Effects Of Bovine Follicular Fluid Low-density Small Extracellular Vesicles-derived Let-7i On Apoptosis Of Granulosa Cells

Extracellular vesicles（EVs）are nano-scale lipid bilayer vesicles that are released outward from the cell and are widely found in extracellular biological fluids,participating in the exchange of information between cells and the regulation of the body’s physiological functions by carrying a variety of bioactive substances including nucleic acids and proteins.In mammalian ovarian follicle development,less than 1%of the follicles can mature and reach ovulation,while the others are atresia at different stages.Apoptosis of granulosa cells is a key factor in mammalian follicular atresia.EVs in follicular fluid have been found to play an important regulatory role in the proliferation,metabolism and apoptosis of follicular granulosa cells,and these EVs may perform their function of intercellular information exchange by transferring the bioactive substances they carry.However,EVs are a highly heterogeneous group,and there are few studies on the function of EVs subtypes.In this study,we investigated the effects of LD-sEVs,one of the EVs subtypes,on granulosa cell apoptosis,and explored the key components and related mechanisms to provide a theoretical basis for further revealing the regulatory role of LD-sEVs on follicular development and follicular atresia.In this study,the subtype LD-sEVs of sEVs was successfully isolated and identified from the follicular fluid of bovine small follicles by differential hypercentrifugation and density gradient centrifugation with iodixanol,and we characterized them.After treatment of follicular granulosa cells with LD-sEVs,the effect of LD-sEVs on follicular granulosa cell apoptosis was examined using flow cytometry,RT-q PCR and WB.The mi RNA component highly expressed in LD-sEVs,let-7i,was screened and the targeting of let-7i in granulosa cells was validated.The results obtained are as follows:（1）The isolated LD-sEVs were identified using transmission electron microscopy,nano-particle size analysis and WB technique.The results showed that the isolated LD-sEVs showed a teatrope shape,98.98%of the vesicles were between 30-150 nm in size,and the EVs marker protein CD63 and TSG101 were positive,while the l EVs marker protein GP96 was negative.It indicates that the LD-sEVs obtained by separation had high purity and no contamination by l EVs;the granulocytes isolated were identified using the FSHR staining method,and the results showed that 97.53%of the cells were granulocytes.It indicates that pure granulocytes were isolated.（2）Co-culture with PKH67-labelled LD-sEVs and granulocytes revealed that granulocytes could uptake LD-sEVs.apoptosis rate of granulocytes decreased significantly after LD-sEVs treatment,mainly manifested as a significant decrease in early apoptosis rate of granulocytes;Bcl2 gene expression was upregulated in granulocytes,Caspase3,Caspase8and CHOP gene expression was downregulated;Bcl2 protein The expression of Bcl2 protein was increased,the expression of Bax and FADD protein was decreased,the Bcl2/Bax ratio was increased and the Caspase3 enzyme activity of granulosa cells was decreased.（3）After LD-sEVs treatment of granulosa cells,the expression of let-7i in granulosa cells was significantly increased.When let-7i mimic was transfected into granulosa cells,the apoptosis rate of granulosa cells was significantly decreased,mainly in the early stage.The expression of Caspase3 gene in granulosa cells was significantly down-regulated,and the protein expressions of Bax and FADD were significantly decreased,the ratio of Bcl2/Bax was increased,and the activity of Caspase3 enzyme in granulosa cells was significantly down-regulated.（4）According to the results of bioinformatics analysis,Fas L was selected as the candidate target gene of let-7i for follow-up research.Constructed Psi CHECK^TM-2 reporter vector of WT-Fas L and MUT-Fas L.Double luciferase reporter gene results showed that the RLU1/RLU2 ratio of 293T cells co-transfected with WT-Fas L-Psi CHECK^TM-2 reporter vector and let-7i mimic was significantly lower than the control group.This result was also verified in bovine follicular granulosa cells.After transfection with let-7i mimic,the protein level of Fas L in granular cells decreased significantly.Using si RNA to interfere with the expression of Fas L in granulosa cells can significantly inhibit the apoptosis of granulosa cells,which is manifested as a significant decrease in the early apoptosis rate of granulosa cells.At the same time,the expression of Bax and FADD proteins in granulosa cells decreased significantly,the ratio of Bcl2/Bax increased,and the activity of Caspase3 enzyme was significantly down-regulated.Treatment of granulosa cells with LD-sEVs can significantly down-regulate the expression of Fas L protein,and the expression of Fas L protein in granulosa cells treated with LD-sEVs transfected with let-7i inhibitor was significantly higher than that in LD-sEVs-treated group.This suggests that let-7i plays a key role in the negative regulation of granulosa cell apoptosis by LD-sEVs.In conclusion,bovine fetal follicular fluid LD-sEVs and their let-7i inhibit the apoptosis of granulosa cells by targeting FasL.