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。     

邻苯二甲酸二乙基己酯对新生小鼠睾丸及Leydig细胞影响的实验研究

目的:探讨邻苯二甲酸二乙基己酯(DEHP)对新生小鼠睾丸及Leyd ig细胞形态结构及功能的影响。方法:DEHP分别以低、中、高3组剂量[100、200、500 mg/(kg.d)]灌胃作用于怀孕12 d到产后3 d(GD12~PND3)的KM母鼠,观察DEHP对新生雄性仔鼠体重、睾丸重量、Leyd ig细胞形态结构和3β-羟基类固醇脱氢酶(3-βHSD)活性、酶反应面积的影响。结果:DEHP作用于母鼠后,其雄性子代幼鼠体重和睾丸重量减轻,睾丸Leyd ig细胞形态、超微结构发生改变;高剂量组Leyd ig细胞数量明显增多;低、中剂量组睾酮合成关键酶3β-HSD酶活性下降,酶反应面积减小,但高剂量组在仔鼠出生后15 d时酶活性降低[(吸光度值(0.154±0.011)vs空白对照组(0.222±0.013),P<0.01],而酶反应面积增大[(6 303.0±745.6)μm2vs空白对照组(5 091.4±214.4)μm2,P<0.01)]。结论:DEHP能影响新生雄性小鼠体重、睾丸重量、Leyd ig细胞的形态结构和3β-HSD活性,具有抗雄激素效应。     

小鼠胚胎睾丸Leydig细胞培养、纯化及其功能研究

目的:探讨小鼠胚胎睾丸Leyd ig细胞体外培养、鉴定、纯化的方法,并进行形态学观察、分泌睾酮能力等生物学特性检测。方法:选择胎龄16 d的胎鼠睾丸,0.03%胶原酶Ⅰ消化,3β-羟基类固醇脱氢酶(HSD)染色鉴定及纯度测定,锥虫蓝染色检测细胞活率。放免法测定Leyd ig细胞不同培养时间及密度下分泌睾酮的水平。结果:Leyd ig细胞培养前和培养72 h后纯度分别为(45.10±1.66)%和(81.17±2.32)%;培养液中可检测到睾酮,睾酮水平与Leyd ig细胞数、培养时间相关,单个Leyd ig细胞睾酮分泌能力逐日下降。结论:该法分离的胚胎Leyd ig细胞纯度较高,生长性好,保持增殖和分泌睾酮的生物学特性,可应用于相关的研究。     

ATP50荧光表达载体构建及其在TM3小鼠睾丸Leydig细胞定位研究

目的:构建ATP50的荧光表达载体,研究其在原代培养的小鼠睾丸Leyd ig细胞中的表达和在TM3小鼠睾丸Leyd ig细胞中的定位。方法:原代培养小鼠睾丸Leyd ig细胞并利用3β-HSD染色法鉴定,应用PCR方法研究ATP50在小鼠睾丸Leyd ig细胞中的表达。利用BamH I和EcoR I酶切位点把ATP50克隆到pEYFP-N1。细胞转染和细胞荧光显微镜技术观察YFP-ATP50在TM3小鼠睾丸Leydig细胞的定位。结果:ATP50表达在原代培养的小鼠睾丸Leydig细胞中。TM3小鼠睾丸Leydig细胞中,绿色荧光的YFP-ATP50和红色荧光的线粒体标记物M ito-tracker有完全的共定位。结论:ATP50在小鼠睾丸Leydig细胞表达,成功构建了ATP50真核荧光蛋白表达载体,明确YFP-ATP50定位在Leydig细胞的线粒体。这些结果将对老年男性睾丸间质细胞睾酮合成功能障碍的研究提供重要的信息,有利于进一步深入研究。     

环境污染物三丁基氯化锡和氯化三苯锡对大鼠睾丸Leydig细胞的影响

目的:探讨环境污染物三丁基氯化锡(TBT)和氯化三苯锡(TPT)对大鼠睾丸Leyd ig细胞的影响。方法:①用0~80 nmol/L浓度的TBT和TPT处理大鼠睾丸Leyd ig(LC-540)细胞24~96 h,用四唑蓝(MTT)法确定细胞的存活率;②用DNA片段法确定是否存在细胞凋亡;③观察细胞内Ca2+螯合剂氨基苯乙烷四乙酸(BAPTA)和蛋白激酶A(PKA)抑制剂H-89、蛋白激酶C(PKC)抑制剂GF109203X、酪氨酸蛋白激酶(TPK)抑制剂Gen iste in是否可以阻断TBT所致的细胞凋亡;④检测TBT处理的原代大鼠睾丸Leyd ig细胞分泌睾酮的变化。结果:①TBT和TPT影响Leyd ig细胞存活率的强度基本相同,在20~80 nmol/L浓度时细胞存活率呈剂量和时间依赖性降低;②DNA片段法研究证实TBT和TPT可引起细胞凋亡;③BAPTA可阻断20 nmol/L的TBT所致的细胞凋亡,而PKA、PKC和TPK抑制剂对细胞存活率没有影响;④TBT可减少Leyd ig细胞分泌睾酮,并使其对人绒毛膜促性腺激素(hCG)刺激的反应性降低。结论:环境污染物TBT和TPT能直接导致睾丸Leyd ig细胞凋亡,并抑制睾酮分泌,这种致凋亡作用可能与细胞内Ca2+浓度增加有关。     

内源性睾酮抑制对大鼠睾丸内α-catenin表达的影响

目的:检测十一酸睾酮注射致内源性睾酮抑制的大鼠睾丸内α-caten in的表达情况。方法:成年雄性SD大鼠10只,随机分为对照组(肌注生理盐水)和睾酮组[肌注十一酸睾酮19 mg/(kg.15 d)],共130 d。制作睾丸石蜡切片,采用抗α-caten in多克隆抗体进行免疫组化染色。结果:在对照组中,α-caten in主要表达于精子细胞顶体、管周肌样细胞和Leyd ig细胞胞质。睾酮组的Leyd ig细胞明显萎缩,-αcaten in的表达明显减弱或消失,而精子细胞顶体和管周肌样细胞胞质的-αcaten in表达无明显改变。结论:内源性睾酮抑制所致生精细胞排列疏松或脱落与粘附分子-αcaten in的表达无关。-αcaten in有可能成为识别Leyd ig细胞的标记物。     

重组蛋白Cox7a2原核表达及鉴定

目的:从原代培养的小鼠睾丸Leyd ig细胞中克隆Cox7a2基因,构建pGEX4T-1-Cox7 a2载体,表达和鉴定重组蛋白。方法:应用RT-PCR方法从原代培养的小鼠睾丸Leyd ig细胞中克隆Cox7 a2,利用BamH I和EcoR I酶切位点把Cox7a2克隆到pGEX4T-1载体,酶切和测序鉴定后,诱导重组蛋白的表达,进行纯化后,利用蛋白免疫印迹进行鉴定。结果:从原代培养的小鼠睾丸Leyd ig细胞克隆到Cox7a2完整的编码序列,构建了pGEX4T-1-Cox7 a2载体,表达了预期相对分子质量的融合蛋白,并经免疫印迹检测鉴定。结论:成功克隆Cox7a2,表达纯化了其融合蛋白,为基因的功能研究奠定了前期基础。     

雌激素与雄性生殖

雌激素与男(雄)性生殖密切相关。人和哺乳动物体内芳香化酶(Ar)参与合成雌激素,雌激素与特异受体(ERα、ERβ)结合,行使其生物学效应。雌激素受体和Ar在雄性生殖器官所有发育阶段几乎都有表达,并通过对生殖细胞、Sertoli细胞、Leyd ig细胞和附睾的多方面调节而发挥作用。     

有机磷农药-敌敌畏诱导大鼠尿道下裂动物模型睾丸组织的病理学初步观察

目的:探讨环境内分泌干扰物-有机磷农药敌敌畏导致尿道下裂发生的可能机制。方法:30只SD大鼠随机分为两组:实验组(20只)和对照组(10只)。于孕12~17 d分别以敌敌畏10 mg/(kg.d)和生理盐水灌胃。实验组产雄仔鼠88只,其中22只发生尿道下裂,对照组产雄仔鼠33只,无尿道下裂发生。实验组(尿道下裂鼠)和对照组各5只仔鼠喂养至性成熟,取其睾丸组织作常规石蜡切片分别用HE染色和用抗大鼠Calretin in抗体作SP免疫组化染色检测。结果:尿道下裂大鼠睾丸组织中间质细胞(Leyd ig)数量明显减少,生精小管数及形态无明显改变;尿道下裂大鼠睾丸组织中Calretin in阳性的Leyd ig细胞数明显少于对照组。结论:孕SD大鼠染毒敌敌畏农药后能导致雄性仔鼠睾丸组织中的Leyd ig细胞数量减少,推测环境内分泌干扰物质敌敌畏诱导大鼠尿道下裂发生的可能机制是敌敌畏的毒性作用致使胎鼠睾丸中的Leyd ig细胞受损,导致胎鼠睾丸产生的睾酮水平降低,在尿道形成过程中发生障碍而产生尿道下裂。     

衰老雄性大鼠睾丸组织脂质过氧化与睾酮水平和bcl-2基因表达的研究

目的:探讨衰老雄性大鼠睾丸组织脂质过氧化对血清睾酮(T)水平和睾丸间质(Leyd ig)细胞bcl-2基因表达的影响。方法:由D-半乳糖建立亚急性衰老模型,SD大鼠20只随机均分为对照(C)组和D-半乳糖(D)组,用分光光度计检测睾丸组织超氧化物歧化酶(SOD)和丙二醛(MDA)的含量、放免法检测血清中T水平以及免疫组化检测Leyd ig细胞bcl-2基因表达情况。结果:①SOD活性:D组(116±18.09)U/mg.prot显著低于C组(156±31.02)U/mg.prot(P<0.01);②MDA含量:D组(1.77±0.41)nmol/mg.prot明显高于C组(1.19±0.15)nmol/mg.prot(P<0.05);③血清T:D组(2.39±0.90)nmol/L显著低于C组(8.95±2.53)nmol/L(P<0.01);④bcl-2基因表达:D组(35.1±3.6)%明显低于C组(49.6±7.4)%(P<0.01)。结论:衰老大鼠睾丸组织脂质过氧化的变化影响T水平和Leyd ig细胞bcl-2基因表达。     

睾丸间质干细胞分化和移植

睾丸间质细胞是男性体内合成雄激素的主要细胞,胚胎发育期中肾胚的间质细胞及生精小管周成纤维样细胞可能是睾丸间质细胞的干细胞。在胚胎期间质干细胞分化为胎儿型间质细胞;出生后间质干细胞经间质祖细胞、未成熟间质细胞分化为成熟间质细胞。老年期间质细胞数量可能不变,但雄激素合成下降。间充质干细胞及脂肪干细胞等干细胞经诱导可分化为分泌雄激素的睾丸间质细胞,因此,间质干细胞移植可望成为治疗男性性腺功能不全和中老年雄激素缺乏的创新方法,本文对睾丸间质干细胞的分化及移植方面研究进行综述。     

睾丸Leydig干细胞分离、纯化及鉴定的体外研究

目的 改进Leydig干细胞的分离及纯化方法,观察其生物学特性并进行鉴定.方法　通过胶原酶消化、差速贴壁法及双抗体免疫磁珠分选法,从大鼠睾丸内获得Leydig干细胞.以条件培养液进行培养,采用CCK法测定增殖能力.通过PDGFRα、LIFR及3β-HSD免疫组织化学染色鉴定Leydig干细胞.经定向诱导分化后,检测Leydig细胞的睾酮分泌能力.结果　每个睾丸约可获得83 000个干细胞,经免疫组织化学染色分析,PDGFRα阳性率为(98.0±0.8)％.在条件培养液中培养的SLCs增殖明显,在1个月内能稳定维持其干性而未向Leydig细胞系分化.免疫组织化学染色结果表明,PDGFRα、LIFR阳性表达,3β-HSD为阴性表达.Leydig干细胞在含有T3和HCG的培养液中培养后,第5天即可测到睾酮分泌,分化后的细胞3β-HSD+.结论　 由差速贴壁法及双抗体免疫磁珠分选法能获得纯化的PDGFRα+/LHRˉ细胞,这些细胞符合SLCs所必须具有的特征.采用该方法获取SLCs,操作简便,细胞损伤小并且获取纯度高.     

Evidence that testosterone regulates Leydig cell 3 beta-hydroxysteroid dehydrogenase-isomerase activity by a trans-acting factor distal to the androgen receptor.

Leydig cells are a target for their own steroid product, testosterone, and thus could be subject to short-loop feedback regulation by androgens. The authors previously reported that 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) activity was higher in freshly isolated Leydig cells from C57BL/6J than those from C3H/HeJ inbred mice. To determine whether this strain-related difference in 3 beta HSD activity could be mediated by differential sensitivity to feedback effects of testosterone, Leydig cells from the two strains were cultured in the presence or absence of testosterone, the synthetic androgen receptor agonist, mibolerone, or the nonaromatizable androgen, dihydrotestosterone. After 7 days of treatment, all three androgens significantly decreased 3 beta HSD activity in Leydig cells from C57BL/6J, but not from C3H/HeJ mice. When Leydig cells were cultured with hydroxyflutamide, an androgen receptor antagonist, the effect of testosterone was negated. To determine whether the strain-related difference in sensitivity to testosterone was mediated by a difference in the androgen receptor protein, Leydig cells from reciprocal F1 hybrid lines of mice were cultured in the presence or absence of testosterone. Testosterone treatment inhibited 3 beta HSD activity in both F1 lines to the same extent as observed for Leydig cells from C57BL/6J mice. Thus, there is a strain-related difference in the response to testosterone, but it cannot account for the strain-related difference in Leydig cell 3 beta HSD activity because the high 3 beta HSD strain (C57BL/6J) is the sensitive strain. Although the effect on C57BL/6J Leydig cells is androgen receptor-mediated, the dominant effect of testosterone on both F1 lines rules out a difference in the androgen receptor protein per se. However, the data are consistent with the difference being in a trans-acting factor distal to the androgen receptor.     

Sites of Action of Cyproterone and Cyproterone Acetate in the Immature Male Rat

Plasma testosterone (Leydig cell function), LH and FSH (pituitary function), the epididymal content of androgen binding protein (ABP) (Sertoli cell function) and plasma corticosterone (adrenal cortical function) were determined after 10 daily injections of varying doses of cyproterone and cyproterone acetate, beginning at 21 days of age. Daily doses of 0.5 mg or greater of either antiandrogen resulted in a marked depression in the levels of plasma testosterone and intra-testicular testosterone (measured only in the cyproterone group) with a dose-dependent decrease in testis and epididymal weights; both effects occurring with only minor changes in the levels of circulating gonadotrophins. In addition, these compounds caused a marked decrease in Sertoli cell secretion as reflected in a significant fall in the epididymal content of ABP.
A more detailed examination of the apparently direct effects of cyproterone on Leydig cell function revealed: 1) no major effects of in vivo treatment for 6 days (5 mg/day) on [I¹²⁵]hLH binding to testis membrane particles or on the in vitro response of enriched Leydig cell suspensions to hCG, 2) a dose-dependent inhibition of the hCG responsiveness of normal Leydig cells in the presence of the drug in vitro . The inhibitory effects on steroidogenesis in vivo can well be explained by a direct inhibition of the 3β-steroid-dehydrogenase-isomerase exerted by both antiandrogens.     

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.     

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。     

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。     

Decreased steroidogenesis and cAMP production in vitro by leydig cells isolated from rats made hypothyroid during adulthood

The Leydig cell function of adult male rats made hypothyroid with 6-propyl-2-thiouracil (6-PT, 0.1% w/v in drinking water for 1 month) was studied and compared with that of age-matched controls. After 6-PT treatment, a slight, non-significant decrease in serum testosterone was observed, but no changes in testis weight or number of Leydig cells were noted. The in vitro function of Leydig cells was therefore investigated during incubation for 3 h in the presence or absence of several stimuli: LH (30 mIU/mL), forskolin (FK 1 microM), isobutylmethylxanthine (IBMX, 100 microM), GnRH (100 nM) or FK 1 microM + IBMX 100 microM. Irrespective of the stimulus, cells from hypothyroid rats secreted less cyclic AMP, 17-hydroxyprogesterone, androstenedione and testosterone. No differences in LH receptors were noted between the groups. Prolonged incubation with triiodothyronine (5-250 ng/mL) or thyroxine (5-250 ng/mL) for 3, 16, 24 or 48 h did not affect testosterone secretion in either group; however, administration of IGF-I (8 ng/mL for 24 h) resulted in increased spontaneous and stimulated testosterone production in both groups. However, when hypothyroid animals were supplemented in vivo with thyroxine a full recovery of Leydig cell function in vitro was noted. In conclusion: (1) Leydig cells from rats made hypothyroid during adulthood produce less testosterone in vitro, both spontaneously and in response to cAMP and non-cAMP-mediated stimuli; (2) this is due to a reduction in cAMP production and in the activity of the enzymes in the androgen biosynthetic pathway, and not to changes in LH receptors; (3) direct administration of thyroid hormones did not improve testosterone secretion in either group, while incubation with IGF-I did.