J.B. Lloyd

Sources of amino acids for protein synthesis during earlyorganogenesis in the rat. 3. Methionine incorporation

Rat conceptuses at 9.5 days post-conception were cultured for 27 h in whole rat serum. [3H]Methionine, or rat serum proteins containing [3H]methionine, was introduced at 24 or 6 h before the termination of the culture. The total clearance of radioactivity into the embryo and the visceral yolk sac from the two sources was measured; also the extent to which the accumulated radioactivity was acid-insoluble. Similar experiments, but using [3H]leucine, were performed for comparison. The results indicate that free amino acid and protein can both serve as sources of amino acids for incorporation into the embryo and yolk sac, and it is estimated that in vivo over 95 per cent of the methionine (and the leucine) incorporated into these tissues arises from protein captured and digested by the yolk sac. Almost all the leucine accumulated into the conceptus is present as protein, but a larger fraction of the methionine accumulated is found in acid-soluble form. When the amino acids were delivered in the form of plasma proteins, the incorporation of methionine was two to three times more efficient than that of leucine, an observation most readily explained by leucine being provided in excess of requirements. In the light of reports that an adequate concentration of free methionine is important for the normal development of rat embryos in vitro, it is concluded that, although most of the amino acid required by the embryo is supplied as protein, the small fraction supplied as free amino acid may be critical for methionine but probably not for leucine.

Sources of amino acids for protein synthesis during earlyorganogenesis in the rat. 4. Mechanisms before envelopment of the embryo by the yolk sac

It was previously shown that uptake and digestion of protein by the visceral yolk sac supplies almost all of the amino acid needed by the 9.5-11.5-day rat conceptus cultured in vitro. Our aim was to test the hypothesis that protein uptake and digestion may not be as important as an amino acid source in the 8.5-9.5-day period, a stage of development before the yolk sac placenta envelops the embryo and before the vitelline circulation is established. Eight and a half-day rat conceptuses were cultured in serum supplemented with trace amounts of free [3H]leucine, [3H]leucine-containing serum proteins, free [3H]methionine or [3H]methionine-containing serum proteins. The incorporation of radiolabelled amino acid into acid-soluble and acid-insoluble fractions of the conceptus was determined. Leucine from either source was incorporated principally into proteins of the conceptus, but a greater proportion of the methionine incorporated was found in the low molecular weight fraction. It is estimated that 88 per cent of the leucine and 96 per cent of the methionine used by the conceptus was derived from protein in the culture serum; free amino acid comprised a minor supply source. We conclude that, despite the different anatomic relationships, the majority of amino acid incorporated into newly synthesized proteins of the conceptus very early in organogenesis is supplied by the digestion of protein in extraembryonic tissue, most likely the visceral yolk sac.

Sources of amino acids for protein synthesis during early organogenesis in the rat. 2. Exchange with amino acid and protein pools in embryo and yolk sac

Tenth-day rat conceptuses were cultured in whole rat serum containing [3H]leucine and harvested after 24 or 48 h. Hydrolysates of the acid-precipitable fraction of embryo or yolk-sac homogenates were prepared and subjected to paper chromatography. Liquid scintillation counting of the separated amino acids showed that leucine was the only amino acid with above-background radioactivity. This established that radiolabel was not transferred from leucine to other amino acids in the cultured rat conceptus. Tenth-day rat conceptuses were cultured in whole rat serum containing [3H]leucine, as above. After 19 h, some conceptuses were harvested; other conceptuses were rinsed, transferred to culture medium without [3H]leucine, and after a further 24 h of culture the embryos and yolk sacs were harvested. A comparison of the protein-associated radioactivity of embryo and yolk sac before and after culturing for the further 24-h period showed that these structures quantitatively conserve radiolabelled leucine incorporated into their proteins. Further experiments involved culturing the rat conceptus for 24 h as above but in the presence of either [3H]leucine or [3H]leucine-labelled serum proteins. After harvesting the conceptuses, the specific radioactivity of [3H]leucine was determined in the acid-soluble and acid-precipitable fractions prepared from embryo and yolk-sac homogenates. The specific radioactivity of [3H]leucine in the acid-soluble fraction of embryos or yolk sacs from conceptuses grown in the presence of radiolabelled protein was about 120 per cent of that in the culture medium, while that in the acid-precipitable fractions was about 70 per cent of that in the culture medium. By comparison, the specific radioactivity of [3H]leucine in the acid-soluble fraction of embryos and yolk sacs from conceptuses grown in the presence of free [3H]leucine was only 3-4 per cent of that in the culture medium, while that in the acid-precipitable fraction was about 1 per cent of that in the culture medium. If our data on the fate of leucine incorporated into newly synthesized proteins of the early post-implantation rat embryo can be extrapolated to the other amino acids, they suggest that once amino acids have been incorporated into newly synthesized protein in embryo or yolk sac, they are not exchanged to any detectable extent with amino acid pools outside the conceptus. The results also provide independent confirmation of our earlier conclusion that the only significant source of amino acids utilized by the 10th-day embryo is protein taken up by the yolk sac and digested intracellularly.(ABSTRACT TRUNCATED AT 400 WORDS)

Leucine sources for the rat fetus.

Fetal and maternal plasma were assayed for the concentration of free leucine, acid-insoluble radioactivity and acid-soluble radioactivity at intervals after an intravenous bolus injection of [3H]leucine into anaesthetized pregnant rats at 17.5 days post-conception. The concentrations of total free leucine and of free [3H]leucine in maternal and fetal plasma were effectively unchanged from 5 to 180 min post-injection. Plasma free leucine concentrations in the fetus were five times those in the mother. The concentration of free [3H]leucine in fetal plasma was similar to that in maternal plasma. Thus the specific radioactivity of free leucine in fetal plasma is substantially lower than that in maternal plasma, indicating that a significant portion of the free leucine in plasma of the 17.5-day rat fetus comes from a source other than the free leucine in the maternal plasma. The data are consistent with a major contribution of amino acids coming from the degradation of extraembryonic protein in the yolk sac. Other possible sources of unlabelled leucine are discussed.

Pinocytosis in the rat visceral yolk sac: Potential role in amino acid nutrition during the fetal period

At 9.5 to 10.5 days post-conception (p.c.) the rat embryo receives amino acids primarily by pinocytosis of maternal proteins by the visceral yolk sac (VYS) endodermal cells and their subsequent digestion in lysosomes. As this mechanism may also contribute to the nutritional support of the rapidly growing fetus later in gestation, this study determines the potential of the VYS to contribute to the amino acid needs of the rat fetus on 13.5, 15.5 and 17.5 days p.c. Wistar rats were killed on 11.5, 13.5, 15.5, 17.5 and 19.5 days p.c. and the protein content of fetuses, VYSs and serum was determined. Additional VYSs were isolated on the same days p.c. and the uptake of [14C]sucrose, a measure of fluid-phase pinocytosis, was determined. The rate of protein accretion by the fetus and the capacity of the VYS to pinocytose and digest proteins were calculated on 13.5, 15.5 and 17.5 days p.c. There was a decrease in pinocytic capacity, per mg protein, over the period of development studied but, owing to the growth of the tissue, the total capacity of the yolk sac to pinocytose and digest proteins increased from 13.5 to 17.5 days p.c. Comparison of the VYSs capacity to pinocytose and digest proteins and the rate of protein accretion by the fetus suggests that the VYS could support a significant proportion of the increase in protein content of the growing fetus. This conclusion is reinforced by the limited capacity of the maternal plasma to supply free amino acids to the fetus.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid requirements in the early post-implantation rat conceptus

Amino acid analysis has been performed on hydrolysates of embryos/fetuses, visceral yolk sacs and ectoplacental cones/placentae from early post-implantation rat conceptuses. The increments in each amino acid between 10.5 and 11.5 days, between 11.5 and 12.5 days, and between 12.5 and 13.5 days, are expressed as percentages of the total amino acid increment. These three profiles are very similar to each other and also strongly resemble the amino acid composition of hydrolysates of rat serum. The results are discussed in the context of the hypothesis that a transudate of plasma is responsible for the amino acid nutrition of the embryo at this stage of gestation, and that inhibition of this pathway can lead to the induction of congenital defects. The results suggest that an inhibition of pinocytosis or lysosomal proteolysis would affect the supply of all protein-derived amino acids to approximately the same extent: there is no indication that the supply of any particular amino acid would be particularly vulnerable.

Uptake and processing of 59Fe-labelled and 125I-labelled rat transferrin by early organogenesis rat conceptuses in vitro.

The delivery of iron to the early organogenesis rat embryo has been studied, using 59Fe- and 125I-labelled rat transferrin. Rat conceptuses at 9.5 days postconception were cultured for 27 or 51 h in whole rat serum. Rat transferrin labelled with 59Fe was added for the final 0.1, 0.5, 6, 24 or 48 h of culture. Radioactivity accumulated progressively in both the embryo and the visceral yolk sac. Similar results were obtained when unconjugated 59Fe3+ was added to the rat serum used as culture medium. Both acid-soluble and acid-insoluble 59Fe were substantially present in the embryo and yolk sac after all exposure periods. When conceptuses were cultured in the presence of 125I-labelled rat transferrin, acid-soluble radioactivity was progressively released into the culture medium, but accumulation into the embryo and visceral yolk sac was slight and did not change with duration of exposure to the labelled protein. Similar findings were obtained using 125I-labelled bovine serum albumin. In these experiments, there was a close correspondence between the amount of iron accumulated by the embryo and visceral yolk sac in the final 24 h of a 51-h culture and the amount of transferrin converted into acid-soluble products in the same period. Visceral yolk sacs from 17.5-day pregnant rats were explanted and cultured in the presence of 59Fe-labelled rat transferrin, 125I-labelled rat transferrin or 125I-labelled bovine serum albumin, for periods up to 3 h. Again uptake of 59Fe increased with time of incubation, and the 125I-labelled proteins were digested to acid-soluble products which were released into the culture medium. The results indicate that transferrin delivers iron for incorporation into both the embryo and the visceral yolk sac, and are consistent with a mechanism involving receptor-mediated endocytosis of iron-laden transferrin by the cells of the visceral yolk sac. The transferrin itself appears to be quantitatively degraded, following delivery of iron to the yolk sac cells, a result that differs from findings in other cell types, in which the protein is not degraded but returns to the plasma membrane to participate in further cycles of iron acquisition and delivery.