| Background and Objective:Chronic stress refers to a state of long-term stress.With the accelerated pace of life,chronic stress has become a major problem endangering people’s physical and mental health.Studies have found that long-term exposure to stress can affect female fertility and lead to female reproductive dysfunction,but the mechanism has not been clarified.The purinergic receptor P2X7(P2X7R)is an ATP-gated cation channel.Studies have shown that P2X7R is involved in multisystem pathological processes under stress.However,whether P2X7R is involved in the impact of chronic stress on female reproduction has not been studied.In this study,we investigated whether P2X7R mediates ovarian dysfunction caused by chronic stress by establishing a mouse model of chronic cold stress.Research methods:To construct a chronic cold stress mouse model,the mice were randomly divided into two groups:the control group(Control)and the chronic cold stress group(CCS).CCS mice were subjected to chronic cold stress for 8 weeks,while the control group mice were placed indoors at 26 ℃ for the corresponding time.The weight was weigh weekly and the changes of the estrous cycle of the mice were observed through continuous vaginal smears for the last two weeks.After 8 weeks,the mice were killed.Serum cortisol and progesterone levels were detected by Elisa;The morphological changes of ovaries and the number of corpus luteum in the two groups of mice were observed by HE staining;The expression of P2X7R in the ovaries of the two groups of mice was detected by immunohistochemical staining and Western blotting;The two groups of mice were subjected to superovulation treatment and injected with P2X7R inhibitor A-438079 HCL for intervention,to observe the changes in ovulation rate in each group of mice,The changes of mouse ovarian fibrosis was detected by Sirius red staining,and western blotting was used to detect the expression of mouse ovarian fibrosis factor,including IL-1β,TGF-β1 and MMP2.The primary follicles and cumulus complexes were isolated from 14 and 21 day old mice,cultured in vitro and treated with P2X7R agonists and inhibitors.The follicular development and cumulus expansion were dynamically observed.The expression changes of ovulation related factors were detected by q PCR and EIA,including EGF,EGFR,EREG,AREG,BTC,NPPC,NPR2,PTGS2,HAS2,PTX3,and TNFAIP6.Research results:By observing the estrous cycle of mice,it was found that the estrous cycle of chronic cold stress mice was disordered;The content of progesterone in cold stress mice was significantly decreased by ELisa;The results of he staining showed that the number of corpora lutea in the ovary of cold stress mice was significantly reduced.The number of corpora lutea indirectly reflected the ovulation status,indicating that chronic cold stress affected ovulation.Immunohistochemical staining showed an increase in P2X7R expression in follicular granulosa cells,and Western blotting was consistent with the above results.After superovulation treatment,the superovulation rate of mice in the chronic cold stress group was significantly lower than that in the control group.P2X7R agonist BzATP treatment also reduced the superovulation rate of mice,while P2X7R antagonist A-438079 HCL partially alleviated the ovulation disorder caused by the above treatment.Follicular development and cumulus expansion are key steps in oocyte maturation and ovulation.We further observed the effects of P2X7R agonist BzATP on follicular development and ovulation,and found that BzATP promoted early follicular development,but inhibited EGF induced cumulus expansion.Mechanism studies have shown that P2X7R signals promote the expression of the ovulation inhibitory factor NPPC in granulosa cells.In addition,the results of Sirius red staining showed a significant increase in ovarian fibrosis in the chronic cold stress model.Fibrotic inflammatory and fibrosis-related factor IL-1β and TGF-β1 expression increased and MMP2 decreased.We also studied the effect of P2X7R activation on the expression of fibrosis regulatory factors in granulosa cells,and found that P2X7R agonist BzATP treated cells promoted TGF-β1 expression and decreased MMP2 expression.P2X7R antagonist A-438079 HCL alleviates the effects of P2X7R agonist BzATP on TGF-β1and MMP2 expression.Conclusion:1.The expression of P2X7R is increased in the ovary of a chronic cold stress mouse model.The increase in P2X7R affects follicular development and cumulus expansion by regulating NPPC expression,leading to ovulation disorders.2.P2X7R regulates inflammatory factors and fibrosis-related factors(including IL-1β、TGF-β1 and MMP2)expression,which contributes to the ovarian fibrosis... |
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