c-jun对离体睾丸间质细胞睾酮分泌作用的机理研究

目的本研究拟通过c-jun反义寡脱氧核苷酸(ASODNs)观察c-jun在调节hCG促进睾丸间质细胞(leydigcells,LC)睾酮分泌中的作用机制。方法用c-junASODNs拮抗c-jun,再加用cAMP观察其对睾酮分泌的影响,用放射免疫方法检测睾酮水平。结果hCG可刺激LC睾酮分泌,是LC功能研究的有用模型。c-junASODNs呈剂量依赖性地抑制hCG诱导下的离体LC的睾酮分泌(P<0.01)。加用cAMP后睾酮分泌增加。结论c-jun促进hCG诱导的大鼠LC的睾酮分泌,c-jun表达可能与cAMP相关。     

维拉帕米对c-jun调节睾丸间质细胞睾酮分泌的影响

目的通过c-jun反义寡脱氧核苷酸(ASODNs)观察c-jun在调节人绒毛膜促性腺激素(hCG)诱导的睾丸间质细胞(LC)睾酮(T)分泌的作用。方法采用c-jun ASODNs拮抗c-jun,维拉帕米阻断钙通道,hCG诱导体外培养大鼠LC的T分泌,放射免疫方法检测T水平。结果c-jun ASODNs(0~2μmol/L)呈剂量依赖性抑制hCG诱导的离体LC的T分泌(P<0.01)。维拉帕米(10-5mol/L)可加强c-jun ASODNs抑制T分泌。结论c-jun促进hCG诱导大鼠LC的T分泌,c-jun表达可能与钙离子相关。     

内皮素对大鼠睾丸间质细胞睾酮生成的影响

本研究采用大鼠睾丸间质细胞体外培养的技术 ,观察了内皮素 1对离体间质细胞睾酮分泌的影响。研究发现 ,10 -9mol/L的ET 1可显著抑制间质细胞睾酮的基础分泌 (P <0 .0 5 ) ,并且ET 1对人绒毛膜促性腺激素 (hCG)刺激睾丸间质细胞睾酮分泌也有抑制作用 ,其有效抑制浓度为 10 -10 mol/L(P <0 .0 5 )。本实验结果提示 ,ET 1呈剂量依赖性抑制睾丸间质细胞睾酮的基础分泌和hCG诱导的分泌 ,ET 1可能为睾丸内的一种局部调节多肽     

大鼠睾丸Leydig细胞的培养和鉴定

目的:研究体外培养大鼠睾丸Leyd ig细胞的有效方法。方法:原代培养大鼠睾丸Leyd ig细胞,用4 U/m l人绒毛膜促性腺激素(hCG)作用细胞,对照组未用hCG,放射免疫法测定培养液中睾酮浓度,3β羟类固醇脱氢酶(3β-HSD)免疫组化染色观察睾丸Leyd ig细胞形态和生物学特性。结果:培养细胞成分均一、增殖旺盛、分化率高。接种72 h后大鼠睾丸Leyd ig细胞纯度达95%。接种后24 h内,hCG刺激组较对照组睾酮分泌量明显提高(P<0.05)。结论:体外培养的睾丸Leyd ig细胞可分泌高浓度的睾酮;睾丸Leyd ig细胞的纯化和培养方法的建立,可为中老年男性雄激素部分缺乏综合征睾酮替代治疗的基础和临床研究提供一条可行的思路。     

老年大鼠睾丸间质细胞结构和功能变化的实验研究

目的:研究老年SD大鼠(PADAM动物模型)睾丸间质细胞形态、分泌功能变化,探讨老年大鼠睾丸间质细胞的功能状态。方法:分别取青年SD大鼠和老年各20只,静脉血测定血清总睾酮和游离睾酮的浓度,并通过组织切片和透射电镜观察两个年龄组大鼠睾丸间质细胞形态学变化;此外,分别用hCG、Forskolin刺激体外培养的两个年龄组大鼠的睾丸间质细胞,比较培养基中睾酮和孕酮的浓度。结果:老年大鼠的血清总睾酮[(3.07±0.75)nmol/L]和游离睾酮[(0.71±0.65)nmol/L]均比青年大鼠[(10.89±6.11)nmol/L和(2.42±1.02)nmol/L]显著降低(P<0.05);细胞形态有较显著差异;体外培养的大鼠睾丸间质细胞分泌能力显著降低(P<0.05)。结论:老年SD大鼠血清睾酮和游离睾酮浓度显著低于青年SD大鼠,原因在于睾酮合成酶系统整体功能衰退。     

植物雌激素对体外培养的大鼠睾丸间质细胞分泌睾酮的作用

目的:研究植物雌激素(大豆苷元、染料木素)对睾丸间质细胞分泌睾酮的刺激作用,并初步研究其可能的分子作用机制。方法:采用Percoll不连续密度梯度离心,分离获得3月龄Sprague-Dawley(SD)大鼠的睾丸间质细胞。以人绒毛膜促性腺激素(hCG)为阳性对照药物,测定不同浓度(0、0.02、0.1、0.5、1、5μmol/L)大豆苷元、染料木素对睾丸间质细胞分泌睾酮的影响。RT-PCR检测睾酮生物合成过程中胆固醇侧链裂解酶细胞色素P450(P450scc)的表达。结果:0.1μmol/L的染料木素能显著刺激原代大鼠睾丸间质细胞的睾酮分泌,RT-PCR结果显示染料木素能显著上调睾酮合成过程中P450sccmRNA的表达。在较高浓度下(5μmol/L),大豆苷元和染料木素均能抑制睾丸间质细胞分泌睾酮。结论:染料木素在低浓度下(0.1μmol/L)显著促进睾丸间质细胞分泌睾酮,但随着浓度的增加(5μmol/L),大豆苷元和染料木素均显著抑制Leydig细胞的睾酮分泌。     

非酶糖基化终末产物抑制大鼠睾丸Leydig细胞睾酮的生成

目的:研究非酶糖基化终末产物受体(RAGE)在大鼠睾丸Leydig细胞上的表达及非酶糖基化终末产物(AGEs)对大鼠睾丸Leydig细胞睾酮合成的抑制作用。方法:原代培养大鼠睾丸Leydig细胞,RT-PCR和免疫荧光技术检测RACE在大鼠Leydig细胞上表达,不同浓度AGEs处理Leydig细胞(25、50、100、200μg/ml),ELISA法测定睾酮分泌量。结果:RT-PCR和免疫荧光结果表明RAGE在大鼠睾丸Leydig细胞上表达,不同浓度AGEs处理后,人绒毛膜促性腺激素(hCG)诱导的Leydig细胞睾酮合成量呈剂量浓度依赖性下降,与对照组相比,50、100、200μg/ml AGEs处理组差异显著(P0.01)。结论:大鼠Leydig细胞上存在RAGE受体,AGEs显著抑制原代培养大鼠Leydig细胞睾酮的分泌。     

癌基因产物c-jun和c-fos在髓母细胞瘤中的表达及临床意义

目的　了解c jun和c fos在髓母细胞瘤中的表达情况及其与预后的关系。方法　通过免疫组化方法测定c jun和c fos蛋白在 70例髓母细胞瘤和 10例正常小脑组织中的表达情况 ,并结合临床资料 ,对病人生存时间与c jun和c fos表达阳性率之间的关系进行分析。 结果　 (1)正常小脑组织标本不表达c jun和c fos;而髓母细胞瘤中多为不同程度的阳性表达 ,且以高阳性率为主 ,并发现阳性染色主要位于肿瘤细胞核 ,部分胞浆也有染色。 (2 )c jun和c fos表达呈正相关 (r =0 4 93,P <0 0 1) ;二者协同性较强。 (3)生存时间与c jun、c fos表达阳性率呈负相关 (c jun :r =- 0 4 4 7,P <0 0 1;c fos:r =- 0 5 90 ,P <0 0 1) ;c jun、c fos表达强度愈高 ,病人的生存率就愈低 ,生存时间就越短。结论　c jun和c fos在髓母细胞瘤组织中呈高表达。c jun和c fos二者在髓母细胞瘤中表达具有协同性。c jun、c fos在肿瘤中的阳性表达率与髓母细胞瘤病人的预后呈显著负相关     

睾丸间质细胞凋亡及调控

睾丸间质细胞 (Leydigcells,LC)的发生与成熟过程都和细胞凋亡相关 ,一定范围内的凋亡对机体具有积极的生理意义 ,但过度凋亡会使睾酮 (T)分泌明显减少 ,导致生精细胞凋亡增加 ,甚至不育。二甲磺基乙烷 (ethanedimethane sulphonate,EDS) ,糖皮质激素 ,下丘脑 垂体 睾丸轴分泌的促性腺激素释放激素 (GnRH)、卵泡刺激素 (FSH)、黄体生成素 (LH) /绒毛膜促性腺激素 (hCG)、T等激素 ,LC发育阶段及其他一些因素都会影响其凋亡。多个基因参与LC凋亡的调控 ,SCF/c kit、Bcl 2、Bcl xl可抑制其凋亡 ,caspase 3、Fas、Bax和clusterin可促进其凋亡     

植物抗氧化剂TA9901保护臂丛撕脱后运动神经元的实验研究

目的　观察植物抗氧化剂TA990 1对臂丛撕脱后脊髓运动神经元c jun基因的表达和存活的影响。方法　成年SD雌性大鼠 90只分成 2组 ,治疗组行右侧臂丛神经根撕脱术后 ,每天腹腔注射质量浓度为 0 .5%的TA990 1溶液 1ml,对照组注射生理盐水。治疗后 4h～ 6周处死动物行c jun免疫组织化学和中性红染色 ,定量比较两组大鼠损伤侧c jun基因表达阳性和存活运动神经元数目。结果　治疗后 3d ,5d ,1周和 2周 ,治疗组的c jun基因表达阳性运动神经元数目均多于对照组 ,两组间各时间点的差异均具有非常显著意义 (P <0 .0 1)。治疗后 2、4、6周 ,治疗组存活运动神经元数目多于对照组 ,两组间的差异均有非常显著意义 (P <0 .0 1)。结论　TA990 1能增强受损运动神经元c jun基因的表达 ,提高臂丛撕脱后运动神经元的存活率     

Chronic propranolol treatment causes desensitization of the steroidogenic response in testicular interstitial cells but does not alter protein kinase C

We investigated effects of chronic propranolol treatment on the secretory response of rat testicular interstitial cells (testosterone secretion) to subsequent in vitro stimulation with activators of protein kinase-C (PK-C) (L-propranolol, phorbol 12, 13-dibutyrate (PDBu), LHRH) or activators of protein kinase A (PK-A), (hCG or dibutyryl cAMP (dbcAMP)). We determined [3H]PDBu binding and PK-C activity in these cells. Treatment of rats with propranolol (Inderal 500 mg/L of water for 5 weeks) reduced by 48%, 50% and 29% the L-propranolol-, LHRH- or PDBu-induced testosterone secretion, respectively, when compared to cells from controls. This desensitization in testosterone secretion in vitro was also present when the testicular interstitial cells were stimulated with hCG or dbcAMP (secretion decreased by 65%/57%, respectively, when compared to cells from control rats). Challenging the cells originated from rats that received propranolol chronically with the addition in vitro of propranolol resulted in an additional reduction of the hCG/dbcAMP-stimulated testosterone secretion. Chronic propranolol-induced desensitization was not associated with a loss in [3H]PDBu binding or a decrease in PK-C activity. Chronic propranolol-induced desensitization can be uncoupled from down-regulation of protein kinase C. The effector responsible for the desensitization could be distal to the protein kinase C and protein kinase A.     

The temporal response of rat testes to hCG stimulation during sexual maturation

Serum testosterone responses to a single sc injection of hCG (25 IU/100 g body weight) were monitored for 5 days in rats throughout sexual maturation (22-70 days). Two hours after hCG injection serum testosterone levels rose in 22, 37 and 53 day-old animals and remained elevated for 2 days, returning to control levels on day 3. This response differed markedly from the biphasic secretion of testosterone reported for adult animals. In 70 day-old animals the serum testosterone response approached that seen in adult animals. Testosterone levels were elevated 2 h after hCG injection (25.4 +/- 2.5 ng/ml) and declined significantly at 12 and 24 h to 17.1 +/- 1.0 and 16.1 +/- 3.4 ng/ml, respectively. Testosterone levels tended to increase again on days 2 and 3 (19.9 +/- 2.8 and 21.1 +/- 3.5 ng/ml, respectively) but the increase was not statistically significant. This response differed markedly to the biphasic secretion of testosterone reported for adult animals. In vitro patterns of basal and hCG-stimulated testosterone secretion by decapsulated testes following a single hCG injection also changed during sexual maturation. In 22 day-old animals the testes exhibited refractoriness to in vitro hCG stimulation at 12 h, but testes from 37 day old rats were refractory from 2 to 24 h. In vitro testosterone responses of testes from 53 and 70 day-old rats were similar to that reported for adult rats with a period of refractoriness from 12 h to 2 days. This study demonstrates that during sexual maturation in the rat alterations occur in the temporal patterns of testosterone secretion in vivo and in vitro following hCG stimulation.     

Use of a GnRH agonist and hCG to obtain an index of testosterone secretory capacity in the koala (Phascolarctos cinereus)

Testosterone secretion in mammals typically occurs in random pulses such that a single blood sample provides limited information on reproductive endocrine status. However, it has been shown in several species that an index of the prevailing testosterone biosynthetic capacity of the testes can be obtained by measuring the increase in circulating testosterone after injection of a GnRH agonist or human chorionic gonadotrophin (hCG). Hence, the aims of the present study were to examine fluctuations in testosterone secretion in the koala (n = 6) over a 24-hour period and then characterise testosterone secretion after injection of the GnRH agonist buserelin (4 micro g) or hCG (1000 IU). The latter was used to establish an index of the prevailing testosterone biosynthetic capacity of the koala testis. Individual koalas showed major changes in blood testosterone concentrations over 24 hours, but there was no apparent diurnal pattern of testosterone secretion (P > .05). Injection of buserelin and hCG resulted in an increase (P < .05) in blood testosterone concentration. After injection of exogenous hormone, near maximal concentrations of testosterone occurred at around 60 minutes. There was a tendency for plasma testosterone to decline after 90 minutes with buserelin, but concentrations remained close to the upper limit for 240 minutes with hCG. There were strong positive correlations between the average testosterone concentration over 24 hours and the maximum observed testosterone concentration after stimulation with GnRH and hCG (GnRH, r = .772; P = .07 and hCG, r = 1.0; P < .01). The findings in the present study confirmed that individual male koalas can show large fluctuations in blood testosterone concentrations over time and that a GnRH agonist and hCG can be used in the koala to obtain an index of the prevailing steroidogenic capacity of the testes.     

Ethane dimethylsulphonate selectively destroys Leydigcells in the adult bonnet monkeys （Macaca radiata ）

Aim: To study the effect of intratesficular administration of ethane-1,2-dimethylsulphonate (EDS) which has been exten-sively used to selectively destroy Leydig cells in rats and study ~ role of gonadotropin in regulation of differentiation ofLeydig cells (LC) in the adult male bonnet monkey. Methods and Results: In vitro studies with cultured interstitialcells isolated from monkey testis revealed an inhibitory effect of EDS on LC as assessed by decrease in testosterone pro-duction. Intratesticular administration of EDS (5, 10, 20, 50 rag/testis) resulted in a dose-dependent rapid decrease inserum testosterone levels, with a 6.5 % decrease with 5 nag of EDS by the 3rd day, which returned to control levels by the45th day. EDS treatment resulted in a significant decrease in testiculiar testosterone. In addition a significant decrease in[^125 1]hCG binding and phenylesterase activity in the interstitial cells was noticed. Histological analysis of the testes onthe 5th day after administration of EDS revealed an interstitium devoid of LC indicating the destructive action of EDS.Conclusion: The monkey LC are sensitive to destructive action of EDS.     

Effects of inhibitory and stimulatory photoperiods and sexual maturation on the ability of hamster testes to respond to hCG in vitro

The decrease in gonadal weight produced in adult golden hamsters by exposure to short photoperiods was accompanied by a marked reduction in the ability of the testes to produce testosterone from endogenous precursors in vitro, both without and with hCG stimulation. These changes were significant after 4–7 weeks in short photoperiod (5L: 19D) and were even more pronounced after 17–20 weeks. Production of testosterone in vitro by testes of immature hamsters was comparable to values obtained in adult animals with short photoperiod-induced gonadal atrophy. Delay of sexual maturation induced by daily injections of bromocriptine was accompained by a further decrease in testicular testosterone production in vitro. Exposure of gonadally-regressed adult hamsters to a long, stimulatory photoperiod (14L: 10D) produced a rapid and marked increase in testicular testosterone production, which was coincident with the previously demonstrated increase in serum gonadotrophin levels after 1–5 days of photostimulation. Furthermore, testosterone production in vitro by regressed testes of animals exposed to short photoperiod was increased significantly by one large dose of hCG administered 26 h before killing the animals. It is concluded that the suppressive effects of short photoperiods on the ability of the hamster testis to produce testosterone and to respond to hCG stimulation are due to reductions in endogenous LH, FSH and prolactin release, with a consequent loss of testicular LH/hCG receptors and decreased activity of enzymes involved in the biosynthesis of testosterone.     

Effects of follistatin on testosterone secretion of rat Leydig cell in vitro

<正> Objective:To investigate the effects of follistatin(rhFS-288) on biosynthesis andsecretion of testosterone in rat Leydig cell in vitro.Methods:Leydig cells were isolated from Wistar rat testes by a discontinuous Per-coll gradient procedure.Purified cells were incubated in 24-well plate(10~5 cell/ml/well)and maintained for 24 h in a CO_2 incubator,rhFS-288 and Ca~(2+) were added to the wellsindependently or jointly in both baseline (without hCG) and stimulation condition (1.0IU/ml of hCG) to observe the change of testosterone concentration in the media.Results:rhFS-288 showed a dose-dependent inhibiting effect on testosterone releasein baseline and stimulating condition.Ca~(2+) presented inhibitory effect either.Whereas,escape phenomenon emerged while Ca~(2+) concentration reached to 100 mmol/L.A com-bination of rhFS-288 with Ca~(2+) displayed a dose-dependent inhibition on testosterone se-cretion.Conclusion:rhFS-288 inhibits testosterone secretion in a dose-dependent manner.Calcium is thought to be the second messenger of FS action.The mechanism of escapephenomenon during high dose of Ca~(2+) along is unknown.     

Effect of α-chlorohydrin on metabolism and testosterone secretion by rat testicular interstitial cells

The direct effect of alpha-chlorochydrin (alpha-CH) on basic metabolism (glucose utilization and oxygen consumption) and testosterone secretion by isolated rat interstitial cells (I-cells) has been studied. In the range of concentrations between 5 and 100 microliter/ml, only the highest doses of alpha-CH decreased cell vitality and their histochemical stain for 3 beta-HSD. Oxygen consumption of I-cells was depressed at all doses higher than 10 microliter/ml and this effect was reversible only with doses lower than 50 microliter/ml. glucose utilization by I-cells was depressed significantly by alpha-CH and this effect was particularly dramatic with doses higher than 50 microliter/ml. alpha-CH decreased testosterone secretion by I-cells, with maximal effects at 100 microliter/ml. I-cells responded to hCG challenge by increasing testosterone secretion, and hCG prevented the toxic effect of alpha-CH at the lowest dose (10 microliter/ml) of alpha-CH, but failed to overcome the effects of a high dose (100 microliter/ml).