Lorenzo Varano

Morphological and Functional Changes in the Thyroid Gland of Methyl Thiophanate-Injected Lizards, Podarcis sicula

The thyroid has been shown to be a target organ for environmental chemicals, specifically endocrine-disrupting contaminants. Reptiles are particularly suitable as contaminant biomonitors due to their persistence in a variety of habitats, wide geographic distribution, longevity, and, in many cases, site fidelity. Methyl thiophanate is a systemic broad-spectrum fungicide used to prevent and control plant diseases caused by various fungi. The aim of this study was to develop an integrated biological model for monitoring the ecotoxic effects of thiophanate-methyl fungicide on the thyroid of the lizard Podarcis sicula. The results of this study indicate that both structural and functional differences in the thyroid gland of the lizard exist in the animals exposed to methyl thiophanate. Structurally, animals exposed to methyl thiophanate showed decreased epithelial cell height; the nuclei of the thyroid cells were small and elongated with dense chromatin and a greatly reduced cytoplasm. The colloid was retracted with few reabsorption vacuoles. Functionally, the same animals exhibited decreased T4 and T3 plasma levels compared to control animals. Methyl thiophanate administration produced statistically significant inhibition on serum thyroid-stimulating hormone levels and this is the mechanism for altering thyroid function. This study highlights how thyroid gland disruption, both structural and functional, in lizard and other nontarget organisms might also have an environmental aetiology.

Molecular characterization and gene expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) in the lizard brain.

The pituitary adenylate cyclase-activating polypeptide (PACAP) is considered a pleiotropic neuropeptide in vertebrate physiology. The nucleotide sequence, the expression and the distribution of PACAP were determined in the brain of the lizard Podarcis sicula. RT-PCR showed that the brain of this reptile synthesizes an mRNA coding for PACAP. By performing in situ hybridization and immunohistochemistry techniques, a wide distribution of PACAP and its mRNA in neurons, nervous fibers and other cells was found. Phylogenetic sequence analysis indicates that lizard PACAP is highly conserved, resembling the vertebrate PACAP. Our data demonstrate that PACAP is not only highly preserved during vertebrate evolution but also suggest that PACAP could be implicated in a wide number of functions in the physiology of the reptile brain.

An immunocytochemical study of the endocrine pancreas in three genera of lacertids

The comparative morphology of the endocrine pancreas was studied in 11 species of lacertids. Four major cell types were identified immunocytochemically in the endocrine pancreas: glucagon-immunoreactive A-cells, insulin-immunoreactive B-cells, somatostatin-(SRIF)-immunoreactive D-cells, and pancreatic polypeptide(PP)-immunoreactive F-cells. Different distributions of the four cell types were seen in the endocrine tissue within the exocrine parenchyma. F-cells were rare or absent in the splenic lobe and abundant in the duodenal lobe, in which they were usually widespread in the exocrine parenchyma and rarer in the islets. The other three cell types were always present in the islets. The central core consisted of B- and A-cells, with B-cells predominating. The peripheral mantle was formed by A-cells and less abundant D-cells. Rare D-cells were also found in the central core. D- and F-cells showed projections often closely associated with capillaries. The observed arrangements in islets and isolated cells may represent an endocrine network that, in addition to systemic actions, may regulate exocrine function in a paracrine fashion.

Shift from noradrenaline to adrenaline production in the adrenal gland of the lizard, Podarcis sicula, after stimulation with vasoactive intestinal peptide (VIP)

The aim of this study was to investigate the distribution and function of VIP in the adrenal gland of the lizard, Podarcis sicula. We have shown by immunohistochemistry that VIP fibers were localized exclusively around clusters of chromaffin cells in the dorsal ribbon of the lizard adrenal gland. Moreover, a strong positivity for this peptide was observed within ganglial cells and within most chromaffin cells of the gland. To investigate the effects of VIP on the adrenal gland, we have treated lizards with several doses of this peptide and we have shown that injections of exogenous VIP increased plasma levels of catecholamines and corticosteroids, but not of ACTH. This probably suggests a direct effect of VIP on the control of adrenal hormone secretion without the involvement of the hypothalamo-hypophyseal axis. Our results also establish that the increased levels of the hormones were modulated in a time- and dose-dependent manner. Therefore, our morphological studies showed a clear increased function of steroidogenic cells. In the medullary region, VIP administration induced not only a functional enhancement of adrenaline release from adrenergic cells, but also a shift of noradrenaline cells to adrenaline ones.

Different patterns of expression of five neuropeptides in the adrenal gland and kidney of two species of frog.

The aim of this study was to demonstrate in the adrenocortical and renal tissues of two species of frog, Rana italica and Rana esculenta, the presence and distribution of five neuropeptides: atrial natriuretic peptide (ANP), Leu-enkephalin (Leu-ENK), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal peptide (VIP).In anurans, the adrenal medulla is the site for the synthesis, storage and secretion of not only catecholamines but also various peptides. These peptides should not be regarded only as neurotransmitters or modulators for the secretion of catecholamines, but also as hormonal substances that induce systemic effects.All the peptides studied (ANP, Leu-ENK, NPY, SP and VIP) are present in both organs. However, different patterns of expression were observed for some of the peptides in two frogs.Immunopositivity to ANP was found in small clusters of chromaffin cells in both frogs whereas a clear strong positivity was present only in Rana esculenta kidney. Large clusters of chromaffin cells were immunoreactive to Leu-ENK in Rana italica but there were approximately 25% fewer compared to the positive cells present in Rana esculenta. Epithelial cells of renal tubules showed strong immunopositivity to Leu-ENK in Rana esculenta but not in Rana italica. A large number of adrenal cells (70–80%) were immunoreactive to NPY in Rana italica, while in Rana esculenta this peptide was localized in small clusters of chromaffin cells. Both frogs showed many NPY-positive cells in kidney. Many chromaffin cells were found positive to SP and VIP. A strong positivity was also observed in kidney in both frogs. These observations suggest a possible role of these peptides in the control of the physiological functions of adrenal glands and kidney of the two species of frogs studied.

Annual variations of adrenal gland hormones in the lizard Podarcis sicula

The adrenal gland regulates metabolism and maintains normal electrolyte balance. Adrenal hormones are equivalent in all vertebrates; the chromaffin tissue produces adrenaline and noradrenaline and the steroidogenic tissue produces most of the steroid hormones present in mammals. Podarcis sicula belongs to the Squamata family of lizards and it is the most abundant lizard species in southern Italy. This species shows a reproductive annual cycle and the presence of seasonal variations in the activity of the hypothalamus-hypophyseal-thyroid axis. To investigate the existence of an annual cycle of lizard adrenal gland, we have measured plasma concentrations of corticosterone, ACTH, noradrenaline and adrenaline. We have shown that corticosterone rapidly increased from January to March to reach a peak value that persisted until July, then, it slowly decreased until December. ACTH levels increased from January to May and slowly decreased from July to December. Noradrenaline levels were higher in March and then decreased until December. On the contrary, adrenaline levels increased from March to July and slowly decreased until December. Our results demonstrate the existence of an annual cycle of the lizard adrenal gland activity. This is very interesting because its activity is important to rightly regulate the reproductive status of the Podarcis sicula lizard.

Endothelin-B (ETB) receptor distribution in tissues of the lizard Podarcis sicula

Abstract. Although the structural and pharmacological properties of endothelin (ET) receptors have been studied, little is known concerning their physiological significance, even if each subtype is supposed to have a distinct physiological action. Thus, to further elucidate the physiological function of this receptor, we examined the presence and distribution of the endothelin-B receptor (ETB) subtype in tissues of the lizard Podarcis sicula, using immunoblotting and immunohistochemistry. Immunoblotting indicated that, although the ETB receptor appears to be ubiquitous, it is present at different levels in the tissues examined. Furthermore, immunohistochemistry showed that this receptor is very abundant in endothelial cells of all tissues, suggesting that there is an ETB-mediated autocrine system of endothelin, which plays an important role in the regulation of endothelial cell function. On the other hand, the presence of ETB immunoreactivity also in endocrine systems such as adrenal and thyroid glands suggests an involvement also in the paracrine system of these organs.

Distribution of PACAP in the brain of the cartilaginous fish Torpedo Marmorata

Abstract:  In this article, we investigated the distribution of pituitary adenylate cyclase‐activating polypeptide (PACAP) and its mRNA by immunohistochemistry, in situ hybridization, and RT‐PCR techniques, in the central nervous system of the elasmobranch Torpedo marmorata. RT‐PCR analysis showed that the CNS of T. marmorata expresses a messenger encoding PACAP. The immunohistochemistry and in situ hybridization patterns were partly overlapping, with a major expression in the hypothalamo–pituitary region and, surprisingly, in the saccus vasculosus. Our results show that, in T. marmorata, PACAP is synthesized and widely distributed in the CNS, suggesting an as yet unidentified role for this peptide in elasmobranch brain physiology.

Localization and role of serotonin in the adrenal gland of Podarcis sicula (Reptilia, Lacertidae)

The occurrence of serotonin (5-hydroxytryptamine; 5-HT) in the chromaffin cells of Podarcis sicula adrenal gland was demonstrated by immunocytochemical techniques: ABC and immunogold methods. At LM and EM levels, antiserum against 5-HT revealed serotonin immunoreactivity prevalently in noradrenalin (NA) cells, on and around secretory vesicles; adrenalin (A) cells appeared scarcely stained. The role of serotonin in the regulation of adrenal gland activity was studied in vivo using LM and EM techniques coupled to a specific radioimmunoassay for adrenocorticotropic hormone (ACTH) and corticosterone. 5-HT (0.7 mg/100 g body wt)/day for 4 days increased ACTH and corticosterone release; at LM and EM level clear signs of stimulation in the steroidogenic tissue were observed, as evidenced by the variations of lipid/cytoplasm ratio. In the chromaffin tissue, LM observations evidenced a variation of the numeric NA/A cell ratio; at EM level, chromaffin tissue showed intermediate cells with A, NA, and very clear granules with granular elements. The occurrence of these cells might be the result of a process of resynthesis following serotonin-stimulated catecholamine release. These data suggested that serotonin might be involved in the modulation of Podarcis pituitary-adrenal axis, and act as a paracrine factor to modulate corticosteroid production.

The adrenal gland of newt Triturus carnifex (Amphibia, Urodela) following in vivo betamethasone administration

The response of the adrenal gland of Triturus carnifex to betamethasone administration was studied; the effects were evaluated by examination of the ultrastructural morphological features of the tissues as well as the serum levels of aldosterone, corticosterone, norepinephrine and epinephrine. In March and June, betamethasone significantly decreased the serum levels of aldosterone and corticosterone and the lipid droplet content in the steroidogenic cells. Moreover, betamethasone influenced the chromaffin tissue, enhancing in March (when the chromaffin cells produce norepinephrine and epinephrine in almost equal quantities) epinephrine serum levels and the numeric ratio between norepinephrine and epinephrine granules in the chromaffin cells. In June, (when the chromaffin cells contain almost exclusively norepinephrine granules) betamethasone administration raised norepinephrine serum levels, whereas a decrease in the numeric ratio between norepinephrine and epinephrine granules in the chromaffin cells was found. Finally, betamethasone administration did not evoke in June any increase in the mean number of epinephrine granules in the chromaffin cells and/or in epinephrine serum levels, as would be expected if phenyletanolamine-N-methyl transferase (PNMT) enzyme, converting norepinephrine into epinephrine, were activated by corticosteroids. The results of this study showed that betamethasone decreased aldosterone and corticosterone serum levels and enhanced catecholamine serum concentrations. Moreover, the present results suggest that a stimulatory role of glucocorticoids on PNMT enzyme may be ruled out.