Central distribution of kiss2 neurons and peri-pubertal changes in their expression in the brain of male and female red seabream Pagrus major

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Abstract

kisspeptins that are encoded by kiss1 gene are now considered the key regulator of reproduction from a number of studies in mammals. In most vertebrates, a paralogue of kiss1, called kiss2, is also present, and the functional significance of kisspeptins is not known precisely. In the present study, we have cloned kiss2 from a perciform teleost, the red seabream Pagrus major. The amino acid sequence deduced from the red seabream kiss2 contained a highly conserved 10-amino-acid residue, Kiss2(10) or kp-10. A kiss1-like transcript was also identified, but it appears to be non-functional due to the presence of a “premature” stop codon. Neurons that express kiss2 mRNA were distributed in the dorsal (NRLd) and ventral (NRLv) parts of nucleus recessi lateralis in the hypothalamus. In some fish a few kiss2-expressing neurons were detected in the preoptic area and nucleus ventralis tuberis. The number of kiss2-expressing neurons in the NRLd was larger during the first spawning season in both males and females compared with fish in the post-spawning periods. In males the number of kiss2 neurons in the NRLd of maturing fish was also larger than those in the post-spawning periods. In males the number of kiss2 neurons in the NRLv showed a similar pattern of changes to that of NRLd, while significant changes were not detected for females. The numbers of gonadotropin-releasing hormone 1 (GnRH1)-immunoreactive neurons in the preoptic area showed a similar pattern of change as those of kiss2 cells of the NRLd in both males and females (and also the NRLv in males). These results are in good agreement with the hypothesis that kiss2 neurons are involved in pubertal processes via regulatory influences on GnRH1 neurons in red seabream.

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.

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