颗粒细胞端粒酶及相关因子的表达变化对卵巢功能及卵裂球的影响
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摘要
卵巢具有产生卵细胞和分泌激素的功能。卵巢中的卵泡与卵子发育成熟、排卵及合成和分泌激素及某些细胞因子有关,是调控女性生殖的关键。颗粒细胞通过缝隙连接与卵母细胞紧密接触,对卵泡的形成和发育,及卵母细胞的营养和成熟起重要作用。颗粒细胞是卵巢合成和分泌抑制素、雌激素和孕激素的主要功能细胞,有高度分裂增殖的潜能。从原始卵泡生长启动、闭锁或排卵到黄体形成,颗粒细胞在形态、功能等方面发生了各种变化。而其增殖、分化、凋亡机制及其调控非常复杂,牵涉到复杂的分子作用机制和信号传导通路。因而研究颗粒细胞是研究卵巢功能的一个极好的切入点,而对体外受精促排卵过程中获得的黄素化颗粒细胞进行体外培养,则是获得人颗粒细胞并对其进行系统研究的较好的实验方法。
卵巢功能失调可导致多种妇科内分泌疾病及不孕。因而调节卵巢功能、促排卵以及不孕症的相关治疗在妇科临床工作中具有重要意义。年龄增长和/或卵巢功能下降,可以导致卵泡闭锁加剧,促排卵时卵巢反应性差,获得的卵母细胞少,且卵母细胞质量下降,卵裂球质量差,助孕后妊娠率低下。目前激素类药物虽广泛使用,并取得了显著的疗效,但仍有其局限性和副作用,特别是对年龄小而卵巢功能衰退者,其促排卵效果不好。在我们以往的研究中发现,信号转导途径中关键调节因子p53、p19ARF、Rb和p16在HPO轴衰老中协同变化,而预防性应用何首乌饮可通过抑制性腺轴中衰老途径的关键因子而起到抗衰老的作用,何首乌饮能改善下丘脑-垂体-卵巢轴的功能。然而,何首乌饮是否以卵巢颗粒细胞为靶点,直接影响卵巢功能尚无充分证据。
颗粒细胞的凋亡与卵泡闭锁有关,而端粒酶活性及相关因子的表达变化在颗粒细胞的凋亡过程中可能起重要作用,其对卵巢功能、卵裂球的发育的影响还不十分清楚。通过电耦相连的颗粒细胞和卵母细胞之间的联系,可能代表了一种重要的信号传导通路。颗粒细胞的去极化是卵母细胞成熟的重要特征。颗粒细胞电活动的调节为研究其细胞功能提供了一种新方法。钾离子通道是目前发现的亚型数目最多的一类细胞膜离子通道,在调控细胞动作电位的形成、膜复极化、维持静息电位的水平以及在细胞的分泌等一系列细胞生理活动方面起着重要作用。钾离子通道在肿瘤细胞的增殖、分化等中的研究较多,而在人卵巢颗粒细胞上的研究较少,并与颗粒细胞功能有何联系尚不十分清楚。
基于上述,本研究第一部分从进行体外受精患者的卵泡液中分离黄素化颗粒细胞,TRAP-ELISA、RT-PCR、免疫细胞化学方法测定端粒酶的活性及凋亡相关基因及蛋白的表达,探讨端粒酶与凋亡相关因子对卵巢功能和卵裂球的影响;第二部分则在我们以前研究的基础上,为了深入了解何首乌饮对卵巢自身功能的影响,以体外培养的SD大鼠排卵前卵泡颗粒细胞为实验对象,血清药理学的方法为给药手段,TRAP-ELISA、流式细胞术、Western blot观察何首乌饮对颗粒细胞端粒酶活性、凋亡率和凋亡相关蛋白的影响,探讨何首乌饮对其干预作用;第三部分基于少量人卵巢颗粒细胞上钾离子通道表达的研究现状,应用RT-PCR方法证实人卵巢黄素化颗粒细胞存在电压门控钾离子通道mRNA的表达,并用化学发光法、MTT法、流式细胞术、分光光度法分析其被选择性阻滞剂4-氨基吡啶阻滞后,对颗粒细胞分泌及增殖、凋亡的影响,以期对颗粒细胞功能调控与卵泡发育、闭锁及卵母细胞成熟等生理、病理学问题提供一些理论基础,对不孕症的诊断和防治提供新思路。
第一部分人黄素化颗粒细胞端粒酶活性及凋亡相关基因、蛋白的表达变化对卵巢功能及卵裂球的影响
目的
探讨人黄素化颗粒细胞端粒酶的活性、凋亡相关基因bcl-2和p53及蛋白的表达与相应卵巢反应性和卵裂球发育的关系。
方法
收集行体外受精-胚胎移植(in vitro fertilization-embryo transfer, IVF-ET)患者卵泡液中的黄素化颗粒细胞(granulosa cells, GCs)28例,并进行体外培养;根据卵泡数不同将卵巢反应性分组分为:低反应组、中反应组和高反应组;根据优质卵裂球(胚)率进行卵裂球质量分组分为:低质量组和高质量组。端粒重复序列扩增酶联免疫吸附分析法(telomeric repeat amplification protocol-enzyme linked immunoadsordent assay,TRAP-ELISA)测定颗粒细胞端粒酶活性;免疫细胞化学方法检测颗粒细胞bcl-2和p53蛋白表达;逆转录聚合酶链反应(reverse transcriptase- polymerase chain reaction, RT-PCR)测定颗粒细胞bcl-2 mRNA和p53 mRNA表达。分析人黄素化颗粒细胞端粒酶的活性、bcl-2和p53蛋白及mRNA表达与卵巢反应性及相应卵裂球发育的关系。
结果
1.分离提纯的人黄素化颗粒细胞于24h内贴壁生长良好。
2.TRAP-ELISA结果显示:随着患者的年龄、使用促性腺激素(gonadotropin, Gn)量的增加,端粒酶活性ΔA值逐渐降低,随着获卵数的增加端粒酶活性ΔA值逐渐上升,即端粒酶活性与年龄、Gn量呈负相关(r=-0.4931, P<0.05; r=-0.4238, P<0.05),端粒酶活性与获卵数呈正相关(r=0.4906, P<0.05);高反应组、中反应组和低反应组端粒酶活性差异有显著统计学意义(F=6.8693, P<0.01),低反应组与中反应组、高反应组之间差异均有显著统计学意义(q=4.7172, P<0.01; q=4.8770, P<0.01);随着优质卵裂球率的升高,端粒酶活性也逐渐升高,二者呈正相关(r=0.6968, P<0.01);高质量组与低质量组比较,端粒酶活性差异有显著统计学意义(t=3.2983, P<0.01)。
3.免疫细胞化学结果显示:随着获卵数的增加,bcl-2蛋白HSCORE评分(以下简称HSCbcl-2)逐渐上升,HSCbcl-2与获卵数呈显著正相关(r=0.6183, P<0.01),随着Gn量的增加、获卵数的降低,p53蛋白HSCORE评分(以下简称HSCp53)逐渐升高,HSCp53与Gn量呈正相关(r=0.4237, P<0.05),与获卵数呈负相关(r=-0.5954, P<0.05);高反应组、中反应组与低反应组间HSCbcl-2和HSCp53差异均有统计学意义(F=5.2058, P<0.05; F=13.757, P<0.01);随着优质卵裂球率的升高,HSCbcl-2逐渐升高,二者呈显著正相关(r=0.5888, P<0.01),而HSCp53逐渐下降,二者呈显著负相关(r=-0.6343, P<0.01);高质量组与低质量组比较, HSCbcl-2与HSCp53差异均有统计学意义(t=2.2325, P<0.05; t=3.070, P<0.01)。
4.RT-PCR结果显示:bcl-2 mRNA表达与年龄呈显著负相关(r=-0.5667, P<0.01),与获卵数呈正相关(r=0.4584, P<0.05),而p53 mRNA表达与年龄、Gn量呈正相关(r=0.4370, P<0.05; r=0.5305, P<0.05),与获卵数呈负相关(r=-0.4439, P<0.05);高反应组、中反应组与低反应组间bcl-2 mRNA和p53mRNA表达差异均有统计学意义(F=3.5653, P<0.05; F=3.4913, P<0.05);随着优质卵裂球率的升高,bcl-2 mRNA表达逐渐升高,二者呈显著正相关(r=0.6861, P<0.01),而p53 mRNA表达逐渐下降,二者呈显著负相关(r=-0.6238, P<0.01);高质量组与低质量组比较,bcl-2 mRNA与p53 mRNA表达差异均有统计学意义(t=3.2114, P<0.01; t=2.4763, P<0.05)。
结论
1.体外培养的人黄素化颗粒细胞存在端粒酶活性的表达。
2.黄素化颗粒细胞端粒酶活性与年龄、Gn量呈负相关,与获卵数呈正相关,提示端粒酶活性降低与卵巢功能的下降有关。优质卵裂球率与端粒酶活性有明显的相关性,颗粒细胞的凋亡可能影响卵母细胞的发育潜能。3.凋亡相关基因bcl-2、p53及蛋白的表达与卵巢反应性和卵裂球发育有明显的相关性,并与端粒酶活性之间具有相关性。Bcl-2及p53基因可能参与调节端粒酶活性的表达,可作为我们研究端粒酶活性与卵巢功能关系的一座桥梁。
第二部分何首乌饮含药血清对大鼠卵巢颗粒细胞端粒酶活性及凋亡相关蛋白的影响
目的
何首乌饮是具有抗衰老作用的方剂。其疗效和对下丘脑-垂体-卵巢轴的调节效应已被以往的实验研究所证实。本实验拟从卵巢颗粒细胞水平入手,了解中药何首乌饮体内代谢产物在调节卵泡微环境过程中所起的作用,探讨其对卵泡生长、发育、成熟与闭锁的调控机制。
方法
用外源性激素孕马血清促性腺激素制备排卵前卵巢模型,收集颗粒细胞,经预培养后,分别与雌性SD幼鼠空白血清及含不同剂量何首乌饮的药物血清在体外共同培养。TRAP-ELISA方法分析颗粒细胞端粒酶活性;运用流式细胞术分析颗粒细胞凋亡、坏死情况;Western blot检测颗粒细胞凋亡相关基因bcl-2, bax的蛋白表达情况;分光光度法检测颗粒细胞caspase-3活性。
结果
1.TRAP-ELISA结果显示:各剂量组何首乌饮含药血清可上调颗粒细胞端粒酶活性,各组比较差异有显著统计学意义(F=8.4996, P<0.01),与空白组比较,中、高剂量含药血清组能提高端粒酶活性(q=3.9745, P<0.05; q=6.5839, P<0.01)。
2.流式细胞术结果显示:随含药血清药物剂量的增加,颗粒细胞凋亡率逐渐下降,各组凋亡细胞百分率比较差异有显著统计学意义(F=13.2252, P<0.01),各含药血清组细胞坏死率虽较空白组高,但各组比较差异无统计学意义(P>0.05);与空白组比较,中、高剂量组含药血清均能够显著降低颗粒细胞凋亡率(q=5.5814, P<0.01; q=8.3490, P<0.01)。
3.Western blot结果显示:含药血清组颗粒细胞bcl-2蛋白表达上调,bax蛋白表达下调,各组比较差异有显著统计学意义(F=20.3710, P<0.01; F=13.8507, P<0.01);中、高剂量组含药血清与空白组比较,明显上调bcl-2蛋白表达,差异有显著的统计学意义(q=5.4043, P<0.01; q=10.0812, P<0.01),中、高剂量组含药血清与空白组比较,明显下调bax蛋白表达,差异有显著的统计学意义(q=6.1898, P<0.01; q=7.4314, P<0.01)。
4.分光光度法caspase-3活性结果显示:含药血清可下调颗粒细胞caspase-3活性,各组比较差异有显著统计学意义(F= 9.2098, P<0.01),中、高剂量组与空白组比较差异有显著统计学意义(q=4.6755, P<0.01; q=6.7289, P<0.01)。
结论
1.何首乌饮含药血清可上调离体培养的大鼠卵巢颗粒细胞端粒酶活性,并抑制自身的凋亡。
2.何首乌饮含药血清可增加颗粒细胞内凋亡抑制基因bcl-2蛋白表达,减少促凋亡基因bax蛋白表达,这可能是其抑制颗粒细胞凋亡,防止卵泡闭锁的作用机制之一。
3.何首乌饮含药血清可下调颗粒细胞caspase-3蛋白的表达,其可能是在凋亡的较早阶段,通过阻断caspase-3的级联裂解发挥凋亡抑制作用。
4.对卵泡局部微环境的调节,可能是何首乌饮调整下丘脑-垂体-卵巢轴功能的作用靶点之一。
第三部分电压门控钾离子通道对人黄素化颗粒细胞孕酮分泌及增殖凋亡的影响
目的
用RT-PCR方法证实人卵巢黄素化颗粒细胞存在电压门控钾离子通道(voltage-gated K+ channel, Kv)mRNA的表达,分析其阻滞剂4-氨基吡啶(4-Aminopyridine, 4-AP)对人绒毛膜促性腺激素(human chorionic gonadotropin, hCG)诱导的颗粒细胞分泌及增殖、凋亡的作用,探讨Kv对颗粒细胞功能的影响。
方法
收集25例行体外受精患者卵泡液中的黄素化颗粒细胞,采用RT-PCR检测颗粒细胞中Kv mRNA的表达。将培养贴壁后的颗粒细胞按干预方式分为空白组、4-AP组、hCG组和hCG+4-AP组共4组,hCG和4-AP的终浓度分别为1250 IU/L、5 nmol/L。培养24 h后,采用化学发光法测定各组培养液孕酮水平;采用流式细胞术和分光光度法,以膜连蛋白V(annexin V)/碘化吡啶(PI)和caspase-3活性为检测指标,检测各组颗粒细胞的凋亡情况;采用四甲基偶氮唑蓝(methylthiazolyl tetrazolium, MTT)比色法检测各组颗粒细胞的增殖、抑制情况。
结果
1.RT-PCR结果显示:空白组、4-AP组、hCG组和hCG+4-AP组人黄素化颗粒细胞均可以扩增出280 bp的Kv mRNA产物。
2.培养24 h后,空白组、4-AP组、hCG组和hCG+4-AP组培养液中的孕酮水平分别为(173.9933±15.9642)、(64.3867±8.7411)、(628.2333±54.1370)和(240.5867±24.8367)ng/ml。四组间比较差异具有显著的统计学意义(F=421.4680, P<0.01)。4-AP组、hCG组分别与空白组比较,差异均有统计学意义(t= 23.3237, P<0.01; t’= 31.1696, P<0.05);hCG+4-AP组与hCG组比较,差异也有显著的统计学意义(t= 23.3237, P<0.01)。
3.MTT比色法结果显示:空白组、4-AP组、hCG组和hCG+4-AP组A值分别为0.633±0.138、0.508±0.116、0.661±0.159和0.432±0.145。与空白组比较,4-AP组、hCG组和hCG+4-AP组增殖率分别为80.3%、100.4%和68.2%。与空白组比较,4-AP组的抑制率为19.7%,两组比较差异具有统计学意义(t=2.6871, P<0.05);与hCG组比较,hCG+4-AP组的抑制率为34.6%,两组比较差异具有显著统计学意义(t=4.1422, P<0.01)。
4.annexin V/PI凋亡率及caspase-3活性结果显示:4-AP组分别为(40.13±4.85)%和0.0494±0.00939 ,均高于空白组[(17.24±3.69)%和0.0286±0.00759],差异均有显著统计学意义(t=14.5630, P<0.01; t=6.6594,P<0.01);hCG+4-AP组[(24.86±4.13)%和0.0387±0.00794]也均高于hCG组[(14.55±3.19)%和0.0219±0.00663],差异亦有显著统计学意义(t=7.6481, P<0.01; t=6.2742, P<0.01)。
结论
1.人黄素化颗粒细胞存在Kv mRNA的表达。
2.Kv阻滞剂4-AP能够抑制人卵巢黄素化颗粒细胞基础及hCG诱导下的孕酮的分泌。
3.4-AP可抑制颗粒细胞的增殖,促进颗粒细胞凋亡,其抑制颗粒细胞孕酮分泌可能与其抑制增殖、促进凋亡有关。
4.Kv在卵巢黄素化颗粒细胞分泌、增殖及凋亡过程中可能起重要作用。
综上所述,我们的研究发现:(1)体外培养的人卵巢黄素化颗粒细胞存在端粒酶活性的表达;其端粒酶活性、凋亡相关基因bcl-2及蛋白表达与卵巢反应性、卵裂球的发育有关;(2)何首乌饮含药血清上调离体培养的大鼠卵巢颗粒细胞端粒酶活性,上调bcl- 2蛋白表达、下调bax蛋白表达,是其抑制颗粒细胞凋亡,防止卵泡闭锁的作用途径之一;对卵泡局部微环境的调节,可能是何首乌饮调整下丘脑-垂体-卵巢轴功能的作用靶点之一;(3)人卵巢黄素化颗粒细胞存在Kv mRNA的表达;其阻滞剂4-AP能够抑制基础及hCG诱导下的孕酮的分泌,并可能与其抑制增殖,促进凋亡有关;Kv在卵巢颗粒细胞分泌、增殖及凋亡过程中可能起重要作用。
Endocrine function and ovulation are the physiological function of ovarian. Follicle is the basic functional unit in ovarian including not only occyte development, maturity and ovulation but also production of hormones and certain cytokines. Follicle may be the key in reproduction. With the gap junction granulosa cells can contact with oocyte closely and play an important role in follicle development and oocyte maturation. Granulosa cells are the main functional cells in synthesis and secretion of inhibin, estradiol and progesterone with high proliferation potential. From the growth, atresia of primaril follicle or ovulation to corpus luteum, granulosa cells have changed in form and function. And the mechanisms of granulosa cells proliferation, differentiation and apoptosis are extremely complex, including complex molecular mechanisms and signal transduction pathways. So the study in granulosa cells may be an excellent starting point in studying ovarian function and luteinized granulosa cells in vitro cultured may be a good method to value ovarian function and the quality of embryos.
Ovarian dysfunction may lead to a variety of gynecological endocrine diseases and infertility. Adjustment of ovarian function, controlled ovarian hyperstimulation and treatment of infertility may be important in gynecology. Decreased oocytes and embryos quality because of aging or ovarian dysfunction may increase follicular atresia and lead to the lower response and pregnancy rate in controlled ovarian hyperstimulation. Hormone drugs has made significant effect, but still have limitations and side effects, especially in controlled ovarian hyperstimulation with young but lower ovarian response. In previous studies of teacher it was found that in the signal transduction pathway critical regulator p53, p19ARF, Rb and p16 changes in the collaborative in the aging of entire gonadal axis(HPOA). Preventive application of Hswy may play the role of anti-aging by inhibiting the key factor of aging in the gonadal axis. Hswy may improve and adjust the function of HPOA. However, there was no evidence of a direct impact on ovarian function.
Follicular atresia may be correlated with granulosa cells apoptosis. Telomerase activity, apoptosis-related mRNA and protein expression may play important roles in granulosa cells apoptosis. But the effects of them to ovarian function and quality of embryos were not very clear. The electrical activity of granulosa cells may be a new method to study the regulation of cell function. It may represent an important signal transduction pathway through the electrical connected link between granulosa cells and oocyte. Potassium ion channel is the largest number of subtypes found in a class of membrane ion channels. It plays an important role in regulating the formation of cells action potential, membrane depolarization and maintaining the level of resting potential as well as a series of physiological activities such as secretion and nervous of muscle cells. So far, the research of potassium ion channels in proliferation, differentiation in nerve-muscle cells and tumor cell is more fully, but the research in human ovarian granulosa cell and the linked function are still not been very clear.
In this study, we first isolated luteinized granulosa cells from follicular fluid of in vitro fertilization(IVF) patients. By using TRAP-ELISA, RT-PCR, immunocytochemical methods, we have detected telomerase activity and apoptosis-related genes bcl-2 and protein expression, and compared these factors with ovarian response and embryo quality. To understand Hswy on ovarian function deeply, in the second part of this study, we have used the method of serum pharmacology to study SD rats granulosa cells from preovulatory follicles in vitro. TRAP-ELISA, RT-PCR, Western blotting were used to detect telomerase activity, apoptosis rate and apoptosis-related genes bcl-2 and protein expression to explore Hswy’s interventional role. Finally, by RT-PCR we found the expression of voltage-gated potassium channels (Kv) mRNA in human luteinized granulosa cell based on a small number of researches in potassium ion channel in human ovarian granulosa cells. Chemoluminescence method, MTT method, flow cytometry, spectrophotometric analysis methods were used to study the influence of proliferation, differentiation, and apoptosis by 4-aminopyridine(4-AP) through inhibiting Kv in human ovarian luteinized GC with a view to provide some theoretical basis of granulosa cell function in regulating. Our findings may provide an essential background for experimental definition of granulosa cell and may represent novel targets for assisted reproduction.
PartⅠStudy on the relationship between telomerase activity, apoptosis-related mRNA and protein expression of human luteined granulosa cells with ovarian response and embryo quality
Objective
To explore the relationship between telomerase activity, bcl-2, p53 mRNA and protein expression of human luteined granulosa cells with ovarian response and embryo quality.
Methods
Ovarian luteinized granulosa cells were recovered from 28 women underwent in vitro fertilization. The following procedures were carried out: 1) luteined granulosa cells were isolated from follicle fluid and were cultured 24 hours soon. 2) tests of the telomerase activity with TRAP-ELISA. 3) tests of the expression of apoptosis-related bcl-2, p53 proteins by using immuocytochemistry. 4) tests of the expression of apoptosis-related bcl-2, p53 mRNA by using RT-PCR. Comparisons of age, number of ampules of Gn administered, number of retrieved oocytes, ratio of high quality embryo and telomerase activity, protein and mRNA expression of bcl-2, p53 were made in different ovarian response and embryo quality groups.
Results
1.Isolated and purified GCs were cultured and adherenced well in 24 hours.
2.TRAP-ELISA showed: there were negative correlation between telomerase activity with age, number of ampules of Gn administered and positive correlation with number of retrieved oocytes (r=-0.4931, P<0.05; r=-0.4238, P<0.05; r=0.4906, P<0.05 respectively). There were significant difference of telomerase activity in different ovarian response groups (F=6.8693, P<0.01). Low response group had lower telomerase activity compared to medium and high response groups (q=4.7172, P<0.01; q=4.8770, P<0.01 respectively). There were positive correlation between telomerase activity with ratio of high embryo quality (r=0.6968, P<0.01). There were significant difference of telomerase activity in different embryo quality groups (t=3.2983, P<0.01).
3.Immuocytochemistry showed: there were positive correlation between the score of bcl-2 protein expression(HSCbcl-2) and the number of retrieved oocytes (r=0.6183, P<0.01); there were positive correlation between the score of p53 protein expression(HSCp53) with the number of ampules of Gn administered and negative correlation with the number of retrieved oocytes (r=0.4237, P<0.05; r=-0.5954, P<0.05 respectively). There were difference of HSCbcl-2 and HSCp53 in different ovarian response groups(F=5.2058, P<0.05; F=13.757, P<0.01). There were positive correlation between the HSCbcl-2 and negative correlation between the HSCp53 with ratio of high embryo quality(r=0.5888, P<0.01; r=-0.6343, P<0.01 respectively). There were difference of the HSCbcl-2 and HSCp53 in different embryo quality groups (t=2.2325, P<0.05; t=3.070, P<0.01 respectively).
4.RT-PCR showed: there were negative correlation between bcl-2 mRNA expression with age and positive correlation with the number of retrieved oocytes(r=-0.5667, P<0.01; r=0.4584, P<0.05 respectively); there were positive correlation between p53 mRNA expression with age, the number of ampules of Gn administered and negative correlation with the number of retrieved oocytes(r=0.4370, P<0.05; r=0.5305, P<0.05; r=-0.4439, P<0.05 respectively). There were difference of bcl-2 and p53 mRNA expression in different ovarian response groups(F=3.5653, P<0.05; F=3.4913, P<0.05 respectively). There were positive correlation between the HSCbcl-2 and negative correlation between the HSCp53 with ratio of high embryo quality significantly(r=0.6861, P<0.01; r=-0.6238, P<0.01 respectively). There were difference of bcl-2 and p53 mRNA expression in different embryo quality groups (t=3.2114, P<0.01; t=2.4763, P<0.05 respectively).
Conclusions:
1.Telomerase activity could be tested in human luteinized granulosa cells after in vitro cultured.
2.Telomerase activity in granulosa cells were negatively correlated with age, the number of ampules of Gn administered and positively correlated with the number of retrieved oocytes. Those may suggest that telomerase activity decreased with the decline of ovarian function.
3.There were obviously relevant between telomerase activity and ratio of high quality embryo, the apoptosis of granulosa cells directly affect embryo quality. There were clear correlation between apoptosis-related bcl-2, p53 mRNA and protein expression with ovarian response and embryo quality, and significant correlation with telomerase activity. Bcl-2, p53 gene which may regulate the expression of telomerase activity could be used as a bridge to study the relationship between ovarian function in our research.
PartⅡEffects of serum containing Hswy on telomerase activity, apoptosis ratio and apoptosis-related protein expression of rat ovarian granulosa cells
Objective
To investigate the effect of Hswy decoction on function in cultured rat ovarian granulosa cells, which in the purpose to explain the mechanism that the decoction is to promote follicular development.
Methods
PMSG treated female S.D. rats were used as a model and ovarian granulosa cells were prepared. Granulosa cells were cultured in groups. Two groups were set up: study group with rat serum contained Hswy decoction in different dosage and blank group with normal serum. Granulosa cells were cultured in the medium containing 10% different rat serum at 37℃. After 24 hours, telomerase activity were measured by telomeric repeat amplification protocol-enzyme linked immunoadsordent assay(TRAP-ELISA), the percentage of apoptosis and death of granulosa cells were quantitatively assessed by flow cytometry(FCM) analysis, bcl-2 and bax protein expression were assessed by Western blotting and the activity of cellular caspase-3 were measured by colorimetric method.
Results
1.TRAP-ELISA illustrated: the serum with medicine could increase telomerase activity, there were different in groups(F=8.4996, P<0.01 respectively); compared with blank serum group, medium and high dosage groups could increase telomerase activity obviously (q=3.9745, P<0.05; q=6.5839, P<0.01 respectively).
2.FCM showed: the percentage of apoptosis in study group was less than that in blank serum group(F=13.2252, P<0.01); and the percentage of death in study group was same as that in blank serum group(P>0.05); compared with blank serum group, medium and high dosage serum groups could decrease the apoptosis of granulosa cells significantly(q=5.5814, P<0.01; q=8.3490, P<0.01 respectively).
3.Western blotting showed: the serum with medicine could increase the expression of bcl-2 protein and decrease the expression of bax protein (F=20.371, P<0.01; F=13.8507, P<0.01 respectively); compared with blank serum group, medium and high dosage groups could obviously increase bcl-2 protein and decrease bax protein expression (q=5.4043, P<0.01; q=10.0812, P<0.01 and q=6.1898, P<0.01; q=7.4314, P<0.01 respectively).
4.Colorimetric method illustrated: the activity of caspase-3 of granulosa cells in study groups may be lower than the blank serum group(F= 9.2098, P<0.01); compared with blank serum group, medium and high dosage serum groups could decrease the activity of caspase-3 obviously(q=5.4043, P<0.01; q=10.0812, P<0.01).
Conclusions
1. Hswy decoction may increase telomerase activity and decrease ratio of apoptosis of rat granulosa cells in vitro cultured.
2.Hswy decoction may increase the protein expression of bcl-2 and decrease the protein expression of bax. This may be one possible mechanism inhibiting granulosa cells apoptosis and follicle atresia.
3.Hswy decoction may decrease caspase-3 protein expression which may be in the earlier stages of apoptosis, by blocking the cascade of caspase-3. 4.Adjustment of follicular local micro-environment may be one target of Hswy decoction function in hypothalamus - pituitary - ovarian axis.
PartⅢInfluence of 4-aminopyridine on human ovarian luteinized granulosa cells proliferation, differentiation, and apoptosis through inhibiting voltage-gated K+ channel
Objective
To study the influence of proliferation, differentiation, and apoptosis by 4-aminopyridine(4-AP) through inhibiting voltage-gated K+ channel(Kv) in human ovarian luteinized granulosa cells.
Methods
Ovarian luteinized granulosa cells were recovered from 25 women with regular menses who underwent in vitro fertilization programmed. The cultured granulosa cells were divided in 4 groups:blank group, 4-AP treated group, human chorionic gonadotropin(hCG)-induced group and hCG+4-AP co-treated group, and the final concentration of hCG and 4-AP were 1250 IU/L, 5 nmol/L respectively. The progesterone production was detected by chemoluminescence method. The expression of Kv mRNA on human ovarian luteinized granulosa cell was detected by reverse transcription polymerase chain reaction(RT-PCR). The influence on the early apoptosis of granulosa cells by 4-AP was observed by flow cytometry, cellular caspase-3 activities were observed with colorimetric method and the inhibition of the cell proliferation was studied using methyl thiazolyl tetrazolium(MTT) method.
Results
1.Kv mRNA were expressed in human luteinized granulosa cell of the blank group, 4-AP treated group, hCG-induced group and hCG+4-AP co-treated group.
2.The progesterone production of the blank group, 4-AP treated group, hCG-induced group and hCG+4-AP co-treated group were (173.9933±15.9642), (64.3867±8.7411), (628.2333±54.1370) and (240.5867±24.8367)ng/ml respectively after 24 h cultured. Exposure of the granulosa cells to 4-AP reduced the differentiation of progesterone in blank and hCG-induced granulosa cells.
3.24h after treated with 4-AP and hCG, the A value of MTT method of blank group, 4-AP treated group, hCG-induced group and hCG+4-AP co-treated group were 0.633±0.138, 0.508±0.116, 0.661±0.159 and 0.432±0.145 respectively. The inhibitory rate by MTT method of cultured granulosa cells of 4-AP treated group was higher than the blank group (19.7% vs. 0) (t=2.6871, P<0.05), hCG+4-AP co-treated group was higher obviously than hCG-induced group (34.6% vs. 0) (t=4.1422, P<0.01).
4.The flow cytometry analysis and the cellular caspase-3 A405 showed that 4-AP increased the percentage of early phase apoptosis. 4-AP treated group vs. blank group [(40.13±4.85)% and 0.0494±0.00939] vs. [(17.24±3.69)% and 0.0286±0.00759] (t=14.5630, P<0.01; t=6.6594, P<0.01), 4-AP+hCG co-treated group vs. hCG-induced group[(24.86±4.13)% and 0.0387±0.00794] vs. [(14.55±3.19)% and 0.0219±0.00663] (t=7.6481, P<0.01; t=6.2742, P<0.01).
Conclusions
1.The voltage-gated K+ channels mRNA may be expressed in human ovarian luteinized granulosa cell.
2.4-AP may inhibit production of progesterone in base and hCG-induced granulosa cells.
3.4-AP may inhibit the proliferation of granulose cells and induce apoptosis of granulose cells. The effect of 4-AP to production of progesterone may be due to the inhabitation of proliferation and induction of apoptosis.
4.4-AP may play an important role in cell proliferation, differentiation and apoptosis.
To sum up, our study found that: (1) Telomerase activity could be tested in human luteinized granulosa cells after in vitro cultured. These may suggest that telomerase activity, apoptosis-related bcl-2 mRNA and protein expression decreased with the decline in ovarian function and embryo quality. (2) Hswy decoction could improve ovarian function by inhibiting apoptosis, increasing telomerase activity, the protein expression of bcl-2 and decreasing the expression of bax. (3) The voltage-gated K+ channels mRNA may be expressed in human ovarian luteinized granulosa cell. Due to the inhabitation of proliferation and induction of apoptosis of granulosa cells, 4-AP may inhibit production of progesterone in hCG-induced granulosa cells. Voltage-gated K+ channels may play an important role in granulosa cells proliferation, differentiation and apoptosis.
卵巢功能失调可导致多种妇科内分泌疾病及不孕。因而调节卵巢功能、促排卵以及不孕症的相关治疗在妇科临床工作中具有重要意义。年龄增长和/或卵巢功能下降,可以导致卵泡闭锁加剧,促排卵时卵巢反应性差,获得的卵母细胞少,且卵母细胞质量下降,卵裂球质量差,助孕后妊娠率低下。目前激素类药物虽广泛使用,并取得了显著的疗效,但仍有其局限性和副作用,特别是对年龄小而卵巢功能衰退者,其促排卵效果不好。在我们以往的研究中发现,信号转导途径中关键调节因子p53、p19ARF、Rb和p16在HPO轴衰老中协同变化,而预防性应用何首乌饮可通过抑制性腺轴中衰老途径的关键因子而起到抗衰老的作用,何首乌饮能改善下丘脑-垂体-卵巢轴的功能。然而,何首乌饮是否以卵巢颗粒细胞为靶点,直接影响卵巢功能尚无充分证据。
颗粒细胞的凋亡与卵泡闭锁有关,而端粒酶活性及相关因子的表达变化在颗粒细胞的凋亡过程中可能起重要作用,其对卵巢功能、卵裂球的发育的影响还不十分清楚。通过电耦相连的颗粒细胞和卵母细胞之间的联系,可能代表了一种重要的信号传导通路。颗粒细胞的去极化是卵母细胞成熟的重要特征。颗粒细胞电活动的调节为研究其细胞功能提供了一种新方法。钾离子通道是目前发现的亚型数目最多的一类细胞膜离子通道,在调控细胞动作电位的形成、膜复极化、维持静息电位的水平以及在细胞的分泌等一系列细胞生理活动方面起着重要作用。钾离子通道在肿瘤细胞的增殖、分化等中的研究较多,而在人卵巢颗粒细胞上的研究较少,并与颗粒细胞功能有何联系尚不十分清楚。
基于上述,本研究第一部分从进行体外受精患者的卵泡液中分离黄素化颗粒细胞,TRAP-ELISA、RT-PCR、免疫细胞化学方法测定端粒酶的活性及凋亡相关基因及蛋白的表达,探讨端粒酶与凋亡相关因子对卵巢功能和卵裂球的影响;第二部分则在我们以前研究的基础上,为了深入了解何首乌饮对卵巢自身功能的影响,以体外培养的SD大鼠排卵前卵泡颗粒细胞为实验对象,血清药理学的方法为给药手段,TRAP-ELISA、流式细胞术、Western blot观察何首乌饮对颗粒细胞端粒酶活性、凋亡率和凋亡相关蛋白的影响,探讨何首乌饮对其干预作用;第三部分基于少量人卵巢颗粒细胞上钾离子通道表达的研究现状,应用RT-PCR方法证实人卵巢黄素化颗粒细胞存在电压门控钾离子通道mRNA的表达,并用化学发光法、MTT法、流式细胞术、分光光度法分析其被选择性阻滞剂4-氨基吡啶阻滞后,对颗粒细胞分泌及增殖、凋亡的影响,以期对颗粒细胞功能调控与卵泡发育、闭锁及卵母细胞成熟等生理、病理学问题提供一些理论基础,对不孕症的诊断和防治提供新思路。
第一部分人黄素化颗粒细胞端粒酶活性及凋亡相关基因、蛋白的表达变化对卵巢功能及卵裂球的影响
目的
探讨人黄素化颗粒细胞端粒酶的活性、凋亡相关基因bcl-2和p53及蛋白的表达与相应卵巢反应性和卵裂球发育的关系。
方法
收集行体外受精-胚胎移植(in vitro fertilization-embryo transfer, IVF-ET)患者卵泡液中的黄素化颗粒细胞(granulosa cells, GCs)28例,并进行体外培养;根据卵泡数不同将卵巢反应性分组分为:低反应组、中反应组和高反应组;根据优质卵裂球(胚)率进行卵裂球质量分组分为:低质量组和高质量组。端粒重复序列扩增酶联免疫吸附分析法(telomeric repeat amplification protocol-enzyme linked immunoadsordent assay,TRAP-ELISA)测定颗粒细胞端粒酶活性;免疫细胞化学方法检测颗粒细胞bcl-2和p53蛋白表达;逆转录聚合酶链反应(reverse transcriptase- polymerase chain reaction, RT-PCR)测定颗粒细胞bcl-2 mRNA和p53 mRNA表达。分析人黄素化颗粒细胞端粒酶的活性、bcl-2和p53蛋白及mRNA表达与卵巢反应性及相应卵裂球发育的关系。
结果
1.分离提纯的人黄素化颗粒细胞于24h内贴壁生长良好。
2.TRAP-ELISA结果显示:随着患者的年龄、使用促性腺激素(gonadotropin, Gn)量的增加,端粒酶活性ΔA值逐渐降低,随着获卵数的增加端粒酶活性ΔA值逐渐上升,即端粒酶活性与年龄、Gn量呈负相关(r=-0.4931, P<0.05; r=-0.4238, P<0.05),端粒酶活性与获卵数呈正相关(r=0.4906, P<0.05);高反应组、中反应组和低反应组端粒酶活性差异有显著统计学意义(F=6.8693, P<0.01),低反应组与中反应组、高反应组之间差异均有显著统计学意义(q=4.7172, P<0.01; q=4.8770, P<0.01);随着优质卵裂球率的升高,端粒酶活性也逐渐升高,二者呈正相关(r=0.6968, P<0.01);高质量组与低质量组比较,端粒酶活性差异有显著统计学意义(t=3.2983, P<0.01)。
3.免疫细胞化学结果显示:随着获卵数的增加,bcl-2蛋白HSCORE评分(以下简称HSCbcl-2)逐渐上升,HSCbcl-2与获卵数呈显著正相关(r=0.6183, P<0.01),随着Gn量的增加、获卵数的降低,p53蛋白HSCORE评分(以下简称HSCp53)逐渐升高,HSCp53与Gn量呈正相关(r=0.4237, P<0.05),与获卵数呈负相关(r=-0.5954, P<0.05);高反应组、中反应组与低反应组间HSCbcl-2和HSCp53差异均有统计学意义(F=5.2058, P<0.05; F=13.757, P<0.01);随着优质卵裂球率的升高,HSCbcl-2逐渐升高,二者呈显著正相关(r=0.5888, P<0.01),而HSCp53逐渐下降,二者呈显著负相关(r=-0.6343, P<0.01);高质量组与低质量组比较, HSCbcl-2与HSCp53差异均有统计学意义(t=2.2325, P<0.05; t=3.070, P<0.01)。
4.RT-PCR结果显示:bcl-2 mRNA表达与年龄呈显著负相关(r=-0.5667, P<0.01),与获卵数呈正相关(r=0.4584, P<0.05),而p53 mRNA表达与年龄、Gn量呈正相关(r=0.4370, P<0.05; r=0.5305, P<0.05),与获卵数呈负相关(r=-0.4439, P<0.05);高反应组、中反应组与低反应组间bcl-2 mRNA和p53mRNA表达差异均有统计学意义(F=3.5653, P<0.05; F=3.4913, P<0.05);随着优质卵裂球率的升高,bcl-2 mRNA表达逐渐升高,二者呈显著正相关(r=0.6861, P<0.01),而p53 mRNA表达逐渐下降,二者呈显著负相关(r=-0.6238, P<0.01);高质量组与低质量组比较,bcl-2 mRNA与p53 mRNA表达差异均有统计学意义(t=3.2114, P<0.01; t=2.4763, P<0.05)。
结论
1.体外培养的人黄素化颗粒细胞存在端粒酶活性的表达。
2.黄素化颗粒细胞端粒酶活性与年龄、Gn量呈负相关,与获卵数呈正相关,提示端粒酶活性降低与卵巢功能的下降有关。优质卵裂球率与端粒酶活性有明显的相关性,颗粒细胞的凋亡可能影响卵母细胞的发育潜能。3.凋亡相关基因bcl-2、p53及蛋白的表达与卵巢反应性和卵裂球发育有明显的相关性,并与端粒酶活性之间具有相关性。Bcl-2及p53基因可能参与调节端粒酶活性的表达,可作为我们研究端粒酶活性与卵巢功能关系的一座桥梁。
第二部分何首乌饮含药血清对大鼠卵巢颗粒细胞端粒酶活性及凋亡相关蛋白的影响
目的
何首乌饮是具有抗衰老作用的方剂。其疗效和对下丘脑-垂体-卵巢轴的调节效应已被以往的实验研究所证实。本实验拟从卵巢颗粒细胞水平入手,了解中药何首乌饮体内代谢产物在调节卵泡微环境过程中所起的作用,探讨其对卵泡生长、发育、成熟与闭锁的调控机制。
方法
用外源性激素孕马血清促性腺激素制备排卵前卵巢模型,收集颗粒细胞,经预培养后,分别与雌性SD幼鼠空白血清及含不同剂量何首乌饮的药物血清在体外共同培养。TRAP-ELISA方法分析颗粒细胞端粒酶活性;运用流式细胞术分析颗粒细胞凋亡、坏死情况;Western blot检测颗粒细胞凋亡相关基因bcl-2, bax的蛋白表达情况;分光光度法检测颗粒细胞caspase-3活性。
结果
1.TRAP-ELISA结果显示:各剂量组何首乌饮含药血清可上调颗粒细胞端粒酶活性,各组比较差异有显著统计学意义(F=8.4996, P<0.01),与空白组比较,中、高剂量含药血清组能提高端粒酶活性(q=3.9745, P<0.05; q=6.5839, P<0.01)。
2.流式细胞术结果显示:随含药血清药物剂量的增加,颗粒细胞凋亡率逐渐下降,各组凋亡细胞百分率比较差异有显著统计学意义(F=13.2252, P<0.01),各含药血清组细胞坏死率虽较空白组高,但各组比较差异无统计学意义(P>0.05);与空白组比较,中、高剂量组含药血清均能够显著降低颗粒细胞凋亡率(q=5.5814, P<0.01; q=8.3490, P<0.01)。
3.Western blot结果显示:含药血清组颗粒细胞bcl-2蛋白表达上调,bax蛋白表达下调,各组比较差异有显著统计学意义(F=20.3710, P<0.01; F=13.8507, P<0.01);中、高剂量组含药血清与空白组比较,明显上调bcl-2蛋白表达,差异有显著的统计学意义(q=5.4043, P<0.01; q=10.0812, P<0.01),中、高剂量组含药血清与空白组比较,明显下调bax蛋白表达,差异有显著的统计学意义(q=6.1898, P<0.01; q=7.4314, P<0.01)。
4.分光光度法caspase-3活性结果显示:含药血清可下调颗粒细胞caspase-3活性,各组比较差异有显著统计学意义(F= 9.2098, P<0.01),中、高剂量组与空白组比较差异有显著统计学意义(q=4.6755, P<0.01; q=6.7289, P<0.01)。
结论
1.何首乌饮含药血清可上调离体培养的大鼠卵巢颗粒细胞端粒酶活性,并抑制自身的凋亡。
2.何首乌饮含药血清可增加颗粒细胞内凋亡抑制基因bcl-2蛋白表达,减少促凋亡基因bax蛋白表达,这可能是其抑制颗粒细胞凋亡,防止卵泡闭锁的作用机制之一。
3.何首乌饮含药血清可下调颗粒细胞caspase-3蛋白的表达,其可能是在凋亡的较早阶段,通过阻断caspase-3的级联裂解发挥凋亡抑制作用。
4.对卵泡局部微环境的调节,可能是何首乌饮调整下丘脑-垂体-卵巢轴功能的作用靶点之一。
第三部分电压门控钾离子通道对人黄素化颗粒细胞孕酮分泌及增殖凋亡的影响
目的
用RT-PCR方法证实人卵巢黄素化颗粒细胞存在电压门控钾离子通道(voltage-gated K+ channel, Kv)mRNA的表达,分析其阻滞剂4-氨基吡啶(4-Aminopyridine, 4-AP)对人绒毛膜促性腺激素(human chorionic gonadotropin, hCG)诱导的颗粒细胞分泌及增殖、凋亡的作用,探讨Kv对颗粒细胞功能的影响。
方法
收集25例行体外受精患者卵泡液中的黄素化颗粒细胞,采用RT-PCR检测颗粒细胞中Kv mRNA的表达。将培养贴壁后的颗粒细胞按干预方式分为空白组、4-AP组、hCG组和hCG+4-AP组共4组,hCG和4-AP的终浓度分别为1250 IU/L、5 nmol/L。培养24 h后,采用化学发光法测定各组培养液孕酮水平;采用流式细胞术和分光光度法,以膜连蛋白V(annexin V)/碘化吡啶(PI)和caspase-3活性为检测指标,检测各组颗粒细胞的凋亡情况;采用四甲基偶氮唑蓝(methylthiazolyl tetrazolium, MTT)比色法检测各组颗粒细胞的增殖、抑制情况。
结果
1.RT-PCR结果显示:空白组、4-AP组、hCG组和hCG+4-AP组人黄素化颗粒细胞均可以扩增出280 bp的Kv mRNA产物。
2.培养24 h后,空白组、4-AP组、hCG组和hCG+4-AP组培养液中的孕酮水平分别为(173.9933±15.9642)、(64.3867±8.7411)、(628.2333±54.1370)和(240.5867±24.8367)ng/ml。四组间比较差异具有显著的统计学意义(F=421.4680, P<0.01)。4-AP组、hCG组分别与空白组比较,差异均有统计学意义(t= 23.3237, P<0.01; t’= 31.1696, P<0.05);hCG+4-AP组与hCG组比较,差异也有显著的统计学意义(t= 23.3237, P<0.01)。
3.MTT比色法结果显示:空白组、4-AP组、hCG组和hCG+4-AP组A值分别为0.633±0.138、0.508±0.116、0.661±0.159和0.432±0.145。与空白组比较,4-AP组、hCG组和hCG+4-AP组增殖率分别为80.3%、100.4%和68.2%。与空白组比较,4-AP组的抑制率为19.7%,两组比较差异具有统计学意义(t=2.6871, P<0.05);与hCG组比较,hCG+4-AP组的抑制率为34.6%,两组比较差异具有显著统计学意义(t=4.1422, P<0.01)。
4.annexin V/PI凋亡率及caspase-3活性结果显示:4-AP组分别为(40.13±4.85)%和0.0494±0.00939 ,均高于空白组[(17.24±3.69)%和0.0286±0.00759],差异均有显著统计学意义(t=14.5630, P<0.01; t=6.6594,P<0.01);hCG+4-AP组[(24.86±4.13)%和0.0387±0.00794]也均高于hCG组[(14.55±3.19)%和0.0219±0.00663],差异亦有显著统计学意义(t=7.6481, P<0.01; t=6.2742, P<0.01)。
结论
1.人黄素化颗粒细胞存在Kv mRNA的表达。
2.Kv阻滞剂4-AP能够抑制人卵巢黄素化颗粒细胞基础及hCG诱导下的孕酮的分泌。
3.4-AP可抑制颗粒细胞的增殖,促进颗粒细胞凋亡,其抑制颗粒细胞孕酮分泌可能与其抑制增殖、促进凋亡有关。
4.Kv在卵巢黄素化颗粒细胞分泌、增殖及凋亡过程中可能起重要作用。
综上所述,我们的研究发现:(1)体外培养的人卵巢黄素化颗粒细胞存在端粒酶活性的表达;其端粒酶活性、凋亡相关基因bcl-2及蛋白表达与卵巢反应性、卵裂球的发育有关;(2)何首乌饮含药血清上调离体培养的大鼠卵巢颗粒细胞端粒酶活性,上调bcl- 2蛋白表达、下调bax蛋白表达,是其抑制颗粒细胞凋亡,防止卵泡闭锁的作用途径之一;对卵泡局部微环境的调节,可能是何首乌饮调整下丘脑-垂体-卵巢轴功能的作用靶点之一;(3)人卵巢黄素化颗粒细胞存在Kv mRNA的表达;其阻滞剂4-AP能够抑制基础及hCG诱导下的孕酮的分泌,并可能与其抑制增殖,促进凋亡有关;Kv在卵巢颗粒细胞分泌、增殖及凋亡过程中可能起重要作用。
Endocrine function and ovulation are the physiological function of ovarian. Follicle is the basic functional unit in ovarian including not only occyte development, maturity and ovulation but also production of hormones and certain cytokines. Follicle may be the key in reproduction. With the gap junction granulosa cells can contact with oocyte closely and play an important role in follicle development and oocyte maturation. Granulosa cells are the main functional cells in synthesis and secretion of inhibin, estradiol and progesterone with high proliferation potential. From the growth, atresia of primaril follicle or ovulation to corpus luteum, granulosa cells have changed in form and function. And the mechanisms of granulosa cells proliferation, differentiation and apoptosis are extremely complex, including complex molecular mechanisms and signal transduction pathways. So the study in granulosa cells may be an excellent starting point in studying ovarian function and luteinized granulosa cells in vitro cultured may be a good method to value ovarian function and the quality of embryos.
Ovarian dysfunction may lead to a variety of gynecological endocrine diseases and infertility. Adjustment of ovarian function, controlled ovarian hyperstimulation and treatment of infertility may be important in gynecology. Decreased oocytes and embryos quality because of aging or ovarian dysfunction may increase follicular atresia and lead to the lower response and pregnancy rate in controlled ovarian hyperstimulation. Hormone drugs has made significant effect, but still have limitations and side effects, especially in controlled ovarian hyperstimulation with young but lower ovarian response. In previous studies of teacher it was found that in the signal transduction pathway critical regulator p53, p19ARF, Rb and p16 changes in the collaborative in the aging of entire gonadal axis(HPOA). Preventive application of Hswy may play the role of anti-aging by inhibiting the key factor of aging in the gonadal axis. Hswy may improve and adjust the function of HPOA. However, there was no evidence of a direct impact on ovarian function.
Follicular atresia may be correlated with granulosa cells apoptosis. Telomerase activity, apoptosis-related mRNA and protein expression may play important roles in granulosa cells apoptosis. But the effects of them to ovarian function and quality of embryos were not very clear. The electrical activity of granulosa cells may be a new method to study the regulation of cell function. It may represent an important signal transduction pathway through the electrical connected link between granulosa cells and oocyte. Potassium ion channel is the largest number of subtypes found in a class of membrane ion channels. It plays an important role in regulating the formation of cells action potential, membrane depolarization and maintaining the level of resting potential as well as a series of physiological activities such as secretion and nervous of muscle cells. So far, the research of potassium ion channels in proliferation, differentiation in nerve-muscle cells and tumor cell is more fully, but the research in human ovarian granulosa cell and the linked function are still not been very clear.
In this study, we first isolated luteinized granulosa cells from follicular fluid of in vitro fertilization(IVF) patients. By using TRAP-ELISA, RT-PCR, immunocytochemical methods, we have detected telomerase activity and apoptosis-related genes bcl-2 and protein expression, and compared these factors with ovarian response and embryo quality. To understand Hswy on ovarian function deeply, in the second part of this study, we have used the method of serum pharmacology to study SD rats granulosa cells from preovulatory follicles in vitro. TRAP-ELISA, RT-PCR, Western blotting were used to detect telomerase activity, apoptosis rate and apoptosis-related genes bcl-2 and protein expression to explore Hswy’s interventional role. Finally, by RT-PCR we found the expression of voltage-gated potassium channels (Kv) mRNA in human luteinized granulosa cell based on a small number of researches in potassium ion channel in human ovarian granulosa cells. Chemoluminescence method, MTT method, flow cytometry, spectrophotometric analysis methods were used to study the influence of proliferation, differentiation, and apoptosis by 4-aminopyridine(4-AP) through inhibiting Kv in human ovarian luteinized GC with a view to provide some theoretical basis of granulosa cell function in regulating. Our findings may provide an essential background for experimental definition of granulosa cell and may represent novel targets for assisted reproduction.
PartⅠStudy on the relationship between telomerase activity, apoptosis-related mRNA and protein expression of human luteined granulosa cells with ovarian response and embryo quality
Objective
To explore the relationship between telomerase activity, bcl-2, p53 mRNA and protein expression of human luteined granulosa cells with ovarian response and embryo quality.
Methods
Ovarian luteinized granulosa cells were recovered from 28 women underwent in vitro fertilization. The following procedures were carried out: 1) luteined granulosa cells were isolated from follicle fluid and were cultured 24 hours soon. 2) tests of the telomerase activity with TRAP-ELISA. 3) tests of the expression of apoptosis-related bcl-2, p53 proteins by using immuocytochemistry. 4) tests of the expression of apoptosis-related bcl-2, p53 mRNA by using RT-PCR. Comparisons of age, number of ampules of Gn administered, number of retrieved oocytes, ratio of high quality embryo and telomerase activity, protein and mRNA expression of bcl-2, p53 were made in different ovarian response and embryo quality groups.
Results
1.Isolated and purified GCs were cultured and adherenced well in 24 hours.
2.TRAP-ELISA showed: there were negative correlation between telomerase activity with age, number of ampules of Gn administered and positive correlation with number of retrieved oocytes (r=-0.4931, P<0.05; r=-0.4238, P<0.05; r=0.4906, P<0.05 respectively). There were significant difference of telomerase activity in different ovarian response groups (F=6.8693, P<0.01). Low response group had lower telomerase activity compared to medium and high response groups (q=4.7172, P<0.01; q=4.8770, P<0.01 respectively). There were positive correlation between telomerase activity with ratio of high embryo quality (r=0.6968, P<0.01). There were significant difference of telomerase activity in different embryo quality groups (t=3.2983, P<0.01).
3.Immuocytochemistry showed: there were positive correlation between the score of bcl-2 protein expression(HSCbcl-2) and the number of retrieved oocytes (r=0.6183, P<0.01); there were positive correlation between the score of p53 protein expression(HSCp53) with the number of ampules of Gn administered and negative correlation with the number of retrieved oocytes (r=0.4237, P<0.05; r=-0.5954, P<0.05 respectively). There were difference of HSCbcl-2 and HSCp53 in different ovarian response groups(F=5.2058, P<0.05; F=13.757, P<0.01). There were positive correlation between the HSCbcl-2 and negative correlation between the HSCp53 with ratio of high embryo quality(r=0.5888, P<0.01; r=-0.6343, P<0.01 respectively). There were difference of the HSCbcl-2 and HSCp53 in different embryo quality groups (t=2.2325, P<0.05; t=3.070, P<0.01 respectively).
4.RT-PCR showed: there were negative correlation between bcl-2 mRNA expression with age and positive correlation with the number of retrieved oocytes(r=-0.5667, P<0.01; r=0.4584, P<0.05 respectively); there were positive correlation between p53 mRNA expression with age, the number of ampules of Gn administered and negative correlation with the number of retrieved oocytes(r=0.4370, P<0.05; r=0.5305, P<0.05; r=-0.4439, P<0.05 respectively). There were difference of bcl-2 and p53 mRNA expression in different ovarian response groups(F=3.5653, P<0.05; F=3.4913, P<0.05 respectively). There were positive correlation between the HSCbcl-2 and negative correlation between the HSCp53 with ratio of high embryo quality significantly(r=0.6861, P<0.01; r=-0.6238, P<0.01 respectively). There were difference of bcl-2 and p53 mRNA expression in different embryo quality groups (t=3.2114, P<0.01; t=2.4763, P<0.05 respectively).
Conclusions:
1.Telomerase activity could be tested in human luteinized granulosa cells after in vitro cultured.
2.Telomerase activity in granulosa cells were negatively correlated with age, the number of ampules of Gn administered and positively correlated with the number of retrieved oocytes. Those may suggest that telomerase activity decreased with the decline of ovarian function.
3.There were obviously relevant between telomerase activity and ratio of high quality embryo, the apoptosis of granulosa cells directly affect embryo quality. There were clear correlation between apoptosis-related bcl-2, p53 mRNA and protein expression with ovarian response and embryo quality, and significant correlation with telomerase activity. Bcl-2, p53 gene which may regulate the expression of telomerase activity could be used as a bridge to study the relationship between ovarian function in our research.
PartⅡEffects of serum containing Hswy on telomerase activity, apoptosis ratio and apoptosis-related protein expression of rat ovarian granulosa cells
Objective
To investigate the effect of Hswy decoction on function in cultured rat ovarian granulosa cells, which in the purpose to explain the mechanism that the decoction is to promote follicular development.
Methods
PMSG treated female S.D. rats were used as a model and ovarian granulosa cells were prepared. Granulosa cells were cultured in groups. Two groups were set up: study group with rat serum contained Hswy decoction in different dosage and blank group with normal serum. Granulosa cells were cultured in the medium containing 10% different rat serum at 37℃. After 24 hours, telomerase activity were measured by telomeric repeat amplification protocol-enzyme linked immunoadsordent assay(TRAP-ELISA), the percentage of apoptosis and death of granulosa cells were quantitatively assessed by flow cytometry(FCM) analysis, bcl-2 and bax protein expression were assessed by Western blotting and the activity of cellular caspase-3 were measured by colorimetric method.
Results
1.TRAP-ELISA illustrated: the serum with medicine could increase telomerase activity, there were different in groups(F=8.4996, P<0.01 respectively); compared with blank serum group, medium and high dosage groups could increase telomerase activity obviously (q=3.9745, P<0.05; q=6.5839, P<0.01 respectively).
2.FCM showed: the percentage of apoptosis in study group was less than that in blank serum group(F=13.2252, P<0.01); and the percentage of death in study group was same as that in blank serum group(P>0.05); compared with blank serum group, medium and high dosage serum groups could decrease the apoptosis of granulosa cells significantly(q=5.5814, P<0.01; q=8.3490, P<0.01 respectively).
3.Western blotting showed: the serum with medicine could increase the expression of bcl-2 protein and decrease the expression of bax protein (F=20.371, P<0.01; F=13.8507, P<0.01 respectively); compared with blank serum group, medium and high dosage groups could obviously increase bcl-2 protein and decrease bax protein expression (q=5.4043, P<0.01; q=10.0812, P<0.01 and q=6.1898, P<0.01; q=7.4314, P<0.01 respectively).
4.Colorimetric method illustrated: the activity of caspase-3 of granulosa cells in study groups may be lower than the blank serum group(F= 9.2098, P<0.01); compared with blank serum group, medium and high dosage serum groups could decrease the activity of caspase-3 obviously(q=5.4043, P<0.01; q=10.0812, P<0.01).
Conclusions
1. Hswy decoction may increase telomerase activity and decrease ratio of apoptosis of rat granulosa cells in vitro cultured.
2.Hswy decoction may increase the protein expression of bcl-2 and decrease the protein expression of bax. This may be one possible mechanism inhibiting granulosa cells apoptosis and follicle atresia.
3.Hswy decoction may decrease caspase-3 protein expression which may be in the earlier stages of apoptosis, by blocking the cascade of caspase-3. 4.Adjustment of follicular local micro-environment may be one target of Hswy decoction function in hypothalamus - pituitary - ovarian axis.
PartⅢInfluence of 4-aminopyridine on human ovarian luteinized granulosa cells proliferation, differentiation, and apoptosis through inhibiting voltage-gated K+ channel
Objective
To study the influence of proliferation, differentiation, and apoptosis by 4-aminopyridine(4-AP) through inhibiting voltage-gated K+ channel(Kv) in human ovarian luteinized granulosa cells.
Methods
Ovarian luteinized granulosa cells were recovered from 25 women with regular menses who underwent in vitro fertilization programmed. The cultured granulosa cells were divided in 4 groups:blank group, 4-AP treated group, human chorionic gonadotropin(hCG)-induced group and hCG+4-AP co-treated group, and the final concentration of hCG and 4-AP were 1250 IU/L, 5 nmol/L respectively. The progesterone production was detected by chemoluminescence method. The expression of Kv mRNA on human ovarian luteinized granulosa cell was detected by reverse transcription polymerase chain reaction(RT-PCR). The influence on the early apoptosis of granulosa cells by 4-AP was observed by flow cytometry, cellular caspase-3 activities were observed with colorimetric method and the inhibition of the cell proliferation was studied using methyl thiazolyl tetrazolium(MTT) method.
Results
1.Kv mRNA were expressed in human luteinized granulosa cell of the blank group, 4-AP treated group, hCG-induced group and hCG+4-AP co-treated group.
2.The progesterone production of the blank group, 4-AP treated group, hCG-induced group and hCG+4-AP co-treated group were (173.9933±15.9642), (64.3867±8.7411), (628.2333±54.1370) and (240.5867±24.8367)ng/ml respectively after 24 h cultured. Exposure of the granulosa cells to 4-AP reduced the differentiation of progesterone in blank and hCG-induced granulosa cells.
3.24h after treated with 4-AP and hCG, the A value of MTT method of blank group, 4-AP treated group, hCG-induced group and hCG+4-AP co-treated group were 0.633±0.138, 0.508±0.116, 0.661±0.159 and 0.432±0.145 respectively. The inhibitory rate by MTT method of cultured granulosa cells of 4-AP treated group was higher than the blank group (19.7% vs. 0) (t=2.6871, P<0.05), hCG+4-AP co-treated group was higher obviously than hCG-induced group (34.6% vs. 0) (t=4.1422, P<0.01).
4.The flow cytometry analysis and the cellular caspase-3 A405 showed that 4-AP increased the percentage of early phase apoptosis. 4-AP treated group vs. blank group [(40.13±4.85)% and 0.0494±0.00939] vs. [(17.24±3.69)% and 0.0286±0.00759] (t=14.5630, P<0.01; t=6.6594, P<0.01), 4-AP+hCG co-treated group vs. hCG-induced group[(24.86±4.13)% and 0.0387±0.00794] vs. [(14.55±3.19)% and 0.0219±0.00663] (t=7.6481, P<0.01; t=6.2742, P<0.01).
Conclusions
1.The voltage-gated K+ channels mRNA may be expressed in human ovarian luteinized granulosa cell.
2.4-AP may inhibit production of progesterone in base and hCG-induced granulosa cells.
3.4-AP may inhibit the proliferation of granulose cells and induce apoptosis of granulose cells. The effect of 4-AP to production of progesterone may be due to the inhabitation of proliferation and induction of apoptosis.
4.4-AP may play an important role in cell proliferation, differentiation and apoptosis.
To sum up, our study found that: (1) Telomerase activity could be tested in human luteinized granulosa cells after in vitro cultured. These may suggest that telomerase activity, apoptosis-related bcl-2 mRNA and protein expression decreased with the decline in ovarian function and embryo quality. (2) Hswy decoction could improve ovarian function by inhibiting apoptosis, increasing telomerase activity, the protein expression of bcl-2 and decreasing the expression of bax. (3) The voltage-gated K+ channels mRNA may be expressed in human ovarian luteinized granulosa cell. Due to the inhabitation of proliferation and induction of apoptosis of granulosa cells, 4-AP may inhibit production of progesterone in hCG-induced granulosa cells. Voltage-gated K+ channels may play an important role in granulosa cells proliferation, differentiation and apoptosis.
引文
1 Rodgers RJ, Irving-Rodgers HF, van Wezel IL, et al.Dynamics of the membrana granulosa during expansion of the ovarian follicular antrum. Mol Cell Endocrinol, 2001, 171(1-2): 41-48
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