Transfer of iron from uteroferrin (purple acid phosphatase) to transferrin related to acid phosphatase activity.

There is continuing controversy as to whether iron can be exchanged from the purple phosphatase, uteroferrin (Uf), to fetal transferrin (Tf) and whether this process might be of physiological relevance during pregnancy in the pig. Here, iron transfer from Uf to apoTf at pH 7.1 was followed by measuring the loss of acid phosphatase activity from native Uf as a function of incubation conditions and time. In the presence of apoTf and 1 mM ascorbate (but not in the presence of either agent alone), 50% of enzyme activity was lost in about 12 h. Loss of activity was accompanied by bleaching of Uf purple color and the appearance of the characteristic visual absorption spectrum of Fe-Tf. Citrate could replace ascorbate in the reaction. Loss of Uf iron did not occur at pH 5.3, at which pH Tf cannot bind Fe. [59Fe]Uf was prepared and shown to be identical in its enzymatic and physical properties with unmodified Uf. Transfer of 59Fe from Uf to apo-Tf was promoted by conditions identical to those which led to loss of purple color and acid phosphatase activity. However, the results suggested that only one of the two iron atoms at the bi-iron center on Uf was readily lost, and that exchange of the second iron occurred more slowly. Loss of iron made Uf more susceptible to denaturation. A third technique, quantitation of the g' = 4.3 signal of iron specifically bound to Tf by EPR, was also tested as a means assaying accumulation of Fe-Tf, but the method was too insensitive to measure the kinetics of iron transfer at physiological protein concentrations. We conclude that iron can be transferred directly from Uf to apoTf in the presence of low molecular weight chelators, and that the process is likely to be of physiological significance.

The excretion of salt load by the developing chick embryo.

Salt loads (0.17 or 0.34 mmol Na+; 6 M NaCl solution labelled with 24Na) were administered into the amnion of 7-day-old chick embryos. The 24Na distribution in embryonic blood, amniotic and allantoic fluids was measured in 1, 2, 4, 8, 12 and 24 h intervals to assess the kinetics of salt load movements in particular egg compartments. The aim was to estimate the efficiency of the embryonic homeostatic apparatus to maintain ionic balance in the internal environment of the embryonic body. The Na+ concentration in amniotic fluid was expected to rise after salt loading by about 275 and 400 mM, respectively. More than 10% of the salt dose per ml appeared in the embryonic blood 2 h after salt load administration while only 0.2% were found in the urine (collected as allantonic fluid). The maximal rise of 24Na activity in the blood of salt-loaded embryos reached 11%-12% of the dose which corresponded to an increase of Na+ concentration by 19 and 41 mM, respectively. The maximum of 24Na activity appeared in the allantoic fluid with a delay of several hours and indicated an increment of Na+ concentration by 6% and 9% of the dose per ml in the case of salt-loaded embryos. The Na+ concentration in the allantoic fluid (urine) never exceeded that in the blood. The final Na+ activity (estimated in the blood 24 h after salt loading) was equal to 5% of the dose per ml in both cases, indicating a persistent elevation of Na+ concentration by 8.6 and 17.2 mM, respectively.

The origin and fate of hyaluronan in amniotic fluid.

The mechanisms which regulate the steady-state concentration and molecular weight of hyaluronan in the amniotic fluid of sheep at different gestational ages have been investigated. An attempt to trace the origin of the polysaccharide has been made by analyses of various fetal fluids (amniotic fluid, allantoic fluid, tracheal fluid, urine, and serum). The fate has been studied by injection of radioactively labelled hyaluronan into the amniotic cavity and following the tracer in fetal tissues and fluids. The concentration of hyaluronan in amniotic fluid varies considerably but is in the order of 5 mg/l at mid-pregnancy and decreases to 1 mg/l in late pregnancy. The polysaccharide has a Mr-distribution with a weight-average in the order of 10(6) at 10 to 13 weeks of gestation which decreases to 10(5) closer to term. Calculations show that urine contributes 0.1 and 0.5 mg of low-molecular (Mr = 10(4) hyaluronan per day in mid- and late pregnancy, respectively, and the lung 10-20% of that amount in the form of high-molecular weight polymer (Mr greater than 10(6). The hyaluronan disappears from the amniotic cavity by bulk flow due to fetal swallowing. It is taken up and degraded in the fetal intestine. Molecules of Mr = 10(3) can pass the intestinal barrier. Calculations show that about 0.5 mg and 1.0 mg of hyaluronan is eliminated per day from the amniotic fluid at 12 and 17 weeks of gestation, respectively. Thus, the higher rate of elimination and the relatively high urinary contribution in more mature fetuses explain the low concentration and Mr of amniotic hyaluronan in late gestation, whereas a slower elimination combined with a relatively larger contribution of high molecular weight hyaluronan both from lung and urine and possibly from other sources are responsible for the higher concentration and Mr of the compound in early pregnancy.

Modulation of the immunosuppressive activities by blastocoelic fluid during rabbit pregnancy.

Temporal variation in immunosuppressive activity was determined in biological samples such as embryo-foetal fluids (blastocoelic- or amino-allantoic fluid) and blood collected from pregnant and pseudopregnant rabbits. Each of the fluids to be analyzed was pre-incubated with mitogen stimulated human lymphocytes for 48 h and then inhibition of [3H]thymidine incorporation or IL-2 receptor expression was estimated. Both means of assessing immunosuppression indicated variations in the suppressive activity throughout pregnancy. This was observed in embryo-foetal fluids but not in autologous peripheral blood nor in homologous pseudopregnant blood. At days 9-13 of pregnancy, the immunosuppressive effects of blastocoelic fluids were higher than that of the autologous sera, reached a peak at days 12 and 13 and declined thereafter, to reach the lowest levels. In order to further characterize the biological activity of day-12 blastocoelic fluid and autologous serum, they were submitted to ultracentrifugation. No suppressive activity could be demonstrated in the lipoprotein fractions. But all the activity was found in the protein fraction. Precipitation with cold ethanol confirmed that the biologically active compound was a protein. Furthermore, results obtained after ultrafiltration suggest biologically active compounds of high mol. wt (greater than 300 kDa). From the above findings, we can suggest that in the rabbit, there is no pregnancy specific systemic immunosuppression. We can also infer that (1) the immuno-tolerance of the mother towards the embryo is more due to a localized effect; (2) this effect decreases with the progression of gestation and (3) a high mol. wt factor is responsible for the immunosuppression.

Presence of the major progesterone-induced proteins of the sheep endometrium in fetal fluids.

Allantoic and amniotic fluids were collected on Days 60 (n = 3), 100 (n = 4), and 140 (n = 3) of pregnancy. The presence of uterine milk proteins (UTM-proteins) in these samples was evaluated by Ouchterlony immunodiffusion and enzyme-linked immunoabsorbant assay (ELISA). Eight of ten samples of allantoic fluid and three of ten samples of amniotic fluid produced one or two immunoprecipitin bands against antiserum to UTM-proteins. Each band fused with immunoprecipitin bands from UTM-proteins purified from uterine fluid. Data from a semi-quantitative ELISA indicated that allantoic fluid from all ewes and amniotic fluid from six of ten ewes contained immunoreactive UTM-proteins. Concentrations of UTM-proteins in these fluids were not statistically affected by day of gestation (p greater than 0.10), but tended to decline as gestation advanced. Greater concentrations of UTM-proteins were detected in allantoic fluid than in amniotic fluid (p less than 0.05). The physical characteristics of the immunoreactive material in allantoic and amniotic fluids were examined by polyacrylamide gel electrophoresis and Western blotting. The immunoreactive material was found to possess pIs and molecular weights identical to UTM-proteins. These results indicate that fetal fluids contain material that reacts with antiserum to UTM-proteins and has physical properties similar to UTM-proteins. It is likely, therefore, that the UTM-proteins are transported across the placenta during gestation, perhaps to serve some function in the fetal compartment.

Morphology and chemical analysis of the sheep conceptus from the 13th to the 19th day of pregnancy.

Between the 13th and 19th day of pregnancy the sheep conceptus developed into a structure showing considerable differentiation and all the extraembryonic membranes were established. Both length and dried weight of the embryo increased exponentially during this period. A highly significant regression of dried weight on length of embryos was found but measurement of the additional variable, width, did not improve the accuracy of estimating weight from the embryo's dimensions. The mass of the extraembryonic membranes also increased greatly. The dried weight of the trophoblast increased 90-fold over this period; that of the yolk sac increased 17-fold from day 15 to day 19. The protein content of each of the structures making up the sheep conceptus approached 50% of dried weight, which is similar to the proportion in adult soft tissues. The contribution of glycogen to dried weight was low in the sheep embryo and embryonic membranes when compared with estimates in the mouse blastocyst. However, at about the time of implantation the level of this polymer in the embryo was high compared with that in adult soft tissues and approached the level found in adult muscle. Concentrations of DNA and RNA in the sheep conceptus are much higher than the levels in most adult soft tissues and probably reflect higher synthetic rates and a smaller cytoplasmic volume in the embryonic cells.

Antiviral activity of the pregnancy recognition hormone ovine trophoblast protein-1.

Ovine trophoblast protein-1 (oTP-1) is an antiluteolytic protein which plays an important role in maternal recognition of pregnancy. It exhibits a 45-70% amino acid sequence homology with alpha interferons (IFN-alpha s) from various species. We show here that purified oTP-1 has high specific antiviral activity (2-3 X 10(8) units/mg protein) and is thus as potent as any known IFN. oTP-1 is antigenically distinct but does show antigenic relation to both ovine and bovine IFN-alpha s. The antiviral activity of oTP-1 could be demonstrated in Day 12 - Day 16 conceptus culture medium and in allantoic fluid from Day 60 of pregnancy. Our functional and antigenic characterization suggests that oTP-1 is a type of IFN-alpha which performs classic roles associated with IFN-alpha, as well as being the primary conceptus secretory product responsible for maternal recognition of pregnancy.

Embryonic osmoregulation: consequences of high and low water loss during incubation of the chicken egg.

The rates of water loss of domestic chicken eggs were varied during incubation to measure the osmoregulatory ability of the avian embryo. Egg water loss was increased by drilling holes in the eggshell over the airspace on day 13 (I = 21 days) and then placing these eggs in a low relative humidity (r.h.: 0-10%) incubator until hatch. Egg water loss was decreased by placing other eggs in a high-r.h. (85-90%) incubator on day 0. Eggs with low water loss (approximately 6% of initial fresh mass [IFM]) produced embryos and yolks that were not different in wet or dry mass when compared to control eggs that lost approximately 12% of IFM. However, 1-4 gm of excess albumen were left in low-water-loss eggs on day 21. Hatching success was 71% and 89% for low and control eggs, respectively. Low egg water loss did not appear to disturb embryonic growth. The allantoic fluid volume and millimolar allantoic Na+ and Cl- ions declined faster with high and slower with low rates of water loss. Thus, excess water was lost as a result of increased movement of water out of allantoic fluid, which was due to increased active transport of Na+ ions by the chorioallantoic membrane (CAM). Eggs with high water loss had elevated Cl- levels after day 17 in plasma and amniotic fluid, which indicated a period of osmotic stress after depletion of allantoic fluid between day 18 and hatch. The decrease in wet embryo mass measured in embryos from high-water-loss eggs was due principally to dehydration of skin. Embryonic skin may serve as an emergency water reservoir during osmotic stress. Dehydrated chicks produced from high-water-loss eggs were 6 gm less in wet mass at hatch compared to controls. However, these chicks regained the water deficit 7 days after hatch and grew at a rate not different from control chicks through 6 weeks of age. Total egg water loss of 12% of IFM results in highest hatching success. However, water losses between 6% and 20% of IFM do not appear to affect adversely the growth or water content of the chick. Water losses above 20% of IFM cause early depletion of allantoic fluid, prolong the period of osmotic stress, and result in subsequent dehydration of blood, amniotic fluid, and embryonic skin.(ABSTRACT TRUNCATED AT 400 WORDS)

Ovine fetal urine contribution to amniotic and allantoic compartments.

The ovine pregnancy has been extensively studied as a model for amniotic fluid metabolism potentially serving as a model for human oligohydramnios. However, it is recognized that the ovine pregnancy contains an allantoic fluid compartment not present in human pregnancies at term. Earlier studies in sheep suggested that the fetal urine contribution to allantoic fluid diminished at term, but without an explanation for maintenance of the significant volume of the allantoic cavity. In the present study we examined the relative fetal urine excretion of 3H-inulin into the allantoic and amniotic cavities in the near-term ovine pregnancy. Amniotic and allantoic volumes, as determined by technetium-labelled dextran and chromium-tagged erythrocyte dilution, were similar although there were significant differences in composition. The allantoic fluid compartment received an equal or greater proportion of fetal urine than did the amniotic fluid during the study period. These results indicate the importance of the allantoic fluid volume and composition in the study of ovine fluid dynamics.

The basic proteins of bovine allantoic fluid.

This paper reports the separation of highly cationic proteins (i.e. pI greater than 9.0) of bovine allantoic fluid and their possible pathogenic properties. Experimentally, polycations and cationic proteins of pI greater than 10 induced intravascular coagulation and hemolysis, as well as precipitation of fibrinogen and proteinuria. Bovine allantoic fluid collected at the time of calving contains from 0.6 to 1.3 g of proteins per liter (11 samples). Ion-exchange chromatography, followed by either chromatofocusing or heparin-sepharose-6 beta binding, and, finally, gel filtration separated several fractions and subfractions. These were examined later using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and at least ten constituents were visualized. Two components, Mr 34,000, pI 9.7, and Mr 38,000, pI 9.6-9.0, accounted for 33% of the basic proteins present in allantoic fluid and 0.7% of its total protein content. Electrophoretic mobility was unaltered by beta-mercaptoethanol, and periodic acid-Schiff staining was negative. These proteins were not found in ox plasma. The major basic proteins were bound to red cells and platelets. Cell electrophoretic mobility decreased linearly with the logarithm of protein concentration. At concentrations between 10(-6) and 10(-5) M, red cell clumping was rare; hemolysis and platelet agglutination were not observed.

The concentration of bovine placental lactogen and the incidence of different forms in fetal cotyledons and in fetal serum.

The concentration of bovine placental lactogen (bPL) was determined in fetal placentomes, allantoic fluid, amniotic fluid, maternal and fetal plasma throughout pregnancy. In addition, chromatofocusing chromatography was used to separate the different forms of bPL found both in fetal serum and in placental homogenates in order to determine whether the different forms that have been reported to exist in the cotyledon are also found in the fetal circulation. Reproductive tracts were collected from cows between 109 and 247 days of pregnancy. The concentration of bPL in the fetal cotyledonary tissue was measured by both radioreceptor assay and radioimmunoassay, both assays showed that the concentration of bPL in the fetal portion of the placentomes remained constant throughout the period of pregnancy tested. The mass of the placenta increased approximately 10-fold during the period of study but the concentration of bPL in the maternal plasma was low (0.9 +/- 0.1 ng/ml) at all stages of pregnancy tested. The mean concentration of bPL (Mean +/- S.E.M.) in amniotic and allantoic fluid was 0.4 +/- 0.1 and 1.2 +/- 0.2 ng/ml respectively. Fetal blood contained the highest concentrations of bPL, from 11.6 to 18.4 ng/ml, and the concentration tended to decrease with advancing gestation (slope = 0.07, P = 0.001). Several forms of bPL were found in the fetal circulation; however, a higher percentage of forms with more acidic isoelectric points were found in the fetal serum than in placental homogenates. These results suggest that either some forms of bPL are more stable or that the hormone isolated from placental tissue is not representative of the final secreted product.

Calcium regulation in the embryonic chick. III. Calcium and phosphate in serum and allantoic fluid of normal and shell-less embryos.

Calcium metabolism of chicken embryos was profoundly affected by incubation in shell-less culture, but phosphate metabolism was largely undisturbed. Shell-less embryos exhibited hypocalcemia and hypocalciuria relative to normal embryos but had similar levels of phosphate in serum and allantoic fluid. The concentration of calcium in allantoic fluid declined during incubation in both groups, owing largely to accompanying increases in allantoic volume, but total amounts of calcium in the allantois did not vary with time. Both normal and shell-less embryos maintained higher concentrations of calcium in serum than in allantoic fluid, with shell-less embryos maintaining a larger gradient between serum and allantoic compartments. In contrast, serum and allantoic concentrations of inorganic phosphate increased over time in both normal and shell-less embryos, and both groups maintained generally higher concentrations of inorganic phosphate in the allantoic sac than in serum. Treatment of embryos with parathyroid hormone had no effect on calcium and phosphate metabolism. Embryos maintained in shell-less culture grew more slowly than those incubated normally and consequently had a dry mass about half that of normal embryos on day 18. Shell-less embryos also exhibited abnormalities in fluid balance, which were reflected in their inability to maintain normal allantoic volume and in their higher relative hydration compared to embryos incubated in ovo.

Anaerobic metabolism in fowl embryos during normal incubation.

Lactate concentration in blood, liver, yolk, amniotic and allantoic fluid and blood pyruvate was measured in embryos in the final week of incubation. Blood lactate was low up to day 18. The blood lactate/pyruvate ratio and liver lactate increased from day 19 until hatching. From day 14 to 19, lactate concentration in amniotic fluid remained constant, it increased 2-fold in yolk and 10-fold in allantoic fluid. There was only a 48% net accumulation of lactate in the three cavities. In conclusion, fowl embryos do not turn to anaerobic metabolism until the hatching process starts on day 19.

[Biochemical indices of the allantoic fluid of chick embryos infected with the influenza virus]. / Biokhimicheskie pokazateli allantoisnoi zhidkosti kurinykh émbrionov, zarazhennykh virusom grippa.

Virus-containing allantoic fluid (VCAF) serves as raw material for influenza virus manufacture in the USSR. VCAF composition varies greatly from batch to batch. The causes of instability of its composition were studied. The main high molecular admixtures determining VCAF instability were found to include proteins and lipids getting into VCAF with amniotic fluid, blood, and yolk of the embryo when VCAF is collected. Physicochemical characteristics (pH, protein and lipid concentrations, protein composition) for VCAF control at pooling are proposed.

Purification of chick interferon by zinc chelate affinity chromatography and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

An improved purification method for chick interferon from the allantoic fluid of embryonated chick eggs is described. Interferon prepurified by perchloric acid treatment, zinc acetate precipitation, and chromatography on SP-Sephadex C-25 was further enriched by column chromatography on zinc chelate. Analysis on sodium dodecylsulfate polyacrylamide gel electrophoresis of the interferon preparation with a specific activity of 8 X 10(5) units/mg protein shows that the major antiviral activity migrated in a broad band in the range of 20-29 kD molecular weight. Several protein bands were stainable with Coomassie blue and silver nitrate in this molecular weight range. Between 80 and 95% of the total protein charged to the gel could be removed from the interferon containing fractions by sodium dodecylsulfate polyacrylamide gel electrophoresis.