A Study On The Characterization Of Pyroptosis During Ovulation Of Zebrafish(danio Rerio)

Ovulation of a fertilizable oocyte is one of the basic functions of the ovaries.Ovulation is an unique biological phenomenon that is the dynamic process of release of mature oocyte from surrounding follicles.This process involves protein degradation and follicle contraction at specific site of the follicular wall,thereby releasing the oocyte through the follicular rupture site.It is well established that the oocyte maturation and ovulation are mainly induced by Luteinizing hormone(LH)secreted by the pituitary gland,and both processes occur almost simultaneously.Ovulation was also regarded as an inflammatory response due to the involvement of prostaglandins(PGs)and many other immune mediators,such as cytokines and chemokines.During ovulation,follicles undergo complex changes in physiological structure and gene expression regulation,involving cell injury and repair,and tissue remodeling.Studies have shown that there are follicular cell death during ovulation,but it is still unclear which cell death is involved.Therefore,in this study,we systematically analyzed and discussed this question.The main types of programmed cell death includes: apoptosis,necroptosis,autophagy,and pyroptosis.Caspase-1 is an important factor in the organism's natural immunity,can cleave Il1 b.In recent years,Caspase-1 has been shown to regulate pyroptosis,a new mode of programmed cell death.The main specific results are as follows:1.During the ovulation of zebrafish induced by hCG,the number of death of follicular cells gradually increased.PI staining and SYTOX green nucleic acid staining was performed on follicles at different stages of ovulation,It was found that the number of dead follicular cells was increased during ovulation,In situ staining of the dead cells by TUNEL,further revealed that the degraded genomic DNA of the dead cells.2.Apoptosis and autophagy might not be involved in the zebrafish ovulation process.The mRNA and protein expressions of the two death types-related genes were detected by real-time PCR and Western blotting.It was found that there was no significant change in the expression of apoptosis-related genes and protein expressionduring ovulation,we also found that autophagy-related proteins LC3 A and Beclin1 could be activated after ovulation,suggesting that autophagy may be involved in the degradation of follicular cells after ovulation.All these date indicate that apoptosis and autophagy might not be involved in the ovulation process.3.Pyroptosis is involved in the zebrafish ovulation process.In hCG-induced ovulation,Igf3-induced ovulation,and primary cultured follicular cells,gene and protein expression analysis at both tissue and cell levels showed that caspase-1 and Il1 b were significantly activated,and LH/hCG could regulate the expression of il1 b through the cAMP/PKA signaling pathway.4.The pyroptosis occurs in the endothelial cell of the capillaries during the ovulation of zebrafish.The morphological structure of follicular cells during ovulation was analyzed by transmission electron microscopy(TEM).The results showed that in the process of zebrafish ovulation,the endothelial cell of the capillaries became swelling at 1.5 hours of hCG injection.At 2 hours of hCG injection,the nucleus condensed and the cell membrane began to rupture.At 2.5 hours of hCG injection,the cell membrane completely ruptured and the contents leakaged,in accordance with the characteristics of pyroptosis.In summary,this study reveals that Caspase-1-mediated pyroptosis participates in the process of zebrafish ovulation,and this is the first report on the role of pyroptosis in normal physiological processes,These information provide a new perspective on understanding of ovulation mechanism.