Kisspeptin, encoded by the KISS1 gene, was first discovered as a potential metastasis suppressor gene. The prepro-kisspeptin precursor is cleaved into shorter mature bioactive peptides of varying sizes that bind to the G protein-coupled receptor GPR54 (=KISS1R). Over the last two decades, multiple types of Kiss and KissR genes have been discovered in mammalian and non-mammalian vertebrate species, but they are remarkably absent in birds. Kiss neuronal populations are distributed mainly in the hypothalamus. The KissRs are widely distributed in the brain, including the hypothalamic and non-hypothalamic regions, such as the hippocampus, amygdala, and habenula. The role of KISS1-KISS1R in humans and Kiss1-Kiss1R in rodents is associated with puberty, gonadal maturation, and the reproductive axis. However, recent gene deletion studies in zebrafish and medaka have provided controversial results, suggesting that the reproductive role of kiss is dispensable. This review highlights the evolutionary history, localisation, and significance of Kiss-KissR in reproduction and reproductive behaviours in mammalian and non-mammalian vertebrates.
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.
Morphine is a potent analgesic opiate commonly used in treating pain, and it is also a substance of abuse and highly addictive. Hence, it is vital to discover the action sites of morphine in the brain to increase its efficacy of treatment. In the present study, we aimed at identifying comprehensive neuroanatomical locations that are sensitive to morphine in the adult zebrafish (Danio rerio). We performed in situ hybridization to localize the mu opioid receptor (oprm1) gene and to map the morphine sensitive brain areas using neuronal PAS domain-containing protein 4a (npas4a), an early gene marker. Real-time PCR was used to detect changes in mRNA levels of oprm1 and npas4a in control and acute morphine treated fish (2 mg/L; 20 min). Intense positive oprm1 signals were seen in the telencephalon, preoptic area, habenula, hypothalamic area and periventricular gray zone of the optic tectum. Acute morphine exposure significantly increased oprm1 and npas4a mRNA levels in the medial zone of dorsal telencephalon (Dm), ventral region of the ventral telencephalon (Vv), preoptic area, and in the hypothalamus but a decrease in oprm1 and npas4a signals in the dorsal habenula. This study provides a detailed map of oprm1 localization in the brain, which includes previously unreported oprm1 in the habenula of teleost. Presence of oprm1 in multiple brain sites implies multiple action targets of morphine and potential brain functions which could include reward, cognitive and negative emotions.
Kisspeptin is a hypothalamic neuropeptide, which acts directly on gonadotropin-releasing hormone (GnRH)-secreting neurons via its cognate receptor (GPR54 or Kiss-R) to stimulate GnRH secretion in mammals. In non-mammalian vertebrates, there are multiple kisspeptins (Kiss1 and Kiss2) and Kiss-R types. Recent gene knockout studies have demonstrated that fish kisspeptin systems are not essential in the regulation of reproduction. Studying the detailed distribution of kisspeptin receptor in the brain and pituitary is important for understanding the multiple action sites and potential functions of the kisspeptin system. In the present study, we generated a specific antibody against zebrafish Kiss2-R (=Kiss1Ra/GPR54-1/Kiss-R2/KissR3) and examined its distribution in the brain and pituitary. Kiss2-R-immunoreactive cell bodies are widely distributed in the brain including in the dorsal telencephalon, preoptic area, hypothalamus, optic tectum, and in the hindbrain regions. Double-labeling showed that not all but a subset of preoptic GnRH3 neurons expresses Kiss2-R, while Kiss2-R is expressed in most of the olfactory GnRH3 neurons. In the posterior preoptic region, Kiss2-R immunoreactivity was seen in vasotocin cells. In the pituitary, Kiss2-R immunoreactivity was seen in corticotropes, but not in gonadotropes. The results in this study suggest that Kiss2 and Kiss2-R signaling directly serve non-reproductive functions and indirectly subserve reproductive functions in teleosts.
Hypothalamic kisspeptin encoded by KISS1/Kiss1 gene emerged as a regulator of the reproductive axis in mammals following the discovery of the kisspeptin receptor (Kissr) and its role in reproduction. Kisspeptin-Kissr systems have been investigated in various vertebrates, and a conserved sequence of kisspeptin-Kissr has been identified in most vertebrate species except in the avian linage. In addition, multiple paralogs of kisspeptin sequences have been identified in the non-mammalian vertebrates. The allegedly conserved role of kisspeptin-Kissr in reproduction became debatable when kiss/kissr genes-deficient zebrafish and medaka showed no apparent effect on the onset of puberty, sexual development, maturation and reproductive capacity. Therefore, it is questionable whether the role of kisspeptin in reproduction is conserved among vertebrate species. Here we discuss from a comparative and evolutional aspect the diverse functions of kisspeptin and its receptor in vertebrates. Primarily this review focuses on the role of hypothalamic kisspeptin in reproductive and non-reproductive functions that are conserved in vertebrate species.
kcnk10a has been predicted in zebrafish to be a member of the two-pore domain potassium ion (K+) channel-related K+ (TREK) channel family known as a thermoreceptor. Since reproduction is affected by temperature, Kcnk10a could be involved in the regulation of reproduction. However, expression of kcnk10a in the zebrafish brain and association with reproduction has not been identified. In this study, the full length sequence and localization of kcnk10a in the brain was investigated and gene expressions of the TREK channel family were examined to investigate association with reproduction. We initially identified the full length cDNA sequence of kcnk10a using Rapid Amplification of cDNA Ends and localization in the zebrafish brain using in situ hybridization. Furthermore, we examined the gene expression differences of kcnk2b, kcnk10a and kcnk10b mRNA between genders as well as developmental stages by real-time PCR. The deduced amino acid sequence of the identified kcnk10a mRNA contains highly conserved two pore domains and four transmembrane regions and was higher similarity to zebrafish Kcnk10b than zebrafish Kcnk2a and 2b. kcnk10a mRNA was widely distributed in the brain such as the preoptic area, hypothalamus and the midbrain. kcnk10a mRNA expression exhibited significant difference between mature male and female, and increase during puberty. Kcnk10a could be involved in the regulation of reproductive function.
INTRODUCTION: To study the ß-gene mutations spectrum, the genotype/phenotype correlation, the modulatory effect of co-inherited factors such as α-gene mutations and of Xmn1 polymorphism in a large cohort of Malaysian patients.
METHODS: A total of 264 cases clinically diagnosed as Thalassemia major (TM) (111), Thalassemia intermedia (21), HbE-β Thalassemia (131), and 1 HbE homozygous were studied. The detection of α and ß gene mutations and characterization of Xmn1 polymorphism were performed by multiplex PCR, amplification refractory mutation system (ARMS), DNA sequencing, and restriction fragment length polymorphism (RFLP)-PCR.
RESULTS: A total of 19 ß Thalassemia mutations were characterized. CD26 and CD41/42 were the most common found in the Malay and Chinese population, respectively. The sensitivity of the clinical diagnosis for β TM, thalassemia intermedia, and HbE/β thalassemia was 94.0%, 15.2%, and 89.2%, respectively. Patients with Xmn1 heterozygosity [+/-] required less frequent transfusion compared with those without the polymorphism. Co-inheritance of α-thalassemia alleviates the severity of HbE-β thalassemia in our cohort.
CONCLUSION: Molecular analysis should be used for a better diagnosis and management of β thalassemia.
Glioblastoma (GBM) mesenchymal (MES) transition can be regulated by long non-coding RNAs (lncRNAs) via modulation of various factors (Epithelial-to-Mesenchymal (EMT) markers, biological signalling, and the extracellular matrix (ECM)). However, understanding of these mechanisms in terms of lncRNAs is largely sparse. This review systematically analysed the mechanisms by which lncRNAs influence MES transition in GBM from a systematic search of the literature (using PRISMA) performed in five databases (PubMed, MEDLINE, EMBASE, Scopus, and Web of Science). We identified a total of 62 lncRNAs affiliated with GBM MES transition, of which 52 were upregulated and 10 were downregulated in GBM cells, where 55 lncRNAs were identified to regulate classical EMT markers in GBM (E-cadherin, N-cadherin, and vimentin) and 25 lncRNAs were reported to regulate EMT transcription factors (ZEB1, Snai1, Slug, Twist, and Notch); a total of 16 lncRNAs were found to regulate the associated signalling pathways (Wnt/β-catenin, PI3k/Akt/mTOR, TGFβ, and NF-κB) and 14 lncRNAs were reported to regulate ECM components (MMP2/9, fibronectin, CD44, and integrin-β1). A total of 25 lncRNAs were found dysregulated in clinical samples (TCGA vs. GTEx), of which 17 were upregulated and 8 were downregulated. Gene set enrichment analysis predicted the functions of HOXAS3, H19, HOTTIP, MEG3, DGCR5, and XIST at the transcriptional and translational levels based on their interacting target proteins. Our analysis observed that the MES transition is regulated by complex interplays between the signalling pathways and EMT factors. Nevertheless, further empirical studies are required to elucidate the complexity in this process between these EMT factors and the signalling involved in the GBM MES transition.
Kisspeptin has recently been recognized as a critical regulator of reproductive function in vertebrates. During the sexual development, kisspeptin neurons receive sex steroids feedback to trigger gonadotropin-releasing hormone (GnRH) neurons. In teleosts, a positive correlation has been found between the thyroid status and the reproductive status. However, the role of thyroid hormone in the regulation of kisspeptin system remains unknown. We cloned and characterized a gene encoding kisspeptin (kiss2) in a cichlid fish, the Nile tilapia (Oreochromis niloticus). Expression of kiss2 mRNA in the brain was analyzed by in situ hybridization. The effect of thyroid hormone (triiodothyronine, T3) and hypothyroidism with methimazole (MMI) on kiss2 and the three GnRH types (gnrh1, gnrh2, and gnrh3) mRNA expression was analyzed by real-time PCR. Expression of thyroid hormone receptor mRNAs were analyzed in laser-captured kisspeptin and GnRH neurons by RT-PCR. The kiss2 mRNA expressing cells were seen in the nucleus of the lateral recess in the hypothalamus. Intraperitoneal administration of T3 (5 μg/g body weight) to sexually mature male tilapia significantly increased kiss2 and gnrh1 mRNA levels at 24 h post injection (P
BACKGROUND: Beta thalassemia represents a great heterogeneity as over 300 mutations have been identified and each population at-risk has its own spectrum of mutations. Molecular characterization with high accuracy, sensitivity and economics is required for population screening and genetic counseling.
METHODS: We used the MALDI-TOF mass spectrometry (MS) platform to develop novel multiplex assays for comprehensive detection of 27 mutations in beta-thalassemia patients. Six multiplex assays were designed to detect 13 common known ß-mutations, namely CD41/42, CD71/72, IVS1-5, IVS1-1, CD26, IVS2-654, CAP+1, CD19, -28, -29, IVS1-2, InCD (T-G) and CD17; and 14 rare ß-mutations, i.e. InCD (A-C), CD8/9, CD43, -86, CD15, Poly A, Poly T/C, IVS2-1, CD1, CD35/36, CD27/28, CD16, CD37, and 619bpDEL in 165 samples. We compared the efficiencies of genotyping by MS and Amplification Refractory Mutation System (ARMS). Discrepant results were confirmed by sequencing analysis.
RESULTS: A total of 88.7% (260/293 allele) of MS and ARMS results was in agreement. More than fifty percent of the discrepant result was due to the false interpretation of ARMS results. Failed CD19 assay by MS method might be due to the assay design. The MS method detected 5 rare ß-mutations (CD15, CD35/36, CD8/9, Poly A and Poly T/C) presented in 13 alleles, which were not included in the ARMS screening panel.
CONCLUSION: We revealed that the MS method is a sensitive, high-throughput, highly automated, flexible, and cost-effective alternative to conventional ß-thalassemia genotyping methods.