Evaristo L. Mañanós

Perspectives on fish gonadotropins and their receptors.

Teleosts lack a hypophyseal portal system and hence neurohormones are carried by nerve fibers from the preoptic region to the pituitary. The various cell types in the teleost pituitary are organized in discrete domains. Fish possess two gonadotropins (GtH) similar to FSH and LH in other vertebrates; they are heterodimeric hormones that consist of a common alpha subunit non-covalently associated with a hormone-specific beta subunit. In recent years the availability of molecular cloning techniques allowed the isolation of the genes coding for the GtH subunits in 56 fish species representing at least 14 teleost orders. Advanced molecular engineering provides the technology to produce recombinant GtHs from isolated cDNAs. Various expression systems have been used for the production of recombinant proteins. Recombinant fish GtHs were produced for carp, seabream, channel and African catfish, goldfish, eel, tilapia, zebrafish, Manchurian trout and Orange-spotted grouper. The hypothalamus in fishes exerts its regulation on the release of the GtHs via several neurohormones such as GnRH, dopamine, GABA, PACAP, IGF-I, norepinephrine, NPY, kisspeptin, leptin and ghrelin. In addition, gonadal steroids and peptides exert their effects on the gonadotropins either directly or via the hypothalamus. All these are discussed in detail in this review. In mammals, the biological activities of FSH and LH are directed to different gonadal target cells through the cell-specific expression of the FSH receptor (FSHR) and LH receptor (LHR), respectively, and the interaction between each gonadotropin-receptor couple is highly selective. In contrast, the bioactivity of fish gonadotropins seems to be less specific as a result of promiscuous hormone-receptor interactions, while FSHR expression in Leydig cells explains the strong steroidogenic activity of FSH in certain fish species.

Immunohistochemical localization of three different prepro-GnRHs in the brain and pituitary of the European sea bass (Dicentrarchus labrax) using antibodies to the corresponding GnRH-associated peptides

The distribution of the cells expressing three prepro‐gonadotrophin‐releasing hormones (GnRH), corresponding to salmon GnRH (sGnRH), seabream GnRH (sbGnRH), and chicken GnRH‐II (cGnRH‐II) forms, was studied in the brain and pituitary of the sea bass (Dicentrarchus labrax) by using immunohistochemistry. To circumvent the cross‐reactivity problems of antibodies raised to GnRH decapeptides, we used specific antibodies generated against the different sea bass GnRH‐associated peptides (GAP): salmon GAP (sGAP), seabream GAP (sbGAP), and chicken‐II GAP (cIIGAP). The salmon GAP immunostaining was mostly detected in terminal nerve neurons but also in ventral telencephalic and preoptic perikarya. Salmon GAP‐immunoreactive (ir) fibers were observed mainly in the forebrain, although sGAP‐ir projections were also evident in the optic tectum, mesencephalic tegmentum, and ventral rhombencephalon. The pituitary only receives a few sGAP‐ir fibers. The seabream GAP‐ir cells were mainly detected in the preoptic area. Nevertheless, sbGAP‐ir neurons were also found in olfactory bulbs, ventral telencephalon, and ventrolateral hypothalamus. The sbGAP‐ir fibers were only observed in the ventral forebrain, innervating strongly the pituitary gland. Finally, chicken‐II GAP immunoreactivity was only detected in large synencephalic cells, which are the origin of a profuse innervation reaching the telencephalon, preoptic area, hypothalamus, thalamus, pretectum, posterior tuberculum, mesencephalic tectum and tegmentum, cerebellum, and rhombencephalon. However, no cIIGAP‐ir fibers were detected in the hypophysis. These results corroborate the overlapping of sGAP‐ and sbGAP‐expressing cells in the forebrain of the sea bass, and provide, for the first time, unambiguous information on the distribution of projections of the three different GnRH forms expressed in the brain of a single species. J. Comp. Neurol. 446:95–113, 2002.

Regulation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) gene expression by gonadotropin-releasing hormone (GnRH) and sexual steroids in the Mediterranean Sea bass ☆

The secretion of gonadotropins, the key reproductive hormones in vertebrates, is controlled from the brain by the gonadotropin-releasing hormone (GnRH), but also by complex steroid feedback mechanisms. In this study, after the recent cloning of the three gonadotropin subunits of sea bass (Dicentrarchus labrax), we aimed at investigating the effects of GnRH and sexual steroids on pituitary gonadotropin mRNA levels, in this valuable aquaculture fish species. Implantation of sea bass, in the period of sexual resting, for 12 days with estradiol (E2), testosterone (T) or the non-aromatizable androgen dihydrotestosterone (DHT), almost suppressed basal expression of FSHbeta (four to 15-fold inhibition from control levels), while slightly increasing that of alpha (1.5-fold) and LHbeta (approx. twofold) subunits. Further injection with a GnRH analogue (15 microg/kg BW; [D-Ala6, Pro9-Net]-mGnRH), had no effect on FSHbeta mRNA levels, but stimulated (twofold) pituitary alpha and LHbeta mRNA levels in sham- and T-implanted fish, and slightly in E2- and DHT-implanted fish (approx. 1.5-fold). The GnRHa injection, as expected, elevated plasma LH levels with a parallel decrease on LH pituitary content, with no differences between implanted fish. In conclusion, high circulating steroid levels seems to exert different action on gonadotropin secretion, inhibiting FSH while stimulating LH synthesis. In these experimental conditions, the GnRHa stimulate LH synthesis and release, but have no effect on FSH synthesis.

Molecular characterization of sea bass gonadotropin subunits (α, FSHβ, and LHβ) and their expression during the reproductive cycle

Reproduction is controlled by two pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This study reports the cloning, sequence analysis, and gene expression of gonadotropin (GTH) subunits from the European sea bass (Dicentrarchus labrax). The GTH subunits were cloned from a sea bass brain-pituitary cDNA library. The nucleotide sequences of the common alpha, the FSHbeta, and the LHbeta subunit cDNAs were 625, 521, and 591 base pair (bp) long, respectively, encoding for mature peptides of 94, 105, and 115 amino acids (aa), respectively. Sequence analysis showed that sea bass FSHbeta is more similar to higher vertebrate FSHbetas (35-37%) than to LHbetas (26-30%), whereas sea bass LHbeta is more similar to LHbetas (40-53%) than to FSHbetas (26-41%). Phylogenetic analysis of fish GTH sequences grouped the beta subunits into two groups, FSH and LH, distributed into four classes, corresponding to the accepted divisions of Elopomorphs, Ostariophysis, Salmonids, and Percomorphs. A dot-blot technique was developed to analyze GTH pituitary mRNA levels during the reproductive cycle of male sea bass. From October (initiation of gametogenesis) to February (spermiation), the expression of all three subunits in the pituitary increased in parallel, concomitantly with the gonadosomatic index (GSI) and the accumulation of LH protein in the pituitary, all values declining sharply at post-spermiation. This study demonstrates that the pituitary of sea bass contains two gonadotropin hormones and that both gonadotropins are probably involved in the control of gametogenesis, gamete maturation, and spermiation.

Sea bass (Dicentrarchus labrax L.) vitellogenin. II Validation of an enzyme-linked immunosorbent assay (ELISA)

Abstract In this study, a competitive ELISA was developed for the quantification of vitellogenin (VTG) in sea bass. The purity of the antigen (VTG) and the specificity of the antibody (AbVTG) used in the assay, were assessed by ELISA and radial immunodiffusion, and no crossreactivity was observed with the male plasma. The sensitivity range of the ELISA was 1–60 ng/ml (85-20% of binding). The intra- and inter-assay variations (at 50% of binding) were 5.3% ( N = 5) and 9.8% ( N = 9), respectively. Annual VTG levels were quantified in animals reared under fish farming conditions and the VTG levels were correlated with the oocyte development.

Preparation and Administration of Gonadotropin-Releasing Hormone Agonist (GnRHa) Implants for the Artificial Control of Reproductive Maturation in Captive-Reared Atlantic Bluefin Tuna (Thunnus thynnus thynnus)

Abstract Mature migrating Atlantic bluefin tuna (Thunnus thynnus thynnus) were captured in the Mediterranean Sea with a purse seine and reared in floating cages for 2 to 3 years. During the natural spawning period (June–July) of two consecutive years, fish were randomly implanted underwater with a controlled-release delivery system (implant) loaded with gonadotropin-releasing hormone agonist (GnRHa), in order to induce final oocyte maturation (FOM), ovulation/spermiation, and spawning. At the time of sampling, males were significantly larger than females (ANOVA, P < 0.001), having a mean (± SE) fork length and body weight of 190 ± 3 cm and 122 ± 5 kg, compared to 176 ± 3 cm and 94 ± 4 kg of females, respectively. All fish were reproductively mature, with their age ranging between 5 and 12 years and males being a year older, on average. After GnRHa implantation, fish were monitored for spawning and the release of eggs, and were sacrificed at different times after hormone treatment in order to examine the progressive effect of the treatment on gonad maturation. The in vitro GnRHa release from the produced implants was maximal during the first 2 d, with a mean (± SE) release of 525 ± 166 μ g GnRHa implant−1 day−1. The plasma GnRHa profile in vivo reflected the release in vitro, and statistically significant elevations in plasma GnRHa levels were measured until 7 d after treatment (ANOVA, P < 0.01). The underwater implantation procedure was improved between 2004 and 2005, requiring an average (± SD) of 3.1 ± 1.4 min for each fish, and was 64 and 84% successful in 2004 and 2005, respectively. There were no differences between the histological appearance of the testes of GnRHa-treated and control males, and almost all of them contained intra-testicular spermatozoa. However, the proportion of spermiating control males (n = 17) was only 12% compared to 26% for the GnRHa-implanted males (n = 19). Also, there were no differences between controls and GnRHa-implanted fish in sperm concentration, initial spermatozoa motility, or duration of forward motility, which ranged between 29.02–48.54 × 1010 spermatozoa ml−1, 58–63% and 8–9 min, respectively. Final oocyte maturation (FOM) and post-ovulatory follicles (POFs) occurred in 63% and 88%, respectively, of the GnRHa implanted females (n = 16), compared to 0% and 21%, respectively, of the control females (n = 14). In addition, two GnRHa-implanted females in 2005 were found to be ovulated at the time of sacrifice, and their eggs were fertilized in vitro with sperm from spermiating males, which resulted in viable embryos and larvae. Finally, although spawning was not observed, fertilized eggs were collected from the cages. Larvae produced from these eggs were identified as Atlantic bluefin tuna, demonstrating that the present GnRHa implantation method can be used to induce FOM, ovulation/spermiation, and spawning in captive-reared Atlantic bluefin tuna.

Photoperiodic manipulations of the reproductive cycle of sea bass ( Dicentrarchus labrax ) and their effects on gonadal development, and plasma 17ß-estradiol and vitellogenin levels

The annual profile of plasma vitellogenin (VTG) and 17ß-estradiol (E2) levels, as well as gonadal development and spawning characteristics were investigated in captive female sea bass (Dicentrarchus labrax). Endocrine and gonadal changes were studied in fish reared under natural conditions or exposed to manipulated photothermal cycles. In natural conditions of photoperiod and temperature, sea bass spawned from February through March (East coast of Spain, 40°N 0°E). One or two months of constant long-days (15L/9D) in a constant short-day photoperiod regime (9L/15D) all-year-round, given early in the year (March and March–April), advanced spawning by 3 months. The same treatment applied later in the year (September–October) delayed spawning by 1 month, compared to controls.In all groups, changes in plasma VTG levels were correlated with E2 levels, oocyte growth and spawning time. In control females, VTG was low (<100 ng ml-1) during the summer, until its first surge in plasma 4 months before the beginning of spawning. The VTG (3.1 ± 0.3 mg ml-1) and E2 (4.1 ± 0.5 ng ml-1) levels showed a single annual peak during late vitellogenesis, the time of the highest proportion of vitellogenic oocytes in the ovary. Constant high levels of VTG (1–1.4 mg ml-1) and E2 (1.6–1.9ng ml-1) were maintained during the entire spawning time, together with the presence of vitellogenic oocytes, suggesting the existence of several waves of oocyte growth in the ovary and thus, several spawns per female. Endocrine profiles and oocyte development in fish exposed to constant photoperiods were similar to controls, but were shifted in time in relation to the displacement of the spawning time. In the fish showing advanced spawns, the duration of the gametogenic proces was compressed when compared to controls. The differences observed in the evolution of the reproductive-related factors in the advanced groups, which were exposed to a reduction in temperature to 15°C, suggest an influence of the temperature in the early stages of the reproductive cycle in sea bass.

Vitellogenin, steroid plasma levels and spawning performance of cultured female Senegalese sole (Solea senegalensis).

The Senegalese sole (Solea senegalensis) is a high value market flatfish, which aquaculture is compromised by severe reproductive problems; these are mostly found in soles hatched and raised in captivity (F1 generation). To gain knowledge on the reproductive dysfunctions observed in cultured (F1) Senegalese sole, this work aimed at developing a specific vitellogenin (VTG) ELISA, for the measurement of plasma VTG levels in this species. Profiles of VTG were correlated with those of sexual steroids and spawning performance of an F1 broodstock, during three consecutive years. The Senegalese sole VTG (ssVTG) was purified by precipitation with MgCl(2)-EDTA and anion-exchange chromatography and showed a molecular mass of 172 kDa, by SDS-PAGE. Specific antibodies were obtained and used to develop a competitive ELISA, which had a sensitivity of 3.6 ng ml(-1), and inter- and intra-assay coefficients of variation of 9.5% (n=29) and 6.7% (n=12), respectively. Annual profiles of plasma VTG showed a major peak at pre-spawning, and a second minor rise around autumn, which mirrored plasma profiles of both estradiol (E(2)) and testosterone (T) levels. Spontaneous spawning occurred every year in the spring season, but no fertilized eggs were obtained. In conclusion, this study described, for the first time, the purification and development of a sensitive and specific ELISA for Senegalese sole VTG. The endocrine and spawning data suggested that F1 female broodstock showed normal VTG and steroid releasing profiles in captivity with occurrence of spontaneous spawning, but no fertilization of the eggs was recorded.

Spawning induction of individual European sea bass females (Dicentrarchus labrax) using different GnRHa-delivery systems

The present study evaluated the effect of different delivery systems loaded with gonadotropin-releasing hormone agonist (GnRHa) on spawning induction, and egg and larval quality in female European sea bass (Dicentrarchus labrax). Mature females (mean±S.E.M. oocyte diameter=905±10 μm) were treated with [d-Ala6, Pro9 NEt]-mGnRHa using GnRHa-loaded microspheres, Mc (60 μg kg−1 body weight, bw) GnRHa-loaded implants, EVAc (50 μg kg−1 bw) or received a single injection of GnRHa in saline, IN (15 μg kg−1 bw). Controls (C) were injected with saline. Females were allocated individually in 2-m3 tanks with two GnRHa-treated males and were allowed to spawn without further handling. All GnRHa treatments induced spawning 3 days after treatment (d.a.t.), whereas control fish did not spawn during the 21-day experimental period. GnRHa microspheres and implants induced up to four consecutive spawns in one female, whereas GnRHa-injected fish spawned only once. Egg quality seemed relatively lower in all GnRHa-treated groups compared to spontaneously spawning fish (natural spawns, NAT group) maintained in large populations (80 fish in 15-m3 tanks), possibly due to both the isolation of individual females in separate tanks and the stress from handling. Mean egg quality of the first spawns was variable, but similar in all GnRHa treatments (6.6–18.2% buoyancy; 21.7–27.4% hatching). In subsequent spawns, the Mc-treated females produced eggs of higher buoyancy than the EVAc group (20.5±6.0% vs. 2.87±1.14%), and similar to the first spawn of the GnRHa-injected group. Our results indicate that, compared to a single GnRHa injection, GnRHa-loaded microspheres can induce multiple spawns in European sea bass, without a negative effect on egg quality.

Effect of dietary lipid composition on vitellogenin, 17β-estradiol and gonadotropin plasma levels and spawning performance in captive sea bass (Dicentrarchus labrax L.)

Abstract The influence of dietary lipid composition on the reproductive performance of the sea bass ( Dicentrarchus labrax ) and on vitellogenin (VTG), 17 β -estradiol (E2) and gonadotropin II (GtH II) plasma levels has been investigated. The control group was fed with a natural diet consisting of trash fish ( Boops boops ). Two experimental groups were fed with pelleted diets containing different amounts of lipids: Group A was fed with a commercially available diet with a 10% lipid content and Group B fed the base diet enriched to a 22% lipid content using refined fish oil enriched with n −3 fatty acids. The control group exhibited higher egg viability and hatching rate than the experimental groups. The better spawning performance of the control group, with respect to the experimental groups, was associated with differences in endocrine profiles during the reproductive cycle. In the experimental groups, E2 levels were higher than in controls during the period of vitellogenesis. In the profiles of VTG levels, groups A and B exhibited a greater decrease of plasma VTG during the mid spawning time as compared to the control group. Profiles of plasma GtH II levels were determined for the first time in the sea bass and showed a single annual peak during the spawning period. At that time, GtH II levels from groups A and B were higher than in the controls. The present data suggest that dietary lipid composition significantly affects the reproductive performance of the sea bass.