Effect Of Gestational Exposure To Arsenic And/or Fluoride On Puberty In Offspring Female Mice

The timing and pattern of puberty initiation\onset are influenced by individual's health status,nutrition and growth,as well as environmental factors.Most of the studies have shown that puberty initiation is a complex biological process controlled by multiple regulatory systems,executed by"hypothalamus-pituitary-gonad" axis(HPGA).In this study,animals in eight groups were treated with Arsenic[0 mg/L As(III),0.15 mg/L As(III),1.5 mg/L As,15 mg/L(?)],Fluoride,(100 mg/L NaF)and Fluoride and Arsenic[0.15 mg/L As(III)+ 100 mg/L NaF,1.5 mg/L As(III)+ 100 mg/L NaF and 15 mg/L As(?)+ 100 mg/L NaF].A series of experimental tests like vaginal smears,paraffin sections HE staining,Western blotting,real-time qRT-PCR,immunohistochemistry,and ELISA were carried out.The results are as follows:1.After the prenatal exposure to arsenic in the mother,the feeding,drinking water,mental status,pregnancy and number of offspring of the mice were normal during the whole experimental period,and there was no statistically significant differences compared to the control group.The visceral coefficient of ovarian tissue in the arsenic and arsenic+fluoride combined exposed groups was increased significantly during puberty but decreased significantly during the mature stage;however,the uterine organ coefficient in fluoride exposure group decreased significantly in puberty.The liver organ coefficient in high arsenic group and arsenic+fluoride combined exposure group was significantly lower than in prepuberty and puberty,however the arsenic+fluoride combined exposure increased the spleen coefficient of puberty female offspring significantly.Ovarian HE staining in arsenic exposed group showed that the size of the nucleus was uneven with pyknosis,the nucleus was ruptured with decreased cytoplasm,and the gap was widened.The results showed that various concentrations of fluoride,arsenic and their combination caused various degrees of damage to the organs;and the ovary was the most damaged organ compared to other organs.2.The results of the vulva opening test showed that the vulva opening time in each arsenic exposed group significantly advanced compared to the control group,indicating that all arsenic concentrations exposed prenatally to mothers significantly prolonged the puberty onset time of female offspring mice.It is also suggested that the estrogen-like effect of arsenic;the maldevelopment in the appearance of the vulva of some female mice after fluoride and fluoride arsenic exposure may be caused by insufficient availability of Leptin or Lentin.3.The expression levels of kiss-1,GnRH1,Oct2 and Ttf1 in female offspring at different pubertal stages were evaluated by using Real-time PCR for gene expression,Western blot and ELISA techniques for protein expression analysis.The results showed:(1)The mRNA expression levels of Kiss-1 was significantly increased at prepubertal and puberty stage treated in medium and high dose of As(?)at puberty compared to the control.The protein expression levels of Kisspeptin was significantly increased in high dose of As(?)at puberty compared to the control,which was in line with the transcriptional changes of related mRNA expressions.(2)The expression levels of GnRH1 gene was significantly decreased in lactation and was significantly increased compared to their control groups,and the protein expression of GnRH was significantly higher during puberty in arsenic group compared to the control group.There was no significant difference in the expression of gene and protein among the groups treated with As and control group.(3)The gene and protein expression of Oct2 were significantly increased in high As exposed group than control group at puberty,however,significantly decreased at mature.(4)The gene and protein expression of Ttfl was significantly increased at prepubertal and puberty,but significantly increased at adulthood compared to their respective controls.The trend of protein expression was consistent with that of gene expression.All the results showed that the gene and protein expressions of kiss-1,GnRH1,Oct2 and Ttf1 were significantly increased at the puberty,which showed arsenic can significantly advance the onset of puberty in female offspring.However the gene expressions of kiss-1,GnRH1,oct2 and Ttf1 were significantly decreased in female offspring whose mothers were exposed to fluorid and combination compared to control group.The results showed that fluoride and arsenic have antagonistic effect in onset of puberty.4.Real-time PCR,ELISA,and immunohistochemistry were used to evaluate Leptin,Leptin-R,POMC,and NPY genes and hormones at different stages of development.After gestational exposure to fluoride,arsenic,and their combination,the results showed that(1)The mRNA expression levels of leptin was significantly increased in prepuberty and puberty;the corresponding immunohistochemical results of prepuberty and puberty were consistent with the trend of gene expression results,i.e.,the leptin protein expression levels in the As high group was significantly higher than that of the control group;(2)The mRNA expression levels of Leptin-R was significantly increased in the prepuberty and puberty periods in the high-As exposure group;the corresponding immunohistochemical results in the prepuberty and puberty were basically consistent with the trend of gene expression results;(3)The mRNA expression levels of POMC was significantly increased in the prepuberty in the high-As exposed group,however,significantly decreased in puberty;(4)The mRNA expression levels of NPY in exposed groups was not statistically significant compared with the control group.(5)In arsenic+fluoride group,the mRNA expression levels of Leptin,Leptin-R,POMC,and NPY were decreased significantly during puberty;(6)The results of puberty related hormones showed that high concentrations of arsenic significantly increased LH level compared with the control group,however,FSH level was not statistically significant compared with the control group;3?-HSD expression levels were significantly higher in arsenic than the control group in prepuberty and puberty.In conclusion,it can be suggested that in the HPG axis,elevation of leptin levels may be the earliest signal of puberty onset,and it may promote activation of the HPG axis,thereby increasing the secretion of sex steroids and subsequent GH/IGF-I axis activation.To sum up,gestational arsenic exposure can lead to the significant advancement in the vaginal opening time in utero in mice exposed to As compared to the control,the expression levels of kiss pathway genes and Leptin related genes were increased significantly in female offspring at the puberty,and the secretion of stimulating hormone level was increased.However,the expression of kiss pathway genes and Leptin related genes were significantly decreased in arsenic+fluoride group.The results showed that Kiss pathway and Leptin related genes play an important role in the initiation of puberty in female offspring.And moreover,certain amount of arsenic has a catalytic effect,and fluorine has an inhibitory effect.