Study Of X Irradiation Induced Damage In Endometrium And Ovary

I. ObjectivePeople are continuously exposed to low-level ionizing radiation(including X ray and gamma ray) which is found in food, soils, building materials and air and comes from outer space. In addition to natural background radiation as above mentioned, people may be exposed to X-ray from medical radiation tests or treatments(such as CT scan and radiotherapy), occupational exposure or nuleur accident. X ray can travel through the air and penetrate many materials, including human tissues. Exposure to X ray can result in significant damage to organs through generating oxidative stress. Due to the increasing incidence of infertility in women with X ray exposure, it is crucial to understand the potential hazardous effects of X ray on female fertility capability and to search for effectively protective agents.Embryo implantation is an extremely complex biological process. Successful initiation of pregnancy requires the ovulation of a mature oocyte and properly prepared, healthy endometrium. Dysfunction in any one of these complex biological steps can lead to female infertility. Excess ROS generation in ovary can cause ovarian granulosa cell apoptosis, therefore, affects the normal ovulation and hormone secretion. Also, it has been reported that low level of antioxidant capacity inhibits the uterine growth. Till now, the mechanism of how X ray affects the endometrium function through oxidative stress is still not yet detailed reported.Autophagy is a normal physiological phenomenon in eukaryotic cells. Extremely low level of autophagy is observed in cells under normal circumstances. When subjected to external stimuli, autophagy will be induced, increasing the cell survival through phagocytosis, showing its cytoprotect effect. Studies have shown that the ROS is significantly important during that autophagy progress. However, what role the autophagy plays in X ray induced low endometrial receptivity still unknown.N-acetylcysteine, NAC, an intracellular glutathione precursor, is known as an antioxidant and has been widely used in the clinical medicine as mucolytic agent for over 40 years. Studies have found that NAC has significant effect on treatment of cancer, AIDS and Parkinson’s disease. NAC was found to have a role in infertility management which maked NAC a potential candidate for its use in the infertility treatment but not yet put into use in clinical practice. Therefore, the radioprotection of NAC on ovary still needs more exploration.Here, using in vivo and in vitro tests, we found that mitochondrial and lysosomal dysfunction are involved in X irradiation-induced oxidative DNA damage, which lead to low endometrial receptivity to embryos. Proper protective reagents can be helpful in improving endometrial function and female fertility. We identified that X ray induced cytoprotective autophagy was attributed to induction of ROS through activation of JNK pathway. Also, we suggested that NAC can be a potential radioprotector and prevent the ovarian failure occurrence. Our study provides new ideas for the research of ionizing radiation affecting women’s reproductive health and evidences for usage on NAC in the treatment of female infertility. II. Methods 1. Oxidative damage of DNA induced by X-irradiation decreases the uterineendometrial receptivity which involves mitochondrial and lysosomal dysfunction(1) X irradiation(2 Gy) and NAC, NH4 Cl, Pepstatin A pretreatment.(2) Use cell counting assay and CCK-8 to detect the cell proliferation.(3) The external and internal morphological changes were observed by usingtransmission electron microscopy and scanning electron microscopy.(4) The in vitro model was used to measure the adhesion rate.(5) Use rhodamine 123(Rh123) and Lyso-tracker red staining to detect thedysfunction of mitochondria and lysosome; The release of Cyt C and CD weredetected by Western blot.(6) The oxidative stress was detected by DCFH-DA staining and IF of 8-OHdGformation.DNA strand breaks were measured by comet assay; Immunofluorescence,confocal laser scanning and Western blot were used to detect the expression ofnucleiγH2AX. 2. Protective role of autophagy induced by X irradiation through ROS mediatedactivation of JNK signaling pathway in RL95-2 and HEC cells(1) Pretreatment of 3-MA, NAC and treatment of X irradiation(2 Gy, 10 Gy).(2) Western blot was used to detect the expression of autophagy, apoptosis relatedproteins and MAPKs signal molecules.(3) Cell death and apoptosis was detected by flow cytometry.(4) Cell survival curves were determined by colony formation method to evaluatechanges of radiosensitivity of the cells.(5) Autophagosomes were observed and measured by Scanning electronmicroscopy and MDC staining; Lyso tracker red staining was used to detect theautophagy level.(6) The ROS generation was detected by DCFH-DA staining.(7) The in vitro model was used to measure the adhesion rate. 3. The protective effect of N-acetylcysteine(NAC) on ionizing radiation inducedpremature ovarian failure and loss of ovarian reserve in female mouse(1) Pretreatment of NAC and treatment of total body X irradiation(3 Gy).(2) The serum level of estrogen, progesterone, Anti-Müllerian hormone andfollicle-stimulating hormone were measured by ELISA assay.(3) Ovarian weight changes were measured.(4) The morphological changes of ovarian and uterine endometrial epithelium wereobserved by H&E staining.(5) IHC was used to detect the expression of PCNA, caspase-3 and 8-OHdG inovarian granule cells; The expression of PCNA was also detected by Westernblot.(6) The apoptosis index was detected by TUNEL assay.(7) The GSH level of ovarian tissue was measured by using Total GlutathioneAssay Kit.(8) The formation of pinopods on the surface of endometrial epithelium wasobserved by using scanning electron microscopy; Statistical analysis of matingrate, fertilization rates and embryo adhesion rate were performed. III. Results 1. Oxidative damage of DNA induced by X-irradiation decreases the uterineendometrial receptivity which involves mitochondrial and lysosomal dysfunction(1) X irradiation reduced the RL95-2 cell viability; RL95-2 cells showedsenescence tendency, increased cell surface microvilli and low JAR celladhesion.(2) X irradiation damaged the mitochondrial and lysosomal function in RL95-2cells.(3) X irradiation induced the oxidative DNA damage in RL95-2 cells;(4) NAC, NH4 Cl and Pep A pretreatments protected the lysosomal andmitochondrial function, reduced the oxidative stress and DNA damage,promoted adhesion of JAR and RL95-2 cells from X irradiation. 2. Protective role of autophagy induced by X irradiation through ROS mediatedactivation of JNK signaling pathway in RL95-2 and HEC cells(1) X irradiation promoted apoptosis in RL95-2 cells.(2) X irradiation promoted autophagy in RL95-2 cells.(3) NAC pretreatment reduced the autophagy level.(4) NAC pretreatment inhibited the JNK signaling pathway.(5) Inhibition of autophagy by 3-MA increased the radiosensitivity of RL95-2 cellsand decreased the adhesion of JAR cells to RL95-2 cells 3. The protective effect of N-acetylcysteine(NAC) on ionizing radiation inducedpremature ovarian failure and loss of ovarian reserve in female mouse(1) X irradiation caused weight loss in mice ovaries.(2) X irradiation reduced the level of serum estrogen, progesterone andanti-Müllerian hormone, increased the serum level of follicle stimulatinghormone.(3) X irradiation significantly reduced the formation of ovarian primordial, andprimary follicles, the preantral follicles were not significantly affected; Thenumber of atresia follicles was increased by X irradiation.(4) X irradiation caused abnormal morphology of uterine endometrial epitheliumand affected the reproductive capacity of female mice.(5) X irradiation induced the oxidative stress and apoptosis in ovarian granulosacells and inhibited the cell proliferation.(6) NAC pretreatment significantly restored the ovarian follicle formation, hormonesecretion, reduced the oxidative stress and apoptosis, increased the cellproliferation, normalized the ovarian and uterine morphology, and to someextent, recovered the mice reproductive capability. IV. Conclusions 1. Oxidative damage of DNA induced by X-irradiation decreases the uterineendometrial receptivity which involves mitochondrial and lysosomal dysfunction(1) X irradiation caused the lysosomal and mitochondrial dysfunction inRL95-2 cells.(2) X irradiation caused the oxidative DNA damage.(3) Mitochondrial-lysosmal axis was involved in X irradiation induced oxidativestress.(4) Antioxidant NAC, lysosomal protective agents NH4 Cl and Pepstatin A canprotect the endometrial epithelial cells from X irradiation induced damage. 2. Protective role of autophagy induced by X irradiation through ROS mediatedactivation of JNK signaling pathway in RL95-2 and HEC cells(1) X irradiation induced autophagy by ROS generation.(2) X irradiation activated the MAPKs signaling pathway to induced autophagy.(3) ROS mediated the autophagy through activation of JNK signaling pathway.(4) Autophagy played as a cytoprotective role in X irradiation induced lowendometrial recepticity. 3. The protective effect of N-acetylcysteine(NAC) on ionizing radiation inducedpremature ovarian failure and loss of ovarian reserve in female mouse(1) Total body X irradiation can cause POF in mice.(2) NAC improved the follicle development and normalized the disordered hormonesecretion in mice induced by X irradiation.(3) NAC promoted uterine function and fertility capability through reducing theoxidative stress and apoptosis induced by X irradiation.(4) NAC may be capable of being used as a clinical ovarian radioprotector from Xirradiation。...