Two apolipoproteins in salmon louse (Lepeophtheirus salmonis), apolipoprotein 1 knock down reduces reproductive capacity.

The salmon louse, Lepeophtheirus salmonis is an ectoparasite of salmonid fish in the Northern Hemisphere, causing large economical losses in the aquaculture industry and represent a threat to wild populations of salmonids. Like other oviparous animals, it is likely that female lice use lipoproteins for lipid transport to maturing oocytes and other organs of the body. As an important component of lipoproteins, apolipoproteins play a vital role in the transport of lipids through biosynthesis of lipoproteins. Apolipoproteins have been studied in detail in different organisms, but no studies have been done in salmon lice. Two apolipoprotein encoding genes (LsLp1 and LsLp2) were identified in the salmon lice genome. Transcriptional analysis revealed both genes to be expressed at all stages from larvae to adult with some variation, LsLp1 generally higher than LsLp2 and both at their highest levels in adult stages of the louse. In adult female louse, the LsLp1 and LsLp2 transcripts were found in the sub-epidermal tissue and the intestine. RNA interference-mediated knockdown of LsLp1 and LsLp2 in female lice resulted in reduced expression of both transcripts. LsLp1 knockdown female lice produced significantly less offspring than control lice, while knockdown of LsLp2 in female lice caused no reduction in the number of offspring. These results suggest that LsLp1 has an important role in reproduction in female salmon lice.

The FTZ-F1 gene encodes two functionally distinct nuclear receptor isoforms in the ectoparasitic copepod salmon louse (Lepeophtheirus salmonis).

The salmon louse, Lepeophtheirus salmonis, is an ectoparasitic crustacean that annually inflicts substantial losses to the aquaculture industry in the northern hemisphere and poses a threat to the wild populations of salmonids. The salmon louse life cycle consists of eight developmental stages each separated by a molt. Fushi Tarazu Factor-1 (FTZ-F1) is an ecdysteroid-regulated gene that encodes a member of the NR5A family of nuclear receptors that is shown to play a crucial regulatory role in molting in insects and nematodes. Characterization of an FTZ-F1 orthologue in the salmon louse gave two isoforms named αFTZ-F1 and ßFTZ-F1, which are identical except for the presence of a unique N-terminal domain (A/B domain). A comparison suggest conservation of the FTZ-F1 gene structure among ecdysozoans, with the exception of nematodes, to produce isoforms with unique N-terminal domains through alternative transcription start and splicing. The two isoforms of the salmon louse FTZ-F1 were expressed in different amounts in the same tissues and showed a distinct cyclical expression pattern through the molting cycle with ßFTZ-F1 being the highest expressed isoform. While RNA interference knockdown of ßFTZ-F1 in nauplius larvae and in pre-adult males lead to molting arrest, knockdown of ßFTZ-F1 in pre-adult II female lice caused disruption of oocyte maturation at the vitellogenic stage. No apparent phenotype could be observed in αFTZ-F1 knockdown larvae, or in their development to adults, and no genes were found to be differentially expressed in the nauplii larvae following αFTZ-F1 knockdown. ßFTZ-F1 knockdown in nauplii larvae caused both down and upregulation of genes associated with proteolysis and chitin binding and affected a large number of genes which are in normal salmon louse development expressed in a cyclical pattern. This is the first description of FTZ-F1 gene function in copepod crustaceans and provides a foundation to expand the understanding of the molecular mechanisms of molting in the salmon louse and other copepods.

Chitin Synthases Are Critical for Reproduction, Molting, and Digestion in the Salmon Louse (Lepeophtheirus salmonis).

Chitin synthase (CHS) is a large transmembrane enzyme that polymerizes Uridine diphosphate N-acetylglucosamine into chitin. The genomes of insects often encode two chitin synthases, CHS1 and CHS2. Their functional roles have been investigated in several insects: CHS1 is mainly responsible for synthesizing chitin in the cuticle and CHS2 in the midgut. Lepeophtheirus salmonis is an ectoparasitic copepod on salmonid fish, which causes significant economic losses in aquaculture. In the present study, the tissue-specific localization, expression, and functional role of L. salmonis chitin synthases, LsCHS1 and LsCHS2, were investigated. The expressions of LsCHS1 and LsCHS2 were found in oocytes, ovaries, intestine, and integument. Wheat germ agglutinin (WGA) chitin staining signals were detected in ovaries, oocytes, intestine, cuticle, and intestine in adult female L. salmonis. The functional roles of the LsCHSs were investigated using RNA interference (RNAi) to silence the expression of LsCHS1 and LsCHS2. Knockdown of LsCHS1 in pre-adult I lice resulted in lethal phenotypes with cuticle deformation and deformation of ovaries and oocytes in adult lice. RNAi knockdown of LsCHS2 in adult female L. salmonis affected digestion, damaged the gut microvilli, reduced muscular tissues around the gut, and affected offspring. The results demonstrate that both LsCHS1 and LsCHS2 are important for the survival and reproduction in L. salmonis.

Effects of chitin synthesis inhibitor treatment on Lepeophtheirus salmonis (Copepoda, Caligidae) larvae.

The salmon louse (Lepeophtheirus salmonis) is an ectoparasite infecting Atlantic salmon (Salmo salar), which causes substantial problems to the salmon aquaculture and threatens wild salmon. Chitin synthesis inhibitors (CSIs) are used to control L. salmonis in aquaculture. CSIs act by interfering with chitin formation and molting. In the present study, we investigated the action of four CSIs: diflubenzuron (DFB), hexaflumuron (HX), lufenuron (LF), and teflubenzuron (TFB) on larval molt. As the mode of action of CSIs remains unknown, we selected key enzymes in chitin metabolism and investigated if CSI treatment influenced the transcriptional level of these genes. All four CSIs interfered with the nauplius II molt to copepodids in a dose-dependent manner. The EC50 values were 93.2 nM for diflubenzuron, 1.2 nM for hexaflumuron, 22.4 nM for lufenuron, and 11.7 nM for teflubenzuron. Of the investigated genes, only the transcriptional level of L. salmonis chitin synthase 1 decreased significantly in hexaflumuron and diflubenzuron-treated larvae. All the tested CSIs affected the molt of nauplius II L. salmonis larvae but at different concentrations. The larvae were most sensitive to hexaflumuron and less sensitive to diflubenzuron. None of the CSIs applied had a strong impact on the transcriptional level of chitin synthesis or chitinases genes in L. salmonis. Further research is necessary to get more knowledge of the nature of the inhibition of CSI and may require methods such as studies of protein structure and enzymological studies.

Correction to: Entry into puberty is reflected in changes in hormone production but not in testicular receptor expression in Atlantic salmon (Salmo salar).

Following publication of the original article [1], the authors would like to apologize for an error in Fig. 5e, the correct graph is presented below and shows the significant increase in pituitary mRNA levels of fshb in recruited males in the SGA stage.

Entry into puberty is reflected in changes in hormone production but not in testicular receptor expression in Atlantic salmon (Salmo salar).

BACKGROUND: Puberty in male Atlantic salmon in aquaculture can start as early as after the first winter in seawater, stunts growth and entails welfare problems due to the maturation-associated loss of osmoregulation capacity in seawater. A better understanding of the regulation of puberty is the basis for developing improved cultivation approaches that avoid these problems. Our aim here was to identify morphological and molecular markers signaling the initiation of, and potential involvement in, testis maturation. METHODS: In the first experiment, we monitored for the first time in large Atlantic salmon males several reproductive parameters during 17 months including the first reproductive cycle. Since testicular growth accelerated after the Winter solstice, we focused in the second experiment on the 5 months following the winter solstice, exposing fish from February 1 onwards to the natural photoperiod (NL) or to continuous additional light (LL). RESULTS: In the first experiment, testis weight, plasma androgens and pituitary gonadotropin transcript levels increased with the appearance of type B spermatogonia in the testis, but testicular transcript levels for gonadotropin or androgen receptors did not change while being clearly detectable. In the second experiment, all males kept under NL had been recruited into puberty until June. However, recruitment into puberty was blocked in ~ 40% of the males exposed to LL. The first morphological sign of recruitment was an increased proliferation activity of single spermatogonia and Sertoli cells. Irrespective of the photoperiod, this early sign of testis maturation was accompanied by elevated pituitary gnrhr4 and fshb and testicular igf3 transcript levels as well as increased plasma androgen levels. The transition into puberty occurred again with stable testicular gonadotropin and androgen receptor transcript levels. CONCLUSIONS: The sensitivity to reproductive hormones is already established before puberty starts and up-regulation of testicular hormone receptor expression is not required to facilitate entry into puberty. The increased availability of receptor ligands, on the other hand, may result from an up-regulation of pituitary Gnrh receptor expression, eventually activating testicular growth factor and sex steroid release and driving germ and Sertoli cell proliferation and differentiation.

Sequence Variations in pxr (nr1i2) From Zebrafish (Danio rerio) Strains Affect Nuclear Receptor Function.

Regulators of biotransformation are of particular interest in pharmacology and toxicology, determining in part the metabolism, disposition, and toxicity of chemicals. The nuclear receptor NR1I2 (pregnane X receptor, PXR) is a prominent xenosensor that regulates the expression of biotransformation enzymes governing elimination of many exogenous as well as endogenous compounds. Zebrafish (Danio rerio) has only one gene locus for pxr, but different genetic variants have been identified in zebrafish. However, the prevalence and significance of these variants are unknown. We hypothesize that sequence variation occurring in the Pxr gene of zebrafish may affect the action and fate of many chemicals in this species, a key model organism in various fields of research, including environmental toxicology. Here, we examine variation in Pxr sequences from four different strains of zebrafish and assess the responses of each Pxr to clotrimazole and butyl-4-aminobenzoate. The Pxr variants differed in both their ability to bind these structurally different ligands and to regulate reporter gene expression in vitro. We infer that the observed sequence variations in zebrafish Pxrs likely affect the response to putative Pxr agonists in vivo and potentially cause strain-specific biotransformation of xenobiotics in zebrafish. Thus, the choice of zebrafish strain could affect the outcome of downstream toxicological studies.

Chitin synthesis and degradation in Lepeophtheirus salmonis: Molecular characterization and gene expression profile during synthesis of a new exoskeleton.

Animals with exoskeleton need to molt to grow and develop. Molting is well described in some arthropods especially insects. Chitin is a polymer of N-acetylglucosamine, and one of the major components of the exoskeleton of arthropods. Chitin is synthesized and degraded by a series of enzymes during the molting cycle. However, the presence and function of these enzymes are largely unknown in copepods such as the ectoparasite salmon louse (Lepeophtheirus salmonis) a major pest in salmonid aquaculture. Here we describe six genes found in the L. salmonis genome (LsCHS1, LsCHS2, LsGFAT, LsGNA1, LsAGM, and LsUAP) with high homology to enzymes in the chitin synthesis pathway. The transcription profiles of these enzymes together with three chitinases enzymes (LsChi1, LsChi2, and LsChi4), which have been characterized before, were examined during the synthesis of a new exoskeleton and revealed a dynamical expression concurrent with the morphological changes during the molt cycle. Further understanding of chitin metabolism and its regulation may prove useful tool to develop new pesticides.

Molecular characterization of the lipophorin receptor in the crustacean ectoparasite Lepeophtheirus salmonis.

The Salmon louse (Lepeophtheirus salmonis) is a marine ectoparasite of salmonid fish in the Northern Hemisphere and considered as a major challenge in aquaculture and a threat to wild populations of salmonids. Adult female lice produce a large number of lipid-rich eggs, however, the mechanism of maternal lipid transport into developing eggs during salmon louse reproduction has not been described. In the present study, a full-length L. salmonis lipophorin receptor (LsLpR) consisting of 16 exons was obtained by RACE and RT-PCR. The predicted ORF was 952 amino acids and structural analysis showed five functional domains that are similar to LpR of insects and decapods. Phylogenetic analysis placed the LsLpR together with LpRs from decapods and insects. Expression analysis revealed that the relative abundance of LsLpR transcripts was highest in the larvae and adult female lice. In adult females, the LsLpR transcripts and protein were found in the ovary and vitellogenic oocytes whereas, in larvae, the LsLpR transcripts were found in the neuronal somata of the brain and the intestine. Oil Red O stain results revealed that storage of neutral lipids was found in vitellogenic oocytes and ovaries of adult females, and in the yolk of larvae. Moreover, RNA interference (RNAi) was conducted to demonstrate the function of LsLpR in reproduction and lipid metabolism in L. salmonis. In larvae, the transcription of LsLpR was decreased by 44-54% while in an experiment LsLpR knockdown female lice produced 72% less offspring than control lice.

Identification and characterisation of the ecdysone biosynthetic genes neverland, disembodied and shade in the salmon louse Lepeophtheirus salmonis (Copepoda, Caligidae).

The salmon louse is a marine ectoparasitic copepod on salmonid fishes. Its lifecycle consists of eight developmental stages, each separated by a molt. In crustaceans and insects, molting and reproduction is controlled by circulating steroid hormones such as 20-hydroxyecdysone. Steroid hormones are synthesized from cholesterol through catalytic reactions involving a 7,8-dehydrogenase Neverland and several cytochrome P450 genes collectively called the Halloween genes. In this study, we have isolated and identified orthologs of neverland, disembodied and shade in the salmon louse (Lepeophtheirus salmonis) genome. Tissue-specific expression analysis show that the genes are expressed in intestine and reproductive tissue. In addition, levels of the steroid hormones ecdysone, 20-hydroxyecdysone and ponasterone A were measured during the reproductive stage of adult females and in early life stages.

Microsomal triglyceride transfer protein in the ectoparasitic crustacean salmon louse (Lepeophtheirus salmonis).

The salmon louse, Lepeophtheirus salmonis, is an endemic ectoparasite on salmonid fish that is challenging for the salmon farming industry and wild fish. Salmon lice produce high numbers of offspring, necessitating sequestration of large amounts of lipids into growing oocytes as a major energy source for larvae, most probably mediated by lipoproteins. The microsomal triglyceride transfer protein (MTP) is essential for the assembly of lipoproteins. Salmon lice have three L. salmonis MTP (LsMTP) transcript variants encoding two different protein isoforms, which are predicted to contain three ß-sheets (N, C, and A) and a central helical domain, similar to MTPs from other species. In adult females, the LsMTPs are differently transcribed in the sub-cuticular tissues, the intestine, the ovary, and in the mature eggs. RNA interference-mediated knockdown of LsMTP in mature females gave offspring with significantly fewer neutral lipids in their yolk and only 10-30% survival. The present study suggests the importance of LsMTP in reproduction and lipid metabolism in adult female L. salmonis, a possible metabolic bottleneck that could be exploited for the development of new anti-parasitic treatment methods.

The ecdysone receptor (EcR) is a major regulator of tissue development and growth in the marine salmonid ectoparasite, Lepeophtheirus salmonis (Copepoda, Caligidae).

The function of the ecdysone receptor (EcR) during development and molting has been thoroughly investigated in some arthropods such as insects but rarely in crustacean copepods such as the salmon louse Lepeophtheirus salmonis (L. salmonis) (Copepoda, Caligidae). The salmon louse is an ectoparasite on Atlantic salmon that has major economical impact in aquaculture due to the cost of medical treatment methods to remove lice from the fish. Handling of salmon louse infestations is further complicated by development of resistance towards available medicines. Understanding of basic molecular biological processes in the salmon louse is essential to enable development of new tools to control the parasite. In this study, we found L. salmonis EcR (LsEcR) transcript to be present in the neuronal somata of the brain, nuclei of muscle fibres and the immature intestine of the salmon louse. Furthermore, we explored the function of LsEcR during development using RNA interference mediated knock-down and through infection trials. Our results show that knock-down of LsEcR in the salmon louse is associated with hypotrophy of several tissues, delayed development and mortality. In addition, combined knock-down of LsEcR/LsRXR resulted in molting arrest during early larval stages.

Environmental contaminants activate human and polar bear (Ursus maritimus) pregnane X receptors (PXR, NR1I2) differently.

BACKGROUND: Many persistent organic pollutants (POPs) accumulate readily in polar bears because of their position as apex predators in Arctic food webs. The pregnane X receptor (PXR, formally NR1I2, here proposed to be named promiscuous xenobiotic receptor) is a xenobiotic sensor that is directly involved in metabolizing pathways of a wide range of environmental contaminants. OBJECTIVES: In the present study, we comparably assess the ability of 51 selected pharmaceuticals, pesticides and emerging contaminants to activate PXRs from polar bears and humans using an in vitro luciferase reporter gene assay. RESULTS: We found that polar bear PXR is activated by a wide range of our test compounds (68%) but has a slightly more narrow ligand specificity than human PXR that was activated by 86% of the 51 test compounds. The majority of the agonists identified (70%) produces a stronger induction of the reporter gene via human PXR than via polar bear PXR, however with some notable and environmentally relevant exceptions. CONCLUSIONS: Due to the observed differences in activation of polar bear and human PXRs, exposure of each species to environmental agents is likely to induce biotransformation differently in the two species. Bioinformatics analyses and structural modeling studies suggest that amino acids that are not part of the ligand-binding domain and do not interact with the ligand can modulate receptor activation.

Androgens directly stimulate spermatogonial differentiation in juvenile Atlantic salmon (Salmo salar).

We studied the effects of androgens on early stages of spermatogenesis along with androgen receptor binding characteristics and the expression of selected testicular and pituitary genes. To this end, immature Atlantic salmon postsmolts received testosterone (T), adrenosterone (OA, which is converted in vivo into 11-ketotestosterone, 11-KT) or a combination of the two androgens (T+OA). Treatment with OA and T elevated the plasma levels of 11-KT and T, respectively, and co-injection of OA with T lead to high 11-KT levels but prevented plasma T levels to reach the levels observed after injecting T alone. Clear stimulatory effects were recorded as regards pituitary lhb and gnrhr4 transcript levels in fish receiving T, and to a lesser extent in fish receiving OA (but for the lhb transcript only). The two androgen receptors (Ara1 and Ara2) we cloned bound T and 11-KT and responded to these androgens in a similar way. Both androgens down-regulated testicular amh and increased igf3 transcript levels after 1 week of treatment, but effects on growth factor gene expression required sustained androgen stimulation and faded out in the groups with the decreasing T plasma levels. In fish exhibiting a sustained elevation of 11-KT plasma levels (OA and T+OA groups) for 2 weeks, the number of differentiating spermatogonia had increased while the number of undifferentiated spermatogonia decreased. Previous work showed that circulating gonadotropin levels did not increase following androgen treatments of gonad-intact immature male salmonids. Taken together, androgen treatment of immature males modulated testicular growth factor expression that, when sustained for 2 weeks, stimulated differentiation, but not self-renewal, of undifferentiated type A spermatogonia.

Molecular characterisation of the salmon louse, Lepeophtheirus salmonis salmonis (Krøyer, 1837), ecdysone receptor with emphasis on functional studies of female reproduction.

The salmon louse Lepeophtheirus salmonis (Copepoda, Caligidae) is an important parasite in the salmon farming industry in the Northern Hemisphere causing annual losses of hundreds of millions of dollars (US) worldwide. To facilitate development of a vaccine or other novel measures to gain control of the parasite, knowledge about molecular biological functions of L. salmonis is vital. In arthropods, a nuclear receptor complex consisting of the ecdysone receptor and the retinoid X receptor, ultraspiracle, are well known to be involved in a variety of both developmental and reproductive processes. To investigate the role of the ecdysone receptor in the salmon louse, we isolated and characterised cDNA with the 5'untranslated region of the predicted L. salmonis EcR (LsEcR). The LsEcR cDNA was 1608 bp encoding a 536 amino acid sequence that demonstrated high sequence similarities to other arthropod ecdysone receptors including Tribolium castaneum and Locusta migratoria. Moreover, in situ analysis of adult female lice revealed that the LsEcR transcript is localised in a wide variety of tissues such as ovaries, sub-cuticula and oocytes. Knock-down studies of LsEcR using RNA interference terminated egg production, indicating that the LsEcR plays important roles in reproduction and oocyte maturation. We believe this is the first report on the ecdysone receptor in the economically important parasite L. salmonis.

A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio).

The zebrafish (Danio rerio) is a widely used model species in biomedical research. The ZFL cell line, established from zebrafish liver, and freshly isolated primary hepatocytes from zebrafish have been used in several toxicological studies. However, no previous report has compared and characterized these two systems at the level of gene expression. The aim of this study was to evaluate the ZFL cell line in comparison to primary hepatocytes as in vitro models for studying effects of environmental contaminants in zebrafish liver. Using quantitative real-time PCR, the basal level and transcriptional induction potential of key genes involved in toxic responses in the ZFL cell line, primary hepatocytes and whole liver from zebrafish were compared. The study showed that the ZFL cells have lower levels of mRNA of most selected genes compared to zebrafish liver. The induced gene transcription following exposure to ligand was much lower in ZFL cells compared to zebrafish primary hepatocytes at the doses tested. Importantly, oestrogen receptor and vitellogenin genes showed low basal transcription and no induction response in the ZFL cell line. In conclusion, it appears that primary hepatocytes are well suited for studying environmental contaminants including xenoestrogens, but may show large sex-dependent differences in gene transcription. The ZFL cell line shows potential in toxicological studies involving the aryl hydrocarbon receptor pathway. However, low potential for transcriptional induction of genes in general should be expected, especially notable when studying estrogenic responses.

Pituitary gonadotropin and ovarian gonadotropin receptor transcript levels: seasonal and photoperiod-induced changes in the reproductive physiology of female Atlantic salmon (Salmo salar).

In female Atlantic salmon kept at normal light conditions, pituitary follicle-stimulating hormone beta (fshb) transcript levels were transiently elevated one year before spawning, re-increased in February, and remained high during spawning in November and in post-ovulatory fish in December. The first increase in plasma 17b-estradiol (E2), testosterone (T) and gonadosomatic index (GSI) was recorded in January; E2 rose up to one month prior to ovulation, while T and GSI kept increasing until ovulation. Pituitary luteinizing hormone beta (lhb) transcript levels peaked at the time of ovulation. Except for transient changes before and after ovulation, ovarian follicle stimulating hormone receptor (fshr) transcript amounts were relatively stable at a high level. By contrast, luteinizing hormone receptor (lhcgr) transcript levels started out low and increased in parallel to GSI and plasma E2 levels. Exposure to continuous light (LL) induced a bimodal response where maturation was accelerated or arrested. The LL-arrested females showed previtellogenic oil droplet stage follicles or primary yolk follicles only, and fshb and E2 plasma levels collapsed while fshr increased. The LL-accelerated females showed elevated lhb transcript levels and slightly elevated E2 levels during early vitellogenesis, and significantly elevated lhcgr E2 and GSI levels in late vitellogenesis. We conclude that Fsh-dependent signaling stimulates recruitment into and the sustained development through vitellogenesis. Up-regulation of lhcgr gene expression during vitellogenesis may reflect an estrogenic effect, while elevated fshr gene expression following ovulation or during LL-induced arrestment may be associated with ovarian tissue remodeling processes.

Sex differentiation in Atlantic cod (Gadus morhua L.): morphological and gene expression studies.

BACKGROUND: In differentiated gonochoristic species, a bipotential gonad develops into an ovary or testis during sex differentiation. Knowledge about this process is necessary to improve methods for masculinizing genetically female Atlantic cod for the subsequent purpose of producing all-female populations. METHODS: Gonads were examined histologically in juveniles from 14 to 39 mm total body length (TL). Number and size of germ cells were determined in a subset of the samples. Relevant genes were cloned, and mRNA levels determined by qPCR of amh, cyp19a1a; dax1 (nr0b2); shp (nr0b2a) and sox9b in a mixed-sex and an all-female population ranging from 12-49 mm TL. RESULTS: Individuals between 14-20 mm TL could be separated in two subgroups based on gonad size and germ cell number. Ovarian cavity formation was observed in some individuals from 18-20 mm TL. The mixed sex population displayed bimodal expression patterns as regards cyp19a1a (starting at 12 mm TL) and amh (starting at 20 mm TL) mRNA levels. After approximately 30 mm TL, cyp19a1a and amh displayed a gradual increase in both sexes. No apparent, sex-dependent expression patterns were found for dax1, shp or sox9b transcripts. However, shp levels were high until the larvae reached around 35 mm TL and then dropped to low levels, while dax1 remained low until 35 mm TL, and then increased sharply. CONCLUSIONS: The morphological sex differentiation in females commenced between 14-20 mm TL, and ovarian cavities were evident by 18-20 mm TL. Testis development occurred later, and was morphologically evident after 30 mm TL. This pattern was corroborated with sexually dimorphic expression patterns of cyp19a1a from 12-13 mm TL, and a male-specific increase in amh from 20 mm TL.

Involvement of Prop1 homeobox gene in the early development of fish pituitary gland.

When mutated in mammals, paired-like homeobox Prop1 gene produces highly variable pituitary phenotypes with impaired regulation of Pit1 and eventually defective synthesis of Pit1-regulated pituitary hormones. Here we have identified fish prop1 orthologs, confirmed their pituitary-specific expression, and blocked the splicing of zebrafish prop1 transcripts using morpholino oligonucleotides. Very early steps of the gland formation seemed unaffected based on morphology and expression of early placodal marker pitx. Prop1 knock-down reduced the expression of pit1, prl (prolactin) and gh (growth hormone), as expected if the function of Prop1 is conserved throughout vertebrates. Less expectedly, lim3 was down regulated. This gene is expressed from early stages of vertebrate pituitary development but is not known to be Prop1-dependent. In situ hybridizations on prop1 morphants using probes for the pan pituitary gene pitx3 and for the hormone gene markers prl, gh and tshß, revealed abnormal shape, growth and cellular organization of the developed adenohypophysis. Strikingly, the effects of prop1 knock-down on adenohypophysis morphology and gene expression were gradually reversed during late development, despite persistent splice-blocking of transcripts. Therefore, prop1 function appears to be conserved between mammals and fish, at least for the mediation of hormonal cell type differentiation via pit1, but the existence of other fish-specific pathways downstream of prop1 are suggested by our observations.

Pharmacological characterization, localization and quantification of expression of gonadotropin receptors in Atlantic salmon (Salmo salar L.) ovaries.

The gonadotropins Fsh and Lh interact with their receptors (Fshr and Lhr, respectively) in a highly specific manner in mammals with little overlap in biological activities. In fish, the biological activities seem less clearly separated considering, for example, the steroidogenic potency of both Fsh and Lh. Important determinants of the biological activity are the specificity of hormone-receptor interaction and the cellular site of receptor expression. Here, we report the pharmacological characterization of Atlantic salmon Fshr and Lhr, identify receptor-expressing cells in the ovary, and validate receptor mRNA quantification systems. For the pharmacological studies, we used highly purified coho salmon gonadotropins and found that the Fshr preferentially responded to Fsh, but was also activated by approximately 6-fold higher levels of Lh. The Lhr was specific for Lh and did not respond to Fsh. Photoperiod manipulation was used to generate ovarian tissue samples with largely differing stages of maturation. Specific real-time, quantitative (rtq) PCR assays revealed up to 40-fold (fshr) and up to 350-fold (lhr) changes in ovarian expression levels, which correlated well with the differences in ovarian weight, histology, and circulating oestrogen levels recorded in January and June, respectively. Vitellogenic ovaries were used to localise receptor-expressing cells by in situ hybridization. Granulosa cells of small and large vitellogenic follicles were positive for both receptors. Also theca cells of small and large vitellogenic follicles expressed fshr mRNA, while only in large vitellogenic follicles theca cells were (weakly) positive for lhr mRNA. While only ovulatory Lh levels seem high enough to cross-activate the Fshr, expression by both receptors by granulosa and theca cells suggests that homologous ligand receptor interaction will prevail.