骨髓间充质干细胞移植治疗生精细胞损伤模型大鼠的实验研究

目的:探讨骨髓间充质干细胞(MMSCs)在睾丸内定向分化为生精细胞用于治疗实验性雄性不育模型大鼠的可行性。方法:复苏冻存的MMSCs-GFP并培养传代后经睾丸网移植到经白消安处理的生精细胞损伤模型大鼠睾丸曲细精管内,移植后8周采样,观察睾丸质量指数、组织学表现和供体细胞分布,运用免疫荧光检测生殖细胞表面特异标志c-kit的表达。结果:MMSCs-GFP移植组右侧受体睾丸质量指数与模型组大鼠及自体未细胞移植的左侧睾丸相比有差异(P﹤0.01),与正常组大鼠睾丸质量指数相比无差异(P﹥0.05);光镜下显示曲精管内生精上皮明显恢复,荧光显微镜下观察MMSCs-GFP大部分迁移至曲精管基底部,免疫荧光结果显示移植组与自身未移植组相比,睾丸曲细精管内精原细胞表面特异标志基因c-kit表达有差异。结论:细胞冻存不影响MMSCs的增殖能力,MMSCs在体内生精微环境中能够定植并定向分化为生殖细胞。     

Developmental Changes in Testosterone Production by the Rat Testis in Vitro During Late Fetal Life

The age-related evolution of the in vitro capacity of the rat testis to produce testosterone and to respond to luteinizing hormone (LH) during 3 and 24 h incubation was studied from day 18.5 to day 21.5 of fetal life. Basal testosterone production by testes from 18.5-day-old fetuses was significantly higher than production by testes from 20.5- and 21.5-day-old fetuses when secretion was expressed as either per testis or per microgram of testicular protein. When maximal LH-stimulated testosterone secretion was expressed on a per testis basis, it was significantly lower for day 18.5 testes than for day 20.5 and 21.5 testes. However, when it was expressed on the basis of testicular protein content, it decreased significantly between days 18.5 and 20.5. Basal and LH-stimulated secretions displayed the same time-related decrease throughout 24 h of incubation for the three ages studied. The dose-response curves for LH showed that the sensitivity was similar for day 18.5 and 20.5 testes (ED₅₀ = 10 vs. 14 ng/mL, respectively). These results showed an age-related decrease in testicular steroidogenic capacity without a change in the coupling efficiency of LH receptor to testosterone production during late fetal life in rats.     

ACTIVATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE IN CONTRALATERAL TESTIS DURING UNILATERAL TESTICULAR TORSION IN RATS

There are controversies about the injury of the contralateral testis during unilateral testicular torsion (UTT). An autonomic reflex arc between bilateral testes has been proposed. The authors focused on the involvement of nitric oxide (NO) in the contralateral testis during UTT. Eight-week-old male Wistar rats underwent unilateral torsion (1 h)-detorsion (up to 24 h). NO synthase (NOS) activity was detected as NADPH-diaphorase activity after fixation by paraformaldehyde. N -nitro- L -Arginine methyl ester (L-NAME, 20 mg/kg) was injected intravenously to the other group of rats. To evaluate the testicular injury, proteolysis of &#102 -fodrin production was detected by Western blotting. Apoptosis of the germ cells was evaluated by TUNEL. Long-term effect on spermatogenesis was evaluated by flow cytometry at 60 days after UTT. Transient activation of NOS was detected following the proteolysis of &#102 -fodrin in the contralateral testis. L-NAME inhibited these alterations. NADPH-diaphorase activity and eNOS immunoreactivity were co-localized in the endothelial cells. These reactions were not observed in other organs. There was neither enhanced apoptosis nor deteriorated spermatogenesis in the contralateral testis during and 60 days after UTT. In the contralateral testis, eNOS-derived NO regulates the vasomotor function against unilateral testicular torsion, whereas it acts slightly cytotoxic. These results suggest the possible involvement of a testis-specific neurovasomotor reflex between the bilateral testes.     

大剂量PN诱发雄性大鼠性腺损伤的实验研究

雄性成年ＳＤ大鼠随机分为５组,分别于右侧睾丸内注射生理盐水、７０％甘油和ＰＮ。７天后重复１次。第１５天取样,进行精子分析、睾丸及主要副性腺测量和组织病理学观察。结果显示,睾丸内注射ＰＮ引起了性腺、副性腺萎缩,精子性状改变以及组织病理学改变。推测可能是：（１）ＰＮ或ＰＬ与ＰＬＰ竞争结合部位和抑制ＰＬＰ的合成,干扰细胞代谢;（２）通过转化形式或直接作用于Ｓｅｒｔｏｌｉ细胞,损害细胞骨架。（３）直接透过血睾屏障而损害管腔小室内的生精细胞     

邻苯二甲酸二丁酯对F1 代仔鼠睾丸损害的可逆性恢复特性

目的研究和探讨邻苯二甲酸二丁酯(DBP)对F1代仔鼠睾丸损害是否具有可逆性恢复的特性。方法在宫内暴露和哺乳期(妊娠1d至仔鼠出生21d),每日每kg体重0、50、250和500mg DBP灌胃染毒孕鼠(每组10只),从解剖学、病理学等角度观察DBP对不同发育阶段(出生14、21和70d)F1代雄性大鼠的睾丸损害。结果与对照组相比,虽然不同发育阶段大鼠睾丸重量及系数没有明显统计学意义,但与对照组相比较,250、500mg／L组出生14d大鼠生精上皮变薄,Sertoli细胞数量明显减少(分别减少至对照组的84％和86％),并出现空泡样改变;出生21d时,Sertoli细胞数量和形态基本恢复正常,但有少量生精细胞脱落;至出生70d时,个别大鼠睾丸出现曲细精管变性、生精上皮萎缩等不可逆性损害。结论DBP宫内暴露和哺乳期染毒可损害F1代大鼠睾丸Sertili细胞,但随着仔鼠的发育,DBP致Sertoli细胞损害作用具有部分可逆性恢复的特性。     

犬自体睾丸移植的实验研究

目的 采用一种改良术式进行犬自体睾丸移植，探讨血管吻合方法对植睾功能的影响。方法 切取带主干血管瓣的右侧睾丸动、静脉与同侧髂外动、静脉端一侧吻合进行犬自体睾丸移植。观察移植犬生存情况，对移植睾丸进行影像学检查并比较实验组与对照组睾丸组织学改变及术后不同时间点的性激素水平。结果 移植成功率为92．8％（13／14）。术后8周两组犬的睾丸重量、精曲小管直径、间质细胞数及细胞核直径之间无统计学差异（P〉0，05）。与术前比较，两组术后各时间点LH明显升高（P〈0．05），T及FSH变化无统计学意义；组间比较术后各时间点的T、FSH及LH水平无显著性差异（P〉0．05）。彩色多普勒超声及DSA检查提示移植睾丸血供良好。结论 改良的自体睾丸移植术可提高移植成功率，且对移植睾丸生精及内分泌功能无明显影响，可进一步应用于异体睾丸移植的实验及临床研究。     

输精管结扎后家兔睾丸IL-1、TNF与形态学变化关系的研究

为探讨输精管结扎术后睾丸IL-1活性的变化以及与睾丸形态学改变的关系,在复制输精管结扎家兔动物模型的基础上,检测结扎后6、25月家兔睾丸匀浆上清中IL-1活性和TNF含量,同时观察睾丸组织形态学改变。结果:①睾丸组织匀浆上清中IL-1活性和TNF含量,结扎6月组(VG6)显著高于假手术6月组(SOG6)(P<0.01,P<0.05),结扎25月组(VG25)与假手术25月组(SOG25)在同一水平(P>0.05);②睾丸形态学观察发现,VG6有明显生精障碍,间质中可见炎症细胞浸润,VG25可见生精过程恢复的曲细精管及呈灶状分布萎缩的曲细精管,后者被结缔组织包绕。故推测输精管结扎术后睾丸组织IL-1活性和TNF水平的变化与睾丸的炎性变化过程有关。     

Hesperetin, a citrus flavonone, protects potentially cadmium induced oxidative testicular dysfunction in rats

The present study was aimed to evaluate the protective effect of hesperetin (Hp) on cadmium (Cd) induced oxidative testicular toxicity in rats. Subcutaneous administration of Cd (3 mg/kg body weight) for 21 days significantly elevated the levels of oxidative stress markers, Cd concentration in testis and lowered the levels of enzymatic, non-enzymatic antioxidants and membrane bound enzymes in the testicular tissue. Hp administrated orally along with Cd injection for 21 days, significantly revert back the status of oxidative stress markers, Cd concentration in testis, improved status of antioxidant markers and membrane bound enzymes in the testis to near normal level. The histopathological studies in the testis of rats also supported that Hp (40 mg/kg) markedly reduced the toxicity of Cd and preserved the normal histoarchitecture pattern of the testis. Thus, the results suggest that Hp acts as a potent antioxidative agent against Cd induced testicular toxicity in rats.     

Effect of Ultrasound on Testicular Electrolytes (Sodium and Potassium)

The testes of 50 rats were placed in a cup filled with water and received 1 W/cm² of ultrasound for 15 min. Fluid was collected from the seminiferous tubules and rete testis of the treated and control groups at 1, 8, 12, and 24 hr intervals. Ultrasound increased the sodium concentration in the fluid of the seminiferous tubules, decreased the sodium concentration in the fluid of the rete testis, increased the potassium concentration in the fluid of the rete testis, and decreased the potassium concentration in the fluid of the seminiferous tubules.

Fourteen, slightly sedated, monkeys (Macaca fascicularis) were treated with 1/2 W/cm² of ultrasound for 30 min. Water was used as the coupling agent for seven monkeys and 3% NaCl was used as the coupling agent for the other seven monkeys. The efficacy of ultrasound treatment in reducing sperm count to zero and achieving zero motility was increased when 3% NaCl was used. Sperm count was at the level of presonication after 20 weeks when water was used as a coupling agent.     

吸烟对雄性大鼠睾丸组织结构及细胞凋亡的影响

目的通过测定吸烟雄鼠睾丸组织结构及细胞凋亡情况,探讨吸烟对雄性生殖系统的损伤机制。方法将清洁级SD雄性大鼠160只随机分为染毒2、4、6、8、12周的低、中、高剂量染毒组及对照组,每组10只。采用呼吸道静式染毒,每日染毒1次,染毒周期为2、4、6、8、12周。采用HE染色及末端转移酶标记技术(TUNEL法)测定大鼠睾丸组织病理学改变及细胞凋亡情况,用ELISA法检测大鼠血浆抑制素B的表达水平。结果吸烟组大鼠睾丸脏器系数从第8周起逐渐减小,与2周各剂量染毒组相比,差异有统计学意义(P0.05)。吸烟组大鼠随着染毒时间的延长和剂量的增加,睾丸组织病理学改变逐渐明显,染毒12周后大鼠睾丸组织支持细胞呈现空泡化,管腔内观察不到游离精子。吸烟组大鼠睾丸细胞凋亡指数随染毒时间及剂量的增加而升高,染毒4周高剂量组、6周中及高剂量组、8周及12周各剂量组睾丸细胞凋亡指数均高于对照组,差异有统计学意义(P0.05)。随着染毒时间及剂量的增加,大鼠血浆抑制素B水平逐渐降低,染毒第8周起低于对照组,差异有统计学意义(P0.05)。结论香烟烟雾中的有害成分可致大鼠睾丸组织结构损伤及睾丸细胞凋亡率增加,并抑制血浆抑制素B分泌。     

吸入纳米和微米二氧化钛颗粒对雄性大鼠精子及其功能的影响

[目的]比较雄性大鼠吸入纳米二氧化钛（nm-TiO2）与微米二氧化钛（nm-TiO2）对其生精功能的影响并探讨其作用机制。[方法]45只雄性Wistar大鼠随机分为5组,分别为nm-TiO2100 mg/m^3和300 mg/m^3组,μm-TiO2100 mg/m^3和300 mg/m^3组和对照组。动物隔天在装有风扇扬尘的静式染毒柜中吸入染毒2 h,对照组不扬尘,只吸入空气。65 d后,测定附睾尾精子数量、活动度和畸形率,睾丸组织中乳酸脱氢酶同工酶-C4（LDH-C4）、琥珀酸脱氢酶（SDH）、超氧化物歧化酶（SOD）的活性、丙二醛（MDA）含量和血清中MDA、SOD及8-羟基脱氧鸟苷（8-OHdG）水平。[结果]nm-TiO2致大鼠精子数量下降、LDH-C4活力下降和8-OHdG水平增加程度显著高于同剂量μm-TiO2组（P〈0.05）;nm-TiO2可致大鼠精子活动度、睾丸组织SDH和SOD活力下降,精子畸形率和MDA生成量升高（P〈0.05）,但与同剂量μm-TiO2组比较无显著性差异（P〉0.05）。[结论]nm-TiO2和μm-TiO2对雄性大鼠生精功能影响存在差异,nm-TiO2致生殖损伤能力强于同剂量水平的μm-TiO2。     

1-(2,4-Dichlorobenzyl)-1 H-lndazole-3-Carboxylic Acid (DICA), an Exfoliative Antispermatogenic Agent in the Rat

The oral administration of 50 mg DICA/kg at nine weekly or four monthly intervals produced partially reversible infertility in male rats as judged by the results of serial mating and testicular histology. Oral 500 mg DICA/kg doses administered at the same intervals produced permanent sterility. Single oral doses of 50 or 500 mg DICA/kg elevated mean FSH concentrations on days 2, 3, and 7 but did not affect LH or testosterone. Mean plasma concentration peaked at 74 μg/ml 4 hr after a 50 mg/kg dose of uniformly tritiated DICA; 24 hr later, it had declined rapidly to 5.5 μg/ml. The drug did not have a strong affinity for any tissue studied including the testis. DICA-induced exfoliation of immature germ cells was first observed 4 hr after administration and led to significantly reduced testis weights by day 2. Neither single doses of 10-250 mg DICA/kg nor five daily doses of 10-100 mg DICA/kg reduced seminal vesicle, ventral prostate, or body weights of male rats. Chronic weekly DICA administration did reduce mean seminal vesicle weight. These studies have shown that DICA is an effective, partially reversible antifertility agent that directly affects the rat testis.     

Reversibility of testicular atrophy induced by Di(2-ethylhexyl) phthalate in rats

Previous studies have shown that di(2-ethylhexyl) phthalate (DEHP) results in atrophy of testes and accessory sex organs accompanied by a decreased testicular concentration of zinc in rats. An experiment was performed to determine whether those changes in rat testes are reversible changes. Young Wistar male rats were administered 2.0 g/kg of DEHP for 14 days. At that time, one-half of the rats were killed for determination of zinc and testosterone concentrations in the testis, liver, and serum, and organ weights. The remaining rats were maintained for additional 45 days without further DEHP administration. Testicular weight of rats administered DEHP over the 14-day period was significantly less than that of the control animals, and testicular testosterone content and zinc concentrations were less than those of the control animals. At the end of the 45-day recovery phase, serum testosterone concentration returned to the control level, but testicular zinc concentration and testosterone content were still lower than those of the control animals. In addition, testicular weight of DEHP-treated rats was lower than that of the control animals and histologically, only a small number of seminiferous tubules showed spermatogenesis. These results indicate that, morphologically, DEHP-induced testicular atrophy appears to be of limited reversibility.     

用体外器官培养方法研究EDCs对新生小鼠睾丸的影响

目的 通过已建立的小鼠睾丸体外培养系统,研究四种内分泌干扰物（Endocrine Disrupting Chemicals,EDCs）对男性内分泌系统的影响。 方法 将新生小鼠的睾丸组织在体外环境中培养24h,而后在培养基中分别加入浓度为0.1μM, 1μM, 10μM and 100μM的四种（DEHP、MEHP、NP、p, p’-DDE）内分泌干扰物并培养72h,同时设置对照组;培养结束后进行组织学观察,测定冻存培养基中睾酮和抑制素βB (INH-βB)的分泌水平,同时测定细胞色素P450侧链裂解酶(P450Scc)、3β-羟基类固醇脱氢酶(3β-HSD)、细胞色素P45017α-羟化酶(P450C17)和波形蛋白（vimentin）的基因表达情况。 结果 所有剂量组中睾酮的分泌水平均发生改变;P450Scc、3β-HSD、P450C17和INH-βB蛋白质的表达及mRNA水平均受到四种内分泌干扰物的影响（P<0.05）;DEHP和MEHP降低了波形蛋白的mRNA水平（P<0.05）,而NP和p, p’-DDE对波形蛋白没有显著影响（P>0.05）。 结论 本研究建立的体外培养新生小鼠睾丸模型中,所选的四种已知EDCs改变了两种睾丸激素水平,三种类固醇合成酶以及与支持细胞功能相关的波形蛋白的表达。     

硒对氟致大鼠睾丸脂质过氧化和微量元素改变的影响

给ＳＤ雄性大鼠饮用含氟化钠、亚硒酸钠及氟化钠和亚硒酸钠的水溶液８周，观察硒对氟致睾丸脂质过氧化和微量元素铜、锌、铁、硒含量改变的影响．结果表明，氟化物可使睾丸组织中的脂质过氧化物（ＬＰＯ）含量明显增加，铜、锌、铁含量升高而硒含量降低．同时补硒对氟诱导的睾丸脂质过氧化具有明显的拮抗作用，对氟致睾丸组织中的铜升高利硒降低也有一定的拮抗作用，而对锌、铁含量升高则具有明显的协同作用。因此可以认为，氟致睾丸损害的机理与其诱发的脂质过氧化作用有关，而硒对氟致脂质过氧化则具有明显的拮抗作用．     

高脂饮食对Sprague Dawley大鼠睾丸发育的影响

目的 研究高脂饮食对青春期雄性Sprague Dawley大鼠睾丸发育过程的影响,并探究该影响是否具有可逆性。方法 将1月龄Sprague Dawley雄性大鼠60只,应用随机数字表法分为对照组（n=10）和营养性肥胖模型组(n=50),分别用普通饲料和高脂饲料喂养8周后,将10只营养性肥胖模型组大鼠和对照组大鼠处死。采用酶联免疫法检测血清睾酮和雌二醇水平,HE染色观察左侧睾丸的组织学改变,Johnsen评分评价睾丸的生精能力。剩余40只大鼠应用随机数字表法分为高脂饮食组(n=13)、正常饮食组(n=13)和减肥组(n=14),后两组大鼠均用普通饲料喂养,高脂饮食组大鼠用高脂饲料喂养,减肥组大鼠在正常饮食的基础上,每日跑步20 min,喂养6周后处死大鼠进行上述睾丸结构和功能研究。结果 高脂饮食8周后,营养性肥胖大鼠的体重明显高于对照组(O=0.006)。营养性肥胖大鼠的血清睾酮水平明显低于对照组(P=0.024),血清雌二醇水平明显高于对照组(P =0.017)。HE染色显示：与对照组比较营养性肥胖大鼠曲精小管细胞层数减少,部分曲精小管萎缩。营养性肥胖大鼠的Johnsen评分明显低于对照组(P=0.000)。高脂饮食组的睾酮水平明显低于正常饮食组(P=0.001)和减肥组(P=0.000),正常饮食组的睾酮水平也明显低于减肥组(P=0.001)。高脂饮食组的雌二醇水平明显高于正常饮食组(P=0.001)和减肥组（P=0.000）,正常饮食组的雌二醇水平也明显高于减肥组(P=0.001)。HE染色显示：高脂饮食组和正常饮食组大鼠睾丸组织的病变无进行性加重;减肥组大鼠睾丸组织的病变较轻。减肥组的Johnsen评分均明显高于其他两组（P=0.000及0.001）。相关性分析显示,Johnsen评分与大鼠体重呈负相关（r=-0.962,P=0.000）;与血清睾酮水平呈正相关(r=0.916,P=0.000)。结论 高脂饮食能导致青春期雄性Sprague Dawley大鼠营养性肥胖。营养性肥胖大鼠表现为睾丸发育不全,生精功能减弱,内分泌功能障碍,运动减肥后以上改变有所改善。肥胖程度与生精功能呈负相关。     

丙烯腈对雄性大鼠睾丸组织病理和超微结构的影响

目的探讨丙烯腈(ACN)对雄性SD大鼠睾丸组织的病理学损伤作用及超微结构的改变。方法从52只雄性SD大鼠中随机取出4只作为空白对照组(不做任何处理),其余48只随机分为4组,每组12只,分别以0,7.5,15.0,30.0 mg/kg的剂量(用生理盐水作溶剂)腹腔注射ACN染毒,1次/d,每周5次,连续染毒13周。13周末,从各组中随机取6只大鼠处死;其余实验大鼠停止染毒2周后处死。用光镜和电镜观察大鼠睾丸组织的病理损伤及超微结构的改变。结果ACN染毒13周后,7.5 mg/kg组光镜下可见大鼠睾丸曲精小管组织病理学结构有轻微改变,受损程度随染毒剂量的增加而加重;30.0 mg/kg组,光镜下可见大鼠睾丸部分曲精小管出现退行性病变,管腔中生精细胞和精子数量减少;电镜下可看到凋亡的、核畸形的以及坏死的初级精母细胞和畸形精子,生精管上皮空泡样变,腔面无精子,尚存的发育期精子细胞内没有线粒体的相应移动。15周后(停止染毒2周后)各组大鼠睾丸组织病变与同剂量组染毒13周末比较,差异无显著性。结论ACN能够诱导大鼠睾丸损伤,具有性腺毒性,同时提示,生精细胞的核质损伤是其主要毒作用之一。     

Inhibition of testicular steroidogenesis in the neonatal rat following acute ethanol exposure

We have previously reported detrimental effects of in utero ethanol exposure on testicular steroidogenic enzyme activity in newborn rats. It is now reported that in utero ethanol exposure during Day 12 of gestation through birth has no apparent morphological effect on the testes of Day 1 neonatal rats. It appears that the detrimental effects of ethanol on testicular steroidogenesis can be manifested at the biochemical level in the absence of morphological effects. However, it remained unknown as to whether acute exposure to ethanol would elicit similar biochemical effects as chronic ethanol exposure on testicular steroidogenesis. To test this possibility ethanol was injected at 0, 1, or 2 g/kg intraperitoneally (IP) into rats of various postnatal ages. Plasma ethanol and testosterone levels as well as testicular 17 alpha-hydroxylase and C17,20-lyase activities were measured. The results indicate that acute exposure to ethanol significantly (p less than 0.05) inhibits the catalytic activity of testicular 17 alpha-hydroxylase in the newborn rat testis. This inhibition was specific since the activity of testicular C17,20-lyase was not affected. In conjunction with the reduction in testicular enzyme activity, plasma testosterone levels were reduced to 30% of the control levels in newborn animals receiving ethanol. In older animals, i.e., postnatal Day 20 and 40 rats, plasma testosterone levels were reduced, but not significantly, following ethanol treatment. Furthermore, testicular enzyme activity was not significantly reduced following ethanol treatment in these same older animals. These results suggest that the newborn rat testis is especially sensitive to the effects of ethanol.     

香烟烟雾暴露对大鼠睾丸转铁蛋白mRNA及蛋白表达水平的影响

目的探讨香烟烟雾暴露对大鼠睾丸结构及睾丸组织转铁蛋白(Tf)mRNA及其蛋白表达水平的影响。方法清洁级成年雄性SD大鼠160只,随机分为对照组及低(10根香烟)、中(20根香烟)、高(30根香烟)剂量染毒组,各染毒组大鼠分别染毒2、4、6、8、12周,共16组,每组10只。染毒组大鼠采用呼吸道静式染毒,1次/d,30 min/次。观察大鼠睾丸组织结构,采用RT-PCR及Western blot法检测睾丸组织Tf mRNA及其蛋白表达水平。结果染毒组大鼠睾丸病理切片可见生精上皮层次减少,生精细胞排列疏松,支持细胞空泡化。染毒4、8、12周时高剂量组大鼠睾丸组织Tf mRNA表达水平低于对照组和低剂量组,染毒8周时高剂量组大鼠睾丸组织Tf蛋白表达水平低于对照组,染毒12周时中、高剂量组大鼠睾丸组织Tf蛋白表达水平低于低剂量组,差异均有统计学意义(P0.05)。结论香烟烟雾暴露可导致雄性大鼠睾丸组织损伤,并抑制Tf基因及蛋白表达。