The motility of fowl spermatozoa has been studied in vitro under various modifications of pH, osmotic pressure, and chemical composition of diluents. The glucose and sodium chloride content of the diluents has been varied to give tonicities ranging between that of 0�45 and 1�8 per cent. sodium chloride. Tliese diluents were buffered with citric acid-disodium phosphate, sodium phosphates, or sodium carbonate-bicarbonate mixtures which were equally innocuous.
Electrophoretic separation of solubilized embryos incubated for 24 h in the presence of [U-14C]glucose indicated incorporation of glucose carbon into a number of protein bands. Treatment of nitrocellulose blots of electrophoretograms with glucosidases had no effect on incorporated counts, confirming that the labelled bands were not due to protein bound glycogen. Furthermore, addition of 0.1 microgram mL-1 tunicamycin to the incubation medium virtually eliminated incorporation of glucose into the protein bands but had no effect on the pattern or rate of incorporation of labelled amino acids in parallel experiments. Also the pattern of labelling of protein by glucose was reflected in the pattern of binding of Con A to the nitrocellulose blots. There were quantitative and qualitative changes in labelling as development progressed. For embryos cultured from the 2-cell stage, a small amount of label was incorporated in two major bands at relative mobility (Mr) 69 and 97 K. With culture from the 8-cell stage, three additional major bands (33, 44 and 56 K) were labelled. Embryos cultured from the morula stage showed a different profile of incorporation; there was much more active labelling, and eight major and a number of minor radioactive bands were identified. Whilst tunicamycin suppressed glucose incorporation into glycoproteins and inhibited compaction of embryos, it had little effect on other parameters of metabolism during incubation in its presence for 24 h. No significant effects of the metabolite on protein synthesis, glycogen storage, lactate production or overall macromolecular synthesis were evident. By contrast, the anabolic metabolism of embryos decompacted by long periods of exposure to tunicamycin was severely reduced although glycolysis was still unaffected. Amphomycin at very high concentration (500 micrograms mL-1) was toxic to embryos but at concentrations up to 250 micrograms mL-1 had no effect on compaction and development of blastocysts. Addition of monensin to the incubation medium [16 micrograms mL-1] did not interfere with the development of either 2-cell or 8-cell embryos to blastocysts.
Estimates of the volume of fluid and the concentration of cations in the uterus were made by flushing the uterine cavity of mice mated to vasectomized males and measuring the concentrations of sodium and potassium cations in the recovered fluid. On day 1 following mating, large volumes of fluid (greater than 90 microL) were found in the uterus but, from days 2 to 5, only 2-5 microL of fluid were present. The ratio of sodium to potassium fell from 4.5:1 on day 1 to 1.8:1 on days 2 to 5 of pseudopregnancy, indicating that uterine fluid in the mouse has a high K+ content ranging from 35 mEq L-1 on day 1 to 75 mEq L-1 on day 5. Glucose, lactate and pyruvate in uterine flushings were also assayed and their concentration in uterine fluid calculated using the volumes found above. The level of all substrates was low on day 1 after mating. From day 2 onwards approximately 1 mM glucose was present in the fluids. The concentration of lactate was more variable, and peaked at 4 mM on day 2 of pseudopregnancy. In general, the concentration of pyruvate was 10% of the lactate value.
The chemical composition of fluid from the fallopian tube of the ewe was studied in four cycling ewes and in four groups of four ovariectomized ewes receiving oestrogen and progesterone in factorial combination. Both the major electrolytes and some organic constituents in the fluids were examined.
Satisfactory storage of rabbit spermatozoa has not been reported. As cow milk preparations are useful as diluents for spermatozoa of other species, diluents based on reconstituted skim milk were investigated.
The effect of changes in oxygen concentration on the catabolic utilization of glucose by the sheep conceptus at selected periods between Days 3 and 19 of preimplantation development was examined by estimating the production of CO2 and lactate from [U-14C]glucose during a 2.5-h culture in vitro in the presence of 20%, 5% and 1% O2. In general, lowering O2 significantly altered the catabolism of glucose with a changing pattern of response depending on the stage at which the conceptus was explanted. For embryos at Days 3 and 6 post insemination, reduced O2 caused no significant change in oxidative utilization of glucose and a small decrease in conversion of the substrate to lactate. By contrast, lowering O2 concentration during incubation of the structures of the advanced conceptus from Day 13 through to Day 19 of pregnancy significantly restrained oxidative utilization of glucose but stimulated its conversion to lactate. The effects of these changing levels of O2 on the generation of energy from glucose in the form of ATP was estimated. Except for the Day-13 conceptus, reduction in O2 concentration had little influence on the calculated amount of ATP produced from glucose, with glycolysis making up the deficit in energy production when reduced O2 inhibits oxidation of the substrate at the later stages of development. At Day 13, the switch in the metabolism of glucose to glycolysis is not fully effective and energy production fell as O2 concentration was reduced. The results indicate a major shift towards dependence by the preimplantation sheep conceptus on the glycolytic pathway for energy generation from glucose as development progresses. This move to glycolysis is increased by low O2 concentration. As a low concentration of O2 most probably exists in the lumen of the sheep uterus, the results indicate that, in utero, the energy required for the rapid growth of the conceptus depends progressively more on glycolysis than oxidative metabolism of glucose. The finding that the Day-13 conceptus has not fully adapted to this method of ATP generation at low O2 concentrations may make it especially vulnerable during development in utero.
The production of carbon dioxide and lactate from glucose by sheep embryos and samples of extraembryonic membranes was measured during a 2.5 h incubation period. Both embryos and their membranes were active in the glycolytic and oxidative utilization of glucose and, in general, the utilization of glucose per unit weight fell as development progressed from Day 13 to Day 19 of pregnancy. Both oxidation of glucose and glycolysis by the extraembryonic tissues, expressed as activity per microgram dried tissue, fell progressively with development. The rate of decline in CO2 production was greater than the rate for glycolysis and, as a consequence, the contribution of glycolysis to the estimated energy yield from the catabolism of glucose rose with time. In the embryo, both glucose oxidation and glycolysis peaked on Day 15 with estimates of adenosine triphosphate (ATP) production from glucose per microgram dried tissue on this day being 50% above those on Day 13 and 100% above those on Day 17. In general, the estimated yields of ATP from glucose were similar for structures of the same developmental age except that, at Day 19, it was calculated that the rate of ATP production by embryos was double that by the extraembryonic membranes. In incubations using 5.56 mM glucose as sole exogenous energy source, glucose turnover by embryos and embryonic membranes tended to be higher in a bicarbonate-buffered medium than in HEPES (4-(2-hydroxyethyl)-1-piperazincethane sulfonic acid) and phosphate-buffered media. As a result, the estimate of ATP yield plus the contribution of oxidative pathways to this yield were significantly higher in this medium than in the others. Glucose turnover by the embryo and its membranes in bicarbonate-buffered medium containing 0.56 mM glucose plus the alternate substrates, lactate and pyruvate, was severely depressed. Further experiments using samples of trophoblast and yolk sac indicated that both reduction in glucose concentration and the presence of the other substrates contributed to this suppression. Furthermore, an interaction between these factors was evident with the effects of alternative substrates being exaggerated when glucose concentration was low.
Neither insulin nor epinephrine influenced the incorporation of glucose into the acid-soluble or acid-insoluble glycogen pool of mouse embryos at the morula-early blastocyst stage during 5 h culture in the presence of radiolabelled glucose. During a 5 h chase culture of pulse-labelled embryos at this stage of development, acid-soluble glycogen labelled during the pulse was not utilized by the embryo but acid-insoluble glycogen was reduced. Addition of glucagon, insulin, epinephrine, cAMP, theophylline or caffeine during chase culture had no effect on the turnover of label in the glycogen pools of the embryo. These results indicate that the turnover of embryonic glycogen observed in vivo is not due to the direct effect of the hormones that regulate glycogen metabolism in the mother.
The fallopian tubes of four ewes were cannulated so that secretions collected from the isthmus and ampulla of one side could be compared with the secretion from the other entire fallopian tube. Maximum fluid output from all sites occurred at oestrus and was least during the luteal phase of the cycle. Secretion from the ampulla was generally twice that from the isthmus.
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