一氧化氮、总抗氧化能力对大鼠隐睾生殖细胞凋亡的影响

目的 :探讨一氧化氮 (nitricoxide,NO)、总抗氧化能力 (totalantioxidecapacity ,T AOC)对大鼠隐睾生殖细胞凋亡的影响。　方法 :健康SD雄性大鼠 2 0只 ,日龄 2 2d时复制单侧隐睾模型。生物素 dUTP/酶标亲和素测定法检测睾丸生殖细胞凋亡 ,采用硝酸还原酶法测定睾丸组织中NO含量 ,化学比色法测定睾丸组织中T AOC。　结果 :术后第 7d ,与对侧正常睾丸相比 ,隐睾侧睾丸发生凋亡的生殖细胞数显著增加 (P <0 .0 1) ;T AOC显著下降 ;NO含量显著上升 (P <0 .0 1)。　结论 :实验性大鼠隐睾可导致睾丸生殖细胞凋亡增加 ,且与睾丸组织中NO升高及T AOC下降密切相关     

总抗氧化能力和一氧化氮合酶对大鼠隐睾生殖细胞凋亡的影响

目的：探讨总抗氧化能力（T－AOC）和一氧化氮（NO）合物（NOS）对隐睾生殖细胞凋亡的影响。方法：SD雄性大鼠日龄22d时复制单侧隐睾模型。用生物素－dUTP/酶标亲和素测定法检测睾丸生殖细胞凋亡；用硝酸还原酶法测定睾丸组织中NO含量；用化学比色法测定睾丸组织中T－AOC、NOS活性。结果：术后第7天，与对侧睾丸相比，隐睾发生凋亡的生殖细胞数显著增加，NO含量及NOS活性显著上升（P＜0．01），在隐睾组织中两者呈正相关（γ3=0.890,P＜0．01）；T－AOC显著降低（P＜0．01）。结论：隐睾组织中NO和NOS升高、T－AOC下降是隐睾生殖细胞凋亡增加的生化机制之一。     

一氧化氮合酶基因表达对实验性大鼠隐睾生殖细胞凋亡的影响

目的　研究诱生型一氧化氮合酶 (iNOS)及其mRNA表达与实验性大鼠隐睾生殖细胞发育、凋亡的关系。方法　 (1)采用SD雄性健康大鼠 16只 ,日龄 2 2天时复制单侧隐睾模型。 (2 )采用生物素 dUTP/酶标亲和素测定法检测睾丸生殖细胞凋亡。 (3)采用免疫组织化学方法检测大鼠睾丸生殖细胞中iNOS基因表达。 (4 )采用原位杂交法检测大鼠生殖细胞中iNOSmRNA的表达。结果　 (1)术后第 7天 ,与自身对侧正常睾丸相比 ,隐睾侧睾丸发生凋亡的生殖细胞数显著增加 (P <0 .0 1)。 (2 )单侧隐睾模型建立术后第 7天 ,在双侧睾丸的间质细胞、支持细胞和初级精母细胞中均可见iNOS蛋白及iNOSmRNA的弱阳性表达 ,在隐睾侧睾丸曲细精管中脱落的生殖细胞中可见iNOS蛋白及iNOSmRNA的强阳性表达。术后第 7天 ,与自身对侧正常睾丸相比 ,隐睾侧睾丸生殖细胞中iNOSmRNA表达显著增加 (P <0 .0 1)。结论　 (1)实验性大鼠隐睾可以导致睾丸生殖细胞凋亡增加。 (2 )iNOS蛋白及其mRNA的表达增加是隐睾生殖细胞凋亡增加的分子机制之一     

乙醇毒染大鼠睾丸总抗氧化能力、一氧化氮和一氧化氮合酶的变化与生殖细胞凋亡的关系

目的:探讨饮酒大鼠睾丸总抗氧化能力(T-AOC)、一氧化氮(NO)含量和一氧化氮合酶(NOS)活性的变化与生殖细胞凋亡的关系。方法:20只成年健康SD雄性大鼠随机均分为对照组和实验组,实验组和对照组分别用50%的乙醇溶液和蒸馏水按10 m l/kg每晚灌胃1次连续26 d(两个生精周期)后,用硝酸还原酶法测定睾丸组织中NO含量;用化学比色法测定其T-AOC和NOS活性;原位缺口末端标记(TUNEL)法检测生殖细胞凋亡指数(AI)的变化。结果:与对照组相比,实验组生殖细胞AI增加(P<0.01);睾丸组织T-AOC极显著下降(P<0.01),而NO含量明显上升(P<0.01)、NOS活性显著增强(P<0.01)。结论:大量饮酒能诱导生殖细胞凋亡增加,NOS活性增强导致NO的过量产生及T-AOC的显著下降是其重要原因。     

Bcl-2/Bax基因表达对隐睾生殖细胞凋亡的影响

为探讨Ｂｃｌ－２／Ｂａｘ基因表达对隐睾生殖细胞凋亡的影响。采用ＳＤ雄性大鼠日龄２２天时复制单侧隐睾模型;用生物素－ｄＴＵＰ／酣标亲和素测定法检测睾丸生殖细胞凋亡;用免疫组织化学ＳＰ法检测Ｂｃｌ－２／Ｂａｘ基因表达。结果发现术后第７天,与对侧正常睾丸相比,隐睾侧睾丸发生凋亡的生殖细胞显著增加（Ｐ〈０．０１）;Ｂｃｌ－２、Ｂａｘ表达均有显著差异（Ｐ〈０．０１）。提示手术诱导的隐睾生殖细胞凋亡增加;Ｂｃ     

Fas、FasL和C-myc基因表达与隐睾生殖细胞凋亡的关系

1998年 10月至 2 0 0 1年 2月我们利用动物隐睾模型检测生殖细胞凋亡及Ｆａｓ、ＦａｓＬ和Ｃ ｍｙｃ基因在隐睾中的表达 ,探讨Ｃ ｍｙｃ与Ｆａｓ/ＦａｓＬ介导的细胞凋亡途径之间的相关性。报告如下。材料与方法　 2 2日龄ＳＤ雄性健康大鼠 2 0只 ,手术复制单侧隐睾动物模型。模型建立后 ,分别在第 3天 (Ａ组 ,10只 )和第 7天 (Ｂ组 ,10只 )处死两组大鼠 ,取双侧睾丸标本。生物素 ｄＵＴＰ/酶标亲和素测定法检测大鼠睾丸生殖细胞凋亡 (试剂盒购自德国ＢＭ公司 ) ,阳性染色胞核呈棕褐色。免疫组织化学ＳＰ法检测Ｆａｓ、ＦａｓＬ和Ｃ ｍｙｃ…     

大鼠隐睾生殖细胞发育和凋亡与eNOS蛋白与mRNA表达

目的　探讨内皮型一氧化氮合酶 (eNOS)蛋白及其mRNA表达与实验性大鼠隐睾生殖细胞发育和凋亡的关系。　方法　采用生物素 dUTP/酶标亲和素测定法检测SD大鼠单侧隐睾模型睾丸生殖细胞凋亡 ,免疫组化方法检测大鼠睾丸生殖细胞中eNOS基因表达 ,原位杂交法检测大鼠生殖细胞中eNOSmRNA的表达。　结果　术后第 7天隐睾侧睾丸发生凋亡的生殖细胞数与对侧正常睾丸相比显著增加 (P <0 .0 1)。术后第 4 0天 ,对侧正常睾丸的精子细胞中可见eNOS基因表达。术后第 3、7天 ,隐睾侧睾丸生殖细胞中eNOSmRNA表达与对侧正常睾丸相比无明显变化 (P >0 .0 5 )。　结论　在大鼠青春前期 ,eNOS基因表达与雄激素生成和生殖细胞发育有关 ;在成年期 ,eNOS基因表达可能仅与精子的成熟及活力有关而与生殖细胞凋亡无关。     

Fas/FasL和C-myc基因表达与隐睾生殖细胞凋亡的关系

目的：探讨Fas／FasL和C—myc基因表达与隐睾生殖细胞凋亡的关系，以及C—myc与Fas／FasL介导的细胞凋亡途径之间的关系。方法：采用22d龄SD雄性大鼠建立单侧隐睾模型，运用生物素—dUTP／酶标亲和素测定法检测睾丸生殖细胞凋亡，运用免疫组织化学SP法检测Fas／Fasl和C—myc基因表达。结果：①与对侧睾丸相比，术后第3天隐睾侧睾丸发生凋亡的生殖细胞无显著增加(P＞0．05)，Fas／FasL和C—myc基因表达亦无显著增加(P＞0．05)；第7天隐睾侧睾丸发生凋亡的生殖细胞显著增加(P＜0．01)；Fas／FasL和C—myc基因表达均有显著增加(P＜0．01)。②实验性隐睾术后第3天和第7天，C—myc基因表达与相应Fas／FasL基因表达间均存在着正相关关系。结论：手术诱导的隐睾生殖细胞凋亡增加；Fas／FasL和C—myc基因表达上调可能是生殖细胞发生凋亡的重要原因之一；隐睾生殖细胞凋亡可能是通过与C—myc相偶联的Fas／FasL细胞凋亡途径调控的。     

低氧对大鼠睾丸生殖细胞凋亡的影响

目的:研究低氧对大鼠睾丸生殖细胞凋亡和Bax、Bcl-2表达的影响。方法:雄性成年Wistar大鼠随机分为4组:常氧对照组、低氧5 d组、低氧15 d组和低氧30 d组(各组n=6)。常氧对照组在平原喂养;低氧5 d组、15 d组和30 d组分别在低压舱内模拟5 000 m高原喂养5、15、30 d。采用流式细胞术和TUNEL法检测低氧对睾丸生殖细胞凋亡的影响。运用Western印迹技术检测低氧对大鼠睾丸内凋亡相关蛋白Bax、Bcl-2表达的影响。结果:低氧5 d组、15 d组和30 d组睾丸内发生生殖细胞凋亡的生精小管数量[每100个生精小管中,含有凋亡生殖细胞的生精小管数分别为(20.50±5.07)、(21.25±7.85)、(14.00±2.45)个]均非常显著地高于常氧对照组[(6.00±2.16)个,P<0.01]。凋亡的生殖细胞以精原和精母细胞为主。低氧15 d组睾丸组织亚单倍体细胞百分率[(2.18±0.82)%]显著高于常氧对照组[(1.30±0.33)%,P<0.05],低氧30 d组[(3.08±0.93)%]极显著高于常氧对照组(P<0.01)。低氧30 d组睾丸组织Bax表达显著高于常氧对照组(P<0.05),其灰度值分别为17.34±4.54和10.50±2.82。低氧30 d组睾丸组织Bax/Bcl-2比值显著高于常氧对照组(P<0.01),比值分别为0.40±0.10和0.27±0.04。结论:低氧诱导大鼠睾丸生殖细胞凋亡增多,慢性低氧引起的睾丸生殖细胞凋亡增多与Bax表达增加有关。     

SNP对生殖细胞凋亡作用的研究

目的探讨一氧化氮(NO)供体硝普钠(SNP)对大鼠睾丸生殖细胞凋亡的影响。方法采用末端脱氧核苷酸转移酶(TdT)介导的原位末端标记法(TUNEL),透射电镜和琼脂糖凝胶电泳检测生殖细胞凋亡的特征。结果TUNEL标记法检测表明,NO供体SNP可诱导生殖细胞的凋亡,并呈剂量时间依赖性,SNP组凋亡率明显高于对照组(P＜0.01)。透射电镜观察SNP处理15～20h后的生殖细胞,核染色质凝集,附着于核边缘呈新月形,核固缩、碎裂形成凋亡小体。琼脂糖凝胶电泳显示生殖细胞DNA片段呈现凋亡特征性的梯形区带。结论一氧化氮对生殖细胞的凋亡具有促使形成作用,这对不育症的研究有重要的价值。     

Apoptotic cell death and fertility in three unilateral cryptorchid rat models

Three rat strains have been studied, using a sensitive apoptotic detection method for germ-cell degeneration, to resolve the controversy regarding the effect of cryptorchidism on the contralateral descended testis (CDT). Sprague Dawley and Buffalo rats were made cryptorchid by operation at 20–22 days of age, while trans-scrotal (T-S) rats were a congenitally unilateral cryptorchid strain. Sham operated rats or normal T-S littermates were used as controls. Experiments were performed over a period ranging from 2 weeks to 18 months. Testis weight was assayed, as was the detection of apoptosis by agarose gel laddering and immunohistochemistry by using the TUNEL method. Labeled cells in 150 cross-sectioned testis tubules were counted on the TUNEL stained slides and the mean number of labeled cells per tubule was calculated. Paternity studies on Sprague Dawley and T-S rats were carried out at 12 and 24 weeks of age to assess fertility by the resultant number of pregnancies and litter sizes. Both Sprague Dawley and T-S rat models showed a biphasic distribution of apoptosis levels. This biphasic distribution was not observed in Buffalo rats as they were only studied at later time points (12–20 weeks). A significant effect on either testis weight or apoptosis in the CDT compared with the control descended testis (P ≥ 0.1) has not been found in these three cryptorchid models, and the present results are discussed with reference to observations of other researchers in rodents and humans. While the cryptorchid testis showed a high level of labeled apoptotic cells per tubule in all rat strains, fertility was not affected and remained the same as controls at 12 and 24 weeks. There was, however, a marked strain difference in fertility in T-S as compared with Sprague Dawley rats. After 24 weeks of cryptorchidism, both control and cryptorchid T-S rats had a 44% pregnancy incidence compared with a 90% pregnancy incidence in Sprague Dawley rats. In addition, litter size in T-S control and cryptorchid rats were small compared with those of Sprague Dawley rats at 12 and 24 weeks. Received: 11 January 2000 / Accepted: 21 April 2000     

Assessment of germ cell apoptosis in cryptorchid rats

Aim: To investigate the relationship between germ cell degeneration and apoptosis in cryptorchid rats. Methods: Thirteen 21-day-old Wistar rats were made unilaterally cryptorchid by closing the left inguinal canal. At day 30 (Group 1, n=6) and day 60 (Group 2, n=7) after operation, the testes were removed for histopathological examination. The controls (n=8) were sham operated and were sacrificed at day 60. Germ cell apoptosis was assessed by means of the TUNEL method. Results: Spermatogenesis was arrested and the testicular and seminiferous tubular diameters were significantly reduced In the unilateral undescended testes (UUTs) compared with the contralateral descended testes (CDTs) and the control rats. However, atrophic changes, pathological calcification, necrosis of seminiferous tubule, and absence or sloughing of germ cells were not found in all the animals. The spermatocytes were the main type of germ cells undergoing apoptosis in all the groups. In the UUTs, there was a significant and time-depe     

Involvement of Fas in the apoptosis of mouse germ cells induced by experimental cryptorchidism

The role of Fas in the apoptosis of testicular germ cells was investigated in BALB/c mice and Fas-deficient lpr/lpr mice. Spontaneous apoptosis of germ cells was observed in the testes of 40-day-old BALB/c mice, and experimentally induced cryptorchidism increased this apoptosis to such an extent that there was a decrease in the weight of the testis. Flow cytometry and immunohistochemistry using a Fas-specific monoclonal antibody demonstrated expression of Fas on germ cells including spermatogonia, spermatocytes, and spermatids. Furthermore, analysis by flow cytometry suggested that Fas expression on germ cells was increased following cryptorchidism. However, spontaneous and cryptorchidism-induced apoptosis of germ cells were also observed in 40-day-old Fas-deficient lpr/lpr mice. Moreover, testis weight also decreased following cryptorchidism in the mutant mice. The present results may indicate that the expression of Fas on germ cells does not correlate with spontaneous apoptosis or apoptosis induced by cryptorchidism. However, on the contrary, this study shows that Fas are partly involved in cryptorchidism-induced apoptosis, because the decrease in testis weight of lpr/lpr mice was less than that in BALB/c mice. Received: 17 October 1996 / Accepted: 31 July 1997     

Expression of HSP70-2 gene during germ cell apoptosis in rat unilateral cryptorchid testes

To investigate the role of Hsp70-2 gene in germ cell apoptosis induced by heat stress, its expression changes were examined in rat normal and unilateral cryptorchid testes by using in situ hybridization, immunohistochemistry, and northern blot analysis techniques. The results showed that the expression level of Hsp70-2 gene declined slightly at the early stage of germ cell apoptosis, and dropped dramatically when most of the germ cells were undergoing apoptosis on day 7.5 after the induction of cryptorchidism. This report suggests for the first time that Hsp70-2 gene might not inhibit the apoptosis of germ cells at the early stage in cryptorchid testes. Hsp70-2 gene does not belong to the immediate early related genes that are responsible for germ cell apoptosis induced by heat stress.     

阴囊升温对大鼠睾丸生精细胞凋亡及增殖细胞核抗原表达的影响

目的　研究阴囊升温对大鼠睾丸生精细胞凋亡及增殖细胞核抗原 ( PCNA)表达的影响 ,探讨其在升温引起生精细胞变化中的意义。　方法　应用原位末端标记( TUNEL )法和免疫组化法 ,检测大鼠睾丸局部升温至 43℃、3 0 min后 ,经 0 .5、1、3、6、1 0和 50 d观察生精细胞凋亡和 PCNA表达情况。　结果　大鼠睾丸局部升温至 43℃、3 0min后生精上皮受到损伤 ,生精细胞凋亡增加 ( P<0 .0 1 ) ,生精细胞 PCNA表达下降( 0 .5～ 1 0 d组 P<0 .0 1 ,50 d组 P<0 .0 5)。　结论　 43℃、3 0 min作用后生精细胞 PCNA表达与细胞凋亡呈负相关 ,联合检测两者对评估热作用后生精功能的变化有重要意义     

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。