PPARγ Agonist Regulates P450arom Gene Expression In Granulosa Cells Of PCOS Patients

Polycystic ovarian syndrome (PCOS) is a common female reproductiveendocrine abnormalities and metabolic syndrome. The incidence over the worldapproximately is5%-10%and increased gradually. Hyperandrogenism is one of themost important endocrine features of PCOS, could lead to a series of clinicalsymptoms such as ovulation disorders, infertility, obesity, diabetes, seriously affectwomen’s health and mental health. Cytochrome P450arom is one of the mostimportant limiting enzymes catalysis of androgens to estrogens generated in granulosacells, closely associated with hyperandrogenism.If the function or regulation ofP450arom is aberrant, that would lead to changes in androgen levels and ovulationdisorders. Peroxisome proliferator-activated receptor γ is one kind of nuclear receptors,many studies had shown that PPARγ agonist could inhibits the P450arom mRNAexpression and reduce aromatase activity in human ovarian granulosa cells, but themechanism of it has not been elucidated.Transforming growth factor-β/Smad signaling pathway participate in a variety ofbiological processes in the human body, such as oocyte maturation, proliferation ofgranulosa cells, the secretion of estrogen and so on,it played an important role in theabove process. Smad2protein is the most important signal transduction moleculesmediates the signal of this pathway, can regulate multiple cellular processes after be phosphorylated. We usually use the level of p-Smad2protein to represent the degreeof activation of the signal pathway TGF-β/Smads. Many studies have found thatTGF-β/Smads signaling pathways could regulate CYP19gene expression and therebypromoting the expression of P450arom.In other tissues in human body, PPARγ canaffecting the expression of the target gene by affecting Smad2phosphorylation. Ourprevious studies have found: the PPARγ mRNA expression in granulosa cells ofPCOS patients was higher than the control group, and the P450arom mRNA andp-Smad2protein expression were lower, and PPARγ mRNA expression wasnegatively correlated with p-Smad2protein expression, p-Smad2expressionpositively correlated with the expression of P450arom. So in the ovary, PPARγwhether could affect the expression of P450arom by TGF-β/Smads signalling pathway,then resulting in the hyperandrogenism in PCOS patients still need further study.Objective1. Measure the expressions of P450arom mRNA and PPARγ mRNA, the levelsof Smad2and p-Smad2protein in ovarian granulosa cells which by cultured in vitroand add different concentrations of PPARγ agonist (Rosiglitazone) treatmentintervention of NA-PCOS groups, HA-PCOS groups and the control groups.2. To investigate the regulation of PPARγ on P450arom expression in ovariangranulosa cells and whether TGFβ/Smads signaling pathway is involved in.Material and MethodsWe choose30patients who accept IVF-ET treatments in the First AffiliatedHospital of Zhengzhou University’s Reproductive Medical Center from November2013to February2014, and divided into three groups, the first group was normalandrogen PCOS group (NA-PCOS group)with a total of10cases, the second groupwas PCOS with hyperandrogenism (HA-PCOS group) with a total of10cases, thethird group served as the control group with10cases. On day2~4of menstruation andthe day of HCG injection we collected Blood samples. We measured the concentrations of serum sex hormone by full-automatic electrochemistryluminescence immunity analyzer. All patients using the standard long protocol tocontrolled ovarian hyperstimulation. During oocyte pick-up, all granulosa cells werecollected and cultured in vitro. Granulosa cells of each patient were divided into threegroups after18h, add different concentrations of Rosiglitazone(0、1、10nmol/L), thenbe divided into two groups after48h, one group was used to measure mRNAexpression of PPARγ and P450arom by RT-PCR, the other group was used to measureSmad2and p-Smad2protein levels by Western blotting.Results1. Compared three groups, there was no significant difference in infertilityduration, patients’s age, basal serum E2, FSH level, Gn using time and dosage, serum E2level per follicle on the day of HCG injection(P＞0.05). Basal serum LH, T level inHA-PCOS group and NA-PCOS group both were higher than those in the normal group(P＜0.05). The serum LH andT levels of HA-PCOS group was significantly higher thanthe NA-PCOS group (P＜0.05).2. When added to a concentration of0nmol/L rosiglitazone, the PPARγ mRNAexpression in NA-PCOS group and HA-PCOS group were higher than those in normalgroup (P＜0.05), P450arom mRNA and p-Smad2protein level were lower than thosein normal group(P＜0.05). HA-PCOS group compared with the NA-PCOS group, hadhigher PPARγ mRNA expression levels and lower P450arom mRNA expressionlevels and p-Smad2protein levels.3. When added to the rosiglitazone with concentration of1nmol/L and10nmol/L, the expression level of PPARγ mRNA expression in there groups allincreased with concentration of rosiglitazone, P450arom mRNA expression level andp-Smad2protein all decreased with the expression of PPARγ mRNA increase.HA-PCOS group compared with the other two groups, has the highest PPARγ mRNA,the lowest P450arom mRNA expression levels and p-Smad2protein levels. Smad2protein levels were not significantly different among the three groups. Conclusions1. PPARγ may be hindered Smad2proteins phosphorylation to inhibit theexpression of P450arom.2. TGF-β/Smads signaling pathways may be involved in the regulation ofPPARγ on P450arom in ovarian granulosa cells.