Eduardo Rocha | Universidade do Porto (original) (raw)

Papers by Eduardo Rocha

Aquatic Toxicology, 2015

Zebrafish (Danio rerio) is extensively used in research; however the mechanisms that control this... more Zebrafish (Danio rerio) is extensively used in research; however the mechanisms that control this species sex determination are still poorly understood. In the latest decades, it has been established that zebrafish sex is determined by genetic factors on a polygenic basis, as various candidate genes with sex dimorphic expression, as well sex-linked loci have been identified in different zebrafish strains. However, it has been evidenced that sex determination in this species is also influenced by environmental factors. For instance, temperature can have a crucial role in zebrafish sex determination. Likewise, the exposure to endocrine disrupting chemicals (EDCs), the most studied zebrafish sex changing factor, can strongly influence the course of sex differentiation and unbalance the sex ratio of zebrafish populations. Despite this, so far the influence of environmental factors is still less understood and only few studies have addressed this topic. Therefore, this review intends to gather current knowledge on the environmental factors involved in sex determination of zebrafish and identify important gaps in this research area. Briefly, the current understanding on zebrafish sex related genetics is also addressed.

Zygote (Cambridge, England), Jan 14, 2015

The ultrastructural analysis of human oocytes at different maturation stages has only been descri... more The ultrastructural analysis of human oocytes at different maturation stages has only been descriptive. The aim of this study was to use a stereological approach to quantify the distribution of organelles in oocytes at prophase I (GV). Seven immature GV oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. The Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction were used to compare the means of the relative volumes occupied by organelles in oocyte regions: cortex (C), subcortex (SC) and inner cytoplasm (IC). Here we first describe in GV oocytes very large vesicles of the smooth endoplasmic reticulum (SER), vesicles containing zona pellucida-like materials and coated vesicles. The most abundant organelles were the very large vesicles of the SER (6.9%), mitochondria (6.3%) and other SER vesicles (6.1%). Significant differences in organelle distr...

Reproductive BioMedicine Online, 2015

ICBAS-UP. Research in her laboratory focuses on the genetic causes of human infertility and biolo... more ICBAS-UP. Research in her laboratory focuses on the genetic causes of human infertility and biological mechanisms associated to human gametogenesis and embryogenesis. Her current research interests are male fertility preservation, in-vitro spermatogenesis and oocyte dysmorphisms.

Toxicological & Environmental Chemistry, 2012

This article may be used for research, teaching, and private study purposes. Any substantial or s... more This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

Gene, 2012

Acetyl-CoA short chain synthetases (ACSSs) are key enzymes in the activation of fatty acids throu... more Acetyl-CoA short chain synthetases (ACSSs) are key enzymes in the activation of fatty acids through the formation of thioesters with CoA. Three subfamily members are currently recognized in the human genome, ACSS1, ACSS2 and ACSS3, all single copy genes. The mitochondrial isoform, Acss1, plays a key role in the metabolism of acetate for energy production. While the single copy condition has been accurately established in humans, the evolutionary history of the Acss1 subfamily in vertebrates has yet to be elucidated, in particular, the isoform diversity, origin and function. Through genome database mining we analyzed the diversity of Acss1 isoforms in vertebrate classes. We detected the presence of a novel Acss1 isoform, which we name Acss1B. This new gene, Acss1B, has a curious phylogenetic distribution being found in teleosts (except zebrafish), sauropsids (birds and reptiles) and probably chondrichthyes. In contrast Acss1A is found in all the investigated species, except the teleost medaka. By means of comparative genomics and phylogenetics we show that Acss1A and Acss1B were generated in the quadruplication of the vertebrate genome. In effect, we find that amphioxus, a pre-genome duplication chordate, has a single Acss1 gene in a genomic region equally related to a quadrupled vertebrate genomic set. Consequently, Acss1B has been lost in some teleosts, amphibians and mammals, while Acss1A is probably absent in medaka. The reported findings illustrate an especially dynamic pattern of paralogue retention and independent loss in vertebrate species involving the Acss1 subfamily, whose functional consequences in energy metabolism are as yet unknown.

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2013

Dietary long chain polyunsaturated fatty acids (FA) have been recognized of crucial importance 28... more Dietary long chain polyunsaturated fatty acids (FA) have been recognized of crucial importance 28 in early development of vertebrates, contributing to the impressive morphological and 29 physiological changes both as building blocks and to energy production. The importance of 30 lipids along development depends on ontogenetic, phylogenic and environmental parameters. 31 The expression patterns of FA metabolism genes have not been characterized in developing fish 32 embryos nor compared to lipid class profiles. Full lipid metabolism only occurred after hatching, as revealed by alterations in lipid profiles and FA gene expression. Nonetheless, 1 transcriptional changes of some FA genes were already present in embryos at notochord 2 formation. Many genes displayed an expression profile opposed to the decrease of lipids along 3 the development, while others responded solely to starvation. Transcription of most genes 4 involved in FA metabolism had a strong correlation to PPARs mRNA levels (α1, α2, β, γ). The 5 comparison of mRNA expression of the genes with the lipid profiles produced new insights into 6 the FA metabolism and regulation during the development of turbot larvae, providing the basis 7 for future studies including comparative approaches with other vertebrate species.

BMC Evolutionary Biology, 2011

Background: Stearoyl-CoA desaturases (SCDs) are key enzymes involved in de novo monounsaturated f... more Background: Stearoyl-CoA desaturases (SCDs) are key enzymes involved in de novo monounsaturated fatty acid synthesis. They catalyze the desaturation of saturated fatty acyl-CoA substrates at the delta-9 position, generating essential components of phospholipids, triglycerides, cholesterol esters and wax esters. Despite being crucial for interpreting SCDs roles across species, the evolutionary history of the SCD gene family in vertebrates has yet to be elucidated, in particular their isoform diversity, origin and function. This work aims to contribute to this fundamental effort. Results: We show here, through comparative genomics and phylogenetics that the SCD gene family underwent an unexpectedly complex history of duplication and loss events. Paralogy analysis hints that SCD1 and SCD5 genes emerged as part of the whole genome duplications (2R) that occurred at the stem of the vertebrate lineage. The SCD1 gene family expanded in rodents with the parallel loss of SCD5 in the Muridae family. The SCD1 gene expansion is also observed in the Lagomorpha although without the SCD5 loss. In the amphibian Xenopus tropicalis we find a single SCD1 gene but not SCD5, though this could be due to genome incompleteness. In the analysed teleost species no SCD5 is found, while the surrounding SCD5-less locus is conserved in comparison to tetrapods. In addition, the teleost SCD1 gene repertoire expanded to two copies as a result of the teleost specific genome duplication (3R). Finally, we describe clear orthologues of SCD1 and SCD5 in the chondrichthian, Scyliorhinus canicula, a representative of the oldest extant jawed vertebrate clade. Expression analysis in S. canicula shows that whilst SCD1 is ubiquitous, SCD5 is mainly expressed in the brain, a pattern which might indicate an evolutionary conserved function. Conclusion: We conclude that the SCD1 and SCD5 genes emerged as part of the 2R genome duplications. We propose that the evolutionary conserved gene expression between distinct lineages underpins the importance of SCD activity in the brain (and probably the pancreas), in a yet to be defined role. We argue that an expression independent of an external stimulus, such as diet induced activity, emerged as a novel function in vertebrate ancestry allocated to the SCD5 isoform in various tissues (e.g. brain and pancreas), and it was selectively maintained throughout vertebrate evolution.

Aquaculture Nutrition, 2005

A 12-week feeding trial was conducted to evaluate the effects of fish oil replacement by soybean ... more A 12-week feeding trial was conducted to evaluate the effects of fish oil replacement by soybean oil, on lipid distribution and liver histology of two commercially important finfish species: rainbow trout (Oncorhynchus mykiss) and European sea bass (Dicentrarchus labrax). Sea bass (16.2 ± 0.5 g; mean ± SD) and rainbow trout (52.1 ± 0.5 g) juveniles were fed one of three isonitrogenous (500 g kg)1 CP) and isoenergetic (19 kJ g)1) diets, containing 0% (control, diet A), 25% (diet B) and 50% (diet C) soybean oil. At the end of the experiment, lipid deposition was evaluated in muscle, liver and viscera. Cholesterol and triglycerides levels were also determined in plasma. Tissue total, neutral and polar lipid composition (g kg)1 total lipids) showed no significant differences within species, regardless the dietary treatment. The same trend was observed for plasma parameters (P > 0.05). Viscera were the preferential tissue of lipid deposition, with 252-276 and 469-513 g kg)1 total lipid content in trout and sea bass, respectively. Dietary fish oil replacement had no effect on either hepatic lipid droplets accumulation or degree and pattern of vacuolization in the observed liver sections. These data suggest that both sea bass and trout can be fed diets containing up to 50% soybean oil without adverse effects on tissue lipid composition or liver histology.

Journal of Anatomy, 2001

Cortical cerebellar basket cells are stable postmitotic cells ; hence, they are liable to endure ... more Cortical cerebellar basket cells are stable postmitotic cells ; hence, they are liable to endure age-related changes. Since the cerebellum is a vital organ for the postural control, equilibrium and motor coordination, we aimed to determine the quantitative morphological changes in those interneurons with the ageing process, using unbiased techniques. Material from the cerebellar cortex (Crus I and Crus II) was collected from female rats aged 2, 6, 9, 12, 15, 18, 21 and 24 mo (5 animals per each age group), fixed by intracardiac perfusion, and processed for transmission electron microscopy, using conventional techniques. Serial semithin sections were obtained (5 blocks from each rat), enabling the determination of the numberweighted mean nuclear volume (by the nucleator method). On ultrathin sections, 25 cell profiles from each animal were photographed. The volume density of the nucleus, ground substance, mitochondria, Golgi apparatus (Golgi) and dense bodies (DB), and the mean surface density of the rough endoplasmic reticulum (RER) were determined, by point counting, using a morphometric grid. The mean total volumes of the soma and organelles and the mean total surface area of the RER [s-N (RER)] were then calculated. The results were analysed with 1-way ANOVA ; posthoc pairwise comparisons of group means were performed using the Newman-Keuls test. The relation between age and each of the parameters was studied by regression analysis. Significant age-related changes were observed for the mean volumes of the soma, ground substance, Golgi, DB, and s-N (RER). Positive linear trends were found for the mean volumes of the ground substance, Golgi, and DB ; a negative linear trend was found for the s-N (RER). These results indicate that rat cerebellar basket cells endure important age-related changes. The significant decrease in the s-N (RER) may be responsible for a reduction in the rate of protein synthesis. Additionally, it may be implicated in a cascade of events leading to cell damage due to the excitotoxic activity of glutamate, which could interfere in the functioning of the complex cerebellar neuronal network.

BMC Biochemistry, 2003

Background: This study was aimed primarily at testing in the liver of brown trout (Salmo trutta) ... more Background: This study was aimed primarily at testing in the liver of brown trout (Salmo trutta) spectrophotometric methods previously used to measure the activities of catalase and hydrogen peroxide producing oxidases in mammals. To evaluate the influence of temperature on the activities of those peroxisomal enzymes was the second objective. A third goal of this work was the study of enzyme distribution in crude cell fractions of brown trout liver. Results: The assays revealed a linear increase in the activity of all peroxisomal enzymes as the temperature rose from 10° to 37°C. However, while the activities of hydrogen peroxide producing oxidases were strongly influenced by temperature, catalase activity was only slightly affected. A crude fraction enriched with peroxisomes was obtained by differential centrifugation of liver homogenates, and the contamination by other organelles was evaluated by the activities of marker enzymes for mitochondria (succinate dehydrogenase), lysosomes (aryl sulphatase) and microsomes (NADPH cytochrome c reductase). For peroxisomal enzymes, the activities per mg of protein (specific activity) in liver homogenates were strongly correlated with the activities per g of liver and with the total activities per liver. These correlations were not obtained with crude peroxisomal fractions. Conclusions: The spectrophotometric protocols originally used to quantify the activity of mammalian peroxisomal enzymes can be successfully applied to the study of those enzymes in brown trout. Because the activity of all studied peroxisomal enzymes rose in a linear mode with temperature, their activities can be correctly measured between 10° and 37°C. Probably due to contamination by other organelles and losses of soluble matrix enzymes during homogenisation, enzyme activities in crude peroxisomal fractions do not correlate with the activities in liver homogenates. Thus, total homogenates will be used in future seasonal and toxicological studies of brown trout peroxisomes.

Asian Journal of Andrology, 2015

correlated with male infertility, 6 being proposed as a tool for infertility diagnosis. 7 This lo... more correlated with male infertility, 6 being proposed as a tool for infertility diagnosis. 7 This locus is divided into three major regions named AZFa, AZFb, and AZFc. 5 Within the AZFc region is located the Deleted in Azoospermia (DAZ) gene family. 8 Deletions of the DAZ1/DAZ2 gene doublet were found to be responsible for severe oligozoospermia, with possible evolution to secretory azoospermia with different testicular phenotypes, 9 but later demonstrated that only DAZ1 was responsible for this phenotype. 10 Distinct histopathology phenotypes were correlated with the site of the microdeletion, varying from complete absence of germ cells (Sertoli Cell-Only Syndrome, SCOS) in patients with AZFa microdeletions, maturation arrest at meiosis (MA) in cases with AZFb microdeletions, to hypospermatogenesis (HP) in patients with AZFc microdeletions. 5,9,11-16 However, individual variation on the extent of microdeletions has led to the observation of incomplete microdeletions which enable the existence of spermatogenesis foci in SCOS and MA and thus successful testicular sperm retrieval. 17-20 These situations may be referred as variants of classic deletions and were shown to

Tissue and Cell, 2008

The aim of this work was to gain insights into the mechanism of muscle differentiation and growth... more The aim of this work was to gain insights into the mechanism of muscle differentiation and growth in Pagellus bogaraveo, by studying muscle fibre phenotypes identified by immunohistochemistry. At hatching, several layers of deep fast-white fibres were covered by a superficial fibre monolayer. At 5 days, slow-red fibres appeared near the lateral line nerve. At 40 days, the intermediate-pink muscle became visible, and in the slow-red and fast-white muscle layers transitions from larval myosin isoforms to the isoforms typical of adult muscle occurred. Between 70 and 100 days, small fibres with a distinct ATPase profile appeared throughout the fast-white muscle, marking the onset of "mosaic" hyperplasia. The myosin of the original superficial monolayer fibres underwent two myosin transformations, before being slowly replaced by an adult slow-red isoform. In juveniles and adults, the slow-red muscle layer could be resolved into two distinct types. The analysis of fibre phenotypes indicated that post-larval muscle growth occurred by two distinct stages of hyperplasia. This study offers a basis for further comparative and experimental studies with this economically relevant species, namely for identifying factors influencing its muscle growth dynamics and disclosing underlying mechanisms.

The Veterinary Journal, 2014

Canine mammary tumours are highly heterogeneous in morphology and behaviour and successful clinic... more Canine mammary tumours are highly heterogeneous in morphology and behaviour and successful clinical management requires robust prognostic factors. Histological grade, determined by the Nottingham method, has been considered one of these factors. Despite the adoption of this method, it is unknown if inter-observer agreement exists regarding the assessment of its parameters in canine mammary carcinomas (CMC). In this study, the agreement between two observers using the Nottingham method was evaluated in 89 CMC. Histological evidence of vascular invasion and/or lymph node metastases (both early signs of tumour aggressiveness) was recorded. For 48 animals, 2-years follow-up data were available. Nuclear pleomorphism was quantitatively assessed using a stereological method, that allowed for an unbiased estimation of nuclear size and its variability, by determining the volume-weighted mean nuclear volume ( ̅ V). Differences between the  ̅ V estimations and the nuclear pleomorphism scores were evaluated. Additionally, the prognostic significance of clinicopathological features including nuclear score and  ̅ V was evaluated. A poor agreement between the two observers was obtained (Kappa value 0.46). Tumours scored as 1 and 2 presented similar  ̅ V and only tumours scored 3 presented significantly higher estimates. The  ̅ V was not associated with vascular invasion and/or lymph node metastases, but was higher in tumours that progressed during follow-up. In multivariable analysis only tumour size was an independent factor regarding evidence of aggressiveness and an optimal cutoff of 2.9 cm was defined.

Microscopy and Microanalysis, 2009

Ecotoxicology and Environmental Safety, 2009

Stereological methods were used to estimate the volumetric density (V(V)) of the filamentar epith... more Stereological methods were used to estimate the volumetric density (V(V)) of the filamentar epithelium (FE, 39%), lamellae (L, 28%), central venous sinus (CVS, 14%), central axis (16%), mucous cells (MC, 2%) and chloride cells (CC, 1%) in the gill filament of control Nile Tilapia. The relative volumes of FE and L, and the relative volumes of CVS and central axis, varied inversely under exposure to copper, with high copper toxic levels declanching a chronic defence mechanism that was, nevertheless, overcome, and low copper toxic levels causing adaptation within a moderate acute phase type of response. Copper also induced a decrease of the V(V) (MC, gill filament) due to reduction of surface MC, despite the marked increase of stem MC at chronic exposure to high copper toxic levels. Diminution of the numerical density of filamentar CC was responsible for the decreased V(V) (CC, gill filament), although lamellar CC significantly increased at chronic exposure to low copper toxic levels. The present results demonstrate that cell relative volumes, mean volumes and numerical densities are dependent on the variations of the FE and L, which without a quantitative approach may be misinterpreted, thus stressing the importance of using stereological tools for analyzing histopathological patterns.

$Research paper thumbnail of Stereological estimation of Ito cells from rat liver using the optical fractionator-a preliminary report$

Journal of Fish …, 2008

... Monteiro, SM, Rocha, E., Fontaínhas-Fernandes, A. and Sousa, M. (2008), Quantitative histopat... more ... Monteiro, SM, Rocha, E., Fontaínhas-Fernandes, A. and Sousa, M. (2008), Quantitative histopathology of Oreochromis niloticus gills after ... 1013, 5000-911 Vila Real, Portugal, Centre of Marine and Environmental Research (CIMAR); Laboratory of Histology and Embryology ...

Journal of …, 2009

Studies on liver macrophages have elucidated their key roles in immunological, fibrotic and regen... more Studies on liver macrophages have elucidated their key roles in immunological, fibrotic and regenerative responses, and shown that macrophages are not a homogeneous population. In the rat, two sets of liver macrophages coexist, identified by ED1 and ED2 antibodies. Those sets have different quantitative responses in liver injuries and may have different tasks throughout the injury and recovery phases. Nevertheless, the total number (N), number per gram (N g−1) and proportion of those macrophages in relation to other liver cells has never been quantified using design-based stereology. Thus, we combined immunocytochemistry with those tools to produce an unbiased estimate of the N of ED1+ and of ED2+ cells. A smooth fractionator sampling scheme was applied to the liver of five male Wistar rats (3 months old), to obtain systematic uniform random sections (30 µm thick); these were immunostained with the monoclonal antibodies: ED1, a pan-macrophagic marker; and ED2, which identifies the completely differentiated macrophages, i.e. Kupffer cells. The N of ED1+ cells was 340 × 106, estimated with a coefficient of error (CE) of 0.04, and that of ED2+ cells was 283 × 106, with a CE of 0.05. These figures correspond to 10.7% and 8.9%, respectively, of the total liver cells. The new data constitute reference values for correlative inferences. Also, the methodological strategy, by its accuracy and precision, is valuable for future investigations on the liver cell composition in various models of disease, and especially for studying the more subtle variations that occur during the injury and recovery phases.

Microscopy and …, 2006

Abstract: The large variability in kidney morphology among fish makes it difficult to build a un... more Abstract: The large variability in kidney morphology among fish makes it difficult to build a universal model on its function and structure. Therefore, a morphological study of brown trout trunk kidney was performed, considering potential seasonal and sex effects. ...