Effects Of Follicular Fluid Exosomes On Proliferation,Steroid Synthesis,Oxidative Stress Resistance And Chemokines Secretion In Porcine Follicular Cells

The ovary was a highly dynamic organ,and the follicle was the basic functional unit of the ovary.In every estrous cycle,the ovary would undergo a remodeling process,including follicle recruitment,growth,dominance or atresia.The development of ovarian follicles was regulated by endocrine and paracrine.It was of great significance to study the development of porcine ovarian follicles to better understand the reproductive mechanism of pig and further improve their fecundity.Follicular fluid was the environment where oocyte and follicular somatic cells live together.There were a lot of inorganic salts and various nutrients in follicle fluid,and follicular fluid could guarantee the communication between follicle cells.Exosomes were extracellular vesicles between 40 and 200 nm in diameter.Exosomes could carry and transport substances such as proteins,RNA and lipids for intercellular communication.Currently,exosomes had been found in follicular fluid of various large domestic animals,but the influence of exosomes on follicular cells was not completely clear.In this study,porcine follicular granulosa cells and follicular theca cells were taken as experimental objects to explore the influence of follicular fluid exosomes on follicular somatic cells.The main contents and results of this study were as follows:1.Exosome isolation,characterization,tagging,and trackingIn this study,follicular fluid exosomes were isolated from follicular fluid by ultrafast centrifugal filtration method,the purity of the isolated exosomes was determined by TEM,NTA and detection of exosomes marker proteins.The results showed that the exosomes isolated by the ultrafast centrifugal filtration method were consistent with the characteristics of exosomes and had a high purity.Exosomes were labeled with PKH26,and the labeled exosomes were incubated with granulosa cells and follicular theca cells.Laser confocal results showed that exosomes were phagocytic by granulosa cells and follicular theca cells.Further studies showed that this endocytosis could be blocked by chloropromazine.These results indicated that follicular fluid exosomes entered follicular granulosa cells and follicular theca cells via clathrinmediated endocytosis.2.Effects of follicular fluid exosomes on follicular granulosa cell proliferation and hormone synthesisIn this study,gradient-concentration exosomes were used to treat granulosa cells,and CCK-8 was used to detect cell viability.The experimental results showed that120μg/m L of exosomes significantly promoted the viability of follicular granulosa cells.Therefore,the follicular granulosa cells were treated with 120μg/m L of follicular fluid exosomes in subsequent experiments,and then transcriptomic sequencing was performed on follicular granulosa cells,and transcriptomic sequencing was performed.Transcriptome sequencing results showed that some differential expression genes were associated with follicular granulosa cell proliferation and steroid synthesis.In further experiments,follicular fluid exosomes could promote the expression of follicular granulosa cell proliferation-related genes CCND1 and steroid synthesis-related genes HSD3B1,thus significantly promoted follicular granulosa cell proliferation and progesterone synthesis.And the effects of exosomes on CCND1 and HSD3B1 could be block ICG-001,suggesting that the regulation of exosomes on CCND1 and HSD3B1 was mediated by WNT/B-CATENIN signaling pathway.mi RNA sequencing results showed that a large number of mi RNAs were enriched in follicular fluid exosomes,and targeted analysis of mi RNA-m RNA revealed that SFRP4 was a potential target of mi R-31-5p.By overexpression or inhibition of mi R-31-5p in granulosa cells,it was found that mi R-31-5p could activate the WNT/B-CATENIN signaling pathway and promote granulosa cell proliferation and progesterone synthesis by inhibiting the expression of SFRP4.In summary,follicular fluid exosomes promoted follicular granulosa cells proliferation and progesterone synthesis by activating the WNT/B-CATENIN signaling pathway via the mi R-31-5p/SFRP4 axis.3.Effects of follicular fluid exosomes on oxidative stress resistance,proliferation and steroid synthesis of follicular theca cells.In this study,gradient-concentration exosomes were used to treat theca cells,and CCK-8 was used to detect cell viability.The experimental results showed that120μg/m L of exosomes significantly promoted the viability of follicular theca cells.Therefore,the follicular theca cells were treated with 120μg/m L of follicular fluid exosomes in subsequent experiments,and then transcriptomic sequencing was performed on follicular theca cells.Transcriptome sequencing results revealed that some differential genes were related to oxidative stress resistance,proliferation and steroid synthesis.In further experiments,we found that follicular fluid exosomes could enhance the oxidative stress resistance ability of follicular theca cells by directly delivering GPX1 protein and promoting the expression of GPX1 in follicular theca cells.And follicular fluid exosomes could promote the proliferation and the synthesis of steroid hormones(progesterone and androstenedione)of follicular theca cells by promoting the expression of proliferation-related genes CCND1 and PCNA,and steroid synthesis related genes CYP11A1 and HSD3B1.4.Effects of follicular fluid exosomes on the secretion of chemokines in follicular granulosa cellsThe transcriptome sequencing results in Chapter 2 and Chapter 3 showed that follicular fluid exosomes could promote the expression of chemokines CCL2 and CXCL8 m RNA in follicular granulosa cells and theca cells.The effects of follicular fluid exosomes on chemokine secretion of follicular cells were investigated by using follicular granulosa cells as experimental objects.The results showed that follicular fluid exosomes could inhibit the expression of BDNF and promote the secretion of chemokines CCL2 and CXCL8 in granulosa cells.Further experiments showed that the addition of BDNF in vitro could inhibit the secretion of chemokines CCL2 and CXCL8 in granulosa cells.The use of si-BDNF endogenous knockdown of BDNF could promote the secretion of CCL2 and CXCL8 chemokines in granulosa cells.mi RNA sequencing results showed that mi R-10b-5p was enriched in follicular fluid exosomes,and BDNF was found to be a potential target of mi R-10b-5p by targeting mi RNAm RNA analysis.By overexpression or inhibition of mi R-10b-5p in granulosa cells,it was found that mi R-31-5p could promote the secretion of chemokines CCL2 and CXCL8 in granulosa cells by inhibiting BDNF expression.In summary,follicular fluid exosomes promote secretion of granulosa cells chemokines CCL2 and CXCL8 via the mi R-10b-5p/BDNF axis.In conclusion,this study found that follicular fluid exosomes can ensure the normal development of follicles by promoting the proliferation of follicular cells,hormone synthesis and antioxidant functions,and promote the secretion of chemokines by follicular cells to recruit immune cells to prepare for ovulation.The study on the function of exosomes provides some new understanding and new mechanisms for revealing follicular genesis.