The Nasal Exposure Of Aluminium Chloride Leads To Infertility In Female Mice By Inducing The Apoptosis Of Uterus And Ovary Cells

As air pollution is worsening,the exposure of aluminum ion is diversified,the adverse effects on health caused by aluminum ion exposed nasally are worth studying particularly.Previous studies have mainly focused on neurotoxicity,liver and kidney damage,bone damage,and immune system damage.In recent years,the reproductive toxicity of aluminum ions has attracted great attention worldwide.Most studies have focused on the male reproductive systerm,with only a few investigating the female reproductive systerm,so it is necessary for us to assess the reproductive toxicity of aluminum chloride（AlCl₃）exposed nasally in females.The model of C57BL/6 mice exposing to AlCl₃ nasally during pregnancy was established.The 7 weeks old female mice were randomly divided into 4 groups[ddH₂O,AlCl₃（0.1mg/kg,1mg/kg,10mg/kg）]after mating with male mice（1:1）,and the nasal drip was not stopped until17 days after pregnancy.Then the pregnant mice were sacrificed and their organs were sampled,the total aluminum content in serum and organs was analyzed by using ICP-MS（inductively coupled plasma mass spectrometer）;the uterus of the female mice was dissected,and the number of fetal mice and the loss rate of fetal mice in each dose group were described;the expression levels of apoptotic proteins（Fas,Fas-L and Bax）were detected by Western Blot;the accumulation of aluminum in mice after their nasal exposure to aluminium chloride during pregnancy was not found,the embryo loss rate is significantly lower compared with the control and the expression of apoptotic proteins in uterus was abnormal.The model of non-pregnant C57BL/6 mice exposing to AlCl₃ nasally was also established,and then these female mice were grouped into 4groups[ddH₂O,AlCl₃（0.1mg/kg,1mg/kg,10mg/kg）]randomly,the nasal drip was not stopped until 17 days later,some of the female mice were mated with male mice（1:1）to assess their fertility and fecundity,the total aluminum content in serum and organs was analyzed by using ICP-MS（inductively coupled plasma mass spectrometer）.The ovaries of these female mice were dissected,the pathological changes of these ovaries was observed by HE staining;microstructural changes of these ovaries were observed by TEM;ovarian antioxidant enzyme activity was analysed by Elisa;the expression levels of apoptosis-related proteins（Fas,Bax,p53）was detected by Western Blot;the total aluminum content in serum was analyzed by using ICP-MS,finding that the total aluminum content in serum was higher in AlCl₃（10mg/kg）group than the control group significantly,but the total aluminum content in the Hypothalamus,liver,spleen,kidney and uterus of mice showed no significant difference between 4 groups.There was no significant difference in pregnancy rate between female mice exposed to AlCl₃ and the controls;Electron microscopy results showed that some mitochondria in the ovarian granules of mice exposed to AlCl₃（1mg/kg and10mg/kg）swells,presenting cavitated ridge and degenerating matrix;the Elisa results showed that the activity of MDA（malondialdehyde）enzyme in the ovary of mice exposed to AlCl₃（1mg/kg、10mg/kg）increased signigicantly,the activity of SOD（superoxide dismutase）in the ovary showed no significant difference,but the AOR（SOD/MDA）was lower significantly compared with the controls;the Western Blot results showed that the apoptosis of ovarian cells showed significant difference between AlCl₃（1mg/kg、10mg/kg）groups and the control group.Hence,we believe that the nasal exposure of AlCl₃ can change the ultrastructure of the ovary,causing changes in the expression level of apoptosis protein in the ovary,and leading to reproductive toxicity in mice.Part Ⅰ Effects of nasal exposure of aluminum chloride during pregnancy on apoptosis of mouse uterus cellsObjectAluminium chloride was intranasally instilled into a C57BL/6 mouse model during pregnancy to observe the growth,development,and reproduction of pregnant mice,and to analyze the apoptosis of uterine cells in pregnant mice.Methods40 female C57BL/6 mice aged of 7 weeks were housed separately after mating with male C57BL/6 mice aged of 7 weeks,and pregnant mice were randomly divided into 4 groups[ddH₂O,AlCl₃（0.1 mg/kg,1 mg/kg,10 mg/kg）].Throughout the pregnancy,every day at 3 pm nasal instillation was performed until 17 days after pregnancy.The mice were sacrificed and their organs were sampled on the 17th day of pregnancy,the total aluminum content in the serum and organs of these mice was measured by ICP-MS analysis.Their uteruses were dissected on the 17th day of gestation,and the number of pregnant mice was counted to calculate the pregnancy rate,and the embryo loss rate was calculated through the loss of the fetuses.Western Blot was used to analyze the expression of uterine apoptosis-related proteins（Fas,Fas-L,Bax）.ResultsThe results of ICP-MS analysis showed that aluminum did not accumulate in the serum and organs of the pregnant mice.By dissecting the fetus in the mother’s uterus,a 10 mg/kg dose of AlCl₃ was found to lower the pregnancy rate in female mice compared with the control group,but the difference between the groups is not statistically significant.The 1 mg/kg AlCl₃ dose group caused an increase in embryo loss rate.Western Blot results showed that nasal exposure of aluminum chloride to pregnant mice affected the expression of apoptotic proteins in the uterus of pregnant mice,resulting in impaired cell apotosis balance system.ConclusionAfter nasal exposure of AlCl₃ during pregnancy,there was no accumulation in serum and organs,and the growth,development,and reproductive parameters of pregnant mice were affected.Nasal exposure of 10 mg/kg AlCl₃ resulted in decreased fertility in female mice and nasal exposure to 1 mg/kg AlCl₃ can cause obvious embryo loss.The expression of uterine apoptotic protein was significantly different.Part Ⅱ Effects of nasal exposure of aluminum chloride before pregnancy on Ovarian Apoptosis lead to infertility in female MiceObjectTo observe and analyze the effect of nasal exposure of aluminum chloride on the fecundity of mice and the ultrastructural changes of the ovary;to analyze the causes of embryo loss after aluminum chloride exposure during pregnancy;to explore the effects of aluminum chloride nasal exposure on the reproductivity of female mice and their ovarian parameters.MethodsAfter 17 days of nasal exposure to female mice:1:1 caged with the male mice and check the mating condition at 8:00 am the next day,take out the occulted fertilized egg and calculate the detection rate;The ovaries of the mice were placed in paraformaldehyde,and HE staining was used to observe the ovarian pathological changes.The ovaries of the mice was placed in 2.5% glutaraldehyde,and the ovarian structure was observed by TEM.Ovarian protein was extracted and ELISA was used to analyse the activity of ovarian energy metabolic enzyme and oxidative kinase.Ovarian protein was extracted and the expression of apoptosis-related protein was detected by Western Blot.ResultsBy counting the vaginal plugs and whether there is a fertilized egg in the ampulla of the ovaries of female mice,the mating rate and the conception rate of female mice are calculated,finding that there is no significant difference in the mating rate and the conception rate between groups.ICP-MS analysis of total aluminum in serum and organs of female mice showed that aluminum did not accumulate in the organs of mice,and the content of aluminum in the serum of the 10 mg/kg AlCl3 dose group was significantly higher than the control group.The pathological changes and ultrastructural changes of the ovary were observed by HE staining and electron microscopy.It was found that the number of growing follicles in the ovary of exposed mice was significantly lower than that of the controls.The mitochondrion in the ovary of the mice in the 1 mg/kg AlCl3 and 10 mg/kg AlCl3 dose groups swells,showing ambiguous structure.Detection of ovarian antioxidant enzymes MDA,SOD,AOR（SOD/MDA）expression levels showed that ovarian MDA levels in 1mg/kg and 10mg/kg AlCl3 dose groups were lower than that in the control group,ovarian AOR in exposed groups were significantly lower than that In control group,there was no significant difference in SOD among the exposed groups.Western Blot results showed that nasal exposure of aluminum chloride caused apoptosis of mouse ovarian cells through the Bax mitochondria-mediated apoptosis pathway.It was found that nasal exposure of AlCl3 caused increased Bax expression,leading to the increased expression of p53 uptream of Bax,and there was a dose-response relationship.ConclusionAfter nasal exposure to AlCl3,although there was no aluminum accumulation in serum and organs,ovarian apoptosis was affected and the apoptosis was induced through the mitochondria-mediated apoptosis pathway,resulting in impaired mitochondrial structure in the ovary and decreased number of growing follicles.