General and Comparative Endocrinology

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1 General and Comparative Endocrinology 165 (2010) Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: Neuroendocrinology of reproduction in teleost fish Yonathan Zohar a, José Antonio Muñoz-Cueto b, Abigail Elizur c, Olivier Kah d, * a Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD, USA b Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, E-11510, Puerto Real, España, Spain c Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore DC, Maroochydore, Qld., Australia d Neurogenesis and Oestrogens, Université de Rennes 1, UMR CNRS 6026, IFR140, Campus de Beaulieu, Rennes cedex, France article info abstract Article history: Received 15 December 2008 Revised 8 April 2009 Accepted 17 April 2009 Available online 23 April 2009 Keywords: Teleost fish Reproduction Brain Pituitary GABA Neuroendocrine system GnRH Dopamine KISS Sex steroid Gonadotrophin This review aims at synthesizing the most relevant information regarding the neuroendocrine circuits controlling reproduction, mainly gonadotropin release, in teleost fish. In teleosts, the pituitary receives a more or less direct innervation by neurons sending projections to the vicinity of the pituitary gonadotrophs. Among the neurotransmitters and neuropeptides released by these nerve endings are gonadotrophin-releasing hormones (GnRH) and dopamine, acting as stimulatory and inhibitory factors (in many but not all fish) on the liberation of LH and to a lesser extent that of FSH. The activity of the corresponding neurons depends on a complex interplay between external and internal factors that will ultimately influence the triggering of puberty and sexual maturation. Among these factors are sex steroids and other peripheral hormones and growth factors, but little is known regarding their targets. However, very recently a new actor has entered the field of reproductive physiology. KiSS1, first known as a tumor suppressor called metastin, and its receptor GPR54, are now central to the regulation of GnRH, and consequently LH and FSH secretion in mammals. The KiSS system is notably viewed as instrumental in integrating both environmental cues and metabolic signals and passing this information onto the reproductive axis. In fish, there are two KiSS genes, KiSS1 and KiSS2, expressed in neurons of the preoptic area and mediobasal hypothalamus. Pionneer studies indicate that KiSS and GPR54 expression seem to be activated at puberty. Although precise information as to the physiological effects of KiSS1 in fish, notably on GnRH neurons and gonadotropin release, is still limited, KiSS neurons may emerge as the gatekeeper of puberty and reproduction in fish as in mammals. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Neuroendocrinology is the intersection between neurobiology and endocrinology: it is the study of the control exerted by the brain onto the endocrine system and, reciprocally, the study of the effects exerted by the endocrine system onto the brain. These two tightly-linked mechanisms contribute to adapt the response of the organism to changes in the environment and hormonal milieu. Neuroendocrinology, especially in fish, is still a young field. In mammals, the concept that the brain controls the gonadotrophic function of the pituitary was firmly established after the Second World War, when pioneering researchers proposed that a stimulatory brain factor was transported by the blood to the pituitary gland where it affected the gonadotrophic activity (Benoit and Assenmacher, 1952; Donovan and Harris, 1954). These and other reports paved the way to the discovery of GnRH, the main factor controlling the release of pituitary gonadotropins, which was finally in 1971 (Amoss et al., 1971; Matsuo et al., 1971). In fish, most of * Corresponding author. Fax: +33 (0) address: Olivier.kah@univ-rennes1.fr (O. Kah). the early information came from pituitary graft experiments with the aim of looking at the activity of pituitary cells disconnected from the hypothalamus (Olivereau and Ball, 1966). Shortly after, Bernard Breton and colleagues showed for the first time that fish hypothalamic extracts stimulated gonadotrophin release in the carp (Breton et al., 1971; Breton and Weil, 1973). This latter work, together with the publication by the late Dr. Richard E. Peter of the first brain atlas in the goldfish (Peter and Gill, 1975) was the real start of fish reproductive neuroendocrinology. After 35 years, the field has made considerable progress, although there are still many unresolved issues. 2. Organization of the hypothalamo-pituitary complex in fish As in all vertebrates, the activity of the pituitary gland is controlled in large part by a number of neurohormones (neuropeptides, neurotransmitters) that are synthesized by specific neuronal populations and reach the pituitary. Overall, the underlying mechanisms are very similar in fish and tetrapods, but there are significant differences in the way these neurohormones reach their target cells in the pituitary. Because the nature, /$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi: /j.ygcen

2 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) the origin and the localization of these neurohormones provide important information by themselves on how the brain controls the pituitary, it is important to understand well the neuroanatomical organization of the hypothalamo-pituitary complex Organization of the pituitary gland In all vertebrates, the pituitary is attached to the hypothalamus by a short stalk that, in fish, consists of neurosecretory fibers passing from the brain to the pituitary. These are in fact axons from neurons located in the hypothalamus and sending projections to the pituitary. The pituitary gland, or hypophysis, consists of the adenohypophysis and the neurohypophysis. The adenohypophysis contains the different cells secreting the pituitary hormones and thus represents the glandular part of the pituitary. The neurohypophysis mainly consists of bundles of neurosecretory fibers originating from different parts of the brain and secreting various peptides in the vicinity of the pituitary cells. In fish, the adenohypophysis has been divided into the pars distalis (the anterior lobe of terrestrial vertebrates), itself divided into the rostral and proximal pars distalis, and pars intermedia (intermediate lobe of terrestrial vertebrates (Olivereau and Ball, 1964). The posterior lobe of the tetrapod pituitary, which is comprised of nerve fibers emanating from the hypothalamus and secreting vasopressin and oxytocin into the general circulation, is in fish tightly associated with the pars intermedia. This part of the pituitary often receives the name of neurointermediate lobe. In all vertebrates including fish, the anterior lobe contains the corticotrophs (ACTH cells), the mammotrophs (prolactin cells, PRL cells), the somatotrophs (growth hormone cells, GH cells), the thyrotrophs (TSH cells) and the gonadotrophs (LH/FSH cells) (Olivereau and Ball, 1964). In tetrapods, the anterior lobe does not receive a direct innervation due to the existence of a hypothalamo-pituitary portal system, whose primary plexus of capillaries is located in the external zone of the median eminence. The median eminence is located in the floor of the hypothalamus and at this level the neurohormones secreted by the hypophysiotropic neurons are liberated in the vicinity of blood vessels and reach their target cells in the anterior lobe via the blood stream. Compared to terrestrial vertebrates, the pars distalis or anterior lobe of teleosts has two characteristics: (1) cells of a given cell type are often grouped together in a given region of the gland. This is different from mammals in which cells do not form specialized masses. (2) There is no functional hypothalamo-pituitary portal system. In contrast, the different cells of the pars distalis receive an innervation that is more or less direct depending on the fish species (Fig. 1). The pars distalis itself is often divided into two parts, the rostral pars distalis (RPD) usually containing the ACTH, the PRL cells and, in some but not all species, the TSH cells, and the proximal pars distalis (PPD) encompassing the gonadotrophs, GH cells and TSH cells. However, it must be kept in mind that pituitary cells at certain stages can proliferate and that there is a certain level of plasticity in the localization of a given cell type according to the physiological stage. For example, the gonadotrophs in perciforms or salmonids tend to invade the periphery of the pars intermedia during sexual maturation. Originally, before the purification of pituitary hormones by biochemical methods, pituitary cells were identified using classical histophysiological methods based on the staining properties of the hormones. Using such techniques, gonadotrophs were identified as located in the proximal pars distalis in all species. Many pioneering studies on the pituitary of fish were conducted by Madeleine Olivereau who was the first to distinguish two gonadotroph cell types in salmonids using conventional staining techniques (Olivereau, 1976). Following the purification of FSH and LH in coho salmon, this was later confirmed using specific antibodies to the b subunits of the gonadotropins (Dickey and Swanson, 1998; Nozaki et al., 1990; Shimizu et al., 2003) (see gonadotropin chapter). The cloning of the cdna corresponding to these subunits also permitted localization of the gonadotrophs by in situ hybridization, again suggesting that FSH and LH are synthesized by different cells (Fig. 1) The pituitary innervation As stated above, teleost fish are unique among vertebrates in lacking a hypothalamo-pituitary portal system. Instead, the neurohormones controlling the activity of the different cell types in fish are released directly by nerve endings located in close proximity to their target cells. In some species, true neuroendocrine synaptic contacts can be observed. Thus, the pituitary innervation of teleosts must be considered as the functional equivalent of the innervation of the median eminence in terrestrial vertebrates (Fig. 1). In some species, such as salmonids or eels, the neurohypophysis is totally separated from the adenohypophysis by a double basal membrane on which the neurosecretory terminals are apposed (Abraham, 1974; Kaul and Vollrath, 1974). It is believed that the neurosecretory products are released and diffuse within the intracellular spaces to reach their target cells. In other species, such as cyprinids, the basal membrane is interrupted in some places and the nerve fibers invade the adenohypophysis. In general, there is good correspondence between the distribution of the nerve fibers involved in the control of a given cell type and the distribution of this cell type within the pituitary. For example, GnRH-immunoreactive fibers are mostly present in the proximal pars distalis where the gonadotrophs are located. Many neuropeptides and neurotransmitters have been identified in fibers penetrating the pars distalis and shown to modulate gonadotropin release in vivo or in vitro. This is notably the case of GnRH (Gonzalez-Martinez et al., 2002a; Lethimonier et al., 2004; Oka and Ichikawa, 1990), GABA (Kah et al., 1987), neuropeptide Y (Batten et al., 1990; Kah et al., 1989b), dopamine (Kah et al., 1984, 1986b), gamma-amino-butyric acid (GABA; Kah et al., 1987), PACAP (Wong et al., 1998), galanin (Anglade et al., 1994b; Batten et al., 1990), somatostatin, cholecystokinin, substance P, or growth hormonereleasing factor (Batten et al., 1990, 1999) Origin of the pituitary innervation The first attempts to characterize the brain cells sending axons into the pituitary used chemical (Kah et al., 1983) or electrolytic lesions (Kah et al., 1987). More recently, the distribution of the cells directly projecting into the pituitary (hypophysiotropic neurons) was investigated using retrograde transport techniques (Fryer and Maler, 1981; Rao et al., 1993). In particular, the introduction of fluorescent dyes, such as DiI ( dioctadecyl-3,3,3 0,3 0 -tétramethylindocarbocyanine), that are being transported along the membranes of cells fixed by aldehydes, made it quite easy to perform such retrograde tracing in different species (Anglade et al., 1993; Holmqvist and Ekstrom, 1995; Johnston and Maler, 1992; Rao et al., 1993; Weltzien et al., 2006). After fixing the brain (without disrupting the pituitary stalk) with formaldehyde, a small crystal of DiI is introduced into the pituitary. Neighboring fibers will take up the tracer, which is then transported up to the cell body of origin over a certain period of time. The recent emergence of transgenic medaka and zebrafish in which GnRH neurons specifically express fluorescent reporter proteins provides an useful tool for the study of reproductive endocrinology and led to new, highly sensitive tactics to visualize these hypophysiotrophic neurons and their early developmental patterns (Abraham et al., 2008; Okubo et al., 2006). The data of such studies provided a very accurate vision of the entire population of hypophysiotropic neurons. For example, in goldfish,

3 440 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) Fig. 1. The innervation of the pituitary in teleosts. (A) Schematic representation of a lateral view showing projections from hypophysiotropic neurons ending either at the basement membrane (bm) separating the neurohypophysis (NH) from the adenohypophysis (AH) or directly at adenohypophyseal cells. (B) Electron microscopy picture of the pituitary of the mosquito fish (Gambusia affinis) showing several neurosecretory fibers (ne) ending onto the basement membrane (bm) separating the neurohypophysis (NH) from the adenohypophysis (AH). Note the accumulation of small clear vesicles along the membrane (arrows), suggesting release of neurosecretory material. Bar = 1 lm. (C) Electron microscopy picture of the pituitary of the mosquito fish showing a nerve ending establishing synaptic-like contacts (arrows) with two gonadotropes. Bar = 1 lm. (D and E) Adjacent parasagittal sections hybridized with FSHb and LHb probes showing the distribution of FSH (D) and LH (E) expressing cells in the proximal pars distalis (ppd) of a vitellogenic female rainbow trout. Note in (E) that some LHb-expressing cells are observed in the caudal rostral pars distalis (rpd) or in the dorsal pars intermedia (pi). Anterior is to the left as shown by the presence of the saccus vasculosus (sv) on the right. Bar = 1 mm. brown ghost and zebrafish, studies established that most of the pituitary innervation originates from the preoptic area and the mediobasal hypothalamus, which are in all vertebrates the main regions controlling reproductive functions (Johnston and Maler, 1992; Mananos et al., 1999). However, a number of hypophysiotropic neurons were also found in extra hypothalamic regions such as the olfactory bulbs, the ventral telencephalon, the nucleus suprachiasmaticus, the thalamus, or the mesencephalic tegmentum (Anglade et al., 1993; Johnston and Maler, 1992). 3. The main brain factors influencing the pituitary gonadotropic functions While many different brain factors (neuropeptides, neurotransmitters) have been shown to stimulate gonadotropin release in vitro, only a limited number of these are capable of acting in vivo so that the physiological significance of this multifactorial control of the gonadotropic activity is poorly understood. In this review, we shall limit ourselves to those factors having a clearly identified functional significance, mainly GnRH, dopamine, NPY, GABA and KiSS Gonadotropin-releasing hormone The idea that neurohumoral secretions from the brain control pituitary function and gonadotropin secretion took a long time to be accepted (Benoit and Assenmacher, 1952; Donovan and Harris, 1954). In the early seventies, two research groups simultaneously reported the isolation of porcine and ovine hypothalamus LHreleasing factors (Amoss et al., 1971; Matsuo et al., 1971) after struggling with an impressive number of hypothalami from pigs and sheep, respectively. This decapeptide was named LHRH (for Luteinizing Hormone Releasing Hormone) and its primary structure was identified as pglu-his-trp-ser-tyr-gly-leu-arg-pro- Gly-NH 2. Later it became obvious that this decapeptide also stimulates FSH release and thus the name LHRH was replaced by GnRH (for Gonadotropin-Releasing Hormone), which is now widely used. Following the pioneering studies of Breton and colleagues showing that this hypothalamic factor stimulates the release of pituitary gonadotrophic hormone in carp (Breton et al., 1971), the first fish GnRH, salmon GnRH, was (Sherwood et al., 1983). Since then, research on GnRH in teleost fish has attracted increasing attention. This is not only due to the important potential applications of GnRH in fish farming (Zohar and Mylonas, 2001), but also to the fact that teleost fish have turned out to be of special interest to understanding the mechanisms underlying the evolution of GnRH genes in vertebrates. Thus, it now appears that each vertebrate species expresses two or three GnRH forms in multiple tissues and that GnRHs exert pleiotropic actions via several classes of receptors (Kah et al., 2007; Morgan and Millar, 2004). This new vision of the GnRH system arose progressively from numerous comparative studies in all vertebrate classes, but fish in general, and

4 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) teleosts in particular, have often played a leading role in shifting paradigms in reproductive endocrinology. To date, fish still appear as attractive models to decipher the evolutionary mechanisms that led to the diversification of GnRH functions. Not only do teleosts exhibit the highest number of GnRH variants encountered so far (Adams et al., 2002), with a total of 8, but recent data and whole genome analyses indicate that some species possess up to 5 functional GnRH receptors (Kah et al., 2007) GnRH forms, genes and brain distribution The GnRH family has rapidly increased since 1971, now reaching a total of 24 variants found in vertebrates, protochordates and invertebrates (Kah et al., 2007; Lethimonier et al., 2004; Morgan and Millar, 2004; Okubo and Nagahama, 2008). Among vertebrates, teleost fish represent the group with the highest number of different GnRH isoforms. Indeed, following the identification of salmon GnRH (Sherwood et al., 1983), seven other GnRH forms have been purified and sequenced in teleosts (Guilgur et al., 2006). These GnRH variants were traditionally named after the species in which they were first discovered, despite the fact that they can be present in other species. In order to avoid this confusion, a new classification of the GnRH variants on the basis of phylogenetic analysis of known sequences and their respective sites of expression was proposed (Fernald and White, 1999; White et al., 1995). The phylogenetic analysis shows the existence of three main GnRH branches. One branch contains hypophysiotropic variants, mainly expressed in the hypothalamus of amphibians and mammals but also a number of fish hypophysiotropic variants, which were named GnRH-1. Another branch clusters all GnRH forms consistently expressed in the synencephalon/mesencephalon of vertebrates, from fish to mammals, and is referred to as GnRH-2. A third GnRH branch includes only the fish (salmon) GnRH isoforms, mainly expressed in the rostral forebrain, and is named GnRH-3. It is interesting to note that branches 1 and 2 contain sequences from fish through terrestrial vertebrates, indicating that these branches are ancient and emerged before the divergence of these groups, but branch 3 includes only salmon GnRH (fish) sequences. Two hypotheses have been proposed to explain the existence of the third fish GnRH branch. There is the possibility that the gene duplication that gave rise to this branch occurred within the fish lineage, after the divergence between fish and tetrapods (Guilgur et al., 2006; Kah et al., 2007; Okubo and Nagahama, 2008). Because primitive bony fishes such as sturgeons and Polypterus only exhibit two GnRH forms, it could be expected that teleosts contain four different GnRH genes. Therefore, duplication of an ancestral GnRH isoform could give rise to the present GnRH-1 and GnRH-3 variants, which were subjected to subsequent functional specialization and neuroanatomical segregation of cells and projections. In this case, it should be expected that two different GnRH-2 genes also exist, but it is possible that one of these genes was lost after duplication as it occurs with 50 80% of duplicated genes (Lynch and Force, 2000). In turn, other evolutionary and phylogenetic studies suggest that the gene duplication that gave rise to GnRH-1 and GnRH-3 occurred before the divergence of fish and tetrapods but that the corresponding gene has been lost in terrestrial vertebrates or remains to be found (Guilgur et al., 2006; Okubo and Nagahama, 2008). Chromosome analysis of genome regions surrounding GnRH genes in human, chicken, zebrafish, fugu and Tetraodon were considered as support for this putative secondary lost of GnRH-3 gene lineage in tetrapods (Kuo et al., 2005). The GnRH genes of vertebrates share a common structure, being by the presence of 4 exons and 3 introns. Sequence analyses of the different GnRH genes showed that coding regions are highly conserved, but upstream and downstream regions and intron sequences are distinctively divergent (Alestrom et al., 1992; Chow et al., 1998). The decapeptide GnRH issues from a large mrna. The initial mrna product, a precursor peptide called prepro-gnrh, consists of a signal peptide that allows the protein to be transferred to the endoplasmic reticulum, the mature GnRH decapeptide, a processing tripeptide (Gly-Lys-Arg), and a GnRH-associated peptide or GAP. The signal peptide, GnRH decapeptide, processing tripeptide and N-terminal region of GAP are encoded in exon 2. The core and C-terminal parts of GAP are encoded in exons 3 and 4. The and 3 0 -UTR are encoded in exons 1 and 4, respectively (Alestrom et al., 1992; Chow et al., 1998). The signal peptide is cleaved by a signal peptidase and the peptide is transported to the Golgi apparatus where prohormone convertases and exopeptidases liberate the active peptide. The glycine at the C-terminus is amidated and the glutamate at the N-terminus is converted into pyroglutamate by a glutamine cyclotransferase. The mature peptide is concentrated in secretory granules together with the GAP and is transported to the axon terminals for its release (Andersen et al., 1988; Rangaraju et al., 1991). Comparing the different prepro- GnRH sequences, the GAP represents the most divergent region within and between species. In the sea bass, oligonucleotide sequences of the GAPs from GnRH-1, GnRH-2 and GnRH-3 precursors do not exhibit more than 42% identity (Zmora et al., 2002). The sequence of GAPs from sea bass GnRH-2 and GnRH-3 precursors have 88 98% identity with the corresponding sequences in other fish. However, the sea bass GAP GnRH-1 sequence identity dropped to 35 57% when compared with the African cichlid and the seabream. This suggests that the gene coding for the GnRH-1 precursor has evolved faster than those coding for GnRH-2 and GnRH-3, which have been subjected to strong evolutionary pressure. A considerable amount of work has been devoted to the identification and localization of GnRH-expressing neurons in the brain of fish using immunohistochemistry or, more recently, in situ hybridization and transgenesis (Abraham et al., 2008; Guilgur et al., 2006; Kah et al., 2007; Okubo and Nagahama, 2008). Most studies performed up to the early 1990s suggested the existence of two segregated GnRH systems in fish brain: an anterior system extending from the olfactory bulbs to the pituitary through the ventral telencephalon, preoptic area and ventromedial hypothalamus, and a posterior system restricted to the synencephalon/midbrain tegmentum. The anterior system expressed one variable GnRH form, whereas the posterior system consistently expressed GnRH-2 (Kah et al., 1986a; Lepretre et al., 1993; Montero et al., 1995; Okuzawa et al., 1990). However, the situation changed significantly when Powell and coauthors showed for the first time that the brain of a perciform fish, the seabream, expressed three different GnRH forms (Powell et al., 1994) and other labs demonstrated that the anterior GnRH system contained two different neuronal populations each expressing a different GnRH gene (Gothilf et al., 1996; White et al., 1995). The existence of three GnRH forms was confirmed, either by cdna sequencing or biochemical characterization, in other perciform species (Senthilkumaran et al., 1999; Zmora et al., 2002) and there is now evidence on the existence of three different GnRH forms in most teleost orders (Adams et al., 2002; Amano et al., 2002; Andersson et al., 2001; Carolsfeld et al., 2000; Mohamed and Khan, 2006; Montaner et al., 2001; Okubo et al., 2000). The distribution of the cells expressing the different GnRHs was reported originally in the gilthead seabream by Gothilf et al. (1996) and then further clarified in the European sea bass (Gonzalez-Martinez et al., 2001, 2002a). Using specific GAP riboprobes and antibodies it was found, in support of previous studies, that GnRH-2 cells appeared restricted to the dorsal synencephalon but GnRH-1 and GnRH-3 cell distribution overlapped in the olfactory bulbs, ventral telencephalon and preoptic area (Gonzalez-Martinez et al., 2001, 2002a). Similar results were later obtained in other fish species including Coregonus clupeaformis (Vickers et al., 2004), Cichlasoma dimerus (Pandolfi et al., 2005), Micropogonias undulatus (Mohamed et al., 2005) and

5 442 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) Oryzias latipes (Okubo et al., 2006), suggesting that this overlap is the rule and not the exception. Results obtained in zebrafish, a species that has two forms of GnRH (GnRH-2 and GnRH-3), show that in this species GnRH-3 neurons are present in regions occupied by both GnRH-3 and GnRH-1 in three form fish. GnRH-3 neurons in zebrafish are found in the olfactory bulbs, ventral telencephalon, preoptic area and ventromedial hypothalamus (Fig. 2) (Abraham et al., 2008; Palevitch et al., 2009). In addition, studies performed in sea bass provided unambiguous and detailed information on the distribution of fibers immunoreactive for the three different GnRH forms expressed in the brain of a single species (Gonzalez- Martinez et al., 2002a). Thus, GnRH-1 fibers were observed only in the ventral surface of the forebrain, associated with the ventral telencephalon, preoptic area and hypothalamus, whereas GnRH-2 and GnRH-3 fibers exhibited a profuse distribution in the sea bass brain. We also demonstrated that the GnRH-1 neurons represent the main source of pituitary GnRH innervation, arriving at the proximal pars distalis and the border of the pars intermedia where gonadotrophic cells and GnRH receptors were also found (Gonzalez-Martinez et al., 2004b). This result corroborates physiological evidence suggesting a major role for GnRH-1 in the stimulation of the secretion of gonadotropins in three-gnrh perciform species. Although GnRH-3 axons also reach the pituitary of sea bass, this innervation was strongly reduced as compared to GnRH-1 projections. In contrast, no prepro-gnrh-2 axons were detected in the pituitary of sea bass, suggesting that the putative role of GnRH-2 in the control of reproduction does not involve a direct action of cerebral GnRH-2 on gonadotrophic cells, at least in this species (Fig. 2) Ontogeny of fish GnRHs neurons In accordance with data obtained in amphibians, birds and mammals, developing GnRH-2 cells in fish originate in the germinal zone of the third ventricle, at a diencephalic/mesencephalic transitional area identified as the synencephalon (Parhar et al., 1996; White and Fernald, 1998). In the European sea bass, GnRH- 2 neurons represent the earliest detectable GnRH cell population during development, being detected at day 4 after hatching (Gonzalez-Martinez et al., 2004b, 2002b). An early expression of the synencephalic/midbrain GnRH form was also reported in other vertebrate species (Muske and Moore, 1990; White and Fernald, 1998) suggesting an important function of this conserved GnRH form during ontogenesis. Interestingly, using in situ hybridization, Wong and colleagues demonstrated in the gilthead seabream very early GnRH-2 expressing cells in the hindbrain at 1.5 days post-fertilization (DPF), and in the midbrain at 2 DPF. While the midbrain GnRH-2 signal intensified, the hindbrain expression became undetectable at 10 DPF (Wong et al., 2004). Although there was a debate concerning the origin of forebrain GnRH systems and, in particular, of preoptic GnRH neurons in species expressing three GnRH forms, it seems now clear that both GnRH-1 and GnRH-3 neurons develop from the olfactory placode in teleost fish (Gonzalez-Martinez et al., 2004b, 2002b; Kah et al., 2007; Okubo and Nagahama, 2008; Okubo et al., 2006). This is in agreement with what is known in other vertebrates where many studies have shown the olfactory origin of forebrain GnRH neurons (Schwanzel-Fukuda and Pfaff, 1990; Wray et al., 1989). Strong evidence was obtained using transgenic medaka lines in which the green fluorescent protein reporter was genetically targeted to forebrain GnRH neurons. Using this elegant approach, Okubo and colleagues corroborated that both GnRH-1 and GnRH-3 neurons originate at the nasal forebrain junction and migrate to their final positions during embryonic development (Okubo et al., 2006). In fish that have only two forms of GnRH (GnRH-3 and GnRH-2), a similar origin and distribution is true for GnRH-3. Using transgenic zebrafish in which a GnRH-3 promoter drives the expression of GFP reporter gene (Abraham et al., 2008), it was also shown that GnRH- 3 neurons originate in the olfactory region and migrate from this zone to the hypothalamus via the terminal nerve ganglion and the ventral telencephalon (Fig. 2; Abraham et al., 2008). However, in contrast to fish with three GnRH forms, in zebrafish the final location of the GnRH-3 neurons is both in the terminal nerve and in the hypothalamus, and GnRH-3 axons innervate the pituitary as well as multiple other CNS regions (Abraham et al., 2008; Palevitch et al., 2007; Steven et al., 2003), suggesting that GnRH-3 plays the hypophysiotropic role of GnRH-1 as well as the role played by GnRH-3 in three-form fish. It should be noted that fish GnRH-1 forms are orthologs of the tetrapod GnRH-1 gene, which is expressed in hypophysiotrophic neurons and differentiates from the olfactory region (Kah et al., 2007; Lethimonier et al., 2004). The increased availability of very sensitive techniques to detect the GnRHs and the expression of the GnRH genes, and to visualize the GnRH neurons, led to recent progress in understanding the early establishment of the GnRH system and its regulation. The tangential migration during development that forebrain GnRH neurons undertake to their target sites in the brain or pituitary is a complex process. These neurons migrate a long distance, traversing several CNS regions, each with its distinct milieu of cells and factors. Consequently, migration of GnRH soma, as well as GnRH axon targeting, are controlled by multiple factors, including chemo-attractants, chemo-repellents, cell adhesion proteins and more (for review see Cariboni et al., 2007). Gaining a better understanding of the factors that are involved in forebrain GnRH system establishment is important in the context of both human health (Gonzalez-Martinez et al., 2004a; Soussi-Yanicostas et al., 2002) and aquaculture (Zohar and Mylonas, 2001). Transgenic fish in which forebrain GnRH neurons are endogenously marked by fluorescent proteins represent a unique and highly effective model for studying the regulation of GnRH migration. Using transgenic medaka and zebrafish models, several factors have been shown to play a part in various stages of GnRH migration, including GnRH- 3(Abraham et al., 2008), NELF (Palevitch et al., 2009) and Kal1 (Okubo et al., 2006). The medaka, zebrafish and other transgenic fish will continue to develop as effective models for studying the early migration of GnRH neurons, the establishment of the GnRH system and the regulation of these processes in vertebrates GnRH functions While the physiological functions of the GnRHs during early ontogeny are not very clear, a real-time fluorescence-based quantitative polymerase chain reaction study in the gilthead seabream demonstrated several concomitant elevations of mrna levels of all three forms of GnRH, GnRH receptors, FSHb, FSH receptor, LH receptor and Vasa during the first 36 days post-fertilization (Wong et al., 2004). This finding indicates an orchestrated establishment of the main endocrine components of the brain pituitary gonadal axis during the first month of seabream life. In the European sea bass, GnRH-3 and GnRH-1 increase, together with GnRH receptor mrnas, at the time of sexual differentiation in both males and females, indicating possible brain influence on this process and most notably on FSHb gene expression (Moles et al., 2007). It is also important to note that GnRH-expressing cells and axons have also been localized to extra-encephalic regions including the retina, trigeminal ganglion, trunk and gonads (Abraham et al., 2008; Okubo et al., 2006). The functional significance of this peripheral GnRH expression is yet to be discovered. Although the final neuroanatomical organization of the GnRH system in fish is laid out fairly early in development (Gonzalez- Martinez et al., 2004b, 2002b), patterns of GnRH synthesis and secretion change throughout the lifecycle of fish. A detailed characterization of pituitary GnRH levels during juvenile, pubertal and mature stages of reproductive development in the striped bass

6 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) Fig. 2. (A) Schematic representation of the three GnRH systems in the European sea bass. Circles correspond to the cell bodies expressing sgnrh (red), sbgnrh (green) or chicken GnRH II (pink). Arrows correspond to the main projections. (B D) Transverse sections corresponding to the circular regions in (A) showing sgnrh neurons in the nucleus olfactoretinalis (B), sbgnrh neurons in the lateral POA (D) and cgnrh II neurons in the tegmentum of the midbrain (C) (Gonzalez-Martinez et al., 2002a,b; Kah et al., 2007). (E and F) GnRH-3 neurons in a mature transgenic (GnRH-3:EGFP) zebrafish intact adult brain. (E) Ventral view of the ON, OB, Tel and PPa. GnRH-3 somata are located in a continuum from the anteroventral OB to the Vv and are connected by fiber tracts. (F) Enlarged fluorescent image of boxed area in (E). GnRH-3 somata are scattered in the PPa apposed to the anterior commissure. ON-olfactory nerve, OB-olfactory bulb, Tel-telencephalon, PPa-parvocellular preoptic nucleus, Vv-ventral nucleus of the ventral telencephalon. (E) Scale bar: 100 lm. (F) Scale bar = 250 lm. (G) GnRH-3 neurons in a transgenic (GnRH-3:EGFP) zebrafish intact larvae. At 3 dpf, GnRH-3 neurons are clustered bilaterally in the olfactory region (arrows). Scale bar: 50 lm. (H) At 6 dpf GnRH-3 neurons are migrating, forming a loose continuum between the olfactory region and the telencephalon (arrows). Also, clearly visualized are the GnRH-3 fibers that extend from soma into the CNS (arrowheads). Scale bar: 50 lm. CC, corpus of the cerebellum; Hyp, Hypothalamus; MT, midbrain tegmentum; MO, medulla oblongata; OB, olfactory bulb; ON, olfactory nerve; OT, optic tectum; Pit, pituitary; POA, preoptic area; PPa, parvocellular preoptic nucleus; SV, saccus vasculosus; Tel, telencephalon; TNgc, terminal nerve ganglion cells; SC, spinal cord; VC, valvula of the cerebellum; Vv, ventral nucleus of the ventral telencephalon. showed that GnRH-1 and GnRH-2 increased during gonadal recrudescence and peaked around the time of spawning in maturing males and females (Holland et al., 2001). In addition, levels of these two GnRH forms showed a similar seasonal peak in juvenile animals, although absolute abundances tended to increase with the maturational state of the fish. Although low levels of GnRH-3 were present in pituitaries throughout reproductive development, seasonal peaks in this GnRH form were seen only in maturing animals. In spite of a generalized pattern of anatomical localization for the different GnRH neurons, the relative presence of the various GnRH peptides in the pituitary appears to vary between species, regardless of the number of forms present in the brain. Thus,

7 444 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) GnRH-2 is found in the pituitary of siluriforms (Ngamvongchon et al., 1992; Schulz et al., 1993) and cyprinidontiforms (Steven et al., 2003; Yu et al., 1988), which have two forms, but not found in the pituitary of salmonids (Amano et al., 1991; Okuzawa et al., 1990), which also have two forms. The significance of these species differences is unknown. Although secretion of the GnRH peptides is regulated by a variety of factors in accordance with reproductive activity (see sections that follow), regulation of GnRH gene activity is also tightly coordinated with the reproductive cycle. In salmon species, increases in the number and GnRH mrna content of forebrain GnRH neurons coincide with increased gonadosomatic index and are associated with precocious maturation of males (Amano et al., 1997; Ando et al., 2001). In addition, increases in GnRH mrna are seen in forebrain neurons during late stages of spawning migration, coinciding with final gonadal maturation (Onuma et al., 2005; Parhar et al., 1994). In species with three forms of GnRH, such as perciforms, increased mrna levels of all three GnRH genes are associated with adult reproductive activity, although this does not necessarily reflect a direct action at the level of the pituitary. In the gilthead seabream, for example, pituitary contents of the three endogenous GnRH forms were examined at several different stages of the reproductive cycle, using highly specific ELISAs (Holland et al., 2001). Although GnRH-2 was present in pituitaries of first-year spawning males, recrudescent fish undergoing sex determination, and mature, spawning males and females, levels were similarly low at all stages. GnRH-1 in this species was significantly higher in the pituitaries of spawning males and females compared to recrudescent fish, and these levels corresponded precisely with plasma LH content. GnRH-3 was not detected in the seabream pituitary at any stage. In addition, a detailed examination of GnRH mrna levels in the brains of mature females during the spawning season further revealed daily cycles of GnRH gene expression closely related to daily spawning activity (Gothilf et al., 1997). Eight hours prior to spawning, peaks in mrna synthesis for all three forms of GnRH were seen, corresponding with peaks in plasma LH and maturational hormone. Interestingly, copy numbers of GnRH transcripts were highest for GnRH-3, followed by GnRH-2 and GnRH-1. Likewise in the red seabream female, mrna expression of the same three endogenous GnRH forms peak just prior to the spawning season (Okuzawa et al., 2003), although a corresponding increase in GnRH-1 peptide is only seen in the pituitary, while GnRH-3 remains low and GnRH-2 is undetectable (Senthilkumaran et al., 1999). Although all three GnRH forms are found in the pituitary of European sea bass, only GnRH-1 is elevated during the spawning season in mature males (Rodriguez et al., 2000). Considered together, these studies demonstrate a tight link between GnRH-1 expression and adult reproductive activity, and further suggest that GnRH-2 and GnRH-3 likely have neuroendocrine roles in the reproductive cycle other than the known hypophysiotropic functions. The central involvement of GnRH in regulation of LH release has been functionally established in all orders of teleosts. Although assays for quantification of FSH peptide are lacking for most fish species, work in rainbow trout (Breton et al., 1998; Mananos et al., 1999; Weil et al., 1999) and coho salmon (Dickey and Swanson, 1998) have shown that, as in mammals, GnRH also stimulates FSH secretion in fish, at least in salmonids. Molecular assays of gene expression have enabled a much closer examination of the role of GnRH in regulating gonadotropin (GTH) synthesis, and the studies performed to date indicate that the relative effects of GnRH on gene transcription of the FSH and LH subunits depends upon the species, sex and reproductive status of the fish (for review see Yaron et al., 2003). In striped bass, for instance, bolus injection of mammalian GnRH analog (GnRHa) to maturing males undergoing early stages of spermatogenesis results in increased mrna levels of all three gonadotropin subunits, although the increase in FSHb subunit mrna is delayed and significantly less than that of the LHb and GTHa subunit mrnas (Hassin et al., 1998). However, in females undergoing early stages of oocyte maturation, acute treatment with GnRHa results in increased levels of the GTHa and LHb mrnas only. Similarly in common carp, no change in FSHb mrna was seen in post-vitellogenic females, while LHb mrna increased significantly in response to GnRH. Significant increases in both FSHb and LHb mrnas were seen in mature spermiating males (Kandel-Kfir et al., 2002). Differential regulation of GTH gene expression was also demonstrated in reproductively quiescent European sea bass, where GnRHa treatment increased mrna levels of the GTHa and LHb, but not FSHb subunits (Mateos et al., 2002). As one might expect, pituitary responsiveness to GnRH treatment, in terms of GTH subunit synthesis, changes during sexual development. In juvenile common carp, for instance, no change in GTH subunit gene activity was seen in response to GnRH treatment, while first-year maturing males showed an increase in both LHb and FSHb subunit mrnas (Kandel-Kfir et al., 2002). In contrast, chronic administration of GnRHa (in combination with testosterone) in striped bass males resulted in a significant increase in all three GTH subunit mrnas in juveniles, and no effect in maturing animals (Hassin et al., 2000). These data are supported by many in vitro studies generally confirming that GnRH has direct effects on synthesis and release of both FSH and LH. When considered together with the in vivo data, studies using cultured pituitary cells or fragments from goldfish (Hassin et al., 1998; Khakoo et al., 1994) salmon (Ando et al., 2006, 2004; Dickey and Swanson, 2000), trout (Mananos et al., 1999), tilapia (Gur et al., 2002; Melamed et al., 1996) and catfish (Bosma et al., 1997; Rebers et al., 2000a, 2002) demonstrate that the effects of GnRH on GTH activity are varied, tailored to the particular reproductive physiology of the animal, and can be elicited through mechanisms affecting gene transcription, peptide secretion, downstream feedback mechanisms, or a repertoire of all three mechanisms, as has been indicated in experiments in African catfish (Rebers et al., 2000a, 2002). Indeed, studies in striped bass (Holland et al., 1998, 2002) have shown that while chronic in vivo administration of GnRHa is able to stimulate accumulation of LH in the pituitaries of juvenile females and maturing males, a much higher dose of GnRHa, coinjected with testosterone, is needed to elicit LH release in these animals, indicating that the effects of GnRH on the distinct processes of GTH synthesis and secretion are not directly coupled. These studies further demonstrated that LH secretion in response to GnRH is potentiated by gonadal steroids, an effect that has been demonstrated more explicitly in a variety of teleost species using both in vivo and in vitro studies (Weil and Marcuzzi, 1990; Ando et al., 2004; Mateos et al., 2002; Trudeau et al., 1993a; Yen et al., 2002) GnRH receptors in fish Confounding the effects of GnRH on gonadotropin synthesis and release in fish is the presence of multiple isoforms of GnRH in teleosts (Kah et al., 2007; Lethimonier et al., 2004). Studies comparing the relative LH releasing effects of the native GnRH isoforms in teleost species, including goldfish (Chang et al., 1990; Johnson et al., 1999; Khakoo et al., 1994), catfish (Rebers et al., 2000b; Schulz et al., 1993) and seabream (Zohar et al., 1995), as well as in vitro experiments directly testing receptor activation (Alok et al., 2000; Bogerd et al., 2002; Okubo et al., 2001), demonstrated that the most potent GnRH form in terms of bioactivity is GnRH-2, while the species-specific type 1 isoform is generally the least potent. In agreement with the higher potency of GnRH-2 compared to type 1 GnRH, it is worth mentioning that apart from the mammalian type 1 GnRH (Reinhart et al., 1992) receptor, all GnRH receptors cloned in vertebrates have a higher sensitivity to

8 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) GnRH-2, at least in terms of inositol phosphate production (Bogerd et al., 2002; Illing et al., 1999; Kah et al., 2007; Lethimonier et al., 2004; Moncaut et al., 2005; Okubo et al., 2003, 2002a,b; Robison et al., 2001). This is the case in species like goldfish, catfish, medaka or in perciforms in which 5 GnRH receptors have been and cloned (Kah et al., 2007; Lethimonier et al., 2004; Moncaut et al., 2005). One notable exception in the literature was reported for goldfish. In sexually mature, prespawning goldfish, GnRH-2 elicits a more robust LH secretion compared to GnRH-1, yet in sexually regressed animals, GnRH-1 had potent LH releasing activity and GnRH-2 had no effect (Khakoo et al., 1994). Incidentally, these differential effects of the two GnRH forms on LH release in goldfish correspond with relative differences in LH subunit synthesis as measured by changes in mrna levels (Khakoo et al., 1994). This further illustrates the changing responsiveness of the gonadotrophs to GnRH according to the reproductive cycle, and studies in several teleost species suggest that this might be achieved through changing expression patterns of multiple GnRH receptor types or signaling pathways. Aside from such mechanisms governing changes in pituitary responsiveness to the GnRHs, a major factor in neuroendocrine control of GnRH action in fish is likely to be the automodulatory actions of the various forms of GnRH within the pituitary. Although ample data exists describing the changing patterns of GnRH forms in fish pituitaries, few studies have been conducted that examine the effects of coadministration of different GnRH forms on pituitary function. A combination of studies in African catfish examining LH release in vivo and in dispersed pituitary cells, and GnRH receptor activation in isolated gonadotrophs, revealed both inhibitory and stimulatory effects of combinations of the GnRH-1 and GnRH-2 peptides present in the pituitary of this species (Bosma et al., 2000). Low doses of GnRH-2 that were unable to elicit a significant response in terms of LH secretion and receptor activation were able to completely abolish the effects of stimulatory doses of GnRH-1 when coadministered. Conversely, lower doses of GnRH-1 that were insufficient to elicit a response alone were otherwise able to enhance the effects of stimulatory doses of GnRH-2. Coadministration of intermediate doses of both peptides generally had synergistic effects on gonadotroph function. Studies of this sort, in which physiologically-relevant combinations of the different GnRH forms are tested using experimental models that remain faithful to the endogenous physiological milieu of the animal, have the potential to greatly elucidate our understanding of the coordinated effects of multiple GnRHs on pituitary function in fish and other vertebrates Dopamine inhibits gonadotropin release in some but not in all teleosts species In many teleosts, but not all, dopamine was shown to strongly inhibit gonadotropin release through a mechanism that probably has different adaptative significance depending on the species. Dopamine is a small neurotransmitter that is synthesized from tyrosine through a two step reaction involving the step-limiting enzyme tyrosine hydroxylase and DOPA-decarboxylase. Dopamine is known for exerting a wide range of effects in the brain of vertebrates and for acting on several pituitary functions, notably on prolactin secretion in mammals. The distribution of dopamine in the brain of fish has been extensively studied using different techniques showing the existence of a well developed dopaminergic system (Nieuwenhuys et al., 1998). Dopamine receptors belong to the G-protein coupled receptor (GPCR) family. There are two main classes of DA receptors that differ in their ability to activate (D 1 ) or to inhibit (D 2 ) the enzyme adenylyl cyclase, with each class containing various subtypes (Cardinaud et al., 1998; Kebabian and Calne, 1979). The first evidence of the existence of a gonadotropin inhibiting factor was obtained by Richard E. Peter who performed a very elegant series of experiments clearly showing that some brain factors indeed inhibited GTH release and ovulation in female goldfish (Peter and Paulencu, 1980). In these experiments, electrolytic lesions destroyed different brain regions and some of these lesions caused a dramatic increase in GTH plasma levels (GTH2 or LH) and ovulation in female goldfish held at 12 C, a temperature under which a female goldfish would normally not ovulate. By performing lesions in many brain nuclei and looking at their effects on ovulation, the authors were able to show that an inhibitory factor (GRIF) originates from the antero-ventral preoptic region and to trace a pathway from the preoptic region to the pituitary (Peter et al., 1978; Peter and Paulencu, 1980). Several years later, (Chang and Peter, 1983) demonstrated that dopamine (DA) inhibits GTH release from dispersed pituitary cells or pituitary fragments, suggesting that dopamine could be the gonadotropin inhibitory factor. Further investigation established that DA was acting directly at the pituitary cell level, thus indicating that gonadotrophs carry dopamine receptors (Chang and Peter, 1983). Using specific agonists and antagonists in vivo and in vitro, it was shown that dopamine acts directly onto the gonadotrophs through D2 receptors. This was shown first in goldfish (Chang et al., 1990) and then confirmed in other species such as carp (Peng et al., 1994), African catfish (De Leeuw et al., 1986, 1988), trout (Saligaut et al., 1992), tilapia (Levavi-Sivan et al., 2006; Yaron et al., 2003), eel (Dufour et al., 1988, 2005) and gray mullet (Aizen et al., 2005). The rainbow trout is the only species in which dopamine is known to inhibit both LH and FSH release acting through D2 receptors (Vacher et al., 2002, 2000). Dopamine also acts to inhibit GnRH release from GnRH neurons as shown in goldfish. This effect would involve D2 receptors on GnRH pituitary terminals and D1 receptors onto GnRH cell bodies (Yu et al., 1991). Thus, dopamine in goldfish has a double effect, inhibiting gonadotropin release by direct action on the gonadotrophs and reducing GnRH secretion in the vicinity of the gonadotrophs (Trudeau, 1997). In contrast, dopamine inhibition of gonadotropin release was not observed in many other teleost fish, particularly in marine species (Copeland and Thomas, 1989). It is very likely that the dopaminergic inhibition has different physiological and adaptative meanings with respect to the species. In the goldfish, dopamine prevents ovulation when environmental conditions are not appropriate. Early studies showed that the preovulatory surge in female goldfish does not occur at low temperature or in the absence of aquatic vegetation necessary for sexual behavior and spawning (Stacey et al., 1979; see chapter on behavior). In male goldfish, dopamine was shown to prevent spermiation prior to ovulation of potential female partners. Indeed, increased gonadotropin secretion necessary for sperm formation is stimulated by the female pheromone 17a,20b-dihydroxyprogesterone and this is associated with a reduction in pituitary dopamine turnover (Sloley et al., 1992). For this reason, in cyprinids, silurids and tilapia, combined treatment with GnRH agonist and dopamine D2 antagonist, such as pimozide, are extremely efficient to induce ovulation and spermiation (Peng et al., 1994). In contrast, in salmonids, dopaminergic inhibition prevents precocious release of LH during the vitellogenic period. This effect is stimulated by estradiol and thus is strong during vitellogenesis. It decreases at the end of the vitellogenic period, which is the reason why dopamine antagonists are useless for promoting final maturation and ovulation in salmonids (Vacher et al., 2002, 2000). In the European eel, dopamine has been implicated in the blockade of puberty at the silver stage. Removal of DA inhibition is required for triggering GnRH-stimulated LH synthesis and release, as well as ovarian development. Indeed, sustained treatments of silver eels with GnRH agonist (GnRHa), DA-receptor antagonist

9 446 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) (pimozide), and testosterone (T) trigger dramatic increases in LH synthesis and release, as well as an elevation in plasma vitellogenin levels and stimulation of ovarian vitellogenesis (Weltzien et al., 2006) The KISS/GPR54 pair: a new actor in neuroendocrinology In 2003, our understanding of the regulation of reproduction and puberty was revolutionized by the discovery of the KiSS1/ GPR54 system. Pionneering studies (de Roux et al., 2003; Seminara et al., 2003) revealed that inactivating mutations in the G-coupled protein receptor 54 (GPR54, KiSS1r) resulted in idiopathic hypogonadotrophic hypogonadism, where the affected individuals did not undergo puberty. This phenotype is associated with reduced circulating LH levels, yet maintains the ability to respond to GnRH administration. The role of GPR54 in eliciting this phenotype was further confirmed by knockout studies in mice (Seminara et al., 2003). The identification of this new regulatory system has triggered a massive research effort into the role and mechanism of action of GPR54 and its ligands, the kisspeptins. It is now confirmed that KiSS1/GPR54 signaling is central to the regulation of GnRH, and consequently LH and FSH secretion, as well as being implicated in a growing list of key biological functions, including nutrition, metabolism and response to photoperiodicity (Revel et al., 2006; Roa and Tena-Sempere, 2007). The KiSS system is now thought to be the link mediating the response between environmental cues and metabolic signals to the reproductive axis and is considered, in mammalian systems, as the gatekeeper of puberty and reproduction (Tena-Sempere, 2006). The kisspeptins are the products of the KiSS1 gene, which encodes for a 145 amino acid precursor protein that is cleaved to yield a family of four biologically active peptides, 54, 14, 13 and 10 amino acids long (Kotani et al., 2001). They were first identified in non-metastasizing cancer cells (Lee et al., 1996) and were later found to be the ligand for the G-coupled protein receptor 54, now named KiSS1r. The KiSS1 gene and its products have been characterized thus far in a number of mammalian systems, and its key role is considered to be as a modulator of reproduction. Administration of kisspeptins resulted in the secretion of LH and FSH (Navarro et al., 2005), solely through interaction with the GnRH system, although the precise sites of interaction remain to be determined. Indeed, with the exception of the mare, where a few GnRH/KiSS contacts were identified in the median eminence (Decourt et al., 2008), there is little information on this essential aspect. Part of the reason is the difficulty in obtaining proper antibodies against the KiSS1 sequence in mammals (Alain Caraty, personal communication). The brain localization of rodent KiSS1 expression was determined by in situ hybridization, revealing that there are two populations of KiSS1-expressing cells, both located in the hypothalamus. The first is located at the arcuate nucleus (ARC) and the second at the periventricular nucleus (AVPV). These two populations appear to be differentially regulated by sex steroids, with estradiol relaying a negative feedback action on the KiSS1 cells in the ARC and a positive feedback for the cells in the AVPV (Franceschini et al., 2006; Roa et al., 2008a). These feedback mechanisms appear to mediate KiSS1 control of LH and FSH secretion, acting via the GnRH system. The differential distribution of estrogen receptor (ER) a and b is associated with the specific modulations of LH response to kisspeptins and the generation of preovulatory LH surge (Franceschini et al., 2006; Roa et al., 2008b). The kisspeptins and the KiSS1r messengers were found to be also expressed in a range of other tissues including the pituitary, ovary, testes, placenta and adipose tissue, pointing to possible functional pleiotropy, which has yet to be explored. The functional intricacy of the KiSS system is far from being fully elucidated, and the current findings have been extensively reviewed (Dungan et al., 2006; Roa et al., 2008c). For the purpose of this chapter, we will focus on KiSS1 and its receptor in the context of the brain and reproduction KiSS in fish The KiSS1 gene sequence has been quite divergent in evolution, and that delayed its identification and isolation from non-mammalian vertebrates. However, recently a number of laboratories have reported the identification and characterization of a KiSS1 gene from zebrafish, (van Aerle et al., 2008) and medaka, (Kanda et al., 2008). In addition, very recent data indicate the presence of two genes encoding different KiSS peptides in zebrafish, medaka and sea bass (Felip et al., in press; Kitahashi et al., 2008). According to recent synteny analysis, the KiSS gene was duplicated before the divergence of sarcopterygians and actinopterygians, but it seems that the KiSS2 gene was lost in placental mammals. There is also evidence that both genes are lacking from the bird genome (Felip et al., in press). In fish, Figs. 3 and 4 shows that the KiSS1 gene generates a decapeptide with the sequence YNLNSFGLRY while that generated by the KiSS2 gene is FNFNPFGLRF. Thus the two peptides are quite divergent and would certainly result in different efficacies on the KiSS1r(s). For the moment, the only physiological evidence for an effect of KiSSes on gonadotropin release was obtained in the sea bass. It was shown that injection of KiSS2 was more potent than KiSS1 in inducing FSH and LH release in vivo, while the opposite was obtained in rat (Felip et al., in press). However, whether those effects were caused by direct activation of gonadotropin release or indirectly via the GnRH system is unknown. It is however worth pointing out that a potential anatomical association of KiSS1r with the GnRH system was identified through the work of Parhar et al. (2004). Using laser capture technology and RT-PCR in single cells, the co-expression of GnRH and KiSS1r was suggested in tilapia (Parhar et al., 2004). In fish, the anatomical distribution of KiSS1 mrna-expressing neurons was examined in the medaka (Kanda et al., 2008), where two populations of cells expressing KiSS1 were identified in the Fig. 3. Sequences of the KiSS1 and KiSS2 in vertebrates (adapted from Felip et al., in press and Kitahashi et al., 2009).

10 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) Rat Mouse Human Opossum Platypus Xenopus Zebrafish Sea bass Medaka Lamprey Lamprey Lizard Platypus Zebrafish Xenopus Medaka Sea bass Stickleback Fugu Tetraodon KISS1 KISS2 Fig. 4. Phylogenetic analysis of kiss1 and kiss2 cdna sequences (adapted from Felip et al., in press and Kitahashi et al., 2009). hypothalamic nuclei, the nucleus posterioris periventricularis (NPPv) and the nucleus ventral tuberis (NVT). As in mammals, the two populations exhibited a differential response to sex steroids, with only the KiSS1 expressed in the cells at the NVT being positively regulated by estrogens. Sexual dimorphism was also apparent, with males exhibiting an increased number of KiSS1 neurons in the NVT (Kanda et al., 2008). More recently, in situ hybridization and laser capture microdissection coupled with real-time PCR showed KiSS1 mrna expression in the ventro-medial habenula and the periventricular hypothalamic nucleus of zebrafish and medaka (Kitahashi et al., 2008). The KiSS2 mrna expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus (Kitahashi et al., 2008). In the zebrafish, there is preliminary evidence for the presence of a KiSS-immunoreactive system with perikarya in the nucleus posterioris periventricularis and above the lateral recess with extensive projections to the ventral telencephalon, preoptic area, hypothalamus and pituitary. Furthermore, KiSS positive terminals were observed in close association with GnRH-3 perikarya in the olfactory bulbs and ventral telencephalon (M. Vosges and O. Kah, unpublished data) KiSS receptor in fish As is the case for most GPCRs, the sequence of the KiSS1r (GPR54) has been much more conserved throughout evolution than that of its ligand. This fact facilitated the isolation of the KiSS1r transcript from a number of fish species, including tilapia (Parhar et al., 2004), gray mullet (Nocillado et al., 2007), cobia (Mohamed et al., 2007), fathead minnow (Filby et al., 2008) and zebrafish (van Aerle et al., 2008). In the fathead minnow, brain distribution of the KiSSr was examined using RT-PCR (Filby et al., 2008). The majority of expression was found to be in the telencephalon and the olfactory bulbs, where GnRH expression was also detected, consistent with their linked functionality and further supporting the conserved role of the KiSS system in fish. The expression of the KiSS1r was examined in a number of fish species by means of quantitative PCR. In tilapia, Parhar et al. (2004) suggested that KiSS1r is expressed in GnRH-expressing neurons, and expression was found more in cells from mature male tilapia neurons compared with immature male tilapia, indicating it may have a reproduction related role. In the gray mullet, KiSS1r was examined in the brain of females displaying early (perinucleolar oocytes), intermediate (secondary yolk globule stage) and advanced (tertiary yolk globule stage) gonadal development (Nocillado et al., 2007). KiSS1r expression was found to be the highest in the females displaying the early stages of gonadal development. A similar expression profile was also observed for GnRH-2 and GnRH-3. In the cobia, similarly to the situation in mullet, the expression of KiSS1r follows the same pattern as that of GnRH (Mohamed et al., 2007). In this species, expression was detected in larvae from the first day post-hatch, and was also present in fingerlings and fish up to 6 months old, a time during which the males have undergone puberty while the females have not. Sexual dimorphism was observed, with expression of KiSS1r in the males higher than that observed in the females, correlating with the pubertal stage of the males and further supporting the conserved role of KiSS1r in reproductive development. In the fathead minnow, the brain expression of KiSS1r is tightly aligned with that of GnRH-3, which is believed to be the hypophysiotropic form in this species (Filby et al., 2008). Here, as was found in the mullet, expression levels of KiSS1r in females were higher at the early stages of gonadal development/entry into puberty and decreased as the gonad progressed. As was found in the cobia males, KiSS1r expression in the fathead minnow was high at the early stages of spermatogenesis. A similar pattern was also found in the zebrafish, where expression of KiSS1r increased just prior to puberty and decreased once egg development was established (Biran et al., 2008). In this species, two KiSS1r sequences were identified, with the second following a slightly different pattern of expression. Taken together, the results obtained in fish confirms the significance of the KiSS1/KiSS1r system in lower vertebrates (Fig. 6) and the need for additional cross-species studies of a more uniform nature, so that comparisons can be made between expression patterns of KiSS1r and KiSS1 in fish species that display distinct reproductive strategies Other factors Neuropeptide Y Neuropeptide tyrosine or NPY is a 36 amino acid peptide of the pancreatic peptide family, which is strongly implicated in the regulation of feeding in fish and mammals. As with all peptides, NPY is cleaved from a large precursor that is processed by convertase and carboxypeptidase. Its name comes from the fact that the sequence starts and ends with a tyrosine residue (tyrosine abbreviation is Y). NPY is highly effective in stimulating GH in the goldfish (Peng et al., 1993c). However, NPY is also implicated in the regulation of GTH release (Breton et al., 1991; Kah et al., 1989a; Peng et al., 1993b) and could be one of the factors linking the growth, feeding and reproductive axes. In this context, many fish species reduce food consumption during the reproductive period. Thus, the expression of NPY in the hypophysiotrophic preoptic area increases during fasting (Silverstein et al., 1998), and stimulatory effects of NPY on LH secretion are also greater in fasted animals (Cerda-Reverter et al., 1999). NPY in goldfish was shown to be directly involved in the release of LH at the level of the pituitary, however NPY is also able to release GnRH from GnRH terminals in the pituitary or preoptic region slices (Peng et al., 1993a; Trudeau, 1997). Using immunohistochemistry and in situ hybridization, NPY expression was detected mainly in forebrain regions, particularly in the nucleus entopeduncularis of the ventral telencephalon, the preoptic area (POA), the olfactory bulbs and various thalamic regions of the goldfish (Kah et al., 1989a). NPY was originally shown to stimulate GTH release in the trout and the goldfish (Breton et al., 1991; Kah et al., 1989a) and this effect was subsequently found to depend on the steroid environment (Breton et al., 1991; Kah et al., 1989a; Peng et al., 1993c). In the goldfish, pretreatment with testosterone or estradiol induced a 2- to 3-fold increase in NPY mrna

11 448 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) levels in the telencephalon/preoptic area, but not in the optic-tectum/thalamus. In situ hybridization using brains taken from T-implanted fish demonstrated that the site of steroid action is the preoptic region (Peng et al., 1994; Trudeau, 1997) Gamma-aminobutyric acid (GABA) GABA is an inhibitory neurotransmitter that is extremely abundant in the brain of all vertebrates. Following the discovery of a heavy GABA innervation in the goldfish (Kah et al., 1987), GABA effects have been explored mainly in goldfish (see review in Popesku et al., 2008), and to a lesser extent in trout. Initially, GABA was shown to stimulate LH secretion in goldfish through effects that include stimulation of GnRH release (Kah et al., 1992; Sloley et al., 1992) and inhibition of dopamine (Trudeau, 1997; Trudeau et al., 2000). Effects on GTH secretion were studied in the goldfish and rainbow trout. GABA injected intraperitoneally caused an increase of serum GTH levels in regressed or early maturing fish, but not in late maturing animals (Kah et al., 1992). Moreover, injection of a GABA transaminase inhibitor caused a significant increase of GABA within the hypothalamus and pituitary, and a dose-dependent increase in serum GTH levels (Sloley et al., 1992). Using in vitro incubation of pituitary slices, it was found that GABA caused a dose-related stimulation of GnRH release at the level of the pituitary, providing a possible explanation for the observed in vivo stimulatory effect of GABA on GTH release. The stimulatory effect of GABA on GTH release was abolished in estradiol-treated females, but was still observed in testosterone-implanted fish. Moreover, estradiol, but not testosterone, caused a decrease of the GABA concentration within the telencephalon (Kah et al., 1992; Trudeau, 1997). In addition to modulating GABA-stimulated LH release, the gonadal steroids also affect GABA synthesis in both brain and pituitary; for example, testosterone and progesterone decrease and estradiol increases pituitary GABA synthesis rates in sexually regressed goldfish (Trudeau, 1997). These data indicate that GABA neurons in goldfish are very sensitive to changes in circulating sex steroid levels. However, it is still unknown if GABA neurons express estrogen receptor in the goldfish. In the trout, GABA has an overall stimulatory action on FSH and LH secretion in rainbow trout, which depends on the sex and the reproductive stage of the fish. The stimulatory action of GABA may be exerted, at least in part, directly onto the gonadotrophs that receive a heavy innervation by glutamate decarboxylase-positive fibers (Mananos et al., 1999). GABA stimulates both basal and GnRH-induced FSH and LH secretion from dispersed pituitary cells, and this effect is highly dependent on the steroid environment (Mananos et al., 1999). In agreement with an interaction between GABA and estrogens, estrogen receptor a is found in GAD-expressing neurons (I. Anglade and O. Kah, unpublished results) Gonadotropin-inhibitory hormone (GnIH) Gonadotropin-inhibitory hormone is a dodecapeptide recently identified in birds (Tsutsui et al., 2007) that belong to the LPXRF-amide family of peptides also present in amphibian, mammalian and invertebrates (Kriegsfeld et al., 2006; Tsutsui et al., 2007; Tsutsui and Ukena, 2006). This peptide is mainly expressed in hypothalamic and septal neurons from the avian brain and inhibits the synthesis and release of gonadotropins acting directly at the pituitary level (Tsutsui et al., 2007; Tsutsui and Ukena, 2006; Yin et al., 2005). Furthermore, GnIH seems to exert neuromodulatory actions on GnRH cells because preoptic GnRH neurons and GnRH fibers from the median eminence receive a conspicuous GnIH innervation and GnRH cells exhibit GnIH-binding sites (Bentley et al., 2006, 2008). Moreover, GnIH inhibits steroidogenesis and development in avian gonad indicating that this neuropeptide could act at different levels in the reproductive axis (Tsutsui et al., 2007). The synthesis and secretion of hypothalamic GnIH is modulated by melatonin, suggesting that GnIH could be implicated in the transduction of photoperiod information to other endocrine centers involved in the control of reproduction (Tsutsui et al., 2007). The presence of hypothalamic GnIH cells seems to be an evolutionarily-conserved characteristic in vertebrates because the existence of a peptide from the LPXRF-amide family has been recently described in fish (Osugi et al., 2006; Sawada et al., 2002; Tsutsui et al., 2007). In goldfish, these putative GnIH cells are present in the posterior periventricular nucleus of the hypothalamus, as well as in the terminal nerve area, and GnIH-immunoreactive fibers reach the lateral tuberal nucleus, the ventral telencephalon, the optic tectum and the pituitary (Sawada et al., 2002). It remains to be determined whether this LPXRF-amide peptide exerts similar functions in the fish reproductive axis as the avian and mammalian counterparts, but the issue appears of growing interest for reproductive neuroendocrinology in fish. In addition to the above-mentioned factors, a plethora of other neurohormones and neurotransmitters have been shown to stimulate gonadotropin release in vitro from goldfish gonadotrophs. However, the physiological relevance of these factors in other species is not well documented. For review, the reader may refer to the excellent synopsis by Vance Trudeau (Trudeau, 1997). 4. The steroid feedback As mentioned above, there is a permanent communication between the brain/pituitary complex and the periphery of the organism, notably the gonads. This dialogue allows the activity of the different components of the brain pituitary gonad axis to be synchronized at all steps of the life cycle, which is crucial for coordinated responses. Of particular importance are sexual steroids, produced by the gonads, which are used by the brain and pituitary as indicators of the sexual status. Sexual steroids modulate the activity of the neuronal systems influencing the reproductive axis. They notably affect expression of neuropeptides and neurotransmitters, as well as that of their corresponding receptors in the brain and the pituitary. Steroids are also important for the control of sexual behavior and spawning (see chapter on sexual behavior, this issue). These mechanisms are essential components of the hormonal communication along the brain pituitary gonadal axis. However, the precise effects of sex steroids on the above-described neuronal systems are far from being fully deciphered and diverging data have been reported depending on the species, the physiological status, the gonadal steroid examined or the reproductive parameters monitored. Classically, both positive and negative feedback effects have been reported on the synthesis and release of LH in teleosts, using gonadectomy and/or steroid replacement. Negative feedback is documented in many species including salmonids, cyprinids, silurids and perciforms (Aroua et al., 2007; Trudeau, 1997). However, there is also evidence in juvenile fish for a positive feedback of sex steroids on LH content and release (Crim and Evans, 1983). The effects of steroids on FSH release are less documented. A negative effect of estradiol on FSH synthesis was reported in salmonids (Breton et al., 1997; Dickey and Swanson, 1998; Saligaut et al., 1998), whereas estradiol treatment induced an increase in FSHb mrna levels in goldfish in vivo (Huggard-Nelson et al., 2002) and in eel in vitro (Aroua et al., 2007). The mechanisms mediating these effects are likely to be extremely complex and can be caused by direct effects of steroids at the pituitary or the hypothalamic levels. Indeed, both regions contain a high density of estrogen receptors (Fig. 5; Anglade et al., 1994a; Hawkins et al., 2005; Menuet et al., 2004; Navas et al., 1995; Strobl-Mazzulla et al., 2008) and androgen receptors (Blazquez and Piferrer, 2005; Harbott et al., 2007; Sperry and Thomas,

12 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) ). This complexity is even increased by the fact that the brain of fish is well known for its high capacity to convert aromatizable androgens into estrogens (Callard et al., 1990; Pasmanik and Callard, 1988). Recent data have shown that the high brain aromatase activity in fish corresponds to the expression of a specific brain aromatase gene (cyp19a1b), whereas that of the gonads is due to the expression of another aromatase gene (cyp19a1a), encoding a different isoform (Chiang et al., 2001a,b; Tchoudakova and Callard, 1998; Tong and Chung, 2003). This situation is in contrast with the situation in other vertebrates where only one aromatase gene is controlled by tissue-specific promoters. Therefore, in teleost fish, many effects of aromatizable androgens, such as testosterone, can in fact be mediated by ERs after the conversion of testosterone into estradiol at the brain level. In several teleost species, it has been shown that the aromatase activity corresponds mainly to the anterior brain, notably the telencephalon, the preoptic region and the mediobasal hypothalamus (Timmers et al., 1987). Surprisingly, in the brain of teleost fish, aromatase B expression is found exclusively in radial glial cells and not in neurons as observed in mammals (Forlano et al., 2001; Menuet et al., 2003, 2005; Pellegrini et al., 2005, 2007; Strobl-Mazzulla et al., 2005). These cells are non-neuronal cells strongly involved in embryonic neurogenesis in birds and mammals (Pinto and Gotz, 2007). In the developing mammals and birds, radial cells produce new neurons by asymmetrical divisions and these new neurons migrate along the radial process in the deeper layers of the brain. Very recently, similar mechanisms have been reported in fish, showing that aromatase B-expressing radial glial cells are progenitors in the brain of adult fish (Pellegrini et al., 2007). Aromatase B messengers and proteins are also found in all parts of the pituitary, but the precise localization is unknown and seems to vary across species (Menuet et al., 2003). Although they strongly express glucocorticoid receptors (Teitsma et al., 1999), GnRH neurons do not express nuclear estrogen receptor in fish, as in mammals (Navas et al., 1995). Therefore, the documented effect of estrogens on GnRH expression or content is mediated by other neuronal types, possibly the KiSS neurons as shown in rodents (Roa et al., 2008b). In the trout or goldfish, both estradiol and testosterone stimulate GnRH-3 content in the brain and the pituitary of triploid rainbow trout (Breton and Sambroni, 1996; Trudeau, 1997). In the eel, estradiol or the combination of estradiol and androgens caused an increase in brain and pituitary type 1 GnRH levels, whereas androgens administered alone had no significant effect (Montero et al., 1995). Similarly, testosterone, but not 11b-hydroxyandrostenedione, accelerates the development of type 1 GnRH neurons in catfish, suggesting that testosterone effects are mediated by aromatization (Dubois et al., 1998). While androgens appear to have no direct effect on GnRH synthesis, they increase the sensitivity of the LH cells to GnRH (Breton et al., 1997; Trudeau, 1997; Trudeau et al., 1993a). In contrast to GnRH neurons, the dopaminergic neurons of the preoptic area all express estrogen receptor a in the trout (Linard et al., 1996) and probably also in other species with the potential exception of the eel. Indeed, estrogens were shown to deeply affect the dopaminergic inhibitory tone on LH release in several species (De Leeuw et al., 1988; Linard et al., 1995; Trudeau et al., 1993b) and to increase tyrosine hydroxylase expression in trout (Vetillard et al., 2003). Thus, data in goldfish and trout support the assumption that inhibitory dopaminergic neurons are direct targets for estradiol and are probably the principal mediators of negative feedback in fish (Trudeau, 1997). However, the situation may be different in eel, in which estrogens have no effects on tyrosine hydroxylase expression. In contrast, androgens in eel, including the non-aromatizable dihydrotestosterone, strongly up-regulate zferα zferβ1 zferβ2 zferα zferβ1 zferβ2 Dl A Dp Dd Dm Dc Vp Cant DiV PPa Dm Dl Dc Vp Dp D TeO ATN LH Hv TL TPp PTN Hd Pit B C PPa DiV Dl Dm Dc Dp PPa DiV CO E TL TeO TPp PVO Hd PTN LH Hv Pit Fig. 5. Distribution of the three estrogen receptors zfera, zferb1 and zferb2 in the brain of zebrafish. The strongest expression is observed in the preoptic region and the mediobasal hypothalamus, although weaker expression is also present in many brain regions including periventricular areas of the telencephalon and diencephalon. Abbreviations: ATN, anterior tuberal nucleus; Cant, anterior commissure; CO, Optic chiasma; Dc, central zone of dorsal telencephalic area; Dd, dorsal zone of dorsal telencephalic area; DiV, Diencephalic ventricle; Dl, lateral zone of dorsal telencephalic area; Dm, medial zone of dorsal telencephalic area; Dp, posterior zone of dorsal telencephalic area; Hd, dorsal zone of periventricular hypothalamus; Hv, ventral zone of periventricular hypothalamus; LH, lateral hypothalamic nucleus; Pit, pituitary; PPa, parvocellular preoptic nucleus, anterior part; PTN, posterior tuberal nucleus; TeO, tectum opticum; TL, torus longitudinalis; TPp, periventricular nucleus of posterior tuberculum; Vp, postcommissural nucleus of ventral telencephalic area.

13 450 Y. Zohar et al. / General and Comparative Endocrinology 165 (2010) expression of the dopamine synthesizing enzyme (Weltzien et al., 2006). This observation could be related to the fact that the eel brain does not exhibit high aromatase expression similar to other fish (Weltzien et al., 2006). Recently, a detailed study in the striped bass, Morone saxatilis, demonstrated that responsiveness of the neuroendocrine axis to gonadal feedback changes during reproductive development and throughout the adult reproductive cycle, in correlation with the physiological state of the gonads (Klenke, 2006; Klenke and Zohar, unpublished data). Bilateral gonadectomy and steroid replacement, in combination with in vitro experiments utilizing isolated brain slices and pituitary primary cell culture, indicate that a major point of gonadal feedback regulation is at the level of the pituitary. In juvenile females, for example, removal of endogenous gonadal feedback or estradiol replacement has an inhibitory effect on FSHb gene expression during the spring, when adults of this species normally undergo advanced stages of oogenesis. Furthermore, a stimulatory estrogenic effect on expression of the GnRH receptor gene was observed at the same time. Gonadal feedback was absent in juvenile or pubertal females in the fall. However, in pubertal fish, estradiol was able to elicit the same regulatory actions on FSHb and the GnRH receptor gene expression, indicating an increasing responsiveness to steroids of the hypothalamus pituitary axis during puberty. The same regulatory response to steroids was observed for both pituitary genes in adult females, during both the fall and spring. Furthermore, endogenous feedback regulation of FSHb changed from negative in the fall to positive in the spring. These effects are mediated at least partially by direct action on the pituitary, as estradiol has no effect on GnRH gene expression levels in juvenile or pubertal females at any time of year, nor in adult animals undergoing ovarian recrudescence in the fall. Regulation of GnRH gene expression by estradiol appears to be restricted to the spring in adult females, with GnRH-1 being up-regulated while GnRH-2 and GnRH-3 are down-regulated. Thus, there appears to be an absence of direct steroidal feedback control on the GnRH neurons in striped bass females until the stages of advanced oogenesis. This suggests a possible priming effect of low levels of estradiol in juvenile animals that may elicit changes in the GnRH neurons or their environment in the brain, resulting in an adult neuroendocrine profile. In addition, direct, non-steroidal feedback regulation was also seen at the level of the gonadotropin and GnRH receptor genes in the pituitary, implying the complex yet well-coordinated involvement of multiple gonadal factors in regulating the neuroendocrine axis of reproduction (Klenke and Zohar, unpublished data; Klenke, 2006). 5. Conclusions Since the discovery of GnRH in vertebrates over 35 years ago, considerable progress has been made toward deciphering the extremely complex neuroendocrine circuits that control reproduction in mammals and also in fish. The emergence during this time period of molecular and genomic platforms, gene transfer and trangenesis, together with the increased acceptance of fish as vertebrate models in the study of reproductive neuroendocrinology, led to major advances in our basic understanding as well as critical applied contributions in agriculture and medicine. In addition to GnRH and dopamine, considered to be the main modulators of gonadotropin release in fish, we now need to focus our attention on KiSS genes and their receptors (Fig. 6). The field is still in its infancy, but advancing very rapidly. Two KiSS genes have now been in some fish, generating two different peptides, KiSS1 and KiSS2. This adds further complexity to an already very complex system, according to what is known in mammals. The challenge for the future will be to discover the respective sites of expression of these two genes, the organization of the corresponding systems, the localization of their receptors and to decipher the mechanisms mediating their functions. How KiSS neurons interact with the GnRH and dopaminergic circuits remains to be discovered. It is likely that similar to mammals, the KiSS/GPR54 pair is the missing gate to puberty. As such, KiSS neurons could be the final integrator of many environmental and nutritional parameters. In mammals, Fig. 6. Schematic representation of the main circuits controlling gonadotropin release in fish. GnRH and dopamine (in some but not all species), respectively, stimulates and inhibits LH/FSH release directly at the gonadotrophs. These effects are modulated by GABA neurons acting to increase GnRH secretion and dopamine inhibition. Sex steroids modulate the activity of those circuits directly on dopamine and GABA neurons and indirectly, possibly through KiSS neurons, on GnRH circuits. Whether KiSS neurons also integrate photoperiodic information and nutritional status of the animals remains a hypothesis indicated by the question marks.