| Fenvalerate (FEN), a member of the type II synthetic pyrethroid insecticidefamily, is widely used in agricultural and for other domestic applications. As arepresentative environmental endocrinal disrupter, fenvalerate have reported possessestrogenic properties and antiandrogen activities. The current study suggestsfenvalerate has effects in endocrine disrupting and male reproductive functiondamage, but the underlie mechanism of its action is still unknown. In our study, wechoose fenvalerate, a representative of the environmental endocrine disruptors, usinganimal toxin exposure experiment model to analyze the male reproductive toxicityeffects with different exposure levels. Then we use proteomics methods to study themechanism of how does low dose fencalerate exposure affected male reproductivesystem. We choose8-10w male SD rats, pouring fencalerate in stomach, once a day,six days a week, and for eight weeks. Five groups are divided based on differentexposure doses:0,0.00625,0.125,2.5,30mg/kg/d. At the end of fencalerate exposure,we use computer-aided sperm analysis (CASA), acridine orangestaining, CTC staining and HE staining to study the effects of fencalerate on male ratreproductive system. And then we choose testis and sperm protein samples from thecontrol group and low dose exposure group for proteomics study based on themorphology observation. CASA is used to study the amount of sperm obtained fromepididymal tail, the sperm motility and the ratio of forward movement sperm. Theresults show there are no difference among different dose of fencalerate exposuregroups and the control group. Meanwhile acridine orange staining is used to evaluatethe degree of DNA damage; there are also no significant differences among groups.Sperm capacitation and spontaneous acrosome reaction are evaluated by CTCstaining, the capacitation ratios show no significant difference among groups whilethe ratios of spontaneous acrosome reaction show difference between0.00625mgfencalerate exposure group and the control group. Sperm deformity rates are evaluated using HE staining and significant difference can be observed between eachfencalerate exposure group and control group. We classifythe sperm deformity into head, neck and tail. The tail deformity ratesof each exposure group show significant difference compared with control group. Thesperm head and neck deformity rates in2.5mg and30mg groups show significantdifference with control group. There no difference in testis HE staining among allgroups. According to the morphology results of fencalerate exposure experiments,two-dimensional gel electrophoresis and mass spectrometry these two proteomicsmethods are chosen to study the changes of testis and sperm protein between control(0mg) and0.00625mg fencalerate exposure group. Our studies identify55differentprotein points in comparison sperm proteins between these two groups. Among these,38protein points have the differences more than1.5times, which corresponding to34proteins. And in these34proteins, many sperm structure related proteins are included.We choose some of these proteins using Western Blot to verify the difference showedin two-dimensional gel electrophoresis. Meanwhile our studies identify63differentprotein points in comparison testis proteins between these two groups. Among these,46protein points have the differences more than1.5times, which corresponding to43proteins. And in these43proteins, many sperm structure related proteins are included.Also we choose some of these proteins using Western Blot to verify the differenceshowed in two-dimensional gel electrophoresis and do localization researches usinghistochemistry. Based on these results, we further explore the mechanism of spermabnormal caused by low dose fencalerate exposure. According to the literatures, wefind these are several testes different proteins are related to the increase expression ofP53, and it has been proved that transgenic male mice with a moderateoverexpression of P53show a phenotype of teratozoospermia. Real-time PCR showsthat P53mRNA increases in fencalerate exposure group and Western Blot also showsincreased P53in protein level with a significant difference in gray value comparison.The Ccna1mRNA, which is a known gene in downstream of P53pathway, alsoshows increase in Real-time PCR analysis. Therefore, we hypothesizes that thepossible mechanism of low dose fencalerate exposure on rat sperm function is through the up-regulated P53pathway to increase the expression level of P53. Ourresults provide a certain foundation for the further study to the molecular mechanismof male infertility caused by fencalerate exposure. |
PDF Full Text Request