V. Gallego

Molecular and physiological study of the artificial maturation process in European eel males: From brain to testis

European eel males can be artificially matured (1.5IU hCG/g fish), but the regulatory mechanisms of their reproductive development are practically unknown. Spermatogenic stages (S1-S6), biometric characters [eye index (EI), gonadosomatic index (GSI), hepatosomatic index (HSI)] and sperm quality parameters (motility, viability and head spermatozoa morphometry) were analysed. Moreover, the present study evaluated the expression of GnRHs (mammal and chicken II Gonadotropin Release Hormone I) and gonadotrophins (FSHbeta and LHbeta) during hormonal treatment, as well as 11-ketotestosterone (11-KT) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) plasma levels. One week was enough to observe the S2 of gonad development, but it was necessary to reach the 7th week of treatment to obtain animals that presented the most advanced stage of development (S6). Differential regulation of the two GnRH expressions was found, supporting the main role of mGnRH in the control of gonadotrophin release. One hCG injection was enough to dramatically decrease the FSHbeta expression, being close to zero during the rest of the treatment. LHbeta expression and 17,20beta-P registered a significant increase in the same stage of development, S3/4, confirming the role of this gonadotrophin in the last steps of maturation and 17,20beta-P in the spermatozoa maturation. The 11-KT increased with GSI, and the highest 11-KT values coincided with the advanced steps of spermatogenesis prior to spermiation. Being consistent with the known role of the steroid in these processes. Furthermore, this study supports a role for 11-KT in stimulating eye growth, presenting high values when EI increased. Sperm production was obtained from the 4th week of treatment, but it was in the 8th week when a significant increase was observed in sperm quality [viability, high motility (>75%)].

Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software

The development of powerful computer-assisted sperm analysis software has made kinetic studies of spermatozoa possible. This system has been used and validated for several species, but some technical questions have emerged regarding fish sample evaluations (i.e., frame rate, sperm dilution, chamber model, time of analysis, magnification lens, etc.). In the present study, we have evaluated the effects of different procedural and biological settings with the aim to correctly measure sperm quality parameters of the European eel. The use of different chambers did not affect the sperm motility parameters. However, regarding lens magnification, 10× was the most accurate lens, showing the least variation in the acquired data. Similarly, the frame rate setting resulted in a dramatic effect in some sperm kinetic parameters, primarily in terms of curvilinear velocity; we therefore recommend using the cameras highest available frame rate setting. Finally, the reduction in sperm motility over postactivation times suggests that sperm analysis should be performed within the first 60 seconds after activation of the European eel sperm. In conclusion, some protocol variables of sperm analysis by computer-assisted sperm analysis software can affect the measurement of eel sperm quality parameters, and should be considered before directly comparing results obtained by different laboratories. Moreover, because marine fish species show relatively similar features of sperm kinetic parameters, these results could be considered in the evaluation of the motility of sperm from other fish species.

Improvement of European eel sperm cryopreservation method by preventing spermatozoa movement activation caused by cryoprotectants.

Sperm production has been obtained from European and Japanese eels, but its quality and quantity tend to be changeable. So, its cryopreservation has been tried in both species. Dimethyl sulfoxide (Me(2)SO) is the best cryoprotectant for European eel sperm, but increases the medium osmolality, inducing the activation of spermatozoa motility. To avoid this, different combinations of pH (6.5 and 8.5) and NaHCO(3) concentrations (20, 40 and 80mM) were tested with two Me(2)SO concentrations (5% and 10%). Foetal bovine serum (FBS, 25%v/v) was added as a membrane protector to all the freezing media used in the different experiments. The highest Me(2)SO and NaHCO(3) concentrations at pH 6.5 caused the best post-thawing motility (26+/-4%). A second experiment was carried out testing media with Me(2)SO 10% with additional NaHCO(3) concentrations (100 and 120 mM). The highest post-thawing motility (38+/-3%) was found in the media containing NaHCO(3) 100mM, but no significant difference was observed compared with the best in the previous experiment (NaHCO(3) 80 mM). In a parallel experiment, aiming to improve the protection against the cryopreservation process, bovine serum albumin (BSA, 5%w/v) was added instead of FBS. Lower motilities were registered with BSA as membrane protector. Spermatozoa activation caused by addition of Me(2)SO can be prevented using high NaHCO(3) concentrations, improving the cryopreservation process. This effect seems be based on some of the products dissociated from NaHCO(3) in aqueous solution, affecting the intracellular pH, essential in the sperm motility.

Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen

There has been a marked reduction in natural stocks of eels (genus Anguilla) over the past 60 years, and the culture of eels is still based on the capture of very large quantities of juveniles. It is necessary to close the life cycle in captivity in order to ease the pressure on wild populations. The aims of the present study were to evaluate sperm subpopulations (through cluster analysis of computer-aided sperm analysis data) in the European eel (Anguilla anguilla) and to assess the effects of motility acquisition time after activation (i.e. at 30, 60 and 90s), the thermal regimen (i.e. 10°C (T10) or 15°C (T15) and up to 20°C, or constant at 20°C (T20)) and hormonal treatments (i.e. human chorionic gonadotropin (hCG), recombinant (r) hCG or pregnant mare serum gonadotropin (PMSG)) on these subpopulations. In all cases, we obtained three subpopulations of spermatozoa: low velocity and linear (S1); high velocity with low linearity (S2); and high velocity and linear (S3; considered high quality). Total motility and S1 were affected by acquisition time; thus, 30s is recommended as the standard time for motility acquisition. When eels were kept at 20°C (T20), motility data fitted quadratic models, with the highest motility and proportion of S3 between Weeks 8 and 12 after the first injection. Lower temperatures (T10, T15) delayed spermiation and the obtaining of high-quality spermatozoa (S3), but did not seem to alter the spermiation process (similar subpopulation pattern). Conversely, the hormonal treatments altered both the dynamics of the subpopulation pattern and the onset of spermiation (with PMSG delaying it). Total motility and the yield of S3 with the widely used hCG treatment varied throughout the spermiation period. However, using rhCG allowed us to obtain high-quality and constant motility for most of the study (Weeks 7-20), and the S3 yield was also higher overall (61.8±1.3%; mean ± s.e.m.) and more stable over time than the other hormonal treatments (averaging 53.0±1.4%). Using T20 and rhCG would be more economical and practical, allowing us to obtain a higher number of S3 spermatozoa over an extended time.

Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: tissue-distribution and changes in transcript abundance during artificially induced sexual development.

Gonadotropin-releasing hormone receptor (GnRH-R) activation stimulates synthesis and release of gonadotropins in the vertebrate pituitary and also mediates other processes both in the brain and in peripheral tissues. To better understand the differential function of multiple GnRH-R paralogs, three GnRH-R genes (gnrhr1a, 1b, and 2) were isolated and characterized in the European eel. All three gnrhr genes were expressed in the brain and pituitary of pre-pubertal eels, and also in several peripheral tissues, notably gills and kidneys. During hormonally induced sexual maturation, pituitary expression of gnrhr1a (female) and gnrhr2 (male and female) was up-regulated in parallel with gonad development. In the brain, a clear regulation during maturation was seen only for gnrhr2 in the midbrain, with highest levels recorded during early vitellogenesis. These data suggest that GnRH-R2 is the likely hypophysiotropic GnRH-R in male eel, while both GnRH-R1a and GnRH-R2 seems to play this role in female eels.

Comparison of two techniques for the morphometry study on gilthead seabream (Sparus aurata) spermatozoa and evaluation of changes induced by cryopreservation.

The development of powerful software has made possible spermatozoa morphology studies. However, some problems have emerged in relation to protocol standardization to compare results from different laboratories. This study was carried out to compare two techniques commonly used (staining vs phase contrast technique) for the morphometry study of gilthead sea bream spermatozoa using an integrated sperm analysis system (ISAS). Spermatozoa morphometry values were significantly affected by the technique used, and phase contrast technique was found to be the more accurate method, showing lower coefficients of variation on spermatozoa morphometry parameters measurements. Moreover, it has been shown that cryopreservation process produces damage in gilthead sea bream spermatozoa, causing negative effects in sperm parameters as spermatozoa morphometry (a decrease in cell volume), motility (from 95 to 68% motile cells) and viability (from 95 to 87% of live cells), being the addition of freezing medium containing cryoprotectant (DMSO) an important factor that caused the morphometry changes.

Relationship between sperm quality parameters and the fatty acid composition of the muscle, liver and testis of European eel

This study looks at the correlations that fatty acids have with different tissues in the European eel (Anguilla anguilla L.) during hormonally-induced sexual maturation, with different sperm quality parameters. In order to evaluate the different dynamics of the use of fatty acids, a categorization of the results from each sperm quality parameter (volume, concentration, motility and velocity) was performed. Low and moderate correlations were observed between muscle tissue and some sperm quality parameters but no high correlations were found. Eicosapentaenoic acid (20:5n3, EPA) in the liver seems to have a role in determining the volume of sperm produced. This can be explained by the fact that EPA is a major requirement in the early phases of sperm production (probably as a component of the spermatozoal membrane). In addition, the levels of α-linolenic acid (18:3-n3, ALA) and linoleic acid (18:2-n6, LA) in the liver decreased when sperm motility increased. In all the tissues, a negative correlation was observed between arachidonic acid (20:4n-6, ARA) and the different sperm velocity parameters. The fact that an increase in the consumption of ARA coincides with an increase in the speed of spermatozoa, highlights the important role that this fatty acid plays not only in sperm production, but also in sperm velocity. All this information could prove useful in the development of suitable broodstock diets to improve sperm quality and subsequently, the larval development of this species.

Variations in the gene expression of zona pellucida proteins, zpb and zpc, in female European eel (Anguilla anguilla) during induced sexual maturation.

Vertebrate eggs are surrounded by an extracellular glycoprotein coat termed zona pellucida (ZP). Integrity of ZP is critical for a correct embryo development. Two zona pellucida protein genes (zpb and zpc) from European eel were characterized, specific qPCR assays developed and their expression in immature males and females carried out. An experimental group of silver-stage eel females was maintained at 18 °C and hormonally induced to sexual maturation by weekly injections of carp pituitary extract during 12 weeks. Changes in zpb and zpc expression during sexual maturation were studied in liver and ovary by qPCR. In liver, no changes were recorded during hormonal treatment, while in ovary expression of both genes decreased during sexual development. These results are a first step in the characterization of ZP in European eel and in the understanding of the mechanism underlying egg envelope formation.

Heterozygosity-fitness correlations in the gilthead sea bream Sparus aurata using microsatellite loci from unknown and gene-rich genomic locations

Heterozygosity-fitness correlations (HFC) were assessed for a sample of a gilthead sea bream Sparus aurata population. Two hundred and seventy-one fish were genotyped at 22 known and novel microsatellite loci, from which correlations between the multilocus heterozygosity index (I(MLH) ) and various fitness traits (fork length, mass and specific growth rates) were calculated. Significant global HFCs were found in this sample (0·02 ≤r(2) ≤ 0·08). In addition, all the significant correlations found in this work were negative, indicating that heterozygotes had lower fitness than their homozygote counterparts. Marker location could not explain the observed HFCs. Evidence of inbreeding, outbreeding or population and family structuring was not found in this work. The presence of undetected general effects that may lead to the appearance of HFCs, however, cannot be ruled out. These results seem to be best explained by the occurrence of local effects (due to linkage) or even by possible direct locus advantages.

Role of calcium on the initiation of sperm motility in the European eel.

Sperm from European eel males treated with hCGrec was washed in a calcium free extender, and sperm motility was activated both in the presence (seawater, SW) and in the absence of calcium (NaCl+EDTA), and treated with calcium inhibitors or modulators. The sperm motility parameters were evaluated by a computer-assisted sperm analysis (CASA) system, and changes in the [Ca(2+)]i fluorescence (and in [Na(+)]i in some cases) were evaluated by flow cytometry. After sperm motility was activated in a medium containing Ca(2+) (seawater, SW) the intracellular fluorescence emitted by Ca(2+) increased 4-6-fold compared to the levels in quiescent sperm. However, while sperm activation in a Ca-free media (NaCl+EDTA) resulted in a percentage of motility similar to seawater, the [Ca(2+)]i levels did not increase at all. This result strongly suggests that increasing [Ca(2+)]i is not a pre-requisite for the induction of sperm motility in European eel sperm. Several sperm velocities (VCL, VSL, VAP) decreased when sperm was activated in the Ca-free activator, thus supporting the theory that Ca(2+) has a modulatory effect on sperm motility. The results indicate that a calcium/sodium exchanger (NCX) which is inhibited by bepridil and a calcium calmodulin kinase (inhibited by W-7), are involved in the sperm motility of the European eel. Our results indicate that the increase in [Ca(2+)]i concentrations during sperm activation is due to an influx from the external medium, but, unlike in most other species, it does not appear to be necessary for the activation of motility in European eel sperm.