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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.

9 pages,5 figures, 1a table, 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 lM 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 lM 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., 1Supported by the Ministry of Agriculture and Aquaculture of the Region Puglia (Italy) under the program between the Region of Puglia and the University of Salento, and by Spanish Ministry of Education and Science grant AGL2007-60262/ACU to J.C. F.C. was supported by the Marie Curie Research Training Network Aqua(glycero)porins grant MRTN-CT-2006-035995 from the European Commission.

We have previously demonstrated that in sea bream Sparus aurata motility initiation determined changes in the phosphorylation state of some proteins. This paper describes an investigation of the effect of a freezing–thawing procedure on the protein phosphorylation/dephosphorylation pattern. Proteins extracted from fresh and cryopreserved spermatozoa (before and after motility activation) were separated on SDS–PAGE, blotted on nitrocellulose membrane and treated with anti-phosphotyrosine, antiphosphothreonine, or anti-phosphoserine antibodies. The results obtained demonstrate that the cryopreservation protocol has a strong effect on the phosphorylation state of proteins. In general, compared to fresh sperm, phosphorylated proteins are most numerous in both activated and non-activated cryopreserved sperm, and in particular we observed a dramatic increase in threonine phosphorylation. However, frozen–thawed sperm showed a minor number of proteins that changed their phosphorylation state after motility activation. Among these, we identified the acetyl-coenzyme A synthetase that plays a role in sperm motility initiation in both fresh and cryopreserved sperm.

Aim: Sperm of Sparus aurata, like those of other teleosts, become activated when spawned into the external medium. Several extracellular factors (ions, osmotic pressure, O2/CO2, sperm activating peptides) have been reported to control sperm motility activation. These factors act on the flagellar motile apparatus, the axoneme, through signal transduction across the plasma membrane and determining the dynein-mediated sliding of the axonemal outer-doublet microtubules through protein phosphorylation. In the present study we have investigated the role of different proteins involved in this signal transduction cascade using cryopreserved sperm of Sparus aurata. Methods: Studies have been performed by using cryopreserved sperm (Fabbrocini et al., Cryobiology 40:46-53, 2000). To investigate the role played by different proteins involved in the signal transduction cascade we evaluated: (1) the effect of specific protein inhibitors on sperm motility (assessed by CASA), (2) quantitative and qualitative evaluation of phosphorylated proteins (tyrosine-phosphorylated, serine-phosphorylated and threonine-phosphorylated) by western blot analysis in activated and non-activated sperm. Results: Results obtained indicated that: (1) calcium and potassium were not involved in sperm motility activation; (2) osmolality values

The long-term goal of this research project is to set up efficient protocol that can be used to develop a standardized approach for vitrification of marine fish spermatozoa. In particular, the aim of the present study was to develop a vitrification protocol for sea bream (Sparus aurata) spermatozoa. To draw up the protocol, we tested two different dilution media (1% NaCl and Mounib medium), three different vitrification devices (loops, drops and cut straws), different cryoprotectants (CPs) and three different equilibration times (30, 60 and 120 s). The effect of the different vitrification procedures on spermatozoa quality was checked by measuring spermatozoa motility rate and viability, mitochondrial membrane potential and the fertilizing ability of both fresh and post-thawed gametes. The best result was obtained by dropping directly into liquid nitrogen 20 μl of spermatozoa suspension (drop-wise method) diluted with Mounib buffer containing 10% Me2SO + 10% glycerol. The addition of a mixture of anti-freezing proteins, AFPI and AFPIII, to Mounib buffer significantly increases the spermatozoa quality following vitrification so confirming the usefulness of AFPs in improving the quality of gametes subjected to the vitrification process. The present study proves that vitrification offers an alternative to conventional sperm cryopreservation also in this species.

In many fish species, the spermatozoa are immotile in the testes and seminal plasma, and motility is induced when they are released in the aqueous environment. Extracellular factors control the activation of the axoneme through signal transduction across the plasma membrane. The present review examines the existing literature concerning the axoneme activation in fish, with particular emphasis on the role played by the protein phosphorylation/dephosphorylation process, since post-transciptional modifications are involved in the mechanisms of sperm motility activation in many animals with external fertilization (starfish, sea urchins, sea cucumber, fish). We report the current understanding of the role played by the changes of protein phosphorylation state in sperm motility activation of teleost fish and provide additional tools for evaluating the gamete quality before and after cryopreservation procedure and, therefore, improve fish farm management. Statement of relevance The present review examines the existing literature concerning the signaling pathways involved in the axoneme activation in fish, in order to better understand the molecular mechanism regulating sperm motility initiation and to show how the proteins that change their phosphorylation status after spawning can be used as biomarkers for sperm quality and cryodamage.

In the present study, sexual gonadal differentiation and first sexual maturation of Meagre ( Argyrosomus regius ) was studied, based upon the annual changes in gonadosomatic index (GSI), gonadal histology, and the plasma steroid hormones, testosterone (T), 11-ketotestosterone (11-KT), and estradiol (E2). In addition, spermatozoa characteristics were evaluated by measuring sperm motility and morphology. Results demonstrated that Meagre completes sex differentiation at 10 to 12 mo of age, and are group-synchronous spawners, which reach puberty at 2 (mean length 26.8 ± 0.7 cm, mean weight 920 ± 75 g; N = 10) and 3 (mean length 35.8 ± 0.8 cm, mean weight 1610 ± 89 g; N = 10) years of age for males and females, respectively. In males, during the sex differentiation period, T levels were significantly higher with respect to those of 11-KT; this suggests that T has a key role in the early phases of the sex differentiation. During the spawning season an increase in plasma concentrations of all hormones was observed with 11-KT levels being significantly higher that those of T. In females, during the sex differentiation period, there was an increase in E2 plasma levels, while during the first spawning season, a significant increase of T and E2 levels were measured. Regarding sperm characteristics, the measured curvilinear velocity (VCL) and straight-linear velocity (VSL), resulted in the same order of magnitude with respect to those measured in other marine fish, while the average path velocity (VAP) was similar to that measured in the European Eel. The head of Meagre spermatozoa presents as oval shaped with a surface area of approximately 3.66 μm 2 and a perimeter of approximately 6.65 μm. All these findings represent an important basis for further investigation on the reproductive biology of this specie and may assist the farmers to improve seed production in aquaculture.

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 Ca(2+) 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.

In the present study we used two-dimensional polyacryl amide 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 (Pept-Ident, 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.

Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to the procedures of fish sperm cryopreservation.

Fish sperm cryopreservation is considered as a valuable technique for artificial reproduction and genetic improvement (Chao & Liao, 2001; Kopeika et al., 2007; Rana, 1995; Suquet et al., 2000). Semen quality must be monitored when attempts are made to increase the efficiency of artificial fertilization, to cryopreserve only sperm of high quality, and to evaluate frozen-thawed sperm. Cryopreserved sperm usually shows, with respect to fresh sperm, a lower quality, since the freezing–thawing procedure affects DNA and protein integrity (Labbe et al., 2001; Zilli et al., 2003, 2005), membrane lipids (Maldjian et al., 2005; Müller et al., 2008), sperm motility (Linhart et al., 2000; Ritar, 1999; Rodina et al., 2007; Zilli et al., 2005), fertilization ability (Gwo & Arnold, 1992; Rana, 1995), and also larval survival (Suquet et al., 1998). Spermatozoa genome alteration due to cryopreservation may affect only late embryonic development and larval survival (Kopeika et al., 2003a, 2003b, 2004; Suquet et al., 1998), but not the early events in embryonic development, because these are controlled by maternally inherited information (Braude et al., 1988). On the contrary, defects in sperm proteins (degradation and/or change of the phosphorylation state) may compromise sperm motility, fertilization ability, and the early events after fertilization (Cao et al., 2003; Huang et al., 1999; Lessard et al., 2000). The most common parameters used to evaluate sperm quality are fertilization ability, motility (rate and duration) and cellular (chemical and/or biochemical) parameters. Fertilizing capacity is the most conclusive test of sperm quality but the use of this marker is laborious and requires the availability of eggs (McNiven et al., 1992). Motility is normally evaluated as percentage and duration, but some authors also use velocity, flagellum beat frequency, or other parameters measured by computer-assisted sperm analysis (Ciereszko et al., 1996; Cosson et al., 2000; Rurangwa et al., 2001). Cellular bio markers has been used to evaluate spermatozoa quality of different fish species such as Atlantic salmon (Aas et al., 1991; Hwang & Idler, 1969), rainbow trout (Ciereszko & Dabrowski, 1994; Lahnsteiner et al.,1996a, 1998) and sea bass (Zilli et al., 2004). All these parameters have been also used to evaluate the effect of cryopreservation on spermatozoa quality. Here we reviewed data obtained by our group, on the effect of freezing-thawing procedures on sea bass and sea bream sperm. In particular, data concerning the effect of cryopreservation on bio-chemical parameters, DNA integrity, protein profile and phosphorylation state, are reported.

8 páginas, 2 figuras, 3 tablas., 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 HgCl2, 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 HgCl2(AQPs)-independent phosphorylation state after motility activation. The role played by each different signal transduction pathways need to be clarified.

We have previously demonstrated that in sea bream Sparus aurata motility initiation determined changes in the phosphorylation state of some proteins. This paper describes an investigation of the effect of a freezing–thawing procedure on the protein phosphorylation/dephosphorylation pattern. Proteins extracted from fresh and cryopreserved spermatozoa (before and after motility activation) were separated on SDS–PAGE, blotted on nitrocellulose membrane and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. The results obtained demonstrate that the cryopreservation protocol has a strong effect on the phosphorylation state of proteins. In general, compared to fresh sperm, phosphorylated proteins are most numerous in both activated and non-activated cryopreserved sperm, and in particular we observed a dramatic increase in threonine phosphorylation. However, frozen–thawed sperm showed a minor number of proteins that changed their phosphorylation state after motility activation. Among these, we identified the acetyl-coenzyme A synthetase that plays a role in sperm motility initiation in both fresh and cryopreserved sperm.

The aquaporins (AQPs) are a family of small, hydrophobic, integral membrane proteins that allow rapid water transport across the cell membrane. Relatively few reports about AQPs in non-mammalian vertebrates exist. AQP homologous have been reported in some teleosts such as the sea bream, sea bass and eel. At the best of our knowledge, until now there is no information regarding the expression of aquaporins in fish spermatozoa. Here we show that 0.01, 0.1 and 1 mM HgCl2 (known aquaporin inhibitor) completely inhibited sperm motility, a 0.001 mM concentration was ineffective. In addition, using the RT-PCR technique, we demonstrated the presence of aquaglyceroporin mRNA in sea bream and sea bass spermatozoa. Primers used in the RT-PCR were designed for sea bream aquaglyceroporin (AY363261). The sequencing of PCR products of the expected size (213 bp) revealed that this portion of aquaporin of sea bream and sea bass sperm have an homology of 100% with that of gilthead sea bream hind-gut. The presence of AQP in sea bream and sea bass spermatozoa was also confirmed by Western blot experiments. All together our findings allow us to hypothesize that in both sea bream and sea bass sperm AQPs are involved in sperm motility initiation mechanisms.

In many marine fish species, the spermatozoa are immotile in the testis and seminal plasma, and motility is induced when they are released in the aqueous environment. It is well known that the extracellular factors (hyperosmolality or sperm-activating peptides), controlling sperm motility in marine fish, act on the axonemal apparatus through signal transduction across the plasma membrane. To better understand the molecular mechanism regulating axoneme activation in marine fish, the present review examines the existing literature, with particular emphasis on protein phosphorylation/dephosphorylation process. The present review suggests that: (1) there is no single model that can explain the molecular activation and regulation of sperm motility of the marine fish; (2) only in some species (puffer fish, tilapia, gilthead sea bream, and striped sea bream) protein phosphorylation/dephosphorylation has been shown to be involved in flagellar motility regulation; (3) only a few proteins were identified, which show a change in their state of phosphorylation following sperm activation. A model of molecular mechanism controlling the activation of sperm motility in gilthead sea bream is being proposed here, which could be a useful model to clarify the sperm motility activation process in other species.