Activation of a Na+-dependent amino acid transport system in preimplantation mouse embryos

The uptake of l-methionine-methyl-³H and l-leucine-³H from completely defined medium into acid-soluble fractions of preimplantation mouse embryos has been studied. Late four-cell embryos and early blastocysts raised in vitro can concentrate both amino acids by processes which exhibit saturable, Michaelis-Menten type kinetics, characteristic of carrier-mediated active transport systems. This uptake is temperature-sensitive and inhibited by certain amino acids which compete for the same uptake sites. Methionine uptake seems to be mediated by a single transport system (K_m = 6.25 × 10^?5M) at the four-cell stage. Complex kinetics suggest that two distinct transport systems exist at the early blastocyst stage (K_m = 6.25 × 10^?5M; 8.9 × 10^?4M). V_max values (mg/embryo/15 min) for methionine and leucine transport increase significantly from the late four-cell stage to the blastocyst stage, suggesting that additional carriers are produced or activated during development.Most importantly, leucine and methionine transport is Na⁺-independent at the four-cell stage, methionine transport is partially dependent at the morula stage, and both amino acids are completely Na⁺-dependent at the blastocyst stage. The cumulative results suggest that preimplantation embryos accumulate leucine and methionine by specific, chemically mediated, active transport systems. The qualitative and quantitative developmental changes in cell membrane function may represent preparatory steps for subsequent growth of embryonic and/or trophoblastic cells.     

Sodium-dependent amino acid transport in preimplantation mouse embryos. III. Na+-k+-atpase-linked mechanism in blastocysts.

Mouse blastocysts collapse in cytochalasin B (CB), reexpand (accumulate fluid) in control medium, but cannot reexpand in ouabain, an inhibitor of Na⁺K⁺-ATPases. These ATPases, then, seem to be necessary for fluid accumulation in blastocysts. Since intact blastocysts are relatively insensitive to ouabain, CB seems to make it possible for ouabain to reach the Na⁺K⁺-ATPases localized on the blastocoelic surface. CB-Collapsed blastocysts were found to transport alanine and lysine at the same rate as intact blastocysts, indicating that, in 1 hr, amino acids are transported into the cells of the intact blastocyst, and not into the fluid-filled blastocoel. Transport rates in CB-collapsed blastocysts do not exceed those in intact blastocysts, suggesting that hypothetical amino acid carriers are located only on the external blastocyst surface. Most important, ouabain strongly inhibits sodium-dependent alanine transport in CB-collapsed blastocysts, but not in intact blastocysts, providing strong evidence that Na⁺K⁺-ATPases, localized on the blastocoelic surface, are necessary for this transport. Ouabain does not inhibit sodium-independent lysine transport in CB-collapsed blastocysts. Thus, the dependency of both sodium-dependent amino acid transport and fluid accumulation upon Na⁺K⁺-ATPases, and the separate localization of amino acid carriers and these ATPases, provides functional evidence for an epithelial tissue type of mechanism for sodium-dependent amino acid transport in mouse blastocysts.     

Na+-dependent amino acid transport in preimplantation mouse embryos. II. Metabolic inhibitors and nature of the cation requirement.

Experiments were conducted in order to determine the energy source and nature of the cation dependency of [³H]methionine transport in preimplantation mouse embryos. The energy source of methionine transport was studied at the late four-cell and early blastocyst stages. The embryos, raised in vitro, were incubated for 1 hr in inhibitor(s) of energy metabolism and then transferred for 1 hr to medium that contained inhibitor(s) and ³H-methionine. These inhibitor studies suggest that respiration and glycolysis are needed to maintain uptake of methionine in early blastocysts. Late four-cell embryos seem to utilize respiration alone for transport.The cation dependency of methionine transport was studied at the late morula and early blastocyst stages. The kinetics of methionine uptake by early blastocysts in Na⁺-depleted media indicate a competitive type of inhibition. The uptake of methionine by early blastocysts is relatively resistant to ouabain and unaffected by K⁺-free medium. In contrast, methionine uptake by late morula-stage embryos is markedly inhibited by ouabain and K⁺-free medium in 1 hr. These results suggest that 1) Na⁺ serves to increase the affinity of methionine for the carrier in early blastocysts, 2) the cation gradients do not supply a major fraction of the energy required for methionine transport, and/or the gradients are difficult to perturb once the blastocyst has formed, and 3) putative Na⁺ pumps may be localized on the blastocoelic surface of the blastocysts.     

A measurement of the sequence complexity of polysomal messenger RNA in sea urchin embryos

The first measurement has been made of the number of diverse mRNA sequences (mRNA sequence complexity) in the total polysomes of a eucaryotic system, the sea urchin gastrula. mRNA was purified of nuclear RNA and any other heterogeneous RNA contaminants by release from polysomes with puromycin. Trace quantities of labeled nonrepetitive DNA fragments were hybridized with an excess of mRNA. The hybridization reaction followed ideal first order kinetics in mRNA concentration. At completion of the hybridization reaction, 1.35% of the nonrepetitive DNA was present as mRNA-DNA hybrid. The hybridized DNA was extracted and was at least 70% hybridizable with mRNA, demonstrating a 50-fold purification of the expressed sequences. This purified DNA fraction reassociated with excess unfractionated sea urchin DNA at a rate identical to that of the total nonrepetitive DNA tracer. The mRNA had therefore been hybridized to nonrepetitive DNA sequence, and the amount of hybrid could be used as a direct measure of the mRNA sequence complexity.The complexity of the gastrula mRNA can be calculated as about 17 million nucleotides, sufficient to comprise some 14,000 distinct structural genes. This result also provides an estimate of the number of diverse proteins being translated in the gastrula. From the rate of mRNA-DNA hybrid formation, we estimate that about 8% of the mRNA belongs to this complex class, and that less than 500 copies of each species of message in this class exist per embryo. Most of the mRNA population consists of a relatively small number of diverse species represented a much larger number of times.     

The cytoplasmic distribution and characterization of poly(A)+RNA in oocytes and embryos of Drosophilia.

Using the presence of poly(A) tracts as a marker for mRNA, we have examined the distribution of this class of RNA between polysomes and free RNP particles. This has been done in mature oocytes and in embryos aged for various times from fertilization through to hatching of a larva. The proportion of ribosomes that are in polysomes to those that are not has been calculated. In mature oocytes, 58% of the poly(A)⁺ RNA and 72% of the ribosomes are not in polysomes. By 1 hr, this drops to 51% of the poly(A)⁺ RNA and 48% of the ribosomes. By 7 hr, a plateau is reached: 30% of each are not in polysomes. The poly(A)⁺ RNA in the cytoplasm of oocytes and 1-hr embryos is found in particles with an average size of 50S and a range of 30–70S. The poly(A)⁺ RNA ranges in size from 7 to 40S, with an average size of 22S. The polyA from this RNA is 50–200 nucleotides long with an average of 115 nucleotides. These data have allowed us to calculate that 1–2% of the total RNA is poly(A)⁺ RNA.     

Levels and modification of methionyl-transfer RNA in preimplantation rabbit embryos.

Transfer RNA with l-methionine acceptor activity was extracted from preimplantation rabbit embryos and purified on reverse-phase-3 columns. The molar quantity of methionine acylated to RNA increases as embryo development proceeds from the 16-cell stage to the 80,000 cell blastocyst stage. However, the quantity of methionyl-tRNA per genome declines 100-fold as the embryo cell number increases. Formylation of methionyl-tRNA illustrated that approximately one-third of tRNA^Met extracted was tRNA_f^Met. Methylation of purified methionyl-tRNA by an adult rabbit liver methylase extract illustrated that two-day preimplantation embryo tRNA is highly hypomethylated relative to tRNA from later stages of development. The hypomethylated methionyl-tRNA was also less effective in ribosome binding studies than more fully methylated methionyl-tRNA present in the later stages of embryo development.     

Structural gene sets active in embryos and adult tissues of the sea urchin

Structural gene sequences active in a variety of sea urchin adult and embryo tissues are compared. A single-copy ³H-DNA fraction, termed mDNA, was isolated, which contains sequences complementary to the messenger RNA present on gastrula stage polysomes. Gastrula message sequences are 50 fold concentrated in the mDNA compared to total single-copy DNA. mDNA reactions were carried out with excess mRNA from blastula, pluteus, exogastrula, adult ovary, tubefoot, intestine, and coelomocytes, and with excess total mature oocyte RNA. A single-copy ³H-DNA fraction totally devoid of gastrula message sequences, termed null mDNA, was also reacted with these RNAs. Large differences in the extent of both mDNA and null mDNA reaction with the various RNAs were observed, indicating that in each state of differention a distinct set of structural genes is active, generally characterized by several thousand specific sequences. The complexity of gastrula mRNA was shown in previous work to be about 17 × 10⁶ nucleotides. In units of 10⁶ nucleotides, the complexities of the RNA sequence reacting with mDNA and with null mDNA in each tissue are, respectively, as follows: intestine mRNA; 2.1 and 3.7; coelomocyte mRNA: 3.5 and ≤1.4; tubefoot mRNA: 2.7 and ≤0.4; ovary mRNA: 13 and 6.7; oocyte total RNA: 17 and 20; blastula mRNA: 12 and 15; pluteus mRNA: 14 and ≤0.6; exogastrula mRNA: 14 and ≤0.6. The total complexity of each mRNA population is the sum of these values, as verified for several cases by reactions with total single-copy DNA. A relatively small set of mRNAs, the complexity of which is about 2.1 × 10⁶ nucleotides, appears to be shared by several of the tissues studied.     

An extracellular fibrillar matrix in gastrulating sea urchin embryos

Fine structural studies of fractured developing sea urchin embryos revealed the existence of a voluminous, fibrillar, extracellular matrix composed of fine filaments, twisting fibers and granules lining the blastocoel of midgastrula embryos. Glycine disaggregated embryos also exhibited this material. The fibrillar matrix is closely associated with the basal lamina of the ectodermal cells of the embryo and histochemical studies suggest it is composed mostly of sulfated glycosaminoglycans. The position of the matrix within the blastocoel as well as its organized association with embryonic cell surfaces is consistent with the hypothesis that it plays a major role in guiding the invaginating archenteron during gastrulation.     

Questioning of reported evidence for guanosine tetraphosphate synthesis in a ribosome system from mouse embryos.

In 1974, Irr, Kaulenas and Unsworth reported that ppGpp is synthesized by cytosolic ribosomes from mouse embryos and proposed a role for ppGpp in the process of differentiation. This proposal is being challenged because ribosomes of mouse embryos from various stages of development and of mouse embryoid bodies were completely inactive in ppGpp formation.     

Intracellular pH and the metabolic status of dormant and developing Artemia embryos

Encysted embryos of the brine shrimp Artemia salina have long been known to enter profound dormancy under anaerobic conditions. Utilizing in vivo³¹P nuclear magnetic resonance, we show that the reversible transitions between anaerobic dormancy and aerobic development are accompanied by large (>1 unit) intracellular pH changes, the more acid pH being associated with the dormant state. Furthermore, dormant cyst intracellular pH is independent of that of the buffer, suggesting the potential for pH-mediated regulation of enzyme activities during dormancy. An example concerning cyst nucleotide metabolism is discussed.     

On the nature of nuclear envelope-associated RNA

Nuclear envelopes were isolated from rat-liver nuclei. Nuclear envelope-associated RNA was isolated and hybridized to filter-bound DNA in the presence of competing RNA populations. Cytoplasmic RNA did not effectively compete for DNA binding sites, while nuclear RNA did. The results indicate a high degree of complexity for nuclear envelope-associated RNA, and are compatible with the idea that hnRNA may be processed after attachment to the nuclear envelope (or nuclear matrix).     

Structural genes adjacent to interspersed repetitive DNA sequences

The observation that repetitive and single copy sequences are interspersed in animal DNAs has suggested that repetitive sequences are adjacent to single copy structural gene sequences. To test this concept, single copy DNA sequences contiguous to interspersed repetitive sequences were prepared from sea urchin DNA by hydroxyapatite fractionation (repeat-contiguous DNA fraction). These single copy sequences included about one third of the total nonrepetitive sequence in the genome as determined by the amounts recovered during the hydroxyapatite fractionation and by reassociation kinetics. ³H-labeled mRNA from sea urchin gastrula was prepared by puromycin release from polysomes and used in DNA-driven hybridization reactions. The kinetics of mRNA hybridization reactions with excess whole DNA were carefully measured, and the rate of hybridization was found to be 3–5 times slower than the corresponding single copy DNA driver reassociation rate. The mRNA hybridized with excess repeat-contiguous DNA with similar kinetics relative to the driver DNA. At completion 80% of that mRNA hybridizable with whole DNA (approximately 65%) had reacted with the repeat-contiguous DNA fraction (50%). This result shows that 80–100% of the mRNA molecules present in sea urchin embryos are transcribed from single copy DNA sequences adjacent to interspersed repetitive sequences in the genome.     

Atriopeptin release from the isolated perfused rabbit heart

Mammalian atrial extracts have been shown to contain bioactive peptides which exert natruiretic, diuretic, and smooth muscle relaxant effects. These extracts include several low molecular weight (< 5,000 Mr) atrial peptides (atriopeptins) which exhibit identical sequences over a central core region which are derived from the high molecular weight peptide (atriopeptigen) precursor which has been purified and sequenced. In the current study we found that extracts of rabbit atria possess both high and low molecular weight bioactive atrial peptides, however, the coronary venous effluent obtained from the isolated perfused rabbit heart only contained the low molecular weight peptide. This trypsin labile activity causes a dose-dependent relaxation of rabbit aorta and chicken rectum assay strips. Separation of the bioactivity with gel filtration chromatography and reversed phase HPLC indicates the heart releases a single substance similar to atriopeptin III. There was no evidence that atriopeptigen was released from the isolated perfused rabbit heart. We suggest that atriopeptigen is proteolytically processed in the atria to an atriopeptin which is subsequently the released form of the atrial peptide.     

Isolation of globin messenger RNA of Xenopus laevis

The polypeptide chains of Xenopus laevis hemoglobin have been analyzed by sodium dodecyl sulfate (SDS) and acid-urea gel electrophoresis. Four components can be distinguished, each having an approximate molecular weight of 13,000 daltons. Messenger RNA coding for the globin chains has been isolated and characterized. In a denaturing acrylamide gel the mRNA has an approximate molecular weight of 250,000 daltons. The complexity of the RNA is consistent with the presence of four different mRNA molecules, each of this molecular weight. When the mRNA is assayed in a wheat germ in vitro translation system, four polypeptides are synthesized corresponding to the four globin subunits. The relative proportion of the four synthesized polypeptides appears to vary according to the developmental stage of the red blood cells used for mRNA isolation. Hybridization of a complementary DNA (cDNA) copy of the globin mRNA to Xenopus laevis DNA in DNA excess indicates that each of the globin genes is present in one to three copies per haploid genome.     

DNA-dependent RNA-polymerases from Artemia embryos. characterization of polymerases I and II from nauplius larvae.

Partial purification and characterization of DNA-dependent RNA-polymerases from nauplius larvae of the brine shrimp, Artemia salina, are described. Fractionation of solubilized RNA-polymerases on columns of DEAE-cellulose yielded partially purified preparations of RNA polymerases I and II. The properties of these enzymes were found to be similar to properties of corresponding enzymes from other animal sources. A significant change in the relative amounts of polymerases I and II occurs between 36 and 72 hr of development. Polymerase activity obtained from 36-hr nauplii consisted of approximately equal amounts of polymerases I and II, whereas polymerase II accounted for more than 80% of the activity recovered from 72-hr nauplii. Total polymerase activity was lower at 72 than at 36 hr. The significance of these changes in relation to the decrease in RNA synthesis in vivo that occurs after 36 hr is discussed.     

Studies of cloned sequences from four Drosophila heat shock loci.

DNA cloned from the D. melanogaster (Oregon R) heat shock loci at 63BC and 95D codes for the 83,000 and the 68,000 dalton heat shock proteins, respectively. Both coding sequences occur once per haploid genome. Sequences complementary to messenger RNA for the 70,000 dalton heat shock protein are represented five times, twice at 87A and three times at 87 C. The copies at 87A differ characteristically from those at 87C in an interval of a few hundred bp near the 5' end of the messenger sequence, and the corresponding two classes of hsp 70 messenger RNA are found on polysomes after heat shock. Within this differential region, there is about 15% divergence between messenger sequences cloned from the two loci, while in the rest of the messenger region examined the homology is much closer although still imperfect. Unexpectedly, considerable homology is found between the sequence for the 68,000 dalton heat shock protein at 95D and the sequences for the 70,000 dalton protein at 87A and 87C, and between these sequences and a site in 87D. Messenger RNA molecules of 2.4, 2.55 and 3.05 kb code for the 68,000, 70,000 and 83,000 dalton heat shock proteins and hybridize to apparently uninterrupted DNA sequences of 2.1, 2.25 and 2.6 kb, respectively.