Plasma prostaglandin F2 alpha and reproduction in the female Triturus carnifex (Laur.).

Plasma patterns of prostaglandin F2 alpha (PGF2 alpha) and sex hormones (progesterone, androgens and 17 beta-estradiol) have been studied in the female crested newt, Triturus carnifex (Laur.), during the annual sexual cycle. The effects of exogenous PGF2 alpha on sex hormones were determined. In addition, the effects of one week's captivity on plasma PGF2 alpha and sex hormones were reported. PGF2 alpha plasma level peaked in April, was low in summer, and progressively increased during the autumn to peak again in December. The April PGF2 alpha coincided with a 17 beta-estradiol rise, and with a progesterone drop. The autumn PGF2 alpha increase was coupled to a 17 beta-estradiol rise, and therefore it has been tentatively related to ovary and oviduct development. In newts collected in April, moreover, a PGF2 alpha-dependent 17 beta-estradiol synthesis could occur, since PGF2 alpha injection induced a significant 17 beta-estradiol plasma increase. These findings led us to suppose that PGF2 alpha intervenes in spring breeding season termination through the induction of a 17 beta-estradiol synthesis as in other amphibian species. PGF2 alpha injection caused a progesterone decrease, probably by inducing corpora lutea lysis. The patterns of plasma sex hormones were consistent with the results reported for the same newt species.

Sex steroid profile and plasma vitellogenin during the annual reproductive cycle of the crested newt (Triturus carnifex Laur.).

The annual reproductive cycle of the crested newt, Triturus carnifex, has been studied in the field. Temperatures, rainfall, humidity, and photoperiod were recorded throughout the year. Adult male and female newts were sampled monthly; snout vent lengths, crest heights of males, and body ovarian, oviducal, testicular, and abdominal gland weights were recorded. Plasma samples were assayed for androgen, estradiol-17 beta, and progesterone by radioimmunoassay and for vitellogenin by enzyme-linked immunosorbent assay. Air, deep water, water surface, and soil temperatures were low from October to March, but increased in April and May without consistent summer variations. Ovarian and oviducal weights increased in October to reach maximum values between January and March (reproductive period). Crest height and abdominal gland weight in males mirrored the ovarian and oviducal pattern, while testicular weights were maximal in October and November. In females, plasma androgens were high during the reproductive period, and plasma estradiol peaked sharply in March, while plasma progesterone changed little. In the males plasma androgen and estradiol concentrations were similar to those of females, while plasma progesterone was significantly correlated with the cycle in testicular weight. In both sexes androgens showed a significantly negative correlation with air and water surface temperature. Plasma vitellogenin peaked in March but it did not correlate with either ovarian weight or plasma estradiol concentrations. These data support and confirm those previously reported for newts under laboratory conditions. The negative correlation between androgens and temperature suggests that this hormone may trigger the reproductive process. Moreover the correlations between plasma progesterone and testicular weight may indicate that this hormone is involved in male newts reproduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological properties of three types of granulocytes in circulating blood of the newt.

1. The electrophysiological properties of three subtypes of granulocytes obtained from the circulating blood of the newt, Triturus pyrrhogaster, were studied using the whole-cell variation of the patch-electrode voltage-clamp technique. 2. Neutrophils were identified by their multilobulated nucleus in the cytoplasm. Basophils and eosinophils, having characteristic granular structures in their cells, could definitely be distinguished from other leucocytes. The reliability of cellular identification using phase-contrast microscopy was confirmed by fixing and staining the granulocytes with Wright's solution. 3. In neutrophils under current-clamp conditions, a hyperpolarization-induced conductance increase was observed. With depolarization, however, no changes in regenerative potential were detected. When voltage clamped in standard saline (containing 96 mM-NaCl), hyperpolarizing voltage pulses to a potential more negative than -90 mV evoked slowly decaying inward currents. 4. The hyperpolarization-evoked membrane currents in neutrophils were identified as anomalous rectifying K+ currents, since (1) externally applied Cs+ (0.1 or 1 mM) or Ba2+ (1 mM) produced suppressive effects on the currents, (2) replacement of external Na+ with choline ions eliminated the decay of macroscopically observed currents, and (3) both the amplitude and kinetic properties of the currents were strongly dependent on membrane potential as well as on external K+ concentration; the activation of the conductance depended on the electrochemical force for K+ rather than on membrane potential alone. The magnitude of steady-state conductance was roughly proportional to the square root of the external K+ concentration. 5. In basophils and eosinophils, no major time- or voltage-dependent increase in conductance was detected at voltages between +20 and -130 mV. However, under current-clamp conditions, spontaneous fluctuation of zero-current potentials was clearly apparent, presumably due to the activities of some ion channels generating a small amount of current flux through the membranes of these cells. 6. It was concluded that the three subtypes of granulocytes in the newt differ considerably not only in appearance and structure but also in the electrical properties of their membranes. The anomalous rectifying K+ channels in neutrophils may serve to determine the resting potential of the cell at K+ equilibrium potential. The closure of the channels at depolarization might facilitate the maintenance of depolarization triggered by stimuli accompanying current influx.

The first sequenced normal hemoglobin lacking histidine in position 146 of the beta-chains. The primary structures of the major and minor hemoglobin components of the great crested newt (Triturus cristatus, Urodela, Amphibia).

The hemoglobin of the Great Crested Newt (Triturus cristatus), an animal maintaining the gas exchange to about 85% through the skin, consists of a major (HbM = 65%) and a minor (Hbm = 35%) component. The primary structures of the four chains are presented. They could be separated by reversed-phase HPLC and were cleaved with trypsin and additionally by acid hydrolysis. Both the native chains and their peptides were sequenced by liquid and gas phase sequenators. At the N-terminus the alpha M-chains are by one amino-acid residue longer and the beta M-chains by one residue shorter, resulting in a chain length of 142 and 145, respectively. The alpha m-chains are of normal length whereas in the beta m-chains the C-terminal histidine in position 146 is missing. Both alpha-chains differ by 50 residues (35.2%) and the beta-chains by 63 (43.2%). The alpha-chains were compared with those of other salamandroid hemoglobins. The difference to human hemoglobin is marked by 61 (43.3%) amino-acid substitutions in both alpha-chains and by 78 (53.4%) in both beta-chains. Numerous heme contacts and positions involved in the subunit interface are affected by replacements. The most interesting of them were studied by molecular modeling. The importance of the missing beta m-146(HC3)His and of the substitution of several amino-acid residues involved in the binding of organic phosphates is discussed with respect to the reduced Bohr effect of Triturus cristatus hemoglobin.

Sites and trend of erythropoiesis in anemic, normal, and splenectomized newts.

Newts, Triturus cristatus carnifex (Laurenti), were anesthetized by submersion in 2% chlorbutol in tap water for 15 min, splenectomized and then rendered totally anemic two months later by treatment with acetylphenylhydrazine (APH) diluted in their tanks (25 mg/liter for 36 h, changing the solution every 12 h). In the 14 weeks following hemolysis, erythron restoration occurred with the same intermittence as it did in whole animals rendered anemic by APH treatment: Beginning the second week the red blood cell count progressively increases for about one month, followed by a period of stasis which lasts about three weeks, then by a new increase, and then by a final period of stasis. Histological examination shows that erythropoietic activity occurs partly in the circulating blood and partly in erythroblasts nestled in the crypts between the muscular trabeculae of the ventricle as well as in the atrial walls. These cells, which are not part of the freely circulating elements in the blood stream, become very abundant in both whole and splenectomized anemic newts but are also present in normal animals. Newts, thus, have three sites for erythropoiesis: the spleen, the blood stream, and the heart. The other components compensate for the elimination of the spleen without determining any lack of, or delay in, erythropoietic response.

Opening of blood-brain barrier in Triturus cristatus carnifex by hyperosmolar mannitol solutions.

The permeability of the newt cerebral capillaries to lanthanum ion has been studied after perfusion with mannitol solutions of increasing molarity. In the control specimens lanthanum deposits were limited to the luminal side of the capillaries and tracer did not spread to the pericapillary spaces due to the tight junctions. Treatment with hypertonic solutions of mannitol (0.25M, 0.5M, 1M) caused opening of the blood brain barrier with a progressive increase in lanthanum between the endothelial cell edges, in the basal lamina and in the extracellular spaces of the nervous parenchyma in relation to the molarity of the mannitol solution. The spread of lanthanum is probably due to opening of the tight junctions between the endothelial cells, since pinocytotic vesicles labelled with tracer were not evident.

Microcythemia from anemic hypoxia and normal erythropoiesis in the newt.

Specimens of the newt, Triturus cristatus carnifex (Laurenti), rendered totally anemic, restore erythron by cyclic waves of erythropoietic activity that alternate with intervals of stasis. Hemolysis is obtained by administering 25 mg/liter of acetylphenylhydrazine in the breeding water for 36 h. The first cycle of erythropoietic activity produces microcytes, which have completely differentiated by 8 weeks after treatment. However, if the animals are raised in a hyperbaric chamber at a pressure of 1.5 atmospheres, in order to compensate for hypoxia, normocytes are produced. In both cases the hematocrit and hematic concentration of hemoglobin reach analogous values, so microcythemia appears to be the only effect of hypoxia. The hemoglobin, hematocrit values, and normocyte counts in hyperbaric animals are about one-half those of the controls newts. These data, together with those on the life span of red blood cells (RBC) and time span between two successive erythropoietic cycles (2 months and 1 month, respectively), indicate that the newts normally keep only two sets (one new, one old) of RBC in circulation, whose approximate parameters can be defined as RBC count: 60,000/mm3, hematocrit: 17%, and hemoglobin: 5.4 g/100 ml.

Intermittent erythropoiesis in anemic newts.

A group of 88 newts, Triturus cristatus carnifex (Laurenti), was rendered totally anemic by administering acetylphenylhydrazine (APH) in the breeding water for 48 h at a concentration of 25 mg/liter. The course of erythron restoration was followed for 5 months, sacrificing four specimens per week and analyzing the blood and spleen hemopoietic tissue. The return to the normal values of the red blood cell count occurred through marked increases in concentration at fairly regular intervals, which is best explained by a discontinuous, rhythmic erythropoiesis. This fact is strictly correlated with the intermittent mitotic activity observed in the spleen and with the periodic appearance of large quantities of immature elements in the blood smears. The APH-induced synchronization of newt erythropoietic activity revealed the approximate length of each erythropoietic cycle to be 4 to 5 weeks and the erythropoietic life span to be 50 to 60 days.

Multiple hemoglobins in Triturus cristatus: their study by analytical isoelectrofocusing.

Electrophoretic separation of the hemoglobin of healthy adult Triturus cristatus reveals four components. Isoelectric focusing of the same hemolysates in various commercial ampholytes of different chemical composition and pH range results in the separation of eight individual hemoglobin bands. The bands obtained by electrophoresis are not homogeneous as revealed by individual gel electrofocusing. They finally separate into the same eight components, as in the whole hemolysate. From the above findings it is concluded that this species has not four but eight individual hemoglobin molecular forms. Our results demonstrate lack of hemoglobin polymorphism in this species.

Functional properties of hemoglobins from Triturus cristatus.

The functional properties of hemoglobins from Triturus cristatus carnifex have been characterized both from an equilibrium and a kinetic point of view. A special attention has been given to the effect of organic phosphates in view of their role in the modulation of the hemoglobin function. Under stripped conditions newt hemoglobins display a negative Bohr effect which becomes much less pronounced in the presence of 0.2 M inorganic phosphates. The effect of organic phosphates involves not only a drop in the O2 affinity but also a large change in the shape of the O2 Bohr effect which becomes markedly positive in the presence of myo-inositol hexakis(phosphate). The experimental data reported make it possible to give a complete picture of the functional behaviour of newt hemoglobins, making a contribution to clarifying the dependence of the sign of the Bohr effect on the acclimatization temperature of the animals that has been previously reported [Morpurgo, G., Battaglia, P. A. and Leggio, T. (1970) Nature (Lond.) 225, 76--77].

The identification of globin messenger ribonucleic acid in newt erythropoietic cells.

Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts of the different globin mRNA subspecies. Globin mRNA activity was also evident in the 25S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25S poly(A)+-RNA fraction contained equimolar amounts of 9S globin mRNA and 26S rRNA. Translation of the 25S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength.

Three-dimensional reconstruction of the chromatin bodies in the nuclei of mature erythrocytes from the newt Triturus cristatus: the number of nuclear envelope-attachment sites.

The arrangement of the chromatin bodies in the interphase nuclei of 6 erythrocytes has been investigated by means of 3-dimensional reconstruction from electron micrographs of serial sections. When the borders of chromatin bodies are marked on the surface of each model, discrete areas of chromatin in contact with the nuclear envelope are revealed. The number of these areas in approximately equal to the number of chromosomes in the diploid set. The data suggest that each chromatin body corresponds to a condensed interphase chromosome and that each chromosome is attached to one discrete site on the nuclear envelope. The data are insufficient to show whether or not the condensed chromosomes are arranged in any orderly pattern in these nuclei.

Multiple hemoglobins in Triturus cristatus: their degradation by sulfhydryl compounds.

1. Hemolysates of newt (Triturus cristatus) red cells contain four distinct hemoglobin species which have been observed consistently both in individual animals and pooled samples. 2. Hemoglobin heterogeneity in the species arises from existence of multiple hemoglobulins, with no indication of genetic polymorphism. 3. While the relative proportions of the different hemoglobins may vary in different samples, HbII is usually the most abundant, with HbIII and HbIV constituting most of the remainder of the total hemoglobin complement. HbI never exceeds 3-5% of the total hemoglobin. 4. Neither the electrophoretic migration nor the anion exchange properties of the four hemoglobin species are altered by conversion of oxyhemoglobin to the cyanmet derivative, excluding artifacts due to different oxidation states of iron. 5. The average molecular weight of newt total hemoglobulin is 67,182 with no indication of hemoglobin polymers. 6. The use of sulfhydryl-reducing agents (mercaptoethanol, dithiothreitol) as a precaution against aggregation results in extensive degradation of newt hemoglobin through a process similar to "coupled oxidation" by ascorbate. 7. The degradative effects of sulfhydryl-reducing agents on newt hemoglobin suggest that these reagents be used cautiously in any hemoglobin analysis.

The effect of toluene on the hematopoietic system in newt Triturus cristatus.

A macrocytic hypocromic anemia was observed in newts after toluene inhalation, with 2 concentrations, for 6 weeks. Anemia resulted associated with the used dose of toluene. Erythroblast counts, performed on blood smears and on spleen and heart imprints, showed an erythropoietic stimulus during the first weeks of treatment followed by a depressed erythrostimulating action. Differential count of erythroblasts indicated a slow maturation induced by toluene on erythroblasts. An aplasia of the spleen was suggested on the basis of the results from 6 week-treated animals. A reduction in white blood cell count, associated with increased neutrophils and lymphocytopenia, occurred only in the animals treated with the higher concentration of toluene, after 5 and 6 weeks of treatment. A decrease of phagocytic activity of neutrophils, obtained with the in vitro assays, appeared the most relevant finding at the level of lekocyte compartment.