Browsing by Author "Sarasquete, C."
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- Comparing skeletal development of wild and hatchery-reared Senegalese sole (Solea senegalensis, Kaup 1858): Evaluation in larval and postlarval stagesPublication . Gavaia, Paulo J.; Domingues, S.; Engrola, S.; Drake, P.; Sarasquete, C.; Dinis, Maria Teresa; Cancela, LeonorThe Senegalese sole is a marine pleuronectiform that naturally occurs in Southern Europe and Mediterranean region where it is being produced in aquaculture, in particular in Portugal and Spain. The aim of this study was to assess the quality of hatchery-reared larvae in comparison with those reared in the wild, and determine to which extension wild growing larvae are also affected by skeletal deformities. The main structures affected included those forming the axial skeleton, the caudal fin complex and both anal and dorsal fins, with the most prevalent anomalies affecting caudal vertebrae and arches. Hatchery-reared fish presented a higher incidence of deformities (79%) compared with the 19% observed in wild specimens. In wild postlarvae collected in Autumn no deformities were observed. This work clearly shows that wild Senegalese sole present less skeletal deformities than those hatchery-reared during larval stages, indicating a selective mortality of wild deformed fish and/or an effect of aquaculture-related rearing conditions in the development of skeletal deformities in sole.
- Detection and localization of osteocalcin (BGP or Bone Gla Protein) in teleosts by in situ hybridization and immunohistochemistryPublication . Gavaia, Paulo J.; Viegas, C. A.; Pinto, Jorge; Sarasquete, C.; Cancela, LeonorAlthough the presence of osteocalcin (BGP) has been known for a number years, little knowledge on the regulation of expression and tissue localization of this protein in lower vertebrate organisms. In this site we have investigated the site of BGP mRNA expression by in situ hybridization using radiolabeled riboprobes and the tissue localization of the mature protein by immunohistochemistry in the Senegal sole (Solea senegalensis), zebrafish (Danio rerio), and gilthead sea bream (Sparus aurata) using fish BGP polyclonal antibodies.
- Detection of mineralized structures in very early stages of development of Marine Teleostei using a modified Alcian blue-Alizarin red double staining technique for bone and cartilagePublication . Gavaia, Paulo J.; Sarasquete, C.; Cancela, LeonorWe have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100-200 pm in length can be clearly visualized. The method is suitable for both ontogenic studies during early stages of skeletal development in most marine fishes (e.g., Sparus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 1000/0 ethanol for up to two years.
- Factors enhancing fish sperm quality and emerging tools for sperm analysisPublication . Cabrita, Elsa; Martínez-Páramo, S.; Gavaia, Paulo J.; Riesco, M. F.; Valcarce, D. G.; Sarasquete, C.; Herráez, M. P.; Robles, V.With this review we try to give a comprehensive overview of the current methods used in research to assess sperm quality. In addition, we identify some of the most important factors for enhancing sperm production and quality, including, broodstock nutrition, epigenetics and sperm management (cryopreservation). Sperm quality can be assessed by analyzing different parameters fromsimplemethods to very sophisticated approaches involving molecular tools. Parameters related with sperm composition or function (e.g. spermatozoa plasma membrane lipids, seminal plasma composition,motility activation) have successfully characterized a spermsample but could not respond to the causes behind sperm defects. Reactive oxygen species (ROS) are one of the causes of the impairment of sperm traits. High contents of ROS are capable of producing cell apoptosis, DNA strand breakages, mitochondria function impairment, and changes in membrane composition due to sugars, lipids, and amino acid oxidation, affecting at later times sperm fertilization ability. Recently, the importance of spermatozoa RNAs in the fertilization and early embryo development has been clearly demonstrated in different species, including fish. Spermatozoa delivers more than the paternal genome into the oocyte, carrying also remnant mRNA from spermatogenesis. These RNAs have been found in sperm from human, rodent, bovine, and recently in several fish species, demonstrating the important predictive value of spermatozoa transcripts present only in those samples with high motility or from males with higher reproductive performance. The content of those transcripts can be changed during gametogenesis process influencing their content in spermatozoa. Wewill focus this reviewon spermqualitymarkers, in new trends on spermanalysis, and in the use of these tools for the identification of factors enhancing gamete quality. Basic research in this field is helping to develop appropriate quality evaluation methodologies and early biomarkers of reproductive success, with potential future industrial applications.
- Gla proteins as markers for studies of skeletal development and malformations in new aquaculture fish species (Pagrus auriga, Diplodus sargo and Scophtalmus maximus)Publication . S B Viegas, Carla; Gavaia, Paulo J.; Ortiz-Delgado, J. B.; Sarasquete, C.; Cancela, LeonorEvaluation of the onset and the development of skeletal structures during larval ontogeny of commercially important species in southern Iberia Peninsula and Mediterranean area (...) is essential for the successful production of high quality fish.
- Matrix gla protein in turbot (Scophthalmus maximus): gene expression analysis and identification of sites of protein accumulationPublication . Roberto, Vania Palma; Cavaco, S.; S B Viegas, Carla; Simes, D; Ortiz-Delgado, J. B.; Sarasquete, C.; Gavaia, Paulo J.; Cancela, LeonorMatrix Gla protein (Mgp) is a secreted vitamin K-dependent extracellular matrix protein and a physiological inhibitor of calcification whose gene structure, amino acid sequence and tissue distribution have been conserved throughout evolution. In the present work, the turbot (Scophthalmus maximus) mgp cDNA was cloned and the sequence of the deduced protein compared to that of other vertebrates. As expected, it was closer to teleosts than to other vertebrate groups but there was a strict conservation of amino-acids thought to be important for protein function. Analysis of mgp gene expression indicated branchial arches as the site with higher levels of expression, followed by heart, vertebra and kidney. These results were confirmed by in situ hybridization with a strong mgp expression in branchial arch chondrocytes. Mgp was found to accumulate in gills where it appeared to be restricted to chondrocytes from branchial filaments, while in vertebrae it was localized in vertebral end plates, in growth zones, in vertebral arches and spines and in notochord cells. In the soft tissues analysed, Mgp was mainly detected in kidney and heart, consistent with previous data and providing further evidence for a role of Mgp as a calcification inhibitor and a modulator of the mineralization process. Our studies provide evidence that turbot, an important new species for aquaculture, is also a useful model to study function and expression of Mgp.
- Non-invasive assessment of reproductive status and cycle of sex steroid levels in a captive wild broodstock of Senegalese sole Solea senegalensis (Kaup)Publication . Garcia-Lopez, A.; Anguis, V.; Couto, Elsa; Canario, Adelino V. M.; Canavate, J. P.; Sarasquete, C.; Martínez-Rodriguez, G.Senegalese sole, Solea senegalensis, intensive culture is currently limited mainly due to the low control on reproduction in captivity. Comprehensive knowledge of reproductive biology and physiology for this species is needed in order to improve tank spawning success. This work describes for the first time the seasonal profiles of plasma levels of sex steroids (17β-estradiol, testosterone, 11- ketotestosterone, and 17,20β-dihydroxy-4-pregnen-3-one [17,20β-P]) in a S. senegalensis captive wild broodstock held under natural conditions, during two consecutive reproductive cycles. Changes in apparent maturation in females, dynamics of sperm release in males, and the condition factor (K) were monitored. Sixmaturation stages were established for females according to apparent size of the ovary and external abdominal swelling: early, intermediate and final ovarian development (F2+, F3+ and F4+, respectively), and partially spawned, mid spawned and spawned out or regressed (F3−, F2−, and F1−, respectively). During summer, F1− and nonrunning males (NRM) were predominant in association with low K and plasma steroid levels. At the end of summer, a new cycle of gonadal development started, denoted by the increase in reproductive parameters (K and steroid levels) and the appearance of F2+. By middle autumn, some females reached advancedmaturation stages (F3+ and F4+)while the proportion of runningmales (RM) showed a maximum. An occasional spawning could be registered during this season (November 2002). Towards the end of winter and beginning of spring, ovarian development reached its maximum. At this point, the proportion of F3+, F4+ and RM, K (specially in females), and steroid concentrations were the highest in concordance with the starting of the main spawning period (lasting from January to June 2003 and fromMarch to June 2004). Throughout this period, concomitantly with oocyte and sperm release, the proportion of F3−, F2−, F1− and NRMprogressively increased, while steroid levels and K progressively declined (concentration of steroids could fluctuate under a decreasing trend). The relatively elevated levels of 17,20β-P correlating with some parts of the spawning periods makes it a candidate for the role of the maturation-inducing steroid in S. senegalensis. Seasonal variations of measured parameters were consistent with the reproductive cycle of this species in the wild, and comparable to those found in other asynchronous multi-spawning fish.
- Osteocalcin and Matrix Gla Protein in developing teleost teeth. Identification of sites of mRNA and protein accumulation at single cell resolutionPublication . Ortiz-Delgado, J. B.; Simes, D; Gavaia, Paulo J.; Sarasquete, C.; Cancela, LeonorIn this study, the tissue distribution and accumulation of osteocalcin or bone Gla protein (BGP) and matrix Gla protein (MGP) were determined during tooth development in a teleost fish, Argyrosomus regius. In this species, the presence ofBGP andMGPmRNAin teethwas revealed by in situ hybridization. mRNA for BGP was detected in the odontoblasts as well as in its cytoplasmic processes emerging through dentinal tubules,whilemRNA for MGP was expressed in the enamel portion within the apical portion of the elongated cell bodies of enameloblasts, adjacent to the root of the teeth as well as in cells within the pulpal space. Immunolocalization of BGP and MGP demonstrated that these proteins accumulate mainly in the mineralized dentin or in enameloblastic processes, confirming in situ hybridization results. In this study, we examined for the first time the localization of both BGP and MGP gene expression and protein accumulation within the different regions of the vertebrate tooth. We clearly demonstrated that although the overall pattern of BGP andMGPgene expression and protein accumulation in A. regius teeth was in general agreement to what is known for other vertebrates such as rats or rodents, our study provided novel information and highlighted some species-differences between fish and higher vertebrates.
- Osteocalcin and Matrix Gla Protein in zebrafish (Danio rerio) and Senegal sole (Solea senegalensis): comparative gene and protein expression during larval development through adulthoodPublication . Gavaia, Paulo J.; Simes, D; Ortiz-Delgado, J. B.; S B Viegas, Carla; Pinto, Jorge; Kelsh, R. N.; Sarasquete, C.; Cancela, LeonorBone Gla protein (Bgp or osteocalcin) and matrix Gla protein (Mgp) are important in calcium metabolism and skeletal development, but their precise roles at the molecular level remain poorly understood. Here, we compare the tissue distribution and accumulation of Bgp and Mgp during larval development and in adult tissues of zebrafish (Danio rerio) and throughout metamorphosis in Senegal sole (Solea senegalensis), two fish species with contrasting environmental calcium levels and degrees of skeletal reorganization at metamorphosis. Mineral deposition was investigated in parallel using a modified Alizarin red/Alcian blue protocol allowing sensitive simultaneous detection of bone and cartilage. In zebrafish, bgp and mgp mRNAs were localized in all mineralized tissues during and after calcification including bone and calcified cartilage of branchial arches. Through immunohistochemistry we demonstrated that these proteins accumulate mainly in the matrix of skeletal structures already calcified or under calcification, confirming in situ hybridization results. Interestingly, some accumulation of Bgp was also observed in kidney, possibly due to the presence of a related protein, nephrocalcin. Chromosomal localization of bgp and mgp using a zebrafish radiation hybrid panel indicated that both genes are located on the same chromosome, in contrast to mammals where they map to different chromosomes, albeit in regions showing synteny with the zebrafish location. Results in Senegal sole further indicate that, during metamorphosis, there is an increase in expression of both bgp and mgp, paralleling calcification of axial skeleton structures. In contrast with results obtained for previously studied marine fishes, in zebrafish and Senegal sole Mgp accumulates in both calcified tissues and non-mineralized vessel walls of the vascular system. These results suggest different patterns of Mgp accumulation between fish and mammals.
- Ovarian development and plasma sex steroid levels in cultured female Senegalese sole Solea senegalensisPublication . Garcia-Lopez, A.; Couto, Elsa; Canario, Adelino V. M.; Sarasquete, C.; Martínez-Rodriguez, G.Ovarian development was studied in cultured female Senegalese sole Solea senegalensis. Females with regressed ovaries, mainly occupied by perinucleolar oocytes, predominated throughout summer exhibiting low condition factor (K), gonadosomatic index (IG), and plasma 17β-estradiol and testosterone levels. Throughout autumn and winter (ovaries at early and intermediate maturation), oocytes progressed to cortical alveoli and vitellogenic stages accompanied by increasing K, IG, and plasma 17β-estradiol and testosterone levels. At late winter/early spring, ovarian development reached its maximum with the predominance of females at intermediate and final maturation (the latter occupied by late vitellogenic oocytes and few early maturation oocytes) and peak values of K, IG, and 17β-estradiol and testosterone concentrations. Steroid levels were lower (especially testosterone) than those for naturally-spawning females, which might cause extensive atresia without final oocyte maturation (no spawning was observed). This degenerative process reduced de size of the ovary (initial and intermediate phases of regression) in association with declining K, IG, and plasma 17β-estradiol and testosterone levels and increasing proportions of perinucleolar oocytes. The circulating 17,20β-dihydroxy-4-pregnen-3-one levels, the proposed maturation-inducing steroid, remained relatively constant throughout the experimental period, suggesting that oocytes were unable to respond adequately to its stimulation. We propose the inadequate seasonal thermal regime as the main cause of such dysfunction.