Characterization of three GnRH cDNA sequences in the pejerrey fish Odontesthes bonariensis

In this work we have characterized cDNAs fragments of GnRH preprohormones by 3′RACE. This characterization confirms the presence and expression of three GnRH forms in brain tissue and constitutes an initial step in the study of the physiological role of GnRHs during the sexual differentiation, maturation and reproduction of pejerrey.     

Temperature effects on sex differentiation of the reciprocal hybrids of Odontesthes bonariensis and Odontesthes hatcheri (Atherinopsidae)

The pejerrey Odontesthes bonariensis (Valenciennes 1835) and the Patagonian pejerrey Odontesthes hatcheri (Eigenmann 1909) are Atherinopsid species with commercial importance and potential for aquaculture. The hybrids of the two species are viable but their mode of sex determination is unknown. This study examined the gonadal histology and sex ratios of reciprocal hybrids that were reared at 15, 17, 21, 25 or 29 °C during the sex differentiation period. The genetic sex of hybrids from O. hatcheri fathers was inferred from a sex‐linked SNP marker. Both hybrids showed female‐biased sex ratios at the lowest temperature, female‐biased to balanced sex ratios at intermediate temperatures and balanced or male‐biased sex ratios at 29 °C, but unlike in purebred O. bonariensis, the lowest and highest temperatures did not yield monosex populations. The proportion of females in the offspring was affected more by parental genome than by hybrid combination. Female hybrids bearing the O. hatcheri Y chromosome showed temporary arrest of ovarian development that was rescued in adults. These results reveal strong interactions between genotype and temperature for sex determination and differentiation of the hybrids and provide important clues to understand the mechanisms of sex determination in these species.     

Morphological comparison of wild,farmed and hybrid specimens of two South American silversides,Odontesthes bonariensis and Odontesthes hatcheri

In this study, body shape of hybrid and presumptive introgressed South American silversides was studied. Body shape of O. bonariensis and O. hatcheri from wild populations and farmed stocks was compared to provide basic information on the effects of fish farming on morphometric parameters. Subsequently, wild presumptive introgressed individuals and artificially hybridized farmed individuals were morphologically analysed to assess the effects of hybridization on the same parameters. Most farmed purebred individuals were shorter and higher than their wild counterparts, which is probably due to the favourable growth conditions compared to the wild habitat. However, the results evidenced that purebred individuals were more slender than both hybrid (farmed) fish and introgressed (wild) fish. Further studies on the growth performance of hybrid Odontesthes will be required in order to assess whether the combination of hybridization and sterilization could produce, under farming conditions, growth performances which satisfy the requirements of aquaculture.     

Extensive cage culture of pejerrey (Odontesthes bonariensis) in a shallow pampean lake in Argentina

Pejerrey is an important zooplanktivorous native fish of the Argentinean inland waters. It has been traditionally propagated for stocking purposes by relatively costly semi‐intensive and intensive methods. In this study, we evaluated the implementation of an extensive culture method by using floating cages in a shallow pampean lake. Four cages were installed in the Lacombe Lake and stocked with juveniles (16.24 ± 1.69 mm length) at 50 fish m^?3 density for growing until the size of 150 mm, which is considered as a suitable size for stocking. Throughout the experiment, the temperature ranged between 10 and 26 °C and the zooplankton biomass ranged between 12 and 3269 μg dw L^?1. The growth patterns in the length were similar in the four cages and directly related to the lake thermal conditions and zooplankton availability. The average final length after 315 days was 154.4 ± 8.8 mm. The survival rates ranged between 53.5% and 64.7% during the first 110 days and 11.1–25.7% at termination. Growth rate for the first 2 months was the highest documented for pejerrey culture. This simple technique offers the possibility to produce juvenile pejerrey at a low cost and provides the alternative of reinforcing the natural populations with fish already adapted to the natural environmental conditions.     

Gonadotropin-releasing hormone (GnRH) neuronal systems in the pejerrey, Odontesthes bonariensis (Atheriniformes)

The brain of the pejerrey (Odontesthes bonariensis) has recently been shown to contain three forms of gonadotropin-releasing hormone (GnRH): salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and pejerrey GnRH (pjGnRH), nevertheless neuroanatomical studies on the distribution of these peptides are lacking. In this study we investigated the distribution of immunoreactive GnRH in the brain of adult pejerrey. Four different policlonal antisera and a monoclonal antibody against different GnRH variants were applied on cryosections and visualized using the ABC method. Three antisera (PBL#49, sGnRH#2 and cII741) revealed three different immunoreactive areas: the terminal nerve ganglion (at the junction between the olfactory bulbs and the anterior telencephalon), the preoptic area just anterior to the hypothalamus and the midbrain tegmentum. Fibers immunoreactive to GnRH were detected in different brain areas: the olfactory bulbs, the ventral thelencephalon, the hypothalamus, the mesencephalic area and an important innervation entering into the pituitary gland. Two other antibodies (LRH13 and s1668) labeled the two nuclei corresponding to the forebrain but not the midbrain tegmentum. As both antibodies have low crossreactivity to cGnRH-II, the data suggest that this group of cells express cGnRH-II. In summary, three different areas with immunoreactivity to GnRH were detected in the pejerrey brain. The distribution of sGnRH, pjGnRH and cGnRH-II expressing neurons, is discussed.     

Leucocyte profile and growth rates as indicators of crowding stress in pejerrey fingerlings (Odontesthes bonariensis)

Crowding is one of the most common stressors found in intensive aquaculture, compromising growth rates and immune function. Plasmatic cortisol is a classic stress biomarker for fish, but its quantification is expensive and demands blood volumes that small individuals do not provide, constraining the usage of this technique to assess stress in fingerlings. The leucocyte profile is an alternative methodology to quantify stress with reduced costs and volumes of blood. Stress conditions promote neutrophilia and lymphocytopenia as response to elevated glucocorticoids levels. Considering the difficulties to assess stress imposed by intensive fish farming using measurement of glucocorticoid hormones, this study aimed to evaluate the stress‐induced changes in leucocyte profiles and growth rates imposed by crowding in fingerlings of Odontesthes bonariensis, a promising South‐American candidate for freshwater aquaculture. To meet these objectives, fingerlings (initial weight 0.05 ± 0.06 g and length 1.68 ± 0.13 cm) were reared for 45 days under three rearing densities (1, 5 and 10 fingerlings L^?1). At the end of this period, fish were anaesthetized and euthanized to obtain the leucocyte profile, neutrophil:lymphocyte ratio (N:L) and growth rate. Increasing density promoted: significant reduction in growth (final length, weight and specific growth rate); neutrophilia and lymphocytopenia; and increased N:L ratio. Concluding, the tested rearing densities imposed distinct levels of stress characterized by different N:L ratio, demonstrating that the leucocyte profile is a reliable alternative to measure stress levels in O. bonariensis fingerlings and probably in other fish species.     

Increase in milt production by hormonal treatment in the pejerrey fish Odontesthes bonariensis (Valenciennes 1835)

In spite of interest in the cultivation of the pejerrey fish Odontesthes bonariensis (Cuvier & Valenciennes 1835), there are few studies on subjects required to advance this activity. One of the problems is the synchronization of female and male maturation to provide eggs and sperm for larval production. The low volume of expressible milt, either in wild or culture fish, is a major problem. The aim of this work was to study the effectiveness of the administration of different hormones on sperm production in pejerrey. Milt production was enhanced by the injection of human chorionic gonadotropin (hCG) (16.7‐fold increase, 625 IU kg^?1), carp pituitary extracts (13.5‐fold increase, 30 mg kg^?1), salmon pituitary extracts (12.8‐fold increase, 30 mg kg^?1), salmon‐type gonadotropin‐releasing hormone analogue (GnRH) (16.7‐fold increase, 10 μg kg^?1) and mammalian‐type GnRH analogue (10.8‐fold increase, 20 μg kg^?1). Sperm concentration, motility and the fertilization rate were not statistically different compared with control groups. It was also demonstrated that sperm could be obtained off‐season. Taken together, hCG is recommended to stimulate pejerrey spermiation because it is effective in low doses is inexpensive and is widely available.     

Regulation of gonadotropin subunit genes expression by 11-ketotestosterone during early spermatogenesis in male red seabream, Pagrus major

To examine the roles of gonadal steroids in the regulation of expression of gonadotropin subunit genes, male red seabream were gonadectomized and a sub-group was treated with 11-ketotestosterone (11-KT). Castration of males during the early stage of spermatogenesis elicited a significant increase in FSHβ mRNA levels, which was prevented by 11-KT replacement. By contrast, LHβ mRNA levels were not changed by castration or 11-KT replacement. In addition, administration of 11-KT to sham-operated males suppressed the steady-state FSHβ and LHβ mRNA levels. These results indicate that 11-KT may function as a negative feedback regulator of FSHβ gene expression, and may act through the testis to down-regulate LHβ mRNA levels in male red seabream during this period.     

The feedback regulation of pituitary GTH-II secretion in male African catfish (Clarias gariepinus): Participation of 11-ketotestosterone

11-ketotestosterone (OT) is a typical androgen of male teleost fish, but information on the question if it is involved in the feedback regulation of pituitary gonadotropin II (GTH-II) secretion is controversial. We have therefore studied the effects of OT on gonadotropin releasing-hormone (GnRH) stimulated GTH-II secretion in male African catfish Clarias gariepinus). In vivo experiments were carried out with intact and castrated fish. OT plasma levels were increased by implantation of silastic capsules containing 11-ketoandrostenedione (OA) which is converted to OT in both intact and castrated fish. When intact males received OA- or blank-capsules, treatment with salmon gonadotropin releasing-hormone analogue (Des-Gly¹⁰-D-Arg⁶-sGnRH-NEt; 0.2 μg sGnRHa/kg body weight) elevated the plasma GTH-11 levels in both groups. However, the levels were about 2 times higher in blank- than in OA-implanted fish. When castrated fish received either blank-or OA-capsules, sGnRHa treatment led to plasma GTH levels significantly higher than in sham-operated fish. However, there was no difference between the blank- or OA-implanted castrates, though OA implantation led to a restoration of OT plasma levels. This suggests that replacement ofOT is insufficient to reverse castration-induced effects. In vitro experiments were carried out with pituitary tissue fragments using a static culture system. The tissue remained sensitive to sGnRHa (5 × 10^?9M) for 4 days after the beginning of incubation. Preincubation of pituitary tissue for 24 hours with 25 ng OT/ml medium (80 nM) completely abolished the stimulatory effect of sGnRHa on GTH-II secretion. Tritiated OT was not metabolized by pituitary tissue during 6 hours of incubation. We conclude that 11-ketotestosterone, a quantitatively prominent and non-aromatizeable circulating androgen participates, at least in part by direct action on the pituitary, in the negative feedback regulation of GnRH-stimulated GTH-II secretion in male African catfish.     

Molecular cloning of cDNAs encoding FSH-β and LH-β subunits in the pejerrey fish, Odontesthes bonariensis

Fragments of the cDNAs encoding for FSH-β (428 bp) and LH-β (366 bp) subunits were studied in the pejerrey (pj), Odontesthes bonariensis, Pejerrey gonadotropins (GtHs) are close to percomorpha fish GtHs, LH-β has been better conserved than FSH-β during teleosts evolution.     

Spawning induction of pejerrey Odontesthes bonariensis in captivity using sustained‐release gonadotropin releasing hormone agonist implants

The aim of this study was to induce and synchronize spawning of pejerrey Odontesthes bonariensis (Valenciennes, 1835), using gonadotropin releasing hormone agonist (GnRHa) implants. In the first experiment, the ovarian condition was assessed by ovarian biopsies and the measurement of the genital pore width (GPW). Females having the leading clutch of oocytes with a diameter of around 800–900 μm and a GPW between 4.5 and 5.5 mm were treated with GnRHa implants. Eighty per cent of females spawned between 2 and 9 days after treatment, 12 days earlier than 20% of the fish in the control group that presented signs of spawning activity. In order to avoid any possible ovarian injury and/or stress by the catheterization procedure, in a second experiment, females were selected only by visual inspection of the abdomen and GPW measurement. As in experiment 1, 80% of females spawned between 2 and 8 days after treatment, 8 days earlier than 30% of the fish that spawned in the control group. In both experiments, fertilization and hatching success were similar between control and GnRHa‐treated groups. These results clearly demonstrated that GnRHa implantation can advance and synchronize ovulation and spawning in pejerrey without affecting egg quality.     

An 11-ketotestosterone induced kidney-secreted protein: the nest building glue from male three-spined stickleback, Gasterosteus aculeatus

In male three-spined sticklebacks, Gasterosteus aculeatus, the kidney hypertrophies during the breeding season and produces a glue which is used in nest-building. This hypertrophy is androgen dependent with 11-ketotestosterone (11 KT) being most effective. The aim of the present study was to characterize the protein composition of this glue. Threads of glue were collected from stickleback nests and glue material was sampled from the content of urinary bladders of male sticklebacks in breeding condition. The samples were investigated using sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE). One major glycoprotein dominated in both the nest-threads and urinary bladder samples. The identified glycoprotein had a molecular mass of approximately 203 kDa. After deglycosylation the molecular mass was approximately 200 kDa. The amino acid composition of the protein from urinary bladder content was almost identical to the amino acid composition of the protein from the nest-threads. The protein had a relatively high content of cysteine (7.6–8.0%). The glycoprotein was named spiggin. Spiggin was absent in the urinary bladder of untreated castrated fish, but spiggin was present in sham- operated fish and in castrated fish treated with 11 KT. These results demonstrate that spiggin is induced by 11 KT. Spiggin is so far the only protein known to be induced by 11KT and based on the present findings we suggest that spiggin represents a novel structural protein.     

Specific binding of 11-ketotestosterone in an androgen target organ,the kidney of the male three-spined stickleback,Gasterosteus aculeatus

The kidney of male three-spined stickleback,Gasterosteus aculeatus, hypertrophies during the breeding season and produces a glue which is used in the building of the nest. This hypertrophy is androgen dependent, with 11-ketotestosterone (11KT) being more effective than other tested steroids in stimulating this secondary sexual character. In the present study kidneys were excised from stickleback males that had been castrated two days earlier. The purpose of this gonadectomy was to reduce the endogenous levels of androgens without allowing time for the kidney to regress. Tissue fragments were incubated with tritiated 11KT with and without unlabelled steroids at increasing concentrations. Displaceable specific 11KT binding was found in kidney tissue fragments whereas only non-specific binding was observed when liver and muscle were investigated in a similar way. Unlabelled 11KT displaced specifically bound, tritiated 11KT with an ED50-value (50% of displaceable binding) of 28 nM. Similar ED50 values were found for 17\-hydroxy-5-androstane-3,11-dione (29 nM) and 5-dihydrotestosterone (20 nM), whereas higher ED50 concentrations were estimated for testosterone (T; 203 nM) and progesterone (69 nM). No displacement of tritiated 11KT was found for the other investigated substances tested; estradiol, 17,20-dihydroxy-4-pregnen-3-one, flutamide or cyproterone acetate. No specific binding to kidney tissue fragments could be detected when labelled T was used instead of labelled 11KT. Specific binding of 11KT or T was not found either in the kidney cytosol or nuclear extracts. However, using the kidney membrane fraction a displacement of tritiated 11KT with unlabelled 11KT (10^–6M) was observed. In conclusion there is a specific binding of 11KT in the stickleback kidney. The absence of binding in liver and muscle, the ED50 value observed and the displacement with some, but not all steroids are consistent with a receptor function. The presence of binding in membrane fractions, but not in cytosol or nuclear extracts suggests that the binding is not related to classic steroid receptors.     

Effects of sex and season in haematological parameters and cellular composition of spleen and head kidney of pejerrey (Odontesthes bonariensis)

Phylogenetic diversity in fish determines high interspecific variability in morphology as well as in physiological parameters. Moreover, several haematological variables and the organ composition of haemolymphopoietic sites may vary according to sex or season. The aim of this study was to establish the haematological parameters and the cellular composition of haemolymphopoietic organs in Odontesthes bonariensis, a commercially valuable fish species in Argentina, and also to determine gender or seasonal variations. Haematocrit exhibited the highest value in summer, while haemoglobin concentration was greater in summer and autumn. Erythrocyte count was higher in spring than autumn and winter, but did not differ with summer. The increase in these variables in seasons with higher water temperatures might be a compensatory mechanism to compensate the lower level of oxygen in the environment. Leucocyte formula and blast haemolymphopoietic cells in spleen and head kidney also showed annual variations since cells related to specific immune response, i.e., lymphocytes and thrombocytes, decrease in winter, whereas cells of the non-specific immune pathways, such as granulocyte, rise. The elevation of a particular type of circulating leucocyte was preceded by an increase in values of its precursor in blood in the previous season. Both, spleen and head kidney were active in haemolymphopoiesis, although with some differences in their activity during different seasons. Males showed higher values of circulating lymphoblasts and granulocytes than females, whereas females exhibited higher values of thrombocytes. This study corroborates the high interspecific variations in haematological parameters in fish that underlines the needing of basic studies in order to assess fish health status in new promising species for aquaculture.     

Neuroanatomical distribution and partial sequence of cDNA encoding brain cytochrome P450 aromatase (P450aromB) in pejerrey Odontesthes bonariensis

The molecular cloning of a cDNA encoding the pejerrey brain cytochrome P450 aromatase (P450aromB) is described. This form shares higher identity to other brain aromatases than with their respective ovarian counterparts and the self ovarian aromatase. Tissue-specific expression of both aromatases was examined in pejerrey by RT-PCR. The immunocytochemical distribution of P450aromB was described.     

Close association of gonadotropin-releasing hormone fibers and gonadotropin, growth hormone, somatolactin and prolactin expressing cells in pejerrey, Odontesthes bonariensis

The purpose of this study was to determine if there is any association between immunoreactive (ir) gonadotropin-releasing hormone (GnRH) fibers with different pituitary endocrine cell types in the pejerrey, Odontesthes bonariensis. Using a monoclonal antibody raised against mammalian GnRH (mGnRH) (LRH13), ir-GnRH fibers were observed passing through the pituitary stalk and reaching the three areas of the pituitary gland: rostral (RPD) and proximal pars distalis (PPD) and pars intermedia (PI). Double labeled immunocytochemistry showed ir-GnRH fibers in close association with prolactin (PRL)-producing cells in the RPD, growth hormone (GH)-producing cells in the PPD, gonadotropin (GtH)-producing cells in the PPD and the external border of the PI, and with somatolactin (SL)-producing cells in the PI. Our results show, direct morphological evidences of a close association of GnRH fibers with GH, PRL, GtH and SL-expressing cells. These results would suggest that GnRH has a broad role in the regulation of the secretion of different pituitary hormones.     

Molecular cloning of SOX9, DMRT1 and SF1 cDNA partial sequences in the pejerrey fish Odontesthes bonariensis (Atheriniformes)

Three different genes known to be involved in the mammalian sex-determining pathway, SOX9, DMRT1 and SF1, were characterized in a remarkable temperature sex determining species, the pejerrey Odontesthes bonariensis.     

11-ketotestosterone potentiates estrogen-induced vitellogenin production in liver of Japanese eel (Anguilla japonica)

Isolated hepatocytes from male Japanese eel at various stages of human chorionic gonadotropin (HCG)-induced spermatogenesis were cultured in the presence of estradiol-17β (E2). Hepatic vitellogenin (VTG) production in response to E2 increased during testicular development. This indicates that VTG production is influenced by gonadal maturity not only in females but also in males, where serum levels of 11-ketotestosterone (11KT) increase during testicular development. Recently, it has been confirmed that significant levels of 11KT are detected in maturing female eel. Therefore, in a next experiment, effect of 11KT on E2-induced VTG production was examined in vitro. As a result, in both male and female hepatocytes, 11KT enhanced E2-induced VTG production, although 11KT alone failed to induce VTG production.     

Synthesis and possible function of 11-ketotestosterone during oogenesis in eel (Anguilla spp.)

The control of 11-ketotestosterone (11-KT) biosynthesis and its physiological roles were examined in female Japanese eel (Anguilla japonica) and New Zealand longfinned eel (Anguilla dieffenbachii). 11-KT was detected in serum of female eels of both species. Among various tissues from Japanese eel, the ovary had the greatest capacity to synthesize 11-KT in vitro. In addition, the oocyte diameters of eels treated with 11-KT had increased significantly. Furthermore, these oocytes were found to have an increased number of oil droplets. These findings suggest that 11-KT in female eels may be mostly of ovarian origin and that this androgen appears to play an important role in controlling pre-vitellogenic oocyte growth.