| Objective: Polycystic ovary syndrome is the most common endocrine disorder affecting women of reproductive age. It is characterized by chronic anovulation and hyperandrogenism. The underlying etiology and the pathophysiology have yet to be determined. By differential cDNA microarray hybridization in our laboratory, we identify 290 gene differentially expressed between normal and PCOS ovaries, and among this group the most interesting gene is NR4A1, which is down-regulated in PCOS ovary. Numerial studies indicate NR4A1 are expressed in many tissues and involved in various biologic functions, such as cell apoptosis and proliferation, steroid hormone production, insulin resistance and so on. Therefore, we put forward the hypothesis that the downexpression of NR4A1 in PCOS ovary may result in a series of pathophysiological changes in PCOS. To explore the effect of NR4A1 on the pathophysiology of PCOS, this study is divided into two parts. In the first part, we examined the localization of NR4A1 protein in mouse ovary. By construct mouse NR4A1 recombinant adenovirus Ad-CMV-NR4A1 and Ad-H1-SiRNA/NR4A1 to enhance or knockdown the expression of NR4A1 in mouse theca cells. Fundamentally investigate the effect of NR4A1 on testosterone production in theca cells. In the second part, we investigate the effect of androgen on the expression of NR4A1 and the proliferation and apoptosis of immature mouse ovarian granulosa cells. By recombinant adenovirus Ad-CMV-NR4A1 and Ad-H1-SiRNA/NR4A1 to enhance or knockdown the expression of NR4A1 in ovarian granulosa cells, respectively. To explore the effect of androgen induced abnormal expression of NR4A1 on the proliferation and apoptosis of mouse ovarian granulosa cells. This study provide basis for us to further explore the mechanism of NR4A1 on the pathophysiology development of PCOS.MethodsPart One: The Expression of NR4A1 in Mouse Ovary and its Effect on Theca Cell Androgen Production1. Examine the localization of NR4A1 protein in mouse ovary by immunochemical. 2. Constructe recombinant adenovirus Ad-CMV-NR4A1 and Ad-H1-SiRNA/NR4A1 to enhance or knockdown the expression of NR4A1 in theca cells, respectively.3. The expression patterns of StAR, CYP11A1, CYP17A1 and HSD3B2 were subse- quently analyzed by real-time RT-PCR. Moreover, concentrations of testosterone in the spent medium were measured by radioimmunoassay.4. Mouse follicles were treated with Forskolin (an activator of cAMP/PKA pathway) for 1, 2, 4, 6 hours separately, the expression of NR4A1 and androgen producing enzymes was detected by Realtime PCR.Part Two: Effects of Testosterone on the Expression of NR4A1 and the Proliferation and Apoptosis of Ovarian Granulosa Cells1. A histological analysis was performed after mouse preantral follicles were treated with different concentrations of testosterone. To observe androgen induced granulosa cell proliferation and apoptosis by MTT, cell cycle determine, AnnexinV/PI and Caspase-3 activity detection.2. Examine the expression of NR4A1 in normal and PCOS rat model ovarian by real-time RT-PCR, immunochemical and Western blot.3. Mouse preantral follicles were treated with different concentrations of testosterone, the expression of NR4A1 was detected by real-time RT-PCR and Western blot.4. Mouse ovarian granulosa cells were treated with testosterone and LY294002 separately, the expression of P-Akt and NR4A1 were detected by Western blot.5. After mouse ovarian granulosa cells infected with recombinant adenovirus Ad-CMV-NR4A1 or Ad-H1-SiRNA/NR4A1 for 48 h, the effects of NR4A1 over-expression and down-expression on granulosa cell proliferation and apoptosis were subsequently analyzed.ResultsPart One: The Expression of NR4A1 in Mouse Ovary and its Effect on Theca Cell Androgen Production1. NR4A1 protein was expressed in all stages of follicles and stroma, it mainly expressed in the nucleus of thecal cells and granulosa cells.2. The expression of NR4A1 protein in the mouse theca cells of follicles infected with Ad-CMV-NR4A1 was significantly increased compared with the control groups. Overexpression of NR4A1 in theca cells stimulates the expression of StAR, CYP11A1, CYP17A1 and HSD3B2, leading to increased testosterone production. Conversely,the expression of NR4A1 in the theca cells of follicles infected with recombinant adenovirus Ad-H1-SiRNA/NR4A1 was significantly decreased compared with the control groups. Knockdown of the endogenous NR4A1 exhibits a significant decrease in StAR, CYP11A1, CYP17A1 and HSD3B2 expression and testosterone production.3. FSK rapidly increases the NR4A1 mRNA levels followed by an increase in StAR, CYP11A1, CYP17A1 and HSD3B2.Part Two: Effects of Testosterone on the Expression of NR4A1 and the Proliferation and Apoptosis of Ovarian Granulosa Cells1. After ovarian granulosa cells treated with 10-5 mol/L testosterone for 10 min to 30 min, the level of P-Akt was increased obviously(p<0.05). However, when pre-treated with LY294002, the expression of androgen induced P-Akt was significantly inhibited and the expression of NR4A1 notably raised.2. Cultured mouse preantral follicle treated with 10-4mol/L or 10-5mol/L testosterone result in increased follicle size. When treated with 10-5mol/L testosterone, the ovarian granulosa cell proliferation added and apoptosis decreased.3. The results of real-time RT-PCR and Western blot indicate the expression of NR4A1 in PCOS rat model ovarian was significantly decreased compared with the normal rats(p<0.05).4. After mouse follicles treated with 10-5 mol/L and 10-4 mol/L testosterone for 24 h and 48 h, the expression of NR4A1 was significantly inhibited(p<0.05).5. Overexpression of NR4A1 in ovarian granulosa cells stimulates cell apoptosis and decreased cell proliferation(p<0.05). Conversely, knockdown of the endogenous NR4A1 exhibits a significant decrease in granulosa cell apoptosis and promote cell proliferation(p<0.05). Conclusion1. NR4A1 protein was expressed in the nucleus of thecal cells and granulosa cells, which implied NR4A1 may regulate biological process of these cells.2. NR4A1 might be involved in theca cell steroidogenic enzymes transcription and androgen synthesis through cAMP/PKA signaling pathway.3. Over-dose testosterone can promote the growth of immature follicles, and its effects of promoting proliferation and inhibiting apoptosis in granulosa cells may influence the follicle development. In vitro and in vivo experiments demonstrated that over-dose androgen decreased the expression of NR4A1 in ovarian granulosa cells, which may involve in follicular development.4. Over-dose testosterone down-regulated the expression of NR4A1 through PI3-K/Akt signaling pathway in granulosa cells. Hyperandrogen induced down-regulation of NR4A1 might be involved in the process of proliferation and apoptosis in immature follicular granulosa cells, and thus, influence the development of immature follicles.5. This study partly illustrated the effect of NR4A1 on the pathophysiology of PCOS.
|
PDF Full Text Request