis is that the orientations of the first few cleavage divisions are stereotyped. Such a define cleavage pattern, leading to the segregation of developmental determinants, is observed in many species [18]. Recently, it was shown that the first cleavage plane is not predetermined but defined by the topology of the two apposing pronuclei [19]. Because the position of the female pronucleus is dependent upon the site of polar body extrusion and the position of the male pronuclei is dependent upon the sperm entry point [19-20], this observation leaves open the possibility that the sperm may provide some kind of directionality [7]. But, even if asymmetries were set up only after fertilization, a stereotyped cleavage pattern should take place during the following cleavage divisions. Thus, we studied the cleavage pattern of two-cell embryos by videomicroscopy to distinguish between the two hypotheses. After the mitotic spindle formed, its orientation did not change until cleavage. During late metaphase and anaphase, the spindle poles appear to be anchored to the cortex through astral microtubules and PARD6a. Only at the time of cleavage, during late anaphase, do the forming daughter cells change their relative positions. These studies show that cleavage planes are oriented randomly in two-cell embryos. This argues against a prepatterning of the mouse embryo before compaction.     

Effect of six-and ten-day-old chick embryo amniotic fluid on development of two-cell mouse embryos.

The purpose of this study was to investigate the effect of six-and ten-day-old chick embryo amniotic fluid (CEAF) on the development of two-cell mouse embryos. Six- and ten-day-old CEAF (6-AF, 10-AF) were aspirated separately from the amniotic cavity and two experiments were performed. In the first experiment, two-cell mouse embryos were cultured in different supplements of heat-inactivated 6-AF, pure heat-inactivated 6-AF and pure active 6-AF. The second experiment was also carried out in the same manner using the 10-AF. The rate of the development of embryos in all groups were daily determined and statistically compared with that of control (Ham's F-10 supplemented with 5 mg/ml human serum albumin). During the cultivation period, more embryos reached to the blastocyst stage in all groups of CEAF compared to the control. The result hatched blastocysts embryos also increased from days 3 till 5 of cultivation in all groups of CEAF. The developmental potential of embryos appears to be almost identical in six- and ten-day-old CEAF. In conclusion, CEAF as a supplement or a natural medium could support the development of two-cell mouse embryos.     

Delay of polarization event increases the number of Cdx2-positive blastomeres in mouse embryo

During preimplantation mouse embryo development expression of Cdx2 is induced in outer cells, which are the trophectoderm (TE) precursors. The mechanism of Cdx2 upregulation in these cells remains unclear. However, it has been suggested that the cell position and polarization may play a crucial role in this process. In order to elucidate the role of these two parameters in the formation of TE we analyzed the expression pattern of Cdx2 in the embryos in which either the position of cells and the time of polarization or only the position of cells was experimentally disrupted. Such embryos developed from the blastomeres that were isolated from 8-cell embryos either before or after the compaction, i.e. before or after the cell polarization took place. We found that in the embryos developed from polar blastomeres originated from the 8-cell compacted embryo, the experimentally imposed outer position was not sufficient to induce the Cdx2 in these blastomeres which in the intact embryo would form the inner cells. However, when the polarization at the 8-cell stage was disrupted, the embryos developed from such an unpolarized blastomeres showed the increased number of cells expressing Cdx2. We found that in such experimentally obtained embryos the polarization was delayed until the 16-cell stage. These results suggest that the main factor responsible for upregulation of Cdx2 expression in outer blastomeres, i.e. TE precursors, is their polarity.     

How many blastomeres of the 4-cell embryo contribute cells to the mouse body?

The aim of this study was to estimate how many blastomeres of the 4-cell mouse embryo contribute cells to the embryo proper and finally to the animal. To this end, 4-cell embryos of pigmented and albino genotypes were disaggregated and single blastomeres (henceforth called '1/4' or 'quarter' blastomeres) were reaggregated in the following combinations: one 'pigmented' blastomere + three 'albino' blastomeres or vice versa (henceforth called '1+3') and two pigmented blastomeres + two albino blastomeres (henceforth called '2+2'). The aggregations were cultured in vitro and transferred as blastocysts either to the oviduct or uterus of pseudopregnant females. Recipients were allowed to litter naturally, or the foetuses were removed by Caesarian section and raised by lactating foster mothers. Chimaerism was assessed on the basis of coat (adults) or eye pigmentation (dead neonates). Among 28 '1+3' animals, there were 13 chimaeric and 15 non-chimaeric individuals. The pigmentation of non-chimaeras was always concordant with the genotype of the three 1/4 blastomeres and not with the genotype of the single blastomere in the given aggregation. These results make rather unlikely the possibility that the mouse is built of cells derived either from one or all four 1/4 blastomeres. Both two remaining options (2 or 3 1/4 blastomeres) are conceivable but the observed ratio of chimaeras to non-chimaeras among '1+3' animals (13:15) fits better the assumption of two 1/4 blastomeres contributing cells to the animal body. This assumption finds additional support in the observation that among '2+2' animals there were non-chimaeras (5 out of 7) and these would not have been expected should three 1/4 blastomeres contribute cells to the mouse body.     

Polarization of blastomeres in the cleaving rabbit embryo

Cellular polarization is believed to be a crucial event in the differentiative divergence of the two cell lineages leading to the blastocyst in rodent embryos. This study was undertaken to determine if rabbit embryos exhibited cellular polarization prior to blastocyst formation and to define the embryonic stage at which polarization was first apparent. Polarity was assayed by observation of the pattern of binding of FITC-Con A to dissociated blastomeres from three stages of rabbit embryos. Scanning electron microscopy on the dissociated cells confirmed the fluorescence results. Fifty-one percent of blastomeres in 38- to 66-cell rabbit embryos exhibited an intense pole of FITC-Con A binding and a single pole of microvilli. Only 2% of blastomeres at the 17- to 34-cell stage were similarly polarized and none were polarized at the 8- to 16-cell stage. In addition, during attempts to remove the mucin coat and zona pellucida from the rabbit embryos prior to their dissociation, it was found that the protease sensitivity of these coats also changed at the 38- to 66-cell stage. Prior to this time, although the mucin coat disappeared after 30 min in 0.5% pronase, the zona required approximately 1.5-2.5 hr in pronase for even partial removal. At the 38- to 66-cell stage, pronase dissolved the mucin coat within 10 min and the zona pellucida within 20 min. The zona was resistant to 0.1% proteinase K at all stages examined.     

Effects of various electric fields on the fusion and in vitro development of mouse two-cell embryos

This study was undertaken to examine the effects of various electric fields such as alternating current (a.c.) voltage, fusion pulse strength, pulse duration, pulse number and electrode geometry on blastomere fusion and developmental rates of mouse two-cell embryos. The a.c. voltages (6 and 12 V/mm) did not affect the fusion and developmental rates. High fusion and developmental rates were obtained when pulse strengths of 1.0 to 2.5 kV/cm, pulse durations of 30 to 90 mu sec and pulse numbers of 1 to 6 were applied using a wire chamber. Comparison of electrode geometries showed that fusion rates were similarly high (93 to 98%) when pulse strengths of 1.0 to 2.5 kV/cm were applied, regardless of the electrode geometry. However, significantly lower developmental rates were observed in a rectangular chamber compared with those in a wire chamber, except when the pulse strength was 1.0 kV/cm. It was further observed that in a rectangular chamber, the developmental rate decreased with increasing pulse strength from 1.0 to 2.0 and 2.5 kV/cm. The results of this study indicate that by using a wire chamber, electric fields can be successfully applied across a relatively wide range of pulse strength, duration and number to provide sufficiently high fusion and subsequent developmental rates. The fusion conditions did, however, vary with chambers of different electrode geometries.