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Cross-talk between light and temperature in reproduction
thesisposted on 2017-02-20, 23:33 authored by Loganathan, Kavinash
Reproduction occurs only under specific, suitable environmental conditions. Recently, the mechanisms regulating reproduction via environmental cues have been clarified with the advent of new molecular techniques. Despite environmentally-sensitive molecules such as melatonin and TREK channels being identified, there are very few studies investigating the interaction between these cues in reproductive function. This study aims to elucidate the molecular-molecular interactions and understand how those mechanisms regulate reproduction. A search on GenBank revealed three Trek channel subtypes in the zebrafish brain, Trek1, Trek2a and Trek2b. However, the full length sequence of trek2a was not identified. In Chapter 2, we identified the full length cDNA sequence of trek2a and localization in the zebrafish brain. Deduced amino acid sequence of the identified cDNA consisted of two pore domains and four transmembrane regions and was 69.3% homologous with zebrafish Trek2b and 61.5% homologous with zebrafish Trek1. High expression of trek2a mRNA was observed in the preoptic area and midbrain, as well as the caudal and dorsal zone of the periventricular hypothalamus. trek2a gene expressions exhibited diurnal rhythms, but no diurnal rhythm was exhibited in trek1 and trek2b gene expression. All three Trek channels exhibited significantly high gene expression in mature females, compared to mature males, whilst trek1 and trek2a expressions were increased during puberty. This suggests that Trek channels play a role in reproduction. In Chapter 3, we first investigated the effects of different light and temperature conditions on brain melatonin concentration. We found that brain melatonin was significantly high under constant darkness, as well as both high and low temperature. From this experiment, we then analyzed the effects of high brain melatonin condition via constant darkness and high temperature on gene expressions of melatonin receptors, Trek channels, GnRH and kiss2 in the adult zebrafish brain. mt1, mel1c and trek2b genes were decreased, whilst trek2a, kiss2 and gnrh3 levels were increased under constant darkness. High temperature decreased gene expressions of mt1, mt2, mel1c and gnrh3 in the preoptic area, whereas other genes remained unchanged. This suggests that melatonin receptors and Trek channels are separately involved in kiss2 and gnrh3 gene expression in the brain. In Chapter 4, molecular interactions among melatonin receptors, Trek channels, α2-adrenoceptor, kisspeptin and GnRH were investigated. Melatonin administration significantly reduced trek2a and gnrh3 expression. Luzindole administration also suppressed trek2a and gnrh3 expression. trek2a-specific siRNA administration significantly reduced expression levels of trek2a, gnrh3 and mt1, whilst increasing kiss2 expression. Finally, prazosin injection lead to an overall reduction in trek2a expression, but gnrh3 expression, which was initially increased, was significantly reduced after 48 hours. In this study, melatonin receptors and Trek2a channel are suggested to affect kiss2 gene expressions separately, while the α2-adrenoceptor is suggested to regulate trek2a and gnrh3 gene expressions. In conclusion, from these findings, we proposed a molecular interaction via the melatonin system, Trek channels and α2-adrenoceptor by constant darkness and high temperature. This mechanism plays an important role in regulation of reproductive function in the zebrafish brain. Furthermore, we also propose that melatonin and α2-adrenoceptors are two cross-talk sites in the brain, integrating both ambient light and temperature cues to regulate reproductive function at the kisspeptin and GnRH levels.