| Extracellular vesicles(EVs)are double-layered membrane vesicles that carry macromolecular substances outside the cell to perform biological functions.The release and uptake of EVs is a new mechanism of cell-to-cell communication,and the study of its separation and purification and the function of its contents(main constituent substances)is a hot issue in the field of modern biology.The important function of the ovary is to produce follicles,maintain follicle development,and produce eggs.The process of follicle formation involves complex intercellular communication,cell metabolism,and cell proliferation.Follicular membrane cells,granulosa cells and oocytes secrete a variety of cytokines and protein factors throughout the growth and maturation of the follicle,and participate in the development of the follicle.There are EVs secreted by granulosa cells,cumulus cells,oocytes and so on in follicular fluid,which affect follicular development and ovarian biological functions through some special transport pathways.The proliferation of ovarian cortical stromal cells and the production of steroid hormones are closely related to follicular development and atresia.The regulatory effect of follicular fluid EVs on the proliferation and steroid hormone production of ovarian cortical interstitial cells is still unclear.This study explored the effects of follicular fluid EVs on the proliferation and steroid hormone production of ovarian cortical interstitial cells in order to clarify the effect of follicular fluid EVs on follicle Lay the foundation for developmental influence and regulation.The research results are as follows:1.Separation and purification of EVs from bovine follicular fluidFollicular fluid is extracted from small follicles(3-5mm)of bovine ovary,and EVs are extracted from follicular fluid by differential ultracentrifugation and multi-step centrifugation.The purity of the extracted follicular fluid EVs was identified by electron microscope observation,Western blot detection and nanometer particle size technology.It was observed under the electron microscope that the extracted follicular fluid EVs showed a typical cup-shaped double-layered membrane vesicle structure with a size of 50～150nm;Western blotting confirmed that the extracted follicular fluid EVs contained the marker proteins CD63 and TSG101,and no internal content was detected.Plasma reticulum marker protein GP96;nanometer particle size test proved that the extracted follicular fluid EVs is consistent with the size described by EVs,and the diameter of EVs is mainly concentrated in 50-150nm.It shows that the extracted follicular fluid EVs has high purity and no endoplasmic reticulum pollution.2.Isolation,culture and purification of bovine ovarian cortical mesenchymal cellsUnder aseptic conditions,the ovarian cortical mesenchymal cells were separated from the bovine ovarian tissue by a combination of mechanical separation and enzymatic digestion,and purified by the differential adhesion method and identified for purity.The results showed that the isolated cells were positive for wave protein and negative for keratin.The purity of the mesenchymal cells was 98.1%.3.Uptake of EVs in bovine follicular fluid by cortical interstitial cellsIn order to prove that EVs in follicular fluid can be taken up by ovarian cortical interstitial cells,PKH67-labeled EVs were used to treat ovarian cortical interstitial cells,and the cell location of PKH67 with green fluorescence was observed with a confocal microscope.The results showed that the green fluorescence of the PKH67-EVs-treated group was distributed in the cytoplasm of cortical interstitial cells,indicating that PKH67 entered the cortical interstitial cells through EVs,proving that follicular fluid EVs can be taken up by ovarian cortical interstitial cells.4.The effect of EVs in bovine follicular fluid on the proliferation of ovarian cortical interstitial cellsTreat ovarian cortical interstitial cells with EVs containing 0μg/mL,10μg/mL,30μg/mL and 100μg/mL follicular fluid,perform CCK detection for 5 consecutive days and use the formula to calculate the proliferation rate to evaluate the proliferation of ovarian cortical interstitial cells.The results show that compared with the 0μg/mL and 10μg/mL follicular fluid EVs group,30μg/mL and 100μg/mL follicular fluid EVs can promote the proliferation of ovarian cortical interstitial cells.TUNEL was used to detect cell apoptosis,and the results showed that compared with 0μg/mL and 10μg/mL follicular fluid EVs group,30μg/mL and 100μg/mL follicular fluid EVs could inhibit the apoptosis of ovarian cortical interstitial cells.After treating ovarian cortical interstitial cells with EVs containing 0μg/mL,10μg/mL,30μg/mL and 100μg/mL follicular fluid for 24h,RT-qPCR was performed for apoptosis-related genes(BAX and CASPASE3)and anti-apoptotic genes(BCL2)The test results showed that compared with the 0μg/mL,10μg/mL and 30μg/mL follicular fluid EVs groups,100μg/mL follicular fluid EVs can significantly inhibit the expression of ovarian cortical interstitial cells BAX and promote the expression of BCL2(P<0.05),while the effect on the expression of CASPASE3 was not significant(P>0.05),and it could increase the ratio of BCL2 and BAX(P<0.05),which was consistent with the results of the TUNEL test.It shows that EVs can inhibit the apoptosis of cortical stromal cells and promote their proliferation.5.The effect of EVs in bovine follicular fluid on the production of steroid hormones in ovarian cortical interstitial cells.The ovarian cortical interstitial cells were treated with EVs containing 0μg/mL 10μg/mL,30μg/mLand 100μg/mL follicular fluid.After 24 hours,the hormone expression of androstenedione and progesterone was detected by the method of Elisa.The results showed that compared with 0μg/mL and 10μg/mLfollicular fluid EVs group,30μg/mL and 100μg/mL follicular fluid EVs significantly promoted the synthesis of androstenedi one and progesterone in ovarian cortical interstitial cells(P<0.05).Treat ovarian cortical interstitial cells with EVs containing 0μg/mL,10μg/mL,30μg/mL and 100μg/mL follicular fluid.After 24 hours,RT-qPCR was used to detect the STAR,CYP11A1,3β-HSD and CYP17A1 genes.The results showed that compared with the 0μg/mL and 10μg/mL follicular fluid EVs group,30μg/mL and 100μg/mL follicular fluid EVs could promote the expression of CYP11A1,3β-HSD and CYP17A1 genes in ovarian cortical interstitial cells(P<0.05),but has no effect on STAR gene expression(P>0.05).Conclusion:EVs in bovine follicular fluid can act on ovarian cortical interstitial cells,promote cell proliferation,and promote the production of androstenedione and progesterone. |
PDF Full Text Request