Molecular Mechanism Of Melatonin Synthesis And Secretion In Rat Pineal Gland Regulated By Norepinephrine Through CircR-WNK2/AANAT Pathway

Melatonin,secreted by the mammalian pineal gland,plays an important regulatory role in animal life activities.Its secretion shows an obvious trend of low day and high night,which can directly act on the reproductive endocrine system composed of hypothalamic-pituitary-gonadal axis and accurately regulate the reproductive cycle of animals.Melatonin and its preparations have been widely used in livestock production.Studies have shown that melatonin administration can reduce oxidative stress of bovine ovaries,improve embryonic development environment and ability,and improve pregnancy rate and reproductive efficiency.During oocyte maturation,fertilization and early embryo culture in vitro,melatonin can significantly improve the embryonic development rate and blastocyst quality of pig,cow,sheep and other species.Therefore,it is of great theoretical and practical value to explore the molecular mechanism of melatonin synthesis and regulation in vivo and to analyze its regulatory effect on animal reproduction.The synthesis and secretion of melatonin are regulated by light and hypothalamic neural pathways.At night,norepinephrine released by the hypothalamus through the nerve endings of the superior cervical ganglion promotes the expression of melatonin synthesis-rate-limiting enzyme Aanat gene in pineal cells,thus increasing the synthesis and secretion of melatonin.As a member of epigenetic regulation,it has been shown that the expression of non-coding RNA in pineal gland is also regulated by norepinephrine signaling.However,it is not clear whether non-coding RNA is involved in the regulation of melatonin synthesis and secretion.Therefore,clarifying the physiological function of non-coding RNA in pineal gland and systematically analyzing the regulatory mechanism of norepinephrine and non-coding RNA on melatonin synthesis and secretion are of great significance for enriching the basic theory of animal reproductive endocrine and improving the application effect of melatonin in animal husbandry production.In this study,daytime and nighttime pineal bodies were collected for transcriptome analysis.circRNA,miRNA and m RNA were differentially expressed day and night based on the constructed whole-transcriptome differential expression profile.Based on the ce RNA mechanism,the interaction between circRNA,miRNA and m RNA was predicted.Based on the bioinformatics analysis,circRNA which may regulate the synthesis of melatonin was selected.The regulatory function of circRNA was verified by dual luciferase reporting system,RNA pull down and FISH experiments.A rat model of superior cervical ganglion resection was established to explore the regulatory effect of norepinephrine on melatonin secretion through circRNA/miRNA/m RNA interaction network in the pineal gland in vivo.The results show that:1.The RNA expression profile of rat pineal gland at diurnal time points was constructed successfully,and 20 circRNAs,37 miRNAs and 259 m RNAs were identified to be highly expressed during the day,while 20 circRNAs,56 miRNAs and 450 m RNAs were highly expressed at night.By constructing the ce RNA regulatory network of circRNA-miRNA-m RNA,enrichment analysis showed that the regulatory role of circRNA was involved in light signal transduction,circadian rhythm formation and the processing and synthesis of melatonin.2.The dual luciferase reporting system,RNA pull down and FISH experiments were used to prove the targeted binding effect between miR-328a-3p and circ R-WNK2 and Aanat m RNA.Through cell transfection,ELSA,WB and other experiments,it was proved that circ R-WNK2 could release transcription inhibition of Aanat by competitive binding to miR-328a-3p in pineal cells,and further promote the expression of melatonin synthetase Aanat,thus promoting the synthesis of melatonin.3.The superior cervical ganglion resection model was established,and the norepinephrine injection recovery experiment was conducted to verify in vivo that the superior cervical ganglion regulates the diurnal differential expression of non-coding RNA and melatonin synthetase in the pineal gland through the secretion of norepinephrine.It was found that norepinephrine can induce the expression of circ R-WNK2 in pineal cells,increase the transcription of Aanat,and promote the synthesis and secretion of melatonin.This study clarified the regulatory role of non-coding RNA in melatonin synthesis in pineal cells,which provided a data basis for the development of molecular targets for hormone regulation and the verification of the regulatory functions of non-coding RNA.The neuroendocrine model of norepinephrine on the regulation of non-coding RNA and melatonin in pinecone was analyzed,which improved the neuroendocrine regulation model of melatonin,provided a new research idea for the neural regulation of melatonin synthesis,and provided a new theoretical basis for the development of a novel and efficient animal reproductive regulation hormone and the improvement of animal reproductive regulation technology.