Study On The Subtype Characteristics Of Small Extracellular Vesicles And The Uptake By Granulosa Cells In Bovine Follicular Fluid

Extracellular vesicles(EVs)are nanoscale encapsulated vesicles that can be released by almost all cells,and they play an important role in cell-to-cell communication.Follicular fluid containsEVs,which can regulate the development of follicles and the maturation of oocytes.So far,the research on EVs in follicular fluid has basically focused on small extracellular vesicles(sEVs).Since sEVs are a mixed heterogeneous group,sEVs subtypes from different intracellular sources may have different functions and clinical applications.Therefore,the heterogeneity of different sEVs subtypes in follicular fluid has become one of the main problems to be solved urgently in the field of EVs.This study used differential ultracentrifugation combined with iodixanol density gradient flotation to separate two subtypes of sEVs from the follicular fluid of bovine small follicles(3-5 mm),namely low-density sEVs(sEVs-F6)and high-density sEVs(sEVs-F8).In this study,we analyzed and identified two sEVs subtypes,studied the uptake characteristics of two sEVs subtypes by ovarian granulosa cells,compared and analyzed the miRNA expression profiles of two sEVs subtypes,and studied the effects of two sEVs subtypes on the proliferation and apoptosis of ovarian granulosa cells.The results of the study were as follows:(1)Isolation and identification of sEVs subtypes in bovine follicular fluid.This study separated EVs from bovine follicular fluid using differential ultracentrifugation,obtained large extracellular vesicles(l EVs)and sEVs,and then evaluated the expression of the marker proteins,vesicle morphology,and size distribution of the two EVs populations.The results showed that CD63 and tumor susceptibility gene 101(TSG101)were significantly overexpressed in sEVs,and glucose regulatory protein 94(GRP94/GP96)was significantly overexpressed in l EVs;both EVs had a typical cup-shaped morphology;the vesicle size of sEVs was smaller.This study separated sEVs subtypes from bovine follicular fluid using iodixanol density gradient flotation,obtained low-density sEVs(sEVs-F6)and high-density sEVs(sEVs-F8),and then evaluated the expression of the marker proteins,vesicle morphology,size distribution,and RNA content,the number of vesicles and protein content of two sEVs subtypes.The results showed that CD63 is significantly enriched in sEVs-F8,and TSG101 is significantly enriched in sEVs-F6;both sEVs have a typical cup-shaped morphology;the size of vesicles in sEVs-F6 is smaller;the RNA content in sEVs-F8 is significantly higher than that in sEVs-F6,but the number of vesicles is significantly lower than that in sEVs-F6;the protein content of the two sEVs subtypes is basically the same.(2)Study on the uptake characteristics of two sEVs subtypes by follicular granulosa cells.PKH67 was used to fluorescently label the two sEVs subtypes isolated,and the uptake of the two sEVs subtypes by granulosa cells was observed by confocal microscopy and flow cytometry.We also used specific inhibitors and si RNA interference methods to explore the specific mechanism of the uptake of the two sEVs subtypes by granulosa cells.The results showed that the uptake of the two sEVs subtypes by granulosa cells was time-dependent,and the uptake rate of sEVs-F8 by granulosa cells was higher;the uptake of either s EV subtype by granulosa cells was almost completely inhibited at 4?;the fluorescence intensity of granulosa cells exposed to an excess of unlabeled EVs was significantly decreased;the addition of heparin,M?CD or EIPA can significantly inhibit the uptake of the two subtypes of sEVs by granulosa cells;add genistein or wortmannin can only significantly inhibit the uptake of sEVs-F8 by granulosa cells;interference of caveolin-1(CAV-1)inhibits the uptake of two sEVs subtypes by granulosa cells;interference of Flotillin-1(FLOT1)or P21(RAC1)Activated Kinase 1(PAK1)can only significantly inhibit the uptake of sEVs-F8 by granulosa cells.(3)Analysis and verification of miRNA expression profiles of different sEVs subtypes in bovine follicular fluid.The two sEVs subtypes were analyzed by miRNA sequencing.The potential target genes of differentially expressed miRNAs(DEMs)were analyzed by GO and KEGG.The potential target genes of non-differentially expressed m i R N A s(non-DEMs)were analyzed by KEGG.The miRNA-hub gene regulatory network was constructed in Rap1 signaling pathway and HIF-1 signaling pathway.The results showed that there are a large number of differentially expressed miRNAs between the two sEVs subtypes;the potential target genes of significantly up-regulated miRNAs in sEVs-F6 are mainly enriched in MAPK signaling pathways,Erb B signaling pathways,Fox O signaling pathways,Rap1 signaling pathways and AMPK signaling pathways;the potential target genes of significantly up-regulated miRNAs in sEVs-F8 are mainly enriched in autophagy and HIF-1 signaling pathways;the potential target genes of non-differentially expressed miRNAs are significantly enriched in autophagy,p53 signaling pathway,endocytosis,cell cycle,insulin signaling pathway,MAPK signaling pathway,AMPK signaling pathway,Erb B signaling pathway and Fox O signaling pathway;the potential target genes of highly expressed miRNAs in both subtypes were mainly related to cell proliferation,apoptosis,cell cycle and autophagy.(4)Study on the effect of different sEVs subtypes of bovine follicular fluid on the proliferation and apoptosis of granulosa cells.The two sEVs subtypes were added to granulosa cells culture medium to detect the uptake and expression of miRNAs related to proliferation and apoptosis in granulosa cells,and further detect the proliferation and apoptosis of granulosa cells.The results showed that the expression levels of miRNA in granulosa cells were significantly increased after treatment with both sEVs subtypes.Both sEVs subtypes treatments promoted the proliferation of granulosa cells,and inhibited the apoptosis of granulosa cells.Among them,the inhibitory effect of sEVs-F8 was more obvious.In summary,there were two different subtypes of sEVs in follicular fluid,among which sEVs-F6 was low-density sEVs,and the specifically enriched protein was TSG101;sEVs-F8 was high-density sEVs,specifically enriched protein was CD63.Both sEVs subtypes were taken up by granulosa cells through clathrin-independent pathways.In addition to the co-dependent uptake pathways,the uptake of sEVs-F8 also depends on the activities of tyrosine kinase and phosphoinositide 3-kinase.The miRNA expression profiles of the two sEVs subtypes are significantly different.Except for the common signaling pathways,the target genes of the highly expressed miRNAs were specifically and significantly enriched in the Rap1 signaling pathway in s EV-F6 and the HIF-1 signaling pathway in s EV-F8.Both subtypes of sEVs can carry their highly expressed miRNAs related to proliferation and apoptosis into granulosa cells and promote the proliferation of granulosa cells,and inhibit the apoptosis of granulosa cells.Among them,sEVs-F8 had a more obvious inhibitory effect on apoptosis.