The Relative Contributions of Newly Synthesized and Stored Messages to Hl Histone Synthesis in Interspecies Hybrid Echinoid Embryos

We have measured the ratio of incoropation of ³H-lysine into the maternal and paternal forms of Hl histones synthesized by the interordinal hybrid embryo which results from the fertilization of sand dollar eggs with sea urchin sperm. This ratio has been used to calculate the relative contributions of newly transcribed and stored Hl histone mRNA to the synthesis of Hl histone at five different stages of development. These calculations are based on the assumption that histone mRNA of both parental types is transcribed with equal efficiency from the genome and that these RNAs are translated with equal efficiency in the cytoplasm of the hybrid embryos. On this basis, we have estimated that the contribution of new mRNA respresents 80% of total Hl histone synthesis at the 16–32 cell stage, 54% at the hatching blastula stage, 40% at the mesenchyme blastula stage, and 100% after gastrulation.
These data are discussed in the light of presently known parameters of histone and histone mRNA synthesis in echinoderm embryos.     

The relative contributions of newly synthesized and stored messages to Hl histone synthesis in interspecies hybrid echinoid embryos.

We have measured the ratio of incorporation of 3H-lysine into the maternal and paternal forms of Hl histones synthesized by the interordinal hybrid embryo which results from the fertilization of sand dollar eggs with sea urchin sperm. This ratio has been used to calculate the relative contributions of newly transcribed and stored Hl histones mRNA to the synthesis of Hl histone at five different stages of development. These calculations are based on the assumption that histone mRNA of both parental types is transcribed with equal efficiency from the genome and that these RNAs are translated with equal efficiency in the cytoplasm of the hybrid embryos. On this basis, we have estimated that the contribution of new mRNA represents 80% of total Hl histone synthesis at the 16--32 cell stage, 54% at the hatching blastula stage, 40% at the mesenchyme blastula stage, and 100% after gastrulation. These data are discussed in the light of presently known parameters of histone and histone mRNA synthesis in echinoderm embryos.     

Developmental changes in Hl histones from bovine liver

The liver histones of calf embryos, calves and adult cows were analyzed by use of a high-resolution sodium dodecyl sulfate slab gel electrophoresis. The results show both qualitative and quantitative changes in the Hl histones. The Hl histone of the young embryos appeared as two components, that of the embryo at seven months of age as three components and that of the older embryos, as also that of the calves and adult cows, as four components. In addition, the relative proportion of the Hl histones increased during the whole ontogeny.     

Performance of a kinetic model for intracellular ice formation based on the extent of supercooling.

Cryomicroscopy was used to study the incidence of intracellular ice formation (IIF) in protoplasts isolated from rye (Secale cereale) leaves during subfreezing isothermal periods and in in vitro mature bovine oocytes during cooling at constant rates. IIF in protoplasts occurred at random times during isothermal periods, and the kinetics of IIF were faster as isothermal temperature decreased. Mean IIF times decreased from approximately 1700 s at -4.0 degrees C to less than 1 s at -18.5 degrees C. Total incidence of IIF after 200 s increased from 4% at -4.0 degrees C to near 100% at -15.5 degrees C. IIF behavior in protoplasts was qualitatively similar to that for Drosophila melanogaster embryos over the same temperature ranges (Myers et al., Cryobiology 26, 472-484, 1989), but the kinetics of IIF were about five times faster in protoplasts. IIF observations in linear cooling of bovine oocytes indicated a median IIF temperature of -11 degrees C at 16 degrees C/min and total incidences of 97%, 50%, and 19% at 16, 8, and 4 degrees C/min, respectively. A stochastic model of IIF was developed which preserved certain features of an earlier model (Pitt et al. Cryobiology 28, 72-86, 1991), namely Weibull behavior in IIF temperatures during rapid linear cooling, but with a departure from the concept of a supercooling tolerance. Instead, the new model uses the osmotic state of the cell, represented by the extent of supercooling, as the independent variable governing the kinetics of IIF. Two kinetic parameters are needed for the model: a scale factor tau 0 dictating the sensitivity to supercooling, and an exponent rho dictating the strength of time dependency. The model was fit to the data presented in this study as well as those from Myers et al. and Pitt et al. for D. melanogaster embryos with and without cryoprotectant, and from Toner et al. (Cryobiology 28, 55-71, 1991) for mouse oocytes. In protoplasts, D. melanogaster embryos, and mouse oocytes, the parameters were estimated from IIF times in the early stages of isothermal periods, while the osmotic state of the cell was relatively constant. In bovine oocytes, the parameters were estimated from linear cooling data. Without further calibration, the model was used to predict total IIF incidence under different cooling regimes. For protoplasts, D. melanogaster embryos, and bovine oocytes, the model's predictions were quite accurate compared to the actual data. In mouse oocytes, adjustment of the hydraulic permeability coefficient (Lp) at 0 degree C was required to yield realistic behavior.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of maternal and paternal histone genes during early cleavage stages of the echinoderm hybrid Strongylocentrotus purpuratus × Lytechinus pictus

Interspecific hybrids of the sea urchins Strongylocentrotus purpuratus (♀) and Lytechinus pictus (♂) were used to estimate the contributions of the maternal and paternal genomes to histone mRNA synthesis during early development. Radiolabeled histone mRNAs from the two sea urchin species were identified by hybridization to cloned histone genes from both S. purpuratus and L. pictus and shown to be electrophoretically distinguishable. The synthesis of maternal and paternal histone mRNA in these hybrid embryos is evident as early as the two-cell stage. By at least the 16-cell stage, both maternal and paternal histone mRNAs are associated with polysomes. The relative amounts of the maternal and paternal histone mRNAs synthesized by the zygote appear to be similar.     

The C system of cattle blood groups. 2. Partial genetic map of the system

20 cases of irregular inheritance of phenogroups in the C system of cattle blood groups were used to deduce a partial genetic map of this system, taking into consideration the 11 internationally recognized antigenic factors and the 4 additional factors recently described by Grosclaude et al. (1980). This partial map bears resemblance to that established by Bouw et al. (1974) in Dutch cattle.
The operational length of the DNA sequence coding for the C system was estimated to be 0.3 centimorgan, a value which is approximately half of that obtained for the B system by Grosclaude et al. (1979). It is concluded that the phenogroups of the C system, like those of the B system, are controlled by a cluster of loci.     

Hypothermia: teratogenic and protective effects on the development of mouse embryos in vitro

Hypothermia often occurs in association with clinical conditions involving severe hypoglycemia, but its effect on embryonic development has not been well evaluated. Thus, the whole embryo culture method was used to expose day 9 (neurulating) and day 10 (early limb bud stage) mouse embryos to physiologic levels of hypothermia (35 degrees C and 32 degrees C) for 4 and 24 hr. Embryos were evaluated after 24 hours for growth and malformations and compared with controls grown at 37 degrees C. Lactate production was measured in embryos cultured for 4 hr at 32 degrees C and compared with those cultured at 37 degrees C. A 4-hr exposure to hypothermia produced little effect morphologically but reduced the rate of lactate production at both embryonic stages. A 24-hr exposure to hypothermia at 35 degrees C or 32 degrees C produced growth retardation and dysmorphogenesis in embryos undergoing neurulation. Early limb bud stage embryos were less sensitive to this treatment, with growth retardation produced only at the lower temperature. Since hypothermia is commonly associated with severe hypoglycemia in cases of diabetic insulin overdose, day 9 (neurulating) mouse embryos were exposed concurrently to short periods of hypothermia and hypoglycemia and compared with embryos cultured in hypoglycemic medium at normal temperature. The results demonstrate that hypothermia partially protects embryos against the dysmorphogenic effects of hypoglycemia. A balance of metabolic rate and available substrate is discussed as a possible mechanism for this protective effect.     

The absence of histone H1 from the chromatin fraction obtained by sonication of calf thymus nuclei under "quasiphysiological" ionic conditions.

The minor chromatin fraction was isolated from the sonicated calf thymus nuclei on the basis of its differential solubility in the "quasiphysiological" salt medium (0.1 M KCl-0.05 M NaCl-l mM MgCl2-1 mM CaCl2). Histone Hl is almost completely absent from this fraction. DNA isolated from this fraction occurs in three discrete low mol. wt. fragments. The fraction of chromatin which lacks histone Hl can also be obtained by two other methods. On of them consists in salt precipitation of the chromatin gel and its subsequent sonication. The second method includes precipitation of the sonicated chromatin gel by salts. In the first case the properties of the chromatin fraction which remains in the supernatant after centrifugation closely resemble those of the original salt-soluble nuclear fraction. The second method yields supernatant fraction also lacking histone Hl but containing heterogeneous DNA. Comparisons were also made of the sonically-solubilized nuclear fractions obtained in the complete salt medium and its mono and divalent cationic constituents.     

The C system of cattle blood groups. 2. Partial genetic map of the system

20 cases of irregular inheritance of phenogroups in the C system of cattle blood groups were used to deduce a partial genetic map of this system, taking into consideration the 11 internationally recognized antigenic factors and the 4 additional factors recently described by Grosclaude et al. (1980). This partial map bears resemblance to that established by Bouw et al. (1974) in Dutch cattle. The operational length of the DNA sequence coding for the C system was estimated to be 0.3 centimorgan, a value which is approximately half of that obtained for the B system by Grosclaude et al. (1979). It is concluded that the phenogroups of the C system. like those of the B system, are controlled by a cluster of loci.     

Protein synthesis in microsurgically produced androgenetic and gynogenetic mouse embryos

Summary In order to compare paternal and maternal gene activity at the protein synthesis level during early development, androgenetic and gynogenetic mouse embryos were experimentally produced by microsurgically removing either the female or the male pronucleus from fertilized mouse eggs. The resulting haploid eggs were diploidized in a medium containing cytochalasin B and then cultured under normal conditions to the blastocyst stage. Protein synthesis was analyzed at different stages of preimplantation development using 2-dimensional polyacrylamide gel electrophoresis. Both types of uniparental embryos synthesized a similar set of proteins independent of whether the paternal or the maternal genome was present. The isodiploid embryos expressed a protein pattern that corresponded remarkably to normal embryos at the subsequent cleavage stage. This temporal change is probably due to the fact that the operated haploid eggs were kept overnight in cytochalasin B in order to allow chromosomal replication to occur without cell division, and the resulting eggs therefore corresponded to normal 2-cell embryos with respect to karyokinesis but differed as far as cytokinesis was concerned. Several 2-cell specific proteins appeared in these isodiploid eggs and, similarly, following their first cleavage some 4-cell specific proteins were detected in 2-cell androgenetic and gynogenetic embryos. The discordance between nuclear and cellular division, which was retained through the 4-cell stage, however disappeared during subsequent cleavage divisions. At the blastocyst stage, both kinds of uniparental embryos showed a similar protein pattern compared to normal embryos. Our data suggest that some stage-specific proteins are synthesized during preimplantation development and correspond to nuclear rather than cellular divisions.Some of these results were presented at the 13th Annual Meeting of the Union of Swiss Societies of Experimental Biology in Lausanne, March 1981 (Petzoldt et al. 1981)     

The correlation between the heat resistance of the zygotes of the common frog Rana temporaria and its embryos at different developmental stages

A study has been made of a correlation between the heat resistance of zygotes and embryos of the same clutches at different stages of development of Rana temporaria L. The embryos were incubated at 19 degrees C, the injurious temperature being 37 degrees C. As criterion of heat resistance served the time of the injurious temperature action which leads by the ++stra of cleavage to a 50 per cent elimination of embryos (LD50). The correlation in question has been evaluated by the rank correlation coefficient (rho). The correlation between the heat resistance of zygotes and embryos at the start of gastrulation (stage 11) was moderate (rho = 0.47). At one of neurulation stages (stage 22) this correlation was weak (rho = 0.207), to become rather high just before the cleavage (stage 28, rho = 0.63). It is assumed that the thermal selection of zygotes, i.e. of the organism at the cellular stage of development, may be oriented to result in the survival of embryos, which just before the cleavage will show both elevated heat resistance and high heritability of this character.     

Effect of ambient temperatures during oocyte recovery on in vitro production of bovine embryos

Recovery of oocytes from ovaries collected at slaughter was carried out at three ambient temperatures (25 degrees, 30 degrees and 35 degrees C) to assess the effect on subsequent embryonic production in vitro. Oocytes recovered at each temperature were thereafter maintained at temperatures > or =35 degrees C as they were subjected to in vitro maturation, fertilization and culture (IVM/IVF/IVC). The oocytes and resulting embryos within each temperature group were subsequently evaluated for their rates of fertilization, cleavage and development to blastocysts, as well as for the number of cells/blastocyst. The results demonstrate that exposure of cumulus-ocyte-complexes (COCs) to temperatures below 35 degrees C during oocyte recovery is detrimental to optimal embryo production. Although the fertilization and cleavage rates of oocytes recovered at temperatures below 35 degrees C were not significantly lower than that of the controls, the percentage of oocytes recovered at 35 degrees C that developed to the blastocyst stage following fertilization and culture (33.7%) was significantly greater than those from oocytes recovered at either 25 degrees C (22.4%) or 30 degrees C (19.5%). The mean numbers of blastomeres/embryo were significantly lower in embryos derived from oocytes collected at either 25 degrees or 30 degrees compared with those collected at 35 degrees C. The results of this study suggest that exposure of COCs to temperatures below 35 degrees C during oocyte recovery may significantly decrease both the quantity and quality of embryos produced by in vitro methods.     

Nuclear function during early development of remote fish hybrids

The participation of paternal genome was studied in the development of remote hybrids obtained as a result of artificial insemination of the loach (Misgurnus fossilis) eggs by the sperm of aquarial Cyprinids (Brachydanio rerio, Danio malabaricus, Barbus tetrazona, Razbora heteromorpha, Carassius auratus) and Cobitids (Acanthophtalmus kuhlii). The hybrids obtained differed at the stage of hatching both from each other and from the loach by some morphological features. To study the function of heterologous nuclei, haploid nucleocytoplasmic hybrids were obtained by means of chromosome inactivation in the loach eggs by heavy doses of X-rays. The participation of paternal genome in development was estimated by comparison of the curves of viability of diploid and haploid hybrids with those of diploid, haploid and "anuclear" loach embryos. Patterns of mortality of embryos and larvae in each hybrid combination (percentage, stage) suggest the functioning of paternal genome already at the early stages of development. The activity of hybrid and heterologous nuclei was also estimated by the onset and the intensity of morphogenetic function which was determined by the time of embryonic death following nuclear inactivation at different stages. The onset of nuclear function in all hybrids coinsides with that in the loach, except B. rerio in which it occurs somewhat earlier. The data obtained prove the participation of paternal genes in development and maintenance of viability of embryos at all developmental stages beginning from the early ones (blastula).     

Glutamine synthetase in cultured whole retinas from the embryonic chick. Role of protein and RNA syntheses in 4 degrees C storage enhancement

Glutamine synthetase (GS) activity is enhanced in cultured whole retinas when a 72 h incubation at 37 degrees C is preceded by storage at 4 degrees C for 2-24 h. This enhancement occurs even in the absence of glucocorticoids and is maximal in retinas from 11 to 14 d embryos. In comparison, cortisol-induced increases in retinal GS activity at 37 degrees C are optimal in retinas from 8 to 12 d embryos. This study, using cycloheximide (an inhibitor of protein synthesis) and cordycepin (an inhibitor of RNA synthesis), indicates that both protein and RNA synthesis are required for the 4 degrees C storage enhancement of GS activity. The necessary RNA synthesis occurs within the first 48 h following transfer to 37 degrees C and does not require concomitant protein synthesis. Uridine uptake, but not incorporation into trichloroacetic acid-precipitable material, is increased by initial 4 degrees C storage when compared with whole retina controls incubated at 37 degrees C for the total time. In contrast, both uptake and incorporation of amino acids are increased in 4 degrees C-stored retinas for as long as 72 h subsequent to transfer from 4 to 37 degrees C. This suggests that enhancement GS activity may arise from a combination of elevated general protein synthesis and specific messenger-RNA synthesis following 4 degrees C storage.     

Involvement of a urethane-sensitive system in timing the onset of gastrulation in Xenopus laevis embryos

This paper describes success in delaying the onset of gastrulation in Xenopus laevis embryos without damage to their subsequent development by temporarily arresting cleavage with urethane. Exposure of X. laevis embryos to 150 mM urethane before gastrulation resulted in cleavage arrest and its removal led to cleavage resumption. During cleavage arrest, cyclic activities including nuclear replication and the M-phase-promoting factor cycle continued, although their duration was lengthened to nearly 1.8-fold that of the controls. Because of a 30-min time lag from removal of urethane to resumption of cleavage, as well as the retardation of cyclic activities during cleavage arrest, the development of embryos after a 60-min exposure to urethane lagged two cell cycles behind that of control embryos. Here, the two cell cycle delay is equivalent to 50 min at 22-23 degrees C. The start of gastrulation in exposed embryos was accordingly delayed about 50 min, although the delay in mid-blastula transition was as little as 20-25 min. Consistent results were obtained in embryos exposed to urethane for 90 or 120 min and those exposed to procaine or NH4Cl for 60 min. Although these results imply that delay in the start of gastrulation in exposed embryos is ascribed simply to delay in their development raised by cleavage arrest, at the same time they suggest that the onset of gastrulation is timed by systems sensitive to urethane, procaine and NH4Cl in X. laevis embryos.     

The locus Om,responsible for the DDK syndrome,maps close to Sigje on mouse Chromosome 11

The DDK inbred strain of mouse has a striking particularity: when DDK females are crossed to males of other strains they exhibit a reduced fertility, whereas the reciprocal crosses (non-DDK females x DDK males) are fertile (Wakasugi et al. 1967; Wakasugi 1973). The low fertility results from an early embryonic lethality, the F₁ embryos dying near the late morula-early blastocyst stage. Genetic analyses (Wakasugi 1974) and nuclear and cytoplasmic transfers (Renard and Babinet 1986; Babinet et al. 1990; Mann 1986), have shown that the failure of the embryos to develop is due to an incompatibility between a DDK maternally encoded cytoplasmic product and the non-DDK paternal genome. In order to elucidate the genetic determinism of this embryonic lethality, we have analyzed the fertility of male progeny from a backcross BALB/c females x (BALB/c x DDK)F₁ males and that of males from a set of recombinant inbred (RI) strains, established from DDK and BALB/c progenitors, when mated with DDK females. Our results indicate that a single locus, Om, is responsible for the DDK syndrome and is located on Chromosome (Chr) 11, very close to the Sigje locus.