Functions Of Zebrafish Kisspeptins In Reproduction And Their Regulation By Circadian Clocks And Estrogen

Kisspeptin is a series of small peptides obtained from hydrolysis of precursor protein encoded by Kiss1 gene,and the most potent activator for GnRH secretion.The timing of GnRH is determined by the timing of Kiss1 for the following reasons:Kiss1 neurons express estrogen receptors,mediating the estrogen positive/negative feedback;Kiss1 shows rhythmic expression in mammals under the presence of estrogen;pulsatile Kiss1 synchronize GnRH neuron.Thus GnRH surge,which is the foundation of pubety and female reproduction,shows a timed and estrogen-dependent pattern in the afternoon before ovulation in women.In contrast to mammals,zebrafish hast two kiss homologous genes.Very little is known about the functions and particularly their regulatory mechanisms of these two zebrafish kiss genes.The purpose of this study is to determine the following questions.1.Does the circadian clock regulates kisspeptin gene in zebrafish as well?And how?2.Does the circadian clock interact with the estrogen receptors in regulation of kisspeptin expression?3.Toexamine whether Kiss1 surge is due to the synergistic effects of clock proteins and estrogen receptors inzebrafish.4.How do kisspeptin genes play their roles in zebrafish reproduction?We constructed Tg(hsp:kiss1),Tg(hsp:kiss2)fish-which can overexpress kiss1 and kiss2 after heat shock.Egg counting after mating peptide injectied fish or heat shock treated Tg(hsp:kiss1)and Tg(hsp:kiss2)revealed the possible mechanism of zebrafish Kisspeptin on reproduction.Kiss1 is an activator,and Kiss2 is an inhibitor of gnrh3/lhb expression.By RT-qPCR,we found that the mRNA levels of two kisspeptin genes showed significant oscillations in zebrafish.The peak expression of kiss1 and kiss2 was ZT4 and ZT3,respectively.We compared the promoter regions of human,mouse and zebrafish,and found different regulatory elements.Dual-luciferase reporter assays and ChIP assays found that the circadian clock regulates the two kiss genes in different ways:kiss1 is regulated through E-box,while kiss2 through both D-box and RRE elements.Binding of clock proteins on the functional CCEs(clock control elements)in the promoter regions of the two kiss genes in vivo is rhythmic with different phases(Peak binding of Bmallb on kiss1 E-box at ZT20;Peak binding of Tef on kiss2 D-box and Ror?a on kiss2 RRE in ZT8 and ZT16 respectively.).We constructed Tg(kiss1:dsluc)and Tg(kiss1:dsluc)fish expressing destabilized luciferase driven by kiss1 and kiss2 promoter,respectively.We observed the same expression pattern as in RT-qPCR results by in vivo monitoring bioluminescence.In combination with results from bmal1b-/-mutant,we concluded that circadian clock disruption resulted in the expression changes of the two genes:the amplitude of kiss1 gene altered(Base±AMP from 1.11±0.06 to 1.07±0.08);both the phase and amplitude of kiss2 mRNA level altered(Phase from ZT3 to ZTO,Base±AMP from 0.99±0.08 to 0.99±0.05).Estrogen treatment suggested that estrogen signals effected kisspeptin through Esr2a.Dual luciferase reporter assays showed Esr2a activates kiss1 in the presence of estrogen and acts with the circadian clock by direct binding to Crylaa.Estrogen receptors activate kiss2 in the presence of estrogen,and have synergistic effect with the regulatory clock proteins,although not by direct binding.We also found that estrogen-related receptor a activate kiss2.In summary,our study illustrated that the circadian clock directly regulated the expression of zebrafish kiss1 and kiss2,and interacted with estrogen receptors to regulate kiss gene expression in the presence of estrogen.Our research provides the basis for the specific mechanism of Kiss1 surge timing induced by the synergistic effect of circadian clock proteins and estrogen receptors in zebrafish.At the same time,our findings expand the knowledge of comparative biology.The role of D-box in regulating Kiss1 is common in human and zebrafish,not in mice;while RRE-mediated regulation of kisspeptin gene is unique in zebrafish.In addition,we constructed the kiss1 and kiss2 promoter-driven luciferase transgenic fish,as well as heat shock-induced kiss1 and kiss2 overexpressing transgenic fish,which provides a powerful tool for further study on regulation mechanism of kisspeptin gene by the clock,and the impact of clock disruption on kisspeptin gene functions.