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The effect of a novel recombinant bovine interferon on the rate of protein synthesis, as measured by the rate of leucine incorporation, in endometrial explants from cows and ewes was investigated and compared with the effect of trophoblast secretory proteins and of a recombinant bovine interferon-alpha1. Exposure of cultured bovine endometrial tissue to the novel recombinant bovine interferon mimicked the effect of pregnancy in reducing the rate of protein synthesis in both caruncular and intercaruncular endometrium. This effect was similar to that seen with naturally produced trophoblast secretory proteins and it was more pronounced than that seen when endometrial tissue was treated with a related recombinant bovine interferon-alpha1. Ovine endometrial cultures were unaffected by exposure to this novel recombinant bovine interferon. This novel recombinant interferon exhibited potent antiviral properties (an activity of 1.6 x 10(10) international reference units/mg protein) and may prove to be a valuable reagent in studying the maternal recognition of pregnancy in the cow and reducing embryo mortality.

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Parturition in sheep is initiated by a rapid rise in fetal plasma cortisol. There is some controversy as to the exact nature of the drive for this pre-partum cortisol surge and it is thought that factors other than ACTH may act in concert to stimulate the development of the fetal adrenal gland. We have investigated the concentrations of ACTH and other peptides derived from pro-opiomelanocortin (POMC) in the circulation of fetal sheep during the final part of gestation, using specific 2-site immunoradiometric assays. The expected rise in fetal cortisol was seen with an 880% (p < 0.01) increase in concentration of this hormone between the initial measurement period (110-119 days gestation) and the final period (139-147 days). Fetal plasma ACTH increased less dramatically (137%; p < 0.03) during this time. The most surprising finding was the presence of very high relative concentrations of the N-terminal POMC peptide N-POMC(1-77) in the fetal circulation. Initially the concentration was 289 +/- 66 pmol/l compared to ACTH concentrations of 6.4 +/- 0.8 pmol/l. In the final week of gestation N-POMC(1-77) levels, although still high, had declined to 188 +/- 35 pmol/l (ACTH having increased to 13.7 +/- 2.2 pmol/l). Fetal plasma 3 yen-MSH was found to increase towards the end of gestation when the concentration of N-POMC(1-77) was declining, suggesting some cleavage of the latter. We postulate that the N-POMC(1-77) and its fragments, acting in concert with ACTH, play a vital role in stimulating the development of the fetal adrenal cortex and provide the additional drive to the adrenal gland required to stimulate parturition.

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1. The effects of insulin, glucagon and a supply of exogenous amino acids on protein degradation have been studied in isolated perfused livers from growing chickens by measuring the rate of net valine release in the presence of cycloheximide. 2. Insulin inhibited protein degradation as did a supply of exogenous amino acids. 3. Addition of glucagon increased uric acid release from the livers but had no significant effect on protein degradation. 4. When the effects of the hormones and amino acid mixture are compared with published data for the rat it is evident that the action of glucagon differs in the two species.

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1. The polymerase chain reaction has been used to amplify specifically the cDNA coding for the secreted form of ovine trophoblast protein-one from a preparation of total cellular RNA extracted from sheep embryos removed from ewes 16 days after mating. 2. Cloning and sequencing of the amplified cDNA revealed two new sequence variants: SPW49 having 93% similarity with deduced amino acid sequences from published cDNA data, and SPW27 a variant coding for a deleted form of ovine trophoblast protein-one. 3. The gene for ovine trophoblast protein-one is intronless. 4. This study provides further evidence for the existence of an ovine trophoblast protein-one gene family. 5. Both variants contain a potential N-glycosylation site not apparent in published sequences for ovine trophoblast protein-one.

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The ability of the trophoblast of the ovine preimplantation blastocyst to take up and metabolise proteins has been investigated using two experimental approaches, microscopical and radiochemical. The ultrastructure of the expanded blastocyst obtained from 14 and 17 day pregnant ewes was examined. The morphology of tissues maintained in culture for 24 h has been compared with that of fresh tissues. After culture, the cellular morphology of the explants was well preserved. Fresh and 24 h cultured tissues were incubated with horse-radish peroxidase and ferritin and these proteins subsequently were found to be localized in coated pits, caveolae and secondary lysosomes of the trophoblast. Comparison of the uptake of [3H]dextran and of 125I-labelled bovine serum albumin indicated that proteins could be taken up by cultured tissue by mechanisms in addition to simple fluid phase endocytosis. During culture of explants of blastocyst with 125I-labelled bovine serum albumin, a large fraction of the radioactivity taken up by the tissue appeared in the TCA-soluble fraction of the culture medium indicating that cultured trophoblast hydrolysed proteins. That amino acids released from captured protein could be used for protein synthesis by the trophoblast was indicated by the labelling of tissue and medium proteins after culturing explants with beta-lactamase labelled with [14C]leucine. A major product (Mr approximately 17 x 10(3) present in the medium was likely to have been ovine trophoblast protein-1. It is concluded that, during the expansion of the ovine blastocyst, the trophoblast has the ability to take up proteins, transport them to lysosomes and degrade them to amino acids which are used for protein synthesis. Thus proteins, as well as free amino acids, present in the histotrophe may be an important source of nitrogen for the sheep conceptus in the critical period just prior to implantation.

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Estimates have been made of the irreversible loss of alanine and of glycine in chronically catheterized fetal lambs and in sucking lambs using [U-14C]-labelled radioisotopes. The experiments in the fetal lambs were carried out at least 5 d after implantation of catheters. The mean concentration of glycine in fetal femoral arterial blood between 102 and 129 d conceptual age was 755 mumol/l and this was not significantly different from that in maternal venous blood. The mean concentration of alanine in fetal femoral arterial blood during the same period of gestation was 229 mumol/l and this was significantly greater than that in maternal venous blood. Assuming a catenary model, the mean irreversible loss of glycine, determined using the single-injection technique, in three fetal lambs of 107, 111 and 127 d conceptual age was 17 mumol/min per kg, whereas in two fetal lambs aged 106 and 109 d into which the isotope was infused continuously the mean irreversible loss, calculated from the specific activity of glycine 5 h after the start of infusion of the tracer ('pseudo plateau'), was 12 mumol/min per kg. In a sucking lamb, 9 d after birth, the irreversible loss of glycine was 11 mumol/min per kg. The mean irreversible loss of alanine, determined by the single-injection technique assuming a catenary model in five fetuses between 112 and 121 d conceptual age was 14 mumol/min per kg, and in two sucking lambs, 9 and 11 d after birth, it was 5.1 mumol/min per kg. When a two-pool model was assumed in which entry of metabolite was not directly into the sampling pool but was by way of the second pool, then the mean irreversible loss of glycine in the three fetuses was 23 mumol/min per kg and of alanine in the five fetuses was 32 mumol/min per kg. Calculations based on the alternative two-pool model did not alter appreciably the rates of irreversible loss of either alanine or glycine in the sucking lambs. From a comparison of the specific activities of the amino acids and of carbon dioxide in blood during the course of the experiments, it was found that in the fetuses 0.96% of the CO2 present in blood was derived from alanine and only 0.12% was derived from glycine. It was calculated that not more than 1.6 mumol alanine/min per kg and 0.29 mumol glycine/min per kg could have been converted to CO2 in the fetal lambs.(ABSTRACT TRUNCATED AT 400 WORDS)

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The activities of 3-hydroxybutyrate dehydrogenase, 3-ketoacid CoA-transferase and acetyl-CoA acetyltransferase have been measured in the kidney, heart and brain of fed and four-day fasted ewes. The results indicate tha there is a decrease in the capacity of these organs to catabolise ketone bodies in fasting ketosis.

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In order to assess the extent to which metabolism within the sheep placenta may influence the transfer of metabolites between mother and foetus at different stages of gestation the activities of enzymes concerned with some aspects of carbohydrate, amino acid and keton body metabolism were determined in placental cotyledons resected from ewes during the last three months of pregnancy. The activities of pyruvate kinase (EC 2.7.1.40), lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37), ATP citrate (pro-3S)-lyase (EC 4.1.3.8), citrate (si)-synthase (EC 4.1.3.7), acetyl-CoA synthetase (EC 6.2.1.1), acetyl-CoA acetyltransferase (EC 2.3.1.9) and 3-keto acid CoA-transferase (EC 2.8.3.5) per gram wet weight cotyledon do not change during the period studied. The activities of alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1), isocitrate dehydrogenase (NADP+) (EC 1.1.1.42), ornithine-oxoacid aminotransferase (EC 2.6.1.13) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) show an increase in activity between the third and fourth months of pregnancy whilst the activities of arginase (EC 3.5.3.1) and possibly pyruvate carboxylase (EC 6.4.1.1) show an increase in activity between the fourth and final months of pregnancy. Ornithine decarboxylase (EC 4.1.1.17) activity declines to one tenth of its activity during this later period. The absence of detectable activities of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) and ornithine carbamoyltransferase (EC 2.1.3.3) indicate that gluconeogenesis and urea synthesis from ammonia do not occur in the sheep placenta. It appears that the ability of the placenta to metabolise several substrates is achieved by the time the placenta reaches its maximum size at approximately 90 days.

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1. In order to assess whether the potential ability of heart ventricular muscle and liver to metabolise substrates such as alanine, aspartate and lactate varies as the sheep matures and its nutrition changes, the activities of the following enzymes were determined in tissues of lambs obtained at varying intervals between 50 days after conception to 16 weeks after birth and in livers from adult pregnant ewes: lactate dehydrogenase (EC 1.1.1.27), alanine aminotransferase (EC 2.6.1.2), pyruvate kinase (EC 2.7.1.40), pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (GTP)(EC 4.1.1.32), malate dehydrogenase (EC 1.1.1.37), aspartate aminotransferase (EC 2.6.1.1) and citrate (si)-synthase (EC 4.1.3.7). 2. In the heart a most marked increase in alanine aminotransferase activity was found throughout development. During this period the activities of citrate (si)-synthase, lactate dehydrogenase and pyruvate carboxylase also increased. There were no substantial changes in the activities of aspartate aminotransferase, malate dehydrogenase or pyruvate kinase. Pyruvate kinase activities were five times greater in the heart compared with those found in the liver. No significant activity of phosphoenolpyruvate carboxykinase (GTP) was detected in heart muscle. 3. In the liver the activities of both alanine aminotransferase and aspartate aminotransferase increased immediately following birth although the activity of alanine aminotransferase was lower in livers of pregnant ewes than in any of the lambs. As with alanine aminotransferase the highest activities of lactate dehydrogenase were found during the period of postnatal growth. No marked changes were observed in malate dehydrogenase or citrate (si)-synthase activities during development. A small decline in pyruvate kinase activity occurred whilst the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase (GTP) tended to rise during development.

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