抗孕激素RU486和ZK98734对双池培养的颗粒细胞和内泡膜细胞分泌功能…

本文观察了ＲＵ４８６和ＺＫ９８７３４对培养在羊膜双池培养系统中的猪卵巢颗粒细胞和内泡膜细胞在甾体激素生成过程中的影响。生长在单膜两侧的颗粒细胞和内泡膜细胞在加入或不加ＦＳＨ、ＬＨ及不同浓度ＲＵ４８６或ＺＫ９８７３４的条件下培养４８ｈ。用ＲＩＡ测定内、外池培养液中的孕酮（Ｐ）和雌二醇（Ｅ２）的浓度并与单独培养的结果相比较，同时亦与大鼠颗粒细胞的结果作比较。结果表明：１．有ＦＳＨ刺激时，两种抗孕激素均     

利洛司酮抗着床效应及其对子宫内膜前列腺素含量的影响

本文观察抗孕激素利洛司酮 (lilopristone ZK98734)对大鼠着床、子宫内膜 PGs含量及血浆孕酮含量的影响。结果显示 :(1 ) 5 mg/ kg ZK98734可明显抑制胚泡着床 (对照组着床率 72 .5 0± 5 .6 8% ;ZK98734组 43.30± 3.86 % ) ;(2 ) ZK98734可促使子宫内膜内源性PGE2 和 PGF2α含量增加 ,5 mg/ kg、1 6 .5 mg/ kg和 2 5 mg/ kg三种剂量呈正相关剂量效应关系 ;(3) ZK98734明显降低血浆孕酮水平 ,三种剂量呈负相关剂量效应关系。提示 :ZK98734可能通过改变着床前子宫孕酮和前列腺素之间的精细平衡 ,干扰着床前子宫“静态”的生理环境 ,不利于胚泡在子宫内附着和发育 ,从而导致着床率降低     

米非司酮诱导大鼠PCOS动物模型的实验研究

目的:建立抗孕激素米非司酮诱导成年雌性SD大鼠多囊卵巢综合征(P-COS)动物模型,并探讨其意义。方法:选择4～5天规律动情周期的成年雌性SD大鼠,于动情周期第一天皮下注射米非司酮并阴道涂片监测动情周期,8天后称重大鼠,取出卵巢,称重并观察大体解剖及光镜(HE)改变,测定血清LH、T、FSH、E2水平。结果:(1)用药后第4天动情周期开始失去规律性,阴道上皮呈持续角化,提示无排卵。(2)大鼠体重及卵巢单位重量明显增加(P<0.01)。(3)肉眼观察:大鼠卵巢表面的卵泡明显扩张,卵巢色泽及白膜厚度未见明显异常。镜下观察:大鼠卵巢白膜下见多个呈囊性扩张的卵泡及闭锁卵泡,颗粒细胞层减少至2～3层,卵泡内卵母细胞或放射冠消失,黄体数量明显下降并伴有不完全的黄素化。(4)血清LH、T、E2显著升高(P<0.01),FSH浓度降低,但差异无统计学意义(P=0.094)。结论:米非司酮诱导的成年雌性大鼠PCOS模型在内分泌和卵巢形态学的改变与PCOS患者相似,是较理想的PCOS模型。     

RU486对体外大鼠垂体胞液孕激素受体的作用

Sprague-Dawley去卵巢雌性大鼠,经雌激素刺激6天后,取垂体制成胞液。以3~H-R5020为配基,用羟基磷灰石吸附已结合配基的受体分子来测定垂体胞液中孕激素受体的结合活性;由测得的饱和曲线Scatchard作图法计算孕激素受体和配基结合的平衡解离常数(Kd)等于1.8nM,最大结合位点为138fmoles/mg蛋白。RU486可抑制3~H-R5020与垂体胞液孕激素受体的结合,抑制是竞争性的。非标记的RU486、R5020或孕酮都可竞争3~H-R5020与孕激素受体的结合,三者的竞争能力无明显差异。本实验表明在离体情况下RU486可与垂体胞液孕激素受体相结合,其亲和力与R5020或天然配基孕酮相类似。     

Apelin/APJ在PCOS模型大鼠卵巢中的表达及意义

目的:探讨Apelin/APJ在多囊卵巢综合征(PCOS)模型大鼠卵巢组织中的表达。方法:21日龄SD大鼠肌肉注射脱氢表雄酮(DHEA),建立PCOS大鼠模型(实验组,n=15),并以等量溶剂油代替DHEA为对照组(n=15),观察卵巢相对重量;HE染色观察卵巢改变;测定E2、T、P、FSH、LH水平;采用免疫组织化学法观察Apelin/APJ在PCOS卵巢中的分布与表达;荧光定量RT-PCR(quantitative RT-PCR)检测Apelin/APJ mRNA表达,Western-blotting检测Apelin/APJ蛋白表达。结果:apelin/APJ分别表达于大鼠卵泡膜细胞、颗粒细胞;实验组卵泡内膜、颗粒细胞中Apelin/APJ mRNA及蛋白表达均高于对照组(P<0.05)。结论:apelin/APJ的异常表达可能与PCOS复杂的发病有关。     

瘦素调节人卵巢黄素化颗粒细胞功能的体外研究

目的　探讨瘦素、促卵泡激素 (FSH)和胰岛素样生长因子Ⅰ (IGF Ⅰ )对人卵巢黄素化颗粒细胞雌二醇 (E2 )、孕酮 (P)生成的影响及可能的作用机制。方法　培养人黄素化颗粒细胞 ,分别以瘦素 (3 0ng/ml)、FSH (1 0ng/ml)、IGF Ⅰ (30 0ng/ml)以及相同终浓度的瘦素 +FSH、瘦素 +IGF Ⅰ、FSH +IGF Ⅰ、瘦素 +FSH +IGF Ⅰ对其刺激 2 4h ,对照组不加任何药物。对药物作用后的黄素化颗粒细胞行形态学观察、细胞计数 ;用放射免疫法检测培养液中E2 和P水平 ;同时用逆转录聚合酶链反应法对黄素化颗粒细胞行瘦素受体mRNA检测。结果　瘦素、FSH和IGF Ⅰ对黄素化颗粒细胞生长无影响。瘦素对黄素化颗粒细胞E2 生成量无影响 ,对FSH刺激E2 的生成也无影响 ,E2 水平作用前分别为 (0 10 3± 0 0 36 )pmol/10 0 0细胞、(0 32 3± 0 0 4 2 )pmol/10 0 0细胞 ,作用后分别为 (0 12 0± 0 0 0 8)pmol/10 0 0细胞、(0 343± 0 0 34)pmol/10 0 0细胞 ;而对IGF Ⅰ、FSH +IGF Ⅰ刺激黄素化颗粒细胞生成E2 有显著的抑制作用 ,E2 水平作用前后分别为 (0 318± 0 0 37)pmol/10 0 0细胞与 (0 4 93± 0 0 36 )pmol/10 0 0细胞、(0 193± 0 0 2 5 )pmol/10 0 0细胞与 (0 2 5 1± 0 0 33)pmol/10 0 0细胞 (P <0 0 5 ,<0     

排卵的诱导:卵巢对人类促性腺激素和促卵泡成熟素/促黄体生成激素释放激素(FSH/LH-RH)的反应

在评价FSH/LH-RH治疗不育和不孕的潜在价值时,直接研究卵巢对该激素的反应,比之测定用药后释入血和尿中的FSH和LH的量更加确切。本文以注射长效孕激素所致的无排卵妇女为对象,以卵巢滤泡膜和颗粒细胞的有丝分裂以及排卵反应为指标,估价和比较了人类卵巢对单独的和合并应用人绝经期促性腺激素(HMG)和人绒毛膜促性腺激素(HCG)及FSH/LH-RH的反应。所有受试者为有生育力的健康育龄妇女(20—42岁)。对照组和实验组的无排卵妇女均系每6个月肌注长效甲地孕酮250—300毫克或每3个月肌注150毫克或每2个月肌注长效氯地孕酮250毫克。对照组为41名持久性无排卵妇女,至少观察12     

TGF-β_1在PCOS卵巢间质纤维化形成中的作用

目的:探讨TGF-β1在PCOS大鼠卵巢间质纤维化、包膜硬化形成中的作用。方法:利用脱氢表雄酮(DHEA)皮下注射的方法建立PCOS病理模型大鼠20只,用微粒子酶免分析法测定血清性激素E2、T、LH、FSH、LH/FSH、空腹胰岛素(FINS)水平,及光镜下观察PCOS大鼠卵巢的病理结构,透射电镜观察细胞超微结构来验证模型。采用免疫组化法检测PCOS组(n=20)卵巢细胞因子TGF-β1的表达,并与20只正常大鼠相比照。结果:TGF-β1在PCOS组各阶段卵泡卵母细胞、颗粒细胞的表达强度与对照组相比,差异无统计学意义(P>0.05)。而在窦状卵泡膜细胞和卵巢间质细胞中的表达,PCOS组显著高于对照组(P<0.01,P<0.05)。结论:多囊卵巢中TGF-β1表达异常,可能是导致多囊卵巢间质纤维化、包膜增厚的原因之一,TGF-β1也参与PCOS卵泡发育、闭锁的调控。     

再生基因Reg IV在多囊卵巢综合征(PCOS)模型大鼠卵巢中的表达

目的:探讨再生基因Reg IV在多囊卵巢综合征(PCOS)模型大鼠卵巢组织中的表达情况。方法:采用Poretsky法用胰岛素联合hCG诱导SD大鼠建立PCOS模型,观察大鼠体质量变化及卵巢相对质量;测定血清T、LH、FSH、E2、Ins水平;HE染色观察卵巢形态改变;免疫组织化学方法检测Reg IV蛋白在卵巢组织的定位表达;定量RT-PCR和Western blotting分别检测Reg IV在mRNA水平和蛋白水平的表达。结果:Reg IV分别表达于大鼠颗粒细胞、卵泡膜细胞以及黄体等部位;模型组卵巢中Reg IV mRNA及蛋白表达均低于正常对照组(P<0.05)。结论:Reg IV的表达下调可能与PCOS的发病有关。     

胰岛素增加猪卵泡膜细胞黄体生成素刺激的类固醇生物合成

近来一些研究表明胰岛素可增加人、猪和大鼠的卵巢粒层细胞。猪卵巢的卵泡膜细胞合成和分泌的雄激素对粒层细胞合成雌激素起重要作用,胰岛素对卵泡生物合成类固醇的作用和机理用粒层细胞已广泛进行研究,发现猪粒层细胞有胰岛素特异性受体,在猪粒层细胞的培养基中加入胰岛素起促进作用并引起细胞形态特征不同变化和增加类固醇激素的合成率。另外通过培养猪卵泡组织发现胰岛素还可增加黄体生成素(LH)刺激的雄激素的产     

Flow cytometry of porcine ovarian cells: antiprogestins play an important role in progesterone receptor upregulation.

To investigate the mechanism of antiprogestins in the regulation of ovarian function, a dual-chamber culture system was prepared with the amnion membrane of human placenta. Isolated porcine granulosa and thecal cells were grown on both sides of the amnion and co-cultured with or without RU486 and ZK98, 734. After 48 h incubation, the progesterone receptor (PR) and estrogen receptor (ER) of both cells were detected by flow cytometry. Progesterone and estradiol concentrations in the media were measured by radioimmunoassay. The results showed that antiprogestins increased PR contents in both cells; no significant change was found for ER. At the same time the progesterone and estradiol production by granulosa cells was inhibited; the progesterone production by thecal cells was reduced also. These data suggest that progesterone regulates progesterone synthesis. This autocrine/paracrine action may be the approach through which progesterone controls PR upregulation. It could be one mechanism for the inhibition of follicle development and steroidogenic function by antiprogestins.     

RU486 inhibits expression of lysophosphatidic acid induced glycodelin

OBJECTIVE: This study was undertaken to provide evidence for the mode of action of RU486 on glycodelin produced in K562 cells. To show that histiocytes may be a source of glycodelin in leiomyoma. STUDY DESIGN: With the use of K562, a leukemia cell line, the effect of lysophosphatidic acid (LPA), RU486, antioxidants, and ZK112,993 on glycodelin protein and gene expression was studied. Immunocytochemistry for glycodelin and HAM-56 (macrophage) was performed on leiomyoma and myometrium. RESULTS: Incubation of K562 cells with LPA, progesterone, ZK112,933 and RU486 significantly induced the expression of glycodelin protein and messenger RNA. The addition of RU486 to LPA activated cells markedly reduced expression of glycodelin. Addition of ZK112,993, an antiprogestin without antioxidant properties, to LPA activated cells did not reduce glycodelin. Histiocytes in leiomyoma and myometrium co-localize with glycodelin. CONCLUSION: RU486, partly acting as an antioxidant, markedly reduces LPA stimulated glycodelin production. Histiocytes in leiomyoma and myometrium immunostain for glycodelin and suggests a source for glycodelin in leiomyoma.     

Endocrine responses to long-term administration of the antiprogesterone RU486 in patients with pelvic endometriosis.

OBJECTIVE: To examine endocrine and clinical responses to long-term administration of RU486 in patients with endometriosis. DESIGN: Prospective open trial. SETTING: Faculty practice of the authors. PATIENTS, PARTICIPANTS: Six normally cycling women with endometriosis were recruited. INTERVENTIONS: Subjects received RU486 100 mg/d for 3 months. MAIN OUTCOME MEASURE(S): Hormonal changes during RU486 were compared with control data obtained in the preceding cycle during the early follicular phase. Clinical responses were determined by patient assessment and second-look laparoscopy. RESULTS: All women became amenorrheic, and daily urinary levels of ovarian steroid metabolites remained acyclic. Mean luteinizing hormone (LH) (P less than 0.02) and LH pulse amplitude (P less than 0.05) were increased without changes in LH pulse frequency. An antiglucocorticoid effect was demonstrated by an increase in serum cortisol (P less than 0.01) and adrenocorticotropic hormone (P less than 0.05) levels. Treatment resulted in an improvement in pelvic pain in all subjects without significant change in the extent of disease as evaluated by follow-up laparoscopy. CONCLUSIONS: Daily administration of RU486 results in acyclic ovarian function and improvement in the subjective painful symptoms of endometriosis.     

The effects of the antiprogesterone RU486 (Mifepristone) on an endometrial secretory glycan: an immunocytochemical study.

OBJECTIVE: To examine the effects of progesterone (P) receptor blockade by RU486 (Mifepristone; Roussel-Uclaf, Paris, France) on a secretory endometrial glycan recognized by monoclonal antibody D9B1. DESIGN: Retrospective comparison of endometrial biopsies from treated and untreated women from 2 to 8 days after the luteinizing peak (LH) peak. SETTING: Infertility clinic, Jessop Hospital for Women, Sheffield. PATIENTS: Twenty-two normal fertile women received the RU486. A control group of 44 normal fertile women were also assessed. INTERVENTIONS: RU486 was administered to 22 normal women during the first half of the luteal phase and an endometrial biopsy examined 3 days later. MAIN OUTCOME MEASURES: Immunohistochemistry was used to assess the production and secretion of the D9B1 epitope. RESULTS: When the drug was given 2 days after the LH peak, it prevented appearance of the epitope. When RU486 was administered 5 days after the LH peak, epitope already present in gland cells was subsequently secreted. CONCLUSIONS: These data suggest that production of the sialo-oligosaccharide is P-dependent, but secretion through established intracellular pathways is P-independent.     

Progesterone antagonist,RU486, represses LHCGR expression and LH/hCG signaling in cultured luteinized human mural granulosa cells

Progesterone, the main steroid synthesized by the corpus luteum (CL), prepares the uterus for implantation, maintains the CL survival, and induces progesterone auto-secretion. However, the molecular mechanisms involving the progesterone auto-secretion pathways at the luteal phase are not fully understood, especially in humans. We aim to study the molecular mechanism of the progesterone pathway in human granulosa cells. Our model system consists of luteinized human-mural-granulosa-cells (hmGCs) obtained from follicles aspirated during in vitro fertilization (IVF) procedures. hmGCs were seeded in culture and were subjected to different hormonal treatments. mRNA levels were analyzed by quantitative real-time PCR (qRT-PCR). Progesterone levels were measured by enzyme immunoassay (EIA). We show that exposure of luteinized hmGCs to the progesterone receptor antagonist, RU486 (mifepristone), resulted in inhibition of LHCGR, LH/hCG target genes and progesterone secretion. Exposure of hmGCs to medium that was incubated with hmGCs for 4?d – conditioned medium (CM), which contain 150?±?7.5?nM progesterone, resulted in induction of LHCGR and LH/hCG target genes, which was blocked by RU486. In addition, RU486 inhibited some of the progesterone biosynthesis pathway genes. Our results revealed a novel mechanism of the progesterone antagonist pathway in the luteal granulosa cells and emphasis the fundamental role of progesterone in the early luteal phase.     

RU486-induced growth inhibition of human endometrial cells

OBJECTIVE: To determine the direct action of RU486 on endometrial cell proliferation and to differentiate whether the antioxidant or the antiprogesterone property of RU486 is predominately responsible for its effect on cell growth. DESIGN: In vitro study comparing the effects of RU486 (antiprogesterone and antioxidant), reduced RU486 (antioxidant), ZK112,993 (antiprogesterone), and lazaroid U74,500A (antioxidant) on endometrial cell growth. The human endometrial cell line EM42 was used in transient transfection assays to confirm the relative antiprogesterone potency of the various compounds. SETTING: Academic medical center PATIENT(S): Women presenting with pelvic pain or infertility and diagnosed with endometriosis at time of surgery or women desiring tubal ligation with a normal pelvis (controls). INTERVENTION(S): Endometrial cell cultures were treated with RU486, reduced RU486, lazaroid U74,500A, and ZK112,993. MAIN OUTCOME MEASURE(S): Tritiated thymidine incorporation was used to assess cell growth. Inhibition of progesterone induction of transiently transfected reporter plasmids was used to measure antiprogesterone activity of compounds studied. RESULT(S): RU486 reduced cell growth in a dose-dependent fashion of the endometrial cell lines EM42 and RL95-2 and of endometrial and endometriosis cells from primary culture. After being reduced, RU486 lost most of its antiprogesterone activity but retained its antiproliferative properties. ZK112,993 was similar in potency to RU486 as a progesterone antagonist but did not significantly modify endometrial cell growth. Lazaroid U74,500A was devoid of antiprogesterone activity but was shown to be a potent antiproliferative agent. CONCLUSION(S): RU486 has a direct inhibitory effect on human endometrial cell growth. This activity appears to be at least partly mediated through its antioxidant property.     

Pharmacodynamics of the antiprogesterone RU486 in women after oral administration

The pharmacokinetic characteristics of RU486 and its acute effects on anterior pituitary hormone secretion after oral administration were examined in six normal women. Serum RU486 concentrations were determined by a radioimmunoassay. The absorption of RU486 was rapid with peak serum levels reached approximately 90 minutes after a single oral dose (4 mg/kg). The disappearance of RU486 and its metabolites conformed to a noncompartmental model with a mean apparent half-life of 53.7 +/- 6.9 hours. The mean apparent volume of distribution and clearance rate were 1.47 +/- 0.25 l/kg and 1.04 +/- 0.09 1/hour, respectively. In comparison with a control group of normal women (n = 9), there were significant elevations in transverse mean cortisol levels in the RU486 group (P less than 0.01). However, mean adrenocorticotropic hormone (ACTH) levels and the diurnal pattern of ACTH and cortisol secretion were not changed. RU486 induced a mild prolactin (PRL) elevation (P less than 0.01), whereas thyroid-stimulating hormone (TSH) and luteinizing hormone (LH) levels were not altered. In view of the relatively slow clearance rate for RU486 and its metabolites, our findings suggest that the pharmacologic action of RU486 is prolonged after a single oral dose.     

Effects of the antiprogesterone RU486 in the early follicular phase of the menstrual cycle

To investigate the role of progesterone (P) in the early follicular phase, the antiprogesterone effect of RU486 was examined in five normally cycling women monitored by daily hormonal levels during three consecutive cycles (control, treatment, and recovery). In addition, luteinizing hormone (LH) pulse characteristics were assessed by frequent blood sampling (10 minutes for 10 hours) on day 3 of the control and the corresponding day of treatment cycles. Administration of RU486 (3 mg/kg, orally) for the first 3 days of the menstrual cycle did not significantly alter the length of the follicular phase (13.4 +/- 1.7 to 15.2 +/- 1.3 days), the LH surge, or the luteal phase length (12.2 +/- 0.5 to 12.6 +/- 0.7 days). The intermenstrual length of the treatment cycle (29.8 +/- 1.9 days) did not differ from the control (27.6 +/- 1.8 days) or recovery cycles (29.6 +/- 2.5 days). Integrated secretion of P and estradiol (E2) did not vary during the luteal phase of the control, treatment, or recovery cycles. During RU486 treatment, LH pulse frequency, pulse amplitude, and mean LH were not altered. Whereas mean E2 levels were significantly decreased from 150.5 +/- 15.1 to 110.1 +/- 7.0 pmol/L, follicle-stimulating hormone, P, adrenocorticotropin hormone, and cortisol were not significantly altered. Thus, in spite of the transient decrement in E2 secretion during RU486 treatment, the integrity of the ovulatory menstrual cycle was maintained. We conclude that administration of the antiprogesterone RU486 at the dose used during the first 3 days of the follicular phase does not perturb menstrual cyclicity.     

Progesterone receptor antagonists Org 31710 and RU 486 increase apoptosis in human periovulatory granulosa cells

Objective: To investigate if progesterone receptor (PR)-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated human luteinizing granulosa cells.

Design: Laboratory study.

Setting: Göteborg University and an in vitro fertilization laboratory of a university hospital.

Patient(s): Women undergoing oocyte retrieval for in vitro fertilization after ovulation induction with gonadotropins.

Intervention(s): Luteinizing granulosa cells were isolated from follicular aspirates after oocyte removal. The cells were treated with or without RU 486 (1 μM–100 μM), Org 31710 (1 μM–100 μM), progesterone (1 nM–10 μM), dexamethasone (0.5 μM–100 μM), dihydrotestosterone (1 nM–25 μM), RU 486 (10 μM–100 μM) + dexamethasone (50 μM), and picrotoxin (1 μM–100 μM) and were cultured under serum-free conditions.

Main Outcome Measure(s): Measurement of caspase-3 activity; detection of internucleosomal DNA fragmentation using gel electrophoresis and fluorospectrophotometry; progesterone analysis of spent medium.

Result(s): Addition of the PR antagonists RU 486 or Org 31710 in vitro to human luteinizing granulosa cells caused an increase in caspase-3 activity and a dose-dependent increase in internucleosomal DNA fragmentation. No effect on DNA fragmentation was seen after addition of dexamethasone, dihydrotestosterone, or picrotoxin .

Conclusion(s): Nuclear PR-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated human luteinizing granulosa cells.     

RU486对人早孕绒毛中纤维蛋白溶酶原激活与抑制系统的影响

目的：探讨ＲＵ４８６对离体培养人早孕绒毛滋养细胞分泌ＰＡｓ及ＰＡＩ┐１、ＰＡＩ┐２的影响，比较药物流产与正常绒毛中ｔＰＡ、ｕＰＡ、ＰＡＩ┐１及ｕＰＡ┐Ｒ表达的差异。方法：应用纤维蛋白琼脂糖铺盖及反向铺盖技术分别检测细胞培养液中ＰＡ与ＰＡＩ┐１的活性，采用琼脂糖指示胶直接点样法测定ＰＡＩ┐２活性。分别应用免疫组织化学与原位杂交技术检测绒毛组织细胞中ｔＰＡ、ｕＰＡ、ＰＡＩ┐１与ｕＰＡ┐Ｒ的抗原及ｍＲＮＡ的存在与分布。结果：受ＲＵ４８６刺激，离体培养绒毛滋养细胞分泌的ＰＡ┐ＰＡＩ复合物的水解活性增加，而ＰＡＩ┐１与ＰＡＩ┐２的活性则受抑制。口服米非司酮药流绒毛滋养细胞中ＰＡＩ┐１及ｕＰＡ抗原含量低，而ｕＰＡ┐Ｒ的ｍＲＮＡ含量显著增加。结论：ＲＵ４８６增加早孕绒毛中纤溶活性，从而使细胞外基质蛋白水解作用加强，绒毛的固着性可能因此松动。