| Goose has the characteristics of resistance to rough feeding,strong adaptability,tender meat and rich nutrition,which is widely welcomed by farmers and consumers.However,due to the low egg production of goose,it has seriously hindered the development and growth of China’s goose industry.Therefore,increasing the egg production of goose has become one of the urgent problems in the goose industry.Studies have shown that the performance of poultry egg production is mainly determined by follicle development and selection results,and the programmed death of a large number of Granulosa cells(GCs)is a key factor in the failure of follicle development to enter the hierarchical stage.Therefore,it is important to explore the regulatory mechanism of programmed death of GCs in follicle development and selection to improve goose egg production.Rich in biologically active substances(lipids,miRNAs,mRNA,and proteins)in follicular fluid,whether these bioactives affect the proliferation or programmed death of GCs.Studies have shown that the active substances contained in follicular fluid can be carried into GCs by exosomes,thereby affecting follicle development and the process of follicular atresia.It is unclear whether the follicle development of geese,as poultry with low egg production,is also affected by exosomes of follicular fluid mediating the programmed death of GCs by certain active substances,resulting in massive follicle atresia.In view of this,Yangzhou goose at peak egg production was used as a research object to explore the differences in follicular fluid exosome components between atretic small yellow follicles(ASYF)and normal small yellow follicles(SYF)and their regulatory mechanism on the programmed death of GCs.This study aims to reveal the regulatory effect of follicular fluid exosomes on the programmed death of GCs in follicular atresia,which not only helps to reveal the physiological mechanism of follicular atresia,but also provides a theoretical reference for improving the egg-laying performance of geese.1.Tissue morphology,granular layer cell programmed death markers,sex steroids and antioxidant levels of goose SYF and ASYFIn order to understand the morphological and physiological differences between SYF and ASYF in the goose,the tissue morphology,programmed death markers of granular layer cells,sex steroids and antioxidant levels of SYF and ASYF at peak egg production were detected by HE staining,transmission electron microscopy,immunohistochemistry and RT-qPCR.The results showed that during the hierarchical development of goose follicles during the peak egg production period,a large number of small follicles such as SYFs were blocked,which greatly reduced the number of SYFs entering the hierarchical follicle recruitment pool.Compared with SYF,the granule cell layer of ASYF has loose intercellular connections and falls inward;The number of ASYF apoptotic bodies and autophagic bodies increased,mitochondria were wrinkled,and mitochondrial crest disappeared.The mRNA expression of the apoptosisrelated genes Caspase3,Caspase8,Caspase9 and BAX and the autophagy-related genes Atg12,Atg5,Beclin1 and LC3 in ASYF was significantly higher than that of SYF(P<0.05).The mRNA expression of ferrorptosis-related genes GPX4 was significantly lower than that of SYF(P<0.05),COX2,HMOX1 and PROM2 were significantly higher than SYF(P<0.05),the mRNA expression of NCOA4 and TXNIP were significantly higher than that of SYF(P<0.05),and the expression of GPX4 in ASYF was significantly lower than that of SYF(P<0.05).The ferropotosis inhibitor DFO can improve Erastin-induced decreased follicular cell activity and increased ROS expression.Steroid-related index detection showed that the content of E2 and P4 in ASYF and the sterol-related genes CYP19A and HSD17B were significantly lower than those in SYF(P<0.05).The contents of ROS,H2O2 and MDA in ASYF were significantly higher than those in SYF(P<0.05),and the activities of T-AOC、SOD and GSH were significantly lower than those in SYF(P<0.05).The above results showed that a large number of GCs with follicle atresia experienced programmed death,granular layer shedding,and significantly reduced levels of sex steroids and antioxidants.2.Differential proteomic analysis of follicular fluid exosomes between SYF and ASYF in geese.To investigate the important role of goose follicular fluid exosomes in follicular development and follicular atresia in female geese,this study used TMT labeling technology to screen the extracted and identified SYF and ASYF follicular fluid exosomes for differentially expressed proteins,and the results showed that the exosomes could carry more than 4,000 protein species,compared with SYF,ASYF exosomes carried 1,615 differential proteins,including 905 up-regulated proteins and 710 down-regulated proteins.GO functional analysis showed that the differential proteins were mainly associated with cell communication functions such as cell surface receptor signaling pathway,cell adhesion,bioadhesion,etc.KEGG mainly enriched cellular functions including cell growth and programmed death,cell activity,cell community,signaling molecules and interactions,etc.PPI network analysis revealed that HMOX1 enriched in iron The PPI network analysis revealed that HMOX1,which is enriched in the iron death pathway,is located at the center of the network regulatory node and is only abundantly expressed in ASYF follicular fluid exosomes.The expression of HMOX1 in SYF and ASYF follicular fluid exosomes was quantified by Western blot and found to be consistent with the proteomic sequencing results.The results screened out the differentially expressed proteins in the follicular fluid exosomes of goose SYF and ASYF,and laid the foundation for further investigation of the mechanism of the key differential protein HMOX1 in follicular atresia caused by massive programmed death of granulosa cells.3.Molecular mechanism of exosomal protein HMOX1 from goose follicular fluid regulates granulosa cell iron death.In order to verify the role of exosome protein HMOX1 in regulating the programmed death of GCs,the extracted and identified goose SYF and ASYF follicular fluid exosomes were added to the goose GCs isolated in vitro,and the results showed that follicular fluid exosomes could enter the GCs through endocytosis,Compared with SYF,ASYF follicular fluid exosomes significantly reduced GCs cell activity(P<0.05),significantly increased the MDA and ferrous ion levels(P<0.05)of GCs,and significantly reduced the mitochondrial membrane potential of GCs(P<0.05),and the above changes could be reversed by the iron ion chelator DFO(P<0.05).Studies have shown that ASYF follicular fluid exosomes induce ferroptosis in GCs.In order to further detect whether the occurrence of hemozosis in GCs is related to the massive expression of HMOX1 in ASYF follicular fluid exosomes,the overexpression of HMOX1 recombinant plasmids was further constructed to transfect GCs and add specific inhibitor Znpp,and the effect of exosome differential protein HMOX1 on GCs was analyzed.The results showed that after HMOX1 overexpression,GCs cell activity decreased significantly,ferrous ion content increased significantly,GPX4 protein expression decreased,and MDA level increased(P<0.05).The addition of the inhibitor Znpp significantly alleviated the decrease in cell activity,elevated ferrous ions,and increased MDA levels caused by elevated HMOX1(P<0.05).The above results suggest that the exosomal protein HMOX1 can mediate the hemozotic pathway to regulate the programmed death of GCs involved in follicular atresia.In summary,during the development and selection of goose follicles,the granular layer of the atresia small yellow follicles was shed,and a large number of GCs died programmatically.Follicular fluid exosomes can enter GCs through endocytosis and regulate the programmed death of GCs through the HMOX1-mediated ferroptosis pathway,and participate in the regulation of follicular atresia. |
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