The Effects And Mechanism Of Puberty Onset On Pregestational Exposure Ethinyl Estradiol

Ethinylestradiol,a synthetic estrogen,is widely used in pharmaceutical formulations.In addition to the contraceptive function,EE is used for regulating normal physiological cycle,assisted conception,for the treatment of acne and so on.At present,many animal and population studies have found that maternal environment during periceonceptional especially on pregestational impact on the growth and development of offspring,so we uses SD rats animal model to explore the effects and mechanism of puberty onset on pregestational exposure EE.We observed the vaginal opening and penis stripping time(a marker of rat puberty)of offspring.Using metabonomics method to analy estrogen metabolism on maternal gestational.We found that estrogen metabolism? the m RNA/protein levels of kisspeptin?GPR54?NKB?Leptin R?Insulin R in hypothalamuswas changed.Eventually lead to puberty reproductive and development dysplasia.Sopregestational is also notable important period of endocrine disruptors,women taken drugs which contain EE on pregestational cause children adolescent reproductive development plans for the subsequent pregnancy health problems deserve attentionPart?Effects on puberty onset and developmental of the offspring of rats exposed to EE onpregestationalObjectUsing a female rats exposed to EE on pregestational,study the effects of puberty onset and reproductive development of offspring rats.Methods(1)Establish the rat model: female SD rats were divided into four groupsrandomly,each group of 10,EE lavage exposed to 15 days.Four groups follow: sesame oil in the control group,50?g/kg,200?g/kg,800?g/kg.After the 15 days exposure,they were mated to conceive.Offspring ratswere random exchange between groups,avoid nest as interference factors.(2)Observe the growth of rat in general: weight changes of different time points,each organ coefficient.(3)Observe the rat in puberty sexual development condition: female vaginal opening time,estrus cycles,the male penis stripping time,serum hormone levels.Results1.Impact on puberty onset and reproductive development of female offspring who exposure to EEon pregestational(1)Compared with control group,the vaginal opening time of 200?g/kg group's offspring were significantly delayed,800?g/kg group has a tendency toadvance vaginal opening time.And there was no significant difference on body weight in its puberty starting point,but on 60 days old there is a difference between age groups on weight,especially in high dose group 800?g/kg weight increased significantly.(2)Females in control group have normal estrus cycle,the length of the estrous cycle in treatment group rats significantly increased,dioestrusd increased,oestrum reduce.The different stages of the estrous cycle sequence is disorder,not regular estrus cycles.(3)Compared with control group,female offspring rats in treatment group serum estrogen levels had no significant change,but the serum leptin levels have significant differences,especially the 200?g/kg,800?g/kg dropped significantly.(4)Each organ coefficient,the kidney/body weight and ovary/body weight of the treatment group rats are difference.Treatment group in the rat kidney/body weight dropped significantly,especially in 50?g/kg,800?g/kg group obviously.Ovary/body weight significantly increases,liver/body weight,spleen/body weight,and the uterus/body weight had no significant changes.2.Impact on puberty onset and reproductive development of male offspring who exposure to EEon pregestational(1)Maternal exposure to EEon pregestationalsignificantly affected puberty onset time of the male offspring rat,50?g/kg group male offspring rat penis stripping time significantly delayed,800?g/kg group have significant differences.And there was no significant difference on body weight in its puberty starting point.But at 60 days old,weight is rising,especially in high dose group 800?g/kg body weight increased significantly.(2)Compared with control group,serum testosterone and leptin levels of male offspring rats in treatment group had no significant change.(3)In each organ coefficient of the male offspring,liver/body weight,kidney /body weight and testicles/body weight of treatment group rats were dropped significantly.There is no significant change in the spleen/body weight.Conclusions1.Exposure to EEon pregestational can significantly change the puberty onset of offspring,puberty onset age of the females offspring of 200?g/kg group had a significantly delayed,in the50?g/kgmale offspring puberty onset age is delayed,and 800?g/kg is in advance.It shows the effect between different dosage,different gender is not the same.The estrus cycle of female offspring rats after pubertyonset is disordered,estrus cycle length increased.2.Offspring rats' body weight at puberty onset is no difference,but with the growth,at P60 of age,the weight of the treatment group gain.It shows maternal exposure to EEon pregestational will have an impact on offspring pubertal development after born.3.The treatment group have the influence onliver,kidney and testis and ovary visceraof offspring.Maternal exposure to EEon pregestational will not only have an effect on offspring reproductive organs,but also the metabolic excretory organs like liver,kidney which we should also pay more attention on.Part ? Investigate the mechanism that Pregestational Ethinylestradiol exposedleads to the disorder of the offspring puberty onset.ObjectInvestigate the mechanism thatthe influence of offspring rats puberty onset and reproductive developmentalon maternal pregestationalexposure to EEfrom the metabolic pathways and endocrine pathways.Methods(1)Female EE exposure processing was the same with the first part.Observe and recorder the mother's weight.Glucose tolerance experiment was carried out after the exposure of the female.Observe whether exposed EE will affect mothers of sugar metabolism.Collectingurinewith metabolic cages at 1 day before and after the exposure,the 15 th day after the exposure,the 7th,14 th,21th day of pregnancy.Analyze the metabolism of estrogen in the body with the metabonomics method.Record the number of born rats in every group.(2)Glucose tolerance experiment was carried out at the prepubertal P21 and the postpubertal P50.The time point of determination were respectively 0,15 min,30 min,60 min,120 min.(3)Analyze thekey genes and protein of kiss1,GPR54,Lep R,Ins R,NKB of rat hypothalamus ARC area with RT-PCR/Western Bolt.Results1.Influence of EE exposed to the maternal glucose tolerance test(1)In experimental rats following exposure to EE weight has a downward trend,but no significant difference.(2)Glucose tolerance test results show that the EE had no significant effect on mother's sugar metabolism.(3)The number of female rats litter size of each group with the statistical analysishas no significant difference.2.Influence of offspring glucose toleranceon pregestational EE exposure(1)About female offspring,experimental prepuberty P21 glucose tolerance levels in 0,15 min,60 min,120 min have significant difference,and onlyin30 min have significant difference when P50.(2)About male offspring,The experimental group compared to the control group,prepubertal P21 tolerance levels are significant changes,particularly in the 0,15 min,30min,60 min,50?g / kg group increased significantly,recovery in 120 min consistent with the control group.While in the P50,experimental group compared with the control group has no significant difference.3.Effects of offspring hypothalamus ARC region key genes and protein expression of pregestational exposure to EE(1)PCR results at female offspring ARC zone showed that 200?g/kg group Ins R m RNA significantly drop,and 800?g/kg group NKB m RNA rise significantly.GPR54 m RNA expression did not differ significantly,but it has increase trend between their dose-response.(2)On female offspring ARC zone Western Bolt showed that kisspeptin,the expression of GPR54,Lep R,Ins R,NK3 R were significant differences.The experimental group compared with the control group that Kisspeptin,Ins R protein expression was significantly increased,the Lep R expression is decreased.In the 50?g / kg group was significantly increased GPR54 protein,and in 800?g / kg group was decreased,there was no significant difference in 200?g / kg group.In 50?g / kg and 800?g / kg group NK3 R protein expressing was significantly decreased,while in 200?g / kg group is rising.(3)The male offspring ARC region m RNA expression showed that compared with the control group,the experimental group kiss1,Ins R m RNA expression levels are significantly changed,Lep R,GPR54,NKB m RNA expression levels have little effect.In 200?g / kg group kiss1 m RNA was significantly upregulated,in 50?g / kg group Ins R m RNA was significantly reduced.(4)Western Bolt showed at male offspring ARC region that theprotein of kisspeptin,GPR54 Lep R,Ins R,NK3 R were significant differences of male offspring.The experimental group compared with the control group,the protein expression of Kisspeptin,Ins R and Lep R is significantly decreased.In the 50?g / kg and 200?g / kg group GPR54 protein was significantly increased,and in 800?g / kg group was decreased.NK3 R protein Expressingin 50?g / kg and 800?g / kg group was significantly decreased,while in 200?g / kg group is rising.4.Using metabonomics analysis the effect of abnormal puberty caused by the metabolism of estrogen on Pregestational EE exposureUrine analysis results show that in the first day following exposure to EE,16 alpha OHE1,2-Me OE2,4-OHE2,4-OHE1,4-Me OE2 rise,Pregnenolone decline.After 15 days washout period,16 alpha-OHE1,4-OHE1,2-OHE1 rise,4-Me OE2,Progesterone drops.Exposed effect is obvious.The rise of 16 alpha-OHE1 and 4-OHE1 after 15 days washout period hasn't changed,it showed the EE exposure has greater impact on hydroxy metabolite.16 alpha-OHE1 and 4-OHE1 rise at the end of pregnancy was the same with phenomenon at the begin.Tip may be lingering effects on hydroxyl metabolism.Pregnancy E1 group is falling.It was found that in the generation of pregnancy estrogen E1,4-OHE2,4-Me OE1,2-Me OE1,2-Me OE2,Progesterone and 17 alpha-OHprogesterone metabolism correlated with female offspring puberty onset;E1,4-OHE2,16 alpha-OHE1 metabolism correlated with male offspring puberty onset.Conclusions1.EE exposure did not significantly affect to maternal body weight,glucose metabolism and its litter size,its weight and glucose metabolism in women taking EE and reported in the literature that has not changed is the same.2.EE exposure on pregestationalcan affect offspring early youth glucose tolerance level,but as they grow up glucose tolerance was gradually returned to normal.3.EE exposure on pregestationalcan affect the expression levels of key genes and proteins under the progeny of hypothalamic ARC region.The main influence on the gene level was the expression of kiss1 Ins R NKB,Lep R m RNA,and a slight influence on the expression of GPR54 m RNA.Protein expression level in the experimental group compared to the control group protein levels were significantly different.The difference of the trend of m RNA and protein expression may be due to regulation and modification change of post-transcriptional modification or the translation process.4.That urine estrogen metabolomics analysis is EE exposure to changes on maternal estrogen metabolism is quite significant,especially for the continuation of the metabolic effects of having a hydroxyl group.In the experimental group E1 pregnancy are significantly decreased.We analyzed the mechanismof offspring puberty onset fromthe maternal internal environment estrogen metabolism levels,the expression levels of key genes and proteins under the progeny of hypothalamic ARC region of the offspring.