Appetite regulating genes in zebrafish gut; a gene expression study.

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.

Impaired leptin signaling causes subfertility in female 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, which are known to be regulated by LH signaling. Moreover, subfertility in lepr mutants could be partially restored by administration of human chorionic gonadotropin. 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.

Transcriptional study reveals a potential leptin-dependent gene regulatory network in zebrafish brain.

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.

Gene co-expression network analysis reveals mechanisms underlying ozone-induced carbamazepine toxicity in zebrafish (Danio rerio) embryos.

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).

Transcriptional study of appetite regulating genes in the brain of zebrafish (Danio rerio) with impaired leptin signalling.

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.

Hormonal changes over the spawning cycle in the female three-spined stickleback, Gasterosteus aculeatus.

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 72h post-spawning (hps) with a male. Following spawning, almost all females (15 out of 19) had ovulated eggs again at Day 3 post-spawning (72hps). 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-ß) and luteinizing hormone (lh-ß) 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 72hps. Plasma T levels showed peaks at 24 and 48hps and decreased at 72hps, while E2 levels increased already at 6hps and remained at high levels up to 48hps. There was a strong positive correlation between T and E2 levels over the spawning cycle. Pituitary lh-ß mRNA levels showed a peak at 48hps, while fsh-ß 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.

Developmental exposure to progestins causes male bias and precocious puberty in zebrafish (Danio rerio).

Progestins are aquatic contaminants that in low concentrations can impair fish reproduction. The mechanisms are likely multiple since different progestins interact with other steroid receptors in addition to progesterone receptors. Puberty is the process when animals first acquire the capability to reproduce and it comprises maturation of sperm and eggs. In zebrafish, puberty is initiated around 45days post fertilization (dpf) in females and around 53-55 dpf in males, and is marked by increased production of pituitary gonadotropins. We exposed juvenile zebrafish from 20 to 80 dpf to the androgenic progestin levonorgestrel at concentrations of 5.5, 79 and 834ngL(-1) and to the non-androgenic progestin progesterone at concentrations of 3.7, 77 and 1122ngL(-1), during sexual differentiation and puberty. Levonorgestrel exposure caused 100% males even at the lowest concentration tested whereas progesterone did not affect the sex ratio. Transcript levels of the gonadal genes amh, CYP11B and CYP19a1a indicated that the masculinizing effect of levonorgestrel occurred very rapidly. Transcript concentrations of gonadotropins in pituitaries were low in control fish at 44 dpf, but high at 55 dpf and onward. In fish exposed to levonorgestrel or progesterone gonadotropin transcript concentrations were high already at 44 dpf, indicating that both progestins caused precocious puberty. Gonad histology at 50 dpf confirmed a well advanced sexual maturation, but only in males. Our results show that progestins can affect sexual development in fish and that the androgenic progestin levonorgestrel induces a male phenotype at concentrations similar to those detected in aquatic environments.

Sex steroids stimulate leptin gene expression in Atlantic salmon parr hepatocytes in vitro.

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 17ß-estradiol (E2). E2 treatment significantly up-regulated lepa1 and lepa2 gene expression at doses of 10nM and 1nM 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.

Regulation of the seasonal leptin and leptin receptor expression profile during early sexual maturation and feed restriction in male Atlantic salmon, Salmo salar L., parr.

In mammals, leptin acts as an adiposity signal and is a crucial link between nutritional status and the reproductive axis. So far the link between leptin and energy balance during sexual maturation in teleosts has been poorly investigated. In this study, seasonal gene expression changes in two leptin genes (lepa1 and lepa2) and the leptin receptor were investigated during early sexual maturation in male Atlantic salmon parr under fully fed (control) and feed restricted conditions from April through September. Both Atlantic salmon lepa1 and lepa2 in the liver and lepr in the brain were significantly down-regulated in non-maturing control males in early spring, coinciding with the start of the growth and fat accumulation. In maturing control males, hepatic leptin expression increased during mid-spermatogenesis and lepa1 and lepa2 mRNA levels were up-regulated by 7.7 and 49 times respectively during final maturation. For the first time in a fish species, a significant up-regulation of lepr expression was observed in the testis throughout mid to late spermatogenesis. Feed restriction decreased the incidence of sexual maturation by 53% and highly up-regulated both leptin genes in the liver and the leptin receptor in the pituitary. This study shows that hepatic lepa1 and lepa2 expression and lepr expression in the testis is affected by early sexual maturation in male Atlantic salmon parr. Fast growth and high fat stores are associated with low leptin levels while feed restriction has a stimulatory effect on hepatic leptin and leptin receptor gene expression in the pituitary, suggesting a role for leptin other than that as an adiposity signal.

The mating brain: early maturing sneaker males maintain investment into the brain also under fast body growth in Atlantic salmon (Salmo salar).

It has been suggested that mating behaviours require high levels of cognitive ability. However, since investment into mating and the brain both are costly features, their relationship is likely characterized by energetic trade-offs. Empirical data on the subject remains equivocal. We investigated if early sexual maturation was associated with brain development in Atlantic salmon (Salmo salar), in which males can either stay in the river and sexually mature at a small size (sneaker males) or migrate to the sea and delay sexual maturation until they have grown much larger (anadromous males). Specifically, we tested how sexual maturation may induce plastic changes in brain development by rearing juveniles on either natural or ad libitum feeding levels. After their first season we compared brain size and brain region volumes across both types of male mating tactics and females. Body growth increased greatly across both male mating tactics and females during ad libitum feeding as compared to natural feeding levels. However, despite similar relative increases in body size, early maturing sneaker males maintained larger relative brain size during ad libitum feeding levels as compared to anadromous males and females. We also detected several differences in the relative size of separate brain regions across feeding treatments, sexes and mating strategies. For instance, the relative size of the cognitive centre of the brain, the telencephalon, was largest in sneaker males. Our data support that a large relative brain size is maintained in individuals that start reproduction early also during fast body growth. We propose that the cognitive demands during complex mating behaviours maintain a high level of investment into brain development in reproducing individuals.

Androgen feedback effects on LH and FSH, and photoperiodic control of reproduction in male three-spined sticklebacks, Gasterosteus aculeatus.

Sexual maturation in the stickleback is controlled by photoperiod. The aim of this study was to find out whether changes in feedback effects exerted by sex steroids could mediate the photoperiodic effect, which is regarded to be of an all-or-nothing character. To that end, males were castrated and treated with different doses of testosterone (T) and in one experiment also with the aromatase inhibitor fadrozole (AI) and kept under different photoperiods. In control fish, long day (LD 16:8) stimulated maturation, associated with more hypertrophied kidneys (a secondary sexual character) and higher levels of pituitary lhb and fshb mRNA than under short day conditions (LD 8:16). Under LD 8:16, low doses of T suppressed both lhb and fshb mRNA levels. However, with the use of high doses of T and/or longer photoperiods the inhibitory effects on lhb and fshb mRNA levels became less clear or instead positive effects were observed. Under intermediate photoperiod conditions, the negative feedback effect of a low dose of T on fshb was more prominent with shorter photoperiods, whereas no such shift was observed for lhb mRNA. The inhibitory effect of the low dose of T on lhb mRNA levels under LD 8:16 was abolished by AI, whereas the stimulatory effect of the high dose of T was not. The negative feedback effects were more marked under short days than under long days, whereas positive feedback effects were more marked under long days. The suppression of both fshb and lhb mRNA levels by low androgen levels, especially under short days, may inhibit maturation completely unless a rise of androgens above threshold levels would allow complete maturation.

Schistocephalus solidus infections increase gonadotropins and gonadotropin releasing hormone (GnRH3) mRNA levels in the three-spined stickleback, Gasterosteus aculeatus.

Parasites often impair the reproduction of their hosts, one well known case being the cestode Schistocephalus solidus which is a common parasite in three-spined sticklebacks, Gasterosteus aculeatus. One of the possible ways that this could be exerted is by suppression on the brain-pituitary-gonadal (BPG) axis. In this study, mRNA levels of FSH-ß and LH-ß and of GnRH2 (cGnRH II) and GnRH3 (sGnRH) were measured via Q-PCR in infected and uninfected fish sampled from the field a few weeks before the onset of breeding. The pituitary mRNA levels of both FSH-ß and LH-ß were higher in infected males than in uninfected males. Also in females, FSH-ß mRNA levels were higher in infected individuals than in others, whereas there was no significant difference found in LH-ß expression. Brain mRNA levels of GnRH3 were higher in infected fish than in uninfected fish in both sexes, but no difference was found in GnRH2 mRNA levels. Thus, infection by S. solidus was able to alter the expressions not only of gonadotropins (GtHs), but also of GnRH which has not been observed previously. However, the effects are opposite to what should be expected if the parasite suppressed reproduction via actions on the brain-pituitary level. The gonads are perhaps more likely to be impaired by the parasites in other ways, and changed feedbacks on the BPG axis could then lead to the increases in GtHs and GnRH.

Effects of long-term restricted feeding on plasma leptin, hepatic leptin expression and leptin receptor expression in juvenile Atlantic salmon (Salmo salar L.).

Leptin is a pleiotropic hormone and plays a key role in body weight regulation, energy homeostasis and lipid store utilization in mammals. In this study, we investigated the effect of feed-restriction on leptin genes (lepa1 and lepa2), leptin receptor (lepr) gene expression and plasma leptin levels in juvenile Atlantic salmon parr. Feed restriction was performed from late April to mid-June, in order to gain insight into the role of the leptin system in energy balance regulation and adiposity in juvenile salmon. A significant increase in lepa1 expression as well as higher levels of plasma leptin was found in feed-restricted fish in June compared to fully fed controls, while lepa2 gene expression decreased in both groups during the treatment period. Lepa2 was, however significantly higher in the feed-restricted group in June. Leptin receptor expression was up regulated during the period of enhanced growth and lipid deposition in the fully fed control, indicating a seasonal effect on the receptor expression in the brain. Both lepa1 and lepa2 genes very mainly expressed in the liver in juvenile salmon, while lepr was expressed in the brain but showed also considerable expression in various peripheral tissues. The study provides evidence that the leptin system is sensitive to the metabolic status of the fish as both season and restricted feeding affect lepa1 and lepa2 gene expression in the liver and brain leptin receptor expression, however, for lepa1 expression and leptin plasma level in an opposite way as that observed in the mammalian system.

Gene expression profiling during spermatogenesis in early maturing male Atlantic salmon parr testes.

The initiation of sexual maturation and spermatogenesis are complex processes that require the highly coordinated regulation of a number of key genes. The endocrine system plays crucial roles in these processes, but the precise mechanisms involved in sexual maturation of fish are poorly understood. We investigated the expression of genes encoding proteins involved in sex steroid biosynthesis (Ff1b (FTZ-F1 homolog), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17), cytochrome P450 11beta-hydroxylase (P45011beta) and 11beta-hydroxysteroid dehydrogenase (11beta-HSD)) and the anti-Müllerian hormone (AMH) homolog during early sexual maturation of one-summer-old male Atlantic salmon parr by RT-PCR. Genes encoding Ff1b, StAR, 3beta-HSD, P450c17 and 11beta-HSD were upregulated during spermatogonial proliferation. During the course of spermatogenesis expression profiles of Ff1b, StAR, 3beta-HSD, P450scc, P450c17, P45011beta, and 11beta-HSD were similar; transcript levels being low during early stages, then strongly increasing during spermiogenesis. These results indicate that coordinated de novo transcription of genes encoding StAR as well as 3beta-HSD, P450c17 and 11beta-HSD might be required for sex steroids production during the initiation of spermatogenesis in salmon. In contrast, transcription levels of AMH were comparatively high in immature testes, decreased when spermatogenesis was initiated, and were lowest during spermiogenesis, suggesting that AMH suppression plays a crucial role in the process of spermatogenesis in salmonids. Correlation analyses show that FSH and LH might be differentially involved in the regulation of several of these genes studied.

Effects of castration and androgen-treatment on the expression of FSH-beta and LH-beta in the three-spine stickleback, gasterosteus aculeatus--feedback differences mediating the photoperiodic maturation response?

In many animals, including the three-spine stickleback (Gasterosteus aculeatus), photoperiod strongly influences reproduction. The aim of this study was to investigate if feedback mechanisms on the brain-pituitary-gonadal axis play a role in mediating the photoperiodic response in the stickleback. To that end, stickleback males, exposed to either non-stimulatory short photoperiod (light/dark 8:16) or under stimulatory long photoperiod (LD 16:8), were subjected to either sham-operation, castration, castration combined with treatment with the androgens 11-ketoandrostenedione (11KA) and testosterone (T), and the effects on levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH)-beta mRNA were analyzed. During breeding season the kidney of the stickleback male hypertrophies and produces a glue used for building nests. Kidney weight and expression of both LH-beta and FSH-beta were higher in sham-operated fish kept under long than under short photoperiod. Under both photoperiods, LH-beta mRNA levels were lower in castrated males compared to sham-operated males and treatment with 11KA and T increased expression, indicating a positive feedback. A positive feedback was also found on FSH-beta expression under long photoperiod, where castration decreased, and androgen replacement restored FSH-beta mRNA expression. On the contrary, castration under short photoperiod instead increased FSH-beta levels whereas treatment with 11KA and T decreased FSH-beta expression, indicating a negative feedback on FSH-beta under these conditions. The positive feedback on FSH-beta expression under stimulatory photoperiod may accelerate maturation, whereas the negative feedback under inhibitory photoperiod may suppress maturation. This could be part of the mechanisms by which photoperiod controls maturation.

Expression of gonadotropin and gonadotropin receptor genes during early sexual maturation in male Atlantic salmon parr.

Atlantic salmon males may mature already as small parr in freshwater. Sexual maturation in teleosts as in vertebrates is characterized by the activation of the brain-pituitary-gonad axis. The endocrine regulation of early puberty is still not well understood. In the present study, one-summer-old male Atlantic salmon parr were sampled regularly from December several months prior to the beginning of spermatogenesis until spawning in October. Pituitary expression levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) beta subunit genes were analyzed in parallel with testis expression of FSH receptor (FSHR) and LH receptor (LHR) genes by RT-PCR and plasma 11-ketostestosterone (11-KT) was measured. Expression levels of FSHbeta, low during winter and spring started to increase prior to the onset of gonadal growth at the end of May while LHbeta mRNA levels were hardly detectable. Both gonadotropin receptor genes were expressed in immature testis with FSHR transcripts being more abundant (8-fold). FSHR transcript levels increased in parallel to FSHbeta levels from early spermatogenesis onwards, while LHR mRNA started to increase prior to any large changes in LHbeta expression. Both transcript levels of LHbeta and LHR were highest during spermiation. Plasma 11-KT increased at the beginning of spermatogenesis reaching highest levels at spermiogenesis suggesting a possible role of FSH in inducing 11-KT production during early spermatogenesis while LH stimulates via its specific receptor 11-KT production at spermiogenesis. The commitment into sexual maturation appears to be dependant on both the presence of FSHR in immature testis and the increase of FSH expression.

Comparable long-term survival after bone marrow versus peripheral blood progenitor cell transplantation from matched unrelated donors in children with hematologic malignancies.

Despite the increasing use of peripheral blood progenitor cells (PBPC) instead of bone marrow (BM) for allogeneic hematopoietic stem cell transplantation (allo HSCT) from human leukocyte antigen (HLA)-matched unrelated donors in children, the relative benefits and risks of both stem cell sources in the pediatric setting remain largely unknown. Recently, the only larger study comparing the value of the 2 stem cell sources in a young patient group was confined to transplantation from HLA-identical sibling donors in older children and adolescents with acute leukemia. Based on the paucity of data in pediatric HLA-matched unrelated donor transplantation, we analyzed the outcome of 23 BM and 38 PBPC transplantations performed at our center. Neutrophil and platelet engraftment were achieved significantly faster in PBPC compared to BM recipients (18 versus 22 days and 26 versus 33 days; P < .001 and P = .03) whereas the risk for grade II-IV acute graft-versus-host disease (aGVHD) (62% versus 55%; P = .53) and chronic GVHD (cGVHD 65% versus 59%; P = .54) was comparable. As overall survival (OS; PBPC versus BM: 47.5% +/- 8.6% versus 51.8% +/- 10.5%; P = .88) and relapse-free survival (43.3% +/- 8.3% versus 51.8% +/- 10.5%; P = .60) are without detectable difference, PBPC and BM appear both as a valid stem cell source for HLA-matched unrelated donor transplantation in children with hematologic malignancies.

Molecular cloning and characterization of FSH and LH receptors in Atlantic salmon (Salmo salar L.).

Two cDNAs encoding the FSH receptor (FSHR) and the LH receptor (LHR) from Atlantic salmon (Salmo salar) were cloned and characterized. The predicted protein sequence for FSHR comprises a mature protein of 635 amino acids (aa) and a signal peptide of 23aa, and for LHR a mature protein of 701aa and a signal peptide of 27aa. Multiple sequence alignment of Atlantic salmon FSHR and LHR with gonadotropin receptor sequences of available teleosts and representative vertebrates revealed high sequence homology with other salmonids (97-98% for both receptors); amino acid identities ranged from 59 to 67% for FSHR and 47-79% for LHR compared with other teleosts, and between 50 and 52% compared with other vertebrates. The salmon FSHR and LHR showed the typical characteristics of glycoprotein receptors, including a long N-terminal extracellular domain (ECD), seven transmembrane domains and a short C-terminal intracellular domain. The ECD of the Atlantic salmon FSHR and LHR were composed of nine imperfect leucine-rich repeats forming the potential recognition sites for the corresponding hormone. The comparative analysis of the recognition sites in the Atlantic salmon gonadotropin receptors with the corresponding sites in the human receptors showed that the nature of the residues involved in the key contacts with the glycoprotein alpha-subunit were highly conserved. In contrast the recognition sites for the specific beta-subunits showed clear differences between the two salmon gonadotropin receptors and the human receptors. In the salmon LHR the recognition sites for the LH beta-subunit were relatively conserved, while the recognition sites for the FSH beta-subunit in the salmon FSHR showed a higher divergence, suggesting different evolution rates for the two teleost gonadotropin receptors. Both FSHR and LHR were mainly expressed in the ovary and testis, but were also detected at low abundance in extra-gonadal tissues such as gills, brain, liver and heart.

Seasonal changes in expression of LH-beta and FSH-beta in male and female three-spined stickleback, Gasterosteus aculeatus.

In teleost fishes, like in other vertebrates, the gonadal development is stimulated by two gonadotropic hormones; luteinizing hormone (LH) and follicle-stimulating hormone (FSH). To achieve a better understanding of the role of gonadotropins in teleost reproduction; expression of LH-beta and FSH-beta mRNA and the status of gonads and secondary sexual characters were analyzed over the annual cycle in male and female three-spined sticklebacks, a species in which the development of male secondary sexual characters and spermatogenesis are separated in time. The kidney in the male stickleback hypertrophies during the breeding season and produces a glue used when building nests. Kidney weights, as well as levels of 11-ketotestosterone (11KT), reached a peak in May. Both testosterone (T) levels and the gonadosomatic index (GSI, gonad weight/body weight x 100) in females started to increase in April, and peaked in May as well. Later in summer, after the breeding season, these features declined. In females, LH-beta expression followed the GSI and T levels closely, levels were low during winter and early spring, increased to a peak in late May and declined to low levels again in July. FSH-beta expression peaked earlier, in January and declined slowly over spring. In males, LH-beta expression peaked in May. During June-September, when spermatogenesis was active, LH-beta levels were very low. FSH-beta expression peaked in January, earlier than LH-beta expression did, and reached the lowest levels in July. Thus, when spermatogenesis started at the end of summer, the expression of both GTH-beta mRNAs, and circulating 11KT, displayed their lowest levels.

Differential regulation of luteinizing hormone and follicle-stimulating hormone expression during ovarian development and under sexual steroid feedback in the European eel.

Pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are, in teleosts as in mammals, under the control of hypothalamic factors and steroid feedbacks. In teleosts, feedback regulations largely vary depending on species and physiological stage. In the present study the regulation of FSH and LH expression was investigated in the European eel, a fish of biological and phylogenetical interest as a representative of an early group of teleosts. The eel FSHbeta subunit was cloned, sequenced and together with earlier isolated eel LHbeta and glycoprotein hormone alpha (GPalpha) subunits used to study the differential regulation of LH and FSH. In situ hybridization indicated that FSHbeta and LHbeta are expressed by separate cells of the proximal pars distalis of the adenohypophysis, differently from the situation in mammals. The profiles of LHbeta and FSHbeta subunit expression were compared during experimental ovarian maturation, using dot-blot assays. Expression levels for LHbeta and GPalpha increased throughout ovarian development with a positive correlation between these two subunits. Conversely, FSHbeta mRNA levels decreased. To understand the role of sex steroids in these opposite variations, immature eels were treated with estradiol (E2)and testosterone (T), both steroids being produced in eel ovaries during gonadal development. E2 treatment induced increases in both LHbeta and GPalpha mRNA levels, without any significant effect on FSHbeta. In contrast, T treatment induced a decrease in FSHbeta mRNA levels, without any significant effect on the other subunits. These data demonstrate that steroids exert a differential feedback on eel gonadotropin expression, with an E2-specific positive feedback on LH and a T-specific negative feedback on FSH, leading to an opposite regulation of LH and FSH during ovarian development.