The gonadotropin-inhibitory hormone system of fish: The case of sea bass (Dicentrarchus labrax).

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide belonging to the RFamide peptide family that was first discovered in quail by Tsutsui and co-workers in the year 2000. Since then, different GnIH orthologues have been identified in all vertebrate groups, from agnathans to mammals. These GnIH genes synthesize peptide precursors that encompass two to four C-terminal LPXRFamide peptides. Functional and behavioral studies carried out in birds and mammals have demonstrated a clear inhibitory role of GnIH on GnRH and gonadotropin synthesis and secretion as well as on aggressive and sexual behavior. However, the effects of Gnih orthologues in reproduction remain controversial in fish with both stimulatory and inhibitory actions being reported. In this paper, we will review the main findings obtained in our laboratory on the Gnih system of the European sea bass, Dicentrarchus labrax. The sea bass gnih gene encodes two putative Gnih peptides (sbGnih1 and sbGnih2), and is expressed in the olfactory bulbs/telencephalon, diencephalon, midbrain tegmentum, rostral rhombencephalon, retina and testis. The immunohistochemical study performed using specific antibodies developed in our laboratory revealed Gnih-immunoreactive (ir) perikarya in the same central areas and Gnih-ir fibers that profusely innervated the brain and pituitary of sea bass. Moreover, in vivo studies revealed the inhibitory role of centrally- and peripherally-administered Gnih in the reproductive axis of male sea bass, by acting at the brain (on gnrh and kisspeptin expression), pituitary (on gnrh receptors and gonadotropin synthesis and release) and gonadal (on androgen secretion and gametogenesis) levels. Our results have revealed the existence of a functional Gnih system in sea bass, and have provided evidence of the differential actions of the two Gnih peptides on the reproductive axis of this species, the main inhibitory role in the brain and pituitary being exerted by the sbGnih2 peptide. Recent studies developed in our laboratory also suggest that Gnih might be involved in the transduction of photoperiod and temperature information to the reproductive axis, as well as in the modulation of daily and seasonal rhythmic processes in sea bass.

Gonadotropin-inhibitory hormone in the flatfish, Solea senegalensis: Molecular cloning, brain localization and physiological effects.

Recently, gonadotropin-inhibitory hormone (GnIH) has emerged as an important regulator of reproduction in birds and mammals. This RFamide neuropeptide has neuromodulatory functions and controls the synthesis and/or release of gonadotropin-releasing hormone (GnRH) and gonadotropins. Although teleosts represent about half of all living vertebrates, scientific and technological advances on the Gnih system in fish are scarce, contradictory, and inconclusive. Research on the fish Gnih system appears necessary to better clarify its role in the neuroendocrine and environmental control of vertebrate reproduction. In this study, we cloned a full-length sequence for the Gnih precursor of a flatfish, the Senegalese sole, coding for three putative Gnih peptides (ssGnih). We also generated specific antibodies against these ssGnih peptides, and used them to localize Gnih cells and their projections in the brain and pituitary. The expression of gnih was particularly evident in the diencephalon, but also in the olfactory bulbs/cerebral hemispheres, optic tectum/tegmentum, retina, and pituitary. The three antibodies used provided consistent results and showed that ssGnih-immunoreactive perikarya were present in the olfactory bulbs, ventral telencephalon, caudal preoptic area, dorsal tegmentum and rostral rhombencephalon, and their fibers innervated the brain and pituitary profusely. Intramuscular injection of ssGnih-3 provoked a significant reduction in gnrh-3 and lh expression, whereas ssGnih-2 treatment did not affect transcript levels of the main reproductive genes. Our results reveal the existence of a functional Gnih system in the sole brain, profusely innervating different brain areas and the pituitary gland, which could represent an important factor in the neuroendocrine control of flatfish reproduction.

A Journey through the Gonadotropin-Inhibitory Hormone System of Fish.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that belongs to the RFamide peptide family and was first identified in the quail brain. From the discovery of avian GnIH, orthologous GnIH peptides have been reported in a variety of vertebrates, including mammals, amphibians, teleosts and agnathans, but also in protochordates. It has been clearly established that GnIH suppresses reproduction in avian and mammalian species through its inhibitory actions on brain GnRH and pituitary gonadotropins. In addition, GnIH also appears to be involved in the regulation of feeding, growth, stress response, heart function and social behavior. These actions are mediated via G protein-coupled GnIH receptors (GnIH-Rs), of which two different subtypes, GPR147 and GPR74, have been described to date. With around 30,000 species, fish represent more than one-half of the total number of recognized living vertebrate species. In addition to this impressive biological diversity, fish are relevant because they include model species with scientific and clinical interest as well as many exploited species with economic importance. In spite of this, the study of GnIH and its physiological effects on reproduction and other physiological processes has only been approached in a few fish species, and results obtained are in some cases conflicting. In this review, we summarize the information available in the literature on GnIH sequences identified in fish, the distribution of GnIH and GnIH-Rs in central and peripheral tissues, the physiological actions of GnIH on the reproductive brain-pituitary-gonadal axis, as well as other reported effects of this neuropeptide, and existing knowledge on the regulatory mechanisms of GnIH in fish.

Developmental changes and day-night expression of the gonadotropin-inhibitory hormone system in the European sea bass: Effects of rearing temperature.

The role of rearing temperature on fish development, sex differentiation and puberty has been largely addressed, but the impact of water temperature on the ontogeny of the main neuroendocrine systems controlling reproduction has received little attention. Gonadotropin-inhibitory hormone (GnIH) has been shown to act on gonadotropin-releasing hormone (GnRH) neurons and on the pituitary to inhibit gonadotropin release and synthesis in vertebrates, including sea bass, Dicentrarchus labrax. In the present study we investigated the effects of rearing temperature during the thermosensitive period (5-60days post-fertilization, dpf) on the expression of the GnIH gene (gnih) and its receptor (gnihr). Animals were maintained under two different conditions, low temperature (LT, 15°C) or high temperature (HT, 21°C), throughout the thermosensitive period and sampled from 5 to 360dpf at mid-light (ML) and mid-dark (MD). Our results showed significant effects of temperature on gnih and gnihr expression during the thermosensitive period, with higher transcript levels under LT condition. Some differences were also evident after the completion of the sex differentiation process. Moreover, we revealed daily variations in the developmental expression of gnih and gnihr, with higher diurnal mRNA levels at early stages (until 25dpf), and a shift to higher nocturnal expression levels at 300-360dpf, which corresponded with the beginning of the winter (reproductive season). To the best of our knowledge, this work represents the first study reporting the effects of rearing temperature on the transcription of gnih system genes, as well as its daily variations during the development of a fish species.

Testicular Steroidogenesis and Locomotor Activity Are Regulated by Gonadotropin-Inhibitory Hormone in Male European Sea Bass.

Gonadotropin-inhibitory hormone (GnIH) is a neurohormone that suppresses reproduction by acting at both the brain and pituitary levels. In addition to the brain, GnIH may also be produced in gonads and can regulate steroidogenesis and gametogenesis. However, the function of GnIH in gonadal physiology has received little attention in fish. The main objective of this study was to evaluate the effects of peripheral sbGnih-1 and sbGnih-2 implants on gonadal development and steroidogenesis during the reproductive cycle of male sea bass (Dicentrarchus labrax). Both Gnihs decreased testosterone (T) and 11-ketotestosterone (11-KT) plasma levels in November and December (early- and mid-spermatogenesis) but did not affect plasma levels of the progestin 17,20ß-dihydroxy-4-pregnen-3-one (DHP). In February (spermiation), fish treated with sbGnih-1 and sbGnih-2 exhibited testicles with abundant type A spermatogonia and partial spermatogenesis. In addition, we determined the effects of peripheral Gnih implants on plasma follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) levels, as well as on brain and pituitary expression of the main reproductive hormone genes and their receptors during the spermiation period (February). Treatment with sbGnih-2 increased brain gnrh2, gnih, kiss1r and gnihr transcript levels. Whereas, both Gnihs decreased lhbeta expression and plasma Lh levels, and sbGnih-1 reduced plasmatic Fsh. Finally, through behavioral recording we showed that Gnih implanted animals exhibited a significant increase in diurnal activity from late spermatogenic to early spermiogenic stages. Our results indicate that Gnih may regulate the reproductive axis of sea bass acting not only on brain and pituitary hormones but also on gonadal physiology and behavior.

Gonadotropin Inhibitory Hormone Down-Regulates the Brain-Pituitary Reproductive Axis of Male European Sea Bass (Dicentrarchus labrax).

Gonadotropin-inhibitory hormone (GnIH) inhibits gonadotropin synthesis and release from the pituitary of birds and mammals. However, the physiological role of orthologous GnIH peptides on the reproductive axis of fish is still uncertain, and their actions on the main neuroendocrine systems controlling reproduction (i.e., GnRHs, kisspeptins) have received little attention. In a recent study performed in the European sea bass, we cloned a cDNA encoding a precursor polypeptide that contained C-terminal MPMRFamide (sbGnIH-1) and MPQRFamide (sbGnIH-2) peptide sequences, developed a specific antiserum against sbGnIH-2, and characterized its central and pituitary GnIH projections in this species. In this study, we analyzed the effects of intracerebroventricular injection of sbGnIH-1 and sbGnIH-2 on brain and pituitary expression of reproductive hormone genes (gnrh1, gnrh2, gnrh3, kiss1, kiss2, gnih, lhbeta, fshbeta), and their receptors (gnrhr II-1a, gnrhr II-2b, kiss1r, kiss2r, and gnihr) as well as on plasma Fsh and Lh levels. In addition, we determined the effects of GnIH on pituitary somatotropin (Gh) expression. The results obtained revealed the inhibitory role of sbGnIH-2 on brain gnrh2, kiss1, kiss2, kiss1r, gnih, and gnihr transcripts and on pituitary fshbeta, lhbeta, gh, and gnrhr-II-1a expression, whereas sbGnIH-1 only down-regulated brain gnrh1 expression. However, at different doses, central administration of both sbGnIH-1 and sbGnIH-2 decreased Lh plasma levels. Our work represents the first study reporting the effects of centrally administered GnIH in fish and provides evidence of the differential actions of sbGnIH-1 and sbGnIH-2 on the reproductive axis of sea bass, the main inhibitory role being exerted by the sbGnIH-2 peptide.

LPXRFa peptide system in the European sea bass: A molecular and immunohistochemical approach.

Gonadotropin-inhibitory hormone (GnIH) is a neuropeptide that suppresses reproduction in birds and mammals by inhibiting GnRH and gonadotropin secretion. GnIH orthologs with a C-terminal LPXRFamide (LPXRFa) motif have been identified in teleost fish. Although recent work also suggests its role in fish reproduction, studies are scarce and controversial, and have mainly focused on cyprinids. In this work we cloned a full-length cDNA encoding an LPXRFa precursor in the European sea bass, Dicentrarchus labrax. In contrast to other teleosts, the sea bass LPXRFa precursor contains only two putative RFamide peptides, termed sbLPXRFa1 and sbLPXRFa2. sblpxrfa transcripts were expressed predominantly in the olfactory bulbs/telencephalon, diencephalon, midbrain tegmentum, retina, and gonads. We also developed a specific antiserum against sbLPXRFa2, which revealed sbLPXRFa-immunoreactive (ir) perikarya in the olfactory bulbs-terminal nerve, ventral telencephalon, caudal preoptic area, dorsal mesencephalic tegmentum, and rostral rhombencephalon. These sbLPXRFa-ir cells profusely innervated the preoptic area, hypothalamus, optic tectum, semicircular torus, and caudal midbrain tegmentum, but conspicuous projections also reached the olfactory bulbs, ventral/dorsal telencephalon, habenula, ventral thalamus, pretectum, rostral midbrain tegmentum, posterior tuberculum, reticular formation, and viscerosensory lobe. The retina, pineal, vascular sac, and pituitary were also targets of sbLPXRFa-ir cells. In the pituitary, this innervation was observed close to follicle-stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH) cells. Tract-tracing retrograde labeling suggests that telencephalic and preoptic sbLPXRFa cells might represent the source of pituitary innervation. The immunohistochemical distribution of sbLPXRFa cells and fibers suggest that LPXRFa peptides might be involved in some functions as well as reproduction, such as feeding, growth, and behavior.

The circadian clock machinery during early development of Senegalese sole (Solea senegalensis): effects of constant light and dark conditions.

Circadian rhythms are established very early during vertebrate development. In fish, environmental cues can influence the initiation and synchronization of different rhythmic processes. Previous studies in zebrafish and rainbow trout have shown that circadian oscillation of clock genes represents one of the earliest detectable rhythms in the developing embryo, suggesting their significance in regulating the coordination of developmental processes. In this study, we analyzed the daily expression of the core clock components Per1, Per2, Per3, and Clock during the first several days of Senegalese sole development (0-4 d post fertilization or dpf) under different lighting regimes, with the aim of addressing when the molecular clock first emerges in this species and how it is affected by different photoperiods. Rhythmic expression of the above genes was detected from 0 to 1 dpf, being markedly affected in the next few days by both constant light (LL) and dark (DD) conditions. A gradual entrainment of the clock machinery was observed only under light-dark (LD) cycles, and robust rhythms with increased amplitudes were established by 4 dpf for all clock genes currently studied. Our results show the existence of an embryonic molecular clock from the 1st d of development in Senegalese sole and emphasize the significance of cycling LD conditions when raising embryos and early larvae.

Identification of two arylalkylamine N-acetyltranferase 1 genes with different developmental expression profiles in the flatfish Solea senegalensis.

The existence of two arylalkylamine N-acetyltransferase 1 (Aanat1) genes in the genome of some teleosts has been reported recently by in silico analysis. However, there are no data concerning the similarities and/or differences between them and many questions remain to be answered, such as their expression sites, development, or kinetics. Here, we report the cloning of Aanat1a and Aanat1b cDNAs from the sole retina and show for the first time that at least three Aanat genes are expressed in a vertebrate species. Because melatonin is involved in fish ontogeny, we analyzed the developmental transcript levels of Aanat1a and Aanat1b by quantitative real-time PCR, showing their inverse and stage-specific expression patterns. Aanat1a was more abundant during early than late larval stages. Before metamorphosis, nocturnal expression was higher. At metamorphosis, Aanat1a expression decreased and lost these day-night variations. In contrast, the abundance of Aanat1b transcripts, low during early developing stages, rose significantly throughout metamorphosis. This situation seemed to apply to the adult because Aanat1a expression was lower than Aanat1b expression in the retina of adults, where the former did not exhibit day-night variations, while the latter did so with much higher nocturnal transcript levels. In situ hybridization analysis detected Aanat1a and Aanat1b messengers in the outer and inner nuclear layers of retina. The differences in abundance and distinct day-night expression patterns between Aanat1a and Aanat1b during sole development suggest different functions for these two enzymes as well as the existence of interactions between the melatoninergic and thyroid hormone systems during flatfish metamorphosis.