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The underlying molecular pathophysiology of feeding disorders, particularly in peripheral organs, is still largely unknown. A range of molecular factors encoded by appetite-regulating genes are already described to control feeding behaviour in the brain. However, the important role of the gastrointestinal tract in the regulation of appetite and feeding in connection to the brain has gained more attention in the recent years. An example of such inter-organ connection can be the signals mediated by leptin, a key regulator of body weight, food intake and metabolism, with conserved anorexigenic effects in vertebrates. Leptin signals functions through its receptor (lepr) in multiple organs, including the brain and the gastrointestinal tract. So far, the regulatory connections between leptin signal and other appetite-regulating genes remain unclear, particularly in the gastrointestinal system. In this study, we used a zebrafish mutant with impaired function of leptin receptor to explore gut expression patterns of appetite-regulating genes, under different feeding conditions (normal feeding, 7-day fasting, 2 and 6-hours refeeding). We provide evidence that most appetite-regulating genes are expressed in the zebrafish gut. On one hand, we did not observed significant differences in the expression of orexigenic genes (except for hcrt) after changes in the feeding condition. On the other hand, we found 8 anorexigenic genes in wild-types (cart2, cart3, dbi, oxt, nmu, nucb2a, pacap and pomc), as well as 4 genes in lepr mutants (cart3, kiss1, kiss1r and nucb2a), to be differentially expressed in the zebrafish gut after changes in feeding conditions. Most of these genes also showed significant differences in their expression between wild-type and lepr mutant. Finally, we observed that impaired leptin signalling influences potential regulatory connections between anorexigenic genes in zebrafish gut. Altogether, these transcriptional changes propose a potential role of leptin signal in the regulation of feeding through changes in expression of certain anorexigenic genes in the gastrointestinal tract of zebrafish.

Reproduction is an energetically costly event across vertebrates and tightly linked to nutritional status and energy reserves. In mammals, the hormone leptin is considered as a link between energy homeostasis and reproduction. However, its role in fish reproduction is still unclear. In this study, we investigated the possible role of leptin in the regulation of reproduction in zebrafish, using a loss of function leptin receptor (lepr) strain. Impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. lepr mutant females laid significantly fewer eggs, with low fertilization rates compared to wild-type females. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutants. Interestingly, the expression of luteinizing hormone beta (lhb) in the pituitary was significantly lower in mutant females. Analysis of candidate genes in the ovaries and isolated fully grown follicles revealed differential expression of genes involved in steroidogenesis, oocyte maturation and ovulation in the mutants. In conclusion, our results show that leptin deficiency does not affect early stages of follicular development, but leptin might be essential in later steps, such as in oocyte maturation and ovulation. To our knowledge, this is the first time that leptin is associated to reproductive deficiencies in zebrafish.

Sewage effluent ozonation can reduce concentrations of chemical pollutants including pharmaceutical residues. However, the formation of potentially toxic ozonation byproducts (OBPs) is a matter of concern. This study sought to elucidate toxicity mechanisms of ozonated carbamazepine (CBZ), an anti-epileptic drug frequently detected in sewage effluents and surface water, in zebrafish embryos (Danio rerio). Embryos were exposed to ozonated and non-ozonated CBZ from 3 h post-fertilization (hpf) until 144 hpf. Embryotoxicity endpoints (proportion of dead and malformed embryos) were assessed at 24, 48, and 144 hpf. Heart rate was recorded at 48 hpf. Exposure to ozonated CBZ gave rise to cardiovascular-related malformations and reduced heart rate. Moreover, embryo-larvae exposed to ozonated CBZ displayed a lack of swim bladder inflation. Hence, the expression patterns of CBZ target genes involved in cardiovascular and embryonal development were investigated through a stepwise gene co-expression analysis approach. Two co-expression networks and their upstream transcription regulators were identified, offering mechanistic explanations for the observed toxicity phenotypes. The study presents a novel application of gene co-expression analysis elucidating potential toxicity mechanisms of an ozonated pharmaceutical with environmental relevance. The resulting data was used to establish a putative adverse outcome pathway (AOP).

The signal mediated by leptin hormone and its receptor is a major regulator of body weight, food intake and metabolism. In mammals and many teleost fish species, leptin has an anorexigenic role and inhibits food intake by influencing the appetite centres in the hypothalamus. However, the regulatory connections between leptin and downstream genes mediating its appetite-regulating effects are still not fully explored in teleost fish. In this study, we used a loss of function leptin receptor zebrafish mutant and real-time quantitative PCR to assess brain expression patterns of several previously identified anorexigenic genes downstream of leptin signal under different feeding conditions (normal feeding, 7-day fasting, 2 and 6-h refeeding). These downstream factors include members of cart genes, crhb and gnrh2, as well as selected genes co-expressed with them based on a zebrafish co-expression database. Here, we found a potential gene expression network (GRN) comprising the abovementioned genes by a stepwise approach of identifying co-expression modules and predicting their upstream regulators. Among the transcription factors (TFs) predicted as potential upstream regulators of this GRN, we found expression pattern of sp3a to be correlated with transcriptional changes of the downstream gene network. Interestingly, the expression and transcriptional activity of Sp3 orthologous gene in mammals have already been implicated to be under the influence of leptin signal. These findings suggest a potentially conserved regulatory connection between leptin and sp3a, which is predicted to act as a transcriptional driver of a downstream gene network in the zebrafish brain.

The reproductive performance of farmed Arctic charr exhibits substantial individual variation. The causes behind this variation have not yet been properly explained, despite the species relatively long history in aqua-culture. The aim of the present study was to provide better understanding of the issue by evaluating parent and gamete traits in relation to fertility and offspring viability under routine hatchery conditions. Are losses mainly due to failed fertilisation or mortality? Are maternal or paternal factor more predominant? The study covered sex hormone status of the broodstock, egg traits (egg size and wateriness of egg batches), milt traits (volume, density, and sperm motility characteristics), and symmetry of early cell division.

We found that the majority of offspring loss occurred as embryo mortality, although a significant additional loss could be assigned fertilisation failure. Fertilisation and mortality rates were not inter-correlated. Neither of the evaluated egg traits, egg size and wateriness of newly stripped egg batches, proved to be useful indicators of egg quality. With regard to sperm traits, milt density as well as several computer assisted sperm analysis (CASA) parameters, i.e. swimming speed (VAP, VSL, and VCL) and beat cross frequency (BCF), were correlated with reproductive performance and could potentially be used as quality indicators for Arctic charr. Together, male 17,20β-P levels and BCF explained 69.5% of the between male variation in proportion of successfully eyed eggs.

The results support previous indications that post-ovulatory aging of oocytes is contributing to the impairment of reproductive performance in farmed Arctic charr. In addition, a significant paternal factor to this impairment has been exposed. However, the cause and mechanism involved are still far from clear.

The hormone leptin is a key regulator of body weight, food intake and metabolism. In mammals, leptin acts as an anorexigen and inhibits food intake centrally by affecting the appetite centres in the hypothalamus. In teleost fish, the regulatory connections between leptin and other appetite-regulating genes are largely unknown. In the present study, we used a zebrafish mutant with a loss of function leptin receptor to investigate brain expression patterns of 12 orexigenic and 24 anorexigenic genes under different feeding conditions (normal feeding, 7-day fasting, 2 and 6-hours refeeding). Expression patterns were compared to wild-type zebrafish, in order to identify leptin-dependent differentially expressed genes under different feeding conditions. We provide evidence that the transcription of certain orexigenic and anorexigenic genes is influenced by leptin signalling in the zebrafish brain. We found that the expression of orexigenic genes was not affected by impaired leptin signalling under normal feeding conditions; however, several orexigenic genes showed increased transcription during fasting and refeeding, including agrp, apln, galr1a and cnr1. This suggests an inhibitory effect of leptin signal on the transcription of these orexigenic genes during short-term fasting and refeeding in functional zebrafish. Most pronounced effects were observed in the group of anorexigenic genes, where the impairment of leptin signalling resulted in reduced gene expression in several genes, including cart family, crhb, gnrh2, mc4r, pomc and spx, in the control group. This suggests a stimulatory effect of leptin signal on the transcription of these anorexigenic genes under normal feeding condition. In addition, we found multiple gain and loss in expression correlations between the appetite-regulating genes, in zebrafish with impaired leptin signal, suggesting the presence of gene regulatory networks downstream of leptin signal in zebrafish brain. The results provide the first evidence for the effects of leptin signal on the transcription of various appetite-regulating genes in zebrafish brain, under different feeding conditions. Altogether, these transcriptional changes suggest an anorexigenic role for leptin signal, which is likely to be mediated through distinct set of appetite-regulating genes under different feeding conditions.

Leptin modulates all levels of the reproductive endocrine axis in mammals, and in turn, both leptin and the leptin receptor are regulated by sex steroids. The aim of this study was to investigate if sex steroids regulate the leptin system also in fish. Immature one-year old male Atlantic salmon parr were implanted with Silclear capsules that were either empty or filled with 11-ketoandrostenedione (11 KA) or testosterone (T) and the effects of 35-days treatment were investigated on measures of maturation, gene expression of leptin (lepa1, lepa2), leptin receptor (lepra1) and circulating plasma leptin. Both 11-KA and T stimulated the reproductive axis by increasing testes weight and up-regulated pituitary lh-beta mRNA levels and for T also fsh-beta. T up-regulated transcription levels of lepa1 and lepra1 in the pituitary, while 11-KA had no effect. Leptin receptor expression in the testis was unaltered by either androgen. T up-regulated lepa1 mRNA levels significantly also in the liver, but had no effect on lepa2, and 11 KA did not affect hepatic gene expression of either lepa1 or lepa2. Plasma leptin levels did not differ significantly between treatments. The results indicate that androgens regulate gene expression of leptin and the leptin receptor in different tissues in fish and that the effects of leptin might be tissue specific considering plasma levels remained unaltered. Overall, the results suggest a role for leptin in fish reproduction, where sex steroids are able to regulate components of the leptin system differentially in liver and important tissues of the reproductive axis.

Female three-spined sticklebacks are batch spawners laying eggs in a nest built by the male. We sampled female sticklebacks at different time points, when they were ready to spawn and 6, 24, 48 and 72 h post-spawning (hps) with a male. Following spawning, almost all females (15 out of 19) had ovulated eggs again at Day 3 post-spawning (72 hps). At sampling, plasma, brain and pituitaries were collected, and the ovary and liver were weighed. Testosterone (T) and estradiol (E2) were measured by radioimmunoassay. Moreover, the mRNA levels of follicle-stimulating hormone (fsh-beta) and luteinizing hormone (lh-beta) in the pituitary, and of the gonadotropin-releasing hormones (GnRHs: gnrh2, gnrh3) and kisspeptin (kiss2) and its G protein-coupled receptor (gpr54) in the brain were measured by real-time qPCR. Ovarian weights peaked in "ready to spawn" females, dropped after spawning, before again progressively increasing from 6 to 72 hps. Plasma T levels showed peaks at 24 and 48 hps and decreased at 72 hps, while E2 levels increased already at 6 hps and remained at high levels up to 48 hps. There was a strong positive correlation between T and E2 levels over the spawning cycle. Pituitary lh-beta mRNA levels showed a peak at 48 hps, while fsh-beta did not change. The neuropeptides and gpr54 did not show any changes. The changes in T and E2 over the stickleback spawning cycle were largely consistent with those found in other multiple-spawning fishes whereas the marked correlation between T and E2 does not support T having other major roles over the cycle than being a precursor for E2.

Background: Female threespine sticklebacks, Gasterosteus aculeatus, are batch spawners. As in most teleosts, the ovulated eggs are kept in the ovarian cavity until spawning. If spawning or spontaneous release of the eggs does not take place, they can become overripe and harden, and in most cases remain in the ovary. The overripe eggs are lost for reproduction and also block further spawnings. Reproductive hormones regulate egg production and may be involved in the mechanism of overripening. Question: What are the reproductive endocrinological parameters characterizing overripening of ovulated eggs in the threespine stickleback? Organism: Wild-caught adult threespine sticklebacks from the southern Baltic at Skare in southern Sweden and the island of Asko in northwestern Baltic Proper in Sweden. Experiments: We collected blood samples for hormone measurements, as well as pituitaries and brains for measurement of mRNA from both sexually mature non-overripe (non-ovulated and/or ovulated) and overripe (egg-bound) females. For the Skare fish, sexual maturation was induced under laboratory conditions by exposure to a long photoperiod and we compared the non-overripe (including non-ovulated, with oocytes in different maturing or ripening stages, and ovulated females) with the overripe females. The Asko fish were sampled directly from nature, during the natural summer breeding season and we compared the non-overripe (including non-ovulated, with oocytes in different maturing or ripening stages, and ovulated females) with the overripe females. Methods: In the fish collected from Skare, we used radioimmunoassay to measure the plasma levels of four steroids: testosterone, estradiol, 17,20 beta-dihydroxypregn-4-en-3-one (17,20 beta-P), and 17,20 beta, 21-trihydroxypregn-4-en-3-one (17,20 beta,21-P). We also measured the mRNA levels of gonadotropins [GTHs: follicle-stimulating hormone (fsh-beta) and luteinizing hormone (lh-beta)] in the pituitary, and of gonadotropin-releasing hormones (GnRHs: gnrh2, gnrh3) and kisspeptin (kiss2) and its G protein-coupled receptor (gpr54) in the brain by real-time quantitative PCR. In the fish collected from Asko, we measured only progestogens (17,20 beta-P and 17,20 beta,21-P). Results: In the fish from Skare, overripe female sticklebacks had significantly lower levels of circulating plasma steroid hormones (testosterone, estradiol, 17,20 beta-P), as well as of pituitary lh-beta and brain kiss2 and gpr54 mRNA than the non-overripe females. In the fish caught from Asko, overripe females had lower 17,20 beta-P levels than the non-overripe non-ovulated females, but there was no difference between the non-overripe ovulated and the overripe females. The 17,20 beta,21-P plasma levels were under the limit of detection in all groups.

In mammals, leptin plays an important role in puberty and reproduction and leptin is regulated by sex steroids. Elevated leptin levels have been associated with sexual maturation in some teleosts such as Atlantic salmon. In the present study, primary cultures of Atlantic salmon hepatocytes were used to investigate the direct effects of different sex steroids on expression of the two salmon leptin-a genes, lepa1 and lepa2. Testosterone (T) stimulated both lepa1 and lepa2 in a dose dependent manner after four days of incubation. The stimulatory effect of T on leptin expression was not prevented by co-incubation with the aromatase inhibitor fadrozole, indicating a direct androgen effect on transcription. The non-aromatizable androgen 11-ketotestosterone (11-KT), which is the main androgen in fish, was generally slightly less potent than T in stimulating lepa1 and lepa2. The strongest stimulatory response was seen for 17 beta-estradiol (E2). E2 treatment significantly up-regulated lepa1 and lepa2 gene expression at doses of 10 nM and 1 nM for each gene, respectively. Lepa1, but not lepa2, was stimulated by T and 11-KT in immature male and immature female parr, while E2 stimulated expression of both genes. The sensitivity to sex steroid stimulation differed in maturing males compared to immature. In maturing males, the androgens and E2 stimulated lepa2 but not lepa1, while in immature males, the androgens and E2 stimulated lepa1, but only E2 stimulated lepa2. The differential response of the two leptin paralogues to the sex steroids suggests differences in regulation of the two leptin genes during maturation. Altogether, these results indicate that leptin expression in Atlantic salmon hepatocytes is directly regulated at the transcriptional level by the main teleost androgens and an estrogen, and that the response might depend on the developmental stage of the fish.