Taxonomy of introns and the evolution of minor introns.

Classification of introns, which is crucial to understanding their evolution and splicing, has historically been binary and has resulted in the naming of major and minor introns that are spliced by their namesake spliceosome. However, a broad range of intron consensus sequences exist, leading us to here reclassify introns as minor, minor-like, hybrid, major-like, major and non-canonical introns in 263 species across six eukaryotic supergroups. Through intron orthology analysis, we discovered that minor-like introns are a transitory node for intron conversion across evolution. Despite close resemblance of their consensus sequences to minor introns, these introns possess an AG dinucleotide at the -1 and -2 position of the 5' splice site, a salient feature of major introns. Through combined analysis of CoLa-seq, CLIP-seq for major and minor spliceosome components, and RNAseq from samples in which the minor spliceosome is inhibited we found that minor-like introns are also an intermediate class from a splicing mechanism perspective. Importantly, this analysis has provided insight into the sequence elements that have evolved to make minor-like introns amenable to recognition by both minor and major spliceosome components. We hope that this revised intron classification provides a new framework to study intron evolution and splicing.

Cloning of gonadotropin Gph-alpha, FSH-beta and LH-beta subunits and seasonal profiles of steroid hormones in wild-caught Nile perch, Lates niloticus.

The Nile perch (np; Lates niloticus) is a freshwater teleost species with a potential for aquaculture in freshwater surroundings. However, wild-caught breeders have persistently failed to spawn spontaneously in captivity. Cloning of the gonadotropin subunits and analysing seasonal variation in reproductive hormone levels for a 1-year period were done to gain knowledge on the physiological basis underlying the reproductive biology of np. The ß-follicle-stimulating hormone (FSH-ß) and ß-luteinizing hormone (LH-ß) subunits and their common α-glycoprotein (Gph-α) subunit were cloned using 3' and 5' RACE-PCR. The nucleotide sequences of the npgph-α, npfsh-ß, and nplh-ß subunits were 664, 580 and 675 nucleotides in length, encoding peptides of 124, 120 and 148 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity with its counterparts in other teleost. Sequence analysis showed that npFSH-ß is more similar to higher vertebrate FSH-ßs than to higher vertebrate LH-ßs. Heterologous immunoassay was calibrated to analyse pituitary LH levels. While the LH immunoassay showed parallelism of npLH with that of tilapia (ta), no parallelism for FSH was found. Levels of pituitary LH were higher in females at gonadal stages of vitellogenic oocytes, mature secondary oocytes and mature tertiary oocytes with migrating nucleus than in pre-vitellogenic oocytes and early and late perinucleolus oocytes. Using competitive steroid ELISA, variations in the levels of the steroid hormones 11-ketotestosterone (11-KT) in males and E2 in females were characterized in relation to month and reproductive index of Nile perch. Our findings show that in females, gonadosomatic index and plasma E2 were highly correlated (R2 = 0.699, n = 172) and peaked from September to November while in males, the gonadosomatic index and plasma 11-KT peaked from October to November. In female fish, both steroid hormones were detected in the plasma but greatly varied in concentrations. E2 in particular, increased with the developmental stage of the gonads. The levels of steroid hormones, E2 and 11-KT in females and males respectively increased with fish size (total lengths) and suggest that females mature at a body length of 40-59 cm than their counter part males that mature at a total length of 60-70 cm. Taken together, we describe seasonal endocrine differences in wild-caught adult Nile perch which could potentially be exploited to manipulate the reproductive axis in cultured breeders.

Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction.

The endocrine regulation of vertebrate reproduction is achieved by the coordinated actions of several peptide neurohormones, tachykinin among them. To study the evolutionary conservation and physiological functions of neurokinin B (NKB), we identified tachykinin (tac) and tac receptor (NKBR) genes from many fish species, and cloned two cDNA forms from zebrafish. Phylogenetic analysis showed that piscine Tac3s and mammalian neurokinin genes arise from one lineage. High identity was found among different fish species in the region encoding the NKB; all shared the common C-terminal sequence. Although the piscine Tac3 gene encodes for two putative tachykinin peptides, the mammalian ortholog encodes for only one. The second fish putative peptide, referred to as neurokinin F (NKF), is unique and found to be conserved among the fish species when tested in silico. tac3a was expressed asymmetrically in the habenula of embryos, whereas in adults zebrafish tac3a-expressing neurons were localized in specific brain nuclei that are known to be involved in reproduction. Zebrafish tac3a mRNA levels gradually increased during the first few weeks of life and peaked at pubescence. Estrogen treatment of prepubertal fish elicited increases in tac3a, kiss1, kiss2, and kiss1ra expression. The synthetic zebrafish peptides (NKBa, NKBb, and NKF) activated Tac3 receptors via both PKC/Ca(2+) and PKA/cAMP signal-transduction pathways in vitro. Moreover, a single intraperitoneal injection of NKBa and NKF significantly increased leuteinizing hormone levels in mature female zebrafish. These results suggest that the NKB/NKBR system may participate in neuroendocrine control of fish reproduction.

Cell-based homologous expression system for in-vitro characterization of environmental effects on transmembrane peptide transport in fish.

All organisms encounter environmental changes that lead to physiological adjustments that could drive evolutionary adaptations. The ability to adjust performance in order to cope with environmental changes depends on the organism's physiological plasticity. These adjustments can be reflected in behavioral, physiological, and molecular changes, which interact and affect each other. Deciphering the role of molecular adjustments in physiological changes will help to understand how multiple levels of biological organization are synchronized during adaptations. Transmembrane transporters, which facilitate a cell's interaction with its surroundings, are prime targets for molecular studies of the environmental effects on an organism's physiology. Fish are subjected to environmental fluctuations and exhibit different coping mechanisms. To study the molecular adjustments of fish transporters to their external surrounding, suitable experimental systems must be established. The Mozambique tilapia (Oreochromis mossambicus) is an excellent model for environmental stress studies, due to its extreme salinity tolerance. We established a homologous cellular-based expression system and uptake assay that allowed us to study the effects of environmental conditions on transmembrane transport. We applied our expression system to investigate the effects of environmental conditions on the activity of PepT2, a transmembrane transporter critical in the absorption of dietary peptides and drugs. We created a stable, modified fish cell-line, in which we exogenously expressed the tilapia PepT2, and tested the effects of water temperature and salinity on the uptake of a fluorescent di-peptide, ß-Ala-Lys-AMCA. While temperature affected only Vmax, medium salinity had a bi-directional effect, with significantly reduced Vmax in hyposaline conditions and significantly increased Km in hypersaline conditions. These assays demonstrate the importance of suitable experimental systems for fish ecophysiology studies. Furthermore, our in-vitro results show how the effect of hypersaline conditions on the transporter activity can explain expression shifts seen in the intestine of saltwater-acclimated fish, emphasizing the importance of complimentary studies in better understanding environmental physiology. This research highlights the advantages of using homologous expression systems to study environmental effects encountered by fish, in a relevant cellular context. The presented tools and methods can be adapted to study other transporters in-vitro.

Chronic kisspeptin administration stimulated gonadal development in pre-pubertal male yellowtail kingfish (Seriola lalandi; Perciformes) during the breeding and non-breeding season.

The kisspeptin system is now accepted as a key regulator of vertebrate reproductive function, particularly the onset of puberty. In teleosts, the stimulatory effect of exogenous kisspeptins has been demonstrated mainly at the hypothalamic and pituitary levels of the reproductive axis, with very limited information pertaining to gonadal response. We determined the effect of chronic peripheral administration of the conserved kisspeptin decapeptides (YNLNSFGLRY or Kiss1-10; and FNFNPFGLRF or Kiss2-10) on gonadal development of pre-pubertal yellowtail kingfish (Seriola lalandi), a Perciform teleost, during the breeding and non-breeding season. We utilized slow-release implants to chronically deliver the synthesized peptides, which were based on the yellowtail kingfish kiss1 and kiss2 cDNA sequences that we isolated. The expression level of kiss2r and gnrh1 in the brain or hypothalamus did not vary between treated and control groups. Pituitary expression of fshß and lhß was upregulated only with Kiss1-10 treatment regardless of the season. Based on histological evidence, gonadal development was stimulated in male fish with either Kiss1-10 or Kiss2-10, with Kiss2-10 being more effective during the non-breeding period. Overall, our results suggest that kisspeptins modulate the early gonadal development of male yellowtail kingfish, however that may vary with the breeding season.

Genome editing in East African cichlids and tilapias: state-of-the-art and future directions.

African cichlid fishes of the Cichlidae family are a group of teleosts important for aquaculture and research. A thriving research community is particularly interested in the cichlid radiations of the East African Great Lakes. One key goal is to pinpoint genetic variation underlying phenotypic diversification, but the lack of genetic tools has precluded thorough dissection of the genetic basis of relevant traits in cichlids. Genome editing technologies are well established in teleost models like zebrafish and medaka. However, this is not the case for emerging model organisms, such as East African cichlids, where these technologies remain inaccessible to most laboratories, due in part to limited exchange of knowledge and expertise. The Cichlid Science 2022 meeting (Cambridge, UK) hosted for the first time a Genome Editing Workshop, where the community discussed recent advances in genome editing, with an emphasis on CRISPR/Cas9 technologies. Based on the workshop findings and discussions, in this review we define the state-of-the-art of cichlid genome editing, share resources and protocols, and propose new possible avenues to further expand the cichlid genome editing toolkit.

Differential and gonad stage-dependent roles of kisspeptin1 and kisspeptin2 in reproduction in the modern teleosts, morone 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.

Genome Editing Using the CRISPR-Cas9 System to Generate a Solid-Red Germline of Nile Tilapia (Oreochromis niloticus).

In recent years, there has been increasing demand for red tilapia, which are commercial strains of hybrids of different tilapiine species or red variants of highly inbred Nile tilapia. However, red tilapia phenotypes are genetically unstable and affected by environmental factors, resulting in nonuniform coloration with black or dark-red color blotches that reduce their market value. Solute carrier family 45 member 2 (SLC45A2) is a membrane transporter that mediates melanin biosynthesis and is evolutionarily conserved from fish to humans. In the present study, we describe the generation of a stable and heritable red tilapia phenotype by inducing loss-of-function mutations in the slc45a2 gene. For this purpose, we identified the slc45a2 gene in Nile tilapia and designed highly specific guide RNAs (gRNA) for its genomic sequence. Multiplex microinjection of slc45a2-specific ribonucleoproteins to Nile tilapia zygotes induced up to 97-99% albinism, including loss of melanin in the eye. Next-generation sequencing of the injected zygotes demonstrated that all injected fish carried mutant alleles with variable mutagenesis efficiencies. Sanger sequencing of the genomic target region in the slc45a2 gene from fin clips, sperm, and F1 offspring of a highly mutant male identified various genomic indels and germline transmission of the sperm-identified indels. Overall, this work demonstrates the generation of somatic and germline slc45a2 mutant alleles, which leads to complete albinism in Nile tilapia.

Identification and Characterization of a Non-muscular Myostatin in the Nile Tilapia.

The growth and differentiation factor Myostatin (MSTN, also known as GDF8) negatively regulates skeletal muscle development and growth in vertebrates. Most fish genomes contain two or more mstn genes, which are expressed in muscle and other tissues. Yet, in the genome of Nile tilapia (Oreochromis niloticus), which is one of the world's most important aquaculture fish species, only one mstn gene has previously been identified. Here, we identify a second mstn gene in Nile tilapia. We show that it clusters phylogenetically with other piscine mstn2 genes and that it shares chromosomal synteny with the human and zebrafish orthologs. We further show that mstn2 is not expressed in red or white muscles of Nile tilapia, but rather that its main site of expression is the brain. To determine which physiological functions are correlated with mstn expression, adult Nile tilapia were exposed to various environmental conditions and their effect on mstn1 and mstn2 expression in the brain and muscles was measured using real-time PCR. We found that the centrally- and muscle-expressed mstn genes differ in their responsiveness to diverse challenges, suggesting differential gene- and tissue-specific regulation of their expression. Metabolic and stress marker analyses showed that the altered mstn expression is not regulated by classical stress response. Taken together, our findings expand the understanding of the MSTN system in Nile tilapia and provide evolutionary insight into its function.

Publisher Correction: Splice-specific deficiency of the PTSD-associated gene PAC1 leads to a paradoxical age-dependent stress behavior.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Splice-specific deficiency of the PTSD-associated gene PAC1 leads to a paradoxical age-dependent stress behavior.

The pituitary adenylate cyclase-activating polypeptide receptor (PAC1, also known as ADCYAP1R1) is associated with post-traumatic stress disorder and modulation of stress response in general. Alternative splicing of PAC1 results in multiple gene products, which differ in their mode of signalling and tissue distribution. However, the roles of distinct splice variants in the regulation of stress behavior is poorly understood. Alternative splicing of a short exon, which is known as the "hop cassette", occurs during brain development and in response to stressful challenges. To examine the function of this variant, we generated a splice-specific zebrafish mutant lacking the hop cassette, which we designated 'hopless'. We show that hopless mutant larvae display increased anxiety-like behavior, including reduced dark exploration and impaired habituation to dark exposure. Conversely, adult hopless mutants displayed superior ability to rebound from an acute stressor, as they exhibited reduced anxiety-like responses to an ensuing novelty stress. We propose that the developmental loss of a specific PAC1 splice variant mimics prolonged mild stress exposure, which in the long term, predisposes the organism's stress response towards a resilient phenotype. Our study presents a unique genetic model demonstrating how early-life state of anxiety paradoxically correlates with reduced stress susceptibility in adulthood.

Revealing genes associated with vitellogenesis in the liver of the zebrafish (Danio rerio) by transcriptome profiling.

BACKGROUND: In oviparous vertebrates, including fish, vitellogenesis consists of highly regulated pathways involving 17beta-estradiol (E2). Previous studies focused on a relatively small number of hepatic expressed genes during vitellogenesis. This study aims to identify hepatic genes involved in vitellogenesis and regulated by E2, by using zebrafish microarray gene expression profiling, and to provide information on functional distinctive genes expressed in the liver of a vitellogenic female, using zebrafish as a model fish. RESULTS: Genes associated with vitellogenesis were revealed by the following paired t-tests (SAM) comparisons: a) two-month old vitellogenic (Vit2) females were compared with non-vitellogenic (NV) females, showing 825 differentially expressed transcripts during early stages of vitellogenesis, b) four-month old vitellogenic (Vit4) females were compared with NV females, showing 1,046 differentially expressed transcripts during vitellogenesis and c) E2-treated males were compared with control males, showing 1,828 differentially expressed transcripts regulated by E2. A Venn diagram revealed 822 common transcripts in the three groups, indicating that these transcripts were involved in vitellogenesis and putatively regulated by E2. In addition, 431 transcripts were differentially expressed in Vit2 and Vit4 females but not in E2-treated males, indicating that they were putatively not up-regulated by E2. Correspondence analysis showed high similarity in expression profiles of Vit2 with Vit4 and of NV females with control males. The E2-treated males differed from the other groups. The repertoire of genes putatively regulated by E2 in vitellogenic females included genes associated with protein synthesis and reproduction. Genes associated with the immune system processes and biological adhesion, were among the genes that were putatively not regulated by E2. E2-treated males expressed a large array of transcripts that were not associated with vitellogenesis.The study revealed several genes that were not reported before as being regulated by E2. Also, the hepatic expression of several genes was reported here for the first time. CONCLUSION: Gene expression profiling of liver samples revealed 1,046 differentially expressed transcripts during vitellogenesis of which at least ~64% were regulated by E2. The results raise the question on the regulation pattern and temporal pleiotropic expression of hepatic genes in vitellogenic females.

Transcriptome Analysis Reveals Common and Differential Response to Low Temperature Exposure Between Tolerant and Sensitive Blue Tilapia (Oreochromis aureus).

Tilapias are very important to the world's aquaculture. As befitting fish of their tropical origin, their distribution, and culture practices are highly affected by low temperatures. In this study, we used genetic and genomic methodologies to reveal pathways involved in the response and tolerance of blue tilapia (Oreochromis aureus) to low temperature stress. Cold tolerance was characterized in 66 families of blue tilapia. Fish from cold-tolerant and cold-sensitive families were sampled at 24 and 12°C, and the transcriptional responses to low-temperature exposure were measured in the gills and liver by high-throughput mRNA sequencing. Four hundred and ninety four genes displayed a similar temperature-dependent expression in both tolerant and sensitive fish and in the two tissues, representing the core molecular response to low temperature exposure. KEGG pathway analysis of these genes revealed down-regulation of focal-adhesion and other cell-extracellular matrix (ECM) interactions, and up-regulation of proteasome and various intra-cellular proteolytic activities. Differential responses between cold-tolerant and cold-sensitive fish were found with genes and pathways that were up-regulated in one group and down-regulated in the other. This reverse response was characterized by genes involved in metabolic pathways such as glycolysis/gluconeogenesis in the gills and biosynthesis of amino-acids in the liver, with low temperature down-regulation in tolerant fish and up-regulation in sensitive fish.

Specific inhibition of splicing factor activity by decoy RNA oligonucleotides.

Alternative splicing, a fundamental step in gene expression, is deregulated in many diseases. Splicing factors (SFs), which regulate this process, are up- or down regulated or mutated in several diseases including cancer. To date, there are no inhibitors that directly inhibit the activity of SFs. We designed decoy oligonucleotides, composed of several repeats of a RNA motif, which is recognized by a single SF. Here we show that decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1 and PTBP1, can specifically bind to their respective SFs and inhibit their splicing and biological activities both in vitro and in vivo. These decoy oligonucleotides present an approach to specifically downregulate SF activity in conditions where SFs are either up-regulated or hyperactive.

Pituicyte Cues Regulate the Development of Permeable Neuro-Vascular Interfaces.

The hypothalamo-neurohypophyseal system (HNS) regulates homeostasis through the passage of neurohormones and blood-borne proteins via permeable blood capillaries that lack the blood-brain barrier (BBB). Why neurohypophyseal capillaries become permeable while the neighboring vasculature of the brain forms BBB remains unclear. We show that pituicytes, the resident astroglial cells of the neurohypophysis, express genes that are associated with BBB breakdown during neuroinflammation. Pituicyte-enriched factors provide a local microenvironment that instructs a permeable neurovascular conduit. Thus, genetic and pharmacological perturbations of Vegfa and Tgfß3 affected HNS vascular morphogenesis and permeability and impaired the expression of the fenestral marker plvap. The anti-inflammatory agent dexamethasone decreased HNS permeability and downregulated the pituicyte-specific cyp26b gene, encoding a retinoic acid catabolic enzyme. Inhibition of Cyp26b activity led to upregulation of tight junction protein Claudin-5 and decreased permeability. We conclude that pituicyte-derived factors regulate the "decision" of endothelial cells to adopt a permeable endothelial fate instead of forming a BBB.

Zebrafish Reel in Phenotypic Suppressors of Autism.

Chemical genetics can help decipher novel pathways underlying neurodevelopmental psychiatric impairments. Hoffman et al. (2016) utilized behavioral profiling of psychoactive compounds in zebrafish and identified estrogens as suppressors of a phenotype resulting from loss of an autism risk gene.

Distribution of LPXRFa, a gonadotropin-inhibitory hormone ortholog peptide, and LPXRFa receptor in the brain and pituitary of the tilapia.

In vertebrates, gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH), respectively, regulate reproduction in positive and negative manners. GnIH belongs to the LPXRFa family of peptides previously identified in mammalian and nonmammalian vertebrates. Studying the detailed distribution of LPXRFa as well as its receptor (LPXRFa-R) in the brain and pituitary is important for understanding their multiple action sites and potential functions. However, the distribution of LPXRFa and LPXRFa-R has not been studied in teleost species, partially because of the lack of fish-specific antibodies. Therefore, in the present study, we generated specific antibodies against LPXRFa and its receptor from Nile tilapia (Oreochromis niloticus), and examined their distributions in the brain and pituitary by immunohistochemistry. Tilapia LPXRFa-immunoreactive neurons lie in the posterior ventricular nucleus of the caudal preoptic area, whereas LPXRFa-R-immunoreactive cells are distributed widely. Double immunofluorescence showed that neither LPXRFa-immunoreactive fibers nor LPXRFa-R is closely associated or coexpressed with GnRH1, GnRH3, or kisspeptin (Kiss2) neurons. In the pituitary, LPXRFa fibers are closely associated with gonadotropic endocrine cells [expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH)], with adrenocorticomelanotropic cells [corticotropin (ACTH) and α-melanotropin (α-MSH)], and with somatolactin endocrine cells. In contrast, LPXRFa-R are expressed only in LH, ACTH, and α-MSH cells. These results suggest that LPXRFa and LPXRFa-R signaling acts directly on the pituitary cells independent from GnRH or kisspeptin and could play multiple roles in reproductive and nonreproductive functions in teleosts. J. Comp. Neurol. 524:2753-2775, 2016. © 2016 Wiley Periodicals, Inc.

Role of developmental factors in hypothalamic function.

The hypothalamus is a brain region which regulates homeostasis by mediating endocrine, autonomic and behavioral functions. It is comprised of several nuclei containing distinct neuronal populations producing neuropeptides and neurotransmitters that regulate fundamental body functions including temperature and metabolic rate, thirst and hunger, sexual behavior and reproduction, circadian rhythm, and emotional responses. The identity, number and connectivity of these neuronal populations are established during the organism's development and are of crucial importance for normal hypothalamic function. Studies have suggested that developmental abnormalities in specific hypothalamic circuits can lead to obesity, sleep disorders, anxiety, depression and autism. At the molecular level, the development of the hypothalamus is regulated by transcription factors (TF), secreted growth factors, neuropeptides and their receptors. Recent studies in zebrafish and mouse have demonstrated that some of these molecules maintain their expression in the adult brain and subsequently play a role in the physiological functions that are regulated by hypothalamic neurons. Here, we summarize the involvement of some of the key developmental factors in hypothalamic development and function by focusing on the mouse and zebrafish genetic model organisms.

A novel model for development, organization, and function of gonadotropes in fish pituitary.

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of the reproductive axis in vertebrates. Despite the high popularity of zebrafish as a model organism for studying reproductive functions, to date no transgenic zebrafish with labeled gonadotropes have been introduced. Using gonadotropin regulatory elements from tilapia, we generated two transgenic zebrafish lines with labeled gonadotropes. The tilapia and zebrafish regulatory sequences were highly divergent but several conserved elements allowed the tilapia promoters to correctly drive the transgenes in zebrafish pituitaries. FSH cells reacted to stimulation with gonadotropin releasing hormone by proliferating and showing increased transgene fluorescence, whereas estrogen exposure caused a decrease in cell number and transgene fluorescence. Transgene fluorescence reflected the expression pattern of the endogenous fshb gene. Ontogenetic expression of the transgenes followed typical patterns, with FSH cells appearing early in development, and LH cells appearing later and increasing dramatically in number with the onset of puberty. Our transgenic lines provide a powerful tool for investigating the development, anatomy, and function of the reproductive axis in lower vertebrates.

LPXRFa, the piscine ortholog of GnIH, and LPXRF receptor positively regulate gonadotropin secretion in Tilapia (Oreochromis niloticus).

LPXRFamide (LPXRFa) peptides have been characterized for their ability to inhibit gonadotropin (GTH) release in birds and stimulate growth hormone (GH) release in frogs. However, their involvement in regulating the reproductive hypothalamo-pituitary-gonadal axis in mammals and fish is inconclusive. To study the role of LPXRFa peptides in the regulation of GTH secretion, we cloned tilapia LPXRFa and LPXRF receptor (LPXRF-R). Processing of the tilapia preproLPXRFa liberated three mature LPXRFa peptides that varied in size and post-translational modifications. Phylogenetic analysis of LPXRFa and the closely related RFamide peptide PQRFa showed clear clustering of each peptide sequence with its orthologs from various vertebrates. Signal-transduction analysis of the tilapia LPXRF-R in COS-7 cells showed clear stimulation of CRE-dependent luciferase activity, whereas the human NPFFR1 showed suppression of forskolin-induced CRE-dependent activity in this system. Administration of the tilapia pyroglutaminated LPXRFa-2 peptide to primary cell culture of tilapia pituitaries, or to reproductive female tilapia by ip injection, positively regulated both LH and FSH release in vivo and in vitro. Using double-labeled fluorescent in-situ hybridization and immunofluorescence, ßLH cells were found to co-express both tilapia lpxrf and tilapia lpxrf-r mRNA, whereas some of the ßFSH cells coexpressed only lpxrf-r mRNA. No coexpression of tilapia lpxrf-r was identified in GH-positive cells. These findings suggest that the LPXRFa system is a potent positive regulator of the reproductive neuroendocrine axis of tilapia.