Heterochiasmy and the establishment of gsdf as a novel sex determining gene in Atlantic halibut.

Atlantic Halibut (Hippoglossus hippoglossus) has a X/Y genetic sex determination system, but the sex determining factor is not known. We produced a high-quality genome assembly from a male and identified parts of chromosome 13 as the Y chromosome due to sequence divergence between sexes and segregation of sex genotypes in pedigrees. Linkage analysis revealed that all chromosomes exhibit heterochiasmy, i.e. male-only and female-only meiotic recombination regions (MRR/FRR). We show that FRR/MRR intervals differ in nucleotide diversity and repeat class content and that this is true also for other Pleuronectidae species. We further show that remnants of a Gypsy-like transposable element insertion on chr13 promotes early male specific expression of gonadal somatic cell derived factor (gsdf). Less than 4.5 MYA, this male-determining element evolved on an autosomal FRR segment featuring pre-existing male meiotic recombination barriers, thereby creating a Y chromosome. Our findings indicate that heterochiasmy may facilitate the evolution of genetic sex determination systems relying on linkage of sexually antagonistic loci to a sex-determining factor.

Functional Evolution of Clustered Aquaporin Genes Reveals Insights into the Oceanic Success of Teleost Eggs.

Aquaporin-mediated oocyte hydration is considered important for the evolution of pelagic eggs and the radiative success of marine teleosts. However, the molecular regulatory mechanisms controlling this vital process are not fully understood. Here, we analyzed >400 piscine genomes to uncover a previously unknown teleost-specific aquaporin-1 cluster (TSA1C) comprised of tandemly arranged aqp1aa-aqp1ab2-aqp1ab1 genes. Functional evolutionary analysis of the TSA1C reveals a ∼300-million-year history of downstream aqp1ab-type gene loss, neofunctionalization, and subfunctionalization, but with marine species that spawn highly hydrated pelagic eggs almost exclusively retaining at least one of the downstream paralogs. Unexpectedly, one-third of the modern marine euacanthomorph teleosts selectively retain both aqp1ab-type channels and co-evolved protein kinase-mediated phosphorylation sites in the intracellular subdomains together with teleost-specific Ywhaz-like (14-3-3ζ-like) binding proteins for co-operative membrane trafficking regulation. To understand the selective evolutionary advantages of these mechanisms, we show that a two-step regulated channel shunt avoids competitive occupancy of the same plasma membrane space in the oocyte and accelerates hydration. These data suggest that the evolution of the adaptive molecular regulatory features of the TSA1C facilitated the rise of pelagic eggs and their subsequent geodispersal in the oceanic currents.

The role of multiple vitellogenins in early development of fishes.

Functions of vitellogenins have been in the limelight of fish reproductive physiology research for decades. The Vtg system of acanthomorph teleosts consists of two complete forms of Vtgs (VtgAa and VtgAb) and an incomplete form, VtgC. Insufficient uptake and processing of Vtgs and their yolk proteins lead to inadequate oocyte hydration ensuing failure in acquisition of egg buoyancy and early developmental deficiencies. This review presents a summary of our studies on utilization of multiple Vtgs in species with different egg buoyancy characteristics, as examples. Studies of moronids revealed limited degradation of all three forms of lipovitellin heavy chain derived from their three respective forms of Vtg, by which they contribute to the free amino acid pool driving oocyte hydration during oocyte maturation. In later studies, CRISPR/Cas9 was employed to invalidate zebrafish type I, type II and type III Vtgs, which are orthologs of acanthamorph VtgAa, VtgAb and VtgC, respectively. Results revealed type I Vtg to have essential developmental and nutritional functions in both late embryos and larvae. Genomic disturbance of type II Vtg led to high mortalities during the first 24 h of embryonic development. Despite being a minor form of Vtg in zebrafish and most other species, type III Vtg was also found to contribute essentially to the developmental potential of zebrafish zygotes and early embryos. Apart from severe effects on progeny survival, these studies also disclosed previously unreported regulatory effects of Vtgs on fecundity and fertility, and on embryo hatching. We recently utilized parallel reactions monitoring based liquid chromatography tandem mass spectrometry to assess the processing and utilization of lipovitellins derived from different forms of Vtg in Atlantic halibut and European plaice. Results showed the Lv heavy chain of VtgAa (LvHAa) to be consumed during oocyte maturation and the Lv light chain of VtgAb (LvLAb) to be utilized specifically during late larval stages, while all remaining YPs (LvLAa, LvHAb, LvHC, and LvLC) were utilized during or after hatching up until first feeding in halibut. In plaice, all YPs except LvHAa, which similarly to halibut supports oocyte maturation, are utilized from late embryo to late larval development up until first feeding. The collective findings from these studies affirm substantial disparity in modes of utilization of different types of Vtgs among fish species with various egg buoyancy characteristics, and they reveal previously unknown regulatory functions of Vtgs in maintenance of reproductive assets such as maternal fecundity and fertility, and in embryonic hatching. Despite the progress that has been made over the past two decades by examining multiple Vtgs and their functions, a higher complexity of these systems with much greater diversity between species in modes of Vtg utilization is now evident. Further research is needed to reveal novel ways each species has evolved to utilize these complex multiple Vtg systems and to discover unifying principles for this evolution in fishes of diverse lineages, habitats and life history characteristics.

Quantitative proteome profiling reveals molecular hallmarks of egg quality in Atlantic halibut: impairments of transcription and protein folding impede protein and energy homeostasis during early development.

BACKGROUND: Tandem mass tag spectrometry (TMT labeling-LC-MS/MS) was utilized to examine the global proteomes of Atlantic halibut eggs at the 1-cell-stage post fertilization. Comparisons were made between eggs judged to be of good quality (GQ) versus poor quality (BQ) as evidenced by their subsequent rates of survival for 12 days. Altered abundance of selected proteins in BQ eggs was confirmed by parallel reaction monitoring spectrometry (PRM-LC-MS/MS). Correspondence of protein levels to expression of related gene transcripts was examined via qPCR. Potential mitochondrial differences between GQ and BQ eggs were assessed by transmission electron microscopy (TEM) and measurements of mitochondrial DNA (mtDNA) levels. RESULTS: A total of 115 proteins were found to be differentially abundant between GQ and BQ eggs. Frequency distributions of these proteins indicated higher protein folding activity in GQ eggs compared to higher transcription and protein degradation activities in BQ eggs. BQ eggs were also significantly enriched with proteins related to mitochondrial structure and biogenesis. Quantitative differences in abundance of several proteins with parallel differences in their transcript levels were confirmed in egg samples obtained over three consecutive reproductive seasons. The observed disparities in global proteome profiles suggest impairment of protein and energy homeostasis related to unfolded protein response and mitochondrial stress in BQ eggs. TEM revealed BQ eggs to contain significantly higher numbers of mitochondria, but differences in corresponding genomic mtDNA (mt-nd5 and mt-atp6) levels were not significant. Mitochondria from BQ eggs were significantly smaller with a more irregular shape and a higher number of cristae than those from GQ eggs. CONCLUSION: The results of this study indicate that BQ Atlantic halibut eggs are impaired at both transcription and translation levels leading to endoplasmic reticulum and mitochondrial disorders. Observation of these irregularities over three consecutive reproductive seasons in BQ eggs from females of diverse background, age and reproductive experience indicates that they are a hallmark of poor egg quality. Additional research is needed to discover when in oogenesis and under what circumstances these defects may arise. The prevalence of this suite of markers in BQ eggs of diverse vertebrate species also begs investigation.

Long-term feeding of Atlantic salmon with varying levels of dietary EPA + DHA alters the mineral status but does not affect the stress responses after mechanical delousing stress.

Atlantic salmon were fed diets containing graded levels of EPA + DHA (1·0, 1·3, 1·6 and 3·5 % in the diet) and one diet with 1·3 % of EPA + DHA with reduced total fat content. Fish were reared in sea cages from about 275 g until harvest size (about 5 kg) and were subjected to delousing procedure (about 2·5 kg), with sampling pre-, 1 h and 24 h post-stress. Delousing stress affected plasma cortisol and hepatic mRNA expression of genes involved in oxidative stress and immune response, but with no dietary effects. Increasing EPA + DHA levels in the diet increased the trace mineral levels in plasma and liver during mechanical delousing stress period and whole body at harvest size. The liver Se, Zn, Fe, Cu, and Mn and plasma Se levels were increased in fish fed a diet high in EPA + DHA (3·5 %) upon delousing stress. Furthermore, increased dietary EPA + DHA caused a significant increase in mRNA expression of hepcidin antimicrobial peptide (HAMP), which is concurrent with downregulated transferrin receptor (TFR) expression levels. High dietary EPA + DHA also significantly increased the whole-body Zn, Se, and Mn levels at harvest size fish. Additionally, the plasma and whole-body Zn status increased, respectively, during stress and at harvest size in fish fed reduced-fat diet with less EPA + DHA. As the dietary upper limits of Zn and Se are legally added to the feeds and play important roles in maintaining fish health, knowledge on how the dietary fatty acid composition and lipid level affect body stores of these minerals is crucial for the aquaculture industry.

The stress response in Atlantic salmon (Salmo salar L.): identification and functional characterization of the corticotropin-releasing factor (crf) paralogs.

Corticotropin-Releasing Factor (CRF) is one of the main mediators of the Hypothalamic-Pituitary-Interrenal (HPI) axis to stress response. In Atlantic salmon, a comparative understanding of the crf1 paralogs role in the stress response is still incomplete. Our database searches have identified four crf1 genes in Atlantic salmon, named crf1a1, crf1a2, crf1b1 and crf1b2. Brain distribution analysis revealed that the four crf1 paralogs were widely distributed, and particularly abundant in the telencephalon, midbrain, and hypothalamus of Atlantic salmon post-smolts. To increase the knowledge on crf1-mediated response to stress, Atlantic salmon post-smolts were exposed to either repeated chasing, hypoxia or a combination of chasing and hypoxia for eight days, followed by a novel-acute stressor, confinement. Cortisol, glucose, lactate, and creatinine levels were used as markers for the stress response. The crf1 paralogs mRNA abundance showed to be dependent on the stress exposure regime. Both crf1 mRNA levels in the telencephalon and crf1a1 mRNA levels in the hypothalamus showed similar response profiles to the serum cortisol levels, i.e., increasing levels during the first 24 h after stress exposure followed by a decline during the eight-day exposure. The similar trend between crf1 and cortisol disappeared once exposed to the novel-acute stressor. There was a minor response to stress for both crf1b1 and crf1b2 in the hypothalamus, while no changes at mRNA level were observed in the hypothalamic crf1a2 under the different stress conditions. No or weak relationship was found between the crf1 paralogs mRNA expression and the other serum stress-indicators analysed. In summary, our data provide novel insights on the dynamic of the HPI axis activation in Atlantic salmon, and thus underline the involvement of the crf1 paralogs as additional factors in the regulation of the stress response in this species. Likewise, the data highlight the importance of analysing all crf1 paralogues response to a stress-condition, in particular in this premature knowledge stage of their functionality. Further analysis and a more detailed time-point series will help to elucidate the response of the HPI axis and the link of crf1 paralogs in the stress response mechanism.

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.

Termination of puberty in out-of-season male Atlantic salmon smolts.

Environmental conditions are known to contribute to the phenotypic plasticity in the age of sexual maturation of Atlantic salmon (Salmo salar). Here, we report on an observation of out-of-season male Atlantic salmon initiating puberty as pre-smolts (jacks) but failing to complete maturation as post-smolts. Jacks were identified based on elevated plasma 11-ketotestosterone (range, 3-12 ng/ml) and the occurrence of type B spermatogonia in January 2017. However, these males failed to show running milt as post-smolts at the expected time in May 2017. Subsequently, 6 out of the 21 (32%) suspected "terminated jacks" went on to become grilse, whereas only 1 of the 22 (5%) males that showed no signs of initiating puberty in January became grilse in December 2017. Therefore, "terminated" jacks were more likely to mature as grilse than the males that remained immature. Why these pubertal pre-smolt males did not complete maturation is unclear but could be related to the transfer of fish from conditions of warm water and long days, risk factors for early maturation, to conditions of cold water and short days, which are expected to delay the age of maturation. We provide a description of the conditions under which male Atlantic salmon appear to have terminated the process of sexual maturation.

The use of photoperiods to provide year round spawning in lumpfish Cyclopterus lumpus.

In order to provide year round spawning broodstock, lumpfish (initial size 746 g and 24.9 cm) were reared under four different photoperiod regimes from January 2017 to July 2018. One group was reared under simulated natural photoperiod (LDN, control group) for Tromsø (70°N). The second group was transferred to continuous light (LD240) on 30 January 2017 and reared at LD24:0 throughout the trial period. Two compressed and phase advanced photoperiods were also established. Both groups were moved from LDN to LD24:0 on 30 January 2017, and after that reared at compressed natural photoperiods where the annual photoperiod was compressed down to six months (L6) or nine months (L9) for the duration of the study. Spawning time was shifted in both compressed groups during both years of the study. Spawning activity in the second year of the study was higher and followed more closely the expected spawning period in the compressed and the LDN groups. Spawning in the LD240 group was spread out over the experimental period with no distinct peak in spawning. A seasonal and pronounced drop in condition factor was found for females in the L9, L6 and the LDN groups. This post-spawning loss in condition was closely related to the spawning activity of each group. The current findings suggest that photoperiod has a strong influence on the timing of lumpfish maturation and can be used as an efficient and inexpensive tool to secure lumpfish reproduction operations i.e. year-round supply of egg and milt and/or timing with optimal temperature regimes.

Structural and functional maturation of skin during metamorphosis in the Atlantic halibut (Hippoglossus hippoglossus).

To establish if the developmental changes in the primary barrier and osmoregulatory capacity of Atlantic halibut skin are modified during metamorphosis, histological, histochemical, gene expression and electrophysiological measurements were made. The morphology of the ocular and abocular skin started to diverge during the metamorphic climax and ocular skin appeared thicker and more stratified. Neutral mucins were the main glycoproteins produced by the goblet cells in skin during metamorphosis. Moreover, the number of goblet cells producing neutral mucins increased during metamorphosis and asymmetry in their abundance was observed between ocular and abocular skin. The increase in goblet cell number and their asymmetric abundance in skin was concomitant with the period that thyroid hormones (THs) increase and suggests that they may be under the control of these hormones. Several mucin transcripts were identified in metamorphosing halibut transcriptomes and Muc18 and Muc5AC were characteristic of the body skin. Na+, K+-ATPase positive (NKA) cells were observed in skin of all metamorphic stages but their number significantly decreased with the onset of metamorphosis. No asymmetry was observed between ocular and abocular skin in NKA cells. The morphological changes observed were linked to modified skin barrier function as revealed by modifications in its electrophysiological properties. However, the maturation of the skin functional characteristics preceded structural maturation and occurred at stage 8 prior to the metamorphic climax. Treatment of Atlantic halibut with the THs disrupter methimazole (MMI) affected the number of goblet cells producing neutral mucins and the NKA cells. The present study reveals that the asymmetric development of the skin in Atlantic halibut is TH sensitive and is associated with metamorphosis and that this barrier's functional properties mature earlier and are independent of metamorphosis.

Effects of dietary arachidonic acid on the reproductive physiology of female Atlantic cod (Gadus morhua L.).

The present study was designed to investigate potential effects of arachidonic acid (ARA) on the reproductive physiology of female Atlantic cod (Gadus morhua L.). Two-year old Atlantic cod of both sexes were equally distributed into eight sea cages after completion of their first spawning in May 2005. Four experimental groups were established and fed diets with different levels of ARA corresponding to 0.5, 1, 2 and 4% of total fatty acid. Ovarian growth and development was documented every month. Fatty acid composition was analysed in ovaries, liver and plasma at the beginning of the experiment, one month prior to spawning, and in spent fish, one month after spawning was completed. Plasma concentrations of estradiol-17ß, testosterone and vitellogenin, and ovarian gene transcript levels of steroidogenic acute regulatory protein (star), P450aromatase (cyp19a1a) and 20ß-hydroxy steroid dehydrogenase (20bhsd/cbr1) were monitored every month in fish fed the experimental diets and related to oocyte stage. Potential fecundity was calculated based on ovarian samples taken one month before onset of spawning. Ovarian and plasma ARA levels were highly correlated to dietary ARA levels. There was a net accumulation of ARA compared to other essential fatty acids in ovarian tissue that was reflected in a decrease in EPA:ARA ratio. Plasma concentrations of vitellogenin, estradiol-17ß and testosterone and key gene transcript levels were affected by dietary ARA and stage of maturation. The results show that ARA has a significant influence on the reproductive physiology of female Atlantic cod.

Effects of temperature on the final stages of sexual maturation in Atlantic salmon (Salmo salar L.).

Maturing male and female Atlantic salmon (Salmo salar L.) were held under three temperature regimes for 10 weeks between September and December: warm (constant 14-16 °C), ambient (decreasing from 11 to 5 °C), and cold (decreasing from 7 to 3 °C). Blood samples were analyzed for plasma steroid levels, and the fish were inspected for the presence of expressible milt (total volume and spermatocrit) and ovulation weekly. Samples of eggs were dry-fertilized with milt stripped from three males held at the same temperatures and incubated until the eyed stage. In females, levels of plasma testosterone (T) and 17ß-oestradiol (E2) dropped as ovulation approached, concurrent with a rapid increase in levels of plasma 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P). In males, levels of T and 11-ketotestosterone (11-KT) peaked 2-3 weeks after the first appearance of expressible milt, while levels of 17,20ß-P increased steadily and did not exhibit a definite peak. Exposure of females to cold water amplified and advanced the profiles of all three steroids compared with the ambient group, and increased the survival rates to the eyed egg stage. Cold water had no immediate effect on the male steroid profiles, but later, higher levels of 17,20ß-P were evident compared with both the ambient controls and the warm water group, while the effects on 11-KT and T were more variable. Exposure to warm water completely inhibited both milt production and ovulation. Moreover, warm water modulated the steroid profiles of the males with lower 11-KT levels compared with ambient controls and lower 17,20ß-P level compared with cold-water-treated males. In females, warm water resulted in total inhibition of the peri-ovulatory peak in 17,20ß-P and prevented the normal decline of T and E2 levels associated with ovulation. The findings of the present study are highly relevant for broodstock management in aquaculture, as well in understanding the impact of climate change/temperature variability on wild salmon spawning.

Environmental impacts on the gonadotropic system in female Atlantic salmon (Salmo salar) during vitellogenesis: Photothermal effects on pituitary gonadotropins, ovarian gonadotropin receptor expression, plasma sex steroids and oocyte growth.

The gonadotropic system and ovarian growth and development were studied during vitellogenesis in female Atlantic salmon subjected to either simulated natural photoperiod and ambient water temperature (NL-amb), or an accelerating photoperiod (short day of LD8:16 from May 10) combined with either warmed (ca 2°C above ambient; 8L-warm) or cooled water (ca 2°C below ambient; 8L-cold) from May to September. Monthly samples were collected from 10 females/group for determination of transcript levels of pituitary gonadotropin subunits (fshb and lhb) and ovarian gonadotropin receptors (fshr and lhr), plasma sex steroids (testosterone: T and estradiol-17ß: E2), gonadosomatic index (GSI) and oocyte size. Short day in combination with either warmed or cooled water induced an earlier increase in pituitary fshb and lhb levels compared with NL-amb controls, and advanced ovarian growth and the seasonal profiles of T, E2. By contrast only minor effects were seen of the photothermal treatments on ovarian fshr and lhr. The 8L-cold had earlier increase in fshb, lhb and E2, but similar oocyte and gonadal growth as 8L-warm, suggesting that the 8L-cold group tried to compensate for the lower water temperature during the period of rapid gonadal growth by increasing fshb and E2 production. Both the 8L-warm and 8L-cold groups showed incomplete ovulation in a proportion of the females, possibly due to the photoperiod advancement resulting in earlier readiness of spawning occurring at a higher ambient temperature, or due to some reproductive dysfunction caused by photothermal interference with normal neuroendocrine regulation of oocyte development and maturation.

Genetic analyses verify sexually mature escaped farmed Atlantic cod and farmed cod eggs in the natural environment.

Elucidating the effects of domesticated organisms escaping into the natural environment represents a topic of importance in both evolutionary and conservation biology. However, when excluding the abundant data on salmonids, there is a lack of knowledge on this topic for marine fish aquaculture, which continues to expand globally. In order to bridge this empirical gap, we investigated a suspected escape of sexually mature domesticated Atlantic cod from a commercial marine fish farm in northern Norway. This involved genotyping samples of fish from cages on the farm, putatively identified escapees and wild cod captured in the region and samples of recently spawned eggs collected in the sea. Genetic analyses confirmed a farmed ancestry of the suspected escapees, and significantly, 27% of the sampled cod eggs. Furthermore, statistical analyses revealed a strong reduction in genetic variation in all samples of the farmed cod, including low effective population size and high degree of siblingship. These results thus document the escape of sexually mature adult cod and the release of fertilized domesticated cod eggs into the natural environment. Although it is possible that some of the mature escapees spawned post-escape, the fact that only a single egg of potential hybrid farmed × wild origin was identified, together with the high number of mature cod in the farm, points to within cage spawning as the primary source of these eggs. This suggestion is supported by oceanic particle-drift modelling, verifying that transport of eggs between the farm and the egg sampling locations was plausible. This study represents a rare documentation of interaction between domesticated and wild populations for a marine fish, pointing towards potential impacts on the local wild population.

Loss of Fshr Prevents Testicular Maturation in Atlantic Salmon (Salmo salar L.).

Early puberty poses a significant challenge for male Atlantic salmon in aquaculture due to its negative impact on growth and welfare. The regulation of puberty in vertebrates involves 2 key reproductive hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their gonadal receptors. In male mice lacking FSH receptor, testes size is reduced, but fertility is maintained, while medaka and zebrafish with a disrupted fshr gene exhibit near normal testis size and fertility. In these fishes both Fsh and Lh are present during puberty and Lh may rescue fertility, while in salmonid fish only Fsh is present in the circulation during puberty. Using CRISPR-Cas9, we produced crispants with a high prevalence of fshr mutations at the target site, which remained fertile, although more than half showed a testis development deviating from wild-type (wt) males. Crossing out these F0 crispants to each other produced a viable F1 generation showing frameshift (fshr-/-) or in-frame mutations (fshrif/if). Nearly all wt males matured while all fshr-/- males remained immature with small testes containing A spermatogonia as the furthest developed germ cell type and prepubertal plasma androgen levels. Also, the pituitary transcript levels of gnrhr2bba and lhb, but not for fshb, were reduced in the fshr-/- males compared with maturing males. More than half of the fshrif/if mutant males showed no or a delayed maturation. In conclusion, Atlantic salmon show the unique characteristic that loss of Fshr function alone results in male infertility, offering new opportunities to control precocious puberty or fertility in salmon.

Differential regulation of GnRH ligand and receptor genes in the brain and pituitary of Atlantic cod exposed to different photoperiod.

The onset of puberty and reproduction are tightly controlled by extrinsic and intrinsic inputs combined with genetically determined biological blueprints. Environmental inputs are then mediated by the brain-pituitary-gonad endocrine axis resulting in a unified output. In fish, one of the primary factors controlling the timing of sexual maturation is light, although how these signals are mediated in the brain and pituitary is not well understood. We therefore aimed to elucidate the molecular basis of the control of reproduction during the first spawning season in two year old female Atlantic cod. To this end, we measured GnRH and GnRH-R variant gene expression in brains and pituitaries collected from cod kept under four different photoperiod regimes: natural light (NL), continuous light (LL) and combined treatment of NL-LL and LL-NL. LL inhibited sexual development and spawning and LL-NL delayed sexual development and spawning. LL inhibited the spawning-related increase in brain GnRH3 and pituitary GnRH-R2a gene expression found under NL conditions, and the expression of these genes were delayed in concert with spawning of LL-NL cod. This study indicates that regulation of brain GnRH3 and pituitary GnRH-R2a genes likely mediates photoperiod induced changes in cod gonadal maturation.

Cortisol treatment of prespawning female cod affects cytogenesis related factors in eggs and embryos.

A stable supply of viable eggs and embryos is crucial for successful farming of Atlantic cod. Stress during broodstock rearing can have negative effects on offspring, but little is known about the molecular mechanisms that cause abnormal development. Maternally transferred mRNAs have been shown to be essential for normal development, and stress may therefore influence their expression and the subsequent embryonic development. We investigated if mimicked stress in cod females affects mRNA concentrations in eggs/embryos, and if this can be linked to viability of embryos. Three weeks before peak spawning, 20 fish were intraperitoneally implanted with either cortisol-containing or cortisol-free (sham) osmotic pumps. At peak spawning all individuals were stripped and eggs were fertilized and incubated until hatching. Samples were collected from unfertilized eggs and embryos for analysis of gene expression (microarray), viability, steroids and vitellogenin. Plasma concentration of cortisol (ng/ml) in treated females was significantly higher at spawning (127.1±20.9) than that of sham control (11.3±6.7). This difference was also reflected in eggs and embryos. Percent fertilization, asymmetric cell division and hatching were not affected. However, numerous genes were differentially expressed in eggs and embryos in response to elevated cortisol, especially in maternal (oocyte and blastula) stages. Among these differentially expressed genes, some were found to be linked to cytogenesis (stxbp6, fbxw2, capn12, thbs4, sytl2, coro1c, sel1l3), induction of mesodermal fate (fgfrl1) and import of the glucocorticoid receptor to the cell nucleus (ipo7). Gene ontology overrepresentation analysis on the whole set of differentially expressed genes at maternal stages (539 genes) revealed enriched activity in membrane associated regions, which largely corresponds to cytogenesis related processes. These results suggest that despite no visible phenotypic effects in early embryos, broodstock stress affects the egg/embryonic transcriptome, especially in relation to cytogenesis. Furthermore, effects related to egg/embryo phenotypes are difficult to measure at early stages of development, and instead might become apparent at later life stages.

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.