【作者】 许多；

【导师】 秦达念；

【作者基本信息】 汕头大学 ， 生殖病理学， 2003， 硕士

【摘要】 背景：近年来，男性不育的原因及其相关机制受到学者们的重视。调查中发现，过去五十年中男性精子质量呈下降趋势，环境因素被认为是致病的主要因素，其中温度作为一种外在因素引起男性不育也愈来愈被重视。研究表明：包括人类在内的大部分哺乳动物的睾丸需要一个比腹腔温度低的环境，睾丸暴露在腹腔温度或高于腹腔温度可导致可复性的生精障碍和生精细胞退化，引起暂时的、部分或完全性不育，其机制尚不完全清楚，其中热作用后生精细胞的凋亡可能起了重要作用，已引起学者们关注。如对隐睾患者的研究表明，睾丸处于腹腔温度引起生精细胞的凋亡是导致隐睾患者不育的原因之一。但目前对热作用后生精细胞凋亡的研究尚不足，尤其热作用后各类生精细胞凋亡的敏感性的研究亦不统一，并且对生精细胞受热凋亡的分子机制缺乏深入研究。另一方面，在男性避孕方面的研究至今仍不理想，如广为应用的输精管结扎术因其外科手术的风险，高耗费和再通的困难都限制了它的应用。近年来，学者们致力于探索更安全、有效、可恢复的男性避孕方法。作为探索安全、有效的男性避孕途径之一，温度对精子发生的影响受到重视。所以，建立热处理的动物模型，从细胞凋亡的角度探讨热作用后生精功能变化的机理，分析凋亡相关基因的调控，将为探索和阐明男性不育的机制提供重要和有价值的资料，为临床治疗某些男性不育、加强劳动保护和探索男性避孕打下基础。助头大学曹学院硕士研兑全毕，必触戈尸 目的:1)研究皋丸局部热作用后生精细胞的变化，检测生精细胞的凋亡，以此阐明凋亡可作为热作用后生精细胞变化的一种机制;2)探索各类生精细胞对热的敏感性，对热作用引起生精障碍作更深入研究;3)研究凋亡相关蛋白Fas和FasL在热作用后生精细胞的表达情况，在分子水平上探讨热作用诱导生精细胞凋亡的机制。 材料与方法:雄性SPrague一Dawlcy(SD)大鼠，分笼饲养于标准实验房(22℃)，给予规律光照(12h: 12h)，自由饮食，随机分热处理组和对照组，热处理组按热处理后0.5、l、3、6、10、25、50和80d随机分成8个亚组(n=6)，每亚组相应设立对照组(n二4)。热处理组大鼠阴囊浸于43℃恒温水浴中30min，对照组浸于22℃水浴中30min。分别在处理后0.5、l、3、6、10、25、50和80d采取标本，检测皋丸重量变化，应用HE染色观察生精细胞组织学结构;采用电镜、流式细胞仪和原位末端标记法(TdT一mediated dUDp一X niek end labeling，TUNEL)检测皋丸局部受热后生精细胞凋亡情况;应用流式细胞仪和免疫组化法测定Fas和FasL在热作用后生精细胞的表达情况。 结果: 组织学检测:大鼠翠丸局部受热后3d出现皋丸重量明显减轻 (P<0.01)，热作用后以10d一25d翠丸重量减轻更严重，50d已开始恢复，但直到受热后80d翠丸重量才恢复到基本正常。光镜下热作用后攀丸生精上皮变薄，生精细胞排列紊乱，数量减少，有大量空泡生成，出现染色质边集、呈月牙状，可见多核巨细胞。以10一25d组织学改变最明显。50d部分生精小管上皮恢复，到80d生精小管内生精上皮大多恢复正常。 凋亡的检测:l)电镜下热处理各组可见染色质浓缩、附着于核边缘呈新月形、核染色质固缩、断裂以及凋亡小体出现等生精细胞凋亡的表现，以热处理后0.5一6d组更常见;2)流式细胞仪观察，热处理助兴大学医学院硕哇~班男全软必讨戈尸各组均有亚单倍体细胞百分率明显增高(P<0.01)，尤以0.5一6d组更明显(eontrols:0.5d:2.85士0.66:ld:2.36士0.57;3d:2.71士0.49;6d:2.60士0 .44;exPeriments:o.sd:23.85士5.59:ld:30.9士1.65;3d:21.55士0.42:“:22.98士1.53);对各类生精细胞DNA含量进行分析表明，热处理后0.5一 50d组4C细胞百分比明显低于对照组(P<0.05);6d一SOd组IC细胞的百分比明显低于对照组(P<0.05);3d一SOd组ZC细胞百分比明显高于对照组(P<0.05)。3) TUNEL检测发现，各热处理组生精细胞凋亡指数均明显增多(P<0.01)，尤以0.5一6d组更明显(controls:0 .sd:0.60士0.01;ld:0.55士0.06;3d:0.47士0.11;6d:0.48士0.11;exPeriments:0.5d:16.75士0.48:ld:27.75士0.85:3d:18士0.91:6d:16.25士1.11)。热作用后生精细胞凋亡阳性定位观察:0.5、1d组凋亡阳性主要定位在初级精母细胞;3d组出现大量的精子细胞和精子凋亡;6d、10d组凋亡细胞以大核细胞和精母细胞多见;SOd组多见于精原细胞和精母细胞;80d组生精上皮大部分恢复，凋亡细胞多见于精原细胞。 Fas蛋白表达:流式细胞仪检测显示，热处理各组Fas蛋白表达均明显高于其对照组(P<0.01)，热处理后ld组Fas表达最强。免疫组化染色显示:各组生精细胞、支持细胞和间质细胞均有阳性表达，热处理后各组生精细胞Fas表达均增强。 FasL蛋白表达:流式细胞仪检测显示，热处理后各组FasL蛋白表达均明显高于其对照组(P<0.05)，热处理后ld组FasL表达最强。免疫组化染色显示:各组生精细胞、支持细胞和间质细胞均有阳性表达，热处理后各组生精细胞FasL表达均增强。 结论: l)热作用引起大鼠翠丸生精上皮更多还原

【Abstract】 Background The reason and mechanism of male infertility have attracted the investigator’s attention in recent years. An increasing number of reports have suggested a trend toward decreasing sperm quality in men over the past five decades. Due to the relatively short period of time for this trend to occur, it is believed that decreasing sperm quality is the result of environmental rather than genetic factors. Temperature as an environmental factor brings heat induced male infertility has been attentioned. Several studies have shown that in most mammals, including humans, the testis is always maintained at a lower temperature than that in the abdomen, and exposure of the testis to body temperature or above results in increased death of spermatogenic cells. Mild testicular heating has been established as a safe and reversible approach for suppression of spermatogenesis. In addition, some studies suggest that the one of reasonof infertility with cryptorchidism is spermatogenic cell apoptosis induced by abdomen temperature. Currently, surgical ligation of the vas deferens is the only efficacious male contraceptive method. The high financial cost, surgical risk involved, and relatively high incidence of permanent infertility are some of the inherent disadvantages of this method, hence itslimited use. The search for a self-applied, reliable and reversible method has been intensified in recent years. Thus, safety, efficacy and applicability of thermal manipulation of spermatogenesis as a method of male contraception could either be established or refuted. Futher understanding of the regulation of spermatogenic cell apoptosis may allow new targeted approaches to male contraceptive development and treatment of some forms of male infertility.Objective 1) To determine whether the heat-induced loss of spermatogenic cells in the adult rat occurs via apoptosis. 2) To explore the sensitivety of spermatogenic cells with local testicular heating. 3) To examine the role of Fas and FasL in rat testicular spermatogenic cell apoptosis with local testicular heating.Material and Methods Adult male Sprague-Dawley rats were housed in a standard animal facility under controlled temperature (22℃) and photoperiod(12h:12h), with free access to water and rat chow. The scrotum containing testes were then immersed for 30 min in a thermostatically controlled water bath at 43 ℃. Control rats were treated in the same way, except the testes were immersed in a water bath maintained at 22℃. The rats sacrificed at 0.5, 1,3,6, 10, 25, 50 and 80d after heat exposure and collected samples. The weight of testis was detected and the histology of testis was observed by HE stain; The apoptosis of spermatogenic cells was detected by means of electron microscopy, flow cytometry and TUNEL (TdT-mediated dUDP-X nick end labeling) method; Fas and FasL expression was observed with flow cytometry and immunohistochemistry.Results Rat testicular weight fell beginning at 3 days and reachingminimum values during 10-25 days after heating and it was recover at 50 days post-heating (P<0.01) . But by 80 days following heat treatment testicular weight was almost normal. Histological examination showed that germinal epithelium became thin, the number of spermatogenic cells was decreased, vacuoles were common, many spermatogenic cell nuclei contained highly condensed chromatin and multinucleate cells were appeared. On groups of 10 and 25d, these change were severe. By the day80, germinal epithelium was almost recovered.Spermatogenic cell apoptosis: 1) In all groups of heat treatment, crescent chromatin condensation under nuclear membrane and the apoptotic bodies were detected by electron microscopy. Correspondingly, the features of spermatogenic cell apoptosis were occasional in the control groups. 2) Flow cytometry found that the percentage of cells with sub-haploid was increased in all heat treatment groups compared with respective control group (P<0.01) . Moreover, the change was marked in heat treatment groups of 0.5-6d (controls: 0.5d:2.85更多还原