Knockout of Gnrh2 in zebrafish (Danio rerio) reveals its roles in regulating feeding behavior and oocyte quality.

Many studies on Gnrh1, and the teleost Gnrh3, have elucidated the roles of these peptides in reproductive regulation. However, the role of the midbrain population of Gnrh, Gnrh2, has long been a mystery, despite its ubiquitous conservation in all jawed vertebrates except rodents. Previous behavioral studies in sparrows, musk shrews, mice, zebrafish, and goldfish show that Gnrh2 administrations both increase spawning behaviors and decrease feeding behaviors, suggesting a role of this peptide in metabolism regulation along with the canonical role in regulating reproduction. In order to more deeply explore the roles of Gnrh2, we used a cyprinid teleost, zebrafish, which has 2 forms of Gnrh, Gnrh2 and Gnrh3, to generate a knockout zebrafish line which contains a frameshift mutation and subsequent disruption of the coding for the functional Gnrh2 peptide. We examined differences in reproduction, feeding, growth, and mobility in this line, and discovered major differences in feeding and growth parameters, suggesting that Gnrh2 is a potent anorexigen in zebrafish. Additionally, there were no differences in mobility except for increased distances swam during feeding periods. There were no major differences in reproductive success, however, female gnrh2-/- zebrafish exhibited smaller oocytes and increased embryo mortality, indicating slightly decreased oocyte quality. Additionally, there were changes in the expression levels of many feeding, growth, and reproductive neuropeptides in gnrh2-/- zebrafish. Taken together, these findings suggest a role for Gnrh2 in controlling satiation in zebrafish along with a minor role in maintaining optimal oocyte quality in females.

Probiotics administration following sleeve gastrectomy surgery: a randomized double-blind trial.

BACKGROUND: Probiotics are commonly used after bariatric surgery; however, uncertainty remains regarding their efficacy. Our aim was to compare the effect of probiotics vs placebo on hepatic, inflammatory and clinical outcomes following laparoscopic sleeve gastrectomy (LSG). METHODS: This randomized, double-blind, placebo-controlled, trial of 6-month treatment with probiotics (Bio-25; Supherb) vs placebo and 6 months of additional follow-up was conducted among 100 morbidly obese nonalcoholic fatty liver disease (NAFLD) patients who underwent LSG surgery. The primary outcome was a reduction in liver fat content, measured by abdominal ultrasound, and secondary outcomes were improvement of fibrosis, measured by shear-wave elastography, metabolic and inflammatory parameters, anthropometrics and quality of life (QOL). Fecal samples were collected and analyzed for microbial composition. RESULTS: One hundred patients (60% women, mean age of 41.9±9.8 years and body mass index of 42.3±4.7 kg m-2) were randomized, 80% attended the 6-month visit and 77% completed the 12-month follow-up. Fat content and NAFLD remission rate were similarly reduced in the probiotics and placebo groups at 6 months postsurgery (-0.9±0.5 vs -0.7±0.4 score; P=0.059 and 52.5 vs 40%; P=0.262, respectively) and at 12 months postsurgery. Fibrosis, liver-enzymes, C-reactive protein (CRP), leptin and cytokeratin-18 levels were significantly reduced and QOL significantly improved within groups (P⩽0.014 for all), but not between groups (P⩾0.173 for all) at 6 and 12 months postsurgery. Within-sample microbiota diversity (alpha-diversity) increased at 6-month postsurgery compared with baseline in both study arms (P⩽0.008) and decreased again at 12 months postsurgery compared with 6 months postsurgery (P⩽0.004) but did not reach baseline values. CONCLUSIONS: Probiotics administration does not improve hepatic, inflammatory and clinical outcomes 6- and 12 months post-LSG.

Inflammasomes and intestinal inflammation.

The inflammasome is a cytosolic multi-protein innate immune rheostat, sensing a variety of endogenous and environmental stimuli, and regulating homeostasis or damage control. In the gastrointestinal tract, inflammasomes orchestrate immune tolerance to microbial and potentially food-related signals or drive the initiation of inflammatory responses to invading pathogens. When inadequately regulated, intestinal inflammasome activation leads to a perpetuated inflammatory response leading to immune pathology and tissue damage. In this review, we present the main features of the predominant types of inflammasomes participating in intestinal homeostasis and inflammation. We then discuss current controversies and open questions related to their functions and implications in disease, highlighting how pathological inflammasome over-activation or impaired function impact gut homeostasis, the microbiome ecosystem, and the propensity to develop gut-associated diseases. Collectively, understanding of the molecular basis of intestinal inflammasome signaling may be translated into clinical manipulation of this fundamental pathway as a potential immune modulatory therapeutic intervention.

Crustacean hyperglycemic hormone (CHH) neuropeptidesfamily: Functions, titer, and binding to target tissues.

The removal of the eyestalk (s) induces molting and reproduction promoted the presence of regulatory substances in the eyestalk (ES), particularly medulla terminalis X-organ and the sinus gland (MTXO-SG). The PCR-based cloning strategies have allowed for isolating a great number of cDNAs sequences of crustacean hyperglycemic hormone (CHH) neuropeptides family from the eyestalk and non-eyestalk tissues, e.g., pericardial organs and fore- and hindguts. However, the translated corresponding neuropeptides in these tissues, their circulating concentrations, the mode of actions, and specific physiological functions have not been well described. The profiles of CHH neuropeptides present in the MTXO-SG may differ among decapod crustacean species, but they can be largely divided into two sub-groups on the basis of structural homology: (1) CHH and (2) molt-inhibiting hormone (MIH)/mandibular organ-inhibiting hormone (MOIH)/vitellogenesis/gonad-inhibiting hormone (V/GIH). CHH typically elevating the level of circulating glucose from animals under stressful conditions (hyper- and hypothermia, hypoxia, and low salinity) has multiple target tissues and functions such as ecdysteroidogenesis, osmoregulation, and vitellogenesis. Recently, MIH, known for exclusively suppressing ecdysteroidogenesis in Y-organs, is also reported to have an additional role in vitellogenesis of adult female crustacean species, suggesting that some CHH neuropeptides may acquire an extra regulatory role in reproduction at adult stage. This paper reviews the regulatory roles of CHH and MIH at the levels of specific functions, temporal and spatial expression, titers, their binding sites on the target tissues, and second messengers from two crab species: the blue crab, Callinectes sapidus, and the European green crab, Carcinus maenas. It further discusses the diverse regulatory roles of these neuropeptides and the functional plasticity of these neuropeptides in regard to life stage and species-specific physiology.

Functional studies of crustacean hyperglycemic hormones (CHHs) of the blue crab, Callinectes sapidus - the expression and release of CHH in eyestalk and pericardial organ in response to environmental stress.

The rapid increase in the number of putative cDNA sequences encoding crustacean hyperglycemic hormone (CHH) family in various tissues [either from the eyestalk (ES) or elsewhere] underscores a need to identify the corresponding neuropeptides in relevant tissues. Moreover, the presence of provided structural CHH implies the level of the complexity of physiological regulation in crustaceans. Much less is known of the functions of non-ES CHH than of those of its counterpart present in ESs. In the blue crab, Callinectes sapidus, we know little of CHH involvement in response to the stressful conditions that naturally occur in Chesapeake Bay. We have identified two isoforms of CHH neuropeptide in the sinus gland of the ES and isolated a full-length cDNA encoding CHH from the pericardial organ (PO). The functions of ES-CHH and PO-CHH in this species were studied with regard to expression and release in response to stressful episodes: hypoxia, emersion, and temperatures. Animals exposed to hypoxic conditions responded with concomitant release of both CHHs. In contrast, the mRNA transcripts encoding two CHHs were differentially regulated: PO-CHH increased, whereas ES-CHH decreased. This result suggests a possible differential regulation of transcription of these CHHs.

Identification and developmental expression of mRNAs encoding crustacean cardioactive peptide (CCAP) in decapod crustaceans.

Full-length cDNAs encoding crustacean cardioactive peptide (CCAP) were isolated from several decapod (brachyuran and astacuran) crustaceans: the blue crab Callinectes sapidus, green shore crab Carcinus maenas, European lobster Homarus gamarus and calico crayfish Orconectes immunis. The cDNAs encode open reading frames of 143 (brachyurans) and 139-140 (astacurans) amino acids. Apart from the predicted signal peptides (30-32 amino acids), the conceptually translated precursor codes for a single copy of CCAP and four other peptides that are extremely similar in terms of amino acid sequence within these species, but which clearly show divergence into brachyuran and astacuran groups. Expression patterns of CCAP mRNA and peptide were determined during embryonic development in Carcinus using quantitative RT-PCR and immunohistochemistry with whole-mount confocal microscopy, and showed that significant mRNA expression (at 50% embryonic development) preceded detectable levels of CCAP in the developing central nervous system (CNS; at 70% development). Subsequent CCAP gene expression dramatically increased during the late stages of embryogenesis (80-100%), coincident with developing immunopositive structures. In adult crabs, CCAP gene expression was detected exclusively in the eyestalk, brain and in particular the thoracic ganglia, in accord with the predominance of CCAP-containing cells in this tissue. Measurement of expression patterns of CCAP mRNA in Carcinus and Callinectes thoracic ganglia throughout the moult cycle revealed only modest changes, indicating that previously observed increases in CCAP peptide levels during premoult were not transcriptionally coupled. Severe hypoxic conditions resulted in rapid downregulation of CCAP transcription in the eyestalk, but not the thoracic ganglia in Callinectes, and thermal challenge did not change CCAP mRNA levels. These results offer the first tantalising glimpses of involvement of CCAP in environmental adaptation to extreme, yet biologically relevant stressors, and perhaps suggest that the CCAP-containing neurones in the eyestalk might be involved in adaptation to environmental stressors.

Apoptosis in sea bream Sparus aurata eggs.

In the present study, the sea-bream Sparus aurata, a pelagic egg spawner, was used as experimental model, in order to establish the occurrence of apoptosis in vertebrates with external reproduction. The same female ovulates floating and nonfloating eggs, but only the former, after fertilization, proceed to embryo development. The eggs were divided into floating and nonfloating and both were analyzed for the presence of several apoptosis markers. The results here reported provide evidence that the nonfloating cells present severe shrinkage and highly express both FAS receptor and FAS ligand on their surface. Furthermore, DNA fragmentation and mitochondria swelling were found, suggesting that the nonfloating eggs were cells programmed to die.

Gonadotropin response to GnRH during sexual ontogeny in the common carp, Cyprinus carpio.

This study was designed to reveal whether gonadotropic response to GnRH in the common carp (Cyprinus carpio) changes during sexual ontogeny and whether the response of FSHbeta and LHbeta subunits is uniform or differential. The study comprised fish at the following stages: juveniles (4-month-old females with primary oocytes and early spermatogenic males); maturing (9-month-old previtellogenic females and advanced spermatogenic males); and mature (16-month-old postvitellogenic females and spermiating males). Fish were injected with superactive salmon GnRH analogue (sGnRHa; 25 microg/kg) and blood was sampled 6, 12 and 24 h later for cGtH (LH) and sex steroid levels. Pituitaries were taken for determination of FSHbeta and LHbeta mRNA levels by slot-blot hybridization and for cGTH content in the same glands by radioimmunoassay (RIA). Values were compared with the levels prior to sGnRHa administration and with control fish sampled at the same intervals. Juvenile fish did not respond at all to sGnRHa. In maturing females, FSHbeta mRNA increased by >300%, while that of LHbeta increased by 200%. In maturing males, FSHbeta mRNA did not change and only a slight increase occurred in that of LHbeta. In 16-month-old postvitellogenic females, there was no response of FSHbeta mRNA, while that of LHbeta dramatically increased. In spermiating males of the same age, mRNA of both FSHbeta and LHbeta increased following sGnRHa injection. Immunoreactive cGtH was present in the pituitary and plasma of all fish examined, but in juveniles it did not change following sGnRHa injection. In maturing and mature fish of both genders, sGnRHa administration was followed by a marked increase in circulating cGtH, concomitant with a decrease in its pituitary content, indicating the limited amount of the hormone stored in the gland. In conclusion, the response of the gonadotropin subunit mRNAs in the common carp was found to be differential and dependent on the gender and the phase of sexual ontogeny.

The GnRH system in the European sea bass (Dicentrarchus labrax).

The cDNA sequences encoding three GnRH forms, sea bream GnRH (sbGnRH), salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), were cloned from the brain of European sea bass, Dicentrarchus labrax. Comparison of their deduced amino acid sequences to the same forms in the gilthead sea bream, Sparus aurata, and striped bass, Morone saxatilis, revealed high homology of the prepro-cGnRH II (94% and 98% respectively), and prepro-sGnRH (92% to both species). The sbGnRH exhibited dissimilar identities, with high homology to the striped bass (93%), and lower homology (59%) to the gilthead sea bream. Two transcript types were identified for the GnRH-associated peptide (GAP)-sGnRH as well as for the GAP-cGnRH II, which suggests a possible alternative splicing followed by the addition of an early stop codon. In order to obtain antibodies specific for the three GnRH precursors, recombinant GAP proteins were produced. The differential expression of the three GnRHs previously reported in the brain by means of in situ hybridization, using riboprobes corresponding to the GAP-coding regions, was fully confirmed by immunocytochemistry using antibodies raised against the recombinant GAP proteins, indicating that the transcripts are translated into functional proteins. Moreover, this approach allowed us to follow, for the first time, the specific projections of the different cell groups: sGAP fibers are distributed mainly in the forebrain with few projections reaching the pituitary, sbGAP fibers are mainly present in the preoptic area, mediobasal hypothalamus and predominantly project to the pars distalis of the pituitary, whereas cGnRH II fibers have a widespread distribution primarily in the posterior brain, and do not project to the pituitary. These new tools will be extremely useful to study further the development, regulation and functional significance of three independent GnRH systems in the brain of vertebrate species.

Differential expression of three different prepro-GnRH (gonadotrophin-releasing hormone) messengers in the brain of the european sea bass (Dicentrarchus labrax).

The expression sites of three prepro-gonadotrophin-releasing hormones (GnRHs), corresponding to seabream GnRH (sbGnRH: Ser(8)-mGnRH, mammalian GnRH), salmon GnRH (sGnRH: Trp(7)Leu(8)-mGnRH), and chicken GnRH-II (cGnRH-II: His(5)Trp(7)Tyr(8)-mGnRH) forms were studied in the brain of a perciform fish, the European sea bass (Dicentrarchus labrax) by means of in situ hybridization. The riboprobes used in this study correspond to the three GnRH-associated peptide (GAP)-coding regions of the prepro-GnRH cDNAs cloned from the same species (salmon GAP: sGAP; seabream GAP: sbGAP; chicken GAP-II: cIIGAP), which show little oligonucleotide sequence identity (sGAP versus sbGAP: 42%; cIIGAP versus sbGAP: 36%; sGAP versus cIIGAP: 41%). Adjacent paraffin sections (6 mm) throughout the entire brain were treated in parallel with each of the three anti-sense probes and the corresponding sense probes, demonstrating the high specificity of the hybridization signal. The results showed that both sGAP and sbGAP mRNAs had a broader expression in the olfactory bulbs, ventral telencephalon, and preoptic region, whereas cIIGAP mRNA expression was confined to large cells of the nucleus of the medial longitudinal fascicle. In the olfactory bulbs, both the signal intensity and the number of positive cells were higher with the sGAP probe, whereas sbGAP mRNA-expressing cells were more numerous and intensely stained in the preoptic region. Additional isolated sbGAP-positive cells were detected in the ventrolateral hypothalamus. These results demonstrate a clear overlapping of sGAP- and sbGAP-expressing cells in the forebrain of the European sea bass, in contrast to previous reports in other perciforms showing a clear segregation of these two cell populations.

Gonadotropins beta-GtHI and beta-GtHII from the gilthead seabream, Sparus aurata.

Full-length cDNA sequences encoding the beta-subunits of the gonadotrophins GtHI and GtHII were isolated, cloned, and sequenced from a single gilthead seabream (Sparus aurata) pituitary using RACE PCR. Beta-GtHI and beta-GtHII degenerate PCR primers were designed according to regions of high amino acid sequence homology between the chum salmon and the bonito beta-GtHI or beta-GtHII. DNA sequence analysis of the cloned PCR products confirmed the presence of the predicted complete coding region as well as 5' and 3' untranslated sequences. The deduced amino acid sequences of beta-GtHI and beta-GtHII from the gilthead seabream were compared to GtH sequences from a number of teleosts. Seabream beta-GtHI shows the highest homology to the bonito beta-GtHI (76%) and the striped bass (74%), while seabream beta-GtHII is 98% homologous to the yellow fin porgy, 87% homologous to the bonito, and 93% homologous to the striped bass beta-GtHII. Northern blot analysis showed the transcripts of the gilthead seabream beta-GtHI and beta-GtHII to be about 700 and 740 bases, respectively. During the spawning season, beta-GtHI is expressed at higher levels in males than in females while beta-GtHII is expressed at similar levels in both sexes.

Gonadotropin-releasing activities of the three native forms of gonadotropin-releasing hormone present in the brain of gilthead seabream, Sparus aurata.

Three forms of gonadotropin-releasing hormone (GnRH) have been recently identified in the brain of gilthead seabream (Sparus aurata): salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and a novel form, Ser8-mammalian GnRH, named seabream GnRH (sbGnRH). sbGnRH is the most abundant form in the pituitaries of sexually mature seabream during the spawning season. The present study investigated the gonadotropin-releasing activities of the three native forms of GnRH found in seabream brains, as well as of two structural analogs of sbGnRH. All native forms of GnRH stimulated gonadotropin-II (GtH-II) secretion in preovulatory female seabream. cGnRH-II was found to be 7 to 8 times more potent than sbGnRH and 2 times more potent than sGnRH in inducing GtH-II release. sGnRH was found to be 3.5 to 5 times more potent than sbGnRH in inducing GtH-II secretion. These data demonstrate that cGnRH-II, which is not present in pituitaries of sexually mature seabream, is the most potent GtH-II releaser, whereas sbGnRH, 500 times more abundant than sGnRH in the pituitary of maturing fish, is the least potent. The lower potency of sbGnRH may suggest faster enzymatic breakdown, more rapid clearance from the circulation, or a lower binding affinity to the pituitary GnRH receptor. The lower bioactivity of sbGnRH may be compensated for by its high levels in the pituitary. The two analogs of sbGnRH, [D-Nal(2)6,Pro9-NEt]-sbGnRH and [D-Arg6,Pro9-NEt]-sbGnRH, were equipotent to each other and 5 times more potent than sbGnRH in inducing GtH-II release in preovulatory seabream. However, they were 5 to 6 times less active than the analog of mammalian GnRH, [D-Ala6,Pro9-NEt]-mGnRH. Strategies for designing superactive analogs of sbGnRH are discussed.

In vitro bioactivities of various forms of GnRH in relation to their susceptibility to degradation at the pituitary level in the rainbow trout, Oncorhynchus mykiss.

In vitro potencies of native and modified forms of salmon and mammalian gonadotropin-releasing hormone (GnRH) were studied in relation with their susceptibility to degradation by intact pituitary cells maintained in culture. The kinetics of degradation and the origin of the proteases involved in this process were examined. All the molecules tested (native and modified forms) were equipotent at doses between 10(-6) and 10(-7) M in inducing GtH release by cultured pituitary cells. On the other hand, their effectiveness differed at 10(-9) and 10(-8) M leading to the establishment of the following hierarchy of bioactivity: the native forms, LHRH and sGnRH, were the less potent, the fish analogues (DAla6Pro9Net)sGnRH and (DArg6Pro9Net)sGnRH were the more potent, and mammalian analogues with substitutions at position 6 and/or 10 were intermediate in potency. The native form sGnRH was weakly degraded while no degradation of the modified molecules was observed. The degradation of the native sGnRH occurred after 12 and 24 hr of incubation and the results indicate that the peptidases involved are released from the cells into the incubation medium.