Vincenza Laforgia

The Serine Protease HtrA1 Is Upregulated in the Human Placenta During Pregnancy

The placenta has a dynamic and continuous capacity for self-renewal. The molecular mechanisms responsible for controlling trophoblast proliferation are still unclear. It is generally accepted that the simultaneous activity of proteins involved in cell proliferation, apoptosis, and extracellular matrix degradation plays an important role in correct placental development. We investigated in depth the expression of the serine protease HtrA1 during pregnancy in human placenta by in situ hybridization and immunohistochemistry, we demonstrated that HtrA1 displayed a low level of expression in the first trimester of gestation and a strong increase of HtrA1 expression in the third trimester. Finally, by electron microscopy, we demonstrated that HtrA1 was localized either in the cytoplasm of placental cells, especially close to microvilli that characterized the plasma membrane of syncytiotrophoblast cells, or in the extracytoplasmic space of the stroma of placental villi, particularly in the spaces between collagen fibers and on collagen fibers themselves. The expression pattern of HtrA1 in human placentas strongly suggests a role for this protein in placental development and function. Moreover, on the basis of its subcellular distribution it can be postulated that HtrA1 acts on different targets, such as intracellular growth factors or extracellular matrix proteins, to favor the correct formation/function of the placenta. (J Histochem Cytochem 52:885–892, 2004)

Alteration of the Bcl-2 : Bax Ratio in the Placenta as Pregnancy Proceeds

The placenta is the primary site of nutrient and gas exchange between mother and foetus. During human placental development, proliferation, differentiation and apoptosis occur at different stages. In order to clarify some of the molecular mechanisms underlying these events, we investigated the pattern of expression of two members of the Bcl-2 family in human placenta samples and compared them to the level of apoptosis detected by the TUNEL method.In particular, we evaluated the expression of Bcl-2 and Bax and their ratio during the first and third trimester. We found that Bcl-2 was generally expressed at low levels during the entire gestational period. On the other hand, Bax was low during the first trimester but increased towards the end of gestation. In accordance with the change of ratio of these two molecules, the increase of apoptotic cells was observable in the third trimester. These data indicate that Bcl-2 and Bax are spatio-temporally regulated during placental development and that the different expression of the above mentioned genes is at least in part responsible for the delicate balance between cell proliferation and programmed cell death in the human placenta during pregnancy.

Evaluation of cyclin D1 expression and its subcellular distribution in mouse tissues

Cyclin D1 is a key cell‐cycle regulatory protein required for the cell to progress through G1 to S phase. We have shown by Western blot analysis that cyclin D1 has a wide distribution in adult mouse tissues, with its level of expression being tissue‐dependent. Immunohistochemistry has also shown that cyclin D1 may be present in the cytoplasm, in the nucleus or in both these cell compartments: cytoplasmic staining was observed in both proliferating cells (e.g. kidney, intestine, stomach and salivary gland) and in the non‐dividing cells (the mature neurons of adult brain), while nuclear staining was seen in the neurons of the embryonic nervous system. Immunoelectron microscopy results indicate that, in tissues where cyclin D1 is present in both compartments (e.g. intestinal enterocytes), it may move via nuclear pores from the nucleus to the cytoplasm, and vice versa. The findings as a whole suggest that cyclin D1 may play multiple roles within specific tissues, probably by interacting with different substrates, and that its transit between nuclear and cytoplasmic compartments may help maintain cell homeostasis.

Immunohistochemical distribution of proteins belonging to the receptor-mediated and the mitochondrial apoptotic pathways in human placenta during gestation

The balance between cell death and cell proliferation and its regulation are essential features of many physiological processes and are particularly important in fetal morphogenesis and adult tissue homeostasis. Apoptosis is a type of cell suicide that is activated in two main ways: through a receptor-mediated pathway or through a mitochondrial pathway. We have investigated the immunohistochemical distribution of proteins belonging to these two pathways in human placenta during gestation by comparing their expression levels between the first and third trimester of gestation. In the first trimester, the receptor-mediated pathway prevails over the mitochondrial pathway with a moderate/intense expression of its three components, viz., Fas ligand (FasL), Fas, and caspase-8, and weak positivity of anti-apoptotic FLIP, these proteins being mainly localized in the cytotrophoblast compartment. In the third trimester of gestation, there is an increased expression of mitochondrial pathway proteins, viz., Apaf-1 and caspase-9. We have also investigated the expression level of caspase-3, the primary effector caspase of both pathways, and have observed that it is moderately expressed during gestation, being mainly localized in the cytotrophoblast during the first trimester and in both placental compartments during the third trimester of gestation. Thus, both pathways actively function in human placenta to execute cell death. By means of immunoelectron microscopy, we have further shown that, in human placenta, the two proteins of the mitochondrial pathway together with caspase-3 are localized both in the cytoplasm and in the nucleus. In particular, Apaf-1 and caspase-9 are distributed near to the nuclear envelope suggesting an important role for these two proteins in disrupting the nuclear–cytoplasmic barrier.

Pattern of expression of cyclin D1/CDK4 complex in human placenta during gestation.

Progression through the cell cycle in eukaryotic cells is controlled by a family of protein kinases, termed cyclin-dependent kinases (CDKs), and their specific partners, the cyclins. In particular, the control of mammalian cell proliferation occurs largely during the G1 phase of the cell cycle. Five mammalian G1 cyclins have been enumerated to date: cyclins D1, D2, and D3 (D-type cyclins), and cyclins E and E2. By the use of immunohistochemistry and immunoelectron microscopy, we observed that in the first trimester of gestation of human placenta, cyclin D1 was distributed in the nuclei of the cytotrophoblast compartment together with a weak positivity of endothelial cells surrounding blood vessels. The endothelial positivity of cyclin D1 strongly increased in the third trimester of gestation. Moreover, we observed the subcellular localization of cyclin D1 that was present both in the stroma of placental villi and in the nuclei of syncytiotrophoblast cells. Therefore, we observed that CDK4 was localized in the nuclei of the cytotrophoblast compartment during the first and third trimesters and it also had a nuclear positivity in the endothelial cells of blood vessels at the end of the third trimester of gestation. In conclusion we may hypothesize that cyclin D1/CDK4 complex functions to regulate the cell cycle progression in the proliferative compartment of human placenta, the cytotrophoblast, during the first trimester through interaction with p107 and p130. Therefore, cyclin D1 and CDK4 seem to be involved in the control of placental angiogenesis during the third trimester of gestation.

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.

Endocrine-disrupting effects of nonylphenol in the newt, Triturus carnifex (Amphibia, Urodela)

The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19 μg/L nominal concentration of nonylphenol throughout the periods of December-January and March-April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December-January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March-April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline.

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.

Effects of ACTH and corticosteroids on phenylethanoIamine‐N‐methyltransferase (PNMT) expression as determined by immunocytochemical localization in the adrenal gland of the lizard Podarcis sicula

Abstract This study was undertaken to investigate whether, in the lizard Podarcis sicula, the expression of adrenaline synthesizing enzyme phenylethanolamine‐N‐methyltransferase (PNMT) is influenced by variations in corticosteroid levels. Adrenal glands of specimens treated with ACTH or corticosteroids were collected and stained with avidin‐biotin complex (ABC) and immunogold techniques, for light and electron microscopical observations, respectively. In non treated specimens, positive staining was mainly confined to the catecholamine containing cells (adrenaline cells), located closer to the steroidogenic ones. In treated specimens, instead, positive staining was found in all the cells of the ehromaffin portion of the gland (adrenaline and noradrenaline cells). Immunoelec‐tron microscopy confirmed the distribution of immunoreactive cells observed at the light microscope. These results support the hypothesis that corticosteroids regulate PNMT expression in reptilian adrenal glands similarly to what occurs...