Steroidogenesis and prostaglandin synthesis by cultured bovine blastocysts.

Bovine blastocysts were collected at Days 13, 15 and 16 and placed in TCM-199 supplemented with 5% fetal calf serum; some blastocysts were immediately frozen while the others were cultured for 48 h and then frozen. Samples (tissue + medium, 5--12/group) were thawed, homogenized and analysed by radioimmunoassays. Measurable amounts of progesterone were found in all blastocysts but values were higher (P less than 0.01) after culture. Testosterone was not found in the cultured or uncultured blastocysts at Day 13, but was detectable on Days 15 and 16 and in greater amounts (P less than 0.05) in the cultured blastocysts. PGF and PGE-2 were increased (P less than 0.05) in the cultured blastocysts in all 3 days. Oestradiol was measurable in some but not all blastocysts. It is suggested that PG synthetase and enzymes capable of synthesizing progesterone, testosterone and, possibly, oestradiol are present in these early bovine blastocysts.     

Changes of lipid composition in non-cultured and cultured porcine embryos

The objective of the present study was to evaluate the lipid composition of cultured and non-cultured pig embryos during cleavage using histochemical methods. The authors studied pig zygotes as well as 2-to 4-cell embryos, morulae, and blastocysts that were either non-cultured or cultured in NCSU-23 medium. To detect different types of lipids, the authors used the Churukian method with Oil red O, the Sudan black B method, the Cain method with Nile blue sulfate, and the modified osmium tetroxide-ethanol treatment. In the zygotic lipid droplets, diverse classes of unsaturated and saturated lipids were found, with particularly high levels of unsaturated hydrophobic lipids, mainly triglycerides and other esters, free fatty acids, and phospholipids. In the zygotic cytoplasm, the authors observed high levels of fatty acids and phospholipids. The total lipid content remained constant up to the morula stage, decreasing later at the blastocyst stage, but the overall amount of unsaturated lipids declined earlier, at the 2-to 4-cell stage. The amount of free fatty acids and phospholipids decreased during cleavage in both non-cultured and cultured porcine embryos. The main differences between the non-cultured and cultured embryos were the more pronounced reduction in the amount of unsaturated hydrophobic lipids in droplets and the cytoplasmic free fatty acids observed in the cultured morula and the lower content of phospholipids in the cytoplasm of the cultured 2-to 4-cell embryos relative to the non-cultured embryos. The decrease in the unsaturated lipid, free fatty acid, and phospholipid content during in vivo development and the differences in the amount of these types of lipids between developmentally matched cultured and non-cultured pig embryos correlate well with modifications of lipid and carbohydrate metabolism.     

Protein synthesis and degradation in cultured muscle is altered by a phorbol diester tumor promoter

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was observed to influence the relative rates of synthesis and degradation of several polypeptides in cultured chick embryo myotubes. The direction of influence partially correlated with whether the polypeptide was uniquely expressed in myotubes or also expressed in its proliferating precursors. The synthesis of all but one of the eight myotube-unique polypeptides examined was inhibited and the degradation of all but two was stimulated. The exceptions were intermediate filament subunits. In contrast, the metabolism of several non-myotube-unique polypeptides was either unaffected or influenced in the opposite direction. A method involving saturation of the intracellular leucine precursor pool with extracellular radioactive leucine suggested that the absolute rate of protein synthesis was only minimally, if at all, affected by the promoter. The effects on protein synthesis were at least partially reversible following removal of the promoter. The changes in synthesis and degradation did not reflect normal maturational changes in cultured myotubes and thus they appeared to be induced by TPA. The data suggest that under some circumstances the rates of synthesis and degradation of several proteins are inversely coupled in the myotube. They also provide a partial explanation for earlier ultrastructural observations that TPA caused a loss of myofilaments and an accumulation of intermediate filaments. Since myotubes occupy the G₁ (or G₀) stage of the cell cycle, and the DNA content of the cultures was not affected by TPA, TPA's effects were not mediated through cell cycle-dependent mechanisms.     

Butyrate stimulates fibronectin synthesis in cultured rabbit cornea

To understand corneal wound healing, in which fibronectin plays an important role, we investigated the regulatory mechanism of fibronectin synthesis in cultured rabbit corneal blocks in situ. The amount of fibronectin was determined by enzyme-linked immunosorbent assay. We found that butyrate stimulated fibronectin synthesis in a dose-response fashion, and that butyrate had a greater stimulatory effect than did any of its derivatives examined. This suggests that butyrate stimulates fibronectin synthesis specifically; 8Br-cAMP also stimulated fibronectin synthesis. Additivity of stimulation by butyrate and by 8Br-cAMP was observed at the saturated concentration of each; the present result indicated an independent mechanism of action for these two compounds.     

Hexose transport in preimplantation rabbit blastocysts

Transtrophectodermal 3-0-methyl glucose (3-0MG) transport in the rabbit blastocyst at Days 6 and 7 post coitum was investigated to understand better how the trophectoderm can regulate inner cell mass growth by controlling substrate availability. 3-0MG rapidly traversed the trophectoderm and displayed saturation kinetics (Km = 4.3 +/- 0.5 mM, Vmax = 79 +/- 3.8 nmol.cm-2). The flux of 3-0MG was inhibited nearly 95% by 10(-4) M-phloretin, and only 15% by 10(-4) M-phlorizin. Furthermore, 3-0MG influx was inhibited by cytochalasin B (5 microM) and was unaffected by removal of sodium. The transport system had a high specificity for 2-deoxy-D-glucose and glucose, and a very low specificity for fructose and 4-alpha-methyl glucoside. Western blots probed with a polyclonal antibody to the human erythrocyte glucose transport protein and also with a polyclonal antibody to the C-terminus of the glucose transport protein of the rat brain revealed a broad band with a molecular weight of 55,000. Using immuno-gold labelling techniques, Na(+)-independent glucose transporters were localized to both the apical and basolateral borders of the trophectodermal cell. These results suggest that the mechanism in the trophectoderm responsible for transport of glucose is similar to other sodium-independent glucose transport systems. In addition, 3-0MG influx was unaffected by short-term incubation with progesterone, the progesterone antagonist mifepristone (RU-486), PGF-2 alpha, PGE-2, insulin, or cAMP. Day-7 p.c. embryos also transported hexoses by a similar system because the influx rate and the phlorizin/phloretin sensitivity were the same as in the Day-6 p.c. embryo.     

Efficient production of sex-identified and cryosurvived bovine in-vitro produced blastocysts

To establish a protocol for production of bovine in-vitro produced (IVP) blastocysts that were sex-identified and cryopreserved, we examined the sexing efficiency and accuracy of Day-3 and Day-4 embryos by polymerase chain reaction (PCR), the development of the biopsied embryos into Day-7 blastocysts and the freezability of these blastocysts by vitrification in gel-loading tips. One or two blastomeres were isolated from IVP embryos at either the 8-cell or 16-cell stage (Days 3 and 4, respectively) by a pressing-out method, and were then subjected to primer extension preamplification (PEP)-PCR. The successful sex-identification rate of biopsied samples amplified, purified and analyzed for sex by a second PCR (88.9%) was higher than that of those amplified and analyzed without purification (32.0%). Developmental rates into Day-7 blastocysts of biopsied embryos (Day-3, 65.5%; Day-4, 70.8%) were similar to those of non-biopsied control embryos (Day-3, 74.5%; Day-4, 65.1%). Total cell numbers and the inner cell mass (ICM) ratio of blastocysts derived from biopsied embryos were also comparable with those of control embryos. Blastocysts were vitrified-warmed in the presence of 20% DMSO, 20% ethylene glycol and 0.6M sucrose using gel-loading tips as containers. The proportions of biopsied blastocysts that were hatched or hatching rates after warming were high, regardless of the biopsy time (Day-3, 94.1%; Day-4, 91.9%), similar to the rates for control blastocysts (Day-3, 97.5%; Day-4, 96.9%). In conclusion, a protocol that allows sexing of Day-3 and Day-4 bovine embryos without compromising either the developmental ability to the blastocyst stage or freezability of Day-7 blastocysts was developed.     

Growth and DNA replication in rabbit blastocysts.

DNA content and DNA polymerase activity were measured on rabbit blastocysts removed from the uterus at 24-hr intervals over the period of days 4-7 postcoitum (pc). Median DNA content increased 53 times over the 72-hr period, from 25.3 ng on day 4 to 1,360 ng on day 7. Median DNA polymerase activity (fmole of radiolabeled nucleotide incorporated in 30 min at 37 degrees C) increased 393-fold from day 4 to day 7: 32.8 to 12,900. These embryos also increased in surface area and volume by 334-fold and 6,078-fold, respectively. Litters containing individuals with high DNA content also tended to have similar individuals with high DNA polymerase activity. Therefore, DNA polymerase activity may be a useful measure of the potential for the next cell division. A large amount of variation existed between blastocysts in all parameters measured. An analysis of variance, conducted to partition variation between litters and within litters, determined that within-litter variation was actually greater than that between litters, resulting in intraclass correlation coefficients less than 0.5. There was also a positive regression of DNA content and DNA polymerase activity on surface area in 6- and 7-day-old blastocysts after eliminating variation attributable to litters. The developmental pattern of DNA polymerase activity in the rabbit may be quantitatively different from that described in the mouse. The pattern in mammals is very different from that described in several nonmammalian species.     

Dexamethasone affects phosphatidylinositol synthesis and degradation in cultured human embryonic cells

Dexamethasone (DEX), a glucocorticoid which induces cleft palate, causes marked alterations in the synthesis and degradation of phosphatidylinositol (PI) but not phosphatidylcholine in an established fibroblastic cell line derived from a human embryonic palate. Incorporation of radiolabeled inositol into phosphatidylinositol as well as degradation of prelabeled phosphatidylinositol is stimulated by DEX. The dose-response curves for the DEX-induced effect on PI synthesis and DEX-induced inhibition of cell proliferation are nearly identical, with the maximal responses occurring at 10^?8M DEX. Our results suggest that DEX-induced inhibition of human embryonic palatal mesenchyme cell proliferation and alterations in synthesis and degradation of phosphatidylinositol are related.     

Concomitant synthesis and degradation of glutamine in isolated rabbit kidney tubules

When rabbit kidney tubules were incubated with 1 mM [1-14C]glutamine as substrate, a release of 14CO2 together with a net production of glutamine were observed. That glutamine utilization was masked by higher rates of concomitant glutamine synthesis was demonstrated by: (i) inhibiting glutamine synthesis; and (ii) measuring the specific radioactivity of [1-14C]glutamine which fell during incubation.     

Protein synthesis in tissues cultured from the bovine hoof

Salmon gonadotropin (GTH II) is a heterodimeric glycoprotein hormone ( and II subunits), serving as a maturational GTH, and is produced in a specific gonadotropic cell-type (GTH II-cells) containing small granules and large globules. In trout GTH II-cells, double immunolabeling for the - and II-subunits shows that colocalization of the - and II-immunolabeling is confined to the small granules, indicating storage of functional GTH II. On the other hand, -immunolabeling is absent in the large globules, even though II labeling is abundant throughout the period of seasonal gametogenesis. The -specific antiserum recognizes the intact -subunit as well as the reduced and deglycosylated -subunits by immunoblotting. These results indicate that an accumulation of the II-subunit is specifically generated in the large globules of these cells. In fact, with sexual maturity, the quantity of II-subunits becomes elevated in the trout pituitary due to a marked increase in GTH II-cells containing many large globules. However, the derivation and function of the large globules and the fate of their contained II-subunits remains unknown.     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

Protein degradation and synthesis of cyanophycin granule polypeptide in Aphanocapsa sp

Cyanophycin granule polypeptide content increased by 2- to 3-fold, soluble protein content decreased by 1.5-fold, and carbohydrate content increased by 2-fold within 6 h of chloramphenicol addition to exponentially growing cells of Aphanocapsa sp. strain 6308. Analysis of 14C- and 3H-labeled cells transferred to unlabeled medium and analysis of pulse-labeled cells both suggested cyanophycin granule synthesis from preformed protein breakdown.     

Partly ordered synthesis and degradation of glycogen in cultured rat myotubes.

The following questions concerning glycogen synthesis and degradation were examined in cultured rat myotubes. 1) Is synthesis and degradation of the individual glycogen molecule a strictly ordered process, with the last glucosyl unit incorporated into the molecule being the first to be released (the last-in-first-out principle), or is it a random process? 2) Are all glycogen molecules in skeletal muscle synthesized and degraded in phase (simultaneous order) or out of phase (sequential order)? Basal glycogen stores were minimized by fasting and were subsequently replenished in two intervals, the first (0-0.5 h) with tritium-labeled and the second (0.5-3 h) with carbon-labeled glucose as precursor. Glycogen degradation was initiated by addition of forskolin. The kinetics of glycogen accumulation as well as degradation could be approximated by monoexponential equations with rate constants of 0.81 and 1.39 h(-1), respectively. The degradation of glycogen largely followed the last-in-first-out principle, particularly in the initial period. Analysis of the size of the glycogen molecules and the beta-dextrin limit during glycogen accumulation and degradation showed that both synthesis and degradation of glycogen molecules are largely sequential and the small deviation from this order is most pronounced at the beginning of the accumulation and at the end of the degradation period. This pattern may reflect the number of synthase and phosphorylase molecules and fits well with the role of glycogen in skeletal muscle as a readily available energy store and with the known structure of the glycogen molecule. It is emphasized that the observed nonlinear relation between the change in glycogen concentration and release of label during glycogen degradation may have important practical consequences for interpretation of experimental data.     

Cyclic AMP and cyclic GMP in rabbit blastocysts.

Concentrations of both nucleotides were significantly higher in Day-6 than in Day-5 blastocysts but the ratio of cAMP to cGMP changed from 0.5 to 1.5.