皮质酮诱导大鼠Leydig细胞凋亡是一经caspase-3激活的过程

目的 超生理剂量的皮质酮(大鼠体内的糖皮质激素)能诱导大鼠Leydig细胞凋亡。但有关皮质酮诱导Leydig细胞凋亡的细胞内机制尚不清楚。本研究旨在观察皮质酮是否经caspase-3激活的途径诱导大鼠Leydig细胞凋亡。方法 采用Western Blot方法检测不同时间点上经皮质酮处理的大鼠Leydig细胞中caspase-3酶原及裂解的caspase-3酶表达情况。运用荧光发光法检测不同时间点上经皮质酮处理的人鼠Leydig细胞中caspase-3酶活性。结果 caspase-3酶原表达水平在皮质酮处理6h时开始上升，12h及24h时表达量下降，而具生物活性的、裂解的caspase-3酶于12h时开始出现，24h时的表达水平最为显著。caspase-3酶活性在皮质酮处理12h时明显升高，以24h时最为显著。Caspase-3抑制剂DEVD-CHO对经皮质酮处理12、24及48h的Leydig细胞中的caspase-3酶活性均具有明显的抑制作用，加caspase-3抑制剂的处理组其细胞基因组DNA电泳未见有凋亡特征性的梯状条带。结论 皮质酮诱导的大鼠Leydig细胞凋亡是一经caspase-3激活的过程。     

皮质酮诱导青春期大鼠睾丸间质细胞凋亡的研究

本研究目的旨在观察皮质酮能否诱导青春期大鼠睾丸间质细胞凋亡,用皮质酮分别经体内、外途径处理大鼠睾丸间质细胞。凋亡细胞经碘化丙碇（ＰＩ）标记后用流式细胞仪检测。体外研究结果表明,经１００ｎＭ皮质酮处理２４小时后的睾丸间质细胞,其凋亡量（３６．５％）显著高于对照组（１２．７％。Ｐ〈０．０１）。在体研究得到类似结果,经皮质酮（２．５ｍｇ／１００ｇ体重）处理２４小时后的大鼠,其纯化的睾丸间质细胞调亡量（２     

Involvement of nuclear factor-kappa B on corticosterone- induced rat Leydig cell apoptosis

Aim:To investigate the activation of nuclear factor-kappa B(NF-kappa B)and its function in glucocorticoid-inducedLeydig cell apoptosis.Methods:The Leydig cells were isolated from male Sprague-Dawley rats(90 days of age)andwere incubated with corticosterone(CORT,glucocorticoid in rat)for 6 h,12 h and 24 h,respectively.The P65subunit of NF-kappa B(NF-kappa B/P65)in nuclei and the inhibitor of NF-kappa B(Ikappa B)in cytoplasm wereanalyzed by Western-blotting.The Leydig cells were treated with anti-Fas antibody for 3 h followed by Westernblotting to assay the changes of NF-kappa B/P65 in nuclei and in cytoplasm.The role of NF-kappa B in CORT-induced Leydig cell apoptosis was evaluated by observing the effects of NF-kappa B/P65 overexpression and inhibit-ing activation of NF-kappa B by 100μmol/L Pyrrolidine dithiocarbamate(PDTC)on this apoptosis.Results:Thetreatment of Leydig cells with CORT increased the levels of NF-kappa B/P65 in nuclei and decreased the levels ofIkappa B in cytoplasm.Following the Leydig cells were treated with anti-Fas antibody,the levels of NF-kappaB/P65was increased in nuclei and decreased in cytoplasm.The CORT-induced Leydig cell apoptosis was inhibited byoverexpressed NF-kappaB/P65 and was enhanced by incubation with PDTC.Conclusion:NF-kappa B is activatedby increased FasL/Fas in CORT-induced Leydig cell apoptosis.NF-kappa B may play an anti-apoptotic role in thisapoptosis.(Asian J Androl 2006 Nov;8:693-702)     

皮质酮诱导的大鼠Leydig细胞凋亡中caspase-3活化途径的研究

目的我们新近的研究表明，超生理剂量的皮质酮（大鼠体内的糖皮质激素）可通过激活caspase-3诱导大鼠Leydig细胞凋亡。本研究拟评价在皮质酮诱导的大鼠Leydig细胞凋亡过程中，caspase-3的激活是否有其上游的caspase-8平和 caspase-9的参与。方法采用荧光分光光度法榆测经皮质酮处理的大鼠Leydig细胞中caspase-8活性，以DNA梯状电泳条带作为评价细胞凋亡的指标，观察caspase-8抑制剂是否能够抑制细胞凋亡，采用RTPCR枪测皮质酮诱导的大鼠Leydig细胞中caspase-9的mRNA水平。结果在皮质酮诱导的大鼠Leydig细胞中出现caspase-8活性增高，以12h最为址著，升高的caspase-8的活性可被caspase-8抑制剂抑制，并导致Leydig细胞的凋亡过程被阻断。Leydig细胞中caspase-9的mRNA水平在皮质酮作用下上升，同样以12h最为显著。结论皮质酮诱导的大鼠Leydig细胞调亡与caspase-8和caspase-9有关。     

Ca2+和CaN凋亡信号通路参与皮质酮诱导的Leydig细胞凋亡中FasL的表达

目的观察在皮质酮诱导的大鼠Leydig细胞凋亡中,Ca2 和钙调神经磷酸酶(CaN)依赖的信号通路是否参与FasL表达的调控。方法利用钙定性探针Fluo-3/AM检测皮质酮作用下的Leydig细胞中Ca2 浓度变化。通过酶底物法测定CaN活性。以Westernblot检测FasL表达。用Annexin-Ⅴ-FITC和PI双标评价Leydig细胞凋亡率。结果经超生理剂量皮质酮处理的Leydig细胞中出现Ca2 浓度升高,CaN活性增加及FasL表达增加。环孢菌素A可抑制CaN活性,使FasL表达下调,细胞凋亡率下降。结论Ca2 和CaN依赖的信号通路参与了皮质酮诱导的大鼠Leydig细胞凋亡;CaN介导了由Ca2 引发的FasL表达,Ca2 和CaN在大鼠Leydig细胞凋亡过程中起重要作用。     

Effects of varicocele on testosterone, apoptosis and expression of StAR mRNA in rat Leydig cells

The aim of this study was to explore the effects of varicocele on the morphology and function of Leydig cells in the rat testis. Forty male Sprague-Dawley rats were divided into two groups: the experimental group underwent surgery to create a left varicocele (VC), and the control group underwent a sham operation. Serum testosterone and intratesticular testosterone levels were measured using a radioimmunoassay after 4 and 8 weeks of operation. Leydig cells were studied for apoptosis and expression of steroidogenetic acute regulatory (StAR) protein mRNA levels. Serum testosterone levels declined after 4 and 8 weeks of operation but were not significant (P>0.05). However, the intratesticular testosterone levels after 8 weeks were significantly decreased compared with the control group (P<0.01). The mean apoptosis index of Leydig cells in the experimental group was significantly higher than that in the control group after 4 or 8 weeks (P<0.01). StAR mRNA levels in the Leydig cells of the experimental group were significantly lower compared to those of the control group (P<0.01). Our data show that varicocele did impair Leydig cell function by increasing apoptosis and suppressing the expression of the StAR protein.     

Egr参与调控皮质酮处理的睾丸Leydig细胞中FasL启动子的活性

目的观察转录因子Egr(early growth response factor,早期生长反应因子)是否参与调控皮质酮处理的Leydig细胞中FasL启动子的活性。方法经RT-PCR检测皮质酮处理的Leydig细胞中Egr-2及Egr-3的mRNA的水平,采用双荧光素酶报告基因系统评价Egr-2和Egr-3表达载体对Leydig细胞中FasL启动子活性的影响。结果Leydig细胞经皮质酮处理后,细胞内Egr-2及Egr-3在转录水平呈显著上调。双荧光素酶报告基因检测发现,Egr-2及Egr-3表达载体均可上调皮质酮处理的Leydig细胞中FasL启动子的活性,并以Egr-3的作用为显著。结论Egr-2及Egr-3参与调控皮质酮处理的Leydig细胞中FasL启动子的活性。     

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 Glyphaea brevis twigs extract on cell viability,apoptosis induction and mitochondrial membrane potential in TM3 Leydig cells

Glyphaea brevis twigs (Spreng) Monach. (GBT) are used by local herb healers to manage male sexual fertility disorders. The aim of this study was to examine the effects of G. brevis twigs on TM3 Leydig cells. GBT was extracted using methanol solvent, and Leydig cells were exposed to the respective concentrations of GBT extract (0.1, 1, 10, 100 and 1,000 μg/ml) for 24 and 72 hr respectively. Parameters evaluated include cell morphology, viability (MTT assay), mitochondrial membrane potential (TMRE dye), apoptosis (Annexin V Alexa Fluor 488 binding) and RT‐qPCR analyses of the mRNA expression. Results revealed that GBT had no cytotoxic effect on cell viability and the cell morphology. GBT also revealed a considerable elevation (p < 0.05) in fluorescence intensity, accompanied by intact mitochondria in TM3 Leydig cells. Furthermore, GBT resulted in the reduction of necrotic and apoptotic cells. The mRNA StAR was upregulated markedly with the effect prominent at 100 μg/ml. This study showed that GBT might be useful for managing male infertility ailments.     

14C]-2-deoxyglucose uptake studies in Leydig cells

The present study examines several aspects of [14C]-2-deoxyglucose uptake by rat Leydig cells: the characteristics of uptake by Leydig cells and type of inhibition by cytochalasin B, the effect of other drugs on [14C]-2-deoxyglucose uptake, the specificity of the glucose transporter for other substrates and the effect of various hormones. The apparent Km and V for [14C]-2-deoxyglucose were 0.4 mM and 0.17 mumol/min/10(6) cells, respectively. The inhibition of [14C]-2-deoxyglucose by cytochalasin B was competitive in nature, with an apparent Ki of 0.28 microM. Both phloretin and phlorizin (1.0-50 microM) inhibited [14C]-2-deoxyglucose uptake in a dose-dependent manner. Although D-glucose inhibited [14C]-2-deoxyglucose uptake by Leydig cells, L-glucose was ineffective, reflecting the stereospecificity of the glucose transporter for the former substrate. Various hormones, including: insulin, LH, 17 beta-estradiol or growth hormone which have been reported to stimulate glucose uptake in cells from other tissues, had no effect on [14C]-2-deoxyglucose uptake in Leydig cells under the current conditions. It remains to be determined how glucose uptake in Leydig cells is regulated.     

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.     

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.     

Cyproterone acetate induced changes in the behaviour of hydroxysteroid dehydrogenases in rat Leydig cells during perinatal development

The influence of cyproterone acetate (CA) upon the behaviour of hydroxysteroid dehydrogenases (HSDH) in the Wistar rat Leydig cells was investigated during the perinatal phase with the help of enzymhistochemical cum morphometrical techniques. The pregnant rats as well as their offsprings were injected with CA (dosage: 35 mg/kg body wt) sc, daily from 14 fetal day upto 31 postnatal day (p.n.d.). The animals were killed on 5, 20, and 32 p.n.d.; the enzymhistochemical reactions for 3-beta-HSDH, 11-beta-HSDH, 17-HSDH and 3-alpha-HSDH were performed in the cryostat sections of the testis, and the morphometric evaluation of HSDH positive Leydig cells was carried out. On 5 p.n.d. the activity of 17-beta-HSDH was slightly impaired in the intertubular Leydig cells of the CA treated animals. On 20 p.n.d., CA prevented nearly completely the HSDH activity in the newsly built peritubular Leydig cells; the activities of 3-beta-HSDH, 17-beta-HSDH, and 3-alpha-HSDH resided mainly in the intertubular Leydig cells. On 32 p.n.d. the HSDH activities in the Leydig cells were observed in the control as well as in treated animals. It seems that the differentiation of peritubular Leydig cells, and thereby the steroid production, is delayed by CA, but not entirely blocked.     

Non-tumoural parenchyma in Leydig cell tumours: pathogenetic considerations

Little is known about the pathogenesis of Leydig cell tumours (LCTs) of the testis. The observation of several associated dysgenetic features in the non-tumoural parenchyma and in the contralateral testes of men with testicular germ cell neoplasms has served as the basis to propose that there may be a common mechanism for different male reproductive disorders. However, the possible relationship between LCTs and other testicular lesions has not been explored. Here we describe the presence of primary lesions in the non-tumoural parenchyma of testes with LCT, from which we try to establish possible pathogenetic associations. We studied the non-tumoural parenchyma adjacent to 16 LCT specimens. Parameters as Leydig cell hyperplasia (LCHY), qualitative evaluation of the germinal epithelium and spermatogenesis, the presence of Sertoli cell-only tubules (SCOT), and the Sertoli cell nuclear morphology were consistently assessed in all cases. SCOT associated with Sertoli cell dysgenetic morphology was the most frequent finding, present in 50% of the cases. Another interesting finding was the presence of LCHY in four cases (25%). Abnormal spermatogenesis was found in 81.25% of the cases, and it consisted of lesions of the adluminal or basal compartments of seminiferous tubules. The occurrence of either dysgenetic Sertoli cells or LCHY adjacent to LCTs could represent primary anomalies, resulting from a common insult also involved in tumourigenesis. The abnormalities in spermatogenesis observed here are likely to represent consequences of either tumour compression or abnormal hormonal production. The significance of these associations merits further investigation regarding a common pathogenesis.     

敌敌畏对大鼠睾丸Leydig细胞凋亡的影响

目的:观察敌敌畏对子代雄性大鼠睾丸Leydig细胞凋亡的影响,探讨Leydig细胞变化在泌尿生殖系统畸形发病机制中的意义。方法:将21只孕SD大鼠随机分为对照组和敌敌畏各剂量组,在孕12～17 d期间,对照组每天分别给予灌喂玉米油1.0 ml;敌敌畏各剂量组每天分别给予敌敌畏1、4、8、16、20、24 mg/kg。待孕鼠分娩完毕,每组随机选取5只新生雄性仔鼠睾丸标本行HE染色、caspase-3免疫组化染色和DAPI荧光染色,将余下的雄性仔鼠饲养至90 d后再随机选取5只成年雄性仔鼠睾丸标本行HE染色、caspase-3免疫组化染色和DAPI荧光染色。结果:玉米油对照组雄性仔鼠睾丸组织caspase-3染色阳性的Leydig细胞数量和DAPI标记的Leydig细胞的总分值与敌敌畏1 mg/(kg.d)组比较无统计学意义(P>0.05),与敌敌畏其余剂量组比较有统计学差异(P<0.05),敌敌畏可使雄性仔鼠睾丸caspase-3染色阳性的Leydig细胞数量和DAPI标记的Leydig细胞的总分值增加,并与敌敌畏存在剂量依赖关系。结论:孕期染毒敌敌畏可使雄性仔鼠睾丸Leydig细胞的凋亡增加,这可能影响到Leydig细胞的数量,使胚胎期胎睾产生睾酮的功能受到干扰,从而使泌尿生殖系统的发育受到影响。     

TIGIT regulates apoptosis of risky memory T cell subsets implicated in belatacept-resistant rejection

Belatacept confers increased patient and graft survival in renal transplant recipients relative to calcineurin inhibitors, but is associated with an increased rate of acute rejection. Recent immunophenotypic studies comparing pretransplant T cell phenotypes of patients who reject versus those who remain stable on belatacept identified three potential “risky” memory T cell subsets that potentially underlie belatacept-resistant rejection: CD4⁺ CD28⁺ T_EM, CD8⁺ CD28^null, and CD4⁺ CD57⁺ PD1⁻ subsets. Here, we compared key phenotypic and functional aspects of these human memory T cell subsets, with the goal of identifying additional potential targets to modulate them. Results demonstrate that TIGIT, an increasingly well-appreciated immune checkpoint receptor, was expressed on all three risky memory T cell subsets in vitro and in vivo in the presence of belatacept. Coculture of human memory CD4⁺ and CD8⁺ T cells with an agonistic anti-TIGIT mAb significantly increased apoptotic cell death of all three risky memory T cell subsets. Mechanistically, TIGIT-mediated apoptosis of risky memory T cells was dependent on FOXP3⁺ Treg, suggesting that agonism of the TIGIT pathway increases FOXP3⁺ Treg suppression of human memory T cell populations. Overall, these data suggest that TIGIT agonism could represent a new therapeutic target to inhibit belatacept-resistant rejection during transplantation.