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

Abstract

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.

Introduction

Kisspeptin, a member of the RFamide peptide family, was first identified by its inhibitory effect on cancer metastasis (Lee et al., 1996). In mammals, variants of kisspeptin including kisspeptin-54, kisspeptin-14, kisspeptin-13, and kisspeptin-10 have been identified (Bilban et al., 2004). These variants are the products of Kiss1 gene caused by differential processing of Kiss1 preprohormone (Ohtaki et al., 2001). They all share the common C-terminal 10 amino acid (a.a.) core sequence kisspeptin-10, which allows them to bind to their cognate receptor Kiss1r (or GPR54) with high affinity (Kotani et al., 2001). Kisspeptin, through activation of Kiss1r, plays an important role in the timing of puberty (Colledge, 2004), maintenance of gonadal functions (Roa et al., 2008a), photoperiod control of seasonal breeding (Greives et al., 2007), metabolic gating of fertility (Castellano et al., 2009), and trophoblast invasion during pregnancy (Bilban et al., 2004). In recent years, kisspeptin has been proposed to be a novel gatekeeper for the gonadotropic axis mainly by its potent stimulation on gonadotropin secretion (Roa et al., 2009, Roseweir and Millar, 2009). In the rat, the stimulatory actions on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are mediated by the Kiss1/Kiss1r systems within the periventricular and arcuate nuclei (Gottsch et al., 2004), which can activate gonadotropin-releasing hormone (GnRH) neurons located in the preoptic area (POA) of the hypothalamus (Roseweir and Millar, 2009). In the same animal model, kisspeptin-induced GnRH release has been documented both in vivo (Messager et al., 2005) and in vitro (Nazian, 2006, Quaynor et al., 2007) and is involved in steroid feedback during preovulatory LH surge (Adachi et al., 2007). In ovariectomized cows, sex steroids can also modulate the sensitivity of LH secretion in vivo to kisspeptin stimulation (Whitlock et al., 2008). These findings taken together support the idea that kisspeptin is an integral component of the neuroendocrine control for reproduction.

Although the hypothalamus represents the primary site of action for kisspeptin regulation of reproduction, the direct effect of kisspeptin at the pituitary level is still unclear (Richard et al., 2009). Kiss1r mRNA expression have been reported in the pituitary of the rat (Richard et al., 2008) and human (Ohtaki et al., 2001). In sheep, kisspeptin immunoreactivity can be detected in hypophysial portal blood, despite the fact that its levels do not exhibit noticeable changes during reproductive cycle and preovulatory LH surge (Smith et al., 2008). Although these findings raise the possibility that kisspeptin may also act as a hypophysiotropic factor, in vitro studies on pituitary actions of kisspeptin have yielded conflicting results. In static cultures of rat pituitary fragments (Thompson et al., 2004) and pituitary cells (Matsui et al., 2004, Navarro et al., 2005a), kisspeptin treatment did not alter LH and FSH secretion at the pituitary level. In other studies with rat pituitary cell cultures, however, kisspeptin not only could induce LH release but also stimulate growth hormone (GH) secretion. These hormone-release responses were of lower magnitude compared to that caused by the primary regulators GnRH (for LH) and GHRH (for GH) and occurred with concurrent rises in cytosolic Ca²⁺ in gonadotrophs and somatotrophs (Gutierrez-Pascual et al., 2007). Recently, kisspeptin-induced prolactin (PRL) release have also been reported in bovine pituitary cells (Kadokawa et al., 2008), suggesting that lactotrophs may also serve as the pituitary target for kisspeptin actions.

Similar to mammals, Kiss1 has been cloned in fish models (Biran et al., 2008) and its involvement in gonadotropin release (Felip et al., 2008) and signaling of sexual maturation/puberty (Filby et al., 2008) have been confirmed (e.g., medaka and zebrafish). Besides Kiss1, a second Kiss gene, namely Kiss2, was also identified in lower vertebrates including the bony fish. Apparently, the two Kiss genes arose early in vertebrate evolution by gene duplication and Kiss2 gene might have been lost recently in the mammalian lineage (Felip et al., 2008). Apart from the core sequence for kisspeptin-10, the sequence homology between Kiss1 and Kiss2 is rather low (∼20–25%). When comparing kisspeptin-10 sequences encoded by the two genes, a distinct pattern of a.a. substitutions at position 1 and 10 can also be noted, with the “Y–Y form” (YNL/WNSFGLRY) for Kiss1 and “F–F form” (FNY/FNPFG LRF) for Kiss2, respectively (Kitahashi et al., 2009). In zebrafish, Kiss1 and Kiss2 are expressed in different locations within the hypothalamus (Kitahashi et al., 2009) and may exert differential actions on LH and FSH secretion/expression (Felip et al., 2008, Kitahashi et al., 2009). At present, the studies on pituitary actions of kisspeptin are restricted to mammals and no information is available in fish species. In this study, using goldfish as a model, we seek to examine the pituitary actions of kisspeptin in regulating pituitary hormone secretion and gene expression in modern-day bony fish. The goldfish was used due to the availability a well-established pituitary cell culture system and ample information for endocrine signaling at the pituitary level (Popesku et al., 2008). As a first step, goldfish Kiss1 was cloned by 3′/5′RACE and its tissue expression profile was characterized by Northern blot and RT-PCR. To provide the initial evidence for the hypothesis that the goldfish pituitary indeed can serve as a target for kisspeptin action, RT-PCR for Kiss1r was performed in goldfish lactotrophs, somatotrophs, and gonadotrophs isolated by laser capture microdissection. The results obtained were then confirmed by testing the effects of goldfish kisspeptin-10 on LH, GH, and PRL release and transcript expression in primary cultures of goldfish pituitary cells. To our knowledge, the present study represents the first report in fish models on pituitary hormone regulation by kisspeptin via direct actions at the pituitary cell level.