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In recent years, impaired fertility and endometrium related diseases are increased. Many evidences suggest that environmental pollution might be considered a risk factor for endometrial physiopathology. Among environmental pollutants, endocrine disrupting chemicals (EDCs) act on endocrine system, causing hormonal imbalance which, in turn, leads to female and male reproductive dysfunctions. In this work, we studied the effects of triclosan (TCL) and bisphenol A (BPA), two widespread EDCs, on human endometrial stromal cells (ESCs), derived from endometrial biopsies from woman not affected by endometriosis. Cell proliferation, cell cycle, migration and decidualization mechanisms were investigated. Treatments have been performed with both the EDCs separately or in presence and in absence of progesterone used as decidualization stimulus. Both TCL and BPA did not affect cell proliferation, but they arrested ESCs at G2/M phase of cell cycle enhancing cell migration. TCL and BPA also increased gene expression and protein levels of some decidualization markers, such as insulin growth factor binding protein 1 (IGFBP1) and prolactin (PRL), amplifying the effect of progesterone alone. All together, our data strongly suggest that TCL and BPA might alter human endometrium physiology so affecting fertility and pregnancy outcome.

Endocrine-disrupting chemicals (EDCs) belong to a heterogeneous class of environmental pollutants widely diffused in different aquatic and terrestrial habitats. This implies that humans and animals are continuously exposed to EDCs from different matrices and sources. Moreover, pollution derived from anthropic and industrial activities leads to combined exposure to substances with multiple mechanisms of action on the endocrine system and correlated cell and tissue targets. For this reason, specific organs, such as the prostate gland, which physiologically are under the control of hormones like androgens and estrogens, are particularly sensitive to EDC stimulation. It is now well known that an imbalance in hormonal regulation can cause the onset of various prostate diseases, from benign prostate hyperplasia to prostate cancer. In this review, starting with the description of normal prostate gland anatomy and embryology, we summarize recent studies reporting on how the multiple and simultaneous exposure to estrogenic and anti-androgenic compounds belonging to EDCs are responsible for an increase in prostate disease incidence in the human population.

Alkylphenols (AP) are widespread environmental compounds belonging to the large family of substances known as Endocrine Disrupting Chemicals (EDCs). The present study was carried out to assess the effects of Octylphenol (OP) alone and in combination with Nonylphenol (NP) on the hypothalamus-pituitary-adrenal gland (HPA) axis of the lizard Podarcis sicula. Lizards are good bioindicators due to their features such as wide distribution, large population and good sensitivity to contaminants. Results obtained showed a time and dose-dependent stimulation of the HPA together with a high variation of both catecholamine plasma levels and greater vascularization and hypertrophy of steroidogenic cord of adrenal gland after both OP and OP + NP treatments. Interestingly, the OP + NP mixture treatment has provoked a state of stress of the adrenal gland which in fact appeared to be characterized by the presence of a marked macrophage infiltration which can be seen especially close to the connective capsule surrounding the gland. This macrophage infiltration could be an evidence of a particularly pronounced inflammatory state to indicate, probably, an animal’s response to a non-physiological situation.

Fetal exposure to certain phthalate esters can disrupt testis development in rodents and lead to male reproductive disorders, but with a causal link less certain in humans. Di(2-ethylhexyl) phthalate (DEHP) is one of the most common phthalates found in the environment and in rodents it is known to induce serious testis toxicity, as well as male reproductive disorders including cryptorchidism, hypospadias, impaired spermatogenesis and reduced fertility. In this study, we show that perinatal DEHP exposure disrupts gap junction localization in fetal and postnatal rat testis and correlate these findings to morphological changes. The protein Connexin 43 (CX43), normally expressed strongly in testicular gap junctions, was markedly downregulated in Leydig cells of DEHP-exposed fetal testes. In the postnatal testes, CX43 expression was recovered in the DEHP-exposed animals, even though Leydig cell clusters and malformed cords with intratubular Leydig cells were still present., Graphical abstract Unlabelled Image, Highlights • DEHP disrupts gap junction localization in fetal and postnatal rat testis. • DEHP exposure reduces Cx43-positive gap junctions in Leydig cell clusters in fetal rat testis. • Leydig cell gap junctions recover in postnatal testis after early life DEHP exposure.

Dibutylphthalate (DBP) is an environmental pollutant widely used as plasticizer in a variety of industrial applications worldwide. This agent can be found in personal-care products, children's toy, pharmaceuticals, food products. Exposure to DBP can occur via ingestion and inhalation as well as intravenous or skin contact. DBP belongs to the family of endocrine disrupting chemicals (EDCs) and its effects on reproductive system were demonstrated both in vivo and in vitro. In the present study we evaluated the effects of DBP on human prostate adenocarcinoma epithelial cells (LNCaP) in order to highlight xenoestrogens influence on human prostate. Moreover, we have compared DBP effects with 17β-estradiol action in order to investigate possible mimetical behaviour. We have assessed the effects of both compounds on the cell viability. After then, we have evaluated the expression of genes and proteins involved in cell cycle regulation. Furthermore, we have observed the expression and the cell localization of estrogen (ERs) and androgen (AR) receptors. In conclusion, we have demonstrated that DBP interacts with estrogen hormonal receptor pathway but differently from E2. DBP alters the normal gland physiology and it is involved in the deregulation of prostate cell cycle.

Nonylphenol (NP) is an industrial chemical with estrogenic activity both in vivo and in vitro; estrogens play a critical role in the development of prostate and may be the cause of some pathological states, including cancer. In this study we examined the effects of NP on human prostate non tumorigenic epithelial cells (PNT1A) investigating on cell proliferation, interaction with estrogen receptors (ERs) and gene expression of genes involved in prostate diseases. We found that NP affects cell proliferation at 10(-6)M, promoting a cytoplasm-nucleus translocation of ERα and not ERβ, like the natural estrogen 17β-estradiol (E2). Moreover, we showed that NP enhances gene expression of key regulators of cell cycle. Estrogen selective antagonist ICI182780 in part reverted the observed effects of NP. These results confirm the estrogenic activity of NP and suggest that other transduction pathways may be involved in NP action on prostate.

Hamartin and tuberin are products of the tumor suppressor genes TSC1 and TSC2, respectively. Mutations affecting either gene result in the tuberous sclerosis syndrome, a neurologic genetic disorder characterized by the formation of multiple benign tumors or hamartomas. In this study, we report the identification of TSC2, but not TSC1, as a substrate of HtrA1, a member of the human HtrA family proteins of serine proteases. We show the direct interaction and colocalization in the cytoplasm of HtrA1 and TSC2 and that HtrA1 cleaves TSC2 both in vitro and in vivo. Finally, we show that alterations in HtrA1 expression cause modifications in phosphorylation status of two downstream targets of TSC2: 4E-BP1 and S6K. Our data suggest that, under particular physiologic or pathologic conditions, HtrA1 degrades TSC2 and activates the downstream targets. Considering that HtrA1 levels are significantly increased during embryogenesis, we speculate that one of the targets of HtrA1 activity during fetal development is the TSC2-TSC1 pathway. Mol Cancer Res; 8(9); 1248–60. ©2010 AACR.

The aetiology of endometriosis, a gynaecological disease defined by the histological presence of endometrial glands and stroma outside the uterine cavity, is still open to debate. Research has recently found evidence for endometriosis in human female fetuses at different gestational ages. This paper reports a new case of fetal endometriosis in a 25-week female fetus, deceased due to placental pathology, from a series of 13 female fetuses analysed at autopsy. The exact anatomical localization of this misplaced endometrium, as well as its histopathological and immunohistochemical characteristics are illustrated. The case suggests that endometriosis can be caused by dislocation of primitive endometrial tissue outside the uterine cavity during organogenesis. © 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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.

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.

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 hypothalamushypophyseal- 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.

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.

TheWFS1gene, encoding a transmembrane glycoprotein of the endoplasmic reticulum called wolframin, is mutated in Wolfram syndrome, an autosomal recessive disorder defined by the association of diabetes mellitus, optic atrophy, and further organ abnormalities. Disruption of theWFS1gene in mice causes progressiveβ-cell loss in the pancreas and impaired stimulus-secretion coupling in insulin secretion. However, little is known about the physiological functions of this protein. We investigated the immunohistochemical expression of wolframin in human placenta throughout pregnancy in normal women and diabetic pregnant women. In normal placenta, there was a modulation of wolframin throughout pregnancy with a strong level of expression during the first trimester and a moderate level in the third trimester of gestation. In diabetic women, wolframin expression was strongly reduced in the third trimester of gestation. The pattern of expression of wolframin in normal placenta suggests that this protein may be required to sustain normal rates of cytotrophoblast cell proliferation during the first trimester of gestation. The decrease in wolframin expression in diabetic placenta suggests that this protein may participate in maintaining the physiologic glucose homeostasis in this organ.

Apoptosis is a pathologic feature of cardiomyocytes in acute myocardial infarction (AMI) and heart failure. The temporal course of apoptosis in the peri-infarct area in the weeks following an AMI is still uncompletely defined. In order to study the time course of apoptosis after AMI, 16 rabbits underwent left coronary artery ligation and were sacrificed at 16, 26, 35, and 56 days after surgery. Increased apoptotic rate (AR) was observed at in the peri-infarct region than in remote myocardium (5.4% [2.5-9.6] vs 0.4% [0.1-0.9], respectively, P < 0.001) and than in sham-operated cases (0.01% [0-0.02], P < 0.001). A gradual decrease of AR in the peri-infarct region was observed over time with a 90% reduction at 8 weeks after coronary ligation. © 2006 Elsevier Ireland Ltd. All rights reserved.

Nonylphenol (NP) is an endocrine disruptor widely distributed in the environment. It accumulates in the lipids of living organisms and enters the human food chain. The main source of human exposure is expected to be food, drinking water and foodstuff contaminated through leaching from packaging or pesticide formulation applications. NP acts as an estrogenic compound and it is able to mimic the action of estradiol 17β (E2) by binding to the estrogen receptor (ER). The aim of the present study was to investigate the NP effects on the hypothalamic–pituitary–adrenal gland (HPA) axis of the bioindicator Podarcis sicula lizard. A time-dependent stimulation of the HPA axis and variations of both catecholamine plasma levels were showed. Moreover, NP effects on adrenal gland morphology were evaluated by light and transmission electron microscopy. Clear morphological signs of adrenal gland stimulation such as an increase of steroidogenic cord diameter and vascularization, a strong escalation of adrenaline cell number and a decrease of noradrenaline cells were observed. The notably elevated levels of adrenal hormones suggested a permanent turning on of hypothalamic corticotropin releasing factor (CRF) secretion together with a lack of the negative feedback of HPA axis, perturbing systemic responses of the organism. Our data may help to predict the biological alterations induced by NP and to extend its impact upon adrenal function.

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.

The present work studied the annual and daily aldosterone, norepinephrine and epinephrine patterns in the newt Triturus carnifex. The annual variations of the serum aldosterone were evaluated every month by radioimmunoassay, from September to July; the daily variations of this hormone were evaluated in the months of January, April, July and November. Aldosterone levels were low from September to November (lowest value 187.23 ± 19.7 pg/ml in November), increased in December, remained high until March and peaked in April (587.25 ± 58.2 pg/ml). In May, the levels decreased and remained steady until July. Considering the daily aldosterone pattern, in all the examined months, serum aldosterone levels were lowest at 5:00 am (lowest value 162.51 ± 17.4 pg/ml in November) and peaked at 5:00 pm (maximal value 2075.00 ± 213.00 pg/ml in April). As regards the annual catecholamine pattern as measured by HPLC, serum norepinephrine levels were low from September to November (lowest value 541.13 ± 39.8 pg/ml in September), increased in December, peaked in January (1457.03 ± 106.97 pg/ml) and remained high in February. In March, the levels decreased and remained steady until June, then decreased in July. Serum epinephrine levels increased from September to November, decreased in December and remained low until February. In March, the levels increased and peaked in April (721.55 ± 49.91 pg/ml), then decreased (lowest value 140.00 ± 10.0 pg/ml in June) and remained steady until July. Considering the daily catecholamines levels, in all the examined months, there were no daily variations among serum norepinephrine levels. Otherwise, serum epinephrine levels were always lowest at 5:00 am (lowest value 100.77 ± 9.9 pg/ml in January) and peaked at 11:00 pm (maximal value 3831.05 ± 211.5 pg/ml in November). The annual and daily aldosterone and catecholamine patterns appear correlated to the metabolic activity of this species.

Recently, suggestive evidence for piecemeal degranulation (PMD), a particulate pattern of cell secretion accomplished by vesicle-mediated extracellular transport of granule-stored material, has been provided by electron microscopy investigations in chromaffin cells of different vertebrate species. In this study, chromaffin tissue from the interrenal gland of the amphibian urodede Triturus carnifex has been investigated by quantitative transmission electron microscopy in search for ultrastructural clues indicative of a vesicle-mediated mode of cell degranulation. Interestingly, a single type of chromaffin cell is recognizable in T. carnifex, which undergoes seasonal variations in its adrenaline and noradrenaline granule ratio according to an annual cycle that matches the trend of changes in secondary sexual characters. In this study, we looked for a series of ultrastructural changes regarded as highly specific for PMD. We calculated the percentage of (i) resting, unaltered granules (type 1 granules); (ii) granules with changes indicative of progressive release of secretory materials, that is, granules with lucent areas in their cores, reduced electron density, disassembled matrices, and residual cores (type 2 granules); and (iii) membrane empty containers (type 3 granules) in chromaffin cells of T. carnifex during the annual cycle. We found a significant increase in type 2 and 3 granules, accompanied by a significant decrease in type 1 granules, in the April and November samples. During the same seasonal periods, the number per net cytoplasmic area of 30-100-nm diameter electron-dense vesicles was found to be significantly augmented, and there was also an augmented percentage of chromaffin granules showing blebs or protrusions in their profiles. These ultrastructural data are indicative of an increased vesicle-mediated transport of chromaffin granule products for extracellular release in the amphibian T. carnifex in accordance with the increased rate of catecholamine release. This vesicle-mediated pattern of cell secretion suits the schema of PMD. In an evolutionary perspective, these findings suggest that PMD is a secretory pathway that has been highly conserved throughout vertebrate classes. Anat Rec, 2009. (c) 2008 Wiley-Liss, Inc.

The objective of this study was to investigate the pattern of expression and the localization of Notch-1, Notch-4 and Jagged-1 in physiological and pathological human endometrium and to evaluate the expression levels of two major regulators of the G1 checkpoint, namely cyclin D1 and p21. Sixty samples of physiological endometrium and 60 samples of pathological endometrium were used for the study. Evaluation of the expression level and the distribution of Notch pathway members and cell-cycle proteins was performed by immunohistochemistry. In the physiological endometrium we observed an increase of Notch-1 and Jagged-1 from proliferative to secretory phase and an opposite trend for Notch-4. In menopause, the level of expression of all three members of the Notch pathway decreased. We also observed a cyclin D1 increase from proliferative to secretory phase. By contrast, p21 showed a slight increase from proliferative to secretory phase. In the pathological endometrium, we observed an increase of Notch-1 expression from polyps to carcinoma and decrease for Notch-4 and Jagged-1. Moreover, we observed a higher expression of cyclin D1 in all the endometrial pathologies. By contrast, the expression level of p21 slightly increased from polyps to carcinoma. We concluded that in human endometrium Notch-4 seems to be more involved in controlling proliferation, whereas Notch-1 seems to be more involved in differentiation programming. Deregulation of these functions may induce the onset of several endometrial pathologies from polyps to cancer.

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.

none 11 Heat shock protein B (HspB) is one of the dominant proteins recognized by most Helicobacter pylori-infected persons and is being considered as potential candidates for subunit vaccines. In the present study we describe the generation of an antibody against HspB and its use in immunohistochemical assays on gastric biopsies. We have demonstrated that our rabbit polyclonal antibody against HspB did not recognize any protein in lysates from a lung human epithelial cell (H1299) line and did not cross-react with the other members of human heat shock proteins. Secondly, we have observed that in gastric biopsies, HspB immunostaining was present inside the cytoplasm of human epithelial cells with a particular localization in the apical portion of gastric epithelial cells other than in the extracellular spaces among gastric cells of human stomach. Finally, we have demonstrated a cytoplasmic HspB immunostaining in groups of neoplastic cells of MALT lymphoma. In conclusion, our observations suggest a possible involvement of HspB in the pathogenesis of H. pylori-related pathologies such as gastritis, ulcer and gastric cancer. © 2008 Wiley-Liss, Inc. mixed DE LUCA A; DE FALCO M; MANENTE L; DATTILO D; LUCARIELLO A; ESPOSITO V; GNARINI M; CITRO G; BALDI A; TUFANO MA; IAQUINTO G De Luca A.; De Falco M.; Manente L.; Dattilo D.; Lucariello A.; Esposito V.; Gnarini M.; Citro G.; Baldi A.; Tufano M.A.; Iaquinto G.

a Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, United States b Department of Biological Science, Section of Evolutive and Comparative Biology, University of Naples ‘‘Federico II’’, Italy c Department of Cardiac Physiopathology, University of Buenos Aires, Argentina d Department of Medicine and Public Health, Sect. of Clinical Anatomy, Second University of Naples, Italy e Department of Biochemistry, Sect. of Pathologic Anatomy, Second University of Naples, Italy

Notch proteins are a transmembrane receptor family that is structurally and functionally conserved from worms to humans. The mammalian family of Notch proteins consists of several genes encoding Notch receptors and related Notch ligands. Notch signaling is involved in different aspects of the cell-fate decision tree: differentiation, proliferation, and apoptosis. These three processes are finely regulated in human placenta in order to allow a successful pregnancy and correct fetal growth. Notch and its ligands also participate in vascular remodeling and stabilization. Vasculogenesis and blood regulation are of importance in the human placenta for normal fetal development and growth; any disorder of these systems leads to preeclampsia. Drawing on this background, we have investigated the expression of Notch-1, Notch-4, and Jagged-1, together with two members related to the Notch pathway in angiogenesis: VEGF and p21. Normal and preeclamptic human placentas have been evaluated by immunohistochemistry. In preeclamptic samples, a down-regulation of Notch pathway members occurs with a weak/moderate expression of the Notch protein members in all components of placenta compared with physiological placentas that, at term, exhibit the strong expression of Jagged-1 and a moderate expression of both Notch-1 and Notch-4 in all compartments of the placental villi. Moreover, preeclamptic samples also reveal a down-regulation of VEGF expression, together with a moderate nuclear expression of p21(Cip1) in the syncytiotrophoblast, cytotrophoblast, and endothelial cells. This down-regulation of VEGF in preeclamptic placentas, in turn, probably decreases Notch protein expression in placental compartments and in endothelial cells and could offer an ethiopathogenetic explanation for the onset of this pathology.

Notch signaling is an evolutionarily conserved mechanism used by invertebrates and vertebrates to control cell fates through close-range cell interactions. Four Notch receptors have been identified in vertebrates and different ligands, divided into Delta-like and Serrate-like (Jagged). Several studies have demonstrated that Notch signaling is involved in different branches of the cell fate decision tree: differentiation, proliferation and apoptosis. These three processes are finely regulated in human placenta in order to allow a successful pregnancy and a correct fetal growth. Moreover, Notch and its ligands participate in the vascular remodelling and stabilization, other two processes much important and ticklish in human placenta. So, we decided to investigate the pattern of expression of Notch-1, Notch-4 and Jagged-1, together with two members related to Notch pathway and involved in angiogenesis: VEGF and p21, in human placenta during gestation by immunoblotting and immunohistochemistry. We showed a modulation of Notch proteins throughout the pregnancy; in particular we showed a slight decrease of Notch-1 throughout pregnancy, with a decreased cytoplasmic staining from the first to the third trimester of gestation in cytotrophoblast and syncytiotrophoblast. In contrast Jagged-1 showed an increase throughout pregnancy especially in syncytiotrophoblast and stroma during the third trimester of gestation. In addition, we found by immunoblotting an increase of VEGF expression from the first to the third trimester and an intense VEGF expression inside endothelial cells throughout the gestation as also confirmed by immunohistochemistry. We also showed a decrease of p21 expression during the pregnancy both through immunoblotting and immunohistochemistry assays. Moreover, we observed Notch localization in extravillous trophoblast cells that are able to invade the decidualized endometrium. Our results suggest an involvement of Notch signaling in regulation of placental cell fate decision and in angiogenesis that are dramatically important to maintain a normal physiology of this organ during pregnancy.

Endocrine-disrupting chemicals (EDCs) are a large group of substances able to modulate endocrine-signaling pathways, altering the normal function of the endocrine system. Although the fungicide methyl thiophanate (MT) is not considering a specific reproductive and developmental toxicant, it can induce histopathological damages in rat thyroid and adrenal glands that have a pivotal role in both processes. We investigated the MT effects on adrenal glands of Podarcis sicula lizard, the endemic species of Southern Italy living in open country and in cultivated fields. Reptiles are good bioindicators because they are easily harvested; they have a wide distribution and large populations. Moreover, they have good sensitivity to contaminants, and bioaccumulate and biomagnify pollutants to levels equal to or greater than those of birds and mammals. We used 1.5% MT/water to pollute terraria, food, and water twice a week for 15 and 30 days, and we evaluated adrenal toxicity through biochemical (adrenal and pituitary hormone plasma levels) and histological parameters (adrenal gland histopathology). We demonstrated a time-dependent increase of corticosterone plasma levels and a decrease of ACTH plasma levels, a hypertrophy of the steroidogenic tissue, and an enlargement of blood capillaries. Moreover, we observed a time-dependent increase of adrenaline plasma levels and adrenaline-producing cells, and an opposite trend of noradrenaline plasma concentrations. We also observed lymphocyte and macrophage infiltrations, signs of cell degeneration. Our findings on the bioindicator P. sicula provide an interesting basis to further elucidate the systemic mechanisms of EDCs.

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.

The present work was undertaken in order to investigate the influence of endocrine pancreas on the adrenal gland of Triturus carnifex. Our experiments aimed at studying the effects of intraperitoneal injections of glucagon on ultrastructural morphological and morphometrical features of steroidogenic and chromaffin tissues, as well as serum levels of aldosterone, corticosterone, norepinephrine (NE) and epinephrine (E). With regard to steroidogenic tissue, in January and November, glucagon decreased lipid droplet content in steroidogenic cells, that showed clear signs of increased activity. Moreover, increased corticosteroid serum levels were found. With regard to chromaffin tissue, in January glucagon played a stimulatory role on PNMT enzyme, eliciting an increase in the presence of E granules, and a decrease in the presence of NE granules, in the chromaffin cells. Moreover, increased E serum levels and decreased NE serum levels were found. In November, glucagon gave rise to a decrease in the presence of NE and E granules in the cells; E serum levels were strongly increased, whereas NE serum levels did not undergo any significant change. These findings suggest an involvement of the endocrine pancreas of the newt in the modulation of adrenal gland activity.

The distribution of endothelin-converting enzyme (ECE) in the lizard Podarcis sicula was investigated immunohistochemically using antibodies against endothelin-converting enzyme ECE-1 and endothelin-converting enzyme ECE-2 homologues. In all the tissues examined, immunoreactivity for both antibodies was found, although the distribution and degree of expression varied. Strong immunoreactivity was found in the endothelial cells and chromaffin tissue for both enzymes, whereas other tissues such as nervous tissue, renal tissue and hepatocytes display distinct patterns. Current knowledge does not allow correlation of these distribution patterns to specific functions but the data suggest that, in reptiles as in mammals, ECE is probably involved in physiological functions such as paracrine activity through endothelins and/or other substrates.

The aims of this study were to propose a bioindicator organism, the newt Triturus carnifex , for the assessment of toxicological impact of thiophanate methyl in the Campania region (Italy) and the possible adverse activity on the adrenal gland. In the acute toxicity study, experimental groups of T. carnifex were exposed to 2.40, 4.80, 9.60 and 19.20 μg/L tap water of thiophanate methyl for 2 days; the LD 50 was found to be 9.60 μg/L. To evaluate the effects on the adrenal gland, newts were exposed to a dose of 25% of the LD 50 2 days for 8 days. The ultrastructural features of the tissues as well as the serum levels of aldosterone, corticosterone, norepinephrine (NE) and epinephrine (E) were evaluated. The number of secretory vesicles in the chromaffin cells appeared significantly decreased, whereas NE and E serum levels appeared strongly increased. Moreover, corticosterone and aldosterone serum levels appeared significantly reduced. The results suggest that: 1) T. carnifex has the features of an ideal bioindicator, due to its high sensitivity to thiophanate methyl, 2) thiophanate methyl acts as endocrine disruptor, affecting the adrenal gland at very low doses, 3) thiophanate methyl may be toxic for nontarget organisms, such as newts.

The distribution of neuronal and non-neuronal mRNAs for alpha7 and alpha4 nicotinic acetylcholine receptor subunits was investigated in Triturus carnifex tissues using the in situ hybridization approach. The findings reveal a composite pattern of expression only partially overlapping for the two subunits; subunit alpha7 seems to be expressed widely throughout nervous, gastrointestinal and skin tissues, while alpha4 is present in a restricted number of cells of nervous and gastrointestinal tissue. We also found a specific pattern for each subunit; alpha7 and alpha4 associated exclusively to the epidermal glands and hypophysis, respectively; this is probably due to alternative roles that nicotinic acetylcholine receptors play in regulating physiological functions of non-neuronal amphibian tissues, rather than as mere neurotransmitters in the nervous system.

The effects of dopamine administration on the adrenal gland of a lizard, Podarcis sicula , are described. Dopamine (0.7 mg/100 g body wt/day for 4 consecutive days) raised plasma ACTH and corticosterone levels (ACTH: from the basal level of 4.40 ± 0.05–7.30 ± 0.08 pg/ml 24 h after the fourth dopamine injection; corticosterone: from 3.59 ± 0.03 ng/ml in untreated lizards to 7.40 ± 0.05 ng/ml 24 h after the fourth dopamine injection), showing a stimulatory effect on the pituitary-interrenal axis activity. In the chromaffin tissue dopamine apparently enhanced the activity of PNMT enzyme; in fact a strong raise in the number of adrenaline cells and a decrease in the number of noradrenaline cells were observed, decreasing the numeric NA/A cell ratio, from 1.4/1 of control specimens to 0.5/1 24 h after the fourth dopamine injection. At EM level, chromaffin cells contained both NA and A granules, as well as very clear granules (CG); CG granules showed granular elements ranging between 340 and 347 A in diameter. These cells might be the morphological expression of a process of catecholamine resynthesis, due to a possible increase in catecholamine release, following exposure to dopamine.

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.

The occurrence of substance P (SP) immunoreactivity was investigated in the adrenal gland of the lizard Podarcis sicula by ABC immunocytochemical technique: SP-immunoreactivity was present in both adrenaline and noradrenaline cells, in ganglion cells and nerve fibers in the connective capsule surrounding the gland. The involvement of substance P in the modulation of pituitary–interrenal axis was studied in vivo by intraperitoneal injections of SP. The effects were estimated by means of the morphological and morphometrical features of the tissues, as well as the plasma levels of adrenocorticotropic hormone (ACTH), corticosterone and catecholamines, adrenaline and noradrenaline. Substance P (0.07 mg/100 g body wt) decreased ACTH plasma levels and raised corticosterone release from steroidogenic tissue, that showed clear signs of stimulation. In the chromaffin tissue, the decrease in the number of noradrenaline cells, and the increase in the number of adrenaline cells, lowered numeric noradrenaline/adrenaline cell ratio. Moreover, an increase in adrenaline plasma level and a decrease in noradrenaline plasma level were found. The results suggest that (1) also in Reptiles as in other Vertebrates, SP may affect pituitary–adrenal axis activity, and (2) the chromaffin cells may be involved in the paracrine control of steroidogenic activity.

The human HtrA family of proteases consists of three members: HtrA1, HtrA2, and HtrA3. In bacteria, the chief role of HtrA is recognition and degradation of misfolded proteins in the periplasm, combining a dual activity of chaperone and protease. In humans, the three HtrA homologues appear to be involved in diverse functions such as cell growth, apoptosis, allergic reactions, fertilization, control of blood pressure, and blood clotting. Previous studies using RNA blot hybridization have shown that the expression of HtrA1 is ubiquitous in normal human tissues. Here we show by immunohistochemistry (IHC) that HtrA1 is widely expressed, although different tissue distributions and/or levels of expression were detected in the different tissues examined. In particular, high to medium HtrA1 expression was detected in mature layers of epidermis, in secretory breast epithelium, in liver, and in kidney tubules of cortex, in concordance with its secretory properties. Furthermore, we show a higher protein expression level in the epithelium of proliferative endometrium, in contrast to epithelium of secretory endometrium, which is almost completely negative for this protein. This suggests a possible role for HtrA1 in the modulation of tissue activity in this organ. The various expression levels in human tissues indicate several possible roles for HtrA1 in different cell types.

Wolframin is a transmembrane glycoprotein of 890 aminoacids, encoded by WFS1 gene. WFS1 mutations are responsible for Wolfram syndrome, an autosomal recessive disorder. In the present paper, we first characterized the polyclonal wolframin antibody by dot blot. Secondly, we verified antibody specificity by western blotting using different human cell lines. Thirdly, we studied wolframin localization in human foetal (14-35 weeks) and adult tissues by immunohistochemistry. Wolframin expression was distributed in many organs, with different tissue and cell localization and expression levels. In foetal systems, wolframin expression was faint at 14-16 weeks and increased when development proceeded. In adult human tissues a variable positive staining was observed in both simple and stratified epithelia. A moderate wolframin expression was observed in liver and in the endocrine portion of the pancreas. In conclusion, our data suggest that this protein may have important roles in a number of different tissues, including many that are not known to be affected by WFS1-linked diseases. The immunopositivity in adult human tissues suggests that it may function maintaining physiological cellular homeostasis.

Background Endometriosis is a gynecological disease defined by the histological presence of endometrial glands and stroma outside the uterine cavity. Women with endometriosis have an increased risk of different types of malignancies, especially ovarian cancer and non-Hodgkin's lymphoma. Though there are several theories, researchers remain unsure as to the definitive cause of endometriosis. Our objective was to test the validity of the theory of müllerianosis for endometriosis, that is the misplacing of primitive endometrial tissue along the migratory pathway of foetal organogenesis Methods We have collected at autopsy 36 human female foetuses at different gestational age. We have performed a morphological and immunohistochemical study (expression of oestrogen receptor and CA125) on the pelvic organs of the 36 foetuses included en-block and totally analyzed. Results In 4 out of 36 foetuses we found presence of misplaced endometrium in five different ectopic sites: in the recto-vaginal septum, in the proximity of the Douglas pouch, in the mesenchimal tissue close to the posterior wall of the uterus, in the rectal tube at the level of muscularis propria, and in the wall of the uterus. All these sites are common location of endometriosis in women. Conclusion We propose that a cause of endometriosis is the dislocation of primitive endometrial tissue outside the uterine cavity during organogenesis.