The Effect And Mechanism Of Chronic Overloaded Iron On The Ovarian Function In Female C57BL/6J Mice

Background:With the sharp decline of the birth population and the aggravation of the aging population in China,it has become an issue of increasing social concern to protect the female ovarian function and improve their fertility.The healthy ovary can not only maintain physiological endocrine and fertility function for women,but also protect their cardiovascular?skeletal and neurological systems and keep high quality of life due to the estrogen.As an essential trace element,iron is involved in a plethora of physiological activities such as energy metabolism,protein synthesis,DNA synthesis,oxidative respiration,immune function regulation and so on.It also participates in regulating the follicle development and ovulation in the ovary.So,it is crucial to maintain the iron homeostasis.However,chronic blood transfused diseases in clinic often lead to iron overload,such as Beta thalassemia major,which has a high incidence in southern China,namely Guangdong,Guangxi,Fujian,Hainan,Yunnan and Sichuan provinces.Repeated blood transfusion can deposit excess iron in the body,causing harm to the liver,heart and endocrine system.It also results in hypogonadotropic hypogonadism in women,which is characterized by reduced serum sex hormones such as FSH?LH?E₂and AMH,dwindled number of antral follicles,disordered menstruation or amenorrhea,and even infertility.Those females who can get pregnant spontaneously or by the help of the assisted reproductive techniques may suffer from higher rates of complications,such as miscarriage,intrauterine growth retardation,low birth weight and so on.The secretion of AMH is independent of gonadotropins,so the reduced AMH may indicate that overloaded iron may directly cause damage to the ovaries.At present,studies are lack in exhibiting the mechanisms of overloaded iron in the ovary.Thus,it is of great significance to establish an animal model to mimic female iron overloaded state and to investigate the underlying mechanisms,so as to protect the reproduction of iron overloaded females,avoid the pregnancy related complications and improve their fertility and life quality.Methods:No animal model of chronic iron overload in female mice has been reported.It has been reported that intraperitoneal injection of iron dextran at concentrations of 0.1g/kg,0.5g/kg and 1.0g/kg in male mice can lead to osteoporosis.For women,osteoporosis is inextricably associated with the decrease of estrogen.We hypothesized that osteoporosis in female mice may be caused not only by the deposition of excess iron in bone,but also by the interference of excess iron on estrogen synthesis.Low,middle and high concentrations of iron overloaded model was established by intraperitoneal injection of 0.1g/kg,0.5g/kg and 1.0g/kg of iron dextran(LF,MF,HF group)once a week for consecutive eight weeks.(1)The body weight and food intake of mice were observed to evaluate the effect of iron overload on the health status of mice.Serum iron and ferritin levels and iron deposition levels in liver,hypothalamus,pituitary gland,ovary,uterus and peri-ovarian adipose tissue were detected to validate the iron overloaded model.(2)The effect of iron overload on ovarian endocrine function in mice was evaluated by observing estrus cycle and detecting sex hormone levels.(3)H&E staining and Prussian Blue staining were performed on the ovarian tissue to observe the position and level of iron deposition and the morphological changes of the ovary.The expression of PCNA protein was detected by immunohistochemistry(IHC)and the proteins related to primordial follicle activation(p-YAP/YAP,p-PTEN/PTEN,p-mTOR/mTOR,p-rpS6/rpS6)were detected by western blot assay(WB)in order to evaluate the effect of iron overload on follicle development and cell proliferative capacity in the ovary.(4)Mating experiment was conducted to evaluate the effects of iron overload on ovarian reproductive function.At the end of the mating experiment,iron deposition and ovarian function in old-aged mice were examined to evaluate the long-term effect of iron overload on mice.(5)The effect of iron overload on offspring was detected by observing the iron level,the number of follicles and the fertility of offspring.(6)Oxidative stress was detected in the iron overloaded ovaries.Transcriptome sequencing,PCR and WB test were conducted to preliminarily explore the mechanisms of chronic iron overload on the ovarian function.Results:(1)With the increase of iron concentration,the serum iron and ferritin levels of mice increased.The iron was obviously deposited in the liver,hypothalamus,pituitary,ovary,uterus and peri-ovarian adipose tissue,with changed gross morphology and organ index.(2)There were no significant differences in estrus cycle,sex hormone level,follicle numbers and fertility between LF group and the control group.There was no significant increase in oxidative stress level in ovaries of the LF group,and the transcriptome expression level was similar to that of the control group.(3)Mice in the MF and HF groups showed disturbance of estrous cycle,decreased levels of E₂ and AMH.The number of follicles at each stage decreased,while the apoptotic follicles increased,with reduced cell proliferative capacity in the ovaries.The phosphorylation levels of proteins related to primordial follicle activation were decreased.(4)The 10-month mating experiment result showed that all mice in the MF group could get pregnant,while the incidence of adverse pregnancy outcomes was increased.Forty percent of the mice in the HF group were infertility.The rest sixty percent of the mice could conceive,during which twenty percent of mice died of adverse pregnancy outcomes and forty percent showed lower average number of offspring and average litter number than those in the control group.At the end of the mating experiment,iron deposition in the old mice of MF group was relieved.The ferritin level in pregnant mice of HF group was higher,while the iron level was significantly declined.The serum iron and ferritin levels were still high in the non-pregnant mice of HF group(HF-I group)and more iron deposits in the ovarian tissue,accompanied by fewer residual follicles and no obvious corpus luteum formation.(5)The number of primordial follicles and the cell proliferative capacity in the ovary were decreased in the female offspring of the HF group,with a tendency of lower litter size.(6)The ovaries in the MF and HF groups exhibited accumulative oxidative stress,accompanied by the down-regulation of genes and proteins in ovarian steroidogenesis,ovarian extracellular matrix formation and WNT signaling.Conclusion:(1)Iron overloaded animal model was established and validated successfully in this study.(2)Low concentration of iron(0.1g/kg)wasn't detrimental to the ovary.(3)Middle concentration of iron(0.5g/kg)and high concentration ofiron(1.0g/kg)generated accumulative oxidative stress in the ovaries,accompanied by disrupted ovarian steroidogenesis and inhibited follicle development.(4)Middle concentration of iron impeded the childbearing process,though it could be recovered following the iron excretion.High concentration of iron damaged the process of ovulation and corpus luteum formation,even gave rise to sterility.The infertility of mice in the HF-I group could be contributed to the high level of serum iron and abundant iron deposition in the ovaries.(5)Maternal high concentration of iron overload led to the diminished ovarian reserve in the female offspring.(6)Transcriptome profiling results and further PCR and WB validation indicated that overloaded iron could compromise ovarian function by disrupting ovarian steroidogenesis,interfering with ovarian microenvironment and inhibiting WNT signaling.