停服棉酚后恢复的大鼠精母细胞生物活性的研究

棉酚是一种有效的男用避孕药及抗肿瘤药物,但对长期喂服棉酚后,被抑制的精原细胞的生物活性研究尚少。本实验用成年雄性大鼠,喂服棉酚3、4、6、7、8周,停药15天后,被抑制的精原细胞恢复功能,重新进入减数分裂期和粗线期。用流式离心技术,将大鼠睾丸细胞分离成6个组分,组分6,90％以上为第一期恢复的粗线期精母细胞(RPS)。将RPS和对照组细胞(CPS)在体外进行培养,利用~3H-胸腺嘧啶核苷的掺入测定其DNA合成。结果表明,不同时间喂服棉酚,恢复后的粗线期精母细胞与对照组相比,DNA合成明显低于对照组。喂服棉酚3、4、6、7、8周后,RPS组DNA     

低剂量棉酚与甾体激素联合应用对生精细胞凋亡和iNOS蛋白表达的影响

目的 研究低剂量棉酚与甾体激素联合应用(棉甾联合用药)对大鼠睾丸生精细胞凋亡和诱导型一氧化氮合酶(iNOS)蛋白表达的影响. 方法 本实验采用大鼠喂服棉酚12.5mg/(kg.d)+激素[去氧孕烯125μg/(kg.d)+炔雌醇25μg/(kg.d)+十一酸睾丸酮100mg/(kg.d)]联合用药的方式,与单独喂服相同剂量的棉酚或激素的大鼠及喂服甲基纤维素溶剂的大鼠相对照,通过原位缺口末端标记(TUNEL)染色,观察生精细胞凋亡的情况;通过免疫组织化学染色和免疫印迹法,观察诱导型一氧化氮合酶(iNOS)蛋白表达的变化. 结果 TUNEL染色显示,在正常对照组,阳性着色主要分布在Ⅶ、Ⅷ、Ⅸ期生精小管的残余体中,偶见于粗线期精母细胞中.棉甾联合用药组和激素组,阳性着色主要分布在粗线期精母细胞中,随用药时间的延长,阳性着色的细胞数量增多(P＜0.01).iNOS免疫组织化学显示,在正常对照组,在残余体、精原细胞和粗线期精母细胞中的表达具有期依赖性.在Ⅶ、Ⅷ、Ⅸ期生精小管,iNOS主要在残余体中表达;在IX-XII期生精小管,精原细胞和粗线期精母细胞的核中可见少量表达.在棉甾联合用药组,iNOS在残余体中的表达,随用药时间的延长表达量降低(P＜0.05);iNOS在粗线期精母细胞、精原细胞胞核中的表达,随用药时间的延长表达量增高(P＜0.05).iNOS在正常对照组的间质细胞中持续表达,棉甾联合用药组4周降至最低,3时间点表达量无统计学差别. 结论 iNOS蛋白在低剂量棉酚加甾体激素联合应用大鼠睾丸中各部位的表达变化趋势不一致,分别产生不同的功能;生精细胞凋亡数增加,可能与iNOS的表达增多有关.     

棉酚对大鼠毒性的电子显微镜观察

棉酚对雄性大鼠有明显的抗生育作用。用电镜和同位素放射自显术对服药后大鼠睾丸变化动态的观察表明棉酚主要破坏生精上皮的精子发生过程,其中变态期精子细胞和中、后期初级精母细胞对棉酚的毒性最为敏感。4000多名志愿服药者的临床试验进一步证明了棉酚的抗生育效果肯定可靠。精液脱落细胞的分析亦以精子细胞和精母细胞的脱落为主。棉酚对生精细胞的这种毒性作用,为当前寻找男用避孕药提供了前景。棉酚对其他脏器组织的毒性作用亦已有报道。猪服棉酚后可引起心肌损伤,肝、脾、肾     

低剂量棉酚和甾体激素联合用药对精母细胞凋亡和支持细胞吞噬作用的影响

目的 探讨低剂量棉酚和甾体激素联合用药对睾丸精母细胞数量、凋亡和支持细胞吞噬作用的影响。 方法 本实验采用大鼠喂服棉酚与甾体激素联合用药方式［棉酚125mg/（kg•d）+去氧孕烯125μg/（kg•d）+炔雌醇25μg/（kg•d）+十一酸睾丸酮100mg/（kg•d）］,与单独喂服相同剂量的棉酚或激素的大鼠及喂服载体溶剂的大鼠相对照,用药4、6和8周取睾丸组织,应用体视学、原位缺口末端标记(TUNEL)和油红O染色的方法,观察精母细胞和球形精子细胞数量、凋亡和支持细胞吞噬作用。 结果 联合用药中,甾体激素成分可明显减少精母细胞和球形精子细胞的数量,精母细胞凋亡增加并同时伴有生精上皮油红O染色的增强。 结论 精母细胞凋亡并被支持细胞吞噬是联合用药抗生育机制之一。     

棉酚对大鼠垂体、肾上腺和下丘脑作用的组织化学观察

棉酚对男性是具有一定抗生育作用的,经多方面有关资料的介绍,及根据我们实验材料的观察也得到证实。棉酚的作用环节主要是损伤早期精子细胞和粗线期精母细胞;而对间质细胞可能有轻度影响。睾丸是受垂体促性腺激素的调节,包括促卵泡激素(FSH)和促黄体激素(LH)。这两种激素又受下丘脑分泌的促卵泡素释放激素(FRH)及促黄体素释放激素(LRH)的影响。我们根据棉酚对睾丸作用的情况,认为对垂体和下丘脑有进一步研究和探索的必要。另外,肾上腺是一种高度敏感和应激的器官,对性腺也有密切的关系。因此我们也观察了肾上腺皮质的组织化学变化,作为研究棉酚作用的辅助。     

棉酚对大鼠精子细胞碱性核蛋白合成与更新的影响

大鼠喂服棉酚(30毫克/公斤体重/日)3周、4周和6周后,用特异显示精氨酸的坂口反应及显示富于赖氨酸碱性蛋白的肝素-阿尔新蓝(HAB)染色法,结合并合~3H-精氮酸液闪测定,对棉酚影响精子细胞核蛋白在合成与代谢上的改变,进行分析。结果表明: 1.正常大鼠14期以后的晚期精子细胞核坂口反应阳性,HAB染色阴性。相反,精原细胞、精母细胞及14期以前的精子细胞坂口反应为阴性,而HAB染色较深,表明14期前后精子细胞核蛋白有精蛋白合成的转换过程。 2.服棉酚后,部分曲细精管中14期以后的精子细胞及游离精子坂口反应变弱,并能为HAB染色。这是甲绿-焦宁染色异常的原因。也提示棉酚对晚期精子细胞核蛋白转变可能有干扰作用。 3.睾内注射~3H-精氨酸的活体实验和分离后的精子细胞与同位素温育的体外实验,均未见对照组和实验组之间有明显差异。对精子细胞变态过程中核蛋白合成上的转换的细胞化学显示方法,棉酚对这一转换过程的干扰作用的可能性及其意义予以讨论。     

醋酸棉酚对人体精子的影响——精子的细胞学、细胞化学及电镜的观察

在城市及农村两组连续口服醋酸棉酚分别为15毫克/天和22.5毫克/天的不同时间后,分别取精液作细胞学、细胞化学及超微结构的观察。与服药前自身对照相比较,分析了棉酚的抗精子发生的作用效应。精子计数与细胞学分析结果表明:口服棉酚四周后畸形率增高者不多,而八周后在精子数目剧减的同时,涂片内脱落生精细胞(主要为精母细胞及精子细胞)明显增多,反映了棉酚作用部位主要为精子发生过程中的变态期精子细胞和中、晚期精母细胞。服用棉酚后可引起精子中段SDH活性减弱及线粒体超微结构的损伤(空泡化,排列紊乱到线粒体的全部消化)表明棉酚对精子能源代谢有一定的抑制作用,但对精子顶体PLE的影响则似为继发性性质。口服棉酚后可见精子核内染色质的超微结构产生异常变化,提示了棉酚影响精细胞核酸代谢的可能性。     

醋酸棉酚和高能震波对人膀胱癌细胞系单独和联合作用的研究

膀胱癌细胞系ＢＴ５６３７经醋酸棉酚或高能震波单独作用和两者联合处理后，癌细胞生长增殖受抑制，分裂指数及集落形成率下降。＾３Ｈ－ＴｄＲ掺入减少。Ｆｅｕｌｇｅｎ染色后经显微分光光度计检测显示ＤＮＡ含量降低。流式细胞计分析发现醋酸棉酚组Ｇ０／Ｇ１期比例显著增高，Ｓ、Ｇ２／Ｍ期比例减少；震波组Ｓ期比例下降，本研究提示，醋酸棉酚可阻断癌细胸由Ｇ０／Ｇ１期进入Ｓ期，而高能震波则抑制Ｓ期，电镜观察显示，醋酸棉酚     

HLA-B40交叉反应组高分辨度DNA分型

目的　采用顺序特异引物聚合酶链反应技术（ＰＣＲＳＳＰ） ， 建立汉族人群ＨＬＡＢ４０ 交叉反应组高分辨度ＤＮＡ 分型方法。方法　Ｂ４０ 组标准ＤＮＡ１０ 份，血清学Ｂ４０ 阳性临床样本１６４ 份，快速盐析法提取模板ＤＮＡ。设计合成１３ 个特异引物和１ 对阳性对照引物，组成８ 个ＰＣＲ 反应，建立一步法ＰＣＲＳＳＰ 快速ＨＬＡＢ４０ 组高分辨度ＤＮＡ 分型方法。结果　所有样本和标准ＤＮＡ 采用ＰＣＲＳＳＰ 分型均获得成功，可准确分辨出Ｂ４０ 组所有等位基因。无假阳性和假阴性出现，重复率１００ ％ ，总耗时４ ～５ 小时。分型结果经双盲验证符合。临床应用结果显示： 三分之一的汉族人群存在Ｂ４０组抗原，Ｂ６０ 占绝大多数（６７ ．３ ％ ） ，血清学对Ｂ４０ 组的误差率达１０ ％ 。本方法比血清学更加准确， 对血清学分型存在困难、亚型分辨不清的纯合子或杂合子均能作出准确的分型。结论　本研究建立的ＨＬＡＢ４０ 组ＰＣＲＳＳＰ 高分辨度ＤＮＡ 分型方法， 分辨度高、特异性强、结果精确可靠、明显优于血清学方法，适合于临床应用。     

以Chelex 100为介质抽提DNA

近年来，ＤＮＡ的分析技术已广泛地应用于肿瘤学的研究中。获取ＤＮＡ的传统方法是以新鲜、－７０℃或液氮保存的标本为材料，通过蛋白酶Ｋ消化，酚、氯仿抽提而获取ＤＮＡ。１９８７年，Ｆｅｙ等［１］首先用ＮＰ４０成功地从骨髓涂片中提取到较高分子量的ＤＮＡ，用于Ｓｏｕｔｈｅｒｎ印迹分析。国内的一些学者［２，３］也用相似的方法，从骨髓涂片中提取到ＤＮＡ。但这两种方法均需蛋白酶Ｋ消化，酚、氯仿抽提，乙醇沉淀等步骤，操作复杂，费时。同时，需要在多个离心管之间转移，增加了标本间的交叉污染。由于ＰＣＲ技术对ＤＮＡ样品的…     

Use of seminiferous tubule segments to study stage specificity of unscheduled DNA synthesis in rat spermatogenic cells

DNA repair in spermatogenic cells at various stages of maturity was determined by quantitation of unscheduled DNA synthesis (UDS). Male F-344 rats were exposed (i.p.) to methyl methanesulfonate (MMS, 35 mg/kg); 1 hr later, segments of seminiferous tubules corresponding to spermatogenesis stages II, IV-V, VI, VII, VIII, IX-X, XII, and XIV were isolated with the transillumination pattern of the tubules as a guide. Intact tubule segments were cultured 24 hr in the presence of [3H]thymidine, and UDS was quantitated by autoradiography as net grains/nucleus (NG). In primary spermatocytes from treated rats, NG count increased with increasing maturity from leptotene primary spermatocytes (3.5 NG) up through stage VIII and IX-X pachytene spermatocytes (22 NG), after which NG decreased in stage-XII pachytene and diplotene spermatocytes (to 16 NG and 8 NG, respectively). Round spermatids of steps 2-8 of spermiogenesis all exhibited approximately the same UDS response (8 NG). Elongating spermatids as mature as step 14 underwent UDS after exposure to MMS, but step-15 and later-step spermatids did not. The DNA repair response of pachytene spermatocytes cultured within segments of seminiferous tubule corresponding to stages VIII and IX-X was 4 to 25 times greater, depending on the dose of MMS, than pachytene spermatocytes isolated by enzymatic digestion and cultured in suspension [Bentley and Working, Mutat Res 203:135-142, 1988]. Thus, the use of segments of seminiferous tubule both increased the sensitivity of UDS as an indicator of DNA damage in rat germ cells and enabled the study of UDS in spermatogenic cells at different stages of maturity.     

Effects of alpha-amanitin on RNA synthesis in cultured rat seminiferous tubules.

The effect of the specific RNA polymerase II inhibitor α-amanitin on the cell morphology and RNA synthesis during spermatogenesis was investigated. Light microscopic autoradiography showed that α-amanitin largely decreased the rate of RNA synthesis in the pachytene spermatocytes and in the spermatids. Degenerative changes occurred most rapidly in the mid pachytene spermatocytes and in the intermediate and type B spermatogonia. Already after 14 hr of culture with α-amanitin (10 μg/ml) a part of these cells showed progressive degenerative changes beginning with the clumping together of the chromosomes. This indicates that at least a part of the HnRNA which is synthesized in the lampbrush loops of the pachytene chromosomes is needed to maintain the normal structure and function of the pachytene spermatocytes. Biochemical evidence suggested also that the formation of HnRNA was specifically inhibited in the pachytene spermatocytes by α-amanitin.     

磷酸化的丝裂原活化蛋白激酶在大鼠睾丸的表达

目的研究丝裂原活化蛋白激酶(MAPKs)的3个亚家族成员细胞外信号调节激酶(ERK)、c-Jun N-端激酶(JNK)和p38 MAPK的活化形式在睾丸中的定位,了解MAPKs在生精过程中所起的作用。方法免疫组织化学方法检测正常大鼠睾丸中磷酸化的p-ERKJ、NK、p38 MAPK的表达情况。结果正常大鼠睾丸中p-ERK主要分布于精原细胞、细线前期到粗线期的初级精母细胞以及9~12期长形精子细胞的细胞核,p-JNK则主要位于支持细胞与支持细胞、支持细胞与生精细胞(尤其是19期精子细胞)之间,而p-p38 MAPK除了在生精小管的部分细胞胞质中有分布外,其表达最明显的部位是在间质细胞的细胞质。结论ERKJ、NK和p-38 MAPK分别定位于正常大鼠睾丸内的不同部位,提示MAPKs不同的亚家族成员分别在精子发生的不同环节中发挥主要作用。ERK可能参与生精细胞增殖、分化的信号转导,JNK则可能通过调节细胞的黏附而最终影响生精细胞的迁移与精子释放过程,而p38 MAPK除了可能与JNK一起参与精子释放的调节外,最主要的作用可能是睾酮合成分泌的调节。     

Germ cell degeneration in normal and microwave-irradiated rats: potential sperm production rates at different developmental steps in spermatogenesis

Germ cell degeneration in 14 normal and 14 microwave-irradiated, adult (400-500 gm), Sprague-Dawley rats was compared by evaluating potential sperm production rates at different developmental steps in spermatogenesis. Following 9 days of irradiation at 1.3 GHz (6 hours/day at 6.3 mW/gm using 1-mu sec pulsewidth at 600 pulses/second) or sham treatment, rats were killed at 6.5, 13.0, 26.0, or 52.0 days following treatment. Testes were perfused with 2% glutaraldehyde, embedded in Epon, and sectioned at 0.5 micron for morphometric analyses. Plasma LH and FSH concentrations were determined by radioimmunoassay from blood collected on the day of death. Considering nuclear size, percentage of nuclei in the parenchyma, and life span of different cells, potential daily sperm production was determined for type B spermatogonia, preleptotene or pachytene primary spermatocytes, or spermatids with round nuclei. No differences (P greater than .05) in parameters tested were found among time periods following irradiation. With the possible exception of sperm production per testis (P less than .05) based on pachytene spermatocytes, microwave irradiation had no effect on the parameters evaluated. No degeneration was detected in spermatogenesis when potential sperm production rates were determined either from type B spermatogonia to spermatids or from type B spermatogonia to a posttesticular approximation of sperm production rate. Thus, it appears that regulation of sperm production rates must take place during spermatogonial mitoses, since once the number of type B spermatogonia is determined, there is essentially no subsequent alteration in sperm production potential in normal or irradiated adult rats.     

The Golgi apparatus of rat pachytene spermatocytes during spermatogenesis

A morphological and immunocytochemical study of the Golgi apparatus in pachytene spermatocytes was performed in an effort to correlate the structure and function of this organelle during meiotic prophase. In stages I-III of the cycle, the Golgi complex of pachytene spermatocytes is a flattened discoid, 0.5-1 microns in diameter, composed of vesicles interspersed with classically described Golgi cisternae. During subsequent maturation of pachytene spermatocytes (stages IV-XIII), the size of the Golgi complex increases significantly, attaining a size of 2-3 microns. However, unlike pachytene spermatocytes of stages I-III, the majority of the Golgi complex of more mature spermatocytes is characterized by an abundance of distinct stacks of cisternae interspersed with numerous vesicles and tubules. The composition of the Golgi complex was also studied by using two monoclonal antibodies that recognize either the cis or the trans Golgi cisternae, respectively, and employing biotin-streptavidin-peroxidase immunocytochemistry in 5 micron frozen sections of testes. Immunodetection of the distinct cisternae revealed that the increase in size of the Golgi complex during maturation of pachytene spermatocytes was due predominantly to an accumulation of trans Golgi; the amount of cis Golgi remained unchanged. The morphological data presented in this study are consistent with an heightened secretory activity of pachytene spermatocytes during their maturation. In addition, the increase in size of the Golgi apparatus during the extensive prophase of pachytene spermatocytes may suggest that the mechanism employed by germ cells to partition the Golgi complex during the first division of meiosis varies significantly from that of somatic cells undergoing mitosis.     

棉酚对大鼠下丘脑超微结构影响的观察

本文观察了棉酚对雄性成年大鼠下丘脑弓状核、视上核和室旁核细胞超微结构的影响。棉酚以每日30mg/kg体重给大鼠灌服,连续5周。结果表明服棉酚后,视上核和室旁核变化不明显;而弓状核中,部分细胞呈现一系列神经分泌功能增强的变化,高尔基复合体和粗面内质网轻度扩张,内充均质低电子密度物质;神经分泌颗粒、溶酶体、核仁样体和微管数量增多。本文并对以上超微结构改变的性质、意义和可能原因进行了讨论。     

Apoptosis and blood–testis barrier during the first spermatogenic wave in the pubertal rat

This research explores the initial assembly of the blood–testis barrier (BTB) during puberty, when a massive physiological apoptosis in the first spermatogenic wave takes place. Fragments of testis from 14‐ to 20‐day‐old rats were studied by conventional transmission electron microscopic techniques. Lanthanum hydroxide was used as an intercellular tracer. Light microscopy was used to confirm apoptotic death when paraffin‐embedded sections were studied by TUNEL analysis. When the seminiferous cords reached the zygotene–pachytene spermatocyte level, they exhibited abundant apoptotic figures, whereas the remaining segments showed sporadic apoptosis. We found a BTB not yet assembled in the cords with zygotene–pachytene spermatocytes and abundant apoptosis. The observed apoptosis frequency diminished drastically when BTB was organized, as confirmed by the use of the tracer. Our conclusion is that the massive apoptosis found in the zygotene–pachytene spermatocytes between days 14 and 20 coincides with an open BTB. The absence of BTB could be one of the factors causing massive apoptosis of zygotene–pachytene spermatocytes, at least within the time span analyzed. The zygotene–pachytene spermatocytes are left exposed in an open environment instead of being isolated in the adluminal compartment to which they are destined. Anat Rec 290:206–214, 2007. © 2007 Wiley‐Liss, Inc.     

Differential sensitivities of spermatogonial and postspermatogonial cell stages of the mouse to induction of unscheduled DNA synthesis by ethyl- and methyl-nitrosourea.

An autoradiographic procedure was used to measure unscheduled DNA synthesis (UDS, DNA repair synthesis) in spermatogonial and postspermatogonial cell stages of mice after treatment with two doses of N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU). Significant levels of UDS were measured in type A spermatogonia, meiotic spermatocytes, round spermatids, and early elongating spermatids but not in mature spermatids. The extent of UDS varied according to the germ cell stage and the dose. At equimolar concentrations, MNU was more efficient than ENU in eliciting a UDS response in all germ cells. After ENU treatment, type A spermatogonia showed the highest UDS response, while round and elongating spermatids showed the lowest. After MNU treatment, pachytene spermatocytes exhibited the highest UDS response while type A spermatogonia showed the lowest. The high UDS response of type A spermatogonia to ENU parallels the well-known high mutational sensitivity of spermatogonia to this chemical. Similarly, the high UDS response observed in meiotic spermatocytes and early spermatid stages after MNU treatment correlates with the high mutational sensitivity of postspermatogonial stages to MNU. Thus, the present results, like the specific locus mutation studies, indicate that ENU and MNU each has a unique effect on the spermatogenic cells. This effect is likely due to the different mechanism of action of ENU and MNU at the level of DNA and also to the physiological differences between different germ-cell stages. Teratogenesis Carcinog. Mutagen. 19:339-351, 1999. Published 1999 Wiley-Liss, Inc.