Expression Of Reg IV In Ovary Of PCOS Rat And Its Mechanism Of Effect On Granulosa Cells Of Rat Ovary

Polycystic ovary syndrome (PCOS) is the most commonendocrinopathy in women of reproductive years and is characterized byreproductive dysfunction and metabolic disorders. It increases a woman’srisk of infertility, endometrial pathology, diabetes, hypertension, andcardiovascular disease. Various diagnostic criteria have been proposed,generally centered around the features of hyperandrogenism and/orhyperandrogenemia, oligo-ovulation or anovulation and polycystic ovarianmorphology. PCOS affects6-10%of women and accounts for about30%of patients with amenorrhea, and with a prevalence of up to90%ofinfertility patients with infrequent menstruation and ovulatory dysfunction.At present, the mechanism of PCOS still remains unknown and likelyincludes a combination of genetic factors, insulin resistance, andenvironmental factors. Studies have reported that abnormalities of folliculargrowth and development may be the pathological basis of the anovulationand clinical endocrine changes in PCOS, and the follicular dysplasia maybe associated with the abnormal regulation of apoptosis in ovarian granulosa cells. In addition, the proliferation and apoptosis of ovariangranulosa cells are closely related to the development and atresia of follicle,and many regulatory factors involve in the proliferation and apoptosis ofgranulosa cells.Regenerating gene family belongs to calcium dependent lectin (C-typelectin) gene superfamily. It represents a group of small and multi-functionsecretory proteins, which can function as acute phase reactants,antiapoptotic factors or growth factors for pancreatic β-cells, neural cellsand epithelial cells in the digestive system.It was firstly found in pancreaticjuice of rats with acute pancreatitis by French Keim in1984. According tothe primary structure of Reg gene encoding protein, Reg family can bedivided into four subtypes: I, II, III and IV. Reg family members mainlyinvolve in tissue injury, inflammation and diabetes and the occurrence oftumor. Reg IV gene, as the new member of the Reg family, for the firsttime was isolated and screened in the study of inflammatory bowel diseaseby Hanupee etc. in2001. This gene was located on chromosome1(1q12-q21) and composed of17496base pairs, and contained six exons and fiveintrons. The relative molecular weight of its protein is about17kDa. Anincreasing number of studies showed that Reg IV involved in cellproliferation, apoptosis, regeneration, metaplasia and inflammatoryreaction, and is closely related to the occurrence and development of tumor.However, its biological functions and pathophysiologic roles are still largely unknown. The aim was to examine whether Reg IV expressed in ratovary and participated in the development of follicle and the apoptosis andproliferation of granulosa cells. Therefore, this study first observe theexpression of Reg IV in the ovaries of normal and PCOS model rats byimmunohistochemical staining, quantitative RT-PCR and Westernblotting analysis, to investigate whether Reg IV is associated with thepathogenesis of PCOS. Moreover, the pathways relaying Reg IV triggeredsignals, their targets, potential cross-talk with other cascades and theinteractions with other factors are still largely unknown. So the aim of ourstudy is to investigate the effects of Reg IV on the proliferation andapoptosis of granulosa cells by culturing ovarian granulosa cells of rats invitro. By using exogenous Reg IV protein or small molecular RNAinterference technology (Reg IV-siRNA), we further discuss the effects ofReg IV on EGFR/Akt and IGF-I/Akt signaling pathway and the expressionof apoptosis related genes such as Bax and Bcl-2.Evodia rutaecarpa is the dry ripe fruit of rutaceae plants Evodiarutaecarpa. Its pharmacological effects contain analgesia, sedation,anti-bacterial, anti-hypertensive, anti-hypoxic and so on. Evodiamine (evo),as one of its main alkaloids, has many pharmacological effects, such asweight loss, analgesia, anti-inflammatory, lowering blood sugar, improvinginsulin resistance, regulating body temperature, inhibition of tumor growth,and effects on cardiovascular and endocrine systems. The etiology of polycystic ovary syndrome is still not clear, its pathogenesis is related tomany aspects such as the endocrine, nerve and metabolism factors. The rateof obesity in PCOS patients is more than50%, and the incidence of insulinresistance is50%-70%. For a long time, the treatments of PCOS have a lotof researches and improvements, but the clinical effects are still not ideal.Now the researches about evodiamine in gynecology reproductiveendocrinology are very few, so we will study the effects of evodiamine onPCOS and the expression of Reg IV in ovaries of PCOS rats and itsmechanism. In addition, we also study the influence of Evo on the functionof Reg IV by culturing granulosa cells of normal rats in vitro. These willprovide a new strategy for the development of new drugs used to treatPCOS and the guidance of clinical treatment, and lay the foundation forfurther studying the mechanism of PCOS. This study is divided into fourparts as follows:Part One The Expression Patterns of Reg IV Gene in Normal RatReproduction SystemObjective To reveal the expression patterns of Reg IV in normal ratovary and uterus. Methods The expression of Reg IV was analyzed byreverse transcription-polymerase chain reaction (RT-PCR) and Westernblotting at mRNA and protein levels, respectively. The localization of RegIV protein within rat ovary and uterus was investigated byimmunohistochemistry (IHC). Results The expression of Reg IV in ovary was significantly higher than that in the uterus. The strong immunoreactivesignals of Reg IV was observed in granulosa cells and oocytes of ovarianfollicles, corpus luteum, and interstitial cells in rat ovary; only weak signalswere detected in luminal and gland epithelium of rat endometrium.Conclusion These findings first demonstrate the expression of Reg IV inovary and uterus of the healthy rat at both mRNA and protein levels. Itprovides an evidence of Reg IV expression in rat reproductive system,which may help elucidate a potential role in cell growth and proliferation ofreproductive system. Part Two Expression of Reg IV in Ovarian Tissues of PCOS RatsObjective To explore the expression of Reg IV in ovaries ofPoretsky’s PCOS model Rats. Methods Female SD rats aged85d weredivided into PCOS model group and normal control group．Rat models ofPCOS have been established according to Poretsky’s method. Rat bodyweight, ovarian weight and sex hormone levels in rat blood of both groupswere determined. The morphologic changes of ovaries were observed byHE staining. The expression of Reg IV, EGFR and IGF-I in the ovaries ofrats was detected by immunohistochemical staining. The levels of Reg IV,EGFR and IGF-I mRNA and protein in ovary tissue were measured byquantitative RT-PCR and Western blotting technique. ResultsImmunostaining of Reg IV in the granulosa cells, theca cells and corpus luteum of PCOS rat’s ovaries were obviously weaker than those of thecontrol. Immunostaining of EGFR in the granulosa cells, theca cells,stroma and corpus luteum of PCOS rat’s ovaries were stronger than thoseof the control. Immunostaining of IGF-I in the granulosa cells, theca cellsand corpus luteum of PCOS rat’s ovaries were weaker than those of thecontrol. The levels of Reg IV and IGF-I mRNA and protein in ovary ofPCOS rat were obviously lower than those in the control (P<0.05,respectively), but the levels of EGFR mRNA and protein in ovary of PCOSrat were obviously higher than those in the control (P<0.05,respectively).Conclusion The decrease of Reg IV expression may beinvolved in the pathogenesis of PCOS, and may be related to EGFR andIGF-I. Part Three Effects of Reg IV on Granulosa Cells of Rats and itsMechanismObjective To investigate the effects of Reg IV on the proliferation andapoptosis of ovarian granulosa cells in vitro and its mechanism. MethodsPrimary granulosa cells isolated and collected from SD rat ovarian werecultured with DMEM/F12medium with15%serum. The effects of Reg IVon the proliferation and apoptosis of ovarian granulosa cells were detectedby MTT assay. The expression of Reg IV in granulosa cells was inhibited by Reg IV-siRNA. The effects of Reg IV on expressions of IGF-I、EGFR、pEGFR、pAkt、Bcl-2and Bax proteins were detected by Western blotting.Results Reg IV proteins with various concentrations could promote theproliferation of granulosa cells in rats, and the function was dependent onthe concentration and time. When the granulosa cells were cultured withReg IV protein, the levels of IGF-I、EGFR、pEGFR、pAkt and Bcl-2proteins were obviously increased. After the expression of Reg IV proteinwas blocked by100nM Reg IV-siRNA2, the levels of IGF-I、EGFR、pEGFR、pAkt and Bcl-2proteins were distinctly reduced, and the level ofBax was obviously increased. Conclusion Reg IV protein may promote theproliferation granulosa cells by increasing the expressions of IGF-I andEGFR and activating Akt, and result in the increased expression of Bcl-2.In contrast, the blocking of Reg IV may promote the apoptosis of granulosacells by decreasing the levels of IGF-I and EGFR and resulting in theinactivation of Akt, and leading to the decrease of Bcl-2and the increase ofBax. In a word, Reg IV protein may become a potent activator of theEGFR/Akt and IGF-I/Akt. Part Four Effects of Evodiamine on PCOS Rats and Reg IV Functionin Ovaries and Granulosa CellsObjective To investigate the effects of Evodiamine (Evo) on PCOSrats and the function of Reg IV in ovaries and granulosa cells. Methods The kind of animal model established by Poretsky was used to observebody weight, estrous cycle, serum insulin, sex hormone and examinationalresults of histopathology. The expressions of Reg IV, EGFR and IGF-I inovary tissues were measured by quantitative RT-PCR and Western blottingat mRNA and protein levels, respectively. The localization of Reg IV,EGFR and IGF-I proteins in the ovaries of rats were detected byimmunohistochemical staining. Effects of Evodiamine on Reg IV Functionwere detected by MTT assay. Results Evo could lower the level of serumandrogen, luteinizing hormone and insulin in rats, lighten the body weight,and maintain the estrous cycle. The examinational results of pathologicalhistology showed that Evo could increase the stratum of granulosa cells inovarian follicle and the quantity of corpus luteum. The levels of Reg IVand IGF-I mRNAs and proteins in ovaries of PCOS rats were lower thanthose in the control. The levels of EGFR mRNA and protein in ovaries ofPCOS rats were higher than those in the control. After treating by drugs,the levels of Reg IV and IGF-I gradually increased in4mg/kg Evo,metformin and40mg/kg Evo groups; the levels of EGFR graduallydecreased in4mg/kg Evo,40mg/kg Evo and metformin groups. Whilegranulosa cells were cultured with Reg IV protein and Evo, the function ofReg IV promoting the proliferation of granulosa cells in rats was inhibited.Conclusion Evo had the effect of lowering the level of serum androgen,luteinizing hormone and insulin, improving the syndromes of PCO and insulin resistance, and recovering ovulation. The effects of Evo onexpressions of Reg IV, EGFR and IGF-I in ovaries of rats, and theinhibition of Evo on Reg IV function promoting the proliferation ofgranulosa cells, suggest that Reg IV may become a target of Evo function.