Studies Of Expression Of CYP19, GATA-4, FSHR Genes In Both PCOS Animal Models And PCOS Patients

BACKGROUND & OBJECTIVEPolycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age,occuring in 5%～10% of these women occupying 30%～60% of anovulatory infertile patients.Besides, it is also associated with an increased risk of diabetes mellitus, cardiovascular diseases and endometrial cancer in later life.The diagnosis of PCOS in young women may therefore have major implication for long term health.But the precise etiology,diagnose standards and appropriate individual treatment of PCOS are still unclear.Currently,most of investigators focus on the PCOS,especially its etiology.Because the hyperandrogenaemia is the main pathophysiological features of PCOS, the enzmatic gene linked with androgen synthesize and transformation become the target gene of investigation. P450 aromatase catalyses the final stage of estrogen biosynthesis pathway from androgens. P450 aromatase is the product of CYP19 gene expression. Therefore CYP19 gene become the target gene of investigation of PCOS. At present, the standpoin of expression of CYP19 gene in ovary tissue of PCOS has two opposite concepts. FSH is the main stimulus for the expression of the aromatase encoding gene CYP19. GATA4 mediates, at least in part, the stimulatory effect of FSH on CYP19 expression in granulosa cells. For this reason, transcription factor GATA-4 maybe play an important role in the pathogenesis of PCOS. The GATA family are members of transcription factors, the expression of tissue-specific gene are regulated partly by transcription factors. In recent years,more and more studys indicate as follows:the GATA family plays an essential role in the differentiation of germ cell lineage and modulation of hormones durying reproduction period. Especially the GATA-4 are the focal points of current study. It is not reported whether there is a relevance between the expression of GATA-4 and PCOS. The function of FSH is induced by specific receptor. At present,research indicates that most of PCOS patients have normal serum FSH level, but some datum showed an increase of follicle—stimulating hormone receptor levels.The aim of the present study was investigate the roles of CYP19,GATA-4 and FSHR genes in the mechanism of PCOS by detecting the expression of three candidate gene in PCOS patients PCOS animal models.PartⅠExpression of CYP19,GATA-4 and FSHR protein in human PCOS ovary and in the controlObjective:To investigate the feature of expression CYP19,GATA-4 and FSHR protein in human PCOS ovary and in the control, The present study laid the foundation for the further research of the relation of CYP19,GATA-4 and FSHR and PCOS.Methords:1. Samples16 samples ovary were obtained from PCOS women from Nanfang Hospital, The First Foshan Hospital and Fuzhou General Hospital.9 samples ovary were obtained from women treated with adnexectomy as the control group. The age of the two groups had no significant difference. The specimens were fixed by 10% neutral formaldehyde, embedded in paraffin and slided. Hematoxylin-and-eosin and immunohistochemical stainings were performed.2. MethordImmunohistochemistry were used to detect the expression of CYP19,GATA-4 and FSHR genes in human ovary.Specimens were cut into 2um and performed Hematoxylin-and-eosin stainings. Specimens were cut into 5um and mounted on poly-L-lysine-coated glass slides.After rountine deparaffinizanon,sections were placed in 0.3%H2O2-methyl alcohol for 30 min to block endogenous peroxidase activity.Slides for immunostaining were placed in citric acid buffer,heated in a microwave oven for antigen retrieval,and subsequently allowed to cool down for 2h at room temperature.Sections were treated for 30 min at room temperature with normal goat serum. Sections were incubated with primary antibodies for 18 h at 4℃.They were then incubated for 30 min at room temperature with biotinylatedanti-rabbit immunoglobulin(Ig)G, washed with 0.01 M phosphate-buffered saline(PBS) and incubated with peroxidase-conjugated streptavidin under te same conditions. The reaction product was subsequently detected by immersion in a DABsolution. Hematoxylin staining solution was employed for counterstaining the nuclei.Results:1. The number of preantral follicles in PCOS group increased significantly, especially primary follicle.2. Expression of CYP19 in ovary of PCOS group and control group Positive staining was observed intracytoplasm of granulosa cells. Positive staining in earlier stage follicle of PCOS group was significantly higher than that of control group (P=0.028). In advanced stage, expression of CYP19 in PCOS group was higher than control group,but there was no significant difference (P=0.663)3. Expression of GATA-4 in ovary of PCOS group and control groupPositive staining was observed intranuclear of granulosa cells. Positive staining in earlier stage follicle of PCOS group was significantly higher than that of control group (P=0.010). In advanced stage, expression of GATA-4 in PCOS group was higher than control group,but there was no significant difference (P=1.000)4. Expression of FSHR in ovary of PCOS group and control groupPositive staining was observed intracytoplasm of granulosa cells. In earlier stage follicle and advanced stage follicle, expression of FSHR in PCOS group was lower than control group,but there was no significant difference (P=0.533;P=0.878) Summary:1. Positive staining of CYP19 in earlier stage follicle granulosa cells of PCOS group was significantly higher than that of control group. In advanced stage, expression of CYP19 in PCOS group was higher than control group, but there was no significant difference. The results hint that the gene of CYP19 participate the development process of earlier period follicle. The extra higher expression of CYP19 gene in follicle granulosa cells may be significantly correlated with the pathophysiology mechanism of PCOS.2. Positive staining of GATA-4 in earlier stage follicle granulosa cells of PCOS group was significantly higher than that of control group. In advanced stage expression of GATA-4 in PCOS group was higher than control group, but there was no significant difference. The results hint that the gene of GATA-4 participate the development process of earlier period follicle. The extra higher expression of GATA-4 gene in follicle granulosa cells may be significantly correlated with the pathophysiology mechanism of PCOS.3. There was no significant difference expression of FSHR in earlier and advanced stage follicles between PCOS group and control group,which indicate that the over-expression of GATA-4 and CYP19 in earlier stage follicle granulosa cells was induced by otherwise factors no FSH.PartⅡExpression of CYP19,GATA-4 and FSHR gene in ovary tissue of rat model of letrozole-induced polycystic ovary syndromeObjective:The key feature of PCOS are hyperandrogenaemia and chronic anovulation. The high level of androge in the ovary is the main causes in inducing PCOS.P450 aromatase catalyses the final stage of estrogen biosynthesis pathway from androgens. The lower enzymatic activity of P450 aromatase in the ovary may result in hyperandrogenism.After this theory,femal SD rats were administered letrozole once daily during 21d to establish an ideal PCOS model.Subsequently we detected the level of expression CYP19,GATA-4 and FSHR protein in this model.Methords:1.6-week-old female Sprague-Dawley(SD) rats were divided into two groups randomly, PCOS groups administered letrozole at concentration of 1mg/kg p.o.dissolved in 1% CMC(2mL/kg) once daily during 21 d,and the control group was not treated.2. Gonadal hormone (LH,FSH,E2,P,T) concentrations in serum were determined by radioimmunoassay3. The histologic changes in ovaries were observed by H-E staining.4. Realtime RT-PCR were used to assess the relative mRNA of CYP19,GATA-4 and FSHR. Total RNA was isolated from ovary specimen using the Trizol kits.cDNA,synthesized using SuperScript TM reverse transcriptase and an oligo(dT) primer,was used as the template for real-time PCR using gene-specific primers. P-Actin was used as the internal control in the same cDNAsample. Realtime RT-PCR was performed according to the manufacturer protocol.All PCR products were analyzed on agarose gel stained with ethidium bromide. Made dissociation curve amplification curve. Relative quantification of gene expression was performed by the comparative Ct methord.The relative fold expression changes were calculated.5. The expression of CYP19,GATA-4 and FSHR protein in ovary tissue was detected by immunohistochemistry. Immunohistochemistry were used to detect the expression of CYP19,GATA-4 and FSHR genes in human ovary.Specimens were cut into 2um and performed Hematoxylin-and-eosin stainings. Specimens were cut into 5um and mounted on poly-L-lysine-coated glass slides.After rountine deparaffinizanon,sections were placed in 0.3%H2O2-methyl alcohol for 30 min to block endogenous peroxidase activity.Slides for immunostaining were placed in citric acid buffer,heated in a microwave oven for antigen retrieval,and subsequently allowed to cool down for 2h at room temperature.Sections were treated for 30 min at room temperature with normal goat serum. Sections were incubated with primary antibodies for 18 h at 4℃.They were then incubated for 30 min at room temperature with biotinylatedanti-rabbit immunoglobulin(Ig)G, washed with 0.01 M phosphate-buffered saline(PBS) and incubated with peroxida.se-conjugated streptavidin under te same conditions. The reaction product was subsequently detected by immersion in a DAB solution. Hematoxylin staining solution was employed for counterstaining the nuclei.6. Western blotting The expression of CYP19,GATA-4 and FSHR protein in ovary tissue was detected by Western blot.Results:1. The rat weight in experiment group was lower than that in control group (t=3.035,P=0.004). The ovarian weight in experoment group was a little higher than that in control group (t=0.144, P=0.887).Considering about the influence of body weight,there was no significant difference in ovarian weight between the two groups (t=1.171, P=0.249). The relative ovarian weight in experiment group was a little higher than that in control group.2. Ovary histological anatomy shows that ovaries from model groups showed high incidence of subcapsular ovarian cyst and capsular thickening together with incomplete luteinization and decreased number of corporalutea.Many of the small follicles in the early development stage and atretic follicles were observed in model groups. The abundance of subcaplular ovarian cyst lined with a thin layer of granulosa cells.3. When compared to control group, estradiol(t=3.132, P=0.003) and progesterone (t=7.796, P=0.000) in model group showed a considerable reduction, Serum testosterone (t=5.449, P=0.000) and FSH (t=5.246, P=0.000) and LH (t=4.922,P=0.000) levels of model group were markedly increased than control group (P<0.05)4. Gene expressive difference of CYP19,GATA-4 and FSHR between model group and control group1) The expression of CYP19 (t=7.964, P=0.000),GATA-4 (t=6.515, P=0.000) and FSHR (t=4.496, P=0.000) mRNA were significantly stronger in model groups than those of the control group.2) Correlation of Gene expression of between GATA-4 and FSHR and CYP19 Pearson correlation analizes showed that there was significant positive correlation between GATA-4 and FSHR(r=0.705, P=0.000)and between GATA-4 and CYP 19 (r=0.733, P=0.000)。5. Immunohistochemistry were used to detect the expression of CYP19,GATA-4 and FSHR proteins in ovary.1) Expression of CYP 19 in ovary of model group and control group Positive staining was observed intracytoplasm of granulosa cells. Positive staining in earlier stage follicle of model group was significantly higher than that of control group (P=0.011). In advanced stage, expression of CYP19 in model group was higher than control group,but there was no significant difference (P=0.594)2) Expression of GATA-4 in ovary of model group and control group Positive staining was observed intranuclear of granulosa cells. Positive staining in earlier stage follicle of model group was significantly higher than that of control group (P=0.004). In advanced stage, expression of GATA-4 in model group was higher than control group,but there was no significant difference (P=0.572)3) Expression of FSHR in ovary of model group and control group Positive staining was observed intracytoplasm of granulosa cells. Positive staining in earlier stage follicle of model group was significantly higher than that of control group (P=0.038). In advanced stage, expression of FSHR between model group and control group has no significant difference (P=0.747)6. Western blotting1) The expression of CYP19 (t=8.227, P=0.000),GATA-4 (t=9.258, P=0.000) and FSHR (t=9.172, P=0.000) protein were significantly stronger in model groups than those of the control group.2) Correlation of protein expression of between GATA-4 and FSHR and CYP19 Pearson correlation analizes showed that there was significant positive correlation between GATA-4 and FSHR(r=0.705, P=0.000)and between GATA-4 and CYP19 (r=0.733, P=0.000)。Summary:1. The mRNA transcription and protein expression of CYP 19 were significantly stronger in model group than those in the control group. The outcomes of protein expression of CYP 19 in animal model were in good agreement with those of PCOS patients. The results hint that the gene of CYP19 participate the development process of earlier period follicle. The extra higher expression of CYP19 gene in follicle granulosa cells may be significantly correlated with the pathophysiology mechanism of PCOS. 2. The mRNA transcription and protein expression of GATA-4 were significantly stronger in model group than those in the control group. The outcomes of protein expression of GATA-4 in animal model were in good agreement with those of PCOS patients. The results hint that the gene of GATA-4 participate the development process of earlier period follicle. The extra higher expression of GATA-4 gene in follicle granulosa cells may be significantly correlated with the pathophysiology mechanism of PCOS.3. The mRNA transcription and protein expression of FSHR were significantly stronger in model group than those in the control group. Positive staining in earlier stage follicle of model group was significantly higher than that of control group, the outcomes were not in good agreement with those of PCOS patients. The higher expression of FSHR were associated with the higher level of FSH in the control groups.4. Pearson correlation analizes showed that there was significant positive correlation between GATA-4 and FSHR and between GATA-4 and CYP19, which indicate that the higher expression of CYP19 may be correlated with the higher expression of GATA-4 in PCOS.5. The rat model induced by letrozole were proved to be an ideal PCOS animal model with similar morphological and hormonal changes and the outcomes of CYP19 and GATA-4 to those of PCOS patients. Cconclusions1. Expression of CYP19 protein and GATA-4 protein in earlier stage follicle of PCOS group were significantly higher than that of control group,but there were no significant difference between PCOS group and control group in advanced stage.It implies that CYP19 and GATA-4 plays an important role in the growth and development process of earlier stage follicle, they facilitate the amount of preantral follicles increased. Over-expression of CYP19 and GATA-4 were relevant to PCOS occurrence. There was no significant difference expression of FSHR between PCOS group and control group, which indicate that the over-expression of GATA-4 and CYP19 in earlier stage follicle granulosa cells was induced by otherwise factors no FSH.2. The outcomes of protein expression of CYP19 and GATA-4 in animal model were in good agreement with those of PCOS patients. The results hint that the gene of CYP19 and GATA-4 participate the development process of earlier period follicle. The extra higher expression of GATA-4 gene in follicle granulosa cells may be significantly correlated with the pathophysiology mechanism of PCOS..3. The expression of CYP19,GATA-4 and FSHR gene and protein in ovary were significantly stronger in model groups than those of the control group.The outcomes indicate that he higher expression of CYP19,GATA-4 and FSHR gene may be correlated with the pathophysiology of PCOS.4. Pearson correlation analizes showed that there was significant positive correlation between GATA-4 and CYP19, which indicate that the higher expression of CYP19 may be correlated with the higher expression of GATA-4 in PCOS.5. The rat model induced by letrozole were proved to be an ideal PCOS animal model with similar morphological and hormonal changes and the outcomes of CYP19 and GATA-4 to those of PCOS patients.