摘要
第一章卵泡刺激素受体与脂肪分化、代谢的研究
研究背景
垂体分泌的糖蛋白激素,如卵泡刺激素(FSH)、黄体生成素,是下丘脑-垂体-内分泌轴中的关键信号分子。一直以来,普遍认为垂体糖蛋白类激素受体仅存在于特异的内分泌的靶器官和组织。垂体糖蛋白激素受体在内分泌外组织分布的报道是新近出现还存在争议的。
存在于外周的非经典的糖蛋白激素受体,含量往往很低,甚至只在特殊的发展阶段(如修复或者肿瘤发生)呈阶段性表达。因此假设只在特定的情况下,如激素合成受抑制(如垂体功能衰竭)或者激素合成骤增(内分泌功能衰竭导致的垂体功能亢进)的特殊情况下,这些外周的非经典的糖蛋白激素受体才会被激活并发挥生物学效应。
在女性,卵泡刺激素受体(FSHR)特异性表达于卵巢颗粒细胞细胞膜表面。FSH与FSHR特异结合的复合物,激活胞内信号传导最终决定颗粒细胞增殖、分化。FSHR在卵巢的高表达对卵泡的发生和发育、成熟起关键调控作用。在男性,FSHR特异表达于睾丸的支持细胞,通过促进精子的形成而调控生精功能。FSHR在性腺外的表达:在低等的动物,见于斑马鱼的肾脏和肝脏组织。而在人类,仅明确报道在人破骨细胞上FSHR的存在,FSHR直接参与绝经后骨质疏松的发生和发展。
脂肪组织是人体的能量储存器官,维持着机体的能量代谢的平衡。当机体能量摄入大于能量消耗时,多余的能量便以脂肪的形式储存于体内。脂肪组织的过多堆积,特别是内脏脂肪的增多,会导致肥胖、胰岛素抵抗甚至代谢综合征的发生。脂肪组织也是一个活跃的内分泌器官,分泌的脂源性因子包括瘦素、脂联素、抵抗素近百种。在不同的生理或病理情况下,脂肪组织通过这些脂源性因子实现与机体其他组织如肝脏、肌肉、下丘脑等的对话,从而广泛地影响和调节机体的能量代谢。除外源性的脂肪酸、胰岛素、生长激素、糖皮质激素和甲状腺素等促进脂肪分化成熟的因素外,甾体激素如雌激素、孕激素和雄激素等和促性腺激素如人绒毛膜促性腺激素/黄体生成素也直接参与了脂肪细胞的增殖、分化和代谢。目前已经确定的脂肪细胞上表达的性激素受体包括雌激素、孕激素、雄激素和人绒毛膜促性腺激素/黄体生成素受体。绝经期妇女经历体内激素环境的巨大变化时,脂肪细胞分化、代谢发生异常,往往会出现体重增加、肥胖、糖耐量降低等代谢异常的现象。激素可能是通过与其特异性的受体识别反应并结合于DNA的特定区域,最终影响脂肪分化、代谢重要因子的转录和翻译水平。
FSH具有刺激卵泡分泌雌激素的功能,同时也受雌激素的负反馈调节。当性腺功能下降如绝经期时,卵巢功能衰竭,产生的雌激素水平骤降,同时伴随着FSH水平的升高。因此,FSH也被视为绝经期的诊断标志。我们设想,在绝经期,迅速升高FSH,会不会通过FSH-FSHR参与调控脂肪细胞的增殖分化。而目前尚没有着眼于FSH-FSHR与脂肪分化和代谢的研究。
我们兴奋的发现人脂肪组织上存在FSHR,这是世界上第一次在脂肪组织发现FSHR的报道。更令人激动的是,我们发现FSH直接参与调控体外3T3-L1前脂肪细胞的成脂分化,并证实FSH是通过上调过氧化物酶体增殖物激活受体-γ(PPAR-γ)来实现促进3T3-L1的成脂分化。我们还在C57/BL6小鼠去势模型上成功模拟FSH亢进的绝经期激素环境,完整得演绎FSH对在体脂肪细胞增殖分化和脂质代谢的调控:FSH参与了去势后小鼠内脏脂肪的堆积、血脂代谢的异常和脂源性因子的分泌改变。体内外实验结果一致而充分的解释了脂肪细胞上功能性的FSHR的事实。提示高FSH刺激下,FSHR被激活并且发挥生物学效应,直接参与调控脂肪的合成、代谢和内分泌功能。
我们的研究首次阐述了脂肪组织上FSHR的表达和功能,第一次提出FSH直接调控脂肪分化、代谢的观点,FSH-FSHR信号调控开创了绝经期肥胖症研究的崭新方向,开拓了垂体糖蛋白受体新的研究领域。
第一部分卵泡刺激素受体在人体脂肪组织的分布和表达
研究目的:检测脂肪组织是否存在卵泡刺激素受体(FSHR)的表达,明确不同性别、不同年龄、不同部位的脂肪组织FSHR的分布情况和细胞定位。
资料与方法:取自不同年龄、性别的皮下和内脏脂肪组织,采用逆转录-聚合酶链反应和蛋白质印迹技术,测定脂肪组织、分离的脂肪细胞和前脂肪细胞上FSHR的表达。通过免疫组织化学的方法,明确脂肪组织FSHR蛋白的细胞定位。
结果:1.人脂肪组织存在FSHR,mRNA水平和蛋白水平均有表达;FSHR蛋白在脂肪组织的表达量与颗粒细胞上的相当2.FSHR在脂肪细胞上的表达远高于前脂肪细胞,FSHR主要分布于成熟的脂肪细胞的细胞膜表面3.在不同性别,不同年龄段,皮下脂肪和内脏脂肪FSHR表达无显著差异。
结论:
1.人脂肪组织存在FSHR,FSHR蛋白丰富表达于人脂肪组织。
2.FSHR主要表达于成熟脂肪细胞,在前脂肪细胞上低表达
3.FSHR定位于脂肪细胞的细胞膜表面。
4.在不同年龄、性别、部位的脂肪组织中FSHR表达较恒定。
第二部分卵泡刺激素对3T3-L1前脂肪细胞增殖及分化的影响
研究目的:应用小鼠3T3-L1前脂肪细胞株研究卵泡刺激素(FSH)与前脂肪细胞分化的关系,探讨FSH在脂肪细胞增殖、成脂分化过程的作用。
资料与方法:给予不同浓度FSH(0-300ng/mL)刺激,观察3T3-L1前脂肪细胞的增殖能力和成脂分化能力。MTT法观察24h和48h细胞增殖能力。油红O染色法鉴定分化成熟的脂肪细胞并检测细胞内脂质含量,并用Real-timePCR检测检测过氧化物酶体增殖物激活受体-γ(PPAR-γ)mRNA表达。
结果:1.3T3-L1前脂肪细胞在不同浓度FSH下培养24h-48h,细胞增殖能力没有明显变化。2.诱导分化培养基中FSH浓度≥10ng/mL时,3T3-L1前脂肪细胞可见脂滴堆积的速度明显加快,在30ng/mL浓度时,FSH对脂滴堆积的促进效应达到最大。3.PPAR-γmRNA的表达随成脂分化过程逐渐升高,在10天达到峰值。随诱导分化培养基FSH浓度增加,PPARγ表达增加。
结论:
1.首次发现FSH直接参与3T3-L1细胞的成脂分化,其对成脂的调控呈正向剂量时间依赖效应。
2.FSH对3T3-L1细胞分化的调控可能是通过上调PPARγ基因表达来发挥其促进作用。
3.FSH不影响3T3-L1细胞的增殖能力。
第三部分卵泡刺激素对去势小鼠脂肪合成和代谢功能的影响
研究目的:通过观察FSH刺激下去势小鼠脂肪分化和脂类代谢水平,研究FSH对在体脂肪细胞增殖分化和脂质代谢的调控,揭示FSH参与绝经后脂肪代谢和分布异常的可能机制。
资料与方法:10周龄C57/BL6小鼠随机分为三组,假手术组(SHAM);去势组(OVX)和去势后加用FSH(OVX+FSH)组,给予OVX+FSH组每日3IU人重组FSH刺激14日。比较体重、体脂的分布、脂肪细胞的体积、血脂水平、瘦素和脂联素水平、脂肪特异性基因mRNA表达量。
结果:1、FSH刺激14日后,OVX+FSH小鼠体重增加,脂肪细胞体积增大,体脂和内脏脂肪含量明显高于SHAM组,血清甘油三酯、总胆固醇水平也明显升高。2、FSH刺激下,内脏脂肪组织的重要脂肪细胞特异性基因表达发生明显的变化。其中脂肪合成相关基因ACC、FAS、Perilipin表达升高,而脂肪分解基因HSL表达下降3、FSH刺激下转录因子PPARγ和SREBP1表达增强。4、FSH刺激下,去势小鼠瘦素水平升高而脂联素水平降低。
结论:
1.FSH参与调控去势后小鼠脂肪重分布和脂肪分化及代谢。
2.FSH可能通过上调PPARγ和SREBP1转录因子参与调节脂肪合成和分解代谢的过程。
3.FSH参与调节脂源性因子的分泌。
第二章卵巢颗粒细胞卵泡刺激素受体的表达与超排卵周期卵巢反应性的关系
研究背景
卵巢低反应是目前制约体外受精技术(IVF)成功的最大的难题。在控制性促排卵(COH)过程中,大约有9%-24%的妇女发生卵巢低反应,表现为卵巢对促性腺激素的反应极差:卵泡发育少、可供移植的胚胎更少、临床妊娠率极低。对于多数的卵巢低反应病人,目前临床上仍没有有效的处理方案。
卵巢低反应的发生是卵巢衰老的最早征兆,其具体发病机制目前尚不明确。高龄、卵巢病理损伤、卵巢自身抗体的出现、性腺发育相关的基因突变都被认为是卵巢低反应的发病因素,但这些因素远远不能解释大部分卵巢低反应的发生。
至今临床上仍没有方法来准确评估COH周期卵巢对促性腺激素的反应性,也缺乏明确的检测指标来筛查卵巢低反应。常用年龄、基础激素测定以及卵巢超声测量等方法预测卵巢反应性,通常认为年龄大于35岁、基础卵泡刺激素>10-12IU/L,卵巢窦卵泡计数<5个预示卵巢低反应。但是在临床上,仍有不少的年轻的预测卵巢正常反应的妇女对FSH反应不良,甚至面临周期被迫取消的困境。
我们的研究从FSHR角度来分析卵巢低反应发生的可能机制。我们第一次发现了在预测卵巢正常反应的妇女给予FSH刺激后,卵巢的反应性与卵巢颗粒细胞上FSHR表达相关。首次提出颗粒细胞FSHR的低表达导致卵巢低反应的发生。同时FSHR的过度表达与卵巢过度反应相关。我们还发现在这类因FSHR减少引起的卵巢低反应患者,给予临床上最常见的改善卵巢反应的策略,即增加FSH的剂量,也无法改善卵巢低反应的结局。
研究目的:检测颗粒细胞卵泡刺激素受体(FSHR)的表达,探讨超排卵周期中卵巢的反应性与颗粒细胞FSHR表达的关系。
资料与方法:采用蛋白质印迹(Western blot)法,测定因输卵管性不孕或者男性因素不孕患者60例不孕症患者的卵泡颗粒细胞FSHR蛋白的水平;并测定血清雌二醇(E2)峰值、血清和卵泡液中卵泡刺激素(FSH)。根据超排卵周期中发育卵泡数分组,比较各组FSHR蛋白表达水平,并分析FSHR表达水平与卵泡数、E2峰值、血清和卵泡液FSH水平的相关性。
结果:1、颗粒细胞FSHR蛋白表达在卵巢低反应组为0.22±0.02;低于卵巢正常反应组0.32±0.07及卵巢过度反应组0.46±0.03。2、FSHR蛋白表达量与成熟卵泡数、E2峰值均呈正相关。3、卵泡液中FSH水平卵巢低反应组高于卵巢正常反应和卵巢过度反应组,且与FSH用量呈正相关。
结论:
1.超排卵周期中,颗粒细胞FSHR蛋白表达在不同卵巢的反应者中表达各异;在卵巢低反应者表达量最低;而在卵巢过度反应者表达量最高。
2.卵巢低反应者的颗粒细胞FSHR减少是导致卵巢低反应的重要原因之一。
3.在COH过程中,加大FSH用量并不能够纠正由于FSHR减少造成的卵巢低反应。
Chapter I
Follicle Stimulating Hormone Receptor and Adipocyte Differentiation and Metabolism
Introduction
The glycoprotein hormones are usually regarded as pivotal pituitary-endocrine signals. High expression of selective receptors in the pituitary-endocrine axis is key to mammalian endocrine regulation. The occurrence of glycoprotein hormone receptors in non-endocrine organs is an emerging but controversial area of investigation.
The peripheral expression of glycoprotein hormone receptors is relatively limited and might be restricted to specific developmental stages. Glycoprotein hormone receptors might regulate differentiation and survival under circumstances with abnormally low or high levels of circulating hormones.
FSH is the central glycoprotein hormone receptor of mammalian reproduction, necessary for gonadal development and maturation at puberty and for gamete production during the fertile phase of life. FSH binds to its specific membrane rceptor FSHR, which is located exclusively in granulosa cells. FSH-FSHR interaction elicits intracellular signaling pathways to determint proliferation and differentiation of granulosa cells. In zebrafish, FSHR is expressed in the kidney and liver, in addition to the gonads. In human, FSHR has been found localized to the surface of human osteoblasts. FSH stimulation regulates bone mass by directly increasing osteoclastogensis and resorptin.
Obesity is a disorder that results from excess adipose tissue and is a major for risk diabetes and cardiovascular diseases. Sex steroid hormones are involved in the metabolism, accumulation and distribution of adipose tissues. It is now known that estrogen receptor, progesterone receptor, androgen receptor and hCG/LH receptor exist in adipose tissues, so their actions could be direct. The mechanism for the regulation of the amount and distribution of adipose tissues by sex steroid hormones is not clear. One possible mechanism would be the regulation of key proteins in adipose tissues at the genomic level by transcriptional means.
FSH is considered a stimulus for estrogen production from ovarian follicles. As FSH secretion is negatively regulated by estrogen feedback, a high FSH level accompanies hypogonadism. FSH has thus been regarded as a marker for the onset of menopause. However, the effect of FSH on adiposity has never been explored.
Our study provides the first evidence that FSHR is characterized in human adipocytes. We show that FSH stimulated the differentiation and function of preadipocyte in vitro and in vivo. In 3T3-L1 cells, FSH accelerated adipogenesis in a dose-dependent manner under the control of PPAR-γ. In addition, FSH-treated mice exhibited increased visceral fat mass associated with triglyceride and cholesterol. FSHR might regulate differentiation and metabolism under circumstances with abnormally high FSH in menopause. We anticipate that therapeutic inhibition of adipose FSHR activity in post-menopausal women will reduce lipogenesis and counteract the accumulation of visceral fat and its related metabolic abnormalities.
Part I
Follicle Stimulating Hormone Receptor in Adipose Tissue
Objectives: To examine the possible presence of follicle stimulating hormone receptor (FSHR) in human adipose tissue.
Materials and Methods: Adipose tissue was obtained from various anatomical regions of man and women at different ages. It was assessed for the presence of FSHR by western blot, RT-PCR and immunohistochemistry.
Results: It was clearly demonstrated that the presence of FSHR in human subcutaneous and omental adipose tissue. FSHR was primarily located on the membrane of adipocytes. The expression of FSHR was significantly higher in adipocytes versus preadipocytes. There was no difference of FSHR expression in subcutaneous and omental adipose tissue from premenopausal, perimenopausal and postmenopausal men and women.
Conclusions: For the first time, FSHR was characterized in human mature adipocytes. FSHR was found to be localized on the membrane of adipocyte. FSHR might be invariance of expression in adipose tissue.
Part II
The role of follicle stimulating hormone on proliferarion and differentiation of 3T3-L1 preadipocyte
Objectives: To investigate the relationship between follicle stimulating hormone (FSH) and preadipocyte proliferarion and differentiation.
Materials and Methods: The mouse 3T3-L1 preadipocytes were cultured in the presence of FSH (3-300ng/mL). And FSH (3-300ng/mL) was added during the differentiation of 3T3-L1 in response to dexamethasone, isobutyl methyl xanthine and insulin. The rate of cell proliferation was measured over time (24-48h) by MTT metablism. Adipogenesis was judged by Oil Red 0 staining. The gene expression of peroxisome proliferator-activated receptor gamma (PPARγ) was analyzed by real-time RT-PCR.
Results: The amount of mature adipocytes and PPARγmRNA were gradually cumulative during 3T3-L1 differentiation. Treatment of differentiating 3T3-L1 preadipocytes with FSH resulted in a dose-dependent increase in number of mature adipocytes and PPARγexpression. FSH did not alter the proliferation of 3T3-L1.
Conclusions:
1. In 3T3-L1 cells, FSH accelerates adipogenesis in a dose-dependent manner.
2. FSH acts as a potent positive regulator of PPARγ, a master regulator of adipocyte differentiation, and tightly controls adipogenesis.
3. FSH has no effect on preadipocyte proliferarion.
Part III
Follicle stimulating hormone regulation of adiposity in ovariectomized mice
Objectives: To elucidate metabolic and molecular mechanisms by which follicle stimulating hormone (FSH) regulates fat storage and fat mobilization.
Materials and Methods: C57 BL/6 mice were ovariectomized, randomized to FSH (OVX+FSH) and control saline administration (OVX), and pairfed for 14 days. To compare adipose mass, serum cholesterol and triglyceride content, adipose gene expression and serum leptin and adiponectin levels between OVX, OVX+FSH and sham groups.
Results: FSH injection increased adipose weight, adipocyte size and serum cholesterol and triglyceride. FSH upregulated the expression of lipogenic genes in adipocyte as ACC, FAS; downregulated of HSL. FSH treatment was also associated with increasing expression of sterol-regulatory element-binding protein 1 and peroxisome proliferator-activated receptor gamma. FSH+OVX mice were with increased leptin and decreased adiponectin level.
Conclusions:
1. FSH directly stimulates adipocyte differentiation and metabolism.
2. FSH promotes adiposity by upregulating targets adipose genes under the control of PPAR-γand SREBBP1.
3. FSH regulates secretion of adipokines.
Chapter II
Follicle Stimulating Hormone Receptor and Ovarian Response
Introduction
Approximately 9 to 24 percent of women undergoing IVF respond poorly to standard ovarian stimulation protocol. Such patients, referred to as poor responders, may result in low levels of serum E_2, fewer mature oocytes and reduced pregnancy rates. Although a variety of strategies have been used to improve ovarian response, the optical approach for poor responders still remains controversial. Up to date, poor ovarian response is one of the most difficult and challenging problems in the field of IVF.
Advanced maternal age and diminished ovarian reserve may be associated with poor ovarian response. However, some young women with normal ovarian reserve as indexed by basal serum FSH level and antral follicle count do present poor response to ovarian stimulation. Several hypotheses have been proposed for poor ovarian response alternatively. Poor follicular blood flow, dysfunctions of cytokines and growth factor network, and the presence of ovarian autoantibody have been proposed, but none has been proven.
Recently, the description of cases of mutation of the FSH receptor (FSHR) has highlighted the importance of FSHR in determining the ovarian response. Women with inactivating mutations of FSHR were reported to constitute a defect in ovulation failure and amenorrhoea. In-vitro studies had demonstrated that these novel specific mutations of FSHR dramatically reduced receptor expression and impaired proper signal transduction. In a study of transfection of adenoviral vector to immature granulosa cells, it was found that differential effects of FSH in granulosa cells were highly dependent on FSHR density and FSHR-induced signaling which depended on the dosage of transfected vector.
In the present study, it is revealed that the availability of FSHR in granulosa cells is associated with the individual response to ovarian stimulation. To the best of our knowledge, this is the first report describing the different levels of FSHR in granulosa cells of young women with different response to ovarian stimulation whose basal serum FSH levels and antral follicle counts were normal. We found lower FSHR expression in poor responders, while higher FSHR expression in high responders, as compared with moderate responders. Increasing dose of rFSH could not improve ovarian response. These results suggest that the variability of FSHR necessarily may determine ovarian response to gonadotropin stimulation.
Objectives: To explore the importance of follicle stimulating hormone receptor (FSHR) in granulosa cells in the ovarian response to gonadotropin stimulation.
Materials and Methods: 60 women undergoing ovarian stimulation were divided into three groups: poor, moderate, and high responders, according to the number of follicles with diameter >= 14 mm. FSHR expression at protein levels were determined by Western blot in granulosa cells. Estradiol (E2) concentrations in serum/follicle fluid were measured by electrochemiluminescene immunoassay.
Results: The expression of FSHR was significantly different among three groups with the lowest expression in the poor responders (0.22±0.02), moderate (0.32±0.07), and high responders (0.46±0.03). The level of FSHR protein was positively correlated with peak level of serum E_2 and the number of mature oocytes. FSH levels in follicular fluid and the dosage of rFSH used were significantly different among three groups with highest values in the poor responders.
Conclusions:
1. Different levels of FSHR expression in granulosa cells result in different ovarian response with the lowest expression in the poor responders and highest in high responders.
2. Lower expression of FSHR may account for poor ovarian response to gonadotropin stimulation, suggesting the critical role of FSHR in the ovarian response to gonadotropin stimulation.
3. Increasing dose of rFSH could not improve ovarian poor response.
引文
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