Steroids, aromatase and sex differentiation of the newt Pleurodeles waltl

In the newt Pleurodeles waltl, genetic sex determination obeys female heterogamety (female ZW, male ZZ). In this species as in most of non-mammalian vertebrates, steroid hormones play a key role in sexual differentiation of gonads. In that context, male to female sex reversal can be obtained by treatment of ZZ larvae with estradiol. Male to female sex reversal has also been observed following treatment of ZZ larvae with testosterone, a phenomenon that was called the "paradoxical effect". Female to male sex reversal occurs when ZW larvae are reared at 32 degrees C during a thermosensitive period (TSP) that takes place from stage 42 to stage 54 of development. Since steroids play an important part in sex differentiation, we focussed our studies on the estrogen-producing enzyme aromatase during normal sex differentiation as well as in experimentally induced sex reversal situations. Our results based on treatment with non-aromatizable androgens, aromatase activity measurements and aromatase expression studies demonstrate that aromatase (i) is differentially active in ZZ and ZW larvae, (ii) is involved in the paradoxical effect and (iii) might be a target of temperature. Thus, the gene encoding aromatase might be one of the master genes in the process leading to the differentiation of the gonad in Pleurodeles waltl.     

Nerve dependent sulphated glycosaminoglycan synthesis in limb regeneration of the newt Pleurodeles waltl

Denervation of the amputated limb of newts stops the regeneration process by decreasing blastema cell proliferation. We investigated the effect of the denervation on each of the two compartments (epidermal cap, mesenchyme) in mid-bud blastemas on the level of sulphated glycosaminoglycans (GAGS). Denervation resulted in an increase of about threefold in the incorporation of [³⁵S] sulphate into mesenchyme GAGs but had no effect on the epidermal cap. The increase of GAG synthesis in the mesenchymal part of the blastema involved both heparan sulphates and chondroitin-dermatan sulphates. Gel filtration showed no change in GAGs size after denervation. These results confirm that the mesenchymal part of the mid-bud blastema is the main target of nerves and, as heparan sulphates are known to store acidic fibroblast growth factor (aFGF), a polypeptide found in the blastema (Boilly et al.. 1991), this suggest that the nerves' effect on glycosaminoglycans turnover could be implicated in the control of bioavailability of this growth factor in the blastema.     

Identification of the vitellogenin proteins in the newt Pleurodeles waltl (urodele amphibian)

Vitellogenin derived from the blood of estrogen-treated Pleurodeles waltl was identified by immunochemical and electrophoretic analyses, using an antiserum against plasma vitellogenin isolated by dimethylformamide precipitation. Pleurodeles vitellogenin migrates as four bands on native PAGE, designated alpha-, beta-, gamma- and delta- VTG, with apparent mol. wts of 250,000, 270,000, 280,000 and 520,000 respectively. In the plasma, from estrogen-treated males like from ovariectomized estrogen-treated females, an additional band (mu-VTG) was found by native PAGE, never observed in estrogen-treated female plasma. It has a mol. wt of about 380,000 and shows complete immunological cross-reactivity with the vitellogenin antiserum. At least two polypeptides, termed VTG-I and VTG-II (mol. wt = 180,000 and 210,000) were identified by SDS-PAGE. Rocket immunoelectrophoresis displays three distinct precipitate lines indicating major immunological differences between the plasma vitellogenins.     

Micronucleus test in Vicia faba root tips to detect mutagen damage in fresh-water pollution

We have developed a micronucleus test in the root tips of Vicia faba, as a first alarm system for mutagen pollution of fresh water. The test can also give analytical results in the laboratory.Secondary root tips of Vicia faba were treated with mutagens, chromatoclastic and mitoclastic ones, which are known to have different modes of action (X-rays, methyl methanesulfonate, mitomycin C, ethidium bromide and colchicine). A number of variables (dose, exposure time, interval between treatment and fixing) were considered. The patterns of the micronucleus frequencies and of the ‘generating events’ (chromosome aberrations and mitotic anomalies) were compared.The micronucleus test in Vicia faba root tips showed a broad spectrum of mutagen damage, had low control levels, a good correlation between micronuclei and generating events and a good dose-effect relationship for both.This test has the sensitivity, reliability, low cost and manageability necessary for our purposes.     

The micronucleus test using peripheral blood reticulocytes from methotrexate-treated mice

The induction of micronuclei by methotrexate (MTX) was examined in two laboratories using mouse peripheral blood reticulocytes. MTX was a weak inducer in the micronucleus test using bone marrow cells and single treatments, and was one of the few chemicals showing a multiple-treatment effect (CSGMT/JEMS · MMS, 1990). In our preliminary experiments, the ratio of reticulocytes to total erythrocytes decreased greatly after a single treatment with MTX at 100 mg/kg, so lower dose levels of MTX were selected to carry out the micronucleus test in peripheral blood. Full-scale tests were performed at dose levels of 0, 10, 20, 40, and 80 mg/kg, with five sampling times of 0, 24, 48, 72, and 96 h. Frequencies of micronucleated reticulocytes (MNRETs) increased dose-dependently at 72 h, to a maximum of approximately 1%; some preparations obtained from the animals at higher doses could not be examined because the ratio of reticulocytes to total erythrocytes had decreased severely. At doses of 0.5–4.0 mg/kg, the effect of multiple treatments vs. single treatments was not clear, nor was the maximum level of response much different. Since MTX induced a clear positive response in peripheral blood reticulocytes after a single treatment, the reticulocytes in peripheral blood seem a more sensitive target.     

Possible involvement of an acylation mechanism in thalidomide-induced teratogenesis of the newt (Pleurodeles waltl.)

An acylation reaction of biological polyamines by thalidomide has been postulated to explain the teratogenic activity of this drug ( Fabro et al. 1965 ). In a further study, thalidomide has been reported to acylate polyamines at physiological pH; the teratogenic activity of this drug appears to be linked to its high acylating power towards polyamines ( Audit 1994 ).
In the present study, the action of the thalidomide molecule and its two chemical moieties (phthalimide and glutarimide rings) on Pleurodeles embryonic development has been investigated. The phthalimide moiety, which displays acylating activity, appears to generate Pleurodeles teratogenesis. The occurrence of a correlation between acylating activity and teratogenicity was confirmed using homothalidomide and partially hydrolyzed thalidomide. The glutarimide moiety has been found to act as an enhancer of phthalimide activity and to cause moderate alterations of newt development.
As the acylation of polyamines by thalidomide would deprive the embryo of these essential compounds, the effects of polyamine biosynthesis inhibitors have been compared to those of thalidomide. Both thalidomide and polyamine antimetabolites altered the early cleavage process of the Pleurodeles egg and arrested early development.     

The micronucleus test using peripheral blood reticulocytes from methotrexate-treated mice.

The induction of micronuclei by methotrexate (MTX) was examined in two laboratories using mouse peripheral blood reticulocytes. MTX was a weak inducer in the micronucleus test using bone marrow cells and single treatments, and was one of the few chemicals showing a multiple-treatment effect (CSGMT/JEMS.MMS, 1990). In our preliminary experiments, the ratio of reticulocytes to total erythrocytes decreased greatly after a single treatment with MTX at 100 mg/kg, so lower dose levels of MTX were selected to carry out the micronucleus test in peripheral blood. Full-scale tests were performed at dose levels of 0, 10, 20, 40, and 80 mg/kg, with five sampling times of 0, 24, 48, 72, and 96 h. Frequencies of micronucleated reticulocytes (MNRETs) increased dose-dependently at 72 h, to a maximum of approximately 1%; some preparations obtained from the animals at higher doses could not be examined because the ratio of reticulocytes to total erythrocytes had decreased severely. At doses of 0.5-4.0 mg/kg, the effect of multiple treatments vs. single treatments was not clear, nor was the maximum level of response much different. Since MTX induced a clear positive response in peripheral blood reticulocytes after a single treatment, the reticulocytes in peripheral blood seem a more sensitive target.     

FGF2 signaling pathway components in tissues of the posterior eye sector in the adult newt Pleurodeles waltl

The FGF2 signaling pathway components in tissues of the posterior wall in the normal and regenerating eye of the adult Pleurodeles waltl newt were detected for the first time. The fgf2 gene expression was found in the retina, retinal pigment epithelium, and choroid using polymerase chain reaction (PCR). A high homology of the mRNA nucleotide sequence of the most conservative fgf2 gene region in the P. waltl with the fgf2 orthologs in other vertebrates was proved. The Fgf2 protein amino acid sequence of the P. waltl newt demonstrates even more homology with this growth factor in other vertebrates. The Fgf2 protein with a molecular weight 35 kDa was found in the studied eye tissues using Western blot hybridization. Localization of the Fgf2 protein and its Fgfr receptors was immunohistochemically studied in the pigment epithelium, choroid, central and growth retina regions of the newt native eye, and in the connective cilium of photoreceptors. Using real-time PCR and immunohistochemistry methods, it was found that the fgf2 gene down-regulation and a decrease in the intensity of the immunochemical reaction of its protein product (Fgf2) occur in the early period after the retina removal (in 4–8 days) (as compared with those in the same department of the unoperated eye).     

A study of the localization and accumulation of S-phase cells in the retina of newt Pleurodeles waltl after experimental pigment epithelial detachment

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [3H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15-20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.     

Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene

The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) has been purified to homogeneity from skeletal muscle of the newt Pleurodeles waltl (Amphibia, Urodela). The purification procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulted in a 24-fold increase in specific activity and a final yield of approximately 46%. The native protein exhibited an apparent molecular weight of approximately 146 kDa with absolute specificity for NAD(+). Only one GAPDH isoform (pI 7.57) was obtained by chromatofocusing. The enzyme is an homotetrameric protein composed of identical subunits with an apparent molecular weight of approximately 37 kDa. Monospecific polyclonal antibodies raised in rabbits against the purified newt GAPDH immunostained a single 37-kDa GAPDH band in extracts from different tissues blotted onto nitrocellulose. A 510-bp cDNA fragment that corresponds to an internal region of a GapC gene was obtained by RT-PCR amplification using degenerate primers. The deduced amino acid sequence has been used to establish the phylogenetic relationships of the Pleurodeles enzyme--the first GAPDH from an amphibian of the Caudata group studied so far--with other GAPDHs of major vertebrate phyla.     

Differential expression of P450 aromatase during gonadal sex differentiation and sex reversal of the newt Pleurodeles waltl

A better understanding of vertebrate sexual differentiation could be provided by a study of models in which genetic sex determination (GSD) of gonads can be reversed by temperature. In the newt Pleurodeles waltl, a P450 aromatase cDNA was isolated from adult gonads, and the nucleotide or deduced amino acid sequences showed a high level of identity with various vertebrate species. In adults, aromatase expression was found in gonads and brain. In developing gonads, the expression was found to fit with the thermo-sensitive period (TSP) and was detected in both ZZ and ZW larvae, as well as in ZW submitted during the whole TSP to a masculinizing temperature. In the latter individuals, in situ hybridization and semi quantitative RT-PCR showed that, at the end of TSP, aromatase expression was at the same level than in normal ZZ larvae and was significantly lower than in normal ZW ones. Furthermore, temperature-induced down regulation did not occur when heating was performed at the end of TSP. Our results confirm the importance of aromatase regulation in female versus male differentiation and demonstrate that a down regulation of aromatase expression is involved in the process of sex reversal.     

Use of the micronucleus test in the screening and monitoring of mutagens

Data from literature on the use of micronuclear test to determine mutagenicity in agents of physical, chemical and biological nature are presented. The objects on which this method is used most frequently are enumerated. Great attention is paid to the analysis of micronuclei in blood erythrocytes and bone marrow of animals. It is shown that the animal sex, age and the way of mutagen injection are of great importance in micronuclear testing of mutagens. Methodical papers concerning the peculiarities of fixation colour, analysis and mathematical testing are given. Mutagen factors tested by the method of micronuclear analysis are enumerated. A high resolution and small labour input of the micronuclear test are shown.     

In vivo and in vitro metabolism of benzo(a)pyrene by the larva of the newt, Pleurodeles waltl

1. The larva of the amphibian species, Pleurodeles waltl was shown to metabolize benzo(a)pyrene in vivo into a variety of oxidized products. 2. In vitro, BaP hydroxylase (AHH) activity was found in hepatic microsomes and postmitochondrial fractions from both larvae and adults of the pleurodele. 3. The clastogenic effect of BaP formation of micronuclei in the erythrocytes was shown to be related to the presence of BaP quinones in the tissues of the newt.     

Sex-linked differences in activity of enzymes in the blood of the urodele amphibian Pleurodeles waltl.

Few data are available on enzyme activity in amphibian plasma or erythrocytes. We measured the activity of several blood enzymes in the urodele amphibian Pleurodeles waltl reared under standard laboratory conditions. In subsequent experiments, we will estimate and compare the physiological and biochemical conditions of P. waltl when reared under extreme temperature or microgravity conditions. The enzymes selected were glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, superoxide dismutase, catalase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. In fresh plasma samples, enzyme activity in females was higher than in males, except for aspartate and alanine aminotransferases, which were equivalent in females and males. Glutamate dehydrogenase activity was higher in males than in females. In female erythrocytes, the activity of all enzymes was higher than in male erythrocytes. We have also studied the storage conditions of samples and observed that for most enzymes, the activity in freshly isolated plasma and erythrocyte preparations decreased after storage at -18 or +4 degrees C.     

利用吞豆根尖细胞微核技术检测Cu,As污染的诱变性

应用蚕豆根尖细胞微核技术检测Cu、As及其复合污染的诱变性能,计数了蚕豆根尖细胞微核千分率(MCN‰)和污染指数(PI)并进行了F检验.结果表明,在一定浓度范围内(Cu＜200mg@L,As＜15mg@L-1),随着Cu、As浓度的增加,MCN‰上升;24个试验处理组PI均在2以上,各处理组MCN‰有显著差异,和对照组有极显著差异(a＜0,01),表明Cu、As及其复合处理对蚕豆根尖细胞的分裂活动有明显影响.     

The mouse peripheral blood micronucleus test with 2-acetylaminofluorene using the acridine orange supravital staining method

The peripheral blood micronucleus test using the acridine orange (AO) supravital staining method was validated with the potent bone marrow clastogen 2-acetylaminofluorene (2-AAF). 2-AAF induced micronuclei in peripheral blood reticuiocytes dose-dependently as well as in bone marrow polychromatic erythrocytes. The incidence of micronucleated reticuiocytes (MNRETs) peaked 48 h after a single treatment in both CD-1 and BDF₁ mice, and the incidence of micronucleated polychromatic erythrocytes (MNPCEs) peaked 24 or 48 h after treatment. The maximum incidences of MNRETs were always higher than those of MNPCEs in both mouse strains treated once. In the double-treatment regime, the maximum incidence of MNRETs was observed at 24 h after the second treatment in each strain. The incidences of MNRETs in BDF₁ mice were higher than in CD-1 mice after a single treatment but were comparable after double treatment.These results indicate that the peripheral blood micronucleus test using AO supravital staining is as sensitive as the conventional bone marrow assay. The new staining method can be performed more easily than the original smear method using either bone marrow or peripheral blood cells. Thus, the peripheral blood method using AO supravital staining is a possible alternative to the conventional bone marrow assay.