Ovulation Is Associated With The LH-dependent Induction Of Pla2g4aa In Zebrafish

The effects of the preovulatory luteinizing hormone?LH?surge on the ovulatory process are mediated by prostaglandins?PGs?,the synthesis of which involves prostaglandin synthetase and cytosolic phospholipase A2?cPLA2?.In our previous study,we systematically investigated the function of prostaglandin endoperoxide synthase?ptgs?genes on ovulation in zebrafish.However,the role of cPLA2 in ovulation regulation in zebrafish has not been determined.In this study,we investigated the function of cpla2?in PGs production and ovulation in periovulatory follicles.The main results are as follows:1.Real-time PCR was used to detect the expression and distribution of two genes?pla2g4aa and pla2g4ab?of cpla2?in zebrafish follicular cells and the expression patterns at different developmental stages.Pla2g4aa was highly expressed in follicular layer cells,while pla2g4ab was not significantly expressed in follicular layer cells and oocytes.The expression of pla2g4aa increased from PV stage to FG stage and reached the highest level.Compared with FG stage,the expression of pla2g4aa increased significantly in M stage,while the expression of pla2g4ab decreased significantly in M stage.These results suggest that pla2g4aa may play a more important role in the regulation of oocyte maturation and ovulation in zebrafish,.2.An ideal model of ovulation in zebrafish?npr?was constructed in the early stage of the laboratory,and direct evidence was obtained that npr is a key factor mediating LH regulation of ovulation.We used this model to further verify whether the cpla2?gene played a important role in ovulation regulation.The results showed that pla2g4aa was significantly expressed in wild zebrafish,while the expression level was significantly reduced in npr knockout zebrafish.It is suggested that pla2g4aa may play an important role in the regulation of ovulation in zebrafish.3.In previous studies,the laboratory has established a mature model of zebrafish ovulation induced by hCG in vitro.In order to know whether LH surge will regulate their expression changes.We simulated LH surge with hCG stimulation in vitro,and selected follicular cells of FG phase to set up two groups of experiments:concentration gradient and time gradient.The expression of pla2g4aa and pla2g4ab was detected by Real-Time PCR.Pla2g4aa was significantly up-regulated by hCG stimulation,reaching its peak at the concentration of 50 IU/mL;in the time gradient group,the expression of pla2g4aa was up-regulated from 1 hour to 3 hours.However,there was no significant change in the amount of pla2g4ab in the two groups.It is suggested that pla2g4aa is regulated by LH surge in two genes of cpla2?.4.To further understand how LH surge regulates pla2g4aa,we set up a primary cell stimulation experiment.The results showed that the expression of pla2g4aa was significantly up-regulated by the analogs or activators of cAMP in the PKA signaling pathway,while the selective inhibitors of PKA significantly terminated the up-regulation of pla2g4aa expression stimulated by hCG and began to down-regulate.In PKC signaling pathway,PKC activators also up-regulate the expression of pla2g4aa,while MEK inhibitors also down-regulate the expression of pla2g4aa.It is demonstrated that both the cAMP-PKA signaling pathway and PKC signaling pathway are involved in the regulation of pla2g4aa expression and respond to hCG-induced LH surge.5.In order to know whether the expression of cPLA2?is necessary for the production of PGs?prostaglandins?and has an effect on ovulation efficiency,we injected cPLA2 inhibitor in vivo.The results showed that the concentrations of PGE₂and PGF_2aa were significantly decreased two hours after the injection of the inhibitor.Female zebrafish injected with the inhibitor did not ovulate,and the co-injection of hCG and the inhibitor group showed that the inhibitor could effectively limit the ovulation process induced by hCG.It is suggested that the expression of cPLA2?regulates the concentration of prostaglandins and controls the mechanism of ovulation.Collectively,these findings suggest that pla2g4aa is related to the ovulation process in zebrafish.