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Characterisation of kisspeptin system genes in an ovoviviparous teleost: Sebastes schlegeli
... They observed a first increase at 2wph during the larval period, followed by a second one at 13wph concomitantly with the pubertal period in tilapia, leading the authors to suggest a potential involvement of Kiss2 in sex differentiation (2wph), and puberty (13wph). In the ovoviviparous black rockfish (Sebastes schlegeli), where only the kiss1 gene was characterized, an increase in kiss1 mRNA was observed 20 days after birth, during gonadal development (Song et al., 2015). In chub mackerel (Scomber japonicus), both kiss1 and kiss2 mRNA were detected in larvae and showed a similar decreasing trend during larval development (Selvaraj et al., 2015). ...
... Parmi ces potentiels rôles, il a été observé chez le médaka et le poisson-zèbre que les kisspeptines intervenaient dans le régionalisation de l'encéphale et la formation de l'oeil (Wu et al., 2006 ;Hodne et al., 2013) Dans notre étude, nous avons mesuré l'expression génique de kiss1 et kiss2 au cours du développement larvaire (Fig. 13A). Les résultats obtenus décrivent des profils où seul Kiss1 a été caractérisé, il a été rapporté que l'expression de kiss1 augmente à partir de 20 jours post éclosion, ce qui correspond chez cette espèce au développement des gonades (Song et al., 2015). Chez le maquereau (Scomber japonicus), où les deux formes de kisspeptines ont été décrites et caractérisées, il a été trouvé qu'au cours du développement larvaire, les expressions de kiss1 et kiss2 diminuent (Selvaraj et al., 2015). ...
... Selvaraj et al., 2015) et chez le poisson-zèbre et le médaka, la maturation sexuelle à lieu aux alentours de 45 jours post fécondation(Kitahashi et al., 2009). Chez Sebastes schlegeli, le développement des gonades débute dès 20 jours post éclosion(Song et al., 2015). Chez ces espèces, il est proposé que l'activation de l'ensemble de l'axe gonadotrope est essentielle au déclenchement de la différenciation sexuelle et de la puberté.En revanche, chez les espèces pour lesquelles les étapes de différenciation sexuelle et de puberté interviennent plus tardivement, comme le bar européen chez qui la différenciation sexuelle intervient entre 150 et 250 jours post éclosion pour les femelles et entre 200 et 250 jours pour les mâles (Blazquez et al, 2009), on peut émettre l'hypothèse de rôles additionnels pour ces acteurs. ...
Les perturbateurs endocriniens (PE) sont une préoccupation majeure de par leurs effets sur les grandes fonctions physiologiques des organismes etparticulièrement sur la fonction de reproduction. Chez les vertébrés, la reproduction est sous le contrôle d’un axe neuroendocrinien nommé axegonadotrope. Celui-ci comprend différents acteurs cérébraux (gonadolibérines et kisspeptines), hypophysaires (gonadotropines) et gonadiques(stéroïdes sexuels) dont la mise en place et le fonctionnement sont régulés finement. Parmi les mécanismes de régulation, les stéroïdes vont jouer unrôle important en effectuant des boucles de rétrocontrôle. Les PE mimant ces stéroïdes sont donc potentiellement capables d’impacter ces régulations.L’objectif de ce travail était d’évaluer les effets d’un xénooestrogène sur les mises en place des acteurs cérébraux impliqués dans l’axe gonadotrope chezle bar européen (Dicentrarchus labrax). Nous avons d’abord décrit l’ontogénèse des systèmes à kisspeptine, à gonadolibérine et à gonadotropine aucours du développement larvaire. Nous avons mis en évidence une régulation différentielle de ces acteurs suggérant un rôle durant ce processusdéveloppemental. De plus, nous montrons que le 17α-éthinylestradiol (EE2) est capable de perturber l'ontogenèse des systèmes à GnRH2 et à GnRH3au cours des stades précoces du développement larvaire.Ces données constituent une base permettant l’évaluation des effets des PE sur l’ontogénèse des systèmes neuroendocriniens.
... For several years, kisspeptin has been known as the prime neuroendocrine regulator of reproduction in mammals 10 and fishes 8 as well. Moreover, only a few reports are available that describe sexual dimorphism in the expression of kisspeptin and its receptors in various tissues of fishes 4,5,7,[11][12][13] . However, no report is available on sexual dimorphism in the expression of kiss and kissr in the thymus of fishes, even when KISS1 has been described as a metastasis suppressor gene since its discovery 1 . ...
... On the contrary, no sex-related difference in expression of kiss and kissr was reported in any region of the brain of Odontesthes bonariensis 35 . Several other studies in fishes in which total brain has been used for sex-related differential expression of kisspeptin system reported inconsistent results, from no sex-related difference 11,13,[35][36][37][38][39] to high expression in female 4,7,11,12,20,39 or male 13 . The cause and physiological significance of sex-related differential expression of the kisspeptin 1 and its receptors in different regions of brain need to be explored to address this dichotomy. ...
... When sex-related differential expression was examined in the gonads, expression of kiss1 was found to be significantly (P < 0.05) higher in testis than ovary of C. punctatus (Figure 3 b). Our observation is in concordance with reports in Danio rerio 15 , O. latipes 11,34 and C. auratus 20,41 ; however, no sex-related difference in gonadal kiss1 expression was seen in Dicentrarchus labrax 11 , Scomber japonicas 4 and Sebastes schlegeli 12 . ...
The present study paves the way for novel aspects of kisspeptin in the regulation of fish physiology, importantly, immunity and metabolic activities. The expression level of kisspeptin (kiss1) and its receptors (kiss1r, kiss2r) was observed in different regions of the brain, primary and secondary lymphoid organs, liver and gonads of male and female Channa punctatus, suggesting a wider role of kisspeptin in the regulation of fish physiology. Further, expression profile of kiss1, kiss1r and kiss2r revealed sexual dimorphism depending on tissues. Surprisingly, insignificant correlation was observed between the expression of kiss1 and its receptors.
... For instance, in medaka (Oryzias latipes), zebrafish (Danio rerio), goldfish (Carassius auratus), European sea bass (Dicentrarchus labrax), and chub mackerel (Scomber japonicus), two paralogous kisspeptin genes, namely kiss1 and kiss2 (Biran et al. 2008;Felip et al. 2009;Kitahashi et al. 2009;Li et al. 2009;Selvaraj et al. 2010), and their corresponding receptor genes, namely kiss1ra (GPR54a) and kiss1rb (GPR54b), have been identified. In other fish species, such as the Atlantic halibut (Hippoglossus hippoglossus) (Mechaly et al. 2010), grass puffer (Takifugu niphobles) (Shahjahan and Motohashi 2010), Senegalese sole (Solea senegalensis) (Mechaly et al. 2011), and black rockfish (Sebastes schlegelii) (Song et al. 2015), only one isotype of kiss gene and one corresponding receptor gene were identified. The different kisspeptin genes and their receptors likely emerged from the two rounds of whole-genome duplication that took place during early evolution of vertebrates (Pasquier et al. 2012a(Pasquier et al. , b, 2014. ...
... Cloning and analysis of P. olivaceus kiss2 (Pokiss2) and kissr2 (Pokissr2) genes Total RNA and DNA were extracted and quantified as described elsewhere (Song et al. 2015). For the synthesis of first-strand cDNA template, 1 lg of total RNA was reverse transcribed using the Reverse Transcriptase M-MLV (RNase H -) Kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. ...
... Indeed, limited studies of kisspeptin expression in larval have shown similar results. At the development stage, S. schlegeli kiss1 and kissr expression increased gradually from birth and peaked at 20 days after birth (Song et al. 2015). kissr mRNA of R. canadum increased in larvae from 1 day-after-hatching (DAH) until at 18 DAH and peaked at 26 DAH. ...
The role of kisspeptin in puberty onset has been extensively investigated by neuroendocrinologists in the past decade. In the present study, we first cloned and analyzed Pokiss2 and Pokissr2 genes in Paralichthys olivaceus, a Pleuronectiformes fish. By 5'/3' rapid amplification of cDNA ends (RACE), the P. olivaceus kiss2 gene (Pokiss2) and two isoforms of the P. olivaceus kissr2 gene (Pokissr2) transcripts were cloned. During development, Pokissr2 was maternally inherited but Pokiss2 was not, and their expression reached maximum and minimum levels, respectively, when the gonads began to develop. Analysis of tissue distribution revealed that Pokiss2 and Pokissr2 transcripts were predominantly expressed in the brain and gonads, with expression levels in females higher than those in males. Moreover, Pokiss2 and Pokissr2 both showed significantly higher expression in brains and gonads during puberty. In situ hybridization of the ovary at pre-vitellogenesis stage and testis at spermatogonial proliferation stage revealed that both Pokiss2 and Pokissr2 were expressed in spermatocyte, oocytes, and some somatic cells. Our results also showed significantly stronger Pokiss2 expression in the area of the third ventricle of females than males and no Pokissr2 expression in this region in both sexes. These results lay a strong foundation for understanding the role of kisspeptin in neuroendocrine system in teleosts, in particular in Pleuronectiformes.
... However, differences have been noted in the position of dibasic amino acid residues, upstream to kisspeptin-10 regions in few species. Dibasic amino acid residues (KR) have been found five position upstream to Kiss1-10 in medaka (Kitahashi et al., 2009) [26] , zebrafish (Kitahashi et al., 2009) [26] , European seabass (Felip et al., 2009) [12] , chub mackerel (Selvaraj et al., 2010) [58] , goldfish (Li et al., 2009) [30] , striped bass (Zmora et al., 2012) [83] , black rockfish (Song et al., 2015) [73] , catla (Rather et al., 2016) [55] , rohu (Saha et al., 2016) [56] , golden mahseer (Shahi et al., 2017) [64] , pejerrey (Bohórquez et al., 2017) [5] and Atlantic bluefin tuna [48] , suggesting pyroglutamated Kiss1 pentadecapeptide as mature form in these fish. In chub mackerel and grouper species, it is likely that Kiss1 hexadecapeptides are mature forms, based on the position of dibasic amino acid residues (Kang et al., 2012;Ohga et al., 2013) [22,47] . ...
... However, differences have been noted in the position of dibasic amino acid residues, upstream to kisspeptin-10 regions in few species. Dibasic amino acid residues (KR) have been found five position upstream to Kiss1-10 in medaka (Kitahashi et al., 2009) [26] , zebrafish (Kitahashi et al., 2009) [26] , European seabass (Felip et al., 2009) [12] , chub mackerel (Selvaraj et al., 2010) [58] , goldfish (Li et al., 2009) [30] , striped bass (Zmora et al., 2012) [83] , black rockfish (Song et al., 2015) [73] , catla (Rather et al., 2016) [55] , rohu (Saha et al., 2016) [56] , golden mahseer (Shahi et al., 2017) [64] , pejerrey (Bohórquez et al., 2017) [5] and Atlantic bluefin tuna [48] , suggesting pyroglutamated Kiss1 pentadecapeptide as mature form in these fish. In chub mackerel and grouper species, it is likely that Kiss1 hexadecapeptides are mature forms, based on the position of dibasic amino acid residues (Kang et al., 2012;Ohga et al., 2013) [22,47] . ...
... Total RNA and DNA were extracted and quantified as previously described (Song et al. 2015). In brief, the Reverse Transcriptase M-MLV (RNase H-) Kit (TaKaRa, Dalian, China) and DNaseI (TaKaRa, Dalian, China) were used in accordance with the manufacturer's instructions to prepare cDNA templates. ...
... The gene expression profiles of the kisspeptin system during the larval developmental progress have been examined by a few studies on black rock fish (Song et al. 2015) and cobia (Mohamed et al. 2007). However, reports on the gene expression of kiss2 during gonadal differentiation are still very rare at present. ...
Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic-pituitary-gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5'-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.
... It also appears that Kiss2 is involved in food intake and probably also growth in fish (Mechaly et al., 2011;Escobar et al., 2016;Mechaly et al., 2018) and may act as a link between food intake and reproduction, as food restriction upregulates hypothalamic kiss2 concurrently with increased pituitary fsh and lh and a rise in gonadosomatic index (Mechaly et al., 2011;. Additionally, in teleost fish, kiss genes are expressed in other peripheral tissues, and Kiss variants are detected in different tissues and organs, opening new research possibilities for research on kisspeptin systems in the periphery (Mechaly et al., 2013;Song et al., 2015b;Tovar-Bohórquez et al., 2017a;Tovar-Bohórquez et al., 2017b;Mechaly et al., 2018). It is also possible that teleost Kiss2 could be involved in male sex differentiation as suggested in pejerrey (Tovar-Bohórquez et al., 2017a) and in sex reversal in a sequencial hermaphroditic species (Yi et al., 2019). ...
It is well known that gonadotropin-releasing hormone (Gnrh) has a key role in reproduction by regulating the synthesis and release of gonadotropins from the anterior pituitary gland of all vertebrates. About 25 years ago, another neuropeptide, kisspeptin (Kiss1) was discovered as a metastasis suppressor of melanoma cell lines and then found to be essential for mammalian reproduction as a stimulator of hypothalamic Gnrh and regulator of puberty onset. Soon after, a kisspeptin receptor (kissr) was found in the teleost brain. Nowadays, it is known that in most teleosts the kisspeptin system is composed of two ligands, kiss1 and kiss2, and two receptors, kiss2r and kiss3r. Even though both kisspeptin peptides, Kiss1 and Kiss2, have been demonstrated to stimulate gonadotropin synthesis and secretion in different fish species, their actions appear not to be mediated by Gnrh neurons as in mammalian models. In zebrafish and medaka, at least, hypophysiotropic Gnrh neurons do not express Kiss receptors. Furthermore, kisspeptinergic nerve terminals reach luteinizing hormone cells in some fish species, suggesting a direct pituitary action. Recent studies in zebrafish and medaka with targeted mutations of kiss and/or kissr genes reproduce relatively normally. In zebrafish, single gnrh mutants and additionally those having the triple gnrh3 plus 2 kiss mutations can reproduce reasonably well. In these fish, other neuropeptides known to affect gonadotropin secretion were up regulated, suggesting that they may be involved in compensatory responses to maintain reproductive processes. In this context, the present review explores and presents different possibilities of interactions between Kiss, Gnrh and other neuropeptides known to affect reproduction in teleost fish. Our intention is to stimulate a broad discussion on the relative roles of kisspeptin and Gnrh in the control of teleost reproduction.
... Since their first identification in zebrafish and medaka (9), two kisspeptin types have been identified in several teleost species (24,25), but some fish species, such as tilapia (34), Astatotilapia burtoni (27), red seabream (35), Atlantic halibut (36), flatfish Senegalese sole (37), black rockfish (38), Japanese flounder (39), and puffer fish (40) are likely to possess only one (Kiss2) type. Similar to multiple kisspeptin forms, multiple KissR (KissR1, KissR2, KissR3, and KissR4) types have also been identified in various fish species (25,28,41). ...
Kisspeptin is a neuropeptide, encoded by kisspeptin 1 (KISS1)/Kiss1 gene, which primarily acts as the regulator of reproductive functions via its receptor, kisspeptin receptor (KissR) in vertebrates. In the brain, Kiss1 gene is mainly expressed in the hypothalamic region, but KissR gene is widely distributed throughout the brain, suggesting that kisspeptin–KissR system may be involved in not only reproductive, but also non-reproductive functions. In non-mammalian vertebrates, there are two or more kisspeptin and KissR types. The zebrafish (Danio rerio) possess two kisspeptin (Kiss1 and Kiss2) and their respective receptors [Kiss1 receptor (KissR1) and KissR2]. In the brain of zebrafish, while Kiss2 is expressed in the preoptic-hypothalamic area, Kiss1 is predominantly expressed in the habenula, an evolutionarily conserved epithalamic structure. Similarly, KissR1 is expressed only in the habenula, while KissR2 is widely distributed in the brain, suggesting that the two kisspeptin systems play specific roles in the brain. The habenular Kiss1 is involved in the modulation of the raphe nuclei and serotonin-related behaviors such as fear response in the zebrafish. This review summarizes the roles of multiple kisspeptin–KissR systems in reproductive and non-reproductive functions and neuronal mechanism, and debates the biological and evolutional significance of habenular kisspeptin–KissR systems in teleost species.
... In cobia (Rachycentron canadum), kissr2 expression was detected at 1 dph and peaked at 2 and 3 dph (59). In black rockfish (Sebastes schlegelii), both kiss1 and kissr2 expression displayed an increasing trend during early development (60). ...
Kisspeptin, a novel neuropeptide product of the Kiss1 gene, activates the G protein-coupled membrane receptor G protein-coupled receptor 54 (now termed Kiss1r). Over the last 15 years, the importance of the kisspeptin system has been the subject of much debate in the mammalian research field. At the heart of the debate is whether kisspeptin is an absolute upstream regulator of gonadotropin-releasing hormone secretion, as it has been proposed to be the master molecule in reproductive events and plays a special role not only during puberty but also in adulthood. The teleostean kisspeptin system was first documented in 2004. Although there have been a number of kisspeptin studies in various fish species, the role of kisspeptin in reproduction remains a subject of controversy and has not been widely recognized. There is an extensive literature on the physiological and endocrinological bases of gametogenesis in fish, largely derived from studying small, model fish species, and reports on non-model species are limited. The reason for this discrepancy is the technical difficulty inherent in developing rigorous experimental systems in many farmed fish species. We have already established methods for the full life-cycle breeding of a commercially important marine fish, the chub mackerel (cm), and are interested in understanding the reproductive function of kisspeptins from various perspectives. Based on a series of experiments clarifying the role of the brain–pituitary–gonad axis in modulating reproduction in cm, we theorize that the kisspeptin system plays an important role in the reproduction of this scombroid species. In this review article, we provide an overview of kisspeptin studies in cm, which substantially aids in elucidating the role of kisspeptins in fish reproduction.
... Indeed, in goldfish (Carassius auratus), the administration of Kiss1 but not Kiss2 decapeptide increased the serum LH levels [37]. Furthermore, in a recent study, black rockfish (Sebastes schlegelii) was shown to possess kiss1, but potentially lacked the kiss2 gene [38]. At present, it is known that chub mackerel Kiss1 is an important regulator of reproduction. ...
Here we report that the Kiss1 hexadecapeptide (Kiss1-16) directly regulates the functional form of gonadotropin-releasing hormone (GnRH) in the preoptic area (POA) of a scombroid fish model. In this study, we analyzed the localization of two kisspeptin (kiss1 and kiss2) neurons and two kisspeptin receptors (kissr1 and kissr2) in the brain of adult chub mackerel using in situ hybridization to determine whether the kisspeptin receptors co-localize with GnRH1 neurons. The kiss1- and kiss2-expressing neurons were mainly localized in the nucleus recessus lateralis (NRL) and the nucleus of the posterior recess (NRP) in the hypothalamus. Kissr1 was present in the anterior POA and the habenular nucleus. Kissr2 was widely distributed, including in the POA, lateral tuberal nucleus, NRL, and NRP. Notably, GnRH1 was expressed in neurons in the POA, and these neurons co-expressed kissr1. In contrast, kissr2 was expressed abundantly in the vicinity of GnRH1 neurons, but their co-expression did not seem to occur. We also characterized the endogenous mature form of the Kiss1 peptide. An in vitro reporter gene assay clearly showed that Kiss1-16 (HQDMSSYNFNSFGLRY-NH2) was more potent at receptor activation than Kiss1 pentadecapeptide (Kiss1-15), which is the form of Kiss1 found in other fish species. This study strongly suggests that kisspeptin signaling, especially Kiss1 signaling, is important for regulating reproduction in scombroid fish.
... tis and ovary as well as non-reproductive tissues. Previous studies in many fish species revealed high levels of expressions of kisspeptin genes in the brain and gonads (Selvaraj et al., 2010;Shahjahan et al., 2010;Yang et al., 2010). In these species, the paramount expression has been found in the brain and gonads with sexually dimorphic profiles.Song et al., (2015)have reported the levels of kiss2 mRNA in the female fish brain were above 30-fold higher than those in the ovary. The high kisspeptin mRNA in female may be due to difference neuronal density difference in hypothalamus with a greater number of kisspeptin neurons in females compared with males (Clarkson & Herbison, 2006;Kauffman et al., 2 ...
We report the characterization of kisspeptin gene which is considered to be essential for successful animal reproduction. The full-length cDNA sequence of kiss2 was 583 bp, consisted of 11 bp 5'-UTR (untranslated region) and 194 bp 3'-UTR, respectively. Open reading frame of 378 bp encoding a putative protein of 125 amino acids. The Catla catla kiss2 protein was having a molecular weight of 14.51 kDa and isoelectric point (pI) of 8.46. There were four serine (Ser), four threonine (Thr) and two tyrosine (Tyr) phosphorylation sites and no N-glycosylation sites on the predicted protein. The amino acids on positions 8, 11, 24, 80 and 114 were detected to be ligand binding sites. The signal peptide analysis predicted that C. catla kiss2 is a secretory protein. Kiss2 protein is localized in nuclear region (49.7%) and the extracellular region (38.3%) of the cell. Analysis of tissue distribution revealed that, kiss2 transcripts were predominantly expressed in the brain and gonads, with expression levels in female higher than those of male. Ontogenetic analysis of kiss2 demonstrated that expression level was low during early phase of development stages and more expression was observed during mature stage. Overall present results lay a strong basis for understanding the role of kisspeptin in the neuroendocrine system in teleosts.
... The cells were then stored at − 80°C in Trizol reagents (Thermo Fisher Scientific Inc.) until RNA extraction. Total RNA was extracted and cDNA was prepared in accordance with previously described methods (Song et al., 2014). ...
Kisspeptins have been described as one of the most potent activators of the hypothalamic–pituitary–gonadal axis. Kisspeptins control the onset of reproductive functions during puberty by directly stimulating the neuronal activity and release of gonadotropin-releasing hormone (GnRH). The function of kisspeptins has been investigated in vivo and in vitro. In our study, three kinds of recombinant kisspeptin proteins were expressed in Escherichia coli. Kisspeptin fragments Kp54, Kp44, and Kp10 translated from Paralichthys olivaceus kiss2 gene were then obtained. Kp44 contained 44 amide acids (aa) which are the same as the N-terminal of Kp54; Kp10 shares the same 10 aa with the C-terminal of Kp54 but Kp10 also contains some other amide acids. In the dose course of treatments with prokaryotically expressed peptides, Kp54 and Kp10 could induce the expression of kissr2 and gnrh1; by contrast, Kp44 could not induce a similar expression. These results provided direct evidence that the core decapeptide of kisspeptin is necessary to ensure its biological functions. In the time course of the Kp54 treatments on two kinds of cultured brain cells, different patterns of kissr2 and gnrh1 mRNA suggested that the responses of these cells to kisspeptins depends on cell type and treatment duration. Thus, our research provided alternative methods to investigate the functions of kisspeptin in vitro and to detect biological activities; this research also established basis for kisspeptin applications in production processes.
... Testis expression of kisspeptin system genes has been documented in several fish species . For example, levels of kiss1 mRNA have been shown to fluctuate with both testicular development and seasonal reproductive cycles Selvaraj et al., 2010;Song et al., 2015). Thus, it has been suggested that the kisspeptin system is involved in testis function. ...
The kisspeptin system plays pivotal roles in the regulation of vertebrate reproduction. Classically, kisspeptin produced in the brain stimulates brain gonadotropin-releasing hormone signaling, which in turn activates the pituitary-gonad axis. Expression of the kisspeptin system has also been documented in peripheral tissues, including gonads of mammals and fishes. However, the fish gonadal kisspeptin system remained uncharacterized. Herein we report identification and characterization of four kisspeptin system mRNAs (kisspeptin 1 (kiss1), kiss2, and G protein-coupled receptor 54-1 (gpr54-1) and gpr54-2) in sablefish, Anoplopoma fimbria. Sablefish predicted protein sequences were highly similar to those of other marine teleosts, but less so to freshwater teleosts. Tissue distribution analyses revealed that all four kisspeptin-system transcripts were expressed in both brain and gonad. However, kiss2 was the predominant transcript in the gonads and the only transcript detected in ovulated eggs. Ontogenetic analysis of kiss2 expression in juvenile sablefish gonads demonstrated that levels were low during sex differentiation but increased with fish size and gonadal development. Dramatic increases in kiss2 mRNA occurred during primary oocyte growth, while levels remained relatively low in testes. In situ hybridization revealed that kiss2 mRNA was localized to cytoplasm of perinucleolus stage oocytes, suggesting it could play a local role in oogenesis or could be synthesized and stored within oocytes as maternal mRNA. This represents the first study to focus on the gonadal kisspeptin system in fishes and provides important tools for further investigation of both the gonadal and brain kisspeptin systems in sablefish.
Unlike mammals, two kisspeptins genes encoding, kiss1 and kiss2 are detected in fishes with highly varied and contradictory difference in their reproductive activities. The present study was undertaken to examine the direct action of kisspeptin-10 and its role in gonadal activities in the gonadally quiescent Asian catfish using native mammalian kisspeptin decapeptide (KP-10) involving in vivo and in vitro approaches. The in vivo KP-10 treatment caused precocious onset of gametogenesis and its rapid progression, as was evident from the appearance of advanced stages of ovarian follicles in ovary, and advanced germ cells (spermatocytes/ spermatids) in the testis of the treated Clarias batrachus in comparison to the control gonads. It also elevated the steroid levels in gonads of the catfish in vivo and in vitro conditions. Simultaneously, it increased the expressions of key steroidogenic enzymes like 3β-HSD, 17β-HSD, and StAR protein, responsible for transfer of cholesterol from outer to inner membrane of the mitochondria of steroidogenic cells. Concurrently, it augmented the activities of 3β-HSD and 17β-HSD in the ovarian explants. The expressions of MAPK component (pERK1/2 and ERK1/2) were also up-regulated by KP-10 in gonadal explants. Thus, the data suggest that kisspeptin-10 stimulates gametogenesis by enhancing gonadal steroid production. The study also describes the putative mechanistic cascade of steroidogenic actions of kisspeptin-10 in the catfish so much so in teleost fish. The study also suggests that, kisspeptin may act locally to regulate gonadal activities in an autocrine/paracine manner, independent of known extra-gonadal factors in the catfish.
Neurogenesis is an important progress wherein the neural stem cells (NSCs) differentiate into functional neurons under conductive conditions. Neurogenesis occurs continuously in different areas of the central neural system in adult teleosts compared with adult mammals. Therefore, NSC cell lines must be established to offer a valuable in vitro system for studies on neurogenesis and other related functions. In this study, four cell lines designated as PoB1, PoB2, PoBf and PoBh were established from the brain of the Japanese flounder Paralichthys olivaceus. The cell lines were sub-cultured over 150 times and still grew well in DMEM/F12 medium at 24 °C. PoB1, PoB2, PoBf and PoBh were identified as neural stem/progenitor cell lines on the basis of the mRNA expression of nestin and/or aldh1l1 or slc6a4 and the formation of neurospheres. The cells transfected with the pEGFP-C1 plasmid showed fluorescent signals with distinct reagent dependencies. The established cell lines from the brain of P. olivaceus offer a valuable system in vitro for the study of neurogenesis, fish neural regulation and endocrinology-related functions.
In recent years, the Kiss1 gene has been reported in a number of vertebrate species, and a substantial dataset has been acquired to demonstrate the critical role of kisspeptins in the reproductive system; yet limited information is available for carnivores. In the present study, we identified and characterized feline Kiss1 by isolating and cloning its full-length cDNA in the domestic cat hypothalamus and caracal testis, using the method of rapid amplification of cDNA ends. Additionally, we isolated and cloned the 3′ end of Kiss1 cDNA, containing kisspeptin-10 (Kp10), from the ovaries of a clouded leopard and Siberian tiger. Nucleotide sequencing revealed that domestic cat Kiss1 cDNA is of 711 base pairs and caracal Kiss1 cDNA is of 792 base pairs, both having an open reading frame of 450 base pairs, encoding a precursor protein Kiss1 of 149 amino acids. The core sequence of the feline kisspeptin Kp10 was found to be identical in all species analyzed here and is highly conserved in other vertebrate species. Using an anti-Kp10 antibody, we found the immunoreactive kisspeptin to be localized in the periventricular and infundibular nuclei of the cat hypothalamus. The results show that kisspeptin is highly conserved among different feline families, and its immunoreactive distribution in the hypothalamus may indicate its physiological function in the domestic cat.
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Graphical Abstract
In this study, we analyzed the length-weight relationships (LWRs), total length (TL)—standard length (SL) relationships (LLRs), and Fulton’s condition factor (K) of male and female black rockfish Sebastes schlegelii Hilgendorf, 1880 in Lidao Bay, Yellow Sea, China. Among the 729 S. schlegelii specimens sampled seasonally in February, May, August and November between 2011 and 2015, most were males (sex ratio = 1 male:0.79 female). The LWRs of each season were significant (P < 0.05), and all coefficients of determination (r2) were higher than 0.95. The values of the slope (b) estimated for each LWR regression varied from 2.947 to 3.277 and were lower in spring than those in other seasons, especially in females. The LLRs calculated as the regression of TL on SL and vice versa were linear. The values of K ranged from 0.791 to 2.981 in males (n = 407) and from 0.752 to 2.681 in females (n = 322), and the highest value was found in spring. The present results provide baseline information on biological data of LWRs, LLRs, and K for S. schlegelii in this area, and will be useful in further studies on stock management of S. schlegelii.
In recent years, brain kisspeptin system has been shown to be involved in diverse reproductive function, including sexual differentiation in vertebrates. Our previous reports demonstrated that the chub mackerel (Scomber japonicus) brain expresses two kisspeptin (kiss1, kiss2), two kisspeptin receptor (kissr1, kissr2) and three gonadotropin-releasing hormone (gnrh1, gnrh2, gnrh3) genes. In the present study, using quantitative real-time PCR (qRT-PCR) assays, we analysed expression changes of these genes during early development (0-30dphs) and gonadal sex differentiation periods (37-60dphs). Absolute expression level of kiss-kissr-gnrh in the whole head was higher between 0 and 15dphs, in comparison to later developmental periods. Histological analyses revealed presence of sexually differentiated males and females with testicular and ovarian features at 37, 45, and 60dphs. In both males and females, kiss2, kissr1, and kissr2 levels were higher at 37dph, in comparison to 45 and 60dphs, with kiss1 showing no significant differences. Levels of all three gnrh mRNAs were higher at 45dph, in comparison to 60dph. Changes in the expression levels of kiss-kissr-gnrh mRNAs in different brain regions of sexually differentiated males and females indicated differences in their regional distribution. These results suggest possible involvement of Kiss-KissR-GnRH systems during early development and gonadal sex differentiation in the chub mackerel.
The Dmrt genes encode a large family of transcription factors with a conserved zinc finger-like DNA-binding DM domain. The function of Dmrt1, one of the family members, in sexual development has been well studied in invertebrates and vertebrates. In the present study, the full-length cDNA of Dmrt1 was isolated from the testis of Sebastes schlegeli. The full-length cDNA of S. schlegeli
Dmrt1 (SsDmrt1) was 1,587 bp and contained a 189-bp 5′ UTR, a 489-bp 3′ UTR and a 909-bp open reading frame, which encoded 302 amino acids with a conserved DM domain and an male-specific motif domain. Phylogenetic analysis showed the evolutionary relationships of SsDmrt1 with other known Dmrt genes in fish and tetrapods. Several transcriptional factor-binding sites in the 5′ promoter were identified that might regulate SsDmrt1 expression. Quantitative real-time PCR analysis indicated that SsDmrt1 was expressed in all of the inspected larval developmental stages from 1 to 35 days after birth and that the level of expression gradually decreased. The expression of SsDmrt1 in adult gonads was sexually dimorphic with extremely high expression in the testis, but very low expression in the ovary. No expression was detected in other tissues. Using in situ hybridization, we demonstrated that SsDmrt1 was specifically expressed in the germ cells of both the testis and the ovary. Thus, our results suggest that SsDmrt1 may have an important role in the differentiation of both the testis and the ovary of S. schlegeli.
Kisspeptins (Kiss) are prime players in the control of reproductive function through their regulation of gonadotropin-releasing hormone (GnRH) expression in the brain. The experimental scombroid fish, chub mackerel (Scomber japonicus) expresses two kiss (kiss1 and kiss2) and three gnrh (gnrh1, gnrh2, and gnrh3) forms in the brain. In the present study, we analyzed expression changes of kiss and gnrh mRNAs in the brain and corresponding GnRH peptides in the brain and pituitary during final ovarian maturation (FOM) and ovulation.
Female fish possessing late vitellogenic oocytes were injected with GnRH analogue to induce FOM and ovulation. Fish were observed for daily spawning activities and sampled one week post-injection at germinal vesicle migration (GVM), oocyte hydration, ovulation, and post-ovulatory time periods. Changes in relative mRNA levels of kiss and gnrh forms in the brain were determined using quantitative real-time PCR. Changes in GnRH peptides in the brain and pituitary were analyzed using time-resolved fluoroimmunoassay.
Both kiss1 and kiss2 mRNA levels in the brain were low at late vitellogenic stage and increased significantly during the GVM period. However, kiss1 mRNA levels decreased during oocyte hydration before increasing again at ovulatory and post-ovulatory periods. In contrast, kiss2 mRNA levels decreased at ovulatory and post-ovulatory periods. Levels of gnrh1 mRNA in the brain increased only during post-ovulatory period. However, levels of gnrh2 and gnrh3 mRNAs were elevated during GVM and then, decreased during oocyte hydration before increasing again at ovulatory period. During post-ovulatory period, both gnrh2 and gnrh3 mRNA levels declined. Peptide levels of all three GnRH forms in the brain were elevated during GVM and oocyte hydration; their levels were significantly lower during late vitellogenic, ovulatory, and post-ovulatory periods. In contrast, pituitary GnRH peptide levels did not show any significant fluctuations, with the GnRH1 peptide levels being many-fold higher than the GnRH2 and GnRH3 forms.
The results indicate increased expression of multiple Kiss and GnRH forms in the brain and suggest their possible involvement in the regulation of FOM and ovulation in captive female chub mackerel.
The present study assesses the effects of chronic administration of peptides to fish, termed kisspeptins, which are the products of the KISS1 and KISS2 genes, and have been shown to control the development of puberty in animals. Using ecologically and commercially important species (white bass, Morone chrysops, striped bass, Morone saxatilis, and their hybrid) as comparative models, we determined that repeated bi-weekly injections (over 7 weeks) differentially accelerate puberty, as evidenced by increases in the prevalence of spermatozoa in the testes of juvenile fish. Moreover, in sexually mature fish, kisspeptin treatment led to increased gonad weight, gonadosomatic index, and spermatocrit in some white and striped bass. Additionally, mature white bass treated with kisspeptins showed an advancement in oocyte development as determined by histological examination. These gonadal changes occurred in the absence of any photothermal manipulation or hormone injections. To date, this is the first description of kisspeptin-mediated pubertal initiation in fish, and the first evidence that kisspeptins could modulate gonad maturation. Although it remains to be determined how kisspeptins may best be utilized in practice, our findings are a basis for future studies to characterize the molecular underpinnings of the KISS system in various fish species.
Kisspeptin is an important regulator of reproduction in many vertebrates. The involvement of the two kisspeptins, Kiss1 and Kiss2, and their receptors, Gpr54-1 and Gpr54-2, in controlling reproduction was studied in the brains of the modern teleosts, striped and hybrid basses. In situ hybridization and laser capture microdissection followed by quantitative RT (QRT)-PCR detected coexpression of kiss1 and kiss2 in the hypothalamic nucleus of the lateral recess. Neurons expressing gpr54-1 and gpr54-2 were detected in several brain regions. In the preoptic area, gpr54-2 was colocalized in GnRH1 neurons while gpr54-1 was expressed in cells attached to GnRH1 fibers, indicating two different modes of GnRH1 regulation. The expression of all four genes was measured in the brains of males and females at different life stages using QRT-PCR. The levels of kiss1 and gpr54-1 mRNA, the latter being expressed in minute levels, were consistently lower than those of kiss2 and gpr54-2. While neither gene's expression increased at prepuberty, all were dramatically elevated in mature females. The levels of kiss2 mRNA increased also in mature males. Kiss1 peptide was less potent than Kiss2 in elevating plasma luteinizing hormone levels and in up-regulating gnrh1 and gpr54-2 expression in prepubertal hybrid bass in vivo. In contrast, during recrudescence, Kiss1 was more potent than Kiss2 in inducing luteinizing hormone release, and Kiss2 down-regulated gnrh1 and gpr54-2 expression. This is the first report in fish to demonstrate the alternating actions and the importance of both neuropeptides for reproduction. The organization of the kisspeptin system suggests a transitional evolutionary state between early to late evolving vertebrates.
Splice products of the Kiss1 protein (kisspeptins) have been shown to be involved in a diverse range of functions, including puberty, metastasis and vasoconstriction in large human arteries. Circulating Kisspeptin-10 (Kp-10) plasma levels are low in normal individuals but are elevated during various disease states as well as pregnancy. Here, we investigated the potential of Kp-10, the shortest biologically active kisspeptin, to influence microvascular effects, concentrating on the cutaneous vasculature. Kp-10 caused a dose-dependent increase in oedema formation (0.3-10 nmol/injection site), assessed by Evans Blue albumin dye extravasation, in the dorsal skin of CD1 mice. Oedema formation was shown to be inhibited by the histamine H(1) receptor antagonist mepyramine. The response was characterised by a ring of pallor at the injection site in keeping with vasoconstrictor activity. Therefore, changes in dorsal skin blood flow were assessed by clearance of intradermally injected (99m)technetium. Kp-10 was found to significantly reduce clearance, in keeping with decreased blood flow and providing further evidence for vasoconstrictor activity. The decreased clearance was partially inhibited by co-treatment with the cyclo-oxygenase inhibitor indomethacin. Finally evidence for the kisspeptin receptor gene (Kiss1R), but not the kisspeptin peptide gene (Kiss1), mRNA expression was observed in heart, aorta and kidney samples from normal and angiotensin II induced hypertensive mice, with similar mRNA levels observed in each. We have evidence for two peripheral vasoactive roles for kisspeptin-10. Firstly, plasma extravasation indicative of ability to induce oedema formation and secondly decreased peripheral blood flow, indicating microvascular constriction. Thus Kp-10 has vasoactive properties in the peripheral microvasculature.
Kiss2, a paralogous gene for kiss1, has recently been identified in several vertebrates. However, their relative potencies for the regulation of reproductive functions appear to differ among species. Here we used medaka as a model animal to examine the kiss1 and kiss2 expression dynamics by in situ hybridization under different conditions: breeding or nonbreeding and ovariectomized or sham operated. Medaka kiss1-expressing neurons and kiss2-expressing neurons were mainly localized in two hypothalamic nuclei, nucleus ventralis tuberis (NVT) and nucleus recessus lateralis (NRL), respectively. NRL kiss2 expression did not change according to differences in breeding condition, whereas NVT kiss1 expression was strongly correlated with breeding condition. In addition, ovariectomy did not change kiss2 expression but significantly decreased the kiss1 expression. Moreover, double-label in situ hybridization revealed that NVT Kiss1 neurons coexpress estrogen receptor-alpha, whereas NRL Kiss2 neurons do not. From these results, we conclude that the NVT Kiss1 neurons are positively regulated by ovarian estrogen via their coexpressed estrogen receptor-alpha and are directly involved in the central regulation of reproduction in medaka. In contrast, we argue that the NRL Kiss2 neurons in medaka may serve nonreproductive functions. These functional differences between Kiss1 and Kiss2 neurons are discussed from a phylogenetic viewpoint.
To ascertain the neuroendocrine function of the kisspeptin/GPR54 system in non-mammalian species, full-length cDNAs encoding for Kiss1 and Kiss2 as well as their putative cognate receptors GPR54a and GPR54b, were isolated from goldfish (Carassius auratus). The deduced protein sequences between Kiss1 and Kiss2 in goldfish share very low similarity, but their putative mature peptides (kisspeptin-10) are relatively conserved. RT-PCR analysis demonstrated that the goldfish kiss1 gene (gfkiss1) is highly expressed in the optic tectum-thalamus, intestine, kidney, and testis, while the goldfish kiss2 gene (gfkiss2) is mainly detected in the hypothalamus, telencephalon, optic tectum thalamus, adipose tissue, kidney, heart, and gonads. The two receptor genes (gfgpr54a and gfgpr54b) are highly expressed in the brain regions including telencephalon, optic tectum thalamus, and hypothalamus. Both mature goldfish kisspeptin-10 peptides (gfKiss1-10 and gfKiss2-10) are biologically active as they could functionally interact with the two goldfish receptors expressed in cultured eukaryotic cells to trigger the downstream signaling pathways with different potencies. The actions of gfKiss1-10 and gfKiss2-10 on LH secretion were further investigated in vitro and in vivo. Intraperitoneal administration of gfKiss1-10 to sexually mature female goldfish could increase the serum LH levels. However, this peptide does not significantly influence LH release from goldfish pituitary cells in primary culture, indicating that the peptide does not exert its actions at the pituitary level. On the other hand, gfKiss2-10 appears to be a much less potent peptide as it exhibits no significant in vivo bioactivity and is also inactive on the primary pituitary cells.
Newly discovered kisspeptin (metastin), encoded by the Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of GnRH. In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125 and 115 amino acids, respectively, and its core sequence (FNLNPFGLRF, F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY, Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across nonmammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventromedial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P < 0.002), gnrh2, and gnrh3 (P < 0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased FSH-β (2.7-fold, P < 0.05) and LH-β (8-fold, P < 0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish.
Habenular kisspeptin-1 (Kiss1) and the novel hypothalamic Kiss2 are potential regulators of puberty. Kiss2 is the predominant regulator of gonadotropin synthesis in fish.
Kisspeptin and its receptor GPR54 have recently been identified as key signaling partners in the neural control of fertility in animal models and humans. The gonadotropin-releasing hormone (GnRH) neurons represent the final output neurons of the neural network controlling fertility and are suspected to be the primary locus of kisspeptin-GPR54 signaling. Using mouse models, the present study addressed whether kisspeptin and GPR54 have a key role in the activation of GnRH neurons to generate the luteinizing hormone (LH) surge responsible for ovulation. Dual-label immunocytochemistry experiments showed that 40-60% of kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) expressed estrogen receptor alpha and progesterone receptors. Using an ovariectomized, gonadal steroid-replacement regimen, which reliably generates an LH surge, approximately 30% of RP3V kisspeptin neurons were found to express c-FOS in surging mice compared with 0% in nonsurging controls. A strong correlation was found between the percentage of c-FOS-positive kisspeptin neurons and the percentage of c-FOS-positive GnRH neurons. To evaluate whether kisspeptin and/or GPR54 were essential for GnRH neuron activation and the LH surge, Gpr54- and Kiss1-null mice were examined. Whereas wild-type littermates all exhibited LH surges and c-FOS in approximately 50% of their GnRH neurons, none of the mutant mice from either line showed an LH surge or any GnRH neurons with c-FOS. These observations provide the first evidence that kisspeptin-GPR54 signaling is essential for GnRH neuron activation that initiates ovulation. This broadens considerably the potential roles and therapeutic possibilities for kisspeptin and GPR54 in fertility regulation.
SRY, the testis determining gene, encodes a member of a family of DNA binding proteins characterized by an amino acid sequence motif known as the HMG box. Using degenerate primers and the polymerase chain reaction, we have isolated SRY-related cDNAs from adult murine testis RNA. One of these, Sox-5, encodes a 43 kDa HMG-box protein with similarities to transcription activating proteins. Anti-Sox-5 antibody was used to analyse expression of Sox-5 in pre-pubertal testis and in fractionated spermatogenic cells. Sox-5 is restricted to post-meiotic germ cells, being found at highest levels in round spermatids. Sox-5 is a DNA binding protein and binding site selection assays suggest that it can bind specifically to oligonucleotides containing the consensus motif AACAAT. Sry can also bind to this motif, indicating that the Sry family may have overlapping sequence specificities.
We have isolated two murine homeobox genes, Nkx-2.5 and Nkx-2.6, that are new members of a sp sub-family of homeobox genes related to Drosophila NK2, NK3 and NK4/msh-2. In this paper, we focus on the Nkx-2.5 gene and its expression pattern during post-implantation development. Nkx-2.5 transcripts are first detected at early headfold stages in myocardiogenic progenitor cells. Expression preceeds the onset of myogenic differentiation, and continues in cardiomyocytes of embryonic, foetal and adult hearts. Transcripts are also detected in future pharyngeal endoderm, the tissue believed to produce the heart inducer. Expression in endoderm is only found laterally, where it is in direct apposition to promyocardium, suggesting an interaction between the two tissues. After foregut closure, Nkx-2.5 expression in endoderm is limited to the pharyngeal floor, dorsal to the developing heart tube. The thyroid primordium, a derivative of the pharyngeal floor, continues to express Nkx-2.5 after transcript levels diminish in the rest of the pharynx. Nkx-2.5 transcripts are also detected in lingual muscle, spleen and stomach.
The expression data implicate Nkx-2.5 in commitment to and/or differentiation of the myocardial lineage. The data further demonstrate that cardiogenic progenitors can be distinguished at a molecular level by late gastrulation. Nkx-2.5 expression will therefore be a valuable marker in the analysis of mesoderm development and an early entry point for dissection of the molecular basis of myogenesis in the heart.
Microcell-mediated transfer of chromosome 6 into human C8161 and MelJuSo melanoma cell suppresses their ability to metastasize by at least 95% without affecting their tumorigenicity. This observation demonstrates that the ability to metastasize is a phenotype distinct from tumor formation and suggests that tumorigenic cells acquire metastatic capability only after accumulating additional genetic defects. These results also imply that mutations of genes on chromosome 6 are among those late genetic changes responsible for metastatic potential. They further suggest that a melanoma metastasis-suppressor gene(s) is encoded on chromosome 6 or is regulated by genes on chromosome 6.
Our objective was to identify the gene(s) responsible for the suppression of metastasis in chromosome 6/melanoma cell hybrids.
A modified subtractive hybridization technique was used to compare the expression of messenger RNAs (mRNAs), via an analysis of complementary DNAs (cDNAs), in metastatic cells (C8161 or MelJuSo) and nonmetastatic hybrid clones (neo6/C8161 or neo6/MelJuSo).
A novel cDNA, designated KiSS-1, was isolated from malignant melanoma cells that had been suppressed for metastatic potential by the introduction of human chromosome 6. Northern blot analyses comparing mRNAs from a panel of human melanoma cells revealed that KiSS-1 mRNA expression occurred only in nonmetastatic melanoma cells. Expression of this mRNA in normal heart, brain, liver, lung, and skeletal muscle was undetectable by northern blot analysis. Weak expression was found in the kidney and pancreas, but the highest expression was observed in the placenta. The KiSS-1 cDNA encodes a predominantly hydrophilic, 164 amino acid protein with a polyproline-rich domain indicative of an SH3 ligand (binds to the homology 3 domain of the oncoprotein Src) and a putative protein kinase C-alpha phosphorylation site. Transfection of a full-length KiSS-1 cDNA into C8161 melanoma cells suppressed metastasis in an expression-dependent manner.
These data strongly suggest that KiSS-1 expression may suppress the metastatic potential of malignant melanoma cells.
KiSS-1 may be a useful marker for distinguishing metastatic melanomas from nonmetastatic melanomas.
Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta.
These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis
suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to
rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP2 hydrolysis, Ca2+ mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited
cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role
in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed
this hypothesis.
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a technique widely used for quantification of mRNA transcription. Data normalization is an indispensable process for RT-qPCR and reference genes are most commonly used to normalize RT-qPCR and to reduce possible errors generated in the quantification of genes among several proposed methods. To date, RT-qPCR has been used in terms of gene expression studies in black rockfish (Sebastes schlegeli) but the majority of published RT-qPCR studies still lack proper validation of the reference genes. In the present study, mRNA transcription profiles of eight putative reference genes (18S rRNA, ACTB, GAPDH, TUBA, RPL17, EF1A, HPRT, and B2M) were examined using RT-qPCR in different tissues and larvae developmental stages of black rockfish. Three common statistical algorithms (geNorm, NormFinder, and BestKeeper) were used to assess expression stability and select the most stable genes for gene normalization. Two reference genes, RPL17 and EF1A showed high stability in black rockfish tissue analysis, while GAPDH was the least stable gene. During larvae developmental stages, EF1A, RPL17 and ACTB were identified as the optimal reference genes for data normalization, whereas B2M appeared unsuitable as the reference gene. In summary, our results could provide a useful guideline for reference gene selection and enable more accurate normalization of gene expression data in gene expression studies of black rockfish.
Kisspeptin and its receptor, GPR54, play a pivotal role in vertebrate reproduction. Recent advances in bioinformatic tools combined with comparative genomics have led to the identification of a large number of kisspeptin and GPR54 genes in a variety of vertebrate species. Genome duplications may have produced at least two isoforms of both ligand (KiSS1 and KiSS2) and receptor (GPR54-1 and GPR54-2). Additional isoforms of kisspeptin (KiSS1b) and GPR54 (GPR54-1b) have been found in an amphibian species, Xenopus (Silurana) tropicalis. Here, we describe the evolutionary lineages of these kisspeptin and GPR54 isoforms using genome synteny and phylogenetic analyses, and possible molecular interactions between kisspeptin and GPR54 subtypes based on ligand-receptor selectivity. Together, kisspeptin and GPR54 provide an excellent model for understanding molecular coevolution of the peptide ligand and GPCR pairs.
The discovery that kisspeptin was critical for normal fertility in all mammalian species including humans, ushered in a new chapter in our understanding of the control of GnRH secretion. Kisspeptin, the product of the KISS1 gene, plays an essential role in the regulation of spermatogenesis acting primarily at the hypothalamic level of the gonadotropic axis. Among the many identified substances in human semen, fructose is becoming increasingly significant. Fructose is synthesized and secreted by the seminal vesicles. Its synthesis is regulated by androgens and it is correlated directly with the levels of testosterone. Dose dependent degeneration of seminal vesicle has been described following intraperitoneal kisspeptin treatment; however, effects of kisspeptin administration on the levels of seminal fructose remain elusive till date. The present study, therefore, addresses the effects of 12-day administration of kisspeptin on seminal fructose levels in male mice. Kisspeptin-10 was administered intraperitoneally at different dosage concentrations (1 μg, 1 ng, and 10 ρg) to adult male mice, twice daily for 12 days. Seminal fructose levels were studied photometrically after 12 days of treatment. At the end of the treatment, seminal fructose levels decreased significantly after all tested doses. Chronic intermittent kisspeptin-10 administration negatively regulates seminal fructose levels in adult male mice.
Metastasis is a major cause of death in cancer patients and involves a multistep process including detachment of cancer cells from a primary cancer, invasion of surrounding tissue, spread through circulation, re-invasion and proliferation in distant organs. KiSS-1 is a human metastasis suppressor gene, that suppresses metastases of human melanomas and breast carcinomas without affecting tumorigenicity. However, its gene product and functional mechanisms have not been elucidated. Here we show that KiSS-1 (refs 1, 4) encodes a carboxy-terminally amidated peptide with 54 amino-acid residues, which we have isolated from human placenta as the endogenous ligand of an orphan G-protein-coupled receptor (hOT7T175) and have named `metastin'. Metastin inhibits chemotaxis and invasion of hOT7T175-transfected CHO cells in vitro and attenuates pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. The results suggest possible mechanisms of action for KiSS-1 and a potential new therapeutic approach.
Differences in reproductive endocrinology distinguish Hatano high-avoidance animals (HAA) from low-avoidance animals (LAA). Compared to HAA rats, female LAA rats secrete low levels of basal luteinizing hormone (LH) and a reduced LH surge. To investigate the underlying cause of the differences between the two strains, levels of the following mRNAs were measured in the hypothalamus of intact and ovariectomized (OVX) females treated with vehicle control or estradiol-17β (E2): gonadotropin-releasing hormone (Gnrh), newly isolated rat kisspeptin (Kiss)1 mRNA variant-1 (Kiss1V1) and variant-2 (Kiss1V2) and estrogen receptor (Er) α. In OVX-HAA rats, the levels of Gnrh mRNA in the preoptic area (POA) 30 hours after E2 treatment were significantly higher than in OVX-LAA rats. For HAA rats, the levels of Kiss1V1 and Kiss1V2 mRNA in the anteroventral periventricular nucleus (AVPV) were significantly higher in the E2-treated group than in the vehicle-treated group. In the arcuate nucleus (Arc) of HAA rats, Kiss1V1 and Kiss1V2 expression was significantly lower in E2-treated females compared to vehicle-treated females. Kiss1V2 expression was significantly higher than Kiss1V1 expression in intact HAA rats. In E2-treated OVX-LAA rats, there were no changes in the expression levels of Gnrh, Kiss1V1 or Kiss1V2. In intact LAA rats, no differences were observed in the expression levels of Kiss1V1 or Kiss1V2 in the AVPV, but the expression levels of these mRNAs in the Arc were significantly lower in E2-treated OVX-LAA rats. Additionally, no strain differences were observed for Erα mRNA expression in either the AVPV or Arc. These results indicate that the failure of estrogenic regulation of GnRH neurons in the POA and of kisspeptin neurons in the AVPV of LAA rats causes low LH secretion and reduced reproductive function.
The gonadal development and serum profiles of vitellogenin and sex steroids in rearing Sebastes schlegeli were monitored for one full year. Female fish began vitellogenesis from November and completed it in March. Gestation occurred from April, and parturition occurred in June. A thin chorion and scanty cortical alveoli are oogenetic peculiarities of this fish. Male fish began spermatogenesis from June, and matured in November and December. It appears that copulation occurs in November and December, and that the sperm are stored freely in the ovary during the early vitellogenic period and under the ovigerous lamellae epithelium during the late vitellogenic period. Serum vitellogenin levels in female fish had a good correlation with oocyte growth. Serum estradiol-17β (E2) levels in female fish were elevated from November to February, suggesting that E2 controls vitellogenesis. Serum 17,20β-dihydroxy-4-pregnen-3-one (DHP) in female fish increased in the late vitellogenic period, suggesting that DHP was a maturation-inducing steroid. High levels of serum DHP during gestation suggest that it may be one of the endocrine factors for maintaining gestation. Serum 11-ketotestosterone (11-KT) levels in male fish were elevated from June to October, suggesting that 11-KT controls spermatogenesis. Serum DHP in male fish had a single peak in October, suggesting that DHP plays some role in the late stages of spermatogenesis.
Larval and juvenile stages of kurosoi,Sebastes schlegeli, are described and illustrated from wild specimens. Some ecological aspects of larvae and juveniles are also described. Notochord
flexion occurred between 5.6–7.5 mm SL. Transformation occurred between 13–20 mm SL. Preflexion and flexion larvae ofS. schlegeli can be distinguished from similar larvae by the pigmentation of the dorsal and ventral midlines of the tail and absence of
pigmentation on the ventral portion of the rectum. After notochord flexion, the dorsal and lateral regions in both larvae
and pelagic juveniles were heavily pigmented, suggesting adaptation for neustonic life style. Larvae and juveniles were caught
at many coastal stations, but did not occur in cooler offshore waters. Larvae smaller than 20 mm SL inhabited surface waters.
Until ca. 40 mm SL, juveniles inhabited mainly surface waters (without drifting seaweed), but also used other habitats, such
as the drifting seaweed, and near the sea bed. Small larvae (<7 mm SL) fed mainly on copepod nauplii. Larger larvae fed on
calanoid copepodites andEvadne nordmanni. Pelagic juveniles fed mainly on fish eggs, with fish larvae also being important food items for some individuals. Most food
items taken by juveniles that were associated with drifting seaweed were eggs with attaching filaments (Cololabis saira andHyporhamphus sajori), suggesting that the high density of such food items both attracts and keeps juveniles around drifting seaweed.
Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a vital role in hypothalamus-pituitary-gonad (HPG) axis. In the present study, the GnRH-III gene was isolated from half-smooth tongue sole (Cynoglossus semilaevis). In the 1160 bp genomic sequence, four exons, three introns, and 5'-/3'-flanking sequences were identified. The putative peptide was 92 residues long, including a putative signal peptide containing 23 amino acids, the GnRH decapeptide, a proteolytic cleavage site of three amino acids and a GnRH associated peptide of 56 amino acids. The overall amino acid sequence of C. semilaevis GnRH-III (csGnRH-III) was highly conserved with other teleost GnRH-III genes. Phylogenetic analysis showed the evolutionary relationships of csGnRH-III with other known GnRH genes. A 320 bp promoter sequence of the csGnRH-III was also analyzed, and several potential regulatory motifs were identified which were conserved in the GnRH promoters of other teleosts. Quantitative real-time PCR analysis indicated csGnRH-III was expressed only in brain and gonads. In C. semilaevis, the csGnRH-III transcript was maternally deposited and appeared to be developmentally regulated during embryogenesis and early larval development. Comparing sequence and expression patterns of csGnRH-III with other teleosts GnRH-IIIs suggested that the main function of GnRH-III might be conserved in teleosts.
We have cloned and analysed the partial putative promoter sequences of the Yellowtail Kingfish (Seriola lalandi) Kiss2 and Kiss2r genes (380 and 420 bp, respectively). We obtained in silico 1.5 kb of the zebrafish (Danio rerio) Kiss1,
Kiss2,
Kiss1r and zfKiss2r sequences upstream of the putative transcriptional initiation site. Bioinformatic analysis revealed promoter regulatory elements including AP-1, Sp1, GR, ER, PR, AR, GATA-1, TTF-1, YY1 and C/EBP. These regulatory elements may mediate novel roles of the Kiss genes and their receptors in addition to their established role in reproductive function.
Kisspeptin, the product of Kiss1 gene, is a novel regulator of the gonadotropic axis. In mammals, its stimulatory effect on gonadotropin secretion is well documented and mediated mainly by hypothalamic release of gonadotropin-releasing hormone. Although the pituitary actions of kisspeptin have been reported, the effects of kisspeptin on gonadotropin release via direct action on pituitary cells are still controversial. Using goldfish as a model, here we examined the direct actions of kisspeptin on pituitary functions in modern-day bony fish. As a first step, the structural identity of goldfish Kiss1 was established by 5′/3′RACE and Kiss1 transcript was shown to be widely expressed in various tissues in goldfish. At the pituitary level, Kiss1 receptor (Kiss1r) expression was detected in immuno-identified gonadotrophs, lactotrophs, and somatotrophs. Kiss1 transcript was also located in goldfish somatotrophs but not in lactotrophs or gonadotrophs. In parallel studies, goldfish kisspeptin-10 was synthesized and used to test the pituitary actions of kisspeptin in vitro. In goldfish pituitary cell cultures, 30-min incubation with kisspeptin-10 increased basal release of luteinizing hormone (LH), prolactin (PRL), and growth hormone (GH). Transcript expression of LH, PRL, and GH were also elevated by prolonging kisspeptin-10 treatment to 24 h. These results taken together suggest that kisspeptin via Kiss1r activation can act directly at the pituitary level to trigger LH, PRL, and GH secretion and gene expression in goldfish. Our finding of Kiss1 expression in somatotrophs also rises the possibility that kisspeptin may be produced locally in the fish pituitary and serve as an autocrine/paracrine regulator.
Kisspeptins, the peptide products of the Kiss1 gene, were initially identified in mammals as ligands of the G protein-coupled receptor 54 (GPR54; also termed Kiss1R) with ability to suppress tumor metastasis. In late 2003, the indispensable role of kisspeptins in the control of reproductive function was disclosed by the seminal observations that humans and mice carrying inactivating mutations of GPR54 displayed hypogonadotropic hypogonadism. Since then, numerous experimental studies, conducted initially in several mammalian species, have substantiated the roles of kisspeptins as essential players in the physiologic regulation of key aspects of reproductive maturation and function, including the timing of puberty onset, the dynamic control of gonadotropin secretion via stimulation of GnRH neurons, the transmission of the negative and positive feedback effects of sex steroids, the metabolic regulation of fertility and the control of reproductive function by environmental (photoperiodic) cues. Notably, while studies about kisspeptins in non-mammals appeared initially to lag behind, significant efforts have been devoted recently to define the genomic organization and functional characteristics of kiss/kisspeptins and gpr54 in different non-mammalian species, including fish, reptiles and amphibians. These analyses, which will be comprehensively revised herein, have not only substantiated the conserved, essential roles of kisspeptins in the control of reproduction, but have also disclosed intriguing evolutionary aspects of kisspeptins and their receptors. Such comparative approaches will be instrumental to fuel further studies on the molecular regulation and physiological roles of kisspeptins, thus helping to unveil the complex biology of this system as indispensable regulator of the reproductive axis in a wide diversity of animal species.
The cancer suppressor gene, KISS1, was initially described as having an important role in inhibiting cancer metastasis. Since then, KISS1 and its receptor, KISS1R, have been shown to play a key role in controlling the onset of puberty of reproductive physiology in the human and other species. Recent studies have also linked KISS1/kisspeptin/KISS1R to other processes, such as vasoconstriction, aging, adipocyte physiology, and perhaps as a molecular conduit linking metabolism and reproduction. This article highlights the history of KISS1/kisspeptin/KISS1R biology and proposes a consensus for nomenclature of the key molecules in this signaling pathway.
It is well established that reproductive function is gated by the state of energy reserves of the organism; conditions of metabolic stress and energy insufficiency being frequently coupled to disturbed reproductive maturation and/or infertility. In addition, obesity is also commonly linked to altered puberty onset and reproductive impairment. Such an impact of energy status on the reproductive axis is conveyed through a number of neuropeptide hormones and metabolic cues, whose nature and mechanisms of action have begun to be deciphered only in recent years. In this context, the emergence of kisspeptins, encoded by the KiSS-1 gene, and their receptor, GPR54, as indispensable signals for normal pubertal maturation and gonadal function, has raised the possibility that the KiSS-1/GRP54 system might also participate in coupling body energy status and reproduction. We revise herein the experimental evidence, gathered in rodent models, supporting the contention that the hypothalamic KiSS-1 system operates as a central conduit for conveying metabolic information onto the centers governing reproductive function, through a putative leptin-kisspeptin-GnRH pathway. Admittedly, key aspects of this 'metabolic' network involving the KiSS-1 system, such as its different peripheral regulators and central effectors, have not been fully elucidated. Nonetheless, the proposed hypothalamic circuitry, responsible for transmitting metabolic information onto the reproductive axis through KiSS-1 neurons, might explain, at least in part, the mechanisms for the well-known alterations of fertility linked to conditions of disturbed energy balance in humans, from anorexia nervosa to morbid obesity.
The kiss1 gene product kisspeptin is now considered to be an essential regulator of the hypothalamic-pituitary-gonadal (HPG) axis in most vertebrate species. Recent findings in fishes are beginning to set a new stage for the kisspeptin study; the existence of paralogous kisspeptin genes as well as kisspeptin receptor (formerly called GPR54) genes has quite recently been reported in several fish and amphibian species. The fishes may provide excellent animal models for the study of general principles underlying the kisspeptin and kisspeptin receptor systems of vertebrates from the evolutionary viewpoint. Unlike placental and marsupial mammalian species mainly studied so far, many teleost species have two paralogous genes of kisspeptin, kiss1 and kiss2. Medaka, Oryzias latipes, in which kiss1 and kiss2 are expressed in distinctive hypothalamic neuron populations, is a good model system for the study of central regulation of reproduction. Here, the kiss1 system but not the kiss2 system shows expression dynamics strongly indicative of its direct involvement in the HPG axis regulation via its actions on GnRH1 neurons. On the other hand, the kiss1 gene is missing, and only kiss2 is expressed in some fish species. Also, there are some recent reports that Kiss2 peptide may be a potent regulator of reproduction in some fish species. The ancestral vertebrate probably already had two paralogous kiss genes, and their main function was the HPG axis regulation. In the species that retained both paralogues during evolution, either Kiss1 or Kiss2 predominantly retains its ability for the HPG axis regulation, while the other may assume new non-reproductive functions (neofunctionalization). Alternatively, both the paralogues may assume complementary functions in the HPG axis regulation (subfunctionalization). After the divergence of teleost and tetrapod lineages, either one of the two paralogues, or even both in birds, have been lost (degradation) or became a pseudogene (non-functionalization), but the remaining paralogue retained its original function of HPG axis regulation. The identification of multiple forms of kisspeptin receptors and the rather promiscuous ligand-receptor relationships has led to the further proposal that such promiscuousness may be the basis for the functional robustness of kisspeptin and kisspeptin receptor systems in the HPG axis regulation, when one or both paralogous genes are lost or functionally partitioned during evolution.
Kisspeptins, encoded by the Kiss1 gene, have emerged as key modulators of reproduction in mammals. In contrast to the placental mammals, some teleosts express two Kiss genes, Kiss1 and Kiss2. In the present study, full-length cDNAs of Kiss1 and Kiss2 in the chub mackerel were cloned and sequenced. Chub mackerel Kiss1 and Kiss2 cDNAs encode 105 and 123 amino acids, respectively. A comparison of the deduced amino acid sequences of chub mackerel Kiss1 and Kiss2 with those of other vertebrate species showed a high degree of conservation only in the kisspeptin-10 region (Kp-10). The Kp-10 of chub mackerel Kiss1 (YNFNSFGLRY) and Kiss2 (FNFNPFGLRF) showed variations at three amino acids. Tissue distribution analysis using quantitative real-time PCR (qRT-PCR) revealed that the Kiss1 and Kiss2 transcripts were expressed in different tissues of adult chub mackerel. In addition, their levels in the adipose tissue exhibited sexually dimorphic expression. Further, to have a basic understanding on the involvement of Kiss1 and Kiss2 in the seasonal gonadal development, their relative mRNA expression profiles in the brain, pituitary, and gonads at different gonadal stages were analyzed using qRT-PCR. Kiss1 and Kiss2 levels in the brain showed a differential expression profile between male and female fish. In males, Kiss1 and Kiss2 levels gradually decreased from the immature stage to spermiation and reached a minimal level during the post-spawning period. In contrast, Kiss1 levels in the brain of females did not vary significantly among the different gonadal stages. However, Kiss2 levels fluctuated as that of males, gradually declining from the immature stage to the post-spawning period. The pituitary Kiss1 levels did not show significant fluctuations. However, Kiss1 levels in the gonads were highly elevated during spermiation and late vitellogenesis compared to the immature and post-spawning period. These results suggest the possible involvement of two Kiss genes in the brain and Kiss1 in the gonads of chub mackerel during seasonal gonadal development.
Kisspeptins are a family of neuropeptides encoded by Kiss1 and Kiss2 genes, and participate in neuroendocrine regulation of gonadotropin-releasing hormone (GnRH) secretion through activating their receptor, Kiss1r (or GPR54). Bioinformatic analyses have shown that there is a single gene for each kisspeptin (Kiss2) and its receptor (Kiss1r) in pufferfish, the function of which has yet to be elucidated. We cloned these two genes in grass puffer, which spawns on beach with semilunar cycles, and examined changes in their expression levels in the brain and pituitary at different reproductive stages over the spawning season. The Kiss2 precursor of 104 amino acid residues contains a putative kisspeptin peptide (SKFNLNPFGLRF). Kiss1r consists of 377 amino acid residues containing distinct characteristics of G-protein coupled receptors. Kiss2 and Kiss1r genes were expressed extensively in the brain, pituitary and gonads. The amounts of Kiss2 and Kiss1r mRNAs were significantly elevated during the spawning period in the brain and pituitary of both sexes. There were strong positive correlations between the amounts of Kiss2 and Kiss1r mRNAs in the brain and pituitary over the spawning season. Significant positive correlations were also observed between the amounts of Kiss2/Kiss1r mRNAs and GnRH1 mRNA in the brain. The present results indicate that the Kiss2/Kiss1r system most probably plays an important role in the regulation of reproductive function in the spawning period of grass puffer, possibly through the stimulation of GnRH1 secretion. Furthermore, Kiss2 may have a local action in the pituitary.
Precise spatial and temporal expression of the recently identified G-protein coupled receptor GPR54 is critical for proper reproductive function and metastasis suppression. However, regulatory factors that control GPR54 expression remain unknown. Thus, the identification of these cis-acting DNA elements can provide insight into the role of GPR54 in reproduction and cancer. Using luciferase reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays, we demonstrate that three SP1 sites and a partial estrogen response element modulate mouse GPR54 (mGPR54) promoter activity. Supporting experiments show transcription factor SP1 binds directly to the mGPR54 promoter region and activates gene expression. In conclusion, these novel findings now identify factors that regulate activity of the mGPR54 promoter, and these factors are highly conserved across multiple mammalian species.
The status of the neuroendocrine reproductive axis differs dramatically during various stages of development, and also differs in several critical ways between the sexes, including its earlier pubertal activation in females than males and the presence of neural circuitry that generates preovulatory hormone surges in females but not males. The reproductive axis is controlled by various hormonal and neural pathways that converge upon forebrain gonadotropin-releasing hormone (GnRH) neurons, and many of the critical age and sex differences in the reproductive axis likely reflect differences in the "upstream" circuits and factors that regulate the GnRH system. Recently, the neural kisspeptin system has been implicated as an important regulator of GnRH neurons. Here I discuss the evidence supporting a critical role of kisspeptin signaling at different stages of life, including early postnatal and pubertal development, as well as in adulthood, focusing primarily on information gleaned from mammalian studies. I also evaluate key aspects of sexual differentiation and development of the brain as it relates to the Kiss1 system, with special emphasis on rodents. In addition to discussing recent advances in the field of kisspeptin biology, this paper will highlight a number of unanswered questions and future challenges for kisspeptin investigators, and will stress the importance of studying the kisspeptin system in both males and females, as well as in multiple species.
Kisspeptin and its receptor, Kiss1r, play an essential role in the control of the onset of puberty in vertebrates. We characterized the cDNA and genomic DNA encoding Kiss1r in Atlantic halibut (Hippoglossus hippoglossus). The 1146bp open reading frame predicts a 381 amino acid protein with high homology to the Kiss1r-2 of other teleost fish. Phylogenetic analysis of Kiss1r sequences suggests that the mammalian Kiss1r-1 form arose by way of a gene duplication prior to the emergence of amphibians. Synteny analysis demonstrated the highly conserved nature of the Kiss1r-2 region in teleosts, suggesting that flanking regulatory sequences are also likely to be conserved. Bioinformatic analysis identified six conserved regions in piscine Kiss1r-2 upstream sequences, providing potential targets for future in-depth investigation of Kiss1r-2 regulation. Kiss1r-2 expression in the brain increased coinciding with the onset of puberty. Expression levels in the gonads were two orders of magnitude lower than those of the brain, a characteristic apparently conserved in other fishes, and expression in gonads was only detected in immature fish.
Kisspeptin and its receptor GPR54 play important roles in mammalian reproduction and cancer metastasis. Because the KiSS and GPR54 genes have been identified in a limited number of vertebrate species, mainly in mammals, the evolutionary history of these genes is poorly understood. In the present study, we have cloned multiple forms of kisspeptin and GPR54 cDNAs from a variety of vertebrate species. We found that fish have two forms of kisspeptin genes, KiSS-1 and KiSS-2, whereas Xenopus possesses three forms of kisspeptin genes, KiSS-1a, KiSS-1b, and KiSS-2. The nonmammalian KiSS-1 gene was found to be the ortholog of the mammalian KiSS-1 gene, whereas the KiSS-2 gene is a novel form, encoding a C-terminally amidated dodecapeptide in the Xenopus brain. This study is the first to identify a mature form of KiSS-2 product in the brain of any vertebrate. Likewise, fish possess two receptors, GPR54-1 and GPR54-2, whereas Xenopus carry three receptors, GPR54-1a, GPR54-1b, and GPR54-2. Sequence identity and genome synteny analyses indicate that Xenopus GPR54-1a is a human GPR54 ortholog, whereas Xenopus GPR54-1b is a fish GPR54-1 ortholog. Both kisspeptins and GPR54s were abundantly expressed in the Xenopus brain, notably in the hypothalamus, suggesting that these ligand-receptor pairs have neuroendocrine and neuromodulatory roles. Synthetic KiSS-1 and KiSS-2 peptides activated GPR54s expressed in CV-1 cells with different potencies, indicating differential ligand selectivity. These data shed new light on the molecular evolution of the kisspeptin-GPR54 system in vertebrates.
Kisspeptins, the products of KiSS-1 gene, have recently emerged as fundamental regulators of reproductive function in different mammalian and, presumably, non-mammalian species. To date, a single form of KiSS-1 has been described in mammals, and recently, in several fish species and Xenopus. We report herein the cloning and characterization of two distinct KiSS-like genes, namely, KiSS-1 and KiSS-2, in the teleost sea bass. While KiSS-1 encodes a peptide identical to rodent kisspeptin-10, the predicted KiSS-2 decapeptide diverges at 4 amino acids (FNFNPFGLRF). Genome database searches showed that both genes are present in non-placental vertebrate genomes. Indeed, phylogenetic and genome mapping analyses suggest that KiSS-1 and KiSS-2 are paralogous genes that originated by duplication of an ancestral gene, although KiSS-2 is lost in placental mammals. KiSS-1 and KiSS-2 mRNAs are present in brain and gonads of sea bass, medaka and zebrafish. Comparative functional studies demonstrated that KiSS-2 decapeptide was significantly more potent than KiSS-1 peptide in inducing LH and FSH secretion in sea bass. In contrast, KiSS-2 decapeptide only weakly elicited LH secretion in rats, whereas KiSS-1 peptide was maximally effective. Our data are the first to provide conclusive evidence for the existence of a second KiSS gene, KiSS-2, in non-placental vertebrates, whose product is likely to play a dominant stimulatory role in the regulation of the gonadotropic axis at least in teleosts.
The KISSPEPTIN-1 receptor (KISS1R) and its ligands (KISSPEPTINS) are implicated in the regulation of the onset of puberty. We report the coding region and genomic structure of the kiss1r gene of a modern teleost, the Senegalese sole (Ss). Ss kiss1r cDNA contained an opening frame of 1137 bp, which results in a predicted 378 amino acid protein. Searching genomic databases allowed the identification of kiss1r orthologues in six new species belonging to three vertebrate groups and established the evolutionary relationships of all KISS1R sequences available to date. Analysis of Ss kiss1r revealed for the first time in any vertebrate KISS1R gene the presence of features that are characteristic of a mechanism of alternative splicing. This was confirmed by the identification of two transcripts, Ss kiss1r_v1 and Ss kiss1r_v2. The latter, arising from intron III retention, contained a 27 codons insert in transmembrane region 4 with two stop codons, suggesting it may lead to a truncated protein. The mRNA of the two variants was differently expressed in several tissues. In the brain, levels of the Ss kiss1r_v1 were higher than those of Ss kiss1r_v2. In the gonads, the opposite was observed. Both isoforms exhibited changes depending on sex and maturity stage. The presence of two variants may help to explain some discrepancies observed in past studies regarding KISS1R expression during puberty. Thus, the existence of alternative splicing for the KISS1R gene may contribute to our understanding of the many physiological functions suspected to be mediated by KISSPEPTIN-KISS1R signaling.
A search of a 35-kilobase region of the human Y chromosome necessary for male sex determination has resulted in the identification of a new gene. This gene is conserved and Y-specific among a wide range of mammals, and encodes a testis-specific transcript. It shares homology with the mating-type protein, Mc, from the fission yeast Schizosaccharomyces pombe and a conserved DNA-binding motif present in the nuclear high-mobility-group proteins HMG1 and HMG2. This gene has been termed SRY (for sex-determining region Y) and proposed to be a candidate for the elusive testis-determining gene, TDF.
An initial step in the transcriptional activation of the prolactin gene by estrogen is the binding of the estrogen-receptor complex to a specific nucleotide sequence [estrogen responsive element (ERE)]. Using the gel mobility assay, we examined the binding mechanism of purified estrogen receptor to the ERE contained on a 255-base-pair fragment from the upstream region between nucleotides -1784 to -1531 of the rat prolactin gene. Remarkably, specific high-affinity binding was detected to the dissociated "coding strand" but not to the "noncoding strand" of the ERE-containing fragment. The dissociated strands of this fragment possess unusual secondary structure, as indicated by their anomalous migration in the gel mobility assay. The estrogen receptor binds to the coding strand of the ERE with a 60-fold higher affinity than to the double-stranded ERE. Furthermore, the receptor binds with a 1000-fold greater affinity to the coding strand of the ERE than to a double-stranded nonspecific DNA fragment. We propose that, in vivo, the estrogen receptor initially binds to the double-stranded ERE. Subsequently, the DNA strands separate due to transitory strand separation and supercoiling, allowing folding of the coding strand of the ERE into a structure that is then bound more tightly by the receptor. The formation of this receptor-ERE coding strand complex may be a crucial step in the mechanism of estrogen-stimulated transcription.
Prx1 and Prx2 (previously called MHox and S8, respectively) are the members of a small subfamily of vertebrate homeobox genes expressed during embryogenesis from gastrulation onwards. We directly compared the expression domains of the Prx genes in detail in mouse and in addition some aspects of these patterns in chicken. In addition to the superficially similar expression patterns of Prx1 and Prx2 in cranial mesenchyme, limb buds, axial mesoderm, and branchial arches and their derivatives, we detect major differences at many sites particularly in heart and brain. Our analysis indicated in several cases a correlation with regions developing into connective tissues. From at least day 8.5, Prx-1 expression was observed in the heart, initially in the endocardial cushions and later in the developing semilunar and atrioventricular valves. Prx2 develops early on a diffuse myocardial expression pattern and is later higher expressed in the ventricular septum and in particular in the ductus arteriosus. Prx2 is never expressed in the brain, whereas Prx1 is expressed, from at least day 9.5 onwards, in a unique distinct domain in the ventral part of the hypothalamus, as well as in a broader region of the telencephalon.
An unresolved aspect of current understanding of erythroid cell-specific gene expression relates to how a limited number of transcriptional factors cooperate to direct high-level expression mediated by cis-regulatory elements separated over large distances within globin loci. In this report, we provide evidence that GATA-1, the major erythroid transcription factor, activates transcription in a synergistic fashion with two Krüppel family factors, the ubiquitous protein Sp1 and the erythroid-restricted factor EKLF (erythroid Krüppel-like factor), which recognize GC and/or GT/CACC motifs. Binding sites for both GATA-1 and these Krüppel proteins (especially Sp1) are found in close association in the promoters and enhancers of numerous erythroid cell-expressed genes and appear to cooperate in directing their expression. We have shown that GATA-1 interacts physically with Sp1 and EKLF and that interactions are mediated through their respective DNA-binding domains. Moreover, we show that GATA-1 and Sp1 synergize from a distance in constructs designed to mimic the architecture of globin locus control regions and downstream globin promoters. Finally, the formation of GATA-1-SP1 complexes was demonstrated in vivo by the ability of Sp1 to recruit GATA-1 to a promoter in the absence of GATA-binding sites. These experiments provide the first evidence for functionally important protein-protein interactions involved in erythroid cell-specific expression and suggest a mechanism by which DNA loops between locus control regions and globin promoters (or enhancers) might be formed or stabilized.
Metastasis is a major cause of death in cancer patients and involves a multistep process including detachment of cancer cells from a primary cancer, invasion of surrounding tissue, spread through circulation, re-invasion and proliferation in distant organs. KiSS-1 is a human metastasis suppressor gene, that suppresses metastases of human melanomas and breast carcinomas without affecting tumorigenicity. However, its gene product and functional mechanisms have not been elucidated. Here we show that KiSS-1 (refs 1, 4) encodes a carboxy-terminally amidated peptide with 54 amino-acid residues, which we have isolated from human placenta as the endogenous ligand of an orphan G-protein-coupled receptor (hOT7T175) and have named 'metastin'. Metastin inhibits chemotaxis and invasion of hOT7T175-transfected CHO cells in vitro and attenuates pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. The results suggest possible mechanisms of action for KiSS-1 and a potential new therapeutic approach.
A novel human G protein-coupled receptor named AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous expression of AXOR12 in mammalian cells permitted the identification of three surrogate agonist peptides, all with a common C-terminal amidated motif. High potency agonism, indicative of a cognate ligand, was evident from peptides derived from the gene KiSS-1, the expression of which prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase chain reaction was used to study the expression of AXOR12 and KiSS-1 in a variety of tissues. The highest levels of expression of AXOR12 mRNA were observed in brain, pituitary gland, and placenta. The highest levels of KiSS-1 gene expression were observed in placenta and brain. A polyclonal antibody raised to the C terminus of AXOR12 was generated and used to show localization of the receptor to neurons in the cerebellum, cerebral cortex, and brainstem. The biological significance of these expression patterns and the nature of the putative cognate ligand for AXOR12 are discussed.