Sebastiano Vilella

D-glucose transport in decapod crustacean hepatopancreas.

Physiological mechanisms of gastrointestinal absorption of organic solutes among crustaceans remain severely underinvestigated, in spite of the considerable relevance of characterizing the routes of nutrient absorption for both nutritional purposes and formulation of balanced diets in aquaculture. Several lines of evidence attribute a primary absorptive role to the digestive gland (hepatopancreas) and a secondary role to the midgut (intestine). Among absorbed organic solutes, the importance of D-glucose in crustacean metabolism is paramount. Its plasma levels are finely tuned by hormones (crustacean hyperglycemic hormone, insulin-like peptides and insulin-like growth factors) and the function of certain organs (i.e. brain and muscle) largely depends on a balanced D-glucose supply. In the last few decades, D-glucose absorptive processes of the gastrointestinal tract of crustaceans have been described and transport mechanisms investigated, but not fully disclosed. We briefly review our present knowledge of D-glucose transport processes in the crustacean hepatopancreas. A discussion of previous results from experiments with hepatopancreatic epithelial brush-border membrane vesicles is presented. In addition, recent advances in our understandings of hepatopancreatic D-glucose transport are shown, as obtained (1) after isolation of purified R-, F-, B- and E-cell suspensions from the whole organ by centrifugal elutriation, and (2) by protein expression in hepatopancreatic mRNA-injected Xenopus laevis oocytes. In a perspective, the applicability of these novel methods to the study of hepatopancreatic absorptive function will certainly improve our knowledge of this structurally complex organ.

Inhibition of eel enzymatic activities by cadmium.

The aim of the present work was to study the in vitro effect of cadmium on enzymes, such as intestinal and branchial carbonic anhydrase (CA) and Na(+)-K(+)-ATPase which play a key role in salt- and osmoregulation and acid-base balance in the teleost fish, Anguilla anguilla. Carbonic anhydrase activities in gill and intestinal homogenates were significantly inhibited by CdCl(2), the gill CA being more sensitive to the heavy metal (IC(50) for the branchial CA=9.97+/-1.03x10(-6) M, IC(50) for the intestinal CA=3.64+/-1.03x10(-5) M, P<0.01). With regards to the intestinal CA activity, it has been shown in a previous study (Maffia et al., 1996) that two isoforms exist, a cytosolic and a brush-border membrane bound. These two isoforms show a different sensitivity to cadmium, with the membrane-bound enzyme less sensitive with respect to the cytosolic one, since it showed still an incomplete inhibition at the highest cadmium concentration tested. The inhibition of all the CA activity tested revealed a time-dependence since it required at least 10 min (1 h for the membrane-bound isoform) preincubation with the heavy metal to appear. Na(+)-K(+)-ATPase enzymatic activities, measured in intestinal and branchial homogenates, were inhibited by cadmium in a dose-dependent manner, with the branchial activity being more sensitive to the action of the heavy metal than the intestinal one (IC(50) for the branchial enzyme=1.38+/-0.09x10(-7) M, IC(50) for the intestinal enzyme=2.86+/-0.02x10(-7) M, P<0.01). The most of inhibition of the enzyme appeared without any preincubation with the heavy metal. Mg(2+)-ATPase activity was not significantly altered by the in vitro cadmium exposure either in the gills or in the intestine. These findings observed in vitro could be useful in the understanding of the toxic effects that cadmium elicits on aquatic organisms in vivo. In fact, the impairment of the activity of enzymes which carry out key physiological roles could cause alterations of the physiology of the whole organism.

Adenosine Triphosphate Concentration and β-d-Glucuronidase Activity as Indicators of Sea Bass Semen Quality

Abstract The most common parameters used to evaluate sperm quality are motility rate and duration and fertilization ability. In this study, chemical and biochemical parameters of sea bass (Dicentrarchus labrax) sperm were investigated to find an alternative method for evaluating sperm fertilization ability before and after cryopreservation. The biochemical and chemical analyses were performed with fresh and frozen-thawed sperm and seminal plasma. To cryopreserve sperm, 250-μl straws were used. Fertilization ability was evaluated by inseminating eggs (obtained from hormonally stimulated females) with fresh and cryopreserved sperm. The results revealed a linear relationship (P < 0.05) between semen fertilization capacity and some seminal plasma (β-d-glucuronidase activity, potassium concentration) and sperm (ATP concentration, aspartate aminotransferase activity) parameters. Variations in semen fertilization rate could be best described by two multiple regression models: one including the sperm parameters and another including the seminal plasma parameters. For practical application, the use of simple regression models is of value. Fertilization rate in both fresh and cryopreserved sperm was reliably predicted by determining the ATP concentration or the β-d-glucuronidase activity or both.

Effect of Cryopreservation on Sea Bass Sperm Proteins

Abstract In the present study we used two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to verify whether the protein expression of sea bass sperm was affected by the cryopreservation procedure. The protein profiles differed between fresh and frozen-thawed semen as revealed by visual inspection and by image analysis software. We identified 163 spots in fresh sperm; among these, 13 were significantly decreased and 8 were absent in two-dimensional gel obtained with cryopreserved sperm. Five of these spots were analyzed with MALDI-TOF, but only three showed a significant match in the databases used in bio-informatics analysis (PeptIdent, Mascot, and MS-Fit). In particular, spot 5 showed homology with a novel protein of zebrafish (similar to SKB1 of human and mouse), spot 13 showed homology with amphibian G1/S-specific cyclin E2, and spot 20 showed homology with the hypothetical protein DKFZp566A1524 of Brachidanio rerio. The present work shows that the use of the cryopreservation procedure causes the degradation of sperm proteins and among these, two could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm.

Molecular Mechanisms Determining Sperm Motility Initiation in Two Sparids (Sparus aurata and Lithognathus mormyrus)

Abstract Molecular mechanisms involved in sperm motility initiation in two sparids (Sparus aurata and Lithognathus mormyrus) have been studied. Our comparative study demonstrates that osmolality is the key signal in sperm motility activation in both species, whereas K+ and Ca2+ do not have any role. The straight-line velocity that resulted, however, was significantly different when measured in sperm activated with non-ionic and/or calcium-free solutions with respect to that measured in seawater-activated sperm. In both species, motility initiation depends on cAMP-dependent protein phosphorylation. The phosphorylation/dephosphorylation patterns that resulted in gilthead and striped sea bream were quite different. In gilthead sea bream, the phosphorylated proteins have molecular weights of 174, 147, 138, 70, and 9-15 kDa, whereas the dephosphorylated proteins have molecular weights of 76, 57, and 33 kDa. In striped sea bream, phosphorylation after sperm motility activation occurred on proteins of 174, 147, 103, 96, 61, 57, and 28 kDa, whereas only one protein of 70 kDa resulted from dephosphorylation. Matrix-assisted laser desorption ionization-time of flight analyses allowed identification of the following proteins: In gilthead sea bream, the 9-15 kDa proteins that were phosphorylated after motility activation include an A-kinase anchor protein (AKAP), an acetyl-coenzyme A synthetase, and a protein phosphatase inhibitor, and in striped sea bream, 103- and 61-kDa proteins that were phosphorylated after motility activation were identified as a phosphatase (myotubularin-related protein 1) and a kinase (DYRK3), respectively.

Evidence for the Involvement of Aquaporins in Sperm Motility Activation of the Teleost Gilthead Sea Bream (Sparus aurata)

The expression of aquaporins in the spermatozoa of the marine teleost gilthead sea bream (Sparus aurata) and their involvement in the motility activation process were investigated. Sperm motility was activated by a hyperosmotic shock, but it was completely inhibited by 10 μM HgCl2, such inhibition being partially recovered by beta-mercaptoethanol (ME). Conventional RT-PCR using primers specific for S. aurata aquaglyceroporin (glp) and aquaporin 1a (aqp1a) demonstrated the presence of both mRNAs in spermatozoa. Heterologous expression in Xenopus laevis oocytes showed that 10 and 100 μM HgCl2 equally inhibited water and solute transport through S. aurata aquaporin 1a and S. aurata aquaglyceroporin, but treatment with ME only recovered aquaporin 1a-mediated water permeability. Western blot analysis using isoform-specific antisera on protein extracts from spermatozoa revealed bands that corresponded to the predicted molecular mass of S. aurata aquaglyceroporin (31 kDa) and S. aurata aquaporin 1a (28 kDa). The antisera also demonstrated that both aquaporins were localized in the head and flagellum of the spermatozoa. However, the immunoreaction at the plasma membrane of the spermatozoa head was more intense after the hyperosmotic activation, suggesting the translocation of both aquaporin 1a and aquaglyceroporin into the plasma membrane after the osmotic shock. This study therefore provides the first direct demonstration for the presence of aquaporins in fish sperm. The different sensitivities of S. aurata aquaporin 1a and S. aurata aquaglyceroporin to ME may explain the failure of this reducing agent to fully recover the mercurial inhibition of sperm motility, suggesting that these aquaporins may play different physiological roles during the activation and maintenance of sperm motility in sea bream.

Improving Sperm Cryopreservation with Antifreeze Proteins: Effect on Gilthead Seabream (Sparus aurata) Plasma Membrane Lipids

ABSTRACT Changes in the plasma membrane lipid composition have been related to a decrease in sperm quality during cryopreservation. Antifreeze proteins (AFPs) have been tested in different species because of their ability to depress the freezing point and their potential interaction with membranes, but controversial effects were reported. In the present study we analyzed separately the lipid composition of two sperm membrane domains, head plasma membrane (HM) and flagellar membrane (FM), after cryopreservation with an extender containing 5% dimethyl sulfoxide (DMSO) either alone or with AFPI or AFPIII (1 μg/ml). We used sperm from a teleost, Sparus aurata, because the lack of acrosome avoids changes of lipid profiles due to capacitation process or acrosomal losses during freezing/thawing. Comparing with the control (cryopreservation with 5% DMSO alone), the addition of AFPIII increased the velocity, linearity of movement, and percentage of viable cells. In addition, freezing with DMSO alone increased the phosphatidyl-serine content as well as the saturated fatty acids and decreased the unsaturated ones (mainly polyunsaturated) both in HM and FM. These changes in the lipid components were highly avoided with the addition of AFPIII. HM had a higher amount of saturated fatty acids than FM and was more affected by cryopreservation without AFPs. The percentage of viable cells was positively correlated with the amount of unsaturated fatty acids in the HM, whereas the motility parameters were positively correlated with both FM and HM amount of unsaturated fatty acids. AFPs, especially AFPIII, seem to have interacted with unsaturated fatty acids, stabilizing the plasma membrane organization during cryopreservation and contributing to improve sperm quality after thawing.

Aquaporin inhibition changes protein phosphorylation pattern following sperm motility activation in fish

Our previous studies demonstrated that osmolality is the key signal in sperm motility activation in Sparus aurata spermatozoa. In particular, we have proposed that the hyper-osmotic shock triggers water efflux from spermatozoa via aquaporins. This water efflux determines the cell volume reduction and, in turn, the rise in the intracellular concentration of ions. This increase could lead to the activation of adenylyl cyclase and of the cAMP-signaling pathway, causing the phosphorylation of sperm proteins and then the initiation of sperm motility. This study confirms the important role of sea bream AQPs (Aqp1a and Aqp10b) in the beginning of sperm motility. In fact, when these proteins are inhibited by HgCl(2), the phosphorylation of some proteins (174 kDa protein of head; 147, 97 and 33 kDa proteins of flagella), following the hyper-osmotic shock, was inhibited (totally or partially). However, our results also suggest that more than one transduction pathways could be activated when sea bream spermatozoa were ejaculated in seawater, since numerous proteins showed an HgCl(2)(AQPs)-independent phosphorylation state after motility activation. The role played by each different signal transduction pathways need to be clarified.

Subcellular Localization of Selectively Permeable Aquaporins in the Male Germ Line of a Marine Teleost Reveals Spatial Redistribution in Activated Spermatozoa

ABSTRACT In oviparous vertebrates such as the marine teleost gilthead seabream, water and fluid homeostasis associated with testicular physiology and the external activation of spermatozoa is potentially mediated by multiple aquaporins. To test this hypothesis, we isolated five novel members of the aquaporin superfamily from gilthead seabream and developed paralog-specific antibodies to localize the cellular sites of protein expression in the male reproductive tract. Together with phylogenetic classification, functional characterization of four of the newly isolated paralogs, Aqp0a, -7, -8b, and -9b, demonstrated that they were water permeable, while Aqp8b was also permeable to urea, and Aqp7 and -9b were permeable to glycerol and urea. Immunolocalization experiments indicated that up to seven paralogous aquaporins are differentially expressed in the seabream testis: Aqp0a and -9b in Sertoli and Leydig cells, respectively; Aqp1ab, -7, and -10b from spermatogonia to spermatozoa; and Aqp1aa and -8b in spermatids and sperm. In the efferent duct, only Aqp10b was found in the luminal epithelium. Ejaculated spermatozoa showed a segregated spatial distribution of five aquaporins: Aqp1aa and -7 in the entire flagellum or the head, respectively, and Aqp1ab, -8b, and -10b both in the head and the anterior tail. The combination of immunofluorescence microscopy and biochemical fractionation of spermatozoa indicated that Aqp10b and phosphorylated Aqp1ab are rapidly translocated to the head plasma membrane upon activation, whereas Aqp8b accumulates in the mitochondrion of the spermatozoa. In contrast, Aqp1aa and -7 remained unchanged. These data reveal that aquaporin expression in the teleost testis shares conserved features of the mammalian system, and they suggest that the piscine channels may play different roles in water and solute transport during spermatogenesis, sperm maturation and nutrition, and the initiation and maintenance of sperm motility.

Changes in cell type composition and enzymatic activities in the hepatopancreas of Marsupenaeus japonicus during the moulting cycle.

The goal of the present study was to evaluate the changes in the cell type composition and ATPase activities (total ATPase, ouabain-sensitive Na+/K+-ATPase, furosemide-sensitive Na+-ATPase) that occur during the different stages of the moulting cycle in the hepatopancreas of the Marsupenaeus japonicus. The results clearly suggest that the number of resorptive and fibrillar cell types changes significantly during the different stages. An inverse correlation between resorptive and fibrillar cells is observed during moulting (both in normally fed and fasted animals). Fasting, but not the moulting cycle, affects the number of blister-like cells. In the resorptive cells the enzymatic activities (total ATPases and ouabain-sensitive Na+/K+-ATPase) also change during the moulting in a cyclical manner. All these results are in agreement with and confirm the different functions carried out by the two cell types within the hepatopancreas.