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。     

未羧化骨钙素对老年大鼠睾丸间质细胞睾酮合成功能的影响

目的:通过建立老年大鼠模型,探究未羧化骨钙素对老年雄性大鼠睾丸间质细胞睾酮合成的影响。方法:30只3月龄成年雄性大鼠,随机对半分为正常对照组和实验组,实验组颈背部皮下注射D-半乳糖溶液300 mg·kg^-1·d^-1,对照组颈背部皮下注射等剂量生理盐水,注射时间为8周。验证衰老模型构建成功后,取大鼠的睾丸间质细胞,分别用不同质量浓度0(等量PBS溶液)、1、3 ng/mL的非羧化骨钙素刺激大鼠睾丸间质细胞,24 h后收取培养液采用睾酮ELISA检测试剂盒进行睾酮浓度检测;并在24 h后收集细胞提取RNA,然后采用荧光定量PCR法检测睾丸间质细胞睾酮合成关键酶StAR、Cyp11a、Cyp17的表达。结果:同加入等量PBS溶液的对照组大鼠睾丸间质细胞相比,加入等量PBS溶液的模型组大鼠睾丸间质细胞分泌睾酮水平下降(P<0.05);对模型组大鼠睾丸间质细胞分别进行1、3 ng/mL未羧化骨钙素处理,相较于模型组加入等量PBS溶液处理的睾丸间质细胞,骨钙素处理组睾丸间质细胞培养液上清中睾酮水平均升高(P<0.05);与加入等量PBS溶液的...     

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。     

Leydig细胞睾酮合成功能和自噬调控

Leydig细胞是睾丸间质细胞,主要功能是合成睾酮。睾酮被运输到身体各处的靶器官,通过与受体结合发挥重要的生理功能。自噬(Autophagy)是细胞通过溶酶体降解自身物质的方式,能及时清除细胞的不利物质,有利于细胞适应各种环境。在Leydig细胞中,存在自噬现象,对Leydig细胞合成功能发挥调控,但是具体机制和信号转导过程等都不是很清楚。下面,对Leydig细胞合成和自噬的调节研究现状作一综述。     

睾丸间质细胞瘤1例

病例报告患者男性,26岁,体检发现右侧睾丸肿物2个月.查体:阴茎发育可,无畸形.双侧睾丸位于阴囊内,质软,未触及肿物.无男性乳腺发育表现.实验室检查:精子密度32×106/L,精子活率58%.     

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     

衰老大鼠睾丸间质细胞形态学观察

目的通过对老年SD大鼠睾丸间质组织学和Leydig细胞超微结构的观察,探讨老年大鼠性腺功能减退的机制.方法测定3月龄和24月龄SD大鼠血清睾酮水平,并进一步对不同年龄大鼠睾丸间质和Leydig细胞进行光镜和电镜检查.结果老年大鼠血清T浓度显著低于青年大鼠;老年大鼠睾丸色泽灰暗、质地松弛;睾丸间质中成纤维细胞增生明显而Leydig细胞数量减少,单个Leydig细胞变小,并出现退行性改变;hCG刺激8d后,青年和老年大鼠两组睾丸形态学均无明显变化.结论老年大鼠的Leydig细胞出现明显的形态学异常,可能与睾酮合成功能减退有关.     

线粒体呼吸链相关基因Cox7a2对睾丸Leydig细胞睾酮生成影响研究

目的 探讨线粒体相关基因Cox7a2编码蛋白对睾酮生成的影响.方法 构建Cox7a2-EYFP-N1荧光表达载体,鉴定后转染TM3小鼠睾丸Leydig细胞,荧光显微镜观察转染效率和蛋白表达.ELISA的方法测定上清液中睾酮生成水平,CCK-8细胞增殖和生长测定试剂盒检测Cox7a2-YFP融合蛋白对细胞增殖状态的影响.结果 Cox7a2-YFP在TM3细胞中表达后抑制LH诱导的睾酮分泌,细胞增殖试验显示,Cox7a2融合蛋白表达后能抑制TM3细胞的增殖.结论 线粒体定位蛋白Cox7a2能抑制TM3细胞睾酮合成,与Cox7a2对细胞增殖有抑制作用有关.     

电磁辐射对大鼠睾丸P450scc mRNA表达及睾酮合成的影响和防护

目的:探讨电磁辐射对大鼠睾丸合成睾酮(T)的影响及其机制,评价铜丝网的防护效果。方法:采用Wistar雄性大鼠,随机分为对照组(n=60)、无屏蔽电磁辐照组(n=60)和铜丝网全身屏蔽辐射组(n=60),电磁辐照后分别取3、6、24、72h各时相点各组大鼠15只;采用放射免疫法(RIA)分别测定电磁辐照后3、6、24、72h及对照组血清T含量,反转录聚合酶链反应(RTPCR)测定各组睾丸组织中细胞色素P450胆固醇侧链裂解酶(P450scc)mRNA水平。结果:无屏蔽辐照组辐照后3h血清T含量和P450sccmRNA水平均明显低于对照组(分别较对照组降低83.9%,56.9%;P均<0.01),6h略有回升,但仍明显低于对照组(分别较对照组降低54.8%,27.3%;P均<0.01),24h恢复到正常水平,72h再次出现明显降低(分别较对照组降低60.1%,56.1%;P均<0.01);采用铜丝网全身屏蔽后,血清T和睾丸组织P450sccmRNA表达均未见明显变化。结论:电磁辐射能在转录水平影响睾丸间质细胞P450sccmRNA的表达,从而降低T的合成;铜丝网屏蔽具有较好的防护效果。     

老年男性的睾酮替代疗法

男性正常衰老过程伴随血清睾酮水平下降 ,但是尚不清楚它是否将导致许多男性的睾酮缺乏症。过去 10年中 ,越来越多的研究兴趣转向确认对那些睾酮缺乏的老年男性采用睾酮替代疗法 (TRT)是否有助于阻止或逆转衰老的某些方面。TRT有益效应相关的主要雄性激素靶器官包括 :骨、肌肉、脂肪组织、心血管系统和脑。与此同时 ,TRT对靶器官 (如前列腺 )的潜在不良反应仍需评估。本文的目的是总结有关老年男性TRT问题的最新认识。     

Autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells

Late-onset hypogonadism (LOH) is closely related to secondary androgen deficiency in aged males, but the mechanism remains unclear. In this study, we found that reduced testosterone production in aged rat Leydig cells is associated with decreased autophagic activity. Primary rat Leydig cells and the TM3 mouse Leydig cell line were used to study the effect of autophagic deficiency on Leydig cell testosterone production. In Leydig cells from young and aged rats, treatment with wortmannin, an autophagy inhibitor, inhibited luteinising hormone (LH)-stimulated steroidogenic acute regulatory (StAR) protein expression and decreased testosterone production. In contrast, treatment with rapamycin, an autophagy activator, enhanced LH-stimulated steroidogenesis in Leydig cells from aged, but not young, rats. Intracellular reactive oxygen species (ROS) levels were increased in both young and aged Leydig cells treated with wortmannin but decreased only in aged Leydig cells treated with rapamycin. Furthermore, an increased level of ROS, induced by H(2)O(2), resulted in LH-stimulated steroidogenic inhibition. Finally, knockdown of Beclin 1 decreased LH-stimulated StAR expression and testosterone production in TM3 mouse Leydig cells, which were associated with increased intracellular ROS level. These results suggested that autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells, which might be influenced by intracellular ROS levels.     

Is testosterone essential for maintenance of normal morphology in immature rat Leydig cells?

Selective deprivation of gonadotrophins in prepubertal rats by administration of a GnRH antagonist (Ac-D2Nal¹, D4ClPhe², DTrp³, DArg⁶, DA1a¹⁰-GnRH; GnRH code: 103–289–10, National Institutes of Health, USA) for 3 weeks, initiated at 20–22 days of age, induced morphological changes in the Leydig cells, including thickening and indentation of the nuclear margin, pyknosis and elongation of the nuclei. Mean nuclear diameter was reduced to 22% of that in the controls. Under the electron microscope the cells exhibited reduced volume of the nucleus and cytoplasm and the plasma membrane was irregular. This abnormal appearance of the Leydig cells improved marginally in 20–30% of the Leydig cells and their mean nuclear diameter increased to 39% of the control level after FSH supplementation (20 μg ovine FSH/day). Normal morphological integrity of the Leydig cells consisting of round or oval nuclei, a smooth nuclear and cellular margin and the original mean nuclear diameter was restored completely when testosterone (30 μg/day) was administered to GnRH antagonist-treated rats, with or without simultaneous administration of FSH; in these rats testosterone levels in blood were also restored to normal. These findings indicate that testosterone may be important for the maintenance of normal Leydig cell morphology in the rat.     

培养的大鼠间皮细胞的抗凝与纤溶功能研究

目的 测定培养状态下间皮细胞的抗凝与纤溶作用的强度并与内皮细胞、成纤维细胞作比较，为血管假体内腔铺被细胞的选择提供依据。方法 取SD大鼠大网膜、主动脉、皮下结缔组织作间皮、内皮及成纤维细胞的培养。取3类细胞第3代融合后48h的无血清培养液，放射免疫法测定6-酮-PGF1α(前列环素的代谢产物)的含量，发色底物法测定纤溶酶原激活物活性。结果 间皮细胞培养液中6-酮-PGF1α浓度显著高于成纤维细胞及内皮细胞，纤溶酶原激活物活性也高于成纤维细胞，但与内皮细胞相比差异无显著性。结论 培养状态下的间皮细胞具有与内皮细胞相似的功能特点，可能成为血管假体内腔铺被层的理想材料。     

Effect of cyclosporine on steroidogenesis in rat Leydig cells

Cyclosporine induces hypoandrogenism in adult male rats. In order to assess whether this effect of CsA may be due to a direct inhibitory effect on Leydig cell function, CsA (0, 50, 500, and 5000 ng/ml) was added to a collagenase-dispersed mixed Leydig cell preparation and incubated with and without hCG (0, 0.1, 0.3, 1.0, 3.0, and 10.0 ng/ml). Testosterone (T) production, mitochondrial cholesterol side chain cleavage (CSCC) and microsomal 17,20-desmolase enzyme activities in Leydig cells were determined after 3 hr of incubation. In the absence of CsA, stimulation of T production was maximal (about 16-fold) with 1.0 ng/ml hCG. With 50 and 500 ng/ml CsA there were no changes in either the hCG-stimulated T levels or the two enzymatic activities. However, 5000 ng/ml CsA significantly (P less than 0.05) reduced the hCG (1 ng/ml)-stimulated T levels, CSCC and 17,20-desmolase activities. The high dosage of CsA (5000 ng/ml) also caused a significant decrease in cell viability (P less than 0.05) during the incubation period. These effects of CsA were not due to cremophor EL, the CsA vehicle. This in vitro data indicate that high dosages of CsA (greater than or equal to 5000 ng/ml) appear to have a cytotoxic effect on rat Leydig cells that results in a decrease in T production. However, lower doses of CsA (less than 500 ng/ml) do not have any direct inhibitory effect on the rat Leydig cells, suggesting that the hypoandrogenic effect of in vivo CsA in rats is not due to any direct effect on the testis.     

大鼠睾丸Leydig细胞体外增殖研究

目的:探讨通过优化细胞培养体系,实现Leydig细胞的体外增殖培养。方法:联合应用胶原酶消化、不锈钢滤网过滤及差速贴壁法获得3周龄雄性Wistar大鼠睾丸Leydig细胞,贴壁细胞以DMEM/F12培养液及优化培养体系培养,MTT法、细胞计数法检测培养细胞的增殖能力;分别对原代培养2h、4d细胞进行3β-HSD免疫化学染色及流式细胞术分析,检测细胞成分,同时检测培养细胞睾酮在hCG刺激下分泌能力变化。结果:优化培养体系能够明显促进3周龄大鼠睾丸Leydig细胞大量增殖,群体倍增时间为(2.26±0.31)d,传统培养体系培养细胞群体倍增时间为(16.32±2.14)d,两者差异有显著性(P<0.05);原代细胞经流式细胞术鉴定,3β-HSD阳性细胞所占比例分别为(54.3±7.1)%,培养4d后,增殖细胞3β-HSD阳性细胞率为(93.6±4.6)%。增殖细胞均有睾酮生成功能,在hCG刺激下睾酮分泌均明显上升(P<0.05)。结论:优化培养体系能够促进差速贴壁法获得的睾丸Leydig细胞大量增殖。     

Influence of rat testicular macrophages on Leydig cell function in vitro

The influence of co-cultures of rat testicular macrophages and Leydig cells (LC) on LC morphology and steroidogenesis was investigated with and without macrophage stimulation by a bacterial lipopolysaccharide (LPS). LC showed an elongated form in the presence of stimulated testicular macrophages. In the presence of non-stimulated testicular macrophages a significant inhibition of testosterone production was observed (decrease of 33%) from 48 h in co-culture while an increase of 16% was obtained at the same culture time, after stimulation of macrophages by LPS. When LC were treated with testicular macrophage-conditioned media (MCM) obtained from LPS-treated macrophages, they became fusiform and there was stimulation (78%) of steroid production. After human FSH stimulation (1-1000 mIU ml-1), MCM from testicular macrophages was no more effective in enhancing testosterone production by LC than was media from untreated LC. Similar experiments with LPS were conducted with macrophages of peritoneal origin. Peritoneal macrophages stimulated or not by LPS in co-cultures with LC or peritoneal MCM did not significantly modify testosterone production. However, these cells were able to modify LC morphology when LPS-MCM was added to LC-culture medium. The present results suggest strongly that testicular macrophage-LC interactions could be important in the control of LC steroidogenesis.     

Morphologic response of rat Leydig cells to hemicastration

Hemiorchidectomized rats were followed up to 15 days postsurgery for morphologic evaluation of compensatory testicular response and its correlation to serum testosterone levels. Although gross compensatory testicular hypertrophy (CTH) was not noted, an enlarged interstitium was observed with hypertrophy and hyperplasia of Leydig cells with morphologic changes suggestive of increased cellular activity. These histologic changes were accompanied by compensatory testicular hypersecretion (CTHS) illustrated by the return of the serum testosterone levels to near the intact-control value in the later groups. Ultrastructural studies of the Leydig cells indicated an increase in the amount of smooth endoplasmic reticulum as an underlying mechanism for this response. In view of the previously reported normal serum luteinizing hormone levels after hemicastration, the compensatory hyperactivity/hypersecretion should be considered primarily an intrinsic Leydig cell response, not related to changes in the hypothalamo-hypophyseal axis.     

Local differences in Leydig cell morphology in the adult rat testis: evidence for a local control of Leydig cells by adjacent seminiferous tubules

In order to test the hypothesis that Leydig cell function in the adult rat testis is influenced by the surrounding tubules, Leydig cell morphology was compared in different types of interstitial areas. Triangular interstitial areas surrounded by 3 cross-sectioned tubules in nearly the same stage of spermatogenesis were chosen for quantitative light microscopy. It was found that the volume density of Leydig cells in such areas was about 30%, except when the surrounding tubules were in stages IX-X or XI-XII, when it was only about 20%. This variation in total Leydig cell mass seemed to be due to a variation in Leydig cell size and not in Leydig cell number. The largest Leydig cell profile area, 118 pL 6 μm² (mean pL SE n = 6 rats), was observed when the surrounding tubules were in stages VII-VIII, i.e. just prior to sperm release. The smallest Leydig cells were seen when the surrounding tubules were in stages IX-X and XI-XII (68 pL 3 and 66 pL 4 μm²). The present results indicate that there may be a Leydig cell cycle in the adult rat testis, which is regulated by the adjacent tubules.     

Effects of photoperiod on the ultrastructure of Leydig cells in rat

The aim of this study was to examine effects of photoperiod on the ultrastructure of Leydig cells in rat. For this purpose, 21 male Wistar rats were used. Animals were divided into three groups: Control rats in group I were kept under 12 hrs light: 12 hrs dark conditions (12L: 12D) for 10 weeks. Animals in group II were exposed to long photoperiods (18L: 6D), while rats in group III were exposed to short photoperiods (6L:18D) for 10 weeks. At the end of the experiment, all animals were killed by decapitation and blood samples were obtained. Serum testosterone levels were determined with the use of a chemiluminescent enzyme immunoassay. The testes of all rats were removed and weighed, then processed for light and electron microscopy. For morphometric comparison, diameters of seminiferous tubules in each group were measured. In rats exposed to long photoperiods, testicular weights, diameters of seminiferous tubules and serum testosterone levels were significantly increased as compared to those in control rats, whereas exposure of rats to short photoperiods resulted in a significant decrease of testicular weights, diameters of seminiferous tubules and serum testosterone levels as compared to those in control rats and rats maintained in long photoperiods. The amount of mitochondria and cytoplasmic secretory granules were increased in the cytoplasm of Leydig cells of rats exposed to long photoperiods. Furthermore, an increase in extensiveness of rough endoplasmic reticulum in the cell cytoplasm was noticed in this group, whereas a decrease in mitochondria and cytoplasmic secretory granules of the Leydig cell cytoplasm was seen in rats exposed to short photoperiods. The results of our study indicate that testicular functions increase after exposure to long photoperiods and decrease after exposure to short photoperiods.