摘要
1993年,人们首次从大鼠B49胶质细胞系中分离、纯化并成功克隆了一种新的神经营养因子,即胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF),最初因其可以促进多种神经元的存活、生长及分化,调控着胚胎时期肾脏发育及输尿管分支形成,促进精原细胞分化及更新而展开了一系列的研究。GDNF主要通过结合特异性受体而起作用,GDNF家族特异性受体家族(GDNF family receptorα, GFRα)通过C端的糖磷酯酰肌醇键锚定在细胞表面,但缺乏胞内结构域,因此GFRα只是GDNF功能性受体的一部分。GDNF与特异性受体GFRα-1结合形成二聚体复合物后再引导着另一部分功能性受体即Ret (rearranged during transfection)蛋白与之结合,形成三聚体复合物。Ret是在基因转染过程中可以重排的RET基因的蛋白产物,也是受体酪氨酸激酶家族的新成员,具有典型的酪氨酸激酶结构,GDNF-GFRα-Ret复合物形成之后,Ret发生磷酸化,从而激活下游胞内信号转导的的发生。令人感兴趣的是,利用原位杂交技术检测小鼠体内15个器官发现,卵巢内GDNFmRNA的表达水平最为显著,且研究证实,GDNF可以促进猪始基卵泡向初级卵泡发育,增加小鼠卵母细胞第一极体的排出率及促进胚胎向囊胚期发育,因此研究GDNF在卵巢内的作用对进一步阐明卵泡生发、成熟及胚胎发育过程具有重要意义。但是目前为止,关于GDNF及其受体在人卵巢内表达的研究仍鲜有报道。并且,在小鼠体内,GDNF及GFRα-1的表达量随着卵泡的发育在LH峰出现后达到最高值,敲除了卵泡刺激素受体基因后,小鼠体内GDNF分泌量呈双峰式,提示促性腺激素对GDNF及其受体存在着一定的调控作用,那么,在人卵巢内,促性腺激素是否也存在着对GDNF及其受体表达的调控作用呢?又是如何参与调控的呢?本研究拟以GDNF在生殖方面的研究及其他组织研究为基础,检测GDNF及其受体在人排卵前黄素化颗粒细胞内的表达情况,观察促性腺激素对GDNF及其受体mRNA表达水平的影响,初步探讨GDNF在卵泡发育成熟过程中的作用机制。
第一部分人黄素化颗粒细胞原代培养及鉴定
[研究目的]
通过建立稳定的人黄素化颗粒细胞原代培养系统,观察细胞形态及生长周期,体外鉴定颗粒细胞纯度,为后续研究提供实验基础。
[研究方法]
人黄素化颗粒细胞取自2009年5月至2009年12月因输卵管因素或男方因素不孕而在南方医院生殖中心行体外受精-胚胎移植(in vitro fertilization and embryo transfer, IVF-ET)治疗的妇女8例,均使用黄体期长方案降调节,且年龄位于25-30岁,月经第三天基础卵泡刺激素(follicle stimulating hormone, FSH)≤7.5mIU/ml,人绒毛膜促性腺激素(human chorionic gonadotropin, HCG)日血清雌二醇(estradiol,E2)< 4000pg/ml,19kg/m2<体重指数(body mass index, BMI)<23kg/m2,无排卵功能障碍,月经周期正常。
用Percoll梯度离心法提取卵泡液内的颗粒细胞,按2×105／L接种于35mm培养皿中,每皿加入2ml RPMI-1640培养基,20ul青霉素-链霉素溶液,500ul胎牛血清,置于5%CO2、37℃孵箱,24小时后弃去未贴壁细胞,更换完全培养基,培养基成分与前相同,每隔24h观察细胞形态及拍照。
原代颗粒细胞鉴定时,将原代颗粒细胞提纯后,加入约2ml已配置好的完全培养基后,悬起清洗后的细胞沉淀,在10cm皿中放置经多聚赖氨酸处理的防脱载玻片,小心将细胞悬液均匀滴至载玻片上,勿使细胞悬液滴至载玻片外区域,将培养皿放置孵箱过夜后以1：50稀释兔抗人卵泡刺激素受体(follicle-stimulating hormone receptor, FSHR)多克隆抗体进行免疫细胞化学染色。
[结果]
1、8个病人,8个IVF周期,平均年龄(26.4±1.2)岁,平均BMI为(21.3±1.3)kg／m2,月经第三天平均基础FSH水平为(6.8±0.5) mIU/ml, HCG日平均血清E2水平为(1493.2±755.1) pg/ml。
2、人黄素化颗粒细胞在体外生长缓慢,在倒置显微镜下观察,接种时呈圆形,表面可见深色颗粒状物质,24小时内,颗粒细胞呈现贴壁生长,明显可见细胞质内颜色深,粗大的,较多的颗粒状物质,培养基内可见少量红细胞悬浮其中,换液后,红细胞基本去除干净；48小时后,单层颗粒细胞均匀满布培养皿中,外观呈梭状或星状,伸出细长伪足,细胞与细胞间通过伪足连接紧密,胞质内颗粒状物质丰富,细胞核圆,大,胞浆饱满,透光好；72小时后,细胞各组生长形态无明显改变,未见明显细胞增殖；培养96小时,部分颗粒细胞内颗粒状物质消失,胞浆减少,出现类似成纤维细胞形态；培养120小时,部分颗粒细胞内颗粒状物质增多,增粗,体型肥大,伪足短,无明显张力；培养216小时,部分培养皿中颗粒细胞脱落死亡,集结呈团状,悬浮于培养液中,培养至第15天,颗粒细胞完全脱颗粒、呈纤维样变,失去原来的细胞形态。
3、原代培养人黄素化颗粒细胞鉴定：FSHR仅存在于卵巢颗粒细胞中,FSHR免疫细胞化学染色是鉴定颗粒细胞的特异性染色,FSHR阳性染色定位于细胞胞浆,呈深棕色,细胞核呈蓝色,镜下可见FSHR的阳性率为70%-80%。
[小结]
1、利用45%percoll分离法能有效去除卵泡液中大部分红细胞。
2、利用RPMI-1640完全培养基时,培养72小时,细胞未出现明显增殖,培养至96小时,细胞出现退行性改变,提示96小时之前,细胞呈典型颗粒细胞形态,无明显脱落死亡现象,生长稳定,适宜行体外实验。
3、经鉴定,从卵泡液内分离纯化接种的是人颗粒细胞,纯度达70%以上,符合实验要求。
第二部分GDNF及其受体在人黄素化颗粒细胞中的表达
[研究目的]
通过对体外培养原代黄素化颗粒细胞进行GDNF、GFRα-1、Ret免疫细胞化学染色,证实GDNF、GFRα-1、Ret在人黄素化颗粒细胞中的表达情况,提不GDNF及其受体对卵泡发育起了一定的调控作用,为后续研究提供实验依据。
[研究方法]
人黄素化颗粒细胞标本取自2009年5月至2009年12月因输卵管因素或男方因素不孕而在南方医院生殖中心行IVF-ET治疗的妇女15例,均使用黄体期长方案降调节,且年龄位于25～30岁,月经第三天基础FSH≤7.5mIU/ml, HCG日E2<4000pg/ml,19 kg/m2< BMI< 23 kg/m2,月经周期正常,无排卵功能障碍。
用45%Percoll分离液提取分离颗粒细胞,将细胞悬液直接滴至于经多聚赖氨酸处理过的载玻片上,放置过夜后,将爬片用4%多聚甲醛固定20-30min,分别按1：200稀释兔抗人GDNF多克隆抗体,1：200稀释兔抗人GFRα-1多克隆抗体,鼠抗人Ret单克隆抗体原液进行免疫细胞化学染色。
[结果]
1、15个病人,15个IVF-ET周期,平均年龄(26.7±2.0)岁,平均BMI为(21.5±1.2)kg／m2,月经第三天平均基础FSH水平为(6.7±0.6)mIU/ml, HCG日平均血清E2水平为(2009.6±850.7) pg/ml;
2、GDNF阳性染色定位于细胞胞浆及细胞核内,镜下可见GDNF在胞浆内的染色阳性率几近100%,胞核内阳性率为97%；
3、GFRα-1阳性染色定位于细胞胞浆及细胞核内,镜下可见GFRα-1在胞浆内的染色阳性率几近100%,胞核内阳性率为55%；
4、Ret阳性染色定位于细胞胞浆及细胞核内,镜下可见Ret在胞浆内的染色阳性率为47%,胞核内阳性率为17%。
[小结]
GDNF、GFRα-1、Ret均表达于人黄素化颗粒细胞核及细胞浆内,提示GDNF可能通过自分泌与旁分泌的机制参与卵泡生长发育的过程。
第三部分促性腺激素对GDNF、GFRα-1及Ret在人黄素化颗粒细胞中表达的影响
[研究目的]
通过在体外培养基中添加不同浓度的人绝经期促性腺激素(human menopausal gonadotropin, HMG)及FSH,检测黄素化颗粒细胞内GDNFmRNA、GFRα-1mRNA、RETmRNA转录水平的变化,以期寻找促性腺激素调控GDNF及其受体表达的可能机制,深入认识GDNF在卵泡发育成熟中的作用。
[研究方法]
将5例病人黄素化颗粒细胞进行体外原代培养,培养方法同前,实验采用随机区组设计：分为四个处理组,即空白对照组(简称对照组)、HMG 5IU/L组(简称5IU组)、HMG 10IU/L组(简称10IU组),FSH10IU/L组(简称FSH组),以每个病人为一个配伍组,每个病人卵泡液内分离纯化的细胞,均匀按2×105／L密度接种,共接种于四个培养皿中,随机接受四种不同处理,体外培养至72h,收取细胞的总RNA,采用SYBR Green染料法进行Real time PCR检测,运用2-△△Ct法对各实验组中GDNFmRNA、GFRα-1mRNA、RETmRNA相对表达量进行分析。
用SPSS13.0软件进行统计分析,计量资料结果以均数±标准差((?)±s)表示。当数据符合正态分布时,随机区组设计多个样本比较采用随机单位组设计的方差分析,其中多重比较采用S-N-K法；设双侧检验,P<0.05为差异有统计学意义。
[结果]
1、5例病人,5个IVF-ET周期,平均年龄(27.2±1.9)岁,平均BMI为(21.7±0.7)kg／m2,月经第三天基础平均FSH水平为(6.8±0.5) mIU/ml, HCG日平均血清E2水平为(1892.2±396.4) pg/ml.
2、对照组、5IU组,10IU组、FSH组进行随机单位组设计资料方差分析,四组相比,GDNFmRNA的表达量具有显著性差异(F=22.308,P<0.01),其中10IU组GDNFmRNA表达量最高,比对照组高4.37倍,其次为5IU组表达量比对照组高2.76倍,对照组表达量最低。病人个体间有显著性差异(F=6.283,P=0.006),说明配伍组设计有效,经过S-N-K多重比较,5IU组及10IU组的表达量均显著高于空白组及FSH组,且10IU组GDNFmRNA表达量显著高于5IU组,差异有统计学意义(P<0.05)。
3、各处理组计量资料均满足正态分布,对照组、5IU组,10IU组、FSH组进行随机单位组设计资料方差分析,四组相比,GFRα-1mRNA的表达量具有显著性差异(F=18.985,P<0.01),其中10IU组GFRα-1mRNA表达量最高,比对照组高4.86倍,其次为5IU组表达量比对照组高3.23倍,FSH组表达量最低,病人个体间有显著性差异(F=4.568,P=0.018),说明配伍组设计有效,经过S-N-K组间多重比较,5IU组及10IU组的表达量均显著高于空白组及FSH组,且1 0IU组GFRα-1mRNA表达量显著高于5IU组,差异具有统计学意义(P<0.05)。
4、各处理组计量资料均满足正态分布,对照组、5IU组,10IU组、FSH组进行随机单位组设计资料方差分析,四组相比,RETmRNA的表达量均无统计学差异(F=2.026,P=0.164)。
[小结]
1、当单独在培养基中添加FSH的浓度为10IU／L时,未能显著上调GDNFmRNA、GFRα-1mRNA、RETmRNA的表达水平,提示FSH在此浓度下不能对上述mRNA的表达起上调作用,但不能完全排除FSH在维持卵泡内GDNF表达量动态平衡方面的作用。
2、当使用HMG的浓度为5IU／L时,与对照组相比,可显著上调颗粒细胞GDNFmRNA及GFRα-1mRNA的转录水平,当剂量增加到10IU／L时,GDNFmRNA及GFRα-1m RNA的转录水平随着HMG用量的增长而增长,呈剂量依赖性,提示排卵前LH峰促进卵母细胞的最终成熟及排卵可能是通过上调GDNF的分泌水平来实现的,GDNF可能是介导卵母细胞发育成熟的信号机制之一。
3、HMG及FSH均不能上调黄素化颗粒细胞内RETmRNA的表达水平,提示除了GDNF-GFRα-Ret信号转导机制外,可能存在着其他的GDNF信号转导途径参与卵泡生长发育的过程。
[全文总结]
1、45%percoll分离法能有效分离卵泡液颗粒细胞,接种后细胞纯度>70%。
2、利用RPMI-1640完全培养基培养人黄素化颗粒细胞,培养至96小时之前,颗粒细胞形态典型,无明显脱颗粒及死亡现象,适宜进行体外实验。
3、首次发现GDNF、GFRα-1、Ret表达于人黄素化颗粒细胞核及细胞浆内,提示,GDNF可能通过自分泌与旁分泌途径参与卵母细胞的生长发育成熟过程。
4、首次证实HMG能显著上调人黄素化颗粒细胞内GDNFmRNA、GFRα-1 mRNA的转录水平,并呈剂量依赖性,而单独使用FSH的浓度为10IU／L时未见此上调作用,提示LH促进颗粒细胞分泌GDNF并作用于颗粒细胞及卵母细胞可能是LH调控卵泡发育成熟的机制之一,但不能完全排除FSH在维持卵泡内GDNF表达量动态平衡方面的作用。
5、HMG及FSH均不能上调黄素化颗粒细胞内RETmRNA的表达水平,提示除了GDNF-GFRα-Ret信号转导机制外,可能存在着其他的GDNF信号转导途径参与卵泡生长发育的过程。
Neurotrophic factors include neurotrophins, neurokines and glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs). GDNF was purified and characterized in 1993 as a growth factor promoting the survival of various neurons. It has since become evident that GDNF is also expressed in tissues outside the central nervous system and that its overall expression is significantly higher in peripheral organs than in neuronal tissues. GDNF has been shown to regulate ureteric branching during embryonic kidney development. In the testis, GDNF dose-dependently affects spermatogonial stem cells, triggering differentiation at low levels and stimulating self-renewal at higher doses. The trophic effects of GDNF are mediated by a receptor complex consisting of two components, GDNF family receptor (GFR) and the rearranged during transformation (RET) transmembrane tyrosine kinase receptor. All GFLs share the receptor tyrosine kinase RET as their common signaling receptor. The ligand-binding specificity of GFLs is determined by GFRαproteins that have unique binding affinities for each GFL. First of all, GDNF specifically bind to GFRα-1. The complex containing GDNF and GFRα-1, then brings two molecules of RET together, triggering transphosphorylation of specific tyrosine residues in their tyrosine kinase domains and intracellular signaling. RET activates several intracellular signaling cascades, which regulate cell survival, differentiation, proliferation, migration, chemotaxis, branching morphogenesis, neurite outgrowth and synaptic plasticity. Interestingly, a comparison of mRNA levels in 15 organs of mouse by in situ hybridization revealed that GDNF was the most prominently expressed in the ovary. Several reports have demonstrated the presence of GDNFmRNA in the adult murine ovary within the granular layer of developing follicle. A recent report suggests that GDNF stimulates oocyte nuclear and cytoplasmic maturation and cumulus cell expansion within cumulus-oocyte complexes, also enhancing the developmental competence of porcine oocyte. Another reporter suggests potential paracrine roles of GDNF in the promotion of completion of meiosisⅠand the development of early embryos. However, the expression of GDNF and its receptors in human luteinized granulosa cells, to date, and the mechanism of regulation of GDNF have not been investigated. Here, on the basis of other researches about GDNF we investigate the expression of GDNF, GFRa-1, RET in human preovulatory luteinized granulosa cells and treatment of cultured granulosa cells with gonadotropins demonstrate the regulation of gonadotropins on their mRNA levels to explore mechanism of GDNF in follicular development and maturation.
PartⅠCulture and identification of luteinized granulosa cells from human ovary
OBJECTIVE
To establish a pure and stable experimental cell model, we purify and identify cultured luteinized granulosa cells from human ovary by observation of cell morphology and growth cycle.
MATERIALS AND METHODS
The eight subjects recruited in this study were undergoing IVF for tubal factor or male factor reason at the Nanfang Hospital Reproductive Center from May to December in 2009. The research protocol was approved by the University and informed written consent was obtained from all participants. The patients were subjected to controlled ovarian hyperstimulation (COH) accomplished with a modified long luteal suppression protocol in an age from 25 to 30 years, basic FSH<7.5mIU/L, body mass index (BMI) varied from 19kg/m2 to 23kg/m2 and serum estrodiol(E2) concentration<4000pg/L on the day of human chorionic gonadotropin (HCG), without ovulation disorder and have a normal menstrual cycle.
Luteinized granulosa cells were isolated from follicular aspiration by gradient centrifugation. Briefly, pooled aspirates were layered onto 45% percoll and centrifuged at 1500r/min×20min. The luteinized granulosa cells removed from the gradient-media interface were washed with PBS twice.2×106/L cells were cultured on 35mm plates at 37℃,5%CO2 with 2ml of culture medium containing 20% female fetal calf serum (FCS) and 1% antibiotic-antimycotic cocktail. After 24 hours the medium was replaced and washed by PBS to remove cellular debris and suspended cells. Cell morphology and viability were observed every 24 hours and taken photos at the same time.
Luteinized granulosa cells identification:The 2ml of purified suspension of human granulosa cells was dropped onto the polylysine treated glass slides and put into incubator for 24 hours. Then cell samples fixed in 4% paraformaldehyde were processed for paraffin embedding about 30 minutes. After that these samples were incubated with the primary rabbit polyclonal antibodies against FSHR (diluted:1:50). Slide incubated with the secondary antibody alone served as negative controls. Images were captured by an image analysis computer system.
RESULTS
1. Clinical characteristics of eight patients
There were eight patients recruited in this study and the average age was 26.4±1.2 years and BMI was (21.3±1.3) kg/m2, basic FSH level was (6.8±0.5)mIU/mL. The average serum E2 level on the day of HCG was (1493.2±755.1)pg/ml.
2. Primary human luteinized granulosa cell culture
Human luteinized granulosa cells were slowly growing under the culture contition. These cell aggregates in culture attached to culture dish within 24 hours and maintained a healthy granular appearance throughout the period of culture in contrast to the spherical shape when seeded. A few erythrocytes were visible in the initial cell culture and loosely attached to the bottom of dish but did not persist which were were totally displaced at 24 hours as judged by microscopy.The generation of pseudopodia increased during the subsequent 24 hours. At 48 hours, the monolayer cells appeared to be widespread and multiple interconnections between the cells were noted. The generation of pseudopodia seemed not to proceed after 72 hours of incubation. Cells were flattened and had a fibroblast-like appearance with stress fibres at 96 hours and part of granular material dispeared within a few granulosa cells. At 120 hours, part of granulosa cells were seemed to typical luteal phenotype with hypertrophic cell bodies, more granular material and short pseudopodia. Generally, cell death was observed by microscopy at 216 hours with attached cells suspending. In the culture of 15 days, all granulosa cells were generally smaller and had either irregular or elongated forms resembling fibroblasts which were different from the typical granulosa cell morphology.
3. Identification of human luteinized granulosa cell
FSHR was used for identification of granulosa cells specifically as it present exclusively on granulosa cells of ovarian follicles in females. The red-brown staining indicated FSHR protein expression in the luteinized granulosa cells (70%-80% cytoplasmic without nuclear staining).
CONCLUSION
1. Density gradient centrifugation with Percoll is used widely in granulosa cell purification and erythrocytes are removed effectively as judged by microscopy.
2. In the culture medium of RPMI-1640 containing 20% female fetal calf serum (FCS) and 1% antibiotic-antimycotic cocktail, since degenerative changes did not been observed until 96 hours, primary granulosa cells were appropriate for experiment in vitro before 96 hours.
3. By identification, the purity of human luteinized granulosa cell in culture is up to 80% and appropriate for experiments in vitro.
PartⅡExpression of GDNF、GFR-1、Ret in human luteinized granulosa cells
OBJECTIVE
To investigation the expression of GDNF and its receptors, GFRα-1 and Ret in human luteinized granulosa cells at the protein levels by immunocytochemistry.
MATERIALs AND METHODS
The fiftheen subjects recruited in this study were undergoing IVF for tubal factor or male factor reason at the Nanfang Hospital Reproductive Center. The research protocol was approved by the University and informed written consent was obtained from all participants. The patients were subjected to COH accomplished with a modified long luteal suppression protocol in an age from 25 to 30 years, basic FSH<7.5mIU/L, BMI varied from 19kg/m2 to 23kg/m2 and serum E2<4000pg/L on the day of HCG, without ovulation disorder and have a normal menstrual cycle.
After purification by density gradient centrifugation with 45% Percoll, the 2ml of suspension of human granulosa cells was dropped onto the polylysine treated glass slides and put into incubator for 24 hours. Then cell samples fixed in 4% paraformaldehyde were processed for paraffin embedding about 30 minutes. After that these samples were incubated with the primary antibodies (diluted:1:200,1:200, not diluted) as follows:rabbit polyclonal antibodies against GDNF and GFRα-1, mouse monoclonal antibodies against Ret. Slide incubated with the secondary antibody alone served as negative controls. Images were captured by an image analysis computer system.
RESULTS
1. Clinical characteristics of fifteen patients
There were fifteen patients recruited in this study and the average age was 26.7±2.0 years and BMI was (21.5±1.2)kg/m2, basic FSH level was (6.7±0.6) mIU/mL. The average serum E2 level on the day of HCG was (2009.6±850.7)pg/ml.
2. The characteristics cellular localization of GDNF in luteinized granulosa cells included full cytoplasmic staining in 100% and nuclear staining in 97%.
3. The characteristics cellular localization of GFRa-1 in luteinized granulosa cells included full cytoplasmic staining in 100% and nuclear staining in 55%.
4. The characteristics cellular localization of Ret in luteinized granulosa cells included full cytoplasmic staining in 47% and nuclear staining in 17%.
CONCLUSION
The presence of the GDNF and its receptors in human preovulatory luteinized granulosa cells suggest that GDNF may act as an important autocrine/paracrine regulator during follicular development and maturation process.
PartⅢRegulation of gonadotropins on GDNF、GFR-1、RET mRNA levels in human luteinized granulosa cells
OBJECTIVE
To explore whether FSH, human menopausal gonadotropin (HMG) may regulate the mRNA levels of GDNF and its receptors by treatment of cell culture with gonadotropins. This study is noteworthy and may suggest the mechanism of GDNF in follicular development and maturation process.
MATERIALS AND METHODS
Samples from five patients were recruited in this study by using random blocks design. Luteinized granulosa cells from every one participant were divided into four groups randomly.2×105 cells were cultured on 35mm culture dish for 24 hours, then rinsed two times with PBS. RPMI-1640 was added with the experimental treatment of culture containing HMG5IU/L (5IU/L group), HMG10IU/L (10IU/L group), FSH 10IU/L (FSH group) respectively and without gonadotropins as blank control group. For quantitative real-time RT-PCR to detect transcripts of GDNF, GFRa-1 and RET by SYBR Green staining, human luteinized granulosa cells were collected at 48 hours after treatment. Each result was repeated at least three times.
SPSS 13.0 statistical package was used for date analysis. Relative mRNA level data was expressed by mean±standard deviation and analyzed statistically by using two-way classification ANOVA for multifactorial analysis of vatiance. And data was further analyzed using the Student-Newman-Keuls test for multiple comparisons to determine statistical differences between groups. All values were two-tailed, and P<0.05 was considered statistically.
RESULTS
1. Clinical characteristics of five patients
There were five patients recruited in this study and the average age was 27.2±1.9 years and BMI was (21.7±0.7)kg/m2, basic FSH level was (6.8±0.5)mIU/mL. The average serum E2 level on the day of HCG was (1892.2±396.4)pg/ml.
2. Relative GDNFmRNA levels
Expression of GDNFmRNA are significantly differences between 5IU/Lgroup, 10IU/Lgroup, FSH group, and control group(F=22.308, P<0.01). GDNFmRNA was enhanced up to 4.37 fold by HMG at concentrations of 10IU/L and 2.76 fold by HMG at concentrations of 5IU/L compared to blank control group significantly. Block design was effective as statistical significances between patients (F=6.283, P=0.006). Stimulation of GDNFmRNA by HMG was demonstrated to be dose dependent by further analysis using the SNK test for multiple comparisons. However, FSH failed to stimulate GDNFmRNA by cultured luteinized granulosa cells at the concentration of 10IU/L.
2. Relative GFRα-1mRNA levels
Expression of GFRα-1mRNA are significantly differences between 5IU/Lgroup, lOIU/Lgroup, FSH group, and control group(F= 18.985, P<0.01). GFRα-1mRNA was enhanced up to 4.86 fold by HMG at concentrations of 10IU/L and 3.23 fold by HMG at concentrations of 5IU/L compared to blank control group significantly. Block design was effective as statistical significances between patients (F=4.568, P=0.018). Stimulation of GFRα-1mRNA by HMG was demonstrated to be dose dependent by further analysis using the SNK test for multiple comparisons. However, FSH failed to stimulate GFRα-1mRNA by cultured luteinized granulosa cells at the concentration of 10IU/L
3. Relative RETmRNA levels
Expression of RETmRNA are not significantly different between the four groups. 10IU/L FSH,10IU/L HMG and 5IU/L HMG failed to stimulate RETmRNA by cultured luteinized granulosa cells.
CONCLUSION
1. FSH failed to stimulate GDNFmRNA, GFRα-1mRNA, and RETmRNA by treatment of culture containing 10IU/L FSH. It suggests that FSH at the concentration of 10IU/L can not up-regulate their mRNA levels, but the effect of homeostasis of FSH on GDNF expression can not be ruled out.
2. HMG up-regulated GDNFmRNA and GFRa-1mRNA with increasing dosage of HMG in a dose-dependent manner. As FSH failed to stimulate their mRNA LH contained in HMG may played a major role in up-regulation of GDNFmRNA and GFRa-1mRNA. This suggests that GDNF may be one of the most important regulators which is regulated by LH surge in oocyte maturation and ovulation induction.
3. HMG and FSH can not up-regulate expression of RETmRNA by cultured luteinized granulosa cells at the concentration of 5IU/L,10IU/L and 10IU/L respectively. This suggests that other GDNF signal pathways may involved in the process of follicular development and maturation except for GDNF-GFRα-Ret pathway.
引文
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