A Stereological Study of the Three Types of Ganglia of Male, Female, and Undifferentiated Scrobicularia plana (Bivalvia).

Neurotransmitters modulate gonadal maturation in bivalves. However, it remains unclear whether there are differences in the nervous system structure between sexes, maturation, and ganglia. Therefore, a stereological study was conducted on the ganglia of adult peppery furrow shell (Scrobicularia plana). Equal-sized males, females, and undifferentiated (gamete absence) animals were fixed with 10% formalin and processed for light microscopy. They were serially cut into 35 µm paraffin thick sections and stained with hematoxylin-eosin. Sections with cerebral (cerebropleural), pedal, and visceral ganglia were studied. The parameters estimated were the volumes of the ganglia, the total and relative volumes of their cortex (outer layer) and medulla (neuropil), and the total number of cells (neurons, glia, and pigmented) per ganglia and compartment. The volumes and numbers were estimated, respectively, by the Cavalieri principle and by the optical fractionator. Females show a larger glia to neuron numerical ratio. Further, females have a greater ganglionic volume than undifferentiated adults, with males showing intermediate values. These facts indicate that the ganglia size is related somehow to maturation. The cell size forms the basis of the differences because total cellularity is equal among the groups. The three ganglion types differ in total volumes and the volume ratio of the cortex versus the medulla. The greater volumes of the pedal ganglia (vis-a-vis the cerebral ones) and of the visceral ganglia (in relation to all others) imply more voluminous cortexes and medullae, but more neuronal and non-neuronal cells only in the visceral. The new fundamental data can help interpret bivalve neurophysiology.

Autonomic Disbalance During Systemic Inflammation is Associated with Oxidative Stress Changes in Sepsis Survivor Rats.

Sepsis affects 31.5 million people worldwide. It is characterized by an intense drop in blood pressure driving to cardiovascular morbidity and mortality. Modern supportive care has increased survival in patients; however, after experiencing sepsis, several complications are observed, which may be potentiated by new inflammatory events. Nevertheless, the interplay between sepsis survivors and a new immune challenge in cardiovascular regulation has not been previously defined. We hypothesized that cecal ligation and puncture (CLP) cause persistent cardiovascular dysfunctions in rats as well as changes in autonomic-induced cardiovascular responses to lipopolysaccharide (LPS). Male Wistar rats had mean arterial pressure (MAP) and heart rate (HR) recorded before and after LPS or saline administration to control or CLP survivor rats. CLP survivor rats had similar baseline MAP and HR when compared to control. LPS caused a drop in MAP accompanied by tachycardia in control, while CLP survivor rats had a noteworthy enhanced MAP and a blunted tachycardia. LPS-induced hemodynamic changes were related to an autonomic disbalance to the heart and resistance vessels that were expressed as an increased low- and high-frequency power of pulse interval in CLP survivors after saline and enhancement in the low-frequency power of systolic arterial pressure in control rats after LPS. LPS-induced plasma interferon γ, but not interleukin-10 surges, was blunted in CLP survivor rats. To further access whether or not LPS-induced autonomic disbalance in CLP survivor rats was associated with oxidative stress dysregulation, superoxide dismutase (SOD) activity and thiobarbituric acid reactive substances (TBARS) plasma levels changes were measured. LPS-induced oxidative stress was higher in CLP survivor rats. These findings indicate that key changes in hemodynamic regulation of CLP survivors rats take place in response to LPS that are associated with oxidative stress changes, i.e., reduced SOD activity and increased TBARS levels.

Histology and design-based estimation of hepatocellularity and volumes of hepatocytes in control and ethynylestradiol exposed males of platyfish (Xiphophorus maculatus).

The liver hosts numerous vital functions, such as biotransformation and excretion of xenobiotics. Synthetic oestrogens influence liver structure and function, leading to adaptations or to dysfunctions/injury. They are often stated to induce increases in fish liver weight, but there is controversy regarding how: if by changes in hepatocyte size (hypertrophy) and/or number (hyperplasia). Using platyfish as the experimental model, our primary aim was to assess if/how hepatocytes reacted to a sub-acute oestrogenic exposure. A complementary aim was to generate fundamental structural data for the liver of that model organism. Adult males were injected intramuscularly with 17α-ethinylestradiol (EE2) (25 µg/g), every 72 h for two weeks. Control fish were given solvent only. Body and liver morphometry were registered, and hepatocytes examined through histology and stereology at light microscopy. Immunohistochemistry evaluated hepatocytic vitellogenin (VTG) content. Treated and control fish did not differ as to quantitative parameters. Nevertheless, exposed fish were sensitive to EE2. VTG tagging was positive in their hepatocytes and these tended to be more basophilic, though not fully oestrogenized. We hypothesise that the platyfish liver is not particularly sensitive to the disrupting action of EE2 because of its reproductive mode; with no production peaks of VTG and no huge changes in endogenous sex-steroids. The fish may have had no evolutionary pressure for hepatocytes to be particularly reactive to oestradiol (E2). In the end, this study offers the first unbiased estimation of the liver cellularity in the platyfish, as well of the hepatocytic volume, serving now as a baseline reference.

Central leukotrienes modulate fever tolerance to LPS in rats.

Leukotrienes mediate several inflammatory events such as neutrophil chemoattraction, leukocyte adhesion, and central-release of cytokines and fever. However, there is no information available about their putative role in lipopolysaccharide (LPS) tolerance. The rational of the present study was to find out if central leukotrienes are involved in the development of LPS tolerance. Thus, we inhibited central leukotriene synthesis in tolerant rats using a pharmacological tool, i.e., a selective inhibitor of leukotriene synthesis MK-886 injected into the third ventricle (3V) of rats. Body core temperature (Tb) was measured using a datalogger placed inside the abdominal cavity. A low-dose of LPS (100 µg/kg ip) was given for 4 consecutive days to induce LPS tolerance. At day 4, rats received a microinjection of MK-886 into the 3V immediately before LPS, whereas control groups were treated with vehicle (saline). We observed that LPS failed to induce plasma cytokines surges, increased hypothalamic PGE2 levels and fever 3 days post LPS treatment, aptly characterizing the tolerance. When MK-886 was given to control rats treated with saline, no significant change in Tb was observed. However, a full LPS-induced fever was observed in tolerant rats pretreated with MK-886, which was associated with an enhancement in the hypothalamic PGE2 levels, that were not accompanied by plasma cytokines (IL-1ß, and IL-6) and PGE2 surges. These data are consistent with the notion that central leukotrienes play a role in fever tolerance to LPS.

Patients with Incompetent Valves in Chronic Venous Insufficiency Show Increased Systematic Lipid Peroxidation and Cellular Oxidative Stress Markers.

Chronic venous insufficiency (CVI) is a disease that impacts cellular homeostasis. CVI may occur with a valvular destruction process known as venous reflux or valvular incompetence. One of the cellular processes that may be triggered as a consequence of these events is the production of reactive oxygen species (ROS), which may trigger the production of different cellular markers and cell damage processes, such as lipid peroxidation. Therefore, the present study performed an observational, analytical, and prospective cohort study by reviewing 110 patients with CVI, and the activities and plasma levels of iNOS, eNOS, NOX1, and NOX2 were determined using immunohistochemistry and RT-qPCR. Lipid peroxidation (MDA) was also measured. Patients were distributed according to the presence or absence of valvular incompetence-venous reflux, which was diagnosed clinically as the absence of venous reflux (NR = 29) or presence of venous reflux (R = 81). Each group was divided according to age, with a cutoff point of fifty years (NR < 50 = 13, NR ≥ 50 = 16, R < 50 = 32, and R ≥ 50 = 49). The results showed that R patients exhibited significantly increased plasma MDA levels, and R < 50 patients exhibited the highest statistically significant increase. iNOS, NOX1, and NOX2 exhibited the highest gene and protein expression in R patients. The increased expression was maintained in the R < 50 patients. Our data suggest that young patients with valvular incompetence (venous reflux) show higher levels of lipid peroxidation and oxidative stress, which reflects the characteristics of an aged patient.

Implication of the PI3K/Akt/mTOR Pathway in the Process of Incompetent Valves in Patients with Chronic Venous Insufficiency and the Relationship with Aging.

Chronic venous insufficiency (CVI) is a multifactorial disease, commonly caused by valvular incompetence (clinically diagnosed by venous reflux) and venous hypertension. The incidence of these factors clearly increases with patient age, and aging is one of the risk factors involved. The activity of the PI3K/Akt/mTOR pathway is considered fundamental in vascular pathologies, and understanding its involvement would help in the development of possible therapeutic targets. This is an observational, analytical, and prospective cohort study that reviewed 110 patients with CVI scheduled to undergo stratified saphenectomy. They were distributed according to the presence (R = 81) or absence (NR = 29) of valvular incompetence (venous reflux) diagnosed clinically. Each of the groups was further divided according to age, with a cutoff point of 50 years (NR < 50 = 13, NR ≥ 50 = 16, R < 50 = 32, and R ≥ 50 = 49). The involvement of the PI3K/Akt/mTOR pathway, as well as that of HIF-1α and HIF-2α and of CD4+, CD8+, and CD19+ cells and mastocytes, was assessed. Saphenous vein tissue samples obtained during surgery were processed for RT-qPCR and immunohistochemistry. Patients with venous reflux showed a significant increase in mRNA and protein expression levels for PI3K/mTOR and HIF-1α/HIF-2α. The number of mast cells was significantly elevated in the R group. In distribution by age, PI3K/Akt/mTOR and HIF-1α were significantly higher in R < 50 patients. Furthermore, these patients had a significant increase in the number of CD4+, CD8+, and CD19+ cells and mastocytes in the saphenous vein wall. These findings provide a basis for the possible existence of changes in PI3K/Akt/mTOR pathway expression in young patients, with potential accelerated asynchronous aging that is enhanced by CVI.

Placentas from women with pregnancy-associated venous insufficiency show villi damage with evidence of hypoxic cellular stress.

Lower extremity venous insufficiency (VI) is a complication of pregnancy. The potential association of this venous disease with structural damage of the placenta has not been described. We analyzed the pattern of histopathologic lesions and the gene and protein expression of HIF1-α and apoptosis regulatory proteins. A prospective study was carried out on placenta samples from 43 women with pregnancy-associated VI and 24 age-matched pregnant healthy controls (HCs). Women with VI showed a significant increase in the number of villi (150.77 ± 42.55 VI versus 122.13 ± 27.74 HC) and in syncytial knots compared with those found in the placentas from HCs (67.15 ± 31.08 VI versus 42.49 ± 17.36 HC), and an increase in the number of bridges (32.40 ± 2.67 VI versus 22.73 ± 2.37 HC; P < .05). The mean number of syncytial nodes per villus is 1.37 ± 0.90 in the VI group and 0.49 ± 0.58 in the HC group (P < .001). Significant increases in the expression of Bax and caspase-3 and caspase-9 in the placentas from women with VI were observed compared with those found in HC. The expression of HIF-1α at both the messenger RNA and protein levels was also significantly increased in the placentas from women with VI. Our study demonstrates that placentas from women with pregnancy-associated VI show structural remodeling, with an increase in the number of villi and syncytial knots and enhanced apoptotic cellular death. Interestingly, this placental damage is associated with an increased expression of hypoxia-triggered molecular pathways, such as HIF-1α.

Characterization and spatial relationships of the hepatic vascular-biliary tracts, and their associated pancreocytes and macrophages, in the model fish guppy (Poecilia reticulata): A study of serial sections by light microscopy.

The guppy is a tropical fish that has been used as an experimental model organism in science. It is a species well adapted to the natural environment and that can support adverse environmental conditions, and so, at occasions, its presence can be indicative of environmental disturbances. Moreover, as the liver is very important when studying fish diseases, the knowledge of normal microanatomy is essential to assess histological changes, e.g., related to environmental change or toxic pollutants. The target organ of this histological study is the liver. The main objective is to contribute to the identification of anatomical and structural variations of this organ in different teleost species. We studied the distribution and spatial organization of the different types of blood vessels and biliary ducts and the relationships between them are established. For this, each liver was totally sectioned and the serial sections inspected in detail. The guppy liver presented intra-hepatic pancreatic tissue and so reported its association with the vascular and biliary elements. We observed that the input of afferent vessels (i.e., bringing blood into the liver) occur not only in the hilum but pierce and enter the organ at various points. Within the liver, venous vessels and bile ducts are seen, isolated or associated as venous-arteriolar tracts (VAT), and venous-biliary- arteriolar tracts (VBAT). Sometimes, pancreocytes appear within the liver surrounding isolated veins, forming venous tract with pancreatic acini (VT-P), or dual associations with afferent vessels, forming venous-arteriolar tracts with pancreatic acini (VAT-P). Intrahepatic pancreatic ducts were tiny and rare, putting in question the functional role of the acini. Contrary to other fish species, we did not spot isolated arterioles and associations between these and biliary ducts (BAT).We found aggregates of macrophages, namely associated with afferent and efferent (i.e., draining blood out) venous vessels; the latter fact not commonly reported in other fish species. There was a reduced arterialization of the organ (as arterioles were extremely rare), contrasting with an over predominance of a random distribution of the venous vascularization. The guppy differs to some extent from other previously studied models, highlighting the importance of making this kind of study to offer specific frameworks that can explain specific physiological processes or avoid misinterpretations; for instance about gene expression, as the whole liver specific expression will reflect the presence of hepatocytes and pancreocytes as well.

Remodelling of collagen fibres in the placentas of women with venous insufficiency during pregnancy.

Haemodynamic changes produced during pregnancy lead to elevated venous pressure in the legs and an increased resting consumption of oxygen. These events can cause varicose veins, or venous insufficiency (VI), which by creating an environment of hypoxia could affect the structure and function of the placental barrier. This study assesses the remodelling state of the placental villi by examining differences in collagens with a known role in villus structure and in placental barrier permeability between patients with and without VI. Samples of 67 placentas from women with VI (n=24) and without VI (n=43) during their pregnancy were processed for gene and protein expression analysis of COL-I, COL-III, MMP-2 and MMP-9 by RT-qPCR and immunohistochemistry. While no differences in COL-I expression levels were detected in the samples from women with and without VI, significant differences did emerge in both gene and protein expression levels of COL-III. Importantly, COL-I/III ratios were reduced in the VI group compared to controls. MMP-2 activity was similar in the two groups while MMP-9 levels were significantly elevated in VI with greatest expression differences observed at the level of the decidual cells. Mothers who developed VI during pregnancy showed significantly higher COL-III and MMP-9 levels consistent with a state of remodelling of the placental villi.

Unravelling the Role of MAPKs (ERK1/2) in Venous Reflux in Patients with Chronic Venous Disorder.

Chronic venous disorder (CVeD), is a disorder in which there is a modification in the conditions of blood return to the heart. The disorder may arise from incompetent valves and the resultant venous reflux (chronic venous insufficiency, CVI). The economic burden of CVeD on health systems is high, and research efforts have sought to elucidate the mechanisms involved as possible therapeutic targets. The mitogen-activated protein kinase (MAPK) enzymes mediate a wide array of physiopathological processes in human tissues. In this family of proteins, extracellular signal-regulated kinase (ERK)1/2 plays a direct role in the cell homeostasis that determines the viability of mammalian tissues. This study sought to examine whether ERK1/2 plays a role in venous reflux. This was a prospective study performed on 56 participants including 11 healthy controls. Of the CVeD patients, 23 had venous reflux with CVI (CVI-R) and 22 had no reflux (NR). Distribution by age was: controls <50 years (n = 4) and ≥50 years (n = 7); NR <50 years (n = 9) and ≥50 years (n = 13); CVI-R <50 years (n = 11) and ≥50 years (n = 12). Great saphenous vein specimens were subjected to gene (real-time polymerase chain reaction, RT-qPCR) and protein (immunohistochemistry, IHC) expression techniques to identify ERK1/2. Data was compared between groups using the Mann Whitney U test. Patients with CVI showed significant gene activation of ERK1/2 protein, and, in those with venous reflux, the expression of this gene was significantly greater. The CVI-R group <50 years showed significantly greater ERK1/2 gene expression than their age-matched controls. Expression patterns were consistent with IHC findings. Our studies suggest that ERK1/2 expression is involved in venous vascular disease.

Non-Nutritive Sweeteners: Evidence on their Association with Metabolic Diseases and Potential Effects on Glucose Metabolism and Appetite.

There is ongoing debate concerning non-nutritive sweeteners, their usage, and their effects on metabolism. The association between non-nutritive sweeteners consumption, development of metabolic diseases, and changes in appetite-regulating hormones is not clear. The aim of this article is to present an overview of non-nutritive sweeteners and to examine the scientific evidence of their effects on glucose metabolism and appetite-regulating hormones. Some observational studies suggest an association between non-nutritive sweeteners consumption and development of metabolic diseases; however, adiposity is a confounder frequently found in these studies. Results of the available clinical trials are heterogeneous and not comparable because of major differences between them. Future controlled studies evaluating specific non-nutritive sweeteners, with an appropriate sample size, including a uniform study group, with sufficient exposure time, and considering adjustment for confounder variables, such as anthropometric characteristics, previous consumption of non-nutritive sweeteners, and coexistence of significant metabolic comorbidities, are needed.

Seasonal changes in hepatocytic lipid droplets, glycogen deposits, and rough endoplasmic reticulum along the natural breeding cycle of female ohrid trout (Salmo letnica Kar.)-A semiquantitative ultrastructural study.

This study on wild female Ohrid trout was primarily designed to provide a general overview of the breeding cycle influence upon selected aspects of hepatocytes. According with a semiquantitatively evaluation, some of these cell's structural compartments change during the breeding cycle. Structural modifications were disclosed in the relative occurrence of lipid, glycogen, and RER content during breeding cycle. The relative amount of lipid deposits in the hepatocytes was much greater in previtellogenesis, and decreased postspawning. So, while the seasonal changes in RER were positively related with the ovary maturation status, those of the lipid droplets followed an opposite trend. The hepatocytic glycogen occurred rarely, mainly in late-vitellogenesis and spawning, suggesting that in this species such kind of energy storage is comparatively unimportant. Lipid accumulation and later usage is, probably, the relevant biochemical pathway for Ohrid trout in the wild. While glycogen and RER contents were positively correlated with the gonadosomatic index, lipids were negatively correlated. Additionally, glycogen inclusions were positively correlated with the plasma estradiol levels. When comparing seasonal patterns from wild Ohrid trout with those from well-studied rainbow and brown trout (specimens studied were from aquaculture), there are contradicting results as to lipid and glycogen reserves, and also as to RER loads. The differences among the mentioned trout can result from intrinsic interspecies differences or may be associated with natural feeding conditions versus feeding with commercially prepared diets, or other factors. This study offers new data useful as standard to access liver pathology in wild and aquacultured Ohrid trout. Microsc. Res. Tech. 79:700-706, 2016. © 2016 Wiley Periodicals, Inc.

Frequency of micronuclei and of other nuclear abnormalities in erythrocytes of the grey mullet from the Mondego, Douro and Ave estuaries--Portugal.

Fish are bioindicators of water pollution, and an increased rate of their erythrocyte nuclear morphological abnormalities (ENMAs)-and particularly of erythrocyte micronuclei (EMN)-is used as a genotoxicity biomarker. Despite the potential value of ENMAs and MN, there is scarce information about fish captured in Iberian estuaries. This is the case of the Portuguese estuaries of the Mondego, Douro and Ave, suffering from different levels of environmental stress and where chemical surveys have been disclosing significant amounts of certain pollutants. So, the aim of this study was to evaluate genotoxicants impacts and infer about the exposure at those ecosystems, using the grey mullet (Mugil cephalus) as bioindicator and considering the type and frequency of nuclear abnormalities of erythrocytes as proxies of genotoxicity. Sampling of mullets was done throughout the year in the important Mondego, Douro and Ave River estuaries (centre and north-western Portugal). The fish (total n = 242) were caught in campaigns made in spring-summer and autumn-winter, using nets or fishing rods. The sampled mullets were comparable between locations in terms of the basic biometric parameters. Blood smears were stained with Diff-Quik to assess the frequencies of six types of ENMAs and MN (given per 1,000 erythrocytes). Some basic water physicochemical parameters were recorded to search for fluctuations matching the ENMAs. Overall, the most frequent nucleus abnormality was the polymorphic type, sequentially followed by the blebbed/lobed/notched, segmented, kidney shaped, vacuolated, MN and binucleated. The total average frequency of the ENMAs ranged from 73 ‰ in the Mondego to 108 ‰ in the Ave. The polymorphic type was typically ≥50 % of the total ENMAs, averaging about 51 ‰, when considering all three estuaries. The most serious lesion-the MN-in fish from Mondego and Douro had a similar frequency (≈0.38 ‰), which was significantly lower than that in the Ave (0.75 ‰). No significant seasonal differences existed as to the MN rates and seasonal differences existed almost only in the Douro, with the higher values in AW. In general, the pattern of ENMAs frequencies was unrelated with the water physicochemical parameters. Considering the data for both the total ENMAs and for each specific abnormality, and bearing in mind that values of MN in fish erythrocytes >0.3 ‰ usually reflect pollution by genotoxicants, it is suggested that mullets were likely being chronically exposed to such compounds, even in the allegedly less polluted ecosystem (Mondego). Moreover, data supported the following pollution exposure gradient: Mondego < Douro < Ave. The scenario and inferences nicely agree with the published data from chemical monitoring.

Pex11α in brown trout (Salmo trutta f. fario): Expression dynamics during the reproductive cycle reveals sex-specific seasonal patterns.

A negative correlation between female gonadal maturation kinetics and size variations of hepatic peroxisomes was earlier documented in brown trout, as a probable impact of serum estrogen changes during the reproductive cycle. Herein, we investigated whether the organelle volume/surface dynamics seen in female brown trout liver peroxisomes - without numerical changes within each hepatocyte - is followed by variations in the expression of the membrane peroxisome protein Pex11α gene. For comparison, we also studied males. We find in females a seasonal variation with the highest Pex11α expression in February, which was statistically different from all other tested periods. Overall, the expression of PEX11α had over a fivefold decrease from February to September. This period coincides with the reproductive transition between the earlier post-spawning gonadal remodeling and preparatory staging and the pre-spawning period. Males did not show changes. Our approach allowed the first characterization of a peroxin gene in a teleost, the Pex11α, while offering a correlation scenario were, as we hypothesized, the peroxisomal size kinetics is paralleled by membrane-related gene alterations (measured herein as proxy of Pex11α gene expression). Our data support and expand previous results on the regulation, function and morphology of peroxisome dynamics in brown trout, with a broader interest.

Testing the effects of ethinylestradiol and of an environmentally relevant mixture of xenoestrogens as found in the Douro River (Portugal) on the maturation of fish gonads--a stereological study using the zebrafish (Danio rerio) as model.

In natural environments fish populations are exposed to many potential xenoestrogens, whereby understanding the impacts of mixtures continue to be of great interest. The main objective of this study was, therefore, to understand whether and how an environmentally relevant mixture of xenoestrogens found in the Douro River estuary can disrupt the normal gametogenesis in fish. For this purpose, adult zebrafish of both sexes were exposed for 21 days to an environmental mixture (MIX) of 11 xenoestrogens from diverse sources. A 100 ng/L ethinylestradiol (EE2) positive control was added. A quantitative (stereological) analysis with systematic sampling was made in the gonads, and using light microscopy both the relative and the absolute volumes of the gametogenic stages were estimated. Data point that the EE2 stimulus induced changes in structural compartments; with decreasing trends for the advanced maturation stages both in males and females. There was also a trend for a greater amount of interstitial tissue in males. Along with an interstitial fibrosis increase detected, the presence of a proteinaceous fluid was observed in both sexes and experimental groups (EE2 and MIX). Other histopathologic alterations were observed in the EE2 female group, such as the presence of foci of granulomatous inflammation and follicular mineralization in the germinal parenchyma and luminal areas. The most interesting finding of this study was that the exposure to the MIX caused a decrease of the relative volume of spermatozoa in zebrafish. This kind of estrogenic effect has not earlier been structurally quantified in such a fine detail with unbiased stereology in fish gonads. Despite the ultimate consequences of such disruptions being unknown, it could be logically argued that reduction or slowing-down of the appearance of the most mature cohorts and/or eventual interstitial fibrosis and other pathologic changes can adversely affect breeding. The findings add further explanatory bases for understanding the negative impacts of xenoestrogens.

In vitro exposure of Nile tilapia (Oreochromis niloticus) testis to estrogenic endocrine disrupting chemicals: mRNA expression of genes encoding steroidogenic enzymes.

Many environmental pollutants can exert adverse effects on exposed organisms, including fish, leading to disruption of the endocrine system. Enzymes involved in the sex steroid biosynthesis are potential targets for the toxic action of pollutants. In this context, we investigated the hypothesis that selected estrogenic chemicals-the pharmaceutical estrogen ethinylestradiol (EE2), the phytoestrogen genistein (GEN), and the industrial compound bisphenol A (BPA)-may cause endocrine disruption by directly disturbing steps of fish steroidogenic pathways. We studied the mRNA expression of eight selected genes encoding steroidogenic enzymes (11ß-HSD2, 20ß-HSD, 3ß-HSD1, 17ß-HSD1, 17ß-HSD8, 17ß-HSD12, CYP19a, CYP19b) by quantitative real-time PCR. Testis slices from adult specimens of the model fish Nile tilapia were exposed in vitro for 3 and 8 h either to individual or to mixture solutions of EE2 (100 ng/L), GEN (200 ng/L), and BPA (10 µg/L); all at the peak concentrations observed in the Douro River estuary (Portugal). Our data revealed that only the mixture of the tested chemicals directly induced the expression of 11ß-HSD2, 17ß-HSD1, and 17ß-HSD12, after 8 h, whereas no effect was seen for chemicals tested individually. The gene expression pattern agrees with the concept of dose addition for environmental mixtures, and for the first time an interference of estrogenic EDCs is reported for 17ß-HSD1 and 17ß-HSD12.

The hepatocytes of the brown trout (Salmo trutta fario): a stereological study of some cytoplasmic components with the breeding cycle.

Sex differences exist in fish hepatocytes, but studies for characterizing their cytology throughout the breeding cycle are still scarce; suggesting changes, but most lacking quantitative data. To address this limitation, to complement baseline data generated from the brown trout model, and to prove that sex-specific seasonal changes exist, we made an unbiased stereological evaluation of the hepatocytic cytoplasm. Unprecedentedly for fish liver, the stereological design was exempt from model (biased) assumptions. Five (3 years old) animals per sex were studied in endogenous vitellogenesis, exogenous vitellogenesis, and spawning season end. Liver pieces for analysis were systematically sampled. Stereology was done in transmission electron microscopy (TEM) micrographs. Primary data generated relative volume estimates of the major cytoplasmic components. Such values were used for deriving absolute volumes (per cell and per liver). Lipid droplets did not show changes. As to other targets, trends at cell and liver levels were not always equal. If the hepatocyte was the reference space, the contents in mitochondria, dense bodies, glycogen, and cytosol changed seasonally, in both sexes. If taking the liver as the reference, changes attained the Golgi apparatus and rough endoplasmic reticulum (RER), besides dense bodies, glycogen (in females), and cytosol. The components volumes (namely per liver) were often positively (negatively for glycogen) correlated with the ovary weight, disclosing new associations and implications in fish. While also offering gold-standard data for backing morphofunctional correlations and pathology, we revealed a new process by which females increase the amount of RER and Golgi throughout vitellogenesis, breaking from the idea on how this event happens in fish.