Study On The Function And Mechanism Of DDR2 On Follicular Development And Prognosis

Objective: To elucidate the expression and the role of Discoidin Domain Receptor 2(DDR2)during the follicular development and ovulation in the follicular microenvironment,and to study the mechanism of DDR2 on thecal cells and granulosa cells.To reveal the role of DDR2 regulation of follicular development and ovulation and mechanism.For the clinical unexplained infertility,to provide a new theoretical basis.Methods:(1)We choose 21-days old SPF WT mice to establish mouse sexual maturation model(7d,10 d,14d,21 d,25d,30d),follicular development model(P12,P24,P48),ovulation and corpus luteum formation model(H12,H20,H48,H72),respectively.Immunohistochemistry and indirect immunofluorescence were used to detect the expression of DDR2 at the tissue level.(2)We choose 21-days old SPF SLIE mice ovaries as research object and WT mice ovaries as control group to study the effects of DDR2 on follicular development by HE staining(3)to carry out the primary culture of the mouse granulosa cells and thecal cells to investigate the proliferation and differentiation ability of mice.DDR2 adenovirus and control virus were used to recover the SLIE mice ovarian granulosa cells and thecal cells to examine whether its ability to proliferate is improved The effects of DDR2 on cell differentiation and the key enzymes of steroid hormone synthesis were investigated by transmission electron microscopy,western blot and qPCR.Results:(1)Immunohistochemical results showed that DDR2 was expressed in the whole stage of mouse sexual maturation,and the expression of DDR2 was mainly in granulosa cells,ovarian stroma and thecal cells,and no obvious expression in oocytes.(2)Before adolescence(Day 7,Day 10 and Day 14),DDR2 was expressed in the ovarian surface epithelium.In the follicular development model,the expression of DDR2 was mainly concentrated in the P24 stage and P48 stage.At this stage of P24,the follicles developed mainly to the secondary follicle and the Pre-antral follicle stage;at the P48 stage,follicles were mostly antral follicles.In the ovulation and corpus luteum formation model,the expression of DDR2 at the early stage of the corpus luteum(H20)and the developmental stage(H48)was higher,and the expression of DDR2 decreased with the corpus luteum degradation.Subsequently,we used immunofluorescence to further indicate that the expression of DDR2 on granulosa cells was mainly located in the follicular development stage after secondary follicle and was closely related to proliferation.(2)WT mice and SLIE mice were given exogenous gonadotropin.HE staining show that the follicular development of SLIE mice was slow,and the cumulus complex expanded slowly and the corpus luteum cannot be formed.In the H8 stage,the WT mouse cumulus complex was fully expanded and the follicular development was located in the pre-ovulation follicle stage,while the SLIE mouse oocytes were still tightly surrounded by granulosa cells;furthermore,in the corpus luteum formation models,we can find that WT mice ovarian showed significant distribution of the corpus luteum,and SLIE mice still showed a large follicle,and even atresia follicles.(3)Primary culture of WT mice and SLIE mouse ovarian granulosa cells and thecal cells,respectively,using EDU incorporation experiment to study the effect of DDR2 on the proliferation of follicular cells.Compared with the control group WT,we found that the proliferation of SLIE group granulosa cells decreased(P = 0.0035);thecal cell proliferation was also decreased(P = 0.0010).When the Ad-DDR2 adenovirus were added in ovarian granulosa cells and thecal cells of SLIE mice,the proliferative ability was recovered.Compared with Ad-EGFP,P = 0.0107 and P = 0.0111,respectively.Proliferation rate increased by about 10%.(4)The results of scan electron microscopy showed that the number of intracellular tubular mitochondria increased and the number of lipid droplets was abundant in the WTgranulosa cells compared with SLIE granulosa cells.The cytoplasm was abundant and the gap was intact.However,SLIE granules and thecal cells showed loosely connected to each other Immunohistochemistry,western blot and qPCR showed that the expression of E-cadherin in the WT group decreased,and the difference was statistically significant(P <0.05).The difference between the Vimentin markers was not obvious.At the same time,the expression of 3?-HSD,CYP11A1 and STAR were significantly different(P <0.05),which was statistically significant(5)The results of hormone test showed that the levels of hormones in the serum and in the cell were significantly higher in the WT group than in the SLIE group(P <0.05)Conclusion:(1)DDR2 locate in ovarian stroma,granule cell layer,membrane cell and OSE part,and no expression in oocyte.(2)DDR2 is mainly expressed in the granulosa cells of secondary follicles,and is closely related to proliferation;(3)DDR2 affects the proliferation and differentiation of granulosa cells and thecal cells,and their deletions lead to follicular development retardation,ovulation and corpus luteum formation disorders;(4)DDR2 may regulates the key enzymes of steroid hormone synthesis:3?-HSD,CYP11A1 and STAR,and regulates the synthesis and secretion of follicle hormone,regulating follicular development.