孤儿核受体NR4A1在多囊卵巢综合征病理生理机制中作用的研究
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摘要
研究背景和目的:多囊卵巢综合征(polycystic ovary syndrome,PCOS)是生育期妇女常见的内分泌紊乱性疾病,以持续性无排卵和高雄激素的临床表现为主要特征,其病因和病理机制至今尚未完全阐明。在前期研究工作中,我们通过基因芯片杂交技术筛选出正常人和PCOS患者卵巢组织中290个差异表达的基因,NR4A1是该差异表达谱内代表性基因之一,在PCOS患者卵巢中表达下调(正常/PCOS=0.4)。既往研究表明,NR4A1在许多重要组织中均有表达,其功能涉及甾体激素代谢、细胞凋亡/增殖、胰岛素抵抗等诸多方面。因此,我们提出NR4A1可能与PCOS一系列病理生理改变相关的科研假说。为了探讨NR4A1在PCOS发生、发展过程中的作用,本研究分为两大部分:第一部分首先在蛋白水平证实了NR4A1在小鼠卵巢中的表达与定位,通过构建小鼠NR4A1重组腺病毒载体Ad-CMV-NR4A1及Ad-H1-SiRNA/NR4A1,分别实现NR4A1在小鼠卵泡膜细胞内的超表达和表达沉默,从而观察NR4A1在卵泡膜细胞内对雄激素合成的影响;第二部分探讨高雄激素作用下NR4A1的表达变化及小鼠未成熟卵泡颗粒细胞增殖、凋亡的情况,分别观察NR4A1超表达和表达沉默后,对颗粒细胞增殖、凋亡的影响。通过该研究,阐明NR4A1在PCOS病理生理机制中的作用,为多囊卵巢综合征研究提供新的思路。
研究方法
第一部分:NR4A1在小鼠卵巢中的表达及其对卵泡膜细胞雄激素生成的影响
1.通过免疫组化方法研究NR4A1蛋白在小鼠卵巢中的表达和定位。
2.构建小鼠NR4A1超表达重组腺病毒载体AdCMV-NR4A1及表达NR4A1基因的小分子干扰RNA(small interfering RNA , siRNA)重组腺病毒载体AdH1-NR4A1,重组病毒感染体外培养的小鼠卵泡24 h后,Western印迹验证NR4A1在卵泡膜细胞中的超表达及干涉效果。
3. Real-time RT-PCR检测NR4A1超表达及干涉后对雄激素生成酶(StAR、CYP11A1、CYP17A1、HSD3B2)的转录调节,放射免疫法测定培养液中睾酮的浓度。
4.用福斯高林(Forskolin,cAMP/PKA通路激动剂)分别处理小鼠卵泡1小时、2小时、4小时、6小时后,Realtime PCR检测NR4A1及雄激素生成相关酶的表达水平。
第二部分:高雄激素作用下NR4A1的表达及其对颗粒细胞增殖、凋亡的影响
1.用10~(-4)mol/L、10~(-5)mol/L、10~(-6)mol/L睾酮分别处理小鼠窦前卵泡后,观察卵泡的形态变化;通过MTT、流式细胞仪测定细胞周期、AnnexinV/PI及Caspase-3活性检测等方法分别观察雄激素作用后颗粒细胞的增殖与凋亡情况。
2.通过Real-time RT-PCR、免疫组化、Western blot检测NR4A1在正常大鼠及PCOS大鼠模型卵巢组织中的表达。
3.用不同浓度的睾酮分别处理体外培养的小鼠窦前卵泡,Real-time RT-PCR、Western blot检测NR4A1的表达变化。
4.用睾酮及LY294002处理未成熟小鼠卵巢颗粒细胞后,Western blot检测Akt的蛋白磷酸化水平及NR4A1的表达。
5.用Ad-CMV-NR4A1及Ad-H1-SiRNA/NR4A1重组腺病毒载体感染小鼠卵巢颗粒细胞48h后,检测NR4A1超表达及表达沉默后对颗粒细胞增殖、凋亡的影响。
研究结果
第一部分:NR4A1在小鼠卵巢中的表达及其对卵泡膜细胞雄激素生成的影响
1. NR4A1蛋白在小鼠卵巢各级卵泡和间质内均有表达,其主要定位于卵泡膜细胞和颗粒细胞的细胞核。
2.重组病毒Ad-CMV-NR4A1感染卵泡膜细胞后,与对照组相比,NR4A1超表达组NR4A1蛋白的表达水平显著升高,两组间差异显著(p<0.05)。NR4A1在卵泡膜细胞内超表达后明显促进了卵泡膜细胞雄激素生成相关酶(StAR、CYP11A1、CYP17A1、HSD3B2)mRNA的转录,增加睾酮合成。相反,重组病毒Ad-H1-siRNA/NR4A1感染卵泡膜细胞后,与对照组相比,NR4A1干涉组NR4A1蛋白的表达水平显著降低(p<0.05)。NR4A1在卵泡膜细胞内干涉后明显地抑制了StAR、CYP11A1、CYP17A1、HSD3B2 mRNA的表达,降低睾酮合成。
3.在卵泡培养液中加入福斯高林后,NR4A1的表达迅速增加,随后StAR、CYP11A1、CYP17A1、HSD3B2的表达增加。
第二部分:高雄激素作用下NR4A1的表达及其对颗粒细胞增殖、凋亡的影响
1.在小鼠窦前卵泡体外培养的过程中,10~(-4)mol/L和10~(-5)mol/L睾酮能够显著促进卵泡体积的增加(p<0.05);在10~(-5)mol/L睾酮的作用下,小鼠卵巢颗粒细胞的增殖增加,凋亡减少(p<0.05)。
2. Real-time RT-PCR及Western blot结果显示NR4A1在PCOS大鼠模型卵巢中的表达明显低于其在正常大鼠卵巢中的表达(p<0.05)。
3.用10~(-5) mol/L和10~(-4) mol/L睾酮处理卵泡24h及48h后均明显抑制NR4A1的表达(p<0.05)。
4.用10~(-5)mol/L睾酮处理颗粒细胞10min至30min,Akt蛋白磷酸化水平显著升高(p<0.05); LY294002预处理后,明显抑制睾酮作用下的Akt的磷酸化(p<0.05),增加NR41的表达(p<0.05)。
5. NR4A1在颗粒细胞内超表达后促进颗粒细胞的凋亡,抑制其增殖(p<0.05);相反,NR4A1在颗粒细胞内表达沉默后抑制颗粒细胞的凋亡,促进其增殖(p<0.05)。
结论
1. NR4A1在小鼠卵巢的卵泡膜细胞和颗粒细胞内表达,提示NR4A1参与卵泡膜细胞和颗粒细胞内某种(些)生物学过程的调控。
2.在cAMP/PKA信号通路作用下,NR4A1在卵泡膜细胞内促进雄激素生成酶的转录,进而促进雄激素生成。
3.高雄激素促进颗粒细胞增殖、抑制颗粒细胞凋亡,从而促进早期卵泡发育。体内体外实验证明,高雄激素降低卵巢颗粒细胞中NR4A1的表达,可能与此相关。
4.高雄激素作用下,通过PI3-K/Akt通路下调颗粒细胞内NR4A1的表达,NR4A1表达下调参与了颗粒细胞的增殖、凋亡调控过程,从而影响未成熟卵泡发育。
5.本研究部分阐明了NR4A1在PCOS病理生理机制中的作用。
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.
Methods
Part One: The Expression of NR4A1 in Mouse Ovary and its Effect on Theca Cell Androgen Production
1. 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 Cells
1. 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.
Results
Part One: The Expression of NR4A1 in Mouse Ovary and its Effect on Theca Cell Androgen Production
1. 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 Cells
1. 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~(-4)mol/L or 10~(-5)mol/L testosterone result in increased follicle size. When treated with 10~(-5)mol/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).
Conclusion
1. 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.
研究方法
第一部分:NR4A1在小鼠卵巢中的表达及其对卵泡膜细胞雄激素生成的影响
1.通过免疫组化方法研究NR4A1蛋白在小鼠卵巢中的表达和定位。
2.构建小鼠NR4A1超表达重组腺病毒载体AdCMV-NR4A1及表达NR4A1基因的小分子干扰RNA(small interfering RNA , siRNA)重组腺病毒载体AdH1-NR4A1,重组病毒感染体外培养的小鼠卵泡24 h后,Western印迹验证NR4A1在卵泡膜细胞中的超表达及干涉效果。
3. Real-time RT-PCR检测NR4A1超表达及干涉后对雄激素生成酶(StAR、CYP11A1、CYP17A1、HSD3B2)的转录调节,放射免疫法测定培养液中睾酮的浓度。
4.用福斯高林(Forskolin,cAMP/PKA通路激动剂)分别处理小鼠卵泡1小时、2小时、4小时、6小时后,Realtime PCR检测NR4A1及雄激素生成相关酶的表达水平。
第二部分:高雄激素作用下NR4A1的表达及其对颗粒细胞增殖、凋亡的影响
1.用10~(-4)mol/L、10~(-5)mol/L、10~(-6)mol/L睾酮分别处理小鼠窦前卵泡后,观察卵泡的形态变化;通过MTT、流式细胞仪测定细胞周期、AnnexinV/PI及Caspase-3活性检测等方法分别观察雄激素作用后颗粒细胞的增殖与凋亡情况。
2.通过Real-time RT-PCR、免疫组化、Western blot检测NR4A1在正常大鼠及PCOS大鼠模型卵巢组织中的表达。
3.用不同浓度的睾酮分别处理体外培养的小鼠窦前卵泡,Real-time RT-PCR、Western blot检测NR4A1的表达变化。
4.用睾酮及LY294002处理未成熟小鼠卵巢颗粒细胞后,Western blot检测Akt的蛋白磷酸化水平及NR4A1的表达。
5.用Ad-CMV-NR4A1及Ad-H1-SiRNA/NR4A1重组腺病毒载体感染小鼠卵巢颗粒细胞48h后,检测NR4A1超表达及表达沉默后对颗粒细胞增殖、凋亡的影响。
研究结果
第一部分:NR4A1在小鼠卵巢中的表达及其对卵泡膜细胞雄激素生成的影响
1. NR4A1蛋白在小鼠卵巢各级卵泡和间质内均有表达,其主要定位于卵泡膜细胞和颗粒细胞的细胞核。
2.重组病毒Ad-CMV-NR4A1感染卵泡膜细胞后,与对照组相比,NR4A1超表达组NR4A1蛋白的表达水平显著升高,两组间差异显著(p<0.05)。NR4A1在卵泡膜细胞内超表达后明显促进了卵泡膜细胞雄激素生成相关酶(StAR、CYP11A1、CYP17A1、HSD3B2)mRNA的转录,增加睾酮合成。相反,重组病毒Ad-H1-siRNA/NR4A1感染卵泡膜细胞后,与对照组相比,NR4A1干涉组NR4A1蛋白的表达水平显著降低(p<0.05)。NR4A1在卵泡膜细胞内干涉后明显地抑制了StAR、CYP11A1、CYP17A1、HSD3B2 mRNA的表达,降低睾酮合成。
3.在卵泡培养液中加入福斯高林后,NR4A1的表达迅速增加,随后StAR、CYP11A1、CYP17A1、HSD3B2的表达增加。
第二部分:高雄激素作用下NR4A1的表达及其对颗粒细胞增殖、凋亡的影响
1.在小鼠窦前卵泡体外培养的过程中,10~(-4)mol/L和10~(-5)mol/L睾酮能够显著促进卵泡体积的增加(p<0.05);在10~(-5)mol/L睾酮的作用下,小鼠卵巢颗粒细胞的增殖增加,凋亡减少(p<0.05)。
2. Real-time RT-PCR及Western blot结果显示NR4A1在PCOS大鼠模型卵巢中的表达明显低于其在正常大鼠卵巢中的表达(p<0.05)。
3.用10~(-5) mol/L和10~(-4) mol/L睾酮处理卵泡24h及48h后均明显抑制NR4A1的表达(p<0.05)。
4.用10~(-5)mol/L睾酮处理颗粒细胞10min至30min,Akt蛋白磷酸化水平显著升高(p<0.05); LY294002预处理后,明显抑制睾酮作用下的Akt的磷酸化(p<0.05),增加NR41的表达(p<0.05)。
5. NR4A1在颗粒细胞内超表达后促进颗粒细胞的凋亡,抑制其增殖(p<0.05);相反,NR4A1在颗粒细胞内表达沉默后抑制颗粒细胞的凋亡,促进其增殖(p<0.05)。
结论
1. NR4A1在小鼠卵巢的卵泡膜细胞和颗粒细胞内表达,提示NR4A1参与卵泡膜细胞和颗粒细胞内某种(些)生物学过程的调控。
2.在cAMP/PKA信号通路作用下,NR4A1在卵泡膜细胞内促进雄激素生成酶的转录,进而促进雄激素生成。
3.高雄激素促进颗粒细胞增殖、抑制颗粒细胞凋亡,从而促进早期卵泡发育。体内体外实验证明,高雄激素降低卵巢颗粒细胞中NR4A1的表达,可能与此相关。
4.高雄激素作用下,通过PI3-K/Akt通路下调颗粒细胞内NR4A1的表达,NR4A1表达下调参与了颗粒细胞的增殖、凋亡调控过程,从而影响未成熟卵泡发育。
5.本研究部分阐明了NR4A1在PCOS病理生理机制中的作用。
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.
Methods
Part One: The Expression of NR4A1 in Mouse Ovary and its Effect on Theca Cell Androgen Production
1. 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 Cells
1. 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.
Results
Part One: The Expression of NR4A1 in Mouse Ovary and its Effect on Theca Cell Androgen Production
1. 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 Cells
1. 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~(-4)mol/L or 10~(-5)mol/L testosterone result in increased follicle size. When treated with 10~(-5)mol/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).
Conclusion
1. 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.
引文
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