The Effects Of Low-dose Exposure To Mixture Of Pyrethroids At Different Stages On Ovarian Function In Female Mice

Pyrethroid insecticides have been widely used in agricultural pest killings and public health due to their high efficiency and broad-spectrum.There are nearly 80commercial pyrethroid insecticides in the market.The residues of pyrethroids in various environmental media can enter the human body through dietary intake and other routes,which poses potential health risks to human.Our previous epidemiological study has found that the concentrations of pyrethroid metabolites in women were positively associated with the risk of primary ovarian insufficiency in women,indicating that pyrethroids may be a potential risk factor for female ovarian health.Most studies on ovarian toxicity focused on high-dose exposure of a single pyrethroid,while it was difficult to reveal the realistic effects of low-dose exposure of multiple pyrethroids in the environment on ovarian function.In this study,female mice at gestational,lactational or pubertal stages were exposed to eight widely used pyrethroids at the acceptable daily intake（ADI）doses recommended by the World Health Organization（WHO）.This study aimed to evaluate the effects of low-dose exposure of pyrethroids on ovarian function.Furthermore,this study aimed to explore the molecular mechanism of pyrethroid exposure during the sensitive exposure window by regulating miRNA expression to interfere with follicular development.The main results and conclusions of this study were listed as follows:（1）The low-dose pyrethroid exposures at different developmental stages could affect the ovarian follicular development of female mice.The gestational exposure to pyrethroids resulted in a significant decrease by 50.9%in the amount of ovarian primary follicles in female mice on postnatal day 3（PND 3）and a significant increase by 78.7%and 64.7%in the number of primordial follicles and total follicles,respectively,in female mice on PND 5.These results indicated that pyrethroid exposure accelerated the early development of ovarian follicles.The number of total follicles in the ovaries of 16-week-old mice decreased and the number of atretic follicles significantly increased by 40.2%,indicating a trend of reduction in follicular reserve.Exposure to pyrethroids also altered the levels of reproductive hormones in female mice.The gestational exposure to pyrethroids resulted in a decrease by 33.2%in levels of estradiol（E₂）and an increase by 20.3%in levels of follicle-stimulating hormone（FSH）in adult female mice,which are similar to the clinical symptoms of primary ovarian insufficiency in women.The gestational exposure to pyrethroids induced the apoptosis of granulosa cells in the ovaries of 16-week-old mice.The lactational exposure to pyrethroids led to an increase in the number of total follicles in female mice on PND22 and the up-regulation of FSH levels in animals.The pubertal exposure to pyrethroids had no effects on follicular development,but increased AMH levels in adult female mice.（2）The results of high-throughput sequencing showed that 23 microRNAs（miRNAs）were differentially expressed in the ovaries of newborn female mice（PND1）gestationally exposed to pyrethroids.Twelve differentially expressed miRNAs with a relative expression level higher than 100 were selected to predict a total of 491 target genes.These target genes were mainly involved in the neurotrophin signaling pathways,c AMP pathways and necroptosis,as well as the positive regulation of transcription by RNA polymerase II and other biological processes.Among these 12 miRNAs,6miRNAs related to ovarian function were verified by q PCR.The expression of mi R-152-3p,mi R-450b-3p and mi R-196a-5p was significantly up-regulated.The results of bioinformation suggest that these three miRNAs may be the upstream regulators of key genes involving in the PI3K-Akt signaling pathways,which regulates the development of ovarian follicles.In conclusion,our results suggest that the exposure of multiple pyrethroids at a dose considered to be"safe"（ADI）during the sensitive window（the gestational stage）would have irreversible adverse effects on the ovaries in female mice thorough altering the expression of miRNAs during early developmental stages,which may cause changes in the important pathways,especially PI3K-Akt signalings,and biological processes involving in follicular development,leading to primary ovary insufficiency after adulthood.