4-vinylcyclohexene Diepoxide Function On Follicular Maturation And Ovulation

Part Ⅰ Effects of short-term-low-dose VCD exposure on ovarian development in young miceBackground:Mammalian ovary contains the maximum number of follicles to be used in a lifetime at birth.With the development of the ovary,a part of the follicles are activated from the primordial follicle pool and further develop into primary follicles,secondary follicles and eventually turn to mature pre-ovulatory follicles before ovulation.The activation of primordial follicles is not affected by hormone levels and is a spontaneous process.The Pten-PI3K-Akt pathway and apoptotic pathway play an important role in the process of primordial follicle activation and apoptosis.The follicular development following the secondary follicles begins the periodic regulation of hormones,aromatase can convert androgens in granular cells to estrogen to promote the development and maturation of follicles.4-vinylcyclohexene diepoxide(VCD)is a chemical solvent that is classified as an environmental pollutant because it can cause ovarian failure in long-term exposed people.Previous studies have suggested that the killing effect of VCD on the ovaries is mainly specific target the primordial follicles and primary follicles in the ovary,but has no killing effect on the large follicles after the secondary follicles.However,the mechanism of VCD’s killing effect on follicles is not clear.And previous researchers believed that the killing effect of VCD on ovaries must under continuous high concentration treatment conditions,while short term-low dose VCD was considered to have no effect on the ovaries.Objective:In order to investigate the effect of lower concentration and shorter term of VCD treatment on follicular development in the ovary,and further analyze its possible mechanism of action,explore the beneficial effects of VCD in addition to its killing effect.Methods:Ovarian culture was used to observe the effects of short-term-low-dose VCD on mouse ovaries during in vitro processing,and the ability of follicles to develop to the next stage was observed by ovarian transplantation experiments Immunohistochemistry,Western blot,and single-cell PCR were used to observe changes in the apoptotic pathway and Pten-PI3K-Akt pathway in mouse ovarian tissue during in vitro processing.Expression levels of Aromatase in primary granulosa cells and KGN cell lines were detected.Through in vivo experiments,the effects of VCD treatment at different doses and different time points on mouse ovarian follicular development were examinedResults:We found that short-term-low-dose VCD can affect the Pten-PI3K-Akt pathway and inhibiting the Bax/Bcl2 apoptosis pathway,which can maintain the primordial follicles pool in the ovaries of PD2 mice cultured in vitro and further increase the number of primary follicles.And,it can further promote the development of secondary follicles in the ovary of PD 12 mice cultured in vitro.We further verified in ovarian transplantation experiments of PD2 and PD 12 mice treated with VCD,and found that there were more developing follicles in the ovaries of the treatment group,and the number of primordial and primary follicles did not decrease significantly.In vivo experiments,the effects of high-dose-short-term and low-dose-long-term treatment on follicular development are mainly to promote follicular maturation.We also observed that the expression of aromatase in mouse primary granulosa cells and KGN cell lines was significantly increased under VCD treatmentConclusion:Short-term-low-dose VCD can maintain the ovarian reserve(Ovarian Reserve,OR)in the mouse ovary by affecting the Pten-PI3K-Akt pathway and inhibiting the apoptosis pathway in the ovary.It also has an FSH-like effect in promoting aromatase expression,which may be related to its role in promoting follicular development and further maturationPart Ⅱ Effects of short-term-low-dose VCD exposure on ovarian follicular development and early embryo development in aged miceBackground:The ovarian reserve in women gradually decreases with age,and the quality of remaining oocytes will also decrease.The ability of the ovaries to secrete estrogen also decreases with age.Women’s fertility decreases significantly after the age of 35.For mice,fertility will also decrease significantly after 7-8 months.Decreased oocyte quality can cause oocyte maturation disorders,as well as impair fertility and early embryo development after fertilization.Therefore,increasing age will cause a series of problems such as impaired follicle development and maturity,ovulation disorders,and early embryonic development disorders.We have previously observed that VCD plays a positive role in young mice follicular development.With 10-12 month old mice,we observed the possible effects of VCD on the ovary of aged mice.Objective:To explore the role of VCD in follicular development,oocytes maturation and ovulation,and early embryonic development in aged mice,and provide a basis for the further use of VCD.Methods:We observed the changes of ovaries in aged mice after VCD injection,and examined the difference between hormone levels in the treatment group and the control group.We also observed the changes of ovulation in the aged mice after treatment through ovulation stimulating experiments.We collected GV-stage oocytes from the ovaries of aging mice and cultured them in vitro to observe the effects of VCD treatment on mouse oocyte in vitro maturation(IVM).Finally,we observed the effects of VCD on in vitro fertilization(IVF).Results:Five days after intraperitoneal injection of VCD(80mg/kg),there were more near ovulation follicles in the ovaries of the aged mice in the treatment group,and the number of corpus luteum increased significantly.After VCD treatment,the estrogen level in the aged mice increased significantly,accompanied by an increase in FSH levels.Ovulation-stimulating experiments showed that mice in the VCD treatment group could collect more mature oocyteIn the IVM experiment,we observed that 51.3%of the GV stage oocytes in the VCD treatment group turn to the mature MII stage,a 16%increase compared to the control group(35.3%).In the IVF experiment,65.4%of the fertilized oocytes turn to the blastocyst stage after VCD treatment,which was an increase of 15%compared with the control group(50.5%).We found that the role of VCD in promoting early embryonic development was most significant during the morula stageConclusion:VCD can promote the development and maturation of oocytes in aged mice and increase the number of ovulated MII oocyte.In IVF experiments,it can also promote the early embryogenesis in aged mice.The effect of VCD may have potential use value in elderly women with clinical fertility requirements and PCOS patients with ovulation disorders.Part Ⅲ Research on High-dose VCD Induced POF Model in Mice Background:Premature ovarian failure(POF)is a symptom in which the ovaries fail before the age of 40.Women with ovarian failure not only face changes in the reproductive system,but may also be accompanied by symptoms of other systems,such as osteoporosis,cardiovascular disease,and neurological disease.The main causes of POF include genetic factors,iatrogenic factors,and immune factors.The establishment of a mouse model of POF is promotive to further study the progress of POF and other systemic diseases caused by POF.Different animal models have been established to simulate POF for different causes.Previous studies have confirmed that VCD can specifically kill primordial and primary follicles in the ovary,destroy the follicular reserve in the ovary and cause POF.Compared with other POF models,the VCD-induced POF model has certain advantages.However,there are still some details in the establishment and application of VCD models that deserve further study.Objective:The systematic research on the POF model caused by VCD provides a more detailed basis for better use of the model.The changes of intestinal microorganisms in VCD model and their possible effects were studied.Methods:According to previous studies,changes in mouse ovaries at lower VCD doses were studied and changes in follicle-stimulating hormone,estradiol,and anti-Muller hormone were measured.Explore bone tissue changes and behavior changes in VCD models other than the reproductive system.Innovatively studied the changes of gut microorganisms in the VCD model,and conducted a preliminary study on the possible mechanism of gut microorganisms using the C-elegans model.Results:A mouse model induced by traditional modeling methods(injected continuously at a concentration of 160 mg/kg for 15 days)reached a state of ovarian failure in mice at 30 days after injection.In the same case of 15 days of injection,the effect of VCD on the ovaries has a significant dose-dependent effect.The VCD in concentrations ranging from 80 mg/kg to 160 mg/kg have significantly killing effect on primordial follicles.There is no adverse effect of VCD on small follicles at doses of 40 mg/kg.However,the number of pre-ovulatory follicles was found to increase significantly after doses of 80 mg/kg and 120 mg/kg.About 30 days after the end of the administration,VCD model mice has significant anxiety-like behavioral changes in the Open Field experiment,but at the same time,the bone density of the mice did not change significantly.Anxiety-like behaviors were reduced 60 days after the end of the administration,but bone density of the mice was significantly reduced.Analysis of the gut microbes of the mice revealed that the microbes of the mice in the VCD treatment group were significantly different from those of the control group,and the proportion of the various bacterial flora in the dominant flora had changed significantly.The results of gut microbe feeding on C-elegans in two groups of mice showed that gut microbes in VCD treated mice could induce C-elegans to ovulate faster and more.Conclusion:30 days after modeling is a window period for ovarian changes in a VCD-induced POF mouse model,30 days to 60 days is the window period for other tissues or behavioral changes caused by POF.Gut microbial changes may be closely related to POF.