Chiara Maria Motta

Responses to cadmium intoxication in the liver of the wall lizard Podarcis sicula.

This study examined the cytological and molecular effects of cadmium, a toxic heavy metal, in the liver of the Italian wall lizard Podarcis sicula. Cadmium was administered in single dose, by diet, to induce a concentration comparable with that measured in animals living in contaminated sites. For comparison, cadmium was also administered in multiple doses by food (chronic) or in a single dose intraperitoneally (i.p.); the effects were followed at regular time intervals up to 30 days post treatments. Atomic absorption spectrometry analysis demonstrated cadmium ion uptake and accumulation in the parenchyma with an estimated half-life of approximately 8 days. Cytological analyses revealed that the metal induced oedema, activated metallothionein expression in Kupffer cells and extracellular matrix production in fat storing cells. It also caused swelling and alteration in lipid and sugar metabolism in hepatocytes. In conclusion, in the wall lizard cadmium is toxic to the liver even at very low concentrations, the response is not strictly dose and time dependent and almost no recovery occurs in short (30 days) time periods.

How the ovarian follicle of Podarcis sicula recycles the DNA of its nurse, regressing follicle cells

In Podarcis sicula specialized follicle cells send reserve materials to the previtellogenic oocyte via intercellular bridges. Immediately before the onset of vitellogenesis this transferring becomes particularly massive so that the cell volume significantly reduces, meanwhile in the nucleus the morphological alterations typical of apoptosis appear. To clarify why these follicle cells are not simply fully resorbed by the oocyte and to determine whether their DNA is discarded or recycled, we carried out a series of morphological and biochemical investigations. The finding that large macromolecular scaffolds are formed and that these are able to retain the DNA until it is extensively cut by two different endonucleases suggests that regression of the follicle cells is programmed and that the fate of their DNA is strictly controlled. Following its genetical neutralization via fragmentation, the DNA is apparently recycled by being transferred into the oocyte via the intercellular bridges, that, in fact, remain open until the very late stages of cell regression. The small DNA fragments reaching the oocyte cytoplasm would not interfere with meiosis completion but could significantly contribute to the stock of reserve materials to the advantage of the growing oocyte and/or developing embryo. Mol. Reprod. Dev. 51:421–429, 1998.

Cadmium-induced teratogenicity in lizard embryos: correlation with metallothionein gene expression.

Cadmium teratogenic effects and metallothionein expression were studied in tissues of lizard embryos at different stages of development. Incubation of eggs in cadmium contaminated soil had no effect on embryo survival, but strongly affected cranial morphogenesis. Cytological analyses demonstrated abnormalities in the development of proencephalic vesicles, mesencephalon and eyes. No defects were observed in somite or limb development. Northern blot analysis demonstrated that MT expression was much stronger in embryos developed in cadmium contaminated soil. In situ hybridization showed an early induction of MT gene expression in developing liver and gut, whereas in brain and eyes the spatial and temporal localization of MT transcripts did not change. A possible correlation between inability to induce MT expression and abnormalities observed in the head region of lizard developing embryos is suggested.

Regulation of oocyte number during oocyte differentiation in the lizard Podarcis sicula

This paper concerns the differentiation process of germ cells from oogonia to primary follicles in the lizard Podarcis sicula. The study was carried out at the morphological level and using a cytophotometric analysis for determining the number of differentiating germ cells undergoing degeneration. The progressive disorganization of the germ cell clusters during the early diplotene stage and the role played by the prefollicular cells during this process are described. Oocyte degeneration has been observed between the mid-zygotene and the early diplotene stages. When the primary follicle (oocyte plus follicular cells) is being formed, the degeneration process stops and the oocyte undergoes regular growth and ovulation.

Structural and functional changes in the zebrafish (Danio rerio) skeletal muscle after cadmium exposure.

This report describes the alterations induced by an environmentally realistic concentration of cadmium in skeletal muscle fibre organization, composition, and function in the teleost zebrafish. Results demonstrate that the ion induces a significant quantitative and qualitative deterioration, disrupting sarcomeric pattern and altering glycoprotein composition. These events, together with a mitochondrial damage, result in a significant reduction in swimming performance. In conclusion, the evidence here collected indicate that in presence of an environmental cadmium contamination, important economic (yields in fisheries/aquaculture), consumer health (fish is an important source of proteins), and ecological (reduced fitness due to reduced swimming performance) consequences can be expected.

Cadmium impairment of reproduction in the female wall lizard Podarcis sicula

The exposure to environmental toxicants such cadmium (Cd) is an important research area in wildlife protection. In this study, the effect of Cd oral administration on the ovarian structure and function and on reproductive performance of the Italian wall lizard Podarcis sicula was studied. In vivo, adult female lizards were randomly assigned to three groups. Cd was given with food in single dose and in multiple doses 3 days/week for 4 weeks at dose of 1.0 μg/g body weight. Following euthanasia, the ovaries were removed and analyzed for morpho‐functional changes. Results demonstrated that Cd increases prefollicular germ cells number; the evaluation of the number of follicles detects significantly higher number of atretic growing follicles, whereas primary follicles remain unchanged with respect to controls. After Cd treatments, follicles are deformed by the presence of large protrusions and a general dysregulation in the follicle organization is observed. The zona pellucida is also affected. Cd causes alteration in sugar metabolism and in metallothionein gene expression. Finally, Cd administration significantly reduces clutch size and dramatically increases embryo mortality. In conclusion, data here described show that Cd induces morpho‐functional alterations in lizard follicles and indicates that these are responsible for a significant impairment of oogenesis. The effects of the dose are time independent, persisting essentially unchanged regardless of single or multiple administration, so it can be concluded that even occasional, sublethal Cd contamination may significantly impair reproductive performance in these animals.

Spatiotemporal changes in metallothionein gene expression during embryogenesis in the wall lizard Podarcis sicula

Lizard embryos are nutritionally independent from their environment. During the early phases of oogenesis, the egg prepares for development by storing reserve organelles, proteins, and RNAs sufficient to allow the zygote to transform into a juvenile. This preparation also includes the storage of metallothionein (MT) transcripts. This study investigated the localization of these transcripts by in situ hybridization throughout Podarcis sicula developmental stages. Our data show that MT expression undergoes shifts in both regional and cellular localization. MT transcripts were detected early in the central nervous system, later in tissues implicated in metabolic processes. Results are discussed highlighting differences in lizard embryonic spatial and temporal MT expression compared with piscine, amphibian, and mammalian embryos. We hypothesize that, under natural conditions, the nutritionally closed system represented by the lizard egg protects the developing embryo from an unwanted excess of metals. This mechanism would make MT expression and accumulation in detoxifying organs in developing animals unnecessary until hatching and food intake begins. Conversely, the presence of MT transcripts during brain development may ensure the correct final architecture of this organ.

Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii.

The presence and production of IgM in the skin of the Antarctic teleost Trematomus bernacchii were investigated in this study. Immunoglobulins purified from cutaneous mucus and analysed by SDS-PAGE run under non-reducing and reducing conditions, were composed of heavy and light chains of 78 kDa and 25 kDa respectively, with a relative molecular mass of 830 kDa indicating that mucus IgM are tetramers as the serum IgM. Mature transcripts encoding the constant domains of both the secretory and membrane-bound Igμ chain were seen in T. bernacchii skin using a PCR strategy and the expression of the secretory Igμ chain in the skin was compared with that in other tissues by Real-time PCR. Cytological investigations revealed the presence of either immunoglobulins or their transcripts in occasional lymphocytes distributed close to the basal membrane. IgM once produced here, enters the filament-containing cells and is released into the mucus when these cells degenerate and detach from the epidermis. Our findings indicate that a cutaneous defence mechanism, functioning as anatomical and physiological barrier under subzero conditions, is present in this Antarctic species as an important component of the immune system.

Cadmium effects on the retina of adult Danio rerio.

The aim of this work is to describe the effects of cadmium pollution on the vision of adult zebrafish, Danio rerio. Retinal morpho-cytological alterations were investigated by light and electron microscopy, while the functionality of cadmium-exposed retinae was assessed by re-illumination behavioral tests with white or colored light. Our results demonstrate that cadmium toxicity causes significant degeneration and loss of organization at both macro and microscopic levels. These alterations impair functional responses particularly through an increase in light sensitivity. Metallothioneins were not seen to be up-regulated, while the recovery of visual acuity is due to a regenerative process by Müller cells.

Cadmium Contaminated Soil Affects Retinogenesis in Lizard Embryos

Lizards are soil surface animals that represent an important link between invertebrates and higher predators. Being part of wild fauna, they can be affected by contamination from anthropic activities and in particular, pesticides and chemical substances of various nature that reach the soil surface directly or through fall out. Among these substances, heavy metals such as cadmium may exert particularly marked toxic effect on both adult and embryos. In lizards, recent studies show that cadmium may cause developmental defects, including alteration of eye development, with appearance of unilateral microphthalmia and retinal folding. In the present study, the effects of cadmium incubation on retinal development were investigated demonstrating that cadmium interferes with cell cycle regulation by increasing proliferation. An increased expression of Otx2 and Pax6 genes, markers of retinal differentiation, was also found. However, the cellular localization of Pax6 and Otx2 transcripts did not change in treated embryos: in the early stages of retinogenesis, the two genes were expressed in all retinal cells; in the differentiated retina, Otx2 remained in the cellular bodies of retinal cells forming the nuclear and the ganglion layers, whereas Pax6 was expressed only in the cells of the inner nuclear and the ganglion layers. Data suggest that the increased expression of Pax6 and Otx2 could be ascribed to the hyperproliferation of retinal cells rather than to an effective gene overexpression.