Gonadotropin-inhibitory hormone and its receptors in teleosts: Physiological roles and mechanisms of actions

Gonadotropin-inhibitory hormone (GnIH) was the first reported hypothalamic neuropeptide inhibiting reproduction in vertebrates. Since its discovery in the quail brain, its orthologs have been identified in a variety of vertebrate species and even protochordates. Depending on the species, the GnIH precursor polypeptides comprise two, three or four mature peptides of the RFamide family. It has been well documented that GnIH inhibits reproduction at the brain-pituitary–gonadal levels and participates in metabolism, stress response, and social behaviors in birds and mammals. However, most studies in fish have mainly been focused on the physiological roles of GnIH in the control of reproduction and results obtained are in some cases conflicting, leaving aside its potential roles in the regulation of other functions. In this manuscript we summarize the information available in fish with respect to the structural diversity of GnIH peptides and functional roles of GnIH in reproduction and other physiological processes. We also highlight the molecular mechanisms of GnIH actions on target cells and possible interactions with other neuroendocrine factors

Spexin in the European sea bass, Dicentrarchus labrax: Characterization, brain distribution, and interaction with Gnrh and Gnih neurons

Spexin (Spx) is a recently characterized neuropeptide implicated in multiple physiological processes in vertebrates, including reproduction, food intake, and regulation of anxiety and stress. Two orthologs (Spx1 and Spx2) are present in some nonmammalian vertebrates, including teleosts. However, information on the distribution of Spx in the brain and its interactions with other neuroendocrine systems in fish is still scarce. In this work, we cloned and sequenced the sea bass (Dicentrarchus labrax) Spx1, which included a 27 aa signal peptide and a mature peptide of 14 aa that is C-terminal amidated. spx1 transcripts were higher in the diencephalon/caudal preoptic area/hypothalamus and medulla but were also detected in the olfactory bulbs, telencephalon/rostral preoptic area, optic tectum/tegmentum, cerebellum/pons, and pituitary. The immunohistochemical study revealed Spx1-immunoreactive (ir) cells in different nuclei of the preoptic area, habenula, prethalamus, mesencephalic tegmentum and in the proximal pars distalis (PPD) and pars intermedia of the pituitary. Spx1-ir fibers were widely distributed throughout the brain being particularly abundant in the midbrain and hindbrain, in close contact with tegmental gonadotropin-releasing hormone 2 (Gnrh2) cells and isthmic gonadotropin-inhibitory hormone (Gnih) cells of the secondary gustatory nucleus. Moreover, Gnih fibers were observed innervating Spx1-ir cells lying in several subdivisions of the magnocellular preoptic nucleus and in the lateral nucleus of the valvula, whereas ventrolateral prethalamic Spx1-ir cells received immunopositive Gnrh2 fibers. In the pituitary, Gnrh1-ir fibers were observed closely associated with Spx1-ir cells of the PPD. These results suggest that Spx1 could be involved in both reproductive and nonreproductive (i.e., food intake, behavior) functions in sea bass

Differential activation of neuropeptide FF receptors by gonadotropin-inhibitory hormone peptides in the European sea bass

Neuropeptide FF (NPFF) and gonadotropin-inhibitory hormone (GnIH) are thought to be paralogous, and a recent study has revealed that both NPFF and GnIH peptides can activate the GnIH receptor (GnIHR, also called NPFFR1) in the European sea bass (Dicentrarchus labrax). However, whether GnIH can bind to the NPFF receptor (NPFFR2) is still yet unknown in this species. Accordingly, we further investigated the potential interactions between GnIH and NPFFR2 (two NPFFR2 forms present in sea bass, namely NPFFR2-1 and NPFFR2-2) on the intracellular signaling pathways. Neither GnIH1 nor GnIH2 had any effect on basal CRE-luc activity, while forskolin-stimulated CRE-luc activity was significantly reduced when COS-7 cells expressing sea bass NPFFR2-1 and NPFFR2-2 were challenged with these two GnIH peptides. NPFF and NPAF also inhibited forskolin-induced CRE-luc activity via their cognate receptors. An evident stimulation of SRE-luc activity was observed when COS-7 cells transfected with NPFFR2-1 and NPFFR2-2 were treated with NPFF and NPAF, whereas GnIH peptides had no effect, except a slight but significant increase elicited by 1000 nM of GnIH1 in COS-7 cells expressing NPFFR2-2. Moreover, only GnIH2 exerted an inhibitory action on NFAT-RE-luc activity in COS-7 cells expressing NPFFR2-1. None of GnIH or NPFF peptides altered ERK phosphorylation levels via NPFFR2 receptors. Our results provide new evidence that sea bass GnIH peptides may exert their functions partially via NPFFR2, and PKA, PKC and Ca2+ routes are potential mediators

An autofluorescence-based survey of late follicular atresia in ovaries of a teleost fish (Thunnus thynnus)

In this study, the authors examined late atretic follicles in the ovaries of Atlantic bluefin tuna, Thunnus thynnus (Linn AE us 1758), at different times of the year using transmitted light and epifluorescence microscopy. Atresia (degeneration and resorption of developing ovarian follicles) is a natural process involved in fecundity downregulation in teleosts and is substantially enhanced in stressful conditions. Early (alpha and beta) atretic stages of yolked oocytes have a relatively short duration in seasonally reproducing species, whereas later (gamma and delta) atretic follicles (LAF) persist for longer time in the ovary, serving as a sign of previous vitellogenic activity. LAF can thus be used as reliable markers of maturity during non-reproductive periods. Lipofuscin granules accumulate in the cytoplasm of LAF cells as a result of lysosomal digestion of oocyte components. Taking advantage of the well-known autofluorescent properties of lipofuscins, LAF may be identified in unstained histological sections under fluorescence microscopy using appropriate excitation and emission wavelengths. The authors explore in this study the applicability of fluorescence microscopy to provide a fast and effective method to assess late atresia in fishes. This method may be particularly useful to determine sexual maturity in individuals sampled long after the spawning season, where LAF are difficult to detect in standard histological sections. Furthermore, LAF autofluorescence provides a rapid way to quantify late atresia in fishes using image analysis.This research was partly funded by the CEI MAR Foundation (project #CEIJ19-C02.1), the Spanish Ministry of Economy and Competitiveness (project #CTM2017-82808-R) and the Andalusian Government (P12-RNM733, FEDER UCA18-107069)

From Embryo to Adult Life: Differential Expression of Visual Opsins in the Flatfish Solea senegalensis Under Different Light Spectra and Photoperiods

Visual photoreceptors in fish are usually adjusted to the light environment to ensure the highest efficiency and best adaptation. In the Senegalese sole, metamorphosis determines migration from pelagic to benthic habitats, with marked differences in both light intensity and spectrum. Here, we analysed the ontogeny of six visual photopigments, namely, rod opsin (rh1), short wavelength-sensitive (sws1 and sws2), medium wavelength-sensitive (rh2.3 and rh2.4), and long wavelength-sensitive (lws) cone opsins, in sole specimens maintained in light-dark cycles of white (LDW), blue (LDB), red (LDR), and continuous white (LL) lights by using RT-qPCR and in situ hybridisation. Most of the opsins displayed a similar developmental expression pattern under all tested conditions. However, lower transcripts were detected under LDR and LL compared to LDW and LDB. A significant increase in gene expression was detected before and after metamorphosis, reaching minimum transcript levels at hatching and during metamorphosis. Interestingly, green opsins (rh2.3 and rh2.4) displayed a significant increase only before metamorphosis, with their expression remaining low during and after metamorphosis. The rod opsin and short-, medium-, and long-wavelength sensitive cone opsins were detected in retinal photoreceptors of the sole from pre-metamorphic to adult stages by in situ hybridisation. In adults, the short-wavelength cone opsins (sws1 and sws2) were found in single cones, whereas the medium- (rh2.4) and long-wavelength (lws) cone opsins were present in double cones. The results obtained by in situ hybridisation in the retina of developing sole, in terms of number of positive cells and/or intensity of labelling, were consistent with the ontogenetic transcript patterns found by RT-qPCR, suggesting that most of the visual opsin expressions detected in the whole specimens could correspond to retinal expression. Taken together, our results pointed out that the ontogeny of the Senegalese sole is accompanied by remodelling in opsin gene expression, with the green-cone opsins being the most abundant photopigments in pre-metamorphosis and rod opsin the dominant visual photopigment from the completion of metamorphosis onwards. These results enlarge our knowledge of flatfish metamorphosis and ecology and provide useful information to develop light protocols adapted to different ontogenetic stages that could improve welfare and production in sole aquaculture

Daily rhythms of acute stress responses and antioxidant systems in the European sea bass (Dicentrarchus labrax): Effects of the time of the year

Fish reared in aquaculture face various acute stressors, including air exposure during handling. Research on the stress response in fish can provide essential insights into their physiology and help define better aquaculture practices. In this study, we investigated the daily rhythms in the stress-axis response of the European sea bass (Dicentrarchus labrax) subjected to an acute stressor consisting of air exposure (1 min), and how this response is influenced by the time of the day and the season of the year. In addition, rhythms in antioxidant systems were also assessed. The experiments were performed in late Autumn (December) and late Spring (June), with natural photoperiod (10 L:14D and 15 L:9D, respectively) and water temperature (ranging from 19.47 ± 0.17 December to 22.13 ± 0.13 ◦ ◦ C in C in June). Samples were collected throughout a 24-h cycle at Zeitgeber time (ZT) 0.5, 4, 7.5, 12, 16, 20, and 24.5 h at both seasons. At each sampling point, an untreated control (CTRL) group was sampled, while a STRESS group was exposed to air for one minute, returned to the tank, and sampled one hour later. Fish were sacrificed to collect plasma samples, hypothalamus and liver. Plasma samples were analyzed for cortisol, glucose, and lactate. In the hypothalamus, the mRNA expression levels of corticotropin-releasing hormone (crh) and crh-binding protein (crh-bp) were quantified using quantitative RT-PCR (qPCR). In the liver, genes related to antioxidant systems (catalase, superoxide dismutase 1, glutathione peroxidase,and glutathione reductase) and mitochondrial markers of stress (uncoupling protein 1, cytochrome c oxidase IV and peroxiredoxin3) were also analyzed by qPCR. The results revealed that most stress indicators (cortisol, cat, sod1, gsh-px, gsr, ucp1, coxIV) displayed daily rhythms. Furthermore, the stress response was significantly influenced by the time of day and the season in which the stressor was applied. In June, cortisol and glucose responses to stress were higher during the day than at night. The increase observed after stress in genes related to the antioxidant system was more significant in June than in December. Conversely, the response of mitochondrial markers was greater in December. Taken together, these findings highlight that the stress response of the European sea bass is time- dependent, both on a daily and a seasonal basis. This emphasizes the importance of considering cyclic environmental factors and circadian rhythms in aquaculture procedures to enhance fish welfare

Acute Stress in Lesser-Spotted Catshark (Scyliorhinus canicula Linnaeus, 1758) Promotes Amino Acid Catabolism and Osmoregulatory Imbalances

Simple Summary In catsharks (Scyliorhinus canicula), air exposure induces amino acid catabolism altogether with osmoregulatory imbalances. This study describes a novel NHE isoform being expressed in gills that may be involved in ammonium excretion. Acute-stress situations in vertebrates induce a series of physiological responses to cope with the event. While common secondary stress responses include increased catabolism and osmoregulatory imbalances, specific processes depend on the taxa. In this sense, these processes are still largely unknown in ancient vertebrates such as marine elasmobranchs. Thus, we challenged the lesser spotted catshark (Scyliorhinus canicula) to 18 min of air exposure, and monitored their recovery after 0, 5, and 24 h. This study describes amino acid turnover in the liver, white muscle, gills, and rectal gland, and plasma parameters related to energy metabolism and osmoregulatory imbalances. Catsharks rely on white muscle amino acid catabolism to face the energy demand imposed by the stressor, producing NH4+. While some plasma ions (K+, Cl- and Ca2+) increased in concentration after 18 min of air exposure, returning to basal values after 5 h of recovery, Na+ increased after just 5 h of recovery, coinciding with a decrease in plasma NH4+. These changes were accompanied by increased activity of a branchial amiloride-sensitive ATPase. Therefore, we hypothesize that this enzyme may be a Na+/H+ exchanger (NHE) related to NH4+ excretion. The action of an omeprazole-sensitive ATPase, putatively associated to a H+/K+-ATPase (HKA), is also affected by these allostatic processes. Some complementary experiments were carried out to delve a little deeper into the possible branchial enzymes sensitive to amiloride, including in vivo and ex vivo approaches, and partial sequencing of a nhe1 in the gills. This study describes the possible presence of an HKA enzyme in the rectal gland, as well as a NHE in the gills, highlighting the importance of understanding the relationship between acute stress and osmoregulation in elasmobranchs.This studywas partially supported by grant PID2020-117557RB-C22 (funded byMCIN/AEI/10.13039/501100011033 and by the European Union) and Project DISCARDLIFE (Programa Pleamar, Ministerio de Agricultura y Pesca, Alimentacion y Medio Ambiente, Spain) to JMM. I. Ruiz-Jarabo and J.M. Mancera belong to the Fish Welfare and Stress Network (AGL2016-81808-REDT), supported by the Agencia Estatal de Investigacion (MINECO, Spanish Government)

Neuroendocrine Regulation of Reproduction in Sea Bass (Dicentrarchus Labrax)

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Signaling pathways activated by sea bass gonadotropin-inhibitory hormone peptides in COS-7 cells transfected with their cognate receptor

Results of previous studies provided evidence for the existence of a functional gonadotropin-inhibitory hormone (GnIH) system in the European sea bass, Dicentrarchus labrax, which exerted an inhibitory action on the brain-pituitary-gonadal axis of this species. Herein, we further elucidated the intracellular signaling pathways mediating in sea bass GnIH actions and the potential interactions with sea bass kisspeptin (Kiss) signaling. Although GnIH1 and GnIH2 had no effect on basal CRE-luc activity, they significantly decreased forskolin-elicited CRE-luc activity in COS-7 cells transfected with their cognate receptor GnIHR. Moreover, an evident increase in SRE-luc activity was noticed when COS-7 cells expressing GnIHR were challenged with both GnIH peptides, and this stimulatory action was significantly reduced by two inhibitors of the PKC pathway. Notably, GnIH2 antagonized Kiss2-evoked CRE-luc activity in COS-7 cells expressing GnIHR and Kiss2 receptor (Kiss2R). However, GnIH peptides did not alter NFAT-RE-luc activity and ERK phosphorylation levels. These data indicate that sea bass GnIHR signals can be transduced through the PKA and PKC pathways, and GnIH can interfere with kisspeptin actions by reducing its signaling. Our results provide additional evidence for the understanding of signaling pathways activated by GnIH peptides in teleosts, and represent a starting point for the study of interactions with multiple neuroendocrine factors on cell signaling.This work was supported by a grant from PAIDI2020 (Consejería de Economía, Conocimiento, Empresas y Universidad. Junta de Andalucía. Grant no P18-RT-5152) to JAM-C. BW was awarded a scholarship sponsored by the China Scholarship Council (CSC, File No. 201903260004)

The gonadotropin-inhibitory hormone system of sole, Solea senegalensis

Resumen del trabajo presentado en el 11th International Symposium on Reproductive Physiology of Fish, celebrado en Manaus (Brasil) del 03 al 08 de junio de 2018.Gonadotropin-inhibitory hormone (GnIH) belongs to the RFamide family of neuropeptides and exhibits orthologs in different vertebrate groups, including fish, which inhibits the synthesis and/or release of gonadotropin-releasing hormone and gonadotropins. The Senegalese sole, Solea senegalensis, is a flatfish species with increasing interest for Mediterranean aquaculture. However, reproductive problems exhibited by F1 generation of cultured soles are limiting the development of the aquaculture of this species. Therefore, the aim of this study was to obtain pioneer information on this inhibitory GnIH system and elucidate its distribution, function and daily/seasonal regulation in the sole. [Methods]: The distribution of GniH in the brain and pituitary of sole was determined by RT-PCR and immunohistochemistry by using specific antibodies generated against sole GnIH-1 (ssGnIH-1), GnIH-2 (ssGnIH-2) and GnIH-3 (ssGnIH-3). Moreover, ssGnIH-2 and ssGnIH-3 were injected intramuscularly (0.1 and 1 ¿g/g bw) and the effects of both peptides on the expression of gnrh-1, gnrh-2, gnrh-3, kiss2, fsh¿, lh¿ and gh were determined by real time quantitative PCR. Finally, the daily and seasonal expression of gnih was determined by real time quantitative PCR. [Results and Discussion]: The expression of gnih was particularly evident in the diencephalon, but also in the olfactory bulbs/cerebral hemispheres, optic tectum/tegmentum, retina, and pituitary. We showed the presence of ssGnIH-immunoreactive cell bodies in the olfactory bulbs, ventral telencephalon, caudal preoptic area, dorsal tegmentum and rostral rhombencephalon. The ssGnIH fibers innervated the brain and pituitary of sole profusely. Intramuscular injection of ssGnih-3 provoked a significant reduction in gnrh-3 and lh¿ expression. Daily rhythms in gnih expression were found after the completion of metamorphosis, peaking at the beginning of the night. Seasonal variations were also observed in F1 male and female adult specimens, with high levels in April/May, which corresponded with the maturation and spawning season. [Conclusion]: Our results reveal the existence of a functional GnIH system in sole, which could represent an important inhibitory factor, underlying the reproductive dysfunctions observed in this flatfish species. The existence of daily and seasonal variations in the expression of gnih could reflect a role of this neuropeptidergic system in the mediation of the effects of environmental factors