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.

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.

Tumor necrosis factor alpha may act as an intraovarian mediator of luteinizing hormone-induced oocyte maturation in trout.

In fish, like in other vertebrates, luteinizing hormone (Lh) is an essential hormone for the completion of oocyte maturation. In salmonid fish (i.e., salmon and trout), oocyte maturation is induced by Lh through its stimulation of the production of the maturation-inducing steroid, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In mammals, several factors, including ovarian cytokines and growth factors, have been reported to contribute to the regulation of oocyte maturation. In fish, growing evidence suggests that tumor necrosis factor alpha (hereafter referred to as Tnf) could play multiple physiological roles in the control of ovarian function. In the present study, we have investigated the possible involvement of Tnf in the regulation of oocyte maturation in brown trout (Salmo trutta). Our results show that in vitro treatment of brown trout preovulatory follicles with coho salmon (Oncorhynchus kisutch) Lh (sLh) significantly increased oocyte maturation, as assessed by germinal vesicle breakdown (GVBD), and that this effect was blocked by TAPI-1 (an inhibitor of Tnf-converting enzyme or Tace/Adam17). Furthermore, treatment of preovulatory follicles with sLh increased the expression of tnf and tace/adam17 as well as the secretion of the Tnf protein. Importantly, recombinant trout Tnf (rtTnf) significantly increased GVBD in vitro. Our results also show that the stimulatory effects of rtTnf on oocyte maturation may be the result of the direct involvement of rtTnf in stimulating the production of the maturation-inducing steroid as evidenced, first, by the stimulatory effects of rtTnf on 17,20beta-P production in vitro and on the expression of cholesterol side-chain cleavage P450 cytochrome (p450scc) and 20beta-hydroxysteroid dehydrogenase/carbonyl reductase 1 (cbr1), the enzyme responsible for the production of 17,20beta-P, and, second, by the ability of TAPI-1 to block the stimulatory effects of sLh on 17,20beta-P production and cbr1 expression. Furthermore, sLh and rtTnf increased the expression of the Lh receptor (lhr) and decreased the expression of aromatase (cyp19a1), and TAPI-1 completely blocked the effects of sLh. These results strongly suggest that Tnf may contribute to the regulation of oocyte maturation by Lh in trout.

Effects of in vivo treatment with the dopamine antagonist pimozide and gonadotropin-releasing hormone agonist (GnRHa) on the reproductive axis of Senegalese sole (Solea senegalensis).

The Senegalese sole (Solea senegalensis) is a flatfish that exhibits severe reproductive dysfunctions in captivity. This study aimed at investigating the existence of a dopamine (DA) inhibitory tone on the reproductive axis of this species. Four groups of Senegalese sole breeders were treated with, saline (controls, CNT), the DA antagonist pimozide (PIM, 5 mg kg(-1)), gonadotropin-releasing hormone agonist (GnRHa, 40 µg kg(-1)) or a combination of PIM+GnRHa (COMB). Effects were evaluated on pituitary GnRH levels (ELISA), pituitary gonadotropin subunit transcript levels (qPCR), plasma levels of sex steroids and vitellogenin (ELISA), gonad development (histology), spermiation and egg production. The GnRHa treatment induced egg release and stimulated testis maturation. In males, PIM did not affect pituitary GnRH content, but enhanced GnRHa-induced pituitary GPα transcripts and modified plasma androgen levels; moreover, PIM stimulated spermatogenesis and milt production, both alone and combined with GnRHa. In females, PIM did not affect pituitary and plasma endocrine parameters and did not affect egg production and fertilization success of the broodstock, either alone or in the combined treatment. In conclusion, data indicated the existence of a DA inhibition in mature males, which would be absent or weakly expressed in females.

Follicle stimulating hormone (FSH) and luteinizing hormone (LH) gene expression during larval development in Senegalese sole (Solea senegalensis).

The gonadotropins (GTHs), follicle stimulating hormone (FSH) and luteinizing hormone (LH), determine the reproductive competence of adult breeders, but also participate in the establishment of the reproductive axis at early stages of life. The present study aimed at studying, by real-time qPCR, the gene expression levels of GTH subunits (FSHbeta, LHbeta and the common glycoprotein alpha -GPalpha- subunit) during early development in Senegalese sole, from 1 to 100 days post hatching (dph). The FSHbeta, LHbeta and GPalpha transcripts were first detected at 1, 5 and 3 dph, respectively. Transcript levels of FSHbeta, and GPalpha, increased continuously to peak levels at mid metamorphosis (15 dph), decreasing thereafter; levels were maintained low until a second increment detected at 90 and 100 dph. Contrarily, transcript levels of LHbeta were very low and only detectable around metamorphosis. All three subunits were highly expressed in 1-year old soles, with FSHbeta and GPalpha transcript levels 10-fold higher than those of LHbeta. These results suggest, i) activity of the reproductive axis early after hatching (1 dph), which was highest during the metamorphic climax and, ii) a predominant role of FSH, rather than LH, in the early development of the reproductive axis in Senegalese sole.

Vitellogenin, steroid plasma levels and spawning performance of cultured female Senegalese sole (Solea senegalensis).

The Senegalese sole (Solea senegalensis) is a high value market flatfish, which aquaculture is compromised by severe reproductive problems; these are mostly found in soles hatched and raised in captivity (F1 generation). To gain knowledge on the reproductive dysfunctions observed in cultured (F1) Senegalese sole, this work aimed at developing a specific vitellogenin (VTG) ELISA, for the measurement of plasma VTG levels in this species. Profiles of VTG were correlated with those of sexual steroids and spawning performance of an F1 broodstock, during three consecutive years. The Senegalese sole VTG (ssVTG) was purified by precipitation with MgCl(2)-EDTA and anion-exchange chromatography and showed a molecular mass of 172 kDa, by SDS-PAGE. Specific antibodies were obtained and used to develop a competitive ELISA, which had a sensitivity of 3.6 ng ml(-1), and inter- and intra-assay coefficients of variation of 9.5% (n=29) and 6.7% (n=12), respectively. Annual profiles of plasma VTG showed a major peak at pre-spawning, and a second minor rise around autumn, which mirrored plasma profiles of both estradiol (E(2)) and testosterone (T) levels. Spontaneous spawning occurred every year in the spring season, but no fertilized eggs were obtained. In conclusion, this study described, for the first time, the purification and development of a sensitive and specific ELISA for Senegalese sole VTG. The endocrine and spawning data suggested that F1 female broodstock showed normal VTG and steroid releasing profiles in captivity with occurrence of spontaneous spawning, but no fertilization of the eggs was recorded.

[Prolonged total parenteral nutrition in surgery. A case report of severe malnutrition]. / Nutrición parenteral total prolongada en cirugía. Reporte de un caso con desnutrición severa.

We present a 15-years-old female patient severely malnourished due to complications of abdominal surgery, who received total parenteral nutrition during 118 days. The clinical and laboratory parameters are analyzed and a photographic sequence is presented. During the treatment period nitrogen balance was positive and her weight increased in 50%.