双酚A对体外大鼠睾丸支持细胞闭锁蛋白Occludin表达的影响

目的 观察双酚A (BPA)对体外大鼠睾丸支持细胞增殖能力与闭锁蛋白Occludin (OCLN)表达的影响,探讨BPA对精子发生的损伤机制.方法 体外分离培养雄性Wistar大鼠睾丸支持细胞,油红O染色鉴定.实验分为BPA染毒组(25 μM、50 μM和100 μM分别处理细胞24h)、溶剂对照组(无血清DMEM/F12+DMSO+细胞悬液)和空白对照组(无血清DMEM/F 12).CCK-8法测支持细胞增殖活性,Western Blot法检测OCLN表达水平.结果 分离培养大鼠睾丸支持细胞纯度＞90％.CCK-8实验结果显示:BPA对支持细胞增殖活性具有抑制作用,BPA浓度＞103μM时,存活率明显降低,细胞存活率＜44％,其差异有统计学意义(P＜0.05).Western Blot法检测结果显示,OCLN蛋白表达随着BPA染毒剂量的增加而降低,呈剂量依赖性,不同染毒剂量组与对照组比较,差异均有统计学意义(P＜0.05).结论 BPA可抑制睾丸支持细胞增殖活性和OCLN的表达,干扰支持细胞正常的生精过程.     

小鼠单侧睾丸损伤后环孢素A对Fas系统的影响

目的 :研究昆明小鼠 (KM小鼠 )单侧注射冰乙酸致睾丸损伤后环孢素A(CsA)对对侧睾丸生精功能和Fas系统表达的影响。　方法 :6 0只KM小鼠随机分为 4组 :A组为对照组 ,B组为单侧睾丸损伤组 ,C组为单侧睾丸损伤后 6h切除损伤睾丸组 ,D组为单侧睾丸损伤后 6h内开始腹腔注射CsA组。 4周后取对侧附睾尾 ,计数精子及其活率 ,对侧睾丸作石蜡切片苏木精 伊红染色和免疫组化链霉素抗生物素蛋白过氧化物酶连结 (SP)法检测Fas和FasL的表达。　结果 :D组附睾尾精子和活率计数显著高于B组 (P <0 .0 5 ) ,D组的FasL和Fas较B组显著降低 (2 4 .3± 7.0vs37.8± 5 .8和 17.8± 4 .3vs32 .4± 3.6 ,P <0 .0 5 )。　结论 :KM小鼠单侧睾丸损伤后CsA可以通过抑制Fas和FasL的表达 ,降低生精细胞凋亡 ,维持生精功能的稳定     

SET在不同发育阶段小鼠睾丸的表达及其功能

SET蛋白是一个多功能蛋白，在调节包括DNA复制、核小体装配、染色体修饰、DNA转录、细胞周期、细胞凋亡等细胞生物学过程中起重要作用。前期研究发现，SET在卵巢中调节雄激素合成。然而，SET在睾丸组织中的表达及其功能仍然不明确。在此，我们检测不同年龄段小鼠睾丸组织中SET表达，探讨其在精子发生及睾酮生成方面的潜在功能。48只不同年龄段的雄性小鼠（1周龄的ICR雄性小鼠作为幼年期组，4周龄小鼠作为性发育前组，12周龄小鼠作为性成熟期组，12月龄作为老年组）。免疫组织化学方法观察SET各年龄段小鼠的定位表达；qRT-PCR和Western Blot分别检测睾丸中SET mRNA和蛋白水平表达。SET表达定位于生精小管中的精原细胞、精母细胞，在青春期前及成熟期的单倍体和四倍体生殖细胞中高表达；成熟期及老年期的睾丸间质细胞中也有SET的表达；支持细胞中很少量表达。青春期前SET的mRNA与成熟期相比表达量最高（P〈0．05），而SET蛋白在性成熟期小鼠睾丸中表达最高（P〈O．05）。SET主要表达于精原细胞和精母细胞，少量表达于支持细胞，表明SET可能与精子发生有关。SET还表达于睾丸间质细胞，则与睾酮生成有关。     

单侧精索静脉曲张模型大鼠双侧睾丸Claudin-11表达及血-睾屏障变化的实验研究

<正>精索静脉曲张(varicocele,VC)是青年男性常见病,也是导致男性不育的重要原因,目前对VC致男性不育的机制仍未能阐明。Claudin-11是睾丸中的跨膜蛋白,也是构成血-睾屏障最基本的分子结构,通过Claudin-11蛋白间的相互作用可调节生殖细胞的增殖和迁移,对于维持精子生成十分重要[1]。本研究旨在通过建立大鼠左侧VC模型,并检测双侧睾丸间质细胞表达Claudin-11的水平,比较两侧睾     

紧密连接蛋白11在非梗阻性无精子症患者睾丸组织中的表达

目的:分析紧密连接蛋白11(CLAUDIN-11)在非梗阻性无精子症(NOA)患者不同生精障碍类型的睾丸组织中的表达情况,探讨其临床意义。方法:62例NOA患者按不同病理类型分为精子发生能力低下(HS)组和唯支持细胞综合征(SCO)组,其中HS组30例,SCO组32例。免疫组化法检测CLAUDIN-11在睾丸组织中的表达,实时荧光定量PCR检测CLAUDIN-11 mRNA的表达变化,化学发光法检测生殖激素在两组间的差异。结果:免疫组化结果显示,CLAUDIN-11在HS组的表达主要分布于靠近生精小管管壁的支持细胞胞质,在SCO组的表达定位混乱。实时荧光定量PCR结果显示,CLAUDIN-11 mRNA在SCO组表达高于HS组,分别为0.013±0.002、0.008±0.001(t=10.616,P0.01)。血清LH和FSH在HS组和SCO组患者中差异显著[(3.62±1.34)IU/L vs(4.96±3.10)IU/L,(5.36±2.80)IU/L vs(10.65±9.18)IU/L,P均0.05]。结论:CLAUDIN-11在睾丸支持细胞上的表达上调,可能在NOA患者睾丸生精功能障碍的发生和发展中起重要作用。     

五子衍宗复方对睾丸支持细胞氧化应激损伤和细胞凋亡的影响

目的:探讨五子衍宗复方对睾丸支持细胞氧化应激损伤和细胞凋亡的影响。方法:在人睾丸支持细胞系TM4细胞培养液内分别加入H2O2(100 mmol/L)和五子衍宗复方提取液,分为空白组、模型组、低(0.2 mg/ml)、中(1 mg/ml)、高(5 mg/ml)剂量五子衍宗复方加药组。在作用24 h后,用MTT法检测各组的吸光度值,用硫代巴比妥酸比色法和黄嘌呤氧化酶法分别测定各组MDA含量及SOD活性,用流式细胞仪测定各组的细胞凋亡率和存活率,用实时PCR检测caspase-3 mRNA的表达。结果:五子衍宗复方低、中、高剂量组的吸光度值由模型组的0.23±0.01分别增加至0.27±0.02、0.30±0.01和0.35±0.01;SOD活性由(8.32±0.62)U/ml分别增加至(9.69±0.59)U/ml、(10.47±0.74)U/ml和(12.28±0.38)U/ml;MDA含量由(6.99±0.74)mol/L分别降低至(5.78±0.28)mol/L、(4.19±0.43)mol/L和(3.42±0.51)mol/L;细胞凋亡率由(21.63±1.38)%分别降低至(17.87±0.75)%、(14.10±0.50)%和(9.53±1.34)%;存活率由(65.67±7.48)%分别增加至(70.37±1.12)%、(72.57±1.95)%和(81.60±2.46)%;caspase-3 mRNA的相对表达量由2.69±0.16分别降低为2.47±0.12、2.38±0.16和1.96±0.11。结论:五子衍宗复方能改善睾丸支持细胞的氧化应激损伤,抑制细胞凋亡。     

棉酚对睾丸支持细胞间隙连接蛋白表达的影响

目的:探讨药物棉酚对睾丸Sertoli细胞间隙连接蛋白Cx43表达的影响。方法:培养TM4睾丸Sertoli细胞,用1.25、2.5、5、10μmol/L的棉酚分别染毒细胞6、12、24、48 h。CCk-8试剂盒检测细胞毒性,应用细胞免疫荧光化学、RT-PCR检测Cx43在正常TM4细胞和在不同浓度及不同染毒时间的TM4细胞中的表达情况。结果:半定量RT-PCR及免疫荧光结果显示,正常TM4细胞中有较多的Cx43表达;棉酚染毒24 h后,Cx43 mRNA水平开始随时间增加而逐渐下降(P<0.05),且随剂量增加Cx43蛋白表达强度逐渐减弱(P<0.05)。结论:棉酚可抑制TM4细胞表达Cx43,可能是其抗生育的机制之一。     

实验性精索静脉曲张对青春期大鼠睾丸、附睾精子结合抗原11 mRNA及其蛋白异构体表达的影响

目的:研究实验性左侧精索静脉曲张(ELV)对青春期大鼠睾丸和附睾中精子结合抗原11(SPAG11)mRNA及其蛋白异构体SPAG11E表达的影响,并探讨其与精索静脉曲张导致男性不育的关系。方法:40只SD大鼠随机分为ELV2周组、4周组,假手术对照2周组、4周组,每组10只。ELV组行部分结扎左肾静脉建立青春期SD大鼠ELV模型,假手术对照组仅显露左肾静脉过程,但不结扎。应用RT-PCR和免疫组化法(n=5)检测SPAG11 mR-NA及SPAG11E蛋白在ELV2周组和ELV4周组及各自对照组大鼠双侧睾丸、附睾中的表达变化。结果:RT-PCR结果显示,SPAG11基因376bp的特异扩增产物仅见于大鼠附睾组织中。免疫组化结果显示,SPAG11E蛋白主要与睾丸生精上皮的圆形和长形精子细胞的顶体泡和顶体、睾丸间质细胞的胞质相结合;在附睾管上皮主细胞胞质和顶部静纤毛中表达。对RT-PCR的相对吸光度值和免疫组化的灰度值进行统计学分析显示:①左侧附睾ELV2周和4周组SPAG11 mRNA和SPAG11E蛋白的表达与各自右侧组及各自对照组比较均有显著减弱(P<0.05或P<0.01);左侧附睾ELV4周组SPAG11mRNA与SPAG11E的表达较ELV2周组显著减少(P<0.05或P<0.01);右侧附睾ELV4周组SPAG11E的表达也较ELV2周组显著减少(P<0.01);②两实验组双侧睾丸SPAG11E蛋白的表达与相应对照组比较均未见明显差异(P>0.05),且在ELV2周组和ELV4周组之间该蛋白的表达也无显著性差异(P>0.05)。结论:SPAG11是特异表达于附睾的基因,在大鼠睾丸和附睾中均可见其蛋白异构体SPAG11E免疫阳性反应,其定位及表达水平具有细胞特异性和区域特异性,而且SPAG11 mRNA及SPAG11E蛋白在ELV模型鼠附睾中的表达发生了明显变化,这提示SPAG11不仅可能在大鼠精子发生、成熟过程中发挥重要作用,还可能与精索静脉曲张所致的男性不育相关。     

一氧化氮在一侧睾丸扭转对侧睾丸损伤中的作用

目的 研究总抗氧化能力(T-AOC)和一氧化氮(NO)在一侧睾丸扭转对侧睾丸损伤中的作用。方法 SD雄性大白鼠建立左侧睾丸扭转模型，于扭转后6h再分为扭转睾丸复位及切除组，分别于术后1h、1d、1周、2周和4周处死4—5只，取出睾丸用于一氧化氮合酶(NOS)活性、NO、T-AOC及细胞凋亡的检查。结果 UTT复位后对侧睾丸组织NOS活性、NO含量明显升高，T—AOC显著降低。结论 NO过量产生及T-AOC的下降是UTT对侧睾丸损伤的重要原因。     

Berberine ameliorates experimental varicocele‐induced damages at testis and sperm levels; evidences for oxidative stress and inflammation

The present study was performed to show the ameliorative effect of berberine (BBR), as an antioxidant and anti‐inflammatory agent, against experimental varicocele (VCL)‐induced molecular and histological damages. For this purpose, 50 mature Wistar rats were divided into control, control‐sham, VCL‐sole, 50 mg/kg and 100 mg/kg BBR‐treated VCL‐induced groups. The tissue levels of interleukin‐6 (IL‐6), tumour necrosis factor‐α (TNF‐α), nitric oxide (NO), total antioxidant capacity (TAC), malondialdehyde (MDA), superoxide dismutase (SOD) and gluthatione peroxidase (GSH‐px) as well as the mRNA levels of testicular CuZn SOD, MnSOD, EC‐SOD and GSH‐px were evaluated. The serum concentration of testosterone and germ cells mRNA damage were analysed. Finally, the sperm viability, motility, DNA integrity and chromatin condensation were analysed. Observations revealed that, the BBR significantly downregulated VCL‐increased IL‐6, TNF‐α and NO levels, upregulated the CuZn SOD, MnSOD, EC‐SOD and GSH‐px mRNA level, decreased testicular MDA content, enhanced serum testosterone level and ameliorated testicular TAC, SOD and GSH‐px levels. The animals in BBR‐treated groups exhibited diminished mRNA damage versus non‐treated VCL‐induced group. The BBR has significantly (p < 0.05) improved sperm parameters. In conclusion, the BBR by promoting testicular antioxidant potential and by downregulating inflammatory reactions fairly promotes spermatogenesis and upregulates the sperm quality.     

Epigenetics and oxidative stress: A twin‐edged sword in spermatogenesis

Spermatogenesis is a series of complex events involving a delicate balance between cell proliferation and cell differentiation. Aggregation of chromatins and epigenetic modifications play a vital role in spermatogenesis via regulation of molecular pathways to maintain testicular homeostasis. These epigenetic mechanisms consist of histone modification, chromatin remodelling, DNA methylation and miRNA, etc., which reportedly are critical players in spermatogenesis. One such mechanism involves regulation of oxidative stress in the male reproductive system. The fact that testicular cells contain plenty of unsaturated fatty acids and undergo division at a high rate makes spermatogenic cells highly susceptible to oxidative insult leading to deleterious effect on spermatozoa, which may culminate in infertility in men. Although the correlation between ROS‐mediated oxidative stress and epigenetic alterations has been indicated, research in this regard is still in infancy. Further, the fact that environmental and life style factors are critical determinants of spermatogenic potential indicates the importance of epigenetic regulation of key molecular events in spermatogenesis. Therefore, the current review aims to discuss the ROS‐induced epigenetic deregulation of the molecular mechanism(s) involved in spermatogenesis.     

Stage-dependent topographical relationship of spermatogonia and early spermatocytes to Sertoli-Sertoli interspaces in the rat testis

In the rat seminiferous epithelium the Sertoli-Sertoli interspaces can be regarded as ending at the surface of basal germ cells as well as at the basement membrane. The relationship between these basal endings (BEs) of the Sertoli-Sertoli interspaces and the basal germ cells was examined using the electron microscope. The number of BEs in a given area of the seminiferous epithelium was counted and found to be constant throughout the cycle of the seminiferous epithelium. Undifferentiated spermatogonia had contact with very few BEs while differentiating type A spermatogonia at stage VII and later during the cycle seemed to be associated with a maximum of BEs. As type A spermatogonia developed to intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes, the average number of BEs per cell decreased exponentially. Thin, fingerlike cell projections emerged from the differentiating type A and intermediate spermatogonia along the basement membrane. These projections were associated with BEs. It is concluded that a specific stage-dependent topographical arrangement is established between Sertoli cells and germ cells long before the first synchronous division of type A spermatogonia takes place. The significance of this arrangement is discussed.     

Influence of selenium induced oxidative stress on spermatogenesis and lactate dehydrogenase-X in mice testis

Aim: To evaluate the effect of oxidative stress on the spermatogenesis and lactate dehydrogenase-X (LDH-X) activity in mouse testis. Methods: For creating different levels of oxidative stress in mice, three selenium (Se) level diets were fed in separate groups for 8 weeks. Group 1 animals were fed yeast-based Se-deficient (0.02 ppm) diet. Group 2 and Group 3 animals were fed with the same diet supplemented with 0.2 ppm and 1 ppm Se as sodium selenite, respectively. After 8 weeks, biochemical and histopathological observations of the testis were carried out. LDH-X levels in the testis were analyzed by western immunoblot and ELISA. Results: A significant decrease in testis Se level was observed in Group 1 animals, whereas it was enhanced in Group 3 as compared to Group 2. The glutathione peroxidase (GSH-Px) activity was significantly reduced in both the liver and testis in Group 1, but not in Group 2 and 3. A significant increase in the testis glutathione-S-transferase (GST) activity was observed in Group 1,whereas no significant change was seen in Groups 2 and 3. Histological analysis of testis revealed a normal structure in Group 2. A significant decrease in the germ cell population in Group 1 was observed as compared to Group 2 with the spermatids and mature sperm affected the most. Decrease in the lumen size was also observed. In the Se-excess group (Group 3), displacement of germ cell population was observed. Further, a decrease in the LDH-X level in testis was observed in Group 1. Conclusion: Excessive oxidative stress in the Se deficient group, as indicated by changes in the GSH-Px/GST activity, affects the spermatogenic process with a reduction in mature sperm and in turn the LDH-X level. (Asian J Androl 2004 Sep; 6: 227-232)     

Levels of oxidative stress parameters and the protective effects of melatonin in psychosis model rat testis

Aim： To evaluate the effects of melatonin on antioxidant enzyme levels and histopathologic changes in dizocilpine （MK-801）-induced psychosis model rat testis. Methods： A total of 24 adult male Wistar-Albino rats were divided into three groups with 8 in each. Group I was used as control. Rats in Group II were injected with MK-801 （0.5 mg/kg body weight i.p. for 5 days）. In addition to MK-801, melatonin （50 mg/kg body weight i.p. once a day for 5 days） was injected into the rats in Group Ⅲ. The testes were harvested bilaterally for biochemical and histopathological examinations. Antioxidant enzyme activities, malondialdehyde, protein carbonyl and nitric oxide （NO） levels in testicular tissues were analyzed using spectrophotometric analysis methods. Histopathological examinations of the testes were also performed. Results： MK-801 induced testicular damage, which resulted in significant oxidative stress （OS） by increasing the levels of antioxidant enzymes. The malondialdehyde, protein carbonyl and NO levels were increased in testicular tissues of rats. Treatment with melatonin led to significant decrease in oxidative injury. Administration of melatonin also reduced the detrimental histopathologic effects caused by MK-801. Conclusion： The results of the present study showed that MK-801 cause OS in testicular tissues of rats and treatment with melatonin can reduce the harmful effects＇of MK-801. （Asian JAndro12008 Mar; 10： 259-265）     

温度对体外大鼠睾丸支持细胞增殖及occludin蛋白表达的影响

目的:通过观察不同温度对原代大鼠睾丸支持细胞增殖作用与紧密连接蛋白occludin(OCLN)表达的影响,探讨温度因素在男性生殖功能降低或男性不育中的作用机制。方法:体外分离培养雄性Wistar大鼠睾丸支持细胞(Sertoli cell,SC),油红O染色及免疫组化FasL鉴定。实验分为对照组(34℃)和实验组(35℃、36℃、37℃、38℃、39℃)。在相应的温度下培养4 d后,CCK-8法检测SC增殖作用,HE染色观察细胞形态及结构,Western印迹法和免疫荧光法检测OCLN表达水平。结果:体外培养SC的纯度为(96.20±1.95)%,CCK-8结果显示,细胞增殖活性在34～36℃时逐渐增强,36～39℃时逐渐降低,并在镜下观察发现细胞核固缩、裂解明显;免疫荧光及Western印迹显示,36℃时OCLN表达量最高;高于36℃时,OCLN表达量随着温度的增加而降低(P<0.01)。结论:温度升高(>36℃)抑制SC的增殖活性,并降低SC紧密连接蛋白的表达量。因此,高温对SC活性和紧密连接完整性的影响,可能是导致男性生育能力下降或不育的机制之一。     

Effect of transient scrotal hyperthermia on sperm parameters,seminal plasma biochemical markers,and oxidative stress in men

In this experimental prospective study, we aimed to analyze the effect of transient scrotal hyperthermia on the male reproductive organs, from the perspective of sperm parameters, semen plasma biochemical markers, and oxidative stress, to evaluate whether different frequencies of heat exposure cause different degrees of damage to spermatogenesis. Two groups of volunteers (10 per group) received testicular warming in a 43°C water bath 10 times, for 30 min each time: group 1: 10 consecutive days; group 2: once every 3 days. Sperm parameters, epididymis and accessory sex gland function, semen plasma oxidative stress and serum sex hormones were tested before treatment and in the 16-week recovery period after treatment. At last, we found an obvious reversible decrease in sperm concentration (P = 0.005 for Group 1 and P= 0.008 for Group 2 when the minimums were compared with baseline levels, the same below), motility (P = 0.009 and 0.021, respectively), the hypoosmotic swelling test score (P = 0.007 and 0.008, respectively), total acrosin activity (P = 0.018 and 0.009, respectively), and an increase in the seminal plasma malondialdehyde concentration (P = 0.005 and 0.017, respectively). The decrease of sperm concentration was greater for Group 2 than for Group 1 (P = 0.031). We concluded that transient scrotal hyperthermia seriously, but reversibly, negatively affected the spermatogenesis, oxidative stress may be involved in this process. In addition, intermittent heat exposure more seriously suppresses the spermatogenesis compared to consecutive heat exposure. This may be indicative for clinical infertility etiology analysis and the design of contraceptive methods based on heat stress.