生精冲剂对实验性精索静脉曲张大鼠生精细胞凋亡状况的影响

目的:探讨实验性精索静脉曲张大鼠睾丸生精细胞凋亡状况以及生精冲剂对其凋亡的影响。方法:将60只成年雄性W istar大鼠随机抽出20只为对照组,其余按石津和彦改良法制成左精索静脉曲张大鼠模型,再随机分为模型组20只,治疗组20只。用末端脱氧核苷酸转移酶介导的原位缺口末端标记法(TUNEL)检测睾丸生精细胞凋亡。结果:模型组大鼠睾丸生精细胞凋亡指数明显高于对照组(P<0.01)和治疗组(P<0.01),治疗组与对照组比较有明显差异(P<0.01)。结论:实验性精索静脉曲张大鼠睾丸生精细胞凋亡增加,这可能是影响生育力的机制之一;生精冲剂能够减少精索静脉曲张大鼠睾丸生精细胞凋亡,对睾丸生精功能具有保护作用,进而可提高睾丸的生殖能力。     

六味地黄汤抑制大鼠生精细胞凋亡及其促进精子发生

用醋酸棉酚建造大鼠生精障碍模型,用六味地黄汤灌胃治疗,以市售六味地黄丸和甲基睾酮分别为对照和阳性对照,TUNEL法检测实验各组大鼠睾丸生精细胞的凋亡情况,计数实验各组大鼠精子数量,考察实验各组大鼠精子的质量.结果表明,六味地黄汤各治疗组、阳性对照组大鼠的精子数量、精子爬高均与模型组有显著性差异(P<0.01),各治疗组大鼠的精子质量(活力)也显著优于模型组;各实验组大鼠睾丸凋亡的生精细胞明显少于模型组.由此显示,六味地黄汤复方可显著抑制大鼠睾丸生精细胞的凋亡,对生精障碍大鼠有显著的促进生精作用,并可显著提高大鼠精子的质量.     

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     

实验性精索静脉曲张对大鼠睾丸生精细胞凋亡的影响

目的 :探讨大鼠精索静脉曲张对睾丸生精细胞凋亡的影响 ,阐明精索静脉曲张引起不育的病理机制。　方法 :选用雄性SD大鼠 32只 ,随机分为假手术组、4 5d模型组、6 0d模型组及 90d模型组。采用肾静脉缩窄法建立精索静脉曲张动物模型 ,流式细胞术分别检测各组大鼠睾丸凋亡细胞数及各级生精细胞数。　结果 :假手术组未出现明显的凋亡峰 ,各模型组均出现明显的凋亡峰 ,且随着造模时间的延长凋亡峰增高。　结论 :精索静脉曲张可引起睾丸生精细胞大量凋亡 ,各级生精细胞数减少 ,这可能是精索静脉曲张引起睾丸生精功能障碍从而导致不育的根本机制     

精索静脉曲张大鼠生精细胞凋亡的实验研究

目的 :探讨外科所致的精索静脉曲张对大鼠生精细胞凋亡的影响。　方法 :采用成年雄性Wistar大鼠复制左精索静脉曲张模型 ;用原位末端脱氧核苷酸转移酶标记法 (TUNEL)检测睾丸生精细胞凋亡。　结果 :实验组的细胞凋亡率显著高于假手术对照组 (P <0 .0 5 )。　结论 :外科所致的精索静脉曲张大鼠睾丸生长减缓 ,生精细胞凋亡增加 ,这可能是其影响生育能力的机制之一。     

睾丸扭转后生精细胞凋亡与iBOS基因表达

本文研究了睾丸扭转复位后生精细胞凋亡与iBOS基因表达的关系。采用大鼠建立左侧睾丸扭转复位模型（720,2h)。用TUNEL法和免疫组化SP法分别检测扭转复位后第五天生精细胞凋亡和iBOS基因表达。研究发现凋亡主要见于染色质降解的生精细胞（初级精母细胞和圆形精子细胞）。间质细胞和支持细胞未见凋亡发生。iBOS表达见于各级生精细胞,在染色质降解的生精细胞（即凋亡细胞）强表达。本文研究表明睾丸扭转复位后生精细胞凋亡增加与iBOS基因表达密切相关。睾丸局部NO生成异常可能是生精细胞凋亡增加的原因之一。     

己烯雌酚摄入对大鼠生精细胞凋亡及Bax和Bcl-2蛋白表达的影响

目的研究青春期己烯雌酚(diethylstilbestrol,DES)摄入对SD(Sprague-Dawley)大鼠性成熟后睾丸生精细胞凋亡的影响,并初步探讨其机制。方法30只35d龄雄性SD大鼠,随机分为DES 0.01、0.1、1.0、10.0μg/kg·d~(-1)4个实验组和1个对照组(编码为BDa、BDb、BDc、BDd和BC组,每组n=6)。于青春期[出生后第36天(postnatal day 36,PND 36)至49d(PND 49)],实验组每日皮下注射相应剂量的DES,对照组仅注射溶媒。于大鼠性成熟后(PND 64)处死各组大鼠切取双侧睾丸,采用TUNEL法检测大鼠睾丸生精细胞凋亡,用免疫组化方法检测凋亡相关蛋白Bcl-2和Bax在生精细胞中的表达。结果与对照组相比,BDa组大鼠性成熟后生精细胞凋亡无明显变化,BDb、BDc和BDd 3组生精细胞凋亡增加,且随DES摄入剂量增加而有增加趋势。BC、BDa组生精细胞Bax相对弱表达而Bcl-2强表达,伴随DES摄入剂量增加,Bax表达逐渐增强而Bcl-2表达逐渐减弱,BDd组Bax强表达而Bcl-2弱表达。结论青春期较大剂量DES摄入可使大鼠性成熟后睾丸生精细胞凋亡增加,且随DES摄入剂量增加而有加强趋势。凋亡相关蛋白Bax和Bcl-2参与青春期DES摄入所致的生精细胞凋亡过程。     

青春期前己烯雌酚暴露对大鼠性成熟后生精细胞凋亡的影响及其机制初探

目的:研究青春期前己烯雌酚(d iethylstilbestrol,DES)暴露对SD大鼠性成熟后睾丸生精细胞凋亡的影响并初步探讨其机制。方法:30只21日龄雄性SD大鼠,随机分为DES 0.01、0.1、1.0、10.0μg/(kg.d)4个实验组和1个对照组(编码为ADa、ADb、AD c、ADd和AC组,每组6只)。于青春期前[出生后第22 d(postnatal day 22,PND22)至35 d(PND35)],实验组每日皮下注射相应剂量的DES,对照组仅注射溶媒。于大鼠性成熟后(PND 64)处死各组大鼠切取双侧睾丸,采用TUNEL法检测大鼠睾丸生精细胞凋亡,用免疫组化方法检测凋亡相关蛋白Bc l-2和Bax在生精细胞中的表达。结果:与对照组相比,ADa组大鼠性成熟后生精细胞凋亡无明显变化,ADb、AD c和ADd 3组生精细胞凋亡增加,且随DES暴露剂量增加而有增加趋势。AC、ADa组生精细胞Bax相对弱表达而Bc l-2强表达,伴随DES暴露剂量增加,Bax表达逐渐增强而Bc l-2表达逐渐减弱,ADd组Bax强表达而Bc l-2弱表达。结论:青春期前较大剂量DES暴露可使大鼠性成熟后睾丸生精细胞凋亡增加,且随DES暴露剂量增加而有加强趋势。凋亡相关蛋白Bax和Bc l-2参与青春期前DES暴露所致的生精细胞凋亡过程。     

LM23基因沉默引起大鼠睾丸生精细胞凋亡

目的 研究LM23基因敲低后生精细胞的凋亡状况及与凋亡相关基因表达改变.方法 用TUNEL方法检测睾丸细胞的凋亡情况,用大鼠全基因组表达谱芯片检测LM23基因敲低后凋亡相关基因的表达改变.结果 TUNEL结果显示,LM23基因敲低侧大鼠睾丸生精小管内大量生精细胞发生凋亡.大鼠全基因组表达谱芯片分析结果显示,许多促凋亡基因如Bcl-2家族的促凋亡基因表达上调,而抗凋亡基因如Faf1和Zfp91基因的表达明显下调.结论 敲低大鼠的LM23基因,启动了Bcl-2家族介导的线粒体凋亡途径,引发了生精细胞发生大量凋亡.     

睾丸扭转后生精细胞凋亡与iNOS基因表达

本文研究了睾丸扭转复位后生精细胞凋亡与iNOS基因表达的关系。采用大鼠建立左侧睾丸扭转复位模型(720,2h)。用TUNEL法和免疫组化SP法分别检测扭转复位后第五天生精细胞凋亡和iNOS基因表达。研究发现凋亡主要见于染色质降解的生精细胞(初级精母细胞和圆形精子细胞)。问质细胞和支持细胞未见凋亡发生。iNOS表达见于各级生精细胞,在染色质降解的生精细胞(即凋亡细胞)强表达。本研究表明睾丸扭转复位后生精细胞凋亡增加与iNOS基因表达密切相关。睾丸局部NO生成异常可能是生精细胞凋亡增加的原因之一。     

Study on the effect of glucocorticoid on apoptosis of rat spermatogenic cells

The effect of glucocoiticoid on the apoptosis of rat spermato-genic cells labeled with TUNEL was observed in vivo by laser con-focal microscopy and electron microscopy. The results showed thatlarge doses of glucocorticoid increased the apoptosis of rat spermto-genic cell. (Chin J Androl 2001; 2: 98-101)     

糖皮质激素诱导大鼠睾丸间质细胞凋亡的研究

目的 :研究糖皮质激素诱导不同发育阶段培养的大鼠睾丸间质细胞凋亡状况。方法 :以Annexin V FITC和PI双标细胞 ,应用激光共聚焦显微镜和流式细胞仪检测。　结果 :ILC、ALC经皮质酮处理后 ,其凋亡量显著高于对照组 ;PLC经皮质酮处理后 ,其凋亡量与对照组无显著差异。　结论 :糖皮质激素能诱导大鼠睾丸未成熟型和成熟型间质细胞凋亡 ,而不能诱导前体型间质细胞凋亡。     

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     

High-magnification observation of seminiferous tubules through the tunica albuginea by two-photon laser scanning microscopy

Testicular sperm extraction is widely used in the treatment of male infertility in cases of non-obstructive azoospermia. Identifying spermatogenetic foci within the testes is critical for testicular sperm extraction. Two-photon laser scanning microscopy (TPLSM) is an autofluorescence-based microscopy technique that allows observation at a cellular level in the depth of fresh living tissues and does not require any histological processing (fixation or staining). The wavelengths previously used have shown no phototoxicity on sperm. We used TPLSM to detect spermatogenetic foci in fresh mouse testicular parenchyma without disrupting the tunica albuginea. Fresh surgically retrieved testes were observed using TPLSM within 1 h after extraction. Contralateral testes for each animal were observed using standard histology. Using TPLSM we were able to observe and measure the diameter of seminiferous tubules through the tunica albuginea, similar to the histological control. Structures within epithelial tubules were also observed, although their nature has yet to be identified. TPLSM is a real-time microscopy technique that could detect spermatogenetic foci.     

大鼠睾丸局部热作用对生精细胞凋亡的影响

目的 :研究大鼠睾丸局部受热后生精细胞凋亡的情况。　方法 :70只雄性SD大鼠随机分为热处理组 ( 43℃ )和对照组 ( 2 2℃ ) ,分别按睾丸局部处理后 12h、1d、3d、6d、10d、5 0d和 80d分成 7个亚组。应用电镜、流式细胞术和原位末端标记法 (TUNEL) ,检测各亚组大鼠睾丸生精细胞凋亡情况。　结果 :电镜显示热处理各组均出现生精细胞凋亡表现 ;流式细胞术检测发现热处理各组亚单倍体细胞百分率明显升高 (P <0 .0 1) ;TUNEL法显示热处理各组生精细胞凋亡率明显高于对照组 (P <0 .0 1) ,各类生精细胞对热的敏感性不同。　结论 :大鼠睾丸局部受热后生精细胞凋亡增加 ,初级精母细胞受热后最敏感 ,其次为精子细胞和精子 ,并且精原细胞凋亡在受热后一个长时期内均有增加     

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)     

Sperm ultrastructure and meiotic segregation in a group of patients with chronic hepatitis B and C

Little is known about the effect of chronic hepatitis B and hepatitis C on sperm quality. In this study, we analysed sperm quality from selected patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections. Semen samples were examined by light and transmission electron microscopy (TEM). TEM data were elaborated with a mathematical formula able to indicate a fertility index and the presence of the three main sperm pathologies: apoptosis, immaturity and necrosis. Meiotic chromosome segregation was investigated by fluorescence in situ hybridisation carried out on sperm nuclei, using probes for chromosomes 18, X and Y. Despite normal sperm concentration, we observed reduced motility. TEM analysis highlighted that 35.7% of patients showed generally good semen quality. However, significantly higher values of apoptosis and necrosis, compared with controls, were observed, demonstrating spermatogenetic alterations. Regarding meiotic segregation, we found an incidence of disomies similar to that observed in control samples, whereas diploidy resulted higher in HCV patients, without reaching statistical significance. In conclusion, sperm quality in the studied group was not impaired; however, apoptosis and necrosis resulted out of normal range and the fertility index was significantly lower in HCV- and HBV-infected patients versus controls.     

Sperm ultrastructure and meiotic segregation in a group of patients with chronic hepatitis B and C

Little is known about the effect of chronic hepatitis B and hepatitis C on sperm quality. In this study we analysed sperm quality from selected patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections. Semen samples were examined by light and transmission electron microscopy (TEM). TEM data were elaborated with a mathematical formula able to indicate a fertility index and the presence of the three main sperm pathologies: apoptosis, immaturity and necrosis. Meiotic chromosome segregation was investigated by fluorescence in situ hybridisation carried out on sperm nuclei, using probes for chromosomes 18, X and Y. Despite normal sperm concentration, we observed reduced motility. TEM analysis highlighted that 35.7% of patients showed generally good semen quality. However, significantly higher values of apoptosis and necrosis, compared with controls, were observed, demonstrating spermatogenetic alterations. Regarding meiotic segregation, we found an incidence of disomies similar to that observed in control samples, whereas diploidy resulted higher in HCV patients, without reaching statistical significance. In conclusion, sperm quality in the studied group was not impaired, however, apoptosis and necrosis resulted out of normal range and the fertility index was significantly lower in HCV and HBV infected patients versus controls.     

Cryptorchidism and semen quality: a TEM and molecular study

Cryptorchidism is a pathological condition defined as the failure of the testis to descend into the scrotum, the location of the cryptorchid testis can be in the inguinal canal or in the prescrotal and abdominal area, sometimes resulting in atrophic seminiferous tubules. The aim of this study was to analyze semen quality of men who underwent orchidopexy for unilateral or bilateral cryptorchidism during childhood. Semen quality was investigated by light microscopy to evaluate sperm concentration and motility. Sperm morphology was performed by transmission electron microscope (TEM), and the data were mathematically elaborated. The presence of Y microdeletions was investigated by polymerase chain reaction. The effect of cryptorchidism on meiosis was explored by fluorescence in situ hybridization (FISH). The incidence of azoospermia was higher in the group with bilateral compared with unilateral cryptorchidism, and semen parameters were better in the unilateral group. Sperm pathologies detected by TEM indicated a severe deterioration of sperm quality in both groups. Necrosis and apoptosis appeared to be the most frequent pathologies, and their values reached statistical significance compared with those from fertile controls. The presence of chromosome Y microdeletions in patients with cryptorchidism and severe spermatogenetic defects is controversial. No microdeletions were found in this study. FISH values indicated that the mean percentage of gonosome disomies and diploidies were generally out of normal range, indicating a severe disturbance of meiotic segregation. The effects induced by cryptorchidism resolved in childhood seem to include a spermatogenetic impairment, leading to recommendation of detailed ultrastructural and chromosomal sperm analyses before undertaking assisted reproductive techniques.