Functional Analysis Of Pyroptosis In Zebrafish Ovulation

Ovulation,as the most basic reproductive behavior in female animals,is a dynamic process of releasing mature eggs from the follicles to the surface of the ovaries.During the process of ovulation,the follicle needs to undergo complex changes in physiological structure and gene expression regulation,which involve cell death,damage,repair,and tissue reconstruction.During ovulation,the follicles will be highly vascularized,and under the stimulation of LH,the follicular cell layer will produce steroids,prostaglandins,chemokines and cytokines.These mediators are very similar to the substances synthesized in tissue inflammation and wound repair.The view that ovulation is an inflammatory response has gradually been recognized,but the mechanism is still unclear.Pyroptosis is a kind of regulable cell death that depends on the formation of plasma membrane pores by Gasdermin family proteins,and belongs to Inflammatory cell death pathway.The ovulation process is accompanied by cell death.Meanwhile,NLRP3,Caspase-1 and IL-1?have all been shown to be expressed in the mammalian ovaries.However,it is not clear whether the pyroptosis is involved in the ovulation.In this study,zebrafish was used as the research model to systematically study 1)Does LH activate the pyroptosis-related signaling pathway?2)Is pyroptosis important in ovulation?3)How do signal pathways related to pyroptosis regulate ovulation?The main results are as follows:1.The number of cell deaths in the follicular cell layer during zebrafish ovulation increased significantly before ovulation.In this study,a zebrafish h CG artificially induced ovulation model and a natural ovulation model were established.The TUNEL experiment and the SYTOX^TM green nucleic acid dye experiment were used to analyze cell death during the ovulation process of the artificially induced model and the natural ovulation model.It was found that cell death occurred in the follicular layer and the number of cell deaths increased significantly before ovulation.2.Pyroptosis occurred during zebrafish ovulation.In this study,quantitative real-time PCR and western blotting were used to analyze the expression of key factors related to pyroptosis during ovulation.It was found that the inflammasome ASC,Nlrp1 and Nlrp3 are activated during ovulation,which in turn activates Caspase A to cut Gasdermin Eb and release IL-1?.The classic Caspase 1-dependent pyroptosis pathway occurred during ovulation.3.Inhibition of pyroptosis led to the attenuation of the zebrafish ovulation process.In this study,Ac-FEID-CMK,a specific peptide inhibitor of Gasdermin Eb,was prepared and injected with h CG by intraperitoneal injection.It was found that the zebrafish ovulation process was inhibited.Meanwhile,MCC950,an inhibitor of inflammasome Nlrp3,was co-injected with h CG by intraperitoneal injection,and it was found that the ovulation process was inhibited.4.IL-1?could inhibit the maturation and ovulation of zebrafish oocytes.In this study,a recombinant IL-1?mature peptide recombinant protein was prepared using a bacterial system.Using IL-1?and low-concentration h CG(4 IU/fish)co-injection in vivo method,it was found that IL-1?could inhibit zebrafish oocyte maturation and ovulation.Similarly,using IL-1?for incubation in vitro,it was found that IL-1?could inhibit the maturation of zebrafish oocytes.5.In this study,CRISPR/Cas9 system key factors in pyroptosis,including gsdmea and gsdmeb,and the inflammatory factor il1b.This study established a knockout fish line related to pyroptosis.In summary,this study uses zebrafish as a model to reveal that pyroptosis is involved in ovulation of zebrafish and has an important role and function in ovulation of zebrafish.These findings supply an new information on the view that for ovulation is an inflammatory response,as well as a new perspective for understanding the mechanism of ovulation in animals.