密度梯度离心分离大鼠卵巢卵泡膜细胞

探讨用密度梯度离心法快速、有效地分离大鼠卵泡膜细胞.选取23～25 d雌性大鼠卵巢,用Percoll密度梯度离心法将卵泡膜细胞分离纯化,3β-羟基类固醇脱氢酶(3β-hydroxysteroid dehydrogenase,3β-HSD)组织化学染色用于卵泡膜细胞纯度检测.分别用0.1 U/mL和1.0 U/mL卵泡刺激素(Follicle-stimulating hormone,FSH)及黄体生成素(luteinizing hormone,LH)处理细胞,无血清培养48 h后,酶联免疫法检测培养液中雄烯二酮和雌二醇的水平.分离所得细胞中,3β-HSD染色阳性细胞与总细胞数之比大于90％; LH组的雄烯二酮水平显著高于对照组和FSH组(P＜0.05),LH组中1.0 U/mL组的雄烯二酮水平又高于0.1 U/mL组.各组均未检测到雌二醇及孕酮.3β-HSD组织化学染色可快速有效地检验所分离的卵泡膜细胞的纯度,分离所得的卵泡膜细胞可对LH产生反应,且其中几乎没有混杂颗粒细胞.     

褪黑素参与苯并(a)芘诱导的孕早期卵巢功能异常

该研究探讨了孕早期苯并芘[Benzo(a)pyrene,B(a)P]暴露对卵巢雌激素和孕激素的影响及其可能的机制。将SPF级健康成年雌雄昆明小鼠交配合笼,次晨查得阴栓则记为孕第1天(D1)。孕鼠随机分为对照组(n=60)和B(a)P组(n=60),每日称重后以0.1 m L/10 g动物体重分别灌胃给予玉米油和0.2 mg/(kg·d)浓度B(a)P。ELISA(enzyme linked immunosorbent assay)方法检测孕D4小鼠血清雌激素(estrogen,E2)、孕激素(progesterone,P4)和褪黑素水平;q RT-PCR检测孕D1~D4小鼠卵巢组织雌孕激素合成限速酶17β-HSD1(17β-hydroxysteroid dehydrogenase 1)和P450SCC(cholesterol side-chain cleavage enzyme)m RNA水平;Western blot和免疫组化法分别检测卵巢组织褪黑素受体MT1(melatonin receptor 1)、MT2(melatonin receptor 2)蛋白质水平。研究结果发现,与对照组相比,B(a)P暴露导致孕早期小鼠血清中E2、P4明显降低;同时,卵巢雌孕激素合成限速酶17β-HSD1和P450SCC m RNA水平下调(P0.05)。B(a)P组小鼠血清中褪黑素水平明显增高(P0.01),而卵巢黄体细胞MT1、MT2蛋白质水平下降(P0.05)。综上所述,褪黑素及其受体可能参与了B(a)P诱导的孕早期卵巢功能异常。     

多囊卵巢综合征模型鼠颗粒细胞凋亡及TRAIL蛋白的表达

目的通过观察卵巢颗粒细胞凋亡及TRAIL(肿瘤坏死因子相关凋亡诱导配体)蛋白的表达情况,探讨颗粒细胞凋亡与PCOS发病的相关性及凋亡调控蛋白TRAIL在PCOS颗粒细胞凋亡中的作用。方法采用硫酸普拉睾酮钠诱导大鼠PCOS模型,3’-末端原位标记法(TUNEL)检测大鼠卵巢颗粒细胞凋亡情况,免疫组化染色及RT-PCR分析检测TRAIL蛋白及TRAIL mRNA在颗粒细胞的表达。结果PCOS组大鼠卵巢窦状卵泡颗粒细胞凋亡发生率及TRAIL蛋白的表达较对照组明显增强(P<0.01,P<0.05),窦前卵泡颗粒细胞凋亡发生率及TRAIL蛋白的表达两组无显著性差异(P>0.05),两组卵巢始基卵泡颗粒细胞未发现凋亡征象及TRAIL蛋白表达。PCOS组大鼠卵巢颗粒细胞TRAIL mRNA的表达较对照组明显增强(P<0.01)。结论PCOS大鼠卵巢窦状卵泡颗粒细胞凋亡明显增强,TRAIL在PCOS大鼠卵巢颗粒细胞凋亡调控中发挥了作用。     

EGF对大鼠卵巢颗粒细胞增殖与分化影响的研究

许多研究发现,表皮生长因子(EGF)对生殖功能有重要的调节作用。本文用体外细胞培养的方法,研究了EGF对大鼠卵巢颗粒细胞增殖与分化的影响及其作用方式。结果如下:EGF可以明显抑制颗粒细胞DNA的合成,但促进孕酮的生成,后者是因为EGF能显著提高细胞内3β-羟甾脱氨酶(3β-HSD)的活性。放射受体分析表明,颗粒细胞上存在EGF的特异性受体,其K_d为1.83±0.3×10~(-8)mol/L,B_(max)为1.75±0.29×10~4个位点/细胞。卵巢免疫组化结果未发现颗粒细胞有EGF样免疫染色,而卵泡膜、黄体及间质内等均有阳性染色。以上结果提示,EGF可能通过旁分泌机制作用于颗粒细胞的EGF受体,从而调节细胞的生长和性激素的分泌,这对于颗粒细胞的成熟及卵泡的发育有着重要意义。     

HOXA10基因在多囊卵巢综合症患者卵巢中的表达

目的 探讨HOXA10基因在卵巢中颗粒细胞的表达及其与多囊卵巢综合征的关系.方法 采用逆转录聚合酶链反应及免疫印迹法分别测定25例多囊卵巢综合征(PCOS)妇女和32例卵巢功能正常(Non-PCOS)妇女卵巢黄素化颗粒细胞中HOXA10 mRNA和蛋白的表达水平.结果 PCOS妇女黄素化颗粒细胞中HOXA10 mRNA的表达和蛋白的表达均低于Non PCOS妇女(P＜0.01,P＜0.05).结论 HOXA10基因在卵巢中有表达,PCOS妇女卵巢黄素化颗粒细胞HOXA10 mRNA和蛋白表达水平明显低于Non-PCOS妇女,提示该基因可能参与了PCOS的发生及发展过程.     

人参皂甙Rb1对环磷酰胺所致卵巢早衰大鼠的保护作用

目的探讨人参皂甙Rb1对化疗所致卵巢早衰(Premature ovarian failure,POF)大鼠的Bcl-2及Bax基因表达的影响。方法选用30只2-3月龄具有正常动情周期雌性Wistar大鼠随机分3组,分别为对照组,治疗组和模型组。采用免疫组化和免疫印迹方法分别观察和半定量检测POF大鼠卵巢凋亡调节基因Bcl-2和Bax的蛋白表达情况,同时对比观察人参皂甙Rb1对其表达的影响。结果模型组卵巢颗粒细胞Bax蛋白的平均光密度值较对照组明显增高(P0.05);模型组Bcl-2蛋白的表达量较对照组明显下调(P0.05)。Rb1治疗后Bcl-2蛋白表达明显升高,Bax蛋白表达明显下调(P0.05)。结论 Rb1可能是通过下调bax蛋白水平减少卵巢颗粒细胞的凋亡,对化疗所致卵巢早衰起到治疗的作用,进而延缓卵巢衰老。     

顺铂连续和间隔造模诱发的药源性虚证小鼠类固醇激素合成能力的比较研究

目的研究顺铂不同给药方案对小鼠类固醇激素合成及储备功能的影响。方法 40只雌性ICR小鼠分为4组,顺铂连续和间隔造模组以及两个对照组。顺铂连续造模组给药7 d,每天注射3 mg/kg顺铂;间隔造模组每周注射1次10 mg/kg顺铂,共4次。末次造模前进行旷场实验、转棒疲劳实验、抓力测试;次日处死前称体重,摘取小鼠心脏、脾、胸腺和双侧肾并称重; RT-qPCR检测下丘脑GnRH和CRH、垂体Pomc、Fshb、Lhb、Pou1f1基因表达,以及肾上腺和卵巢类固醇合成酶(St AR、Cyp11a1、Cyp21a1、Cyp17a1、Cyp11b1、Hsd3b2、Cyp19a1、Hsd17b1)及受体(Esr1、Esr2和Gper1)基因表达; Western Blot检测肾上腺和卵巢St AR、CYP11A1、CYP21A2和CYP11B1蛋白表达。结果与对照组比较:(1)不同频次顺铂造模均可抑制小鼠体重增长,顺铂累积后可以诱导小鼠气虚证表现,并抑制脏器质量。(2)不同频次顺铂造模均能加速激活原始卵泡分化,产生大量闭锁卵泡,诱导膜细胞及颗粒细胞凋亡,健康的成熟卵泡减少;但对肾上腺组织和细胞形态影响不大。(3)连续造模后肾上腺St AR蛋白表达下调;间隔造模后St AR升高,但下调CYP11B1蛋白表达。(4)连续造模后,卵巢Cyp11a1、Hsd3b2、Esr1和Gper1基因表达上调,Star和Cyp17a1基因表达下调;间隔造模后St AR蛋白表达和Hsd3b2基因表达下调。(5)顺铂连续造模后下丘脑GnRH和CRH基因表达明显下调。(6)顺铂连续造模后垂体Pomc、Fshb、Pou1f1基因表达升高,而间隔造模组Lhb表达下降。结论顺铂能够诱发小鼠气虚、精气不足证的相关表现;合成类固醇激素的卵巢受到选择性损伤大于肾上腺,短期连续给药也进一步抑制HPA轴和HPG轴的高级中枢下丘脑和垂体功能。     

心房钠尿肽(ANP)对大鼠卵巢细胞生成雌激素与孕激素的影响

应用大鼠卵巢黄体细胞、颗粒细胞培养以及放射免疫分析法,观察了α型心房钠尿肽(α-ANP)对性甾体激素孕酮(P)和雌二醇(E_2)分泌的影响,结果发现,0.1—10ng/ml 浓度的α-ANP 促进离体培养的大鼠黄体细胞分泌孕酮,并呈量效关系。α-ANP 也促进大鼠卵泡颗粒细胞分泌孕酮,但对分泌雌二醇没有影响。说明α-ANP 也影响卵巢分泌功能。     

3β-羟基类固醇脱氢酶在甾体激素生成组织中的功能

3β-羟基类固醇脱氢酶(3β-hydroxysteroid dehydrogenase,3β-HSD)是一类依赖于NAD+/NADP+接受质子的氧化还原酶。在胆固醇转化为类固醇激素中起限速酶作用,在体内广泛分布。3β-HSD催化环戊烷并多氢菲结构3号位CH-OH转化为C=O,是参与肾上腺糖皮质激素合成中唯一非细胞色素P450家族中的酶;3β-HSD催化孕烯醇酮生成孕酮、17α-羟孕烯醇酮转化为17α-羟孕酮、雄烯二醇生成睾酮等多步化学反应。3β-HSD通过参与细胞内甾体激素生成,进而在生殖系统中发挥功能。此外,3β-HSD在脑、脂肪组织中也有较强表达,本文就3β-HSD在甾体激素生成组织中的功能及其临床意义进行总结。     

小分子牡蛎多肽对雄性小鼠性功能的影响

探究不同剂量的小分子牡蛎多肽对雄性小鼠性功能的影响。将6周龄的雄性昆明小鼠随机分成4组,连续灌胃6周。分别测定每组小鼠交配能力,性腺器官的重量和脏器指数,精子数目、活力和畸形率;采用分光光度法测定小鼠血清中胆固醇和阴茎中NO含量;采用ELISA法测定小鼠血清中睾酮(testosterone, T)、黄体生成素(luteinizing hormone, LH)和卵泡刺激素(follicle stimulating hormone, FSH)的含量;采用实时荧光定量PCR检测SRPK2及3个雄激素限速酶(类固醇生成急性调节蛋白(StAR)、细胞色素P450胆固醇侧链裂解酶(P450scc)和3β-羟基类固醇脱氢酶(3β-HSD))基因的表达情况。结果表明,小分子牡蛎多肽可以通过提高小鼠的交配能力、性腺器官重量和脏器系数以及精子质量,增加小鼠血清中胆固醇和性激素以及阴茎中NO的含量,提高性功能相关基因的表达量来增强雄性小鼠的性功能。在一定剂量范围内,性功能的增强程度具有一定剂量依赖性。     

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.     

Onset of steroidogenic enzyme gene expression during ovarian follicular development in sheep

Steroidogenesis is a major function of the developing follicle. However, little is known about the stage of onset of steroid regulatory proteins during follicular development in sheep. In this study, several steroidogenic enzymes were studied by immunohistochemistry and/or in situ hybridization; cytochrome P450 side chain cleavage (P450(scc)), cytochrome P450 17alpha-hydroxylase (17alphaOH), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 aromatase (P450(arom)), steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), and LH receptor (LH-R). To define the stages of follicular growth, ovarian maps were drawn from serial sections of ovine ovaries, and follicles were located and classified at specific stages of growth based on morphological criteria. In this way, the precise onset of gene expression with respect to stages of follicular growth for all these proteins could be observed. The key findings were that ovine oocytes express StAR mRNA at all stages of follicular development and that granulosa cells in follicle types 1-3 express 3beta-HSD and SF-1. Furthermore, the onset of expression in theca cells of StAR, P450(scc), 17alphaOH, 3beta-HSD, and LH-R occurred in large type 4 follicles just before antrum formation. This finding suggests that although the theca interna forms from the type 2 stage, it does not become steroidogenically active until later in development. These studies also confirm that granulosa cells of large type 5 follicles express SF-1, StAR, P450(scc), LH-R, and P450(arom) genes. These findings raise new questions regarding the roles of steroidogenic regulatory factors in early follicular development.     

Growth factor modulation of steroidogenic acute regulatory protein and luteinization in the pig ovary.

In vivo and in vitro luteinization were investigated in the porcine ovary, with emphasis on expression of steroidogenic acute regulatory protein (StAR). StAR mRNA and protein as well as cytochrome P450 side-chain cleavage mRNA (P450scc) increased during the luteal phase in the corpus luteum (CL) and were absent in regressed CL. Cytochrome P450 aromatase mRNA (P450arom) was not detectable at any time in CL. In vitro luteinization of granulosa cells occurred over 96 h in culture, during which P450arom mRNA was present at 1 h after cell isolation but not detectable at 6 h; and P450scc and StAR mRNAs were first detectable at 6 h and 48 h, respectively. Incubation of cultures with insulin-like growth factor I (IGF-I, 10 ng/ml), dibutyryl cAMP (cAMP, 300 microM), or their combination, induced measurable StAR mRNA at 24 h (p < 0.05), increased progesterone accumulation at 48 h, and elevated both StAR and P450scc expression through 96 h. Incubation of luteinized granulosa cells with epidermal growth factor (EGF, 10 nM) changed their phenotype from epithelioid to fibroblastic, eliminated steady-state StAR expression, and interfered with cAMP induction of StAR mRNA and progesterone accumulation. EGF had little apparent effect on P450scc mRNA abundance. It is concluded that StAR expression characterizes luteinization, and early luteinization is induced by cAMP and IGF-I in vitro. Further, EGF induces a morphological and functional phenotype that appears similar to an earlier stage of granulosa cell function.     

Mechanisms of digoxin and digitoxin on the production of corticosterone in zona fasciculata-reticularis cells of ovariectomized rats

Previous studies have indicated that digoxin (DG) inhibits testosterone production by rat testicular interstitial cells through both in vivo and in vitro experiments. DG and digitoxin (DT), but not ouabain, inhibit the progesterone, pregnenolone, and corticosterone secretion by rat granulosa cells, luteal cells, and zona fasciculata-reticularis (ZFR) cells, respectively. However, the effect of DG and DT on the enzyme kinetics of cytochrome P450 side chain cleavage enzyme (P450scc), the protein expression of P450scc and steroidogenic acute regulatory protein (StAR), and mRNA expression of StAR are unclear. ZFR cells were prepared from adrenocortical tissues of ovariectomized rats, and then challenged with adrenocorticotropin (ACTH), 8-Br-cAMP, forskolin, A23187, cyclopiazonic acid (CPA), nicotinic acid adenine dinucleotide phosphate (NAADP), trilostane, 25-OH-Cholesterol, progesterone, or deoxycorticosterone in the presence of DG, DT, or ouabain for 1 h. Enzyme kinetics of P450scc, protein expression of acute regulatory protein (StAR) and P450scc, and mRNA expression of StAR were investigated. DG and DT but not ouabain suppressed basal and other evoked-corticosterone release significantly. DG and DT also inhibited pregnenolone production. The Vmax of the DG and DT group was the same as the control group, but the Km was higher in DG- and DT-treated group than in control group. DT and ouabain significant suppressed mRNA expression of StAR. DG and DT had no effect on the P450scc and StAR protein expression at basal state, but diminished ACTH-induced StAR protein expression to basal level. These results indicated that DG and DT have an inhibitory effect on corticosterone production via a Na+, K+-ATPase-independent mechanism by diminishing actions on cAMP-, Ca2+-pathway, competitive inhibition of P450scc enzyme and reduction of StAR mRNA expression.     

Stimulatory effect of LH, PGE2 and progesterone on StAR protein, cytochrome P450 cholesterol side chain cleavage and 3beta hydroxysteroid dehydrogenase gene expression in bovine luteal cells

The aim of these studies was to investigate the effect of LH, progesterone (P4), PGE, noradrenaline (NA) and a nitric oxide donor, S-nitroso-N-acetylpenicillamine (S-NAP), on steroid acute regulatory protein (StAR), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and cytochrome P450 side chain cleavage (P450scc) gene expression and on the synthesis of their protein products. Bovine luteal cells were collected and prepared on days 6-10 of the estrous cycle and preincubated in vitro for 24 h. Thereafter, medium was changed and supplemented with one of six treatments: control medium, LH (100 ng/ml), P4 (10(-5)M), PGE2 (10(-6)M), NA (10(-5)M) or S-NAP (10(-4)M). In Experiment 1, luteal cells (10(6)/well) were incubated for 3, 6, 18 and 24 h. After incubation, total RNA was isolated and P4 concentrations in medium was determined. Semiquantitative RT-PCR was used to measure gene expression. In Experiment 2, luteal cells were preincubated for 24h, then stimulated as in Experiment 1. Total protein was isolated from lysed cells and Western blot analysis was performed using specific antibodies against the StAR, 3beta-HSD and cytochrome P450scc proteins. Bands were analyzed by means of KODAK 1D Image Analysis Software. In Experiment 1, LH and PGE2 stimulated secretion of progesterone from luteal cells. Concentrations of mRNA for StAR, 3beta-HSD, cytochrome P450scc were increased after 6 h in cells stimulated with LH, PGE2 and P4 (P<0.05). Gene expression was not affected by NA. In Experiment 2, LH, P4 and PGE2 induced an increase in the concentration of these three proteins. S-NAP inhibited both concentrations of mRNA and protein for StAR, 3beta-HSD, cytochrome P450scc. Therefore, the increase in secretion of P4 induced by LH and PGE2 is associated with increases in StAR, 3beta-HSD and cytochrome P450scc gene expression. This genomic response may be mediated in part through a positive effect of P4 on the expression of these genes observed in this experiment.