Hematologic reference intervals for Xenopus tropicalis with partial use of automatic counting methods and reliability of long-term stored samples.

BACKGROUND: The African frog, Xenopus tropicalis, is widely used in biomedical and toxicologic research. Reference intervals (RI) for hematologic variables, valuable to research and health status assessment, have not been established. OBJECTIVES: The purpose of the study was to determine hematologic RI of X tropicalis, and establish whether automated cell counting can facilitate routine hematologic assessment in frogs. METHODS: Blood from 41 adult healthy X tropicalis was collected via cardiac puncture, and diluted in Natt-Herrick solution. Complete WBC, RBC, and thrombocyte counts (hemocytometry), differential WBC counts (Wright-Giemsa-stained smears), PCV (centrifugation), total protein (refractometry), and automated total cell counts (WBC + RBC + thrombocytes, Sysmex particle counting) were determined. Concordance correlation coefficients calculated the agreement between total cell counts obtained by hemocytometry and automated particle counting, and between total cell counts at collection and after 2 years of storage. RESULTS: Leukocyte morphology was similar to other amphibians and mammals. PCV was similar to other frogs; RBC counts were higher, and MCV was lower than in other frog species. Neutrophils were the most numerous WBC. Agreement was good between hemocytometry and automated cell counts. Subtracting the hemocytometer WBC and thrombocyte counts from the automated total cell count reliably yielded the RBC count. Cellular integrity evaluated 2 years post collection was good, and automated counts were not clinically different from counts at collection. CONCLUSION: We provide hematologic RI for X tropicalis, suggest how automated cell counts may facilitate hematologic assessments of frogs, and establish that blood in Natt-Herrick solution is stable 2 years post collection.

Nkx2.5 is involved in myeloid cell differentiation at anterior ventral blood islands in the Xenopus embryo.

We have shown previously that two populations of myeloid cells emerge in the anterior and posterior ventral blood islands (aVBI and pVBI) at the different stages in Xenopus laevis embryo. In order to elucidate the regulatory mechanism of myeloid cell differentiation in the aVBI, we examined the role of Nkx2.5, an essential transcription factor for heart differentiation, in regulation of the myeloid cell differentiation in this region. Knockdown of endogenous Nkx2.5 by introducing MO into the dorsal marginal zone (DMZ) suppressed the expression of MHCα as well as that of mpo and spib in the resultant embryos and in DMZ explants made from the injected embryos. Expression of c/ebpα was less affected in the embryos injected with Nkx2.5 MO. The effect of Nkx2.5 MO in myeloid cell differentiation was recovered by coinjection of nkx2.5 or c/ebpα mRNA, indicating that Nkx2.5 functions at the same or the upper level of C/EBPα for the specification of myeloid cells. An attempt to identify transcription factors for myeloid cell differentiation in ventral marginal zone (VMZ) explants demonstrated that coinjection of two transcription factors out of three factors, namely C/EBPα, Nkx2.5 and GATA4, was sufficient to induce a certain amount of mpo expression. We suggest that C/EBPα is an unequivocal factor for myeloid cell differentiation in the aVBI and that Nkx2.5 and GATA4 cooperate with C/EBPα for promotion of myeloid cell differentiation.

Thyroid-stimulating hormone (TSH): measurement of intracellular, secreted, and circulating hormone in Xenopus laevis and Xenopus tropicalis.

Thyroid-stimulating hormone (TSH) is an important regulator of the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis. To evaluate the role of this hormone on developing tadpoles, immunologically-based Western blots and sandwich ELISAs were developed for measuring intracellular (within pituitaries), secreted (ex vivo pituitary culture), and circulating (serum) amounts. Despite the small size of the tadpoles, these methods were able to easily measure intracellular and secreted TSH, and circulating TSH was measurable in situations where high levels were induced. The method was validated after obtaining a highly purified and enriched TSH sample using anti-TSH-ß antibodies conjugated to magnetic beads. Subsequent mass-spectrometric analysis of the bands from SDS-PAGE and Western procedures identified the presence of amino acid sequences corresponding to TSH subunits. The purified sample was also used to prepare standard curves for quantitative analysis. The Western and ELISA methods had limits of detection in the low nanogram range. While the majority of the developmental work for these methods was done with X. laevis, the methods also detected TSH in Xenopus tropicalis. To our knowledge this is the first report of a specific detection method for TSH in these species, and the first to measure circulating TSH in amphibians. Examples of the utility of the methods include measuring a gradual increase in pituitary TSH at key stages of development, peaking at stages 58-62; the suppression of TSH secretion from cultured pituitaries in the presence of thyroid hormone (T4); and increases in serum TSH following thyroidectomy.

Occurrence of a novel fucose-containing pentaglycosylceramide with blood-group-B active determinant in Xenopus blastula cells: its possible involvement in cell-cell adhesion.

For understanding of the biological function of glycoconjugates during embryogenesis and morphogenesis, Xenopus laevis is considered a very useful animal model. We have found that blood-group-active molecules characteristically were distributed in the cell-cell contact region of Xenopus blastula cells. The chemical nature of blood-group-active glycoconjugates, including glycosphingolipids, is little known. T.l.c.-immunostaining using anti-blood-group-antigen antibodies showed that many species of blood group-B-active glycosphingolipids existed in the neutral glycosphingolipid fraction extracted from Xenopus laevis eggs. Among the B-active glycosphingolipids detected, two major components with the fastest mobility on a t.l.c. plate, tentatively termed XN-1 and XN-2, were isolated, and their chemical structures were characterized by gas chromatography-mass spectrometry, immunological anlaysis, fast-atom-bombardment mass spectrometry and 1H-n.m.r. spectroscopy. Both XN-1 and XN-2 had an identical pentaoligosaccharide structure, but differed in their ceramide moiety. The chemical structure is: [table: see text]. This is a novel type of pentaglycosylceramide with blood-group B activity, in that it lacks N-acetylhexosamine in its core carbohydrate structure. In this paper, a possible involvement of the blood-group antigen in the cell-adhesion process of Xenopus embryonic cells is discussed.

Methemoglobin reductase activity in fish erythrocytes.

NADH-methemoglobin reductase activity of erythrocytes from the coho salmon, Oncorhynchus kisutch, sockeye salmon, Oncorhynchus nerka, and the rainbow trout, Salmo gairdneri exhibited a major band of activity that resembled the human enzyme in electrophoretic mobility. No polymorphism was found in 35 samples from rainbow trout, 4 samples from Dolly Varden, 29 samples from sockeye salmon, and 24 samples from coho salmon. All samples differed from the human enzyme in that they appeared to be membrane-bound and required the presence of a detergent, Triton X-100, for solubilization. Rainbow trout and coho salmon enzymatic activity is greater than the human enzyme activity at 15 degrees C.

Comparison of hemoglobins from the genus Xenopus (Amphibia salienta).

A survey of the electrophoretic mobility of hemoglobins of 14 species and subspecies of the genus Xenopus was undertaken. It was found that the variation between the different taxons considered was quite important in the number and the degree of the hemoglobin bands on polyacrylamide slab gels. The profiles of the hemoglobin electrophoretic pattern in tube gels gave corresponding results, confirming the variation between the different Xenopus species. The degree of resolution obtained on polyacrylamide slab gels was insufficient to allow one to distinguish between the subspecies of Xenopus laevis. Specific staining of the gels showed that the protein bands were in fact hemoglobins. From the results obtained it appears that there are relative differences in the number and mobility of hemoglobin bands between the different species, these differences being important enough to stimulate interest in further investigation in this field, but, as far as this preliminary investigation indicates, not clear-cut enough to use as a method of identification for isolated unknown species.

Fine structural distribution of the surface-connected canalicular system in frog thrombocytes.

The existence of the surface-connected canalicular system (SCCS) has been demonstrated in semithick sections of the frog thrombocytes by the use of a high voltage electron microscope. The SCCS of the thrombocytes in Rana catesbeiana and Rana nigromaculata consists of numerous canaliculi and vesicles with a diameter of 250 nm, which join with one another to make a complex network throughout the cytoplasm. Although the SCCS of Xenopus laevis fits well into the pattern described in Rana catebeiana, the diameter of the canaliculi of the SCCS is about 500 nm. The results of this study suggest that the SCCS is a specific organelle of the thrombocyte system common to submammals and mammals.

The origin of yolk-DNA in Xenopus laevis.

Xenopus laevis serum and plasma was found to contain an average of 25 microgram DNA/ml. Isolated X. laevis oocytes incubated in medium containing 25 microgram DNA/ml labeled with either 125I, 32P or 14C and from three different sources (bovine, E. coli and X. laevis), incorporated the label at an average rate of 0.11 ng.mm-2.hr-1. Sucrose gradient fractionation of oocytes revealed that 40-75% of the acid-precipitable label incorporated was associated with the yolk platelets. Additional incubations of oocytes in unlabeled medium demonstrated that the DNA incorporated into the yolk platelets was undergoing turnover; only 20% of the yolk-associated DNA was still present after a one-week incubation. Our data suggest that yolk-DNA arises by the adventitious uptake of DNA present in the maternal serum by vitellogenic oocytes.

Production and fate of erythroid cells in anaemic Xenopus laevis.

Adult Xenopus laevis, rendered anaemic by phenylhydrazine injection, have been studied during the recovery from such anaemia. Electron microscopy of liver and spleen sections indicates that both of these organs are active in the phagocytosis and destruction of the old damaged red blood cells. May-Grunwald and Giemsa staining of liver and spleen cells following anaemia has been used to show that erythropoiesis also occurs in both liver and spleen, and this has been confirmed by electron-microscope studies of these organs. Cell counting and radiolabelling of the new population of circulating erythroid cells in the period following phenylhydrazine injection suggests that a sudden release of basophilic erythroblasts from liver and spleen is followed by mitosis of this new cell population in circulation, and that no further release of erythroid cells from these organs is likely until complete recovery has occurred.

Immunoglobulin evolution: chemical study of clawed toad (Xenopus laevis) heavy and light chains.

NH2 terminal amino acid sequence determinations of clawed toad (Xenopus laevis) immunoglobulins indicate that approximately 30% of the heavy chains and less than 5% of the light chains have unblocked NH2 termini. The major amino acid sequence of the X. laevis 7S immunoglobulin heavy chains is the same as that of the 19S immunoglobulin heavy chains. Thus in the synthesis of the heavy chains, the VH genes coding for unblocked heavy chains can associate with CH genes of both the 19S and 7S classes. This association is particularly important in amphibians because, in contrast to mammals and birds, the majority of amphibian antibody-producing cells synthesize both 19S and 7S immunoglobulins and do not participate in the 'genetic switch' characteristic of lymphocyte differentiation in higher organisms. In X. laevis, the major amino acid sequence at the first twenty-four positions of the unblocked heavy chains shows approximately 54% difference from the prototype amino acid sequence of the mammalian VHIII subgroup. Thus, the VHIII gene(s) must have started to appear after the evolutionary divergence of the common ancestor of mammals and birds from the amphibian line. The amino acid composition of the X. Laevis 7S immunoglobulin heavy chains differs from that of its 19S immunoglobulins as well as those of human IgG and IgA. These data support the concepts (a) that amphibian 7S and 19S immunoglobins belong to distinct classes and (b) that amphibian 7S immunoglobulin does not resemble mammalian IgG or IgA.

A natural heterohaemagglutinin in Xenopus laevis serum.

In most adult Xenopus laevis the serum contains a 'natural' factor capable of lysing the erythrocytes from a wide variety of amniote species. The factor has no effect on the erythrocytes of another amphibian, Ambystoma mexicanum, nor will serum from one animal lyse red cells from another Xenopus individual. No lysing factor was present in the serum of larval (tadpole) Xenopus. Heating of Xenopus serum to 56 degrees for 30 min, absorption of the serum with zymosan or inulin, or removal of calcium and magnesium ions results in loss of lytic activity, although haemagglutinating activity remains, suggesting that the factor can fix complement. The factor elutes from a gel chromatography column in the 19S peak, and is inactivated by thiol reduction and subsequent alkylation. These findings, coupled with immunoabsorption studies suggest that the haemagglutinin is an immunoglobulin of the IgM class. The significance of this suggestion is discussed in the light of previous reports of 'natural' heterohaemagglutinins in other species.

Histone H2B variants from the erythrocytes of an amphibian, a reptile and a bird.

Histones H2B have been isolated from the terminally differentiated diploid erythrocytes of three different classes, amphibia (Xenopus laevis), reptilia (Crocodilus niloticus) and aves (Gallus domesticus). Partial amino acid sequences revealed three regions of sequence variation, each variant involving a single amino acid substitution.

Transcriptional reactivation of isolated Xenopus erythrocyte nuclei: patterns of RNA synthesis.

Nuclei isolated from Xenopus erythrocytes can be transcriptionally reactivated by exposure to certain cytoplasmic proteins. The types of RNA synthesized during this reactivation have been studied and compared with those present in, or synthesized by, isolated nuclei not so reactivated or in entire Xenopus erythrocytes. In all cases, the pattern of transcription indicates the synthesis of a broad range of low molecular weight RNAs. Competitive hybridization demonstrates that the reactivated nuclei synthesize some transcripts not normally produced by the isolated nuclei and we have shown that a proportion of these possess amino acid-accepting activity. The significance of these results is discussed in relation to the control of gene activity in these cells.

Albumin phylogeny for clawed frogs (Xenopus).

Comparisons of albumin indicate that the frogs commonly used by North American molecular and developmental biologists under the name of Xenopus muelleri belong to another species, X. borealis. Phylogenetic analysis of the albumin data reveals two major groups of Xenopus species, one containing only X. tropicalis and the other, called the X. laevis grou, containing the remaining species of the genus. The phylogenetic tree, in conjunction with evidence from chromosomes and DNA content, leads to the hypothesis that total genome duplication occurred in the common ancestor of the X. laevis group.

Adult haemoglobin in developmentally retarded tadpoles of Xenopus laevis.

Tadpoles of Xenopus laevis reared in water containing 0-01% propylthiouracil continue to grow but fail to develop or metamorphose. The haemoglobin of such tadpoles has been extracted in buffer, converted to a cyanmet form, and run on polyacrylamide gels. The developmentally retarded tadpoles are found to possess adult-type haemoglobin rather than the tadpole type which normally characterizes their developmental stage.