Kisspeptin plays an important role in the onset of puberty through stimulation of gonadotropin-releasing hormone (GnRH), a master molecule of reproduction. Furthermore, the existence of multiple kisspeptins is evident in most vertebrate species. Therefore, elucidating the regulatory mechanisms of the kisspeptin genes is important to understand the functions of multiple kisspeptin forms in the brain. This review focuses on the comparative aspects of kisspeptin gene regulation with an emphasis on the role of environmental signals including gonadal steroids, photoperiods and metabolic signals. These environmental signals differently regulate the kisspeptin genes distinctively in each species. In addition, photoperiodic regulation of the kisspeptin genes alters during sexual maturational, suggesting interactions between the gonadal hormone pathway and the photoperiod pathway. Further studies of the regulatory mechanisms of kisspeptin genes especially in teleosts which possess multiple kisspeptin/kisspeptin receptor systems will help to understand the precise role of multiple kisspeptin forms in different species.
The tachykinins are a family of neuropeptides, including substance P (SP), neurokinin A (NKA), and neurokinin B (NKB), that are encoded by the tac1 (SP and NKA) or tac2/3 (NKB) genes. Tachykinins are widely distributed in the central nervous system and have roles as neurotransmitters and/or neuromodulators. Recent studies in mammals have demonstrated the coexpression of NKB and kisspeptin and their comodulatory roles over the control of reproduction. We have recently identified two kisspeptin-encoding genes, kiss1 and kiss2, in teleosts. However, such relationship between tachykinins and kisspeptins has not been demonstrated in non-mammalian species. To determine the involvement of tachykinins in the reproduction in teleosts, we identified tac1 and two tac2 (tac2a and tac2b) sequences in the zebrafish genome using in silico data mining. Zebrafish tac1 encodes SP and NKA, whereas the tac2 sequences encode NKB and an additional peptide homologous to NKB (NKB-related peptide). Digoxigenin in situ hybridization in the brain of zebrafish showed tac1 mRNA-containing cells in the olfactory bulb, telencephalon, preoptic region, hypothalamus, mesencephalon, and rhombencephalon. The zebrafish tac2a mRNA-containing cells were observed in the preoptic region, habenula, and hypothalamus, whereas the tac2b mRNA-containing cells were predominantly observed in the dorsal telencephalic area. Furthermore, we examined the coexpression of tachykinins and two kisspeptin genes in the brain of zebrafish. Dual fluorescent in situ hybridization showed no coexpression of tachykinins mRNA with kisspeptins mRNA in hypothalamic nuclei or the habenula. These results suggest the presence of independent pathways for kisspeptins and NKB neurons in the brain of zebrafish.
The central role of kisspeptin (kiss) in mammalian reproduction is well established; however, its intra-gonadal role is poorly addressed. Moreover, studies investigating intra-gonadal role of kiss in fish reproduction are scanty, contradictory and inconclusive. The expression of kiss1 mRNA has been detected in the fish brain, and functionally attributed to the regulation of reproduction, feeding and behavior. The kiss1 mRNA has also been demonstrated in tissues other than the brain in some studies, but its cellular distribution and role at the tissue level have not been adequately addressed in fish. Therefore, an attempt was made in the present study to localize kiss1 in gonadal cells of the freshwater catfish, Clarias batrachus. This study reports the presence of kiss1 in the theca cells and granulosa cells of the ovarian oocytes and interstitial cells in the testis of the catfish. The role of kiss1 in the ovary and testis of the catfish was also investigated using kiss1 receptor (kiss1r) antagonist (p234). The p234 treatment decreased the production of 17β-estradiol in ovary and testosterone in the testis by lowering the activities of 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase under both, in vivo as well as in vitro conditions. The p234 treatment also arrested the progression of oogenesis, as evident from the low number of advancing/advanced oocytes in the treated ovary in comparison to the control ovary. It also reduced the area and perimeter of the seminiferous tubules in the treated catfish testis. Thus, our findings suggest that kiss is involved in the regulation of gonadal steroidogenesis, independent of known endocrine/ autocrine/ paracine regulators, and thereby it accelerates gametogenic processes in the freshwater catfish.
A cichlid fish, the Nile tilapia (Oreochromis niloticus), is a maternal mouthbrooder, which exhibits minimum energy expenditure and slower ovarian cycles during mouthbrooding. The objective of this study was to observe changes in the gene expression of key neuropeptides involved in the control of appetite and reproduction, including neuropeptide Y a (NPYa), reproductive neuropeptides: gonadotropin-releasing hormone (GnRH1, GnRH2 and GnRH3) and kisspeptin (Kiss2) during mouthbrooding (4- and 12-days), 12-days of food restriction and 12-days of food restriction followed by refeeding. The food restriction regime showed a significant increase in npya mRNA levels in the telencephalon. However, there were no significant alterations in npya mRNA levels during mouthbrooding. gnrh1 mRNA levels were significantly lower in mouthbrooding female as compared with females with food restriction. gnrh3 mRNA levels were also significantly lower in female with 12-days of mouthbrooding, 12-days of food restriction followed by 12-days of refeeding when compared with controls. There were no significant differences in gnrh2 and kiss2 mRNA levels between groups under different feeding regimes. No significant changes were observed in mRNA levels of receptors for peripheral metabolic signaling molecules: ghrelin (GHS-R1a and GHS-R1b) and leptin (Lep-R). These results suggested that unaffected npya mRNA levels in the telencephalon might contribute to suppression of appetite in mouthbrooding female tilapia. Furthermore, lower gnrh1 and gnrh3 mRNA levels may influence the suppression of reproductive functions such as progression of ovarian cycle and reproductive behaviours, while GnRH2 and Kiss2 may not play a significant roles in reproduction under food restriction condition.
Early-life stress can cause long-term effects in the adulthood such as alterations in behaviour, brain functions and reproduction. DNA methylation is a mechanism of epigenetic change caused by early-life stress. Dexamethasone (DEX) was administered to zebrafish larvae to study its effect on reproductive dysfunction. The level of GnRH2, GnRH3, Kiss1 and Kiss2 mRNAs were measured between different doses of DEX treatment groups in adult zebrafish. Kiss1 and GnRH2 expression were increased in the 200mg/L DEX treated while Kiss2 and GnRH3 mRNA levels were up-regulated in the 2mg/L DEX-treated zebrafish. The up-regulation may be related to programming effect of DEX in the zebrafish larvae, causing overcompensation mechanism to increase the mRNA levels. Furthermore, DEX treatment caused negative impact on the development and maturation of the testes, in particular spermatogenesis. Therefore, immature gonadal development may cause positive feedback by increasing GnRH and Kiss. This indicates that DEX can alter the regulation of GnRH2, GnRH3, Kiss1 and Kiss2 in adult zebrafish, which affects maturation of gonads. Computer analysis of 1.5 kb region upstream of the 5' UTR of Kiss1, Kiss2, GnRH2 and GnRH3 promoter showed that there are putative binding sites of glucocorticoid response element and transcription factors involved in stress response. GnRH3 promoter analysed from pre-optic area, ventral telencephalon and ventral olfactory bulb showed higher methylation at CpG residues located on -1410, -1377 and -1355 between control and 2mg/L DEX-treated groups. Hence, early-life DEX treatment can alter methylation of GnRH3 gene promoter, which subsequently affects gene regulation and reproductive functions.
RFamide-related peptide (RFRP)-3 reduces luteinising hormone (LH) secretion in rodents. Stress has been shown to upregulate the expression of the RFRP gene (Rfrp) with a concomitant reduction in LH secretion, but an effect on expression of the gonadotrophin-releasing hormone (GnRH) gene (Gnrh1) has not been shown. We hypothesised that lipopolysaccharide (LPS)-induced stress affects expression of Rfrp, the gene for kisspeptin (Kiss1) and/or Gnrh1, leading to suppression of LH levels in rats. Intracerebroventricular injections of RFRP-3 (0.1, 1, 5 nmol) or i.v. LPS (15μgkg-1) reduced LH levels. Doses of 1 and 5 nmol RFRP-3 were then administered to analyse gene expression by in situ hybridisation. RFRP-3 (5 nmol) had no effect on Gnrh1 or Kiss1 expression. LPS stress reduced GnRH and Kiss1 expression, without affecting Rfrp1 expression. These data indicate that LPS stress directly or indirectly reduces Gnrh1 expression, but this is unlikely to be due to a change in Rfrp1 expression.
The kiss1/gpr54 signaling system is considered to be a critical regulator of reproduction in most vertebrates. However, this presumption has not been tested vigorously in nonmammalian vertebrates. Distinct from mammals, multiple kiss1/gpr54 paralogous genes (kiss/kissr) have been identified in nonmammalian vertebrates, raising the possibility of functional redundancy among these genes. In this study, we have systematically generated the zebrafish kiss1(-/-), kiss2(-/-), and kiss1(-/-);kiss2(-/-) mutant lines as well as the kissr1(-/-), kissr2(-/-), and kissr1(-/-);kissr2(-/-) mutant lines using transcription activator-like effector nucleases. We have demonstrated that spermatogenesis and folliculogenesis as well as reproductive capability are not impaired in all of these 6 mutant lines. Collectively, our results indicate that kiss/kissr signaling is not absolutely required for zebrafish reproduction, suggesting that the kiss/kissr systems play nonessential roles for reproduction in certain nonmammalian vertebrates. These findings also demonstrated that fish and mammals have evolved different strategies for neuroendocrine control of reproduction.
The habenula is an evolutionarily conserved brain structure, which has recently been implicated in fear memory. In the zebrafish, kisspeptin (Kiss1) is predominantly expressed in the habenula, which has been implicated as a modulator of fear response. Hence, in the present study, we questioned whether Kiss1 has a role in fear memory and morphine-induced fear memory impairment using an odorant cue (alarm substances, AS)-induced fear avoidance paradigm in adult zebrafish, whereby the fear-conditioned memory can be assessed by a change of basal place preference (= avoidance) of fish due to AS-induced fear experience. Subsequently, to examine the possible role of Kiss1 neurons-serotonergic pathway, kiss1 mRNA and serotonin levels were measured. AS exposure triggered fear episodes and fear-conditioned place avoidance. Morphine treatment followed by AS exposure, significantly impaired fear memory with increased time-spent in AS-paired compartment. However, fish administered with Kiss1 (10-21 mol/fish) after morphine treatment had significantly lower kiss1 mRNA levels but retained fear memory. In addition, the total brain serotonin levels were significantly increased in AS- and Kiss1-treated groups as compared to control and morphine treated group. These results suggest that habenular Kiss1 might be involved in consolidation or retrieval of fear memory through the serotonin system.
Ambient light and temperature affect reproductive function by regulating kisspeptin and gonadotrophin-releasing hormone (GnRH) in vertebrates. Melatonin and melatonin receptors, as well as the two-pore domain K+ channel-related K+ (TREK) channels, are affected by light and/or temperature; therefore, these molecules could modulate kisspeptin and GnRH against ambient light and temperature. In this study, we investigated the effect of light and temperature, which affect melatonin levels in gene expression levels of TREK channels, kisspeptin, and GnRH. We first investigated the effects of different light and temperature conditions on brain melatonin concentrations by ELISA. Fish were exposed to either constant darkness, constant light, high temperature (35°C), or low temperature (20°C) for 72 h. Brain melatonin levels were significantly high under constant darkness and high temperature. We further investigated the effects of high brain melatonin levels by constant darkness and high temperature on gene expression levels of melatonin receptors (mt1, mt2, and mel1c), TREK channels (trek1b, trek2a, and trek2b), gnrh3, and kiss2 in the adult zebrafish brain by real-time polymerase chain reaction. Fish were exposed to constant darkness or elevated temperatures (35°C) for 72 h. trek2a, kiss2, and gnrh3 levels were increased under constant darkness. High temperature decreased gene expression levels of mt1, mt2, mel1c, and gnrh3 in the preoptic area, whereas other genes remained unchanged. Melatonin receptors, TREK channels, gnrh3, and kiss2 responded differently under high melatonin conditions. The melatonin receptors and the TREK channels could play roles in the regulation of reproduction by environmental cues, especially ambient light and temperature.
Postnatal treatment with selective serotonin reuptake inhibitors (SSRIs) has been found to affect brain development and the regulation of reproduction in rodent models. The normal masculinization process in the brain requires a transient decrease in serotonin (5-HT) levels in the brain during the second postnatal week. Strict regulation of androgen receptor (AR) and gonadotropin-releasing hormone (GnRH) expression is important to control male reproductive activity. Therefore, this study was designed to examine the effects of a potent SSRI (citalopram) on male sexual behavior and expression levels of AR and GnRH in adult male mice receiving either vehicle or citalopram (10mg/kg) daily during postnatal days 8-21. The citalopram-treated male mice showed altered sexual behavior, specifically a significant reduction in the number of intromissions preceding ejaculation compared with the vehicle-treated mice. The citalopram-treated male mice displayed elevated anxiety-like behavior in an open field test and lower locomotor activity in their home cage during the subjective night. Although there was no change in GnRH and AR mRNA levels in the preoptic area (POA), quantified by real-time polymerase chain reaction, immunostained AR cell numbers in the medial POA were decreased in the citalopram-treated male mice. These results suggest that the early-life inhibition of 5-HT transporters alters the regulation of AR expression in the medial POA, likely causing decreased sexual behavior and altered home cage activity in the subjective night.