Effect And Mechanism Of Androgen Excess Interference With Hypothalamus-Pituitary-Ovary Axis And Ovulation Function In Female Rats

Polycystic ovary syndrome(PCOS)is a common disorder of reproductive and metabolic system in women of reproductive age,the clinical features are anovulation/amenorrhea,hyperandrogenism,polycystic ovaries and insulin resistance,which present as individual difference.In study of PCOS pathophysiology,the mechanism of hypothalamus-pituitary-ovary(HPO)axis dysfunction,which induces dysfunction of follicle stimulating hormone/ Luteinizing hormone(FSH/LH)secretion,have not illustrated.Recently,the aetiology study of PCOS have been focused on the regulation of HPO axis in ovulation,and a lot of publications reported that the dysfunction of GnRH(Gonadotropin Releasing Hormone)/LH secretion,which causes androgen excess from ovaries,and interference with FSH secretion,induces development disorders of follicles.The GnRH neurons distribute in hypothalamus of mammals,are regulated by KNDy(Kisspeptin//Neurokinin/Dynorphin)neurons upstream.As some studies showed,normal pulse frequency of GnRH was the basis of regular reproductive cycle,and in mechanism of PCOS development,the disorders of hormone secretion from ovaries(such as androgen excess)feedback to hypothalamus,induced the dysfunction of GnRH secretion and HPO axis regulation in reproductive cycle.Moreover,androgen excess could induce metabolic syndrome such as insulin resistance,obesity,which aggravated endocrine disorders in PCOS patients.Due to the difficult mechanism of neuroendocrine in PCOS,the aetiology of PCOS in GnRH secretion has not been clarified,currently,the treatments of PCOS have included therapy aimed at individual symptoms(such as reducing the level of androgen,ovulation induction)rather than pathogenesis.Therefore,there would be an important effect of researching the relationship between androgen and GnRH secretion as well as HPO axis regulation in PCOS aetiology,treatment and diagnosis.In our study,the PCOS rat model with DHT(5?-dihydrotestosterone)implanted subcutaneously were used to explore the effect of androgen on neuroendocrine system in rats' hypothalamus and anovulation;Meanwhile,the mice hypothalamus neuroma cell line,GT 1-7,was used to research the effect of androgen on neuron signal pathway in vitro,which aimed to clarify the neuroendocrine mechanism of PCOS development.Moreover,our data of in vitro experiments was according with the results of in vivo study,which provides an evidence for aetiology and treatments of PCOS.Part ? Neuroendocrine Disorders of Rats with Ovulation dysfunction Induced by Androgen Excess Objective To observe the neuroendocrine disorders of ovulation dysfunction,the anovulation rat model were estimated,and the in vivo experiments were used to test the androgen effect of secretion in neuron cell.Methods The female rats of 3 weeks aged were implanted subcutaneously the silicone tube containing 7.5 mg DHT or nothing as control.Then the body weight,ovarian morphology,serum steroid hormones and estrus cycle were observed and recorded in pubescent aged(4/6 weeks)and adult aged(12 weeks)female rats,and the expression level of endocrine factors in hypothalamus were tested in both groups.Moreover,to determine the relationship of GnRH secretion of DHT treatment,we observed the effect of DHT and ER stress(Endoplasmic reticulum stress)inducer-TG(Thapsigargin)on GnRH secretion in mice hypothalamus neuroma cell line-GT1-7 cell.Results Comparing to controls,the body weight of DHT rats was significantly higher(p<0.01);And the number of cystic and atretic follicles in DHT rats' ovaries was markedly more than that of controls.Moreover,there was no regular estrus cycle and LH frequency observed in DHT rats of 8-week-aged.Our data showed that the DHT rats of 4-8 weeks aged presented the significant higher expression level of ER stress markers and lower expression level of Kisspeptin in hypothalamus than that of controls(p<0.05),which meant that DHT up-regulated the expression of ER stress signal,and inhibited the Kisspeptin expression.Moreover,in GT1-7 cell line,both of DHT and TG could significantly block the GnRH secretion,which Kisspeptin could up-regulate(p<0.05).All the results suggested that the secretion of GnRH in neuron cell could be inhibited by DHT and ER stress,meanwhile,Kisspeptin could block the effect of DHT and TG.Part ? Mechanism of Androgen Excess Inducing Neuro-endocrine disorders in Female Rats and Dysfunction of\GnRH secretion in GT 1-7 cell Objective To determine the mechanism of secretion dysfunction in neuron cell line,and testify the effect of neuroendocrine function of rat model in vitro experiments.Methods After 24 hours/30 minutes treatment of DHT,DHT+Kisspeptin/TG,TG+Kisspeptin in GT 1-7 cell line respectively,the total RNA/ protein and medium were collected to determine the expression level of ER stress markers and GnRH concentration.Moreover,using transfection by siRNA,the Protein disulfide isomerase(PDI)expression was inhibited,which could activated the ER stress,we observed the Kisspeptin expression and GnRH secretion in GT 1-7 cell line;through a fluorescence microscope,the change of intracellular calcium current was measured.To testify the results of in vitro experiments,the ER stress inhibitor-Salubrinal and Kisspeptin were injected to the lateral ventricles of rats using intracerebroventricular(i.c.v)injection,the change of expression of ER stress and LH secretion was observed.Results The RNA/Protein expression level of ER stress markers and GnRH secretion was dramatically up-regulated after DHT treatment(p<0.05);and down-regulated after DHT+ Kp treatment(p<0.05 or 0.01).The similar results was observed in TG/TG +Kp group.In intracellular calcium measurement,the calcium current was markedly increased after TG treatment,when pre-treat by Kp,the effect of TG was significantly inhibited(p<0.05).Moreover,to testify the results of in vitro experiments,we found that the expression level of ER stress marker in rats' hypothalamus was dramatically decreased after Kp or Salubrinal-I.C.V treatment(p<0.05),and the over-secretio of LH was blocked by Salubrinal in DHT rats.Conclusion 1.According to other studies from domestic and foreign countries,we established aPCOS rat model using silicone tube containing DHT implanted subcutaneously.The rat model exhibited reproductive and metabolic symptom similar to PCOS,including polycystic ovaries,elevated body weight,anovulation,hyperandrogenism,and insulin resistance.This model can be used inneuroendocrinology investigation in PCOS.2.We explored the neuroendocrine mechanism of anovulation in DHT rat modelthrough testifying the phenotype of DHT rats.Our data showed that androgenexcess could induce the dysfunction of hypothalamus in DHT rats,significanthigher expression level of ER stress markers and decreased expression level ofKp/GPR54,which could be the important neuroendocrine factors of anovulation.These results provided an evidence for study of PCOS aetiology.3.In present study,in vitro experiments showed that DHT significantlyup-regulated the expression level of ER stress markers and inhibited the secretionof GnRH in hypothalamus neuroma cell line;Kisspeptin could markedly rescuethe effect of DHT or ER stress inducer,and GnRH secretion.4.In vitro experiments showed that the ER pathway protein-PDI could mediatedthe DHT effect,which induced increased expression level of ER stress;and theintracellular second messager,calcium could play an important role on ER effectof Kisspeptin.It is necessary to clarify the potential machanism of the effect ofandrogen and Kisspeptin on hypothalamus neuron cell,which could associatedwith aetiology in PCOS development.5.In our study,the influence of hypothalamus on neuroendocrine function in ratswas observed.Our data showed that I.C.V of Kisspeptin or other chemicals couldinhibit the expression level of ER stress markers,and affect GnRH/KH secretionin hypothalamus/pituitary in rats.These findings validated that changing of localenvironment in hypothalamus could influence the neuroendocrine function,meanwhile,the in vivo results were according with the in vitro data.6.Through establishment of PCOS rat model,we found that androgen exposure atthe pre-pubertal stage could induce the dysfunction of HPO axis,as well asanovulation.In our study,the mechanism of the typical symptom anovulation inPCOS was explored,the effect of ER stress and Kisspeptin in PCOS aetiologywas emphasized for the first time in these field.These founding provided a basisof pathophysiological mechanisms in PCOS,which could be helpful forprevention,diagnosis and therapy in PCOS development.