Mechanism Of Ferroptosis Regulated By CISD1 On Mouse Oocyte Maturation

The quality of oocytes is the key to affecting the fertility of female animals,and its influencing factors and mechanisms have always been a hot spot in bioscience,especially in animal husbandry research and livestock production in practice.Recent studies have shown that iron overload and its induced ferroptosis are the key factors affecting oocyte maturation and quality,which restricting the effective utilization of female animal germplasm resources.CDGSH iron sulfur domain 1（CISD1）,as a Fe-S protein localized on mitochondria,has a variety of biological functions,such as regulating iron metabolism,energy production and mitochondrial dynamics,and its gene mutation will lead to a decrease in the number of oocytes in ovary,but the specific mechanism is not clear.Therefore,this study took mouse oocytes as the research object to explore the role and mechanism of CIDS1 in affecting oocyte maturation and quality by regulating ferroptosis.This research is mainly divided into three parts,the research content and results are as follows:1.Effects of iron overload and induced ferroptosis on oocyte maturation in mouse（1）Fe²⁺changes dynamically during oocyte maturation in mouse.By means of fluorescence probe and Western blot,it was found that Fe²⁺level in oocytes showed no significant difference between GV stage and GVBD stage,the highest level was in MI stage and the lowest level was in MII stage,and it was related to the dynamic expression of TFRC,FPN,NCOA4 and FTL,which are related to iron metabolism regulation（2）Iron overload inhibited mouse oocyte maturation,fertilization and subsequent embryo development.We set NC group and iron overload treatment group（add Ferric ammonium citrate;FAC group）and ferroptosis inhibition group（add Ferric ammonium citrate and ferroptosis inhibitor Ferrostain-1;FAC+Fer-1 group）,after in vitro maturation culture,the relevant indexes of oocyte maturation were detected.The results showed,compared with NC group,the maturation rate,fertilization rate and blastocyst rate of FAC group were significantly decreased（P<0.01）.Compared with FAC group,the maturation rate and fertilization rate of FAC+Fer-1 group were significantly increased（P<0.05）,but the zygotes could not develop into blastocysts.（3）Iron overload induced ferroptosis and mitochondrial dysfunction of mouse oocytes.The relevant indexes of ferroptosis,apoptosis and mitochondrial function of oocytes were detected after in vitro maturation culture.The results showed that the level of Fe²⁺in FAC group and FAC+Fer-1 group was increased to 1.53±0.01 folds and 1.24±0.01 folds compared with NC group,respectively（P<0.01）.The Lipid peroxide（LPO）level was increased to 2.38±0.05 folds and 1.55±0.04 folds compared with NC group,respectively（P<0.01）.The level of Malondialdehyde（MDA）increased to 3.63±0.35folds（P<0.01）and 1.65±0.12 folds（P<0.05）of compared with NC group,respectively.The level of glutathione peroxidase GPX4 protein was down-regulated to 0.68±0.04folds（P<0.01）and 0.85±0.05 folds（P<0.05）compared with NC group,respectively.The mitochondrial membrane potential of FAC group and FAC+Fer-1 group was reduced to 0.68±0.04 folds and 0.82±0.03 folds of the NC group（P<0.01）,and the ATP level was reduced to 0.73±0.02 folds and 0.86±0.01 folds compared with NC group（P<0.01）.However,there were no significant changes in apoptosis rate and apoptosis related protein Caspase3（P>0.05）.2.Construction and analysis of differential gene transcription profiles induced by CISD1 knockdown in mouse oocytes（1）CISD1 plays a key role in the maturation of mouse oocytes.The expression of CISD1 protein in oocytes was detected by immunofluorescence and Western blot.CISD1 levels were knocked down by microinjection of si RNA at GV stage(NC group was injected with meaningless si RNA;CISD1_KDgroup was injected with CISD1-specific si RNA),and after in vitro maturation culture,the relevant indexes of oocyte maturation were detected.The results showed that CISD1 was expressed at all stages of mouse oocyte maturation and colocalized with mitochondria.The oocyte maturation rates in NC group and CISD1_KD group were 86.00±2.02%and 68.50±2.84%,and fertilization rates were 80.72±2.24%and 62.03±2.57%,and blastocyst rates were66.71±2.07%and 39.01±2.22%（P<0.01）,respectively.CISD1 Knockdown inhibits oocyte maturation,fertilization,and subsequent embryonic development.（2）CISD1 knockdown induced ferroptosis of mouse oocytes.CISD1 knocked down and in vitro maturation culture was performed,and ferroptosis related indexes of oocytes were detected by chemiluminescence and Western blot.The results showed that the levels of Fe²⁺,LPO and MDA in CISD1_KD group were significantly up-regulated compared with the NC group（P<0.01）,and the levels of GPX4 protein were significantly down-regulated（P<0.01）.（3）The differential gene transcription profiles induced by CISD1 knockdown in mouse oocytes were constructed.GV stage oocytes were collected,CISD1 knocked down or ferroptosis inducers（Erastin or RSL3）with different activate mechanisms were added to the culture medium,and in vitro maturation culture was performed.The differential gene transcription profiles of Normal oocytes（NC group）,CISD1knockdown oocytes(CISD1_KD group),Erastin treated oocyte（Erastin group,40μM;Specific action on mitochondrial voltage-dependent anion channels）and RSL3 treated oocyte（RSL3 group,5μM;Broad-spectrum inhibition of GPX4 activity）were constructed using a small amount of RNA sequencing.The results showed that the oocytes gene expression pattern of the CISD1_KD group was closer to that of the Erastin-treated group induced ferroptosis by mitochondria compared with RSL3 treatment.Differentially expressed genes are mainly enriched in the pathways of ferroptosis,oxidative stress,oxidative phosphorylation and mitophagy.3.Mechanism of CISD1 regulating mitochondrial damage and ferritinophagy in mouse oocytes（1）CISD1 knockdown induced mitochondrial iron overload in mouse oocytes.The level of CISD1 was inhibited by microinjection of si RNA.After in vitro maturation culture,mitochondrial iron metabolism and mitochondrial lipid peroxidation related indexes in mouse oocytes were detected by fluorescent probes and Western blot methods.The results showed that the levels of mitochondrial Fe²⁺,mitochondrial reactive oxygen species（ROS）and mitochondrial LPO in CISD1_KD group were significantly increased compared with the NC group（P<0.01）,and the mitochondrial anti-ferroptosis factor FSP1 protein was significantly down-regulated（P<0.01）.（2）CISD1 knockdown resulted in abnormal mitochondrial function and distribution of mouse oocytes.CISD1 protein levels were knocked down and in vitro maturation culture was performed.The mitochondrial function,distribution and dynamics related indexes in mouse oocytes were detected by chemiluminescence and Western blot.The results showed that compared with NC group,mitochondrial membrane potential,ATP level,mitochondrial complex II activity and number of mitochondria associated with spindle migration were significantly down-regulated in CISD1_KD group（P<0.05 or P<0.01）,and mitochondrial fusion-related proteins OPA1,MFN1 and MFN2 were significantly down-regulated（P<0.01）,and mitochondrial fission-related protein DRP1 and mitophagy related protein Parkin were significantly up-regulated（P<0.01）.（3）CISD1 knockdown induced up-regulation of ferritinophagy in mouse oocytes.CISD1 protein levels were knocked down and in vitro maturation culture was performed.Ferritinophagy related indexes in mouse oocytes were detected by Western blot and immunofluorescence.The results showed that the levels of autophagy related protein LC3 and ferritinophagy related protein NCOA4 in CISD1_KD group were significantly up-regulated compared with the NC group（P<0.01）,and the immunofluorescence results showed that the two were significantly co-localized.In summary,this study clarified the effects of iron overload and induced ferroptosis on oocyte maturation and quality by inducing mitochondrial damage,and clarified that inhibition of CISD1 can induce mitochondrial lipid peroxidation and ferroptosis in oocytes by inducing mitochondrial iron ion overload and mitochondrial ROS accumulation,thus driving mitophagy and ferritinophagy,and ultimately inhibit oocyte maturation.The results are helpful to further analyze the occurrence and development of ferroptosis and its influence on oocyte maturation and quality,and provide theoretical basis for improving oocyte in vitro maturation culture system and developing new methods to promote oocyte maturation and improve oocyte quality.