Finn-Arne Weltzien

Control of puberty in farmed fish

Puberty comprises the transition from an immature juvenile to a mature adult state of the reproductive system, i.e. the individual becomes capable of reproducing sexually for the first time, which implies functional competence of the brain-pituitary-gonad (BPG) axis. Early puberty is a major problem in many farmed fish species due to negative effects on growth performance, flesh composition, external appearance, behaviour, health, welfare and survival, as well as possible genetic impact on wild populations. Late puberty can also be a problem for broodstock management in some species, while some species completely fail to enter puberty under farming conditions. Age and size at puberty varies between and within species and strains, and are modulated by genetic and environmental factors. Puberty onset is controlled by activation of the BPG axis, and a range of internal and external factors are hypothesised to stimulate and/or modulate this activation such as growth, adiposity, feed intake, photoperiod, temperature and social factors. For example, there is a positive correlation between rapid growth and early puberty in fish. Age at puberty can be controlled by selective breeding or control of photoperiod, feeding or temperature. Monosex stocks can exploit sex dimorphic growth patterns and sterility can be achieved by triploidisation. However, all these techniques have limitations under commercial farming conditions. Further knowledge is needed on both basic and applied aspects of puberty control to refine existing methods and to develop new methods that are efficient in terms of production and acceptable in terms of fish welfare and sustainability.

Primitive duplicate Hox clusters in the European eel's genome.

The enigmatic life cycle and elongated body of the European eel (Anguilla anguilla L., 1758) have long motivated scientific enquiry. Recently, eel research has gained in urgency, as the population has dwindled to the point of critical endangerment. We have assembled a draft genome in order to facilitate advances in all provinces of eel biology. Here, we use the genome to investigate the eels complement of the Hox developmental transcription factors. We show that unlike any other teleost fish, the eel retains fully populated, duplicate Hox clusters, which originated at the teleost-specific genome duplication. Using mRNA-sequencing and in situ hybridizations, we demonstrate that all copies are expressed in early embryos. Theories of vertebrate evolution predict that the retention of functional, duplicate Hox genes can give rise to additional developmental complexity, which is not immediately apparent in the adult. However, the key morphological innovation elsewhere in the eels life history coincides with the evolutionary origin of its Hox repertoire.

Neuroendocrine control by dopamine of teleost reproduction

While gonadotropin-releasing hormone (GnRH) is considered as the major hypothalamic factor controlling pituitary gonadotrophins in mammals and most other vertebrates, its stimulatory actions may be opposed by the potent inhibitory actions of dopamine (DA) in teleosts. This dual neuroendocrine control of reproduction by GnRH and DA has been demonstrated in various, but not all, adult teleosts, where DA participates in an inhibitory role in the neuroendocrine regulation of the last steps of gametogenesis (final oocyte maturation and ovulation in females and spermiation in males). This has major implications for inducing spawning in aquaculture. In addition, DA may also play an inhibitory role during the early steps of gametogenesis in some teleost species, and thus interact with GnRH in the control of puberty. Various neuroanatomical investigations have shown that DA neurones responsible for the inhibitory control of reproduction originate in a specific nucleus of the preoptic area (NPOav) and project directly to the region of the pituitary where gonadotrophic cells are located. Pharmacological studies showed that the inhibitory effects of DA on pituitary gonadotrophin production are mediated by DA-D2 type receptors. DA-D2 receptors have now been sequenced in several teleosts, and the coexistence of several DA-D2 subtypes has been demonstrated in a few species. Hypophysiotropic DA activity varies with development and reproductive cycle and probably is controlled by environmental cues as well as endogenous signals. Sex steroids have been shown to regulate dopaminergic systems in several teleost species, affecting both DA synthesis and DA-D2 receptor expression. This demonstrates that sex steroid feedbacks target DA hypophysiotropic system, as well as the other components of the brain-pituitary gonadotrophic axis, GnRH and gonadotrophins. Recent studies have revealed that melatonin modulates the activity of DA systems in some teleosts, making the melatonin-DA pathway a prominent relay between environmental cues and control of reproduction. The recruitment of DA neurons for the neuroendocrine control of reproduction provides an additional brain pathway for the integration of various internal and environmental cues. The plasticity of the DA neuroendocrine role observed in teleosts may have contributed to their large diversity of reproductive cycles.

Effects of photoperiod on sexual maturation and somatic growth in male Atlantic halibut (Hippoglossus hippoglossus L.).

A major obstacle in modern, intensive aquaculture is the precocious maturation of male fish, leading to decreased somatic growth and reduced filet quality. Effects of photoperiod on sexual maturation and growth in male Atlantic halibut were therefore examined. In June 1996, 1300 1+ fish of both sexes were distributed in two indoor tanks supplied with continuous light (LL) or a simulated natural photoperiod (SNP). In December 1996 and June 1997, 200 individuals were exchanged between the tanks creating six experimental groups that were followed until June 1998. LL stimulated growth and accelerated timing of first maturation by approximately 3 months. LL also appeared to interrupt circannual rhythmicity in sexual maturation. Sexual maturation led to reduced growth from 3 months pre-spawning and throughout the spawning season. Males that did not mature during the experiment attained the highest final body weight. All males reared on LL from June 1997 reached sexual maturity the following season. In contrast, only 26% of the males matured in the group transferred from LL to SNP in June 1997, and this group also had the highest final body weight. The results indicate a possible route for reducing the problem of precocious maturation in male halibut.

Spermatogenesis and related plasma androgen levels in Atlantic halibut (Hippoglossus hippoglossus L.)

Spermatogenesis in male Atlantic halibut (Hippoglossus hippoglossus L.) was investigated by sampling blood plasma and testicular tissue from 15-39-month-old fish. The experiment covered a period in which all fish reached puberty and completed sexual maturation at least once. The germinal compartment in Atlantic halibut testis appears to be organized in branching lobules of the unrestricted spermatogonial type, because spermatocysts with spermatogonia were found throughout the testis. Spermatogenesis was characterized histologically, and staged according to the most advanced type of germ cell present: spermatogonia (Stage I), spermatogonia and spermatocytes (Stage II), spermatogonia, spermatocytes and spermatids (Stage III), spermatogonia, spermatocytes, spermatids and spermatozoa (Stage IV), and regressing testis (Stage V). Three phases could be distinguished: first, an initial phase with low levels of circulating testosterone (T; quantified by RIA) and 11-ketotestosterone (11-KT; quantified by ELISA), spermatogonial proliferation, and subsequently the initiation of meiosis marked by the formation of spermatocytes (Stage I and II). Secondly, a phase with increasing T and 11-KT levels and with haploid germ cells including spermatozoa present in the testis (Stage III and IV). Thirdly, a phase with low T and 11-KT levels and a regressing testis with Sertoli cells displaying signs of phagocytotic activity (Stage V). Circulating levels of 11-KT were at least four-fold higher than those of T during all stages of spermatogenesis. Increasing plasma levels of T and 11-KT were associated with increasing testicular mass throughout the reproductive cycle. The absolute level of, or the relation between, testis growth and circulating androgens were not significantly different in first time spawners compared to fish that underwent their second spawning season. These results provide reference levels for Atlantic halibut spermatogenesis.

First draft genome sequence of the Japanese eel, Anguilla japonica

The Japanese eel is a much appreciated research object and very important for Asian aquaculture; however, its genomic resources are still limited. We have used a streamlined bioinformatics pipeline for the de novo assembly of the genome sequence of the Japanese eel from raw Illumina sequence reads. The total assembled genome has a size of 1.15 Gbp, which is divided over 323,776 scaffolds with an N50 of 52,849 bp, a minimum scaffold size of 200 bp and a maximum scaffold size of 1.14 Mbp. Direct comparison of a representative set of scaffolds revealed that all the Hox genes and their intergenic distances are almost perfectly conserved between the European and the Japanese eel. The first draft genome sequence of an organism strongly catalyzes research progress in multiple fields. Therefore, the Japanese eel genome sequence will provide a rich resource of data for all scientists working on this important fish species.

Melatonin Activates Brain Dopaminergic Systems in the Eel with an Inhibitory Impact on Reproductive Function

In the eel, a deficit in gonadotrophin‐releasing hormone (GnRH) and a strong dopaminergic (DA) inhibition are responsible for the blockade of gonad development if silver eels are prevented from their reproductive migration. Environmental factors that eels encounter during their oceanic reproductive migration are thought to play an important role in the stimulation of eel pubertal development. We investigated the potential role of melatonin, a known mediator of the effects of external factors on reproductive function in vertebrates. We demonstrated that a long‐term melatonin treatment increased brain tyrosine hydroxylase (TH, the rate limiting enzyme of DA synthesis) mRNA expression in a region‐dependent way. Melatonin stimulated the dopaminergic system of the preoptic area, which is involved in the inhibitory control of gonadotrophin [luteinising hormone (LH) and follicle‐stimulating hormone (FSH)] synthesis and release. Moreover, we showed that the increased TH expression appeared to be consistent with melatonin binding site distribution as shown by 2[125I]‐melatonin labelling studies. On the other hand, melatonin had no effects on the two eel native forms of GnRH (mGnRH and cGnRH‐II) mRNA expression. Concerning the pituitary–gonad axis, we showed that melatonin treatment decreased both gonadotrophin β‐subunit (LHβ, FSHβ) mRNA expression and reduced sexual steroid (11‐ketotestosterone, oestradiol) plasma levels. This indicates that melatonin treatment had a negative effect on eel reproductive function. To our knowledge, the results of the present study provide the first evidence that melatonin enhances TH expression in specific brain regions in a non‐mammalian species. By this mechanism melatonin could represent one pathway by which environmental factors could modulate reproductive function in the eel.

Identification and localization of eight distinct hormone-producing cell types in the pituitary of male Atlantic halibut (Hippoglossus hippoglossus L.)

The eight distinct hormone-producing cell types in the adenohypophysis of male Atlantic halibut (Hippoglossus hippoglossus L.) were identified and localized using immunohistochemistry and in situ hybridization. Lactotropes either occupied most of the rostral pars distalis (RPD) or they were arranged in follicular structures located along the periphery of the RPD. Corticotropes were confined to a thin layer of RPD cells bordering the pars nervosa (PN). The somatotropes were arranged in multicellular layers bordering the highly convoluted PN penetrating the proximal pars distalis (PPD), while thyrotropes, scattered in small islets in between the somatotropes, were located in the centro-dorsal part of the PPD. Gonadotropes were found throughout the PPD. Immunoreactivity to glycoprotein-alpha and luteinizing hormone beta-subunit was also observed along the periphery of the pars intermedia (PI), indicating that a thin extension of the PPD surrounded the PI. In situ hybridization showed that follicle-stimulating hormone and luteinizing hormone were produced in distinct cells of the PPD. PI contained somatolactotropes bordering the highly convoluted PN, and melanotropes that showed positive immunostaining against both anti-alpha-melanocyte-stimulating hormone and anti-beta-endorphin. The general cellular organization was similar to that of other teleost fish. These results lay the basis for future investigations on Atlantic halibut pituitary physiology.

Molecular characterization and expression of FSHβ, LHβ, and common α-subunit in male Atlantic halibut (Hippoglossus hippoglossus)

To elucidate the role of the gonadotropins in the multiple spawner Atlantic halibut (Hippoglossus hippoglossus) full length cDNAs encoding FSHbeta, LHbeta, and the common alpha-subunit were cloned from pituitary glands by RACE-PCR. The three cDNAs consisted of 614, 595, and 666 nucleotides encoding peptides of 131, 146, and 124 amino acids, respectively. Halibut FSHbeta and LHbeta showed unique structural features among the vertebrate glycoprotein hormones. First, in contrast to all known FSHbeta, which contain either one or two conserved N-glycosylation sites, no potential binding site was found in Atlantic halibut FSHbeta. Second, the conserved glycosylation site in the N-terminus of all vertebrate LHbeta has been substituted with a unique C-terminal binding site in Atlantic halibut LHbeta. Furthermore, a specific cysteine residue of importance for the folding and heterodimerization of mammalian FSH is lacking in the FSHbeta from Atlantic halibut as well as many other teleosts. However, teleost FSHbeta is characterized by an additional N-terminal cysteine, which has likely replaced the missing residue, implicating a modified folding pattern of this subunit. In situ hybridization of mature male pituitaries revealed that FSHbeta and LHbeta mRNA were expressed in distinct cell types throughout the proximal pars distalis of the adenohypophysis, while alpha-subunit mRNA was identified in all parts of the proximal pars distalis, and also along the periphery of pars intermedia. Consistently, Northern blot analysis of pituitary RNA from mature males showed that FSHbeta, LHbeta, and alpha-subunit mRNAs were highly expressed. In juvenile male pituitaries very few cells containing FSHbeta, LHbeta, and alpha-subunit mRNA were identified by in situ hybridization. Low mRNA levels encoding LHbeta and the alpha-subunit were also demonstrated by Northern blot analysis of the juvenile pituitaries, while no FSHbeta mRNA was detected using this less sensitive technique. The results suggest that both FSH and LH play a role during both the very early and the final reproductive stages in Atlantic halibut males.

Isolation and characterization of FSH and LH from pituitary glands of Atlantic halibut (Hippoglossus hippoglossus L.)

Two gonadotropins (GtH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), were isolated and characterized from pituitary glands of Atlantic halibut (Hippoglossus hippoglossus L.). Glycoproteins were extracted in 40% ethanol followed by precipitation in 85% ethanol. Subsequently, glycoproteins were fractionated by ion-exchange chromatography on a Whatman DE-52 column using a stepwise gradient of ammonium bicarbonate (50-1000 mM). Intact FSH and LH were finally purified on rpHPLC using an AsahiPak C4P-50 column with an acetonitrile gradient (10-60%). SDS-PAGE showed a molecular mass of 33 and 32 kDa for intact FSH and LH, respectively. Final purification of subunits was performed by a subsequent purification step on rpHPLC using a Phenomenex Jupiter C18 column with an acetonitrile gradient (10-60%). FSHbeta, LHbeta, and the common alpha subunit showed molecular masses of 25, 24, and 19 kDa, respectively. Subunit identity was confirmed by N-terminal amino acid sequencing. Intact FSH and LH showed gonadotropic activity by stimulating release of 11-ketotestosterone from turbot (Scophthalmus maximus L.) testicular tissue in vitro. This provides the first purification of two distinct GtHs from an evolutionary advanced pleuronectiform teleost.