Central distribution of kiss2 neurons and peri-pubertal changes in their expression in the brain of male and female red seabream Pagrus major
Highlights
► kiss2-Expressing cell number in the brain is high during the first spawning period of male and female red seabream. ► GnRH1 cells show a similar pattern of change. ► Kiss2 may be involved in the pubertal processes via the regulatory influences on the GnRH1 system. ► kiss1 may be lacking in red seabream.
Introduction
In spite of the original identification of KISS1 as a metastasis suppressor gene in humans [19], the protein product of this gene or kisspeptin [16] that was first called metastin [29], is now recognized as the key molecule that regulates reproduction. In 2003, a mutation or knockout of the kisspeptin receptor gene (kissr1 or GPR54) was found to be associated with hypogonadotropic hypogonadism [5], [35]. (We follow the terminology for genes and their product as proposed by a recent review [3].) Following these findings that imply critical roles of the kisspeptin-Kissr signaling pathway in the control of pubertal processes, a number of studies suggested that kisspeptin neurons mediate steroid feedback actions to gonadotropin-releasing hormone (GnRH) neurons, which results in an appropriate control on the reproductive functions through the hypothalamo–pituitary–gonadal axis in mammals [2], [9], [10], [14], [22], [25], [27], [39].
In contrast to the situation in mammals where the physiology and functions of kisspeptin neurons are now fairly well understood, much remains to be studied regarding kisspeptins in non-mammalian vertebrates. In addition to kiss1 (medaka [12]; zebrafish and clawed toad [4]; zebrafish [41]; goldfish [42]), a paralogue of kiss1 called kiss2 has been found in a variety of non-mammalian (and one monotreme) species (lamprey, elephant shark, medaka, zebrafish, clawed toad, and platypus [20]; European seabass [6]; zebrafish and medaka [15]; goldfish [21]; chub mackerel [32]). Furthermore, only kiss2 has been found in some vertebrates (stickleback [20]; grass puffer [37]; orange-spotted grouper [38]; Senegalese sole [24]; grass lizard [20]). Such differential phylogenetic distributions of kisspeptins, together with the presence of multiple kisspeptin receptors that interact with both Kiss1 and Kiss2 [20], [21], make it quite difficult to elucidate the significance of kisspeptins in non-mammalian vertebrates, particularly in teleosts with the considerable species differences detailed below.
Studies in medaka and zebrafish have revealed species differences in the central distribution of kiss1 and kiss2 neurons [12], [15], [26], [36]. Different neuroanatomical terminology is adopted in these reports, and we follow the terminology of Kanda et al. [12] in the present article. In medaka kiss1 neurons are present in the habenula and in two hypothalamic nuclei. In zebrafish, however, kiss1 neurons are present only in the habenula. Species differences are also noted regarding the distribution of kiss2 neurons. In medaka, kiss2 neurons are present only in the nucleus recessi lateralis in the hypothalaus, while additional hypothalamic and minor preoptic populations are present in zebrafish.
The effects of kisspeptin administrations [(partial molecules: Kiss1(10) and Kiss2(10)] also show species-specific differences. The administration of Kiss2(10) is more potent than Kiss1(10) in stimulating gonadotrophs in zebrafish and European seabass [6], [15]. However, only Kiss1(10) showed such bioactivity in goldfish [21]. Similarly, hypothalamic kiss1 expression is upregulated by the gonadal steroid and is under the influences of light–dark cycles in medaka [12], [26], suggesting that kiss1 neurons are involved in the steroid feedback system in this species.
Seasonal changes in the kiss1 and kiss2 mRNA levels have been investigated in grass puffer and chub mackerel, which again suggests the presence of species differences. In grass puffer, kiss2 mRNA levels are higher in the brain just after spawning than in fish at different maturation stages [37]. In contrast, in chub mackerel, kiss1 and kiss2 mRNA levels were low in mature fish of both sexes with the exception of kiss2 in females, which remained constant at different gonadal stages [32]. Ontogenetic changes have been studied in zebrafish (kiss1 [4]; kiss1 and kiss2 [15]), which suggested an elevation in the expression of both genes in pubertal periods. Similar ontogenetic data in relation to puberty are lacking in other fish, and also importantly, expression profiles at the level of brain nuclei remain unknown in any fish.
As enumerated above, molecular species, central distribution, and hence the physiological significance of kisspeptins in teleosts show considerable species differences, indicating that further studies in other species are clearly necessary. In the present study, we investigated the kisspeptin system of the red seabream Pagrus major, a fish of great aquacultural importance. The red seabream belongs to the order Perciformes [28], in which species with both kiss1 and kiss2 (European seabass [6] and chub mackerel [32]) as well as species that presumably possesses kiss2 alone (orange-spotted grouper [38]) have been reported. Further studies in this teleost group are clearly necessary to better understand the functions and evolution of multiple kisspeptin molecular types. In addition, considerable knowledge has been accumulated on the reproductive endocrinology of red seabream [7], [8], [11], [17], [18], [23], [30], [31], [33], [34]. When the red seabream is reared in fish pens, their first spawning (puberty) occurs at three years of age from mid-April to early May, i.e. the spawning season of wild fish [31], [33]. A previous study during the peri-pubertal period indicates that the level of GnRH1 in the brain begins to rise in February and peaks during their first spawning period (April–May) when the fish reach the age of three [33]. Furthermore, administration of GnRH analogue to 16-month-old female red seabream induces precocious puberty [17], indicating the involvement of GnRH in the onset of puberty. These previous studies provide very reliable information for interpreting the data obtained in the present study.
In the present study we cloned kiss2 and investigated the central distribution of kiss2-expressing neurons by in situ hybridization in male and female red seabream. We then examined the changes in mRNA expression during the peri-pubertal period in both sexes. We employed in situ hybridization to assess the changes in kiss2 expression rather than the analyses of mRNA amounts in the whole brain (or dissected brain parts), since in situ hybridization experiments suggested the presence of multiple hypothalamic populations of kiss2 neurons that can serve different functions.
To investigate the possible involvement of kiss2 in the regulation of the gonadotropin-releasing hormone 1 (GnRH1 or seabream GnRH) system, we also analyzed the number of GnRH1-immunoreactive neurons in the preoptic area of both sexes, using the same fish analyzed for kiss2 cell numbers. Studies of the changes in GnRH1 (peptide and mRNA contents) during the peri-pubertal period have been conducted only in the female red seabream [31], [33].
Section snippets
Fish
Six red seabream P. major (Sparidae, Perciformes, Teleostei) at 1–4 years of age of both sexes were used for cloning of kiss1 and kiss2 genes. Four red seabream of both sexes at the age of 1–3 years were used for Nissl staining. Twelve red seabream of both sexes at the age of 1–4 years were used for in situ hybridization to examine the central distribution of kiss2 mRNA expressing neurons. These fish were purchased from commercial sources or were sampled from the fish stocks kept in a fish pen at
Sequences
The full-length cDNA of the red seabream kiss2 was 503 bp (479 bp excluding the poly-A tail) that contained an open reading frame of 372 bp encoding a precursor molecule of 124 amino acids (aa) with a putative signal peptide of 18 aa (Fig. 1: AB632369). A presumed polyadenylation signal was recognized at fourteen bp upstream of the poly-A tail. The presumed core-sequence region, Kiss2(10), of the red seabream Kiss2 was FNFNPFGLRF.
An alignment analysis of the deduced amino acid sequence of red
Discussion
In the present study of red seabream, we have cloned kiss2 that has a number of features in common with kiss2 in other teleosts. Neurons that express kiss2 mRNA were present almost exclusively in the NRL of the hypothalamus. In both males and females the number of kiss2 cells in the NRLd was higher in the first spawning period, showing a pattern of change similar to that of GnRH1-immunoreactive cells. Such results suggest that kiss2 neurons in the NRLd are involved in pubertal processes by
Acknowledgments
This work was supported in part by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of Japan. We wish to thank Ms. Narumi Mizuno, Mr. Yusuke Yamada, and Mr. Takeshi Kato (Laboratory of Fish Biology, Graduate School of Bioagricultural Sciences, Nagoya University) for their valuable assistance in the fish sampling.
References (42)
- et al.
Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals
Mol. Cell. Endocrinol.
(2009) - et al.
The metastasis suppressor gene KiSS-1 encodes, kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54
J. Biol. Chem.
(2001) - et al.
Effects of gonadotropin-releasing hormone agonist and dopamine antagonist on hypothalamo-pituitary-gonadal axis of pre-pubertal female red seabream (Pagrus major)
Gen. Comp. Endocrinol.
(2003) - et al.
Effects of gonadotropin-releasing hormone on pituitary-ovarian axis of one-year old pre-pubertal red seabream
Gen. Comp. Endocrinol.
(2004) - et al.
Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat
Biochem. Biophys. Res. Commun.
(2004) - et al.
Gene structure analysis of kisspeptin-2 (Kiss2) in the Senegalese sole (Solea senegalensis): Characterization of two splice variants of Kiss2, and novel evidence for metabolic regulation of kisspeptin signaling in non-mammalian species
Mol. Cell. Endocrinol.
(2011) - et al.
Seasonal variation of the three native gonadotropin-releasing hormone messenger ribonucleic acids levels in the brain of female red seabream
Gen. Comp. Endocrinol.
(2003) - et al.
Molecular characterization, tissue distribution, and mRNA expression profiles of two Kiss genes in the adult male and female chub mackerel (Scomber japonicus) during different gonadal stages
Gen. Comp. Endocrinol.
(2010) - et al.
Elevation of Kiss2 and its receptor gene expression in the brain and pituitary of grass puffer during the spawning season
Gen. Comp. Endocrinol.
(2010) - et al.
GnRH-immunoreactive neuronal system in the presumptive ancestral chordate, Ciona intestinalis (Ascidian)
Gen. Comp. Endocrinol.
(1998)
Evidence for the existence of a functional Kiss1/Kiss1 receptor pathway in fish
Peptides
Goldfish kisspeptin: molecular cloning, tissue distribution of transcript expression, and stimulatory effects on prolactin, growth hormone and luteinizing hormone secretion and gene expression via direct actions at the pituitary level
Gen. Comp. Endocrinol.
Estimation of nuclear population from microtome sections
Anat. Rec.
Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats
J. Reprod. Dev.
Functional and evolutionary insights into vertebrate kisspeptin systems from studies of fish brain
J. Fish Biol.
Molecular identification and functional characterization of the kisspeptin/kisspeptin receptor systems in lower vertebrates
Biol. Reprod.
Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54
Proc. Natl. Acad. Sci. USA
Unique expression of gonadotropin-I and -II subunit genes in male and female red seabream (Pagrus major) during sexual maturation
Biol. Reprod.
Correlation between messenger RNA expression of cytochrmoe P450 aromatase and its enzyme activity during oocyte development in the red seabream (Pagrus major)
Biol. Reprod.
A role for kisspeptins in the regulation of gonadotropin secretion in the mouse
Endocrinology
Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat
Neuroendocrinol.
Cited by (32)
Characterization and distribution of kisspeptins, kisspeptin receptors, GnIH, and GnRH1 in the brain of the protogynous bluehead wrasse (Thalassoma bifasciatum)
2022, Journal of Chemical NeuroanatomyCitation Excerpt :In the preoptic area, it is possible that expression of kiss2 is so limited that we missed expression in four out of six fish due to the use of single slides from a series of six, since each 20 µm section was separated by 100 µm. Kiss2-expressing neurons have also been found in the vicinity of the lateral recess in other teleosts (Kitahashi et al., 2009; Servili et al., 2011; Kanda et al., 2012b; Shimizu et al., 2012; Zmora et al., 2012; Escobar et al., 2013a; Escobar et al., 2013b; Osugi et al., 2013; Ohga et al., 2017). As we found in the bluehead wrasse, an additional and sometimes sporadic population of kiss2-expressing neurons was also detected in the POA of zebrafish (Servili et al., 2011), goldfish (Kanda et al., 2012b), European sea bass (Escobar et al., 2013a; Escobar et al., 2013b), masu salmon (Osugi et al., 2013), and chub mackerel (Ohga et al., 2017).
Kobayashi Award 2019: The neuroendocrine regulation of the mammalian reproduction
2022, General and Comparative EndocrinologyMolecular characterization of kisspeptin receptors and gene expression analysis during oogenesis in the Russian sturgeon (Acipenser gueldenstaedtii)
2021, General and Comparative EndocrinologyCitation Excerpt :Additionally, brain KissR mRNA expression was lower in immature zebrafish compared to that of sexually mature individuals (Biran et al., 2008). The expression of KissR mRNA in the brain were also correlated with pubertal development in the grey mullet (Mugil cephalus) (Nocillado et al., 2007), tilapia (Martinez-Chavez et al., 2008), cobia (Rachycentron canadum) (Mohamed et al., 2007), fathead minnow (Pimephales promelas) (Filby et al., 2008), Senegalese sole (Solea senegalensis) (Mechaly et al., 2012), Atlantic halibut (Hippoglossus hippoglossus) (Mechaly et al., 2010), red seabream (Pagrus major) (Shimizu et al., 2012), blue gourami Trichogaster trichopterus (Degani et al., 2017), and chub mackerel (Scomber japonicas) (Selvaraj et al., 2013). Due to the highly endangered status of the species it was not possible to study the brain expression in these fish.
The Kiss2/GPR54 system stimulates the reproductive axis in male black porgy, Acanthopagrus schlegelii
2019, General and Comparative EndocrinologyKisspeptins and their receptors in the brain-pituitary-gonadal axis of Odonthestes bonariensis: Their relationship with gametogenesis along the reproductive cycle
2017, General and Comparative EndocrinologyCitation Excerpt :However, Kiss2 producing-neurons in the lateral recess of Oryzias latipes (Kanda et al., 2012; Mitani et al., 2010), C. auratus (Kanda et al., 2012), M. saxatilis (Zmora et al., 2012) and D. labrax (Escobar et al., 2013), did not respond to E2, or express estrogen receptors (ERs). The relative abundance of kisspeptins/kisspeptin receptors transcripts during fish gonadal maturation, has been studied in few species belonging to 6 different orders: Scombriformes (Selvaraj et al., 2010; Ohga et al., 2013); Tetraodontiformes (Shahjahan et al., 2010), Spariformes (Shimizu et al., 2012), Pleuronectiformes (Mechaly et al., 2012), Perciformes (Alvarado et al., 2013; Migaud et al., 2012) and Cypriniformes (Saha et al., 2016). In general, all these studies found that kisspeptin(s) increased during or previous to ovulation and/or spermiation.
Kisspeptin2 stimulates the HPG axis in immature Nile tilapia (Oreochromis niloticus)
2016, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular BiologyCitation Excerpt :Additionally, it was reported that the axonal fiber of kisspeptin cells in the brain of zebrafish interacted with the hypothalamic GnRH III cell (Servili et al., 2011). Furthermore, the expression of kiss2 mRNA in female red seabream brains was correlated with the number of GnRH I cells (Shimizu et al., 2012). This suggests, that kisspeptin in teleosts is directly or indirectly related to the control of the GnRH neuron.
- 1
These authors contributed equally to this work.