睾丸组织中的TSP50启动子甲基化差异的研究

目的:检测不同组织中TSP50启动子区域的甲基化状态,研究其表达的机制。方法:分离、纯化鼠睾丸中的精原细胞、精母细胞和精子,分别提取人的睾丸组织、Ntera-2细胞、鼠精原细胞、精母细胞和精子的基因组DNA,用重亚硫酸盐处理基因组DNA,PCR扩增人和鼠的TSP50的启动子CpG岛区域,电泳,提纯,连接于质粒中,转导入细胞中,扩增,提取,纯化质粒,测序。结果:人睾丸组织甲基化水平较低,Ntera-2细胞处于较高水平。鼠的精原细胞和精子细胞甲基化水平较高,精母细胞甲基化水平较低。结论:TSP50启动子区域甲基化位点的甲基化水平调控TSP50蛋白的表达。     

中心体蛋白centrin在大鼠精子发生过程中的表达

目的：研究中心体蛋白centrin在大鼠生精细胞中的表达情况,以深入了解centrin在精子发生过程中的作用。方法：通过重力沉降法分离大鼠不同发育阶段的生精细胞,用免疫荧光和蛋白印迹实验检测各级生精细胞中centrin蛋白的表达,用定量RT－PCR检测centrin同源基因centrin1和centrin2mRNA的表达水平。结果：间接免疫荧光和蛋白印迹显示精母细胞、圆形、长形精子细胞均有centrin蛋白存在,位于中心粒上,而在附睾的成熟精子中centrin则消失。RT－PCR研究发现,centrin在睾丸组织中特异性表达,centrin2在多种组织中均有表达。在睾丸中,centrin1仅在生精细胞进入减数分裂后转录,其mRNA水平在圆形精子细胞中最高,而centrin2在精原细胞中即有表达,减数分裂后其mRNA难以检测到。结论centrin蛋白在大鼠雄性配子的发育过程中最终丢失;该基因家族中同源基因centrin1和centrin2表达呈现组织特异性和发育阶段特性,在精子发生过程中发挥不同功能,centrin1蛋白可能与减数分裂及鞭毛生成相关,centrin2则参与细胞有丝分裂过程。     

二乙基己烯雌酚诱发成年仓鼠睾丸生精异常及对生精细胞表达bcl-2、P53和cadherin的影响

目的探讨在二乙基己烯雌酚(DES)诱发成年动物生精异常过程中,原癌基因bcl-2、p53及黏附分子cadherin在生精细胞中表达的变化,旨在阐明DES诱发生精异常的作用机理。方法成年雄性仓鼠皮下注射DES连续7d后,取其睾丸,进行光镜及电镜的观察和原癌基因bcl-2、p53及黏附分子cadherin的免疫组织化学染色。结果DES处理组的成年仓鼠睾丸生精细胞发育异常,bcl-2和p53表达量均比对照组有显著增加,并以精母细胞和圆形精子细胞较为明显。生精上皮中cadherin的表达比对照组有明显减少。结论DES增加精母细胞和圆形精子细胞表达bcl-2和p53;同时抑制cadherin表达,是诱发成年仓鼠生精异常的原因之一。     

丙酸睾丸酮对生精细胞凋亡影响的流式细胞分析及形态观察

本实验应用流式细胞术 (FCM) ,DNA末端标记(TU NEL) ,及放免技术对生后 37天超剂量注射丙酸睾丸酮15天后的大鼠凋亡生精细胞 DNA含量和血清中性激素水平等进行了研究。结果显示 ,超剂量注射丙酸睾丸酮 ,实验组血清中 FSH浓度明显低于对照组 (P <0 .0 1) ,L H降低但无明显差异。实验组睾丸细胞凋亡率明显高于对照组 3倍(P <0 .0 1)。 FCM直方图显示实验组生精细胞凋亡峰由位于 1C细胞群 (精子细胞和精子 )和 2 C细胞群 (精原细胞和次级精母细胞 )之间 ,而对照组则位于 1C细胞群之前。同时 ,1C细胞群、2 C细胞群明显高于对照组 (P…     

羊睾丸提取液对铅染毒小鼠睾丸生精细胞凋亡及超微结构的影响

目的:探讨羊睾丸提取液对铅染毒小鼠睾丸生精细胞凋亡及超微结构的影响.方法:雄性性成熟小鼠,随机分为对照组(蒸馏水灌胃),模型组(醋酸铅灌胃),给药组(醋酸铅灌胃+腹腔注射羊睾丸提取液),染毒2周.Tunel法观察睾丸生精细胞的凋亡,透射电镜观察生精细胞超微结构的变化. 结果:与对照组相比,模型组睾丸生精细胞凋亡指数明显增高,多数细胞胞质内出现空泡,核固缩,核膜结构模糊,线粒体数目减少、空泡化,溶酶体增多;与模型组相比,给药组睾丸生精细胞凋亡指数有所降低,细胞结构趋于正常,核膜结构清晰,线粒体数量增多.结论:羊睾丸提取液可抑制铅染毒小鼠睾丸生精细胞的凋亡,对精原细胞、精母细胞、精子细胞的超微结构具有显著的保护作用.     

超声波对小白鼠生精上皮的影响

用功率为５Ｗ／ｃｍ２、频率为１．１０ＭＨｚ的超声波照射小白鼠睾丸５分钟（实验组１）、１０分钟（实验组Ⅱ），分别于处理后２４小时，４８小时及７天时间切取睾丸组织，制作石蜡切片，在光镜下观察生精上皮的组织学变化并与对照组进行比较。结果显示：（１）小白鼠睾丸经超声波照射后，曲细精管萎缩，管径变小；生精上皮变薄，精子发生时相消失；生精细胞减少，没有精子形成。（２）超声波照射１０分钟对生精上皮的损伤比照射５分钟更为严重。（３）超声波照射后２４小时，生精上皮即受到破坏，精子细胞减少；照射后４８小时，上皮受损伤的程度增大；照射后７天时间，曲细精管的组织学结构开始恢复，但仍无精子形成。（４）超声波对生精上皮的影响主要限于精母细胞、精子细胞和精子，而精原细胞与支持细胞没有明显变化。上述结果表明，超声波能够抑制小白鼠的精子发生，该抑制作用可能可逆。     

人绒毛膜促性腺激素对切除颌下腺小鼠静止期精母细胞的一些影响

目的：探讨人绒毛膜促性腺激素(HCG)对切除颌下腺小鼠静止期精母细胞表皮生长因子(EGF)的表达和生精功能的影响。方法：采用组织化学和免疫组织化学技术。结果：EGF、分布在小鼠睾丸间质细胞、精原细胞、静止期精母细胞、细线期精母细胞和精子细胞。去颌下腺组EGF阳性反应静止期精母细胞数量明显减少，同时相应的生精小管的各级生精细胞也显著减少。与去颌下腺组相比，去颌下腺给药组EGF阳性反应静止期精母细胞数量明显增多，同时相应的生精小管的生精细胞也显著增多。结论：睾丸间质细胞和部分生精细胞能分泌EGF。EGF在生精小管上皮的分布与生精周期有密切关系。静止期精母细胞EGF表达减弱是切除颌下腺导致小鼠少精症的原因之一。HOG可促进静止期精母细胞表达EGF，以调节精子发生过程。     

pcEgr-hp53稳定转染联合X射线诱导人肺腺癌A549细胞凋亡及相关凋亡蛋白表达变化

目的：探讨辐射诱导表达载体pcEgr-hp53体外稳定转染人肺腺癌A549细胞后联合X射线照射,诱导细胞凋亡的作用及相关凋亡蛋白Bax、Bcl-2和caspase-3蛋白表达的变化。方法：以脂质体介导携有外源野生型p53基因的辐射诱导表达载体pcEgr-hp53和pcDNA3.1,体外转染A549细胞,筛选稳定转染的细胞克隆并扩增培养,所表达的A549-hp53和A549-vect分别接受0、0.5、2和5Gy X射线照射,即8个实验组,采用TUNEL法和流式细胞术检测稳定转染联合辐射对细胞凋亡和Bax、Bcl-2和caspase-3蛋白表达变化的影响。结果：A549-hp53组加不同剂量照射与0 Gy组比较,其百分数均明显增加（0.5-5Gy,P〈0.05-P〈0.01）,Bcl-2蛋白表达在0.5-5Gy时明显下降,而Bax蛋白明显增加（P〈0.05-P〈0.01）,caspase-3蛋白表达在2-5Gy时明显增加（P〈0.01）;A549-hp53照射组不同剂量照射,与A549-vect相应照射剂量组比较,其百分数在0.5Gy时无明显差异,在2-5 Gy时为（P〈0.01）,Bcl-2蛋白表达明显下降（0.5Gy,P〈0.01;2-5Gy,P〈0.05）,Bax和caspase-3蛋白表达0.5-5Gy时明显增加（P〈0.05-P〈0.01）。结论：体外p53基因转染联合X射线照射可诱导肿瘤细胞凋亡明显增多,凋亡相关蛋白Bcl-2表达下调,Bax、caspase-3表达上调,具有显著的肿瘤抑制作用。     

bax基因在小鼠睾丸及实验性隐睾中表达的研究

目的 研究ｂａｘ基因在正常小鼠睾丸中的表达、定位及隐睾所导致的改变。方法 以Ｗｅｓｔｅｒｎ－ｂｏｔｔｉｎｇ印迹法从蛋白水平检测ｂａｘ基因在正常小鼠睾丸及实验性隐睾中的表达、变化;原位杂交技术及Ｎｏｒｔｈｅｒｎ杂交法从ｍＲＮＡ水平检测ｂａｘ基因在小鼠生精上皮中的定位及隐睾所导致的改变。结果 ｂａｘ基因在正常小鼠睾丸中有弱表达,实验性隐睾导致其表达明显增强,表达细胞为主要为精母细胞、精原细胞和精子细胞     

Smad2和Smad4蛋白在成年大鼠睾丸的表达

目的 探讨转化生长因子－β超家族肽类的细胞内信号转导分子Smad2和Smad4蛋白在成年大鼠睾丸的表达及在精子发生中的作用机理。方法 用免疫组织化学ABC法结合葡萄糖氧化酶－DAB－硫酸镍铵增强技术,检测成年大鼠睾丸Smad2和Smad4蛋白的表达和定位。结果Smad2蛋白主要表达在大鼠睾丸生精小管的精原细胞、初级精母细胞、次级精母细胞、精子细胞、支持细胞和间质细胞中,免疫反应物质主要定位于细胞质,胞核为阴性;而Smad4蛋白同主要表达于间质细胞的胞质中,支持细胞呈弱阳性染色,结论 Smad2蛋白主要表达于睾丸生精小管各级生精细胞,Smad4蛋白表达于间质细胞,为揭示TGF－β超家族在精子发生中的分子机理提供了直接证据。     

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.     

Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1 during spermatogenesis in the Japanese eel, a teleost fish.

We cloned A-type cyclins (cyclins A1 and A2) and Dmc1 cDNAs from the eel testis. Cyclin A1 mRNA was predominantly expressed in the livers, ovaries, and testes of the eels. In contrast to cyclin A1 mRNA, a very high expression of cyclin A2 mRNA was observed in the brains, livers, kidneys, spleens, ovaries, and testes of the eels. Dmc1 mRNA was predominantly expressed in the testes and ovaries; expression in the brain was also detected. In the eel testis, a few type-A spermatogonia incorporating 5-bromo-2'-deoxyuridine (BrdU) were seen before the initiation of spermatogenesis by hormonal induction. On day 1 after hormonal induction, the number of BrdU-labeled spermatogonia increased remarkably, and after 3 and 6 days, many labeled type-B spermatogonia were also observed. The expression of cyclin A2 increased 1 day after the induction of spermatogenesis and reached a plateau after 6 days, when many type-B spermatogonia with high proliferative activity were found. In contrast, the expression of cyclin A1 mRNA was detected after 9 days, coincident with the first appearance of spermatocytes. Cyclin A1 mRNA was localized in germ cells of all stages, from primary spermatocytes to round spermatids, whereas cyclin A2 mRNA was specifically localized in spermatogonia, secondary spermatocytes, round spermatids, and testicular somatic cells, including Sertoli cells. Dmc1 was localized only in the earlier stages of primary spermatocytes; before this stage, cyclin A1 mRNA was not detectable. Overall, cyclin A2, Dmc1, and cyclin A1 are expressed in spermatogenic cells sequentially before and during meiosis in the eel testis.     

Validation studies with the micronucleus test for early spermatids of rats. A tool for detecting clastogenicity of chemicals in differentiating spermatogonia and spermatocytes.

Male Wistar rats were given a single i.p. injection with different doses of ethylnitrosourea, mitomycin C, methyl methanesulphonate, cyclophosphamide or vincristine sulphate. Clastogenic damage induced in differentiating spermatogonia and spermatocytes was measured by counting micronuclei in derived early spermatids. At dose levels not resulting in cell death of resting spermatocytes, all chemicals--with the exception of vincristine--induced most of the damage in G1- and S-phase of primary spermatocytes (also called resting, pre-leptotene or pre-meiotic spermatocytes). However, at doses causing death of G1- and S-phase spermatocytes, high frequencies of micronuclei may be observed in early spermatids derived from spermatocytes treated in diplotene, diakinesis and MI and II. This is exemplified by our results with ethylnitrosourea. In our experience, the most sensitive stage of primary spermatocyte development (i.e. G1- and S-phase cells) can best be sampled 20 days after treatment. This is the optimal time interval for demonstrating the clastogenic potential of low or moderate doses of a test chemical in meiotic male germ cells of rats. The optimal sampling time for the detection of typical spindle poisons remains to be established. In general, at low or moderate dose levels, smaller or negligible amounts of chromosomal damage were induced in differentiating spermatogonia, in spermatocytes in meiotic prophase and in dividing primary or secondary spermatocytes. For obvious reasons, the micronucleus test for early spermatids cannot be used to detect clastogens which act exclusively on postmeiotic male germ cells.     

Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis

We investigated the frequency of spontaneous apoptosis and expression of the Bcl-2 family of proteins during normal spermatogenesis in man. Testicular tissue with both normal morphology and DNA content was obtained from necro-donors and fixed in Bouin's solution. A TdT-mediated dUTP end-labelling method (TUNEL) was used for the detection of apoptotic cells. Expression of apoptosis regulatory Bcl-2 family proteins and of p53 and p21(Waf1) was assessed by immunohistochemistry. Germ cell apoptosis was detected in all testes and was mainly seen in primary spermatocytes and spermatids and in a few spermatogonia. Bcl-2 and Bak were preferentially expressed in the compartments of spermatocytes and differentiating spermatids, while Bcl-x was preferentially expressed in spermatogonia. Bax showed a preferential expression in nuclei of round spermatids, whereas Bad was only seen in the acrosome region of various stages of spermatids. Mcl-1 staining was weak without a particular pattern, whereas expression of Bcl-w, p53 and p21(Waf1) proteins was not detected by immunohistochemistry. The results show that spontaneous apoptosis occurs in all male germ cell compartments in humans. Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments suggesting a specific role for these proteins in the processes of differentiation and maturation during human spermatogenesis.     

DOG1 immunohistochemical staining of testicular biopsies is a reliable tool for objective assessment of infertility

Testicular biopsy may be a component of the work-up of male infertility. However, no reliable diagnostic tools are available for objective quantitative assessment of spermatogenic cells. It is well known that MAGE-A4 is selectively expressed in spermatogonia and our group has previously demonstrated that DOG1 differentially stains germ cells. Therefore, we performed DOG1 and a double stain cocktail (DOG1 and 57b murine monoclonal anti-MAGE-A4) immunohistochemical stains on 40 testicular infertility biopsies (10 each with active spermatogenesis, Sertoli cell-only, hypospermatogenesis, and maturation arrest), 25 benign seminiferous tubules from radical orchiectomies, and 5 spermatocytic tumors (ST). In biopsies/resections with active spermatogenesis, DOG1 stained spermatocytes and spermatids and was absent in spermatogonia, while MAGE-A4 stained spermatogonia and primary spermatocytes (weak). In hypospermatogenesis, DOG1 highlighted decreased spermatocytes/spermatids and MAGE-A4 highlighted decreased spermatogonia. DOG1 staining confirmed decreased to absent spermatocytes in maturation arrest and MAGE-A4 staining established the presence of preserved spermatogonia in all cases. All STs were negative for DOG1 and positive for MAGE-A4, while all Sertoli cell-only cases were negative for DOG1 and the double stain cocktail. In conclusion, we confirmed that DOG1 is expressed in spermatocytes and spermatids and MAGE-A4 highlights primarily spermatogonia. Usage of these stains facilitates confirmation of maturation arrest, assessment of the percentage of testis involvement in hypospermatogenesis and identification of mixed patterns. Finally, this study supports that the differentiation of STs is more closely related to spermatogonia than the more mature spermatocytes.     

In vitro responses to known in vivo genotoxic agents in mouse germ cells

Genotoxic compounds have induced DNA damage in male germ cells and have been associated with adverse clinical outcomes including enhanced risks for maternal, paternal and offspring health. DNA strand breaks represent a great threat to the genomic integrity of germ cells. Such integrity is essential to maintain spermatogenesis and prevent reproduction failure. The Comet assay results revealed that the incubation of isolated germ cells with n‐ethyl‐n‐nitrosourea (ENU), 6‐mercaptopurine (6‐MP) and methyl methanesulphonate (MMS) led to increase in length of Olive tail moment and % tail DNA when compared with the untreated control cells and these effects were concentration‐dependent. All compounds were significantly genotoxic in cultured germ cells. Exposure of isolated germ cells to ENU produced the highest concentration‐related increase in both DNA damage and gene expression changes in spermatogonia. Spermatocytes were most sensitive to 6‐MP, with DNA damage and gene expression changes while spermatids were particularly susceptible to MMS. Real‐time PCR results showed that the mRNA level expression of p53 increased and bcl‐2 decreased significantly with the increasing ENU, 6‐MP and MMS concentrations in spermatogonia, spermatocytes and spermatids respectively for 24 hr. Both are gene targets for DNA damage response and apoptosis. These observations may help explain the cell alterations caused by ENU, 6‐MP and MMS in spermatogonia, spermatocytes and spermatids. Taken together, ENU, 6‐MP and MMS induced DNA damage and decreased apoptosis associated gene expression in the germ cells in vitro. Environ. Mol. Mutagen. 58:99–107, 2017. © 2017 Wiley Periodicals, Inc.     

类固醇生成因子-1对青春期小鼠睾丸中睾酮生成及精子发生的调节

目的 探讨类固醇生成因子-1(SF-1)对青春期小鼠睾丸内分泌功能及精子发生过程的调节作用，并推测其可能机制。方法 用免疫组织化学方法定位SF-1在不同年龄小鼠睾丸中的细胞分布，进一步分离有SF-1阳性表达信号的青春期小鼠Leydig细胞在体外进行培养，用反义转染方法抑制细胞内SF-1蛋白质的表达，检测细胞的睾酮分泌量及睾酮生成酶P450scc的mRNA水平变化。结果 1．SF-1在青春期Leydig细胞核有表达；反义抑制细胞内SF-1蛋白质的表达，则细胞的睾酮分泌量及P450scc mRNA水平均显著下降；2．SF-1在青春期小鼠睾丸B型精原细胞及细线期、偶线期、粗线期的初级精母细胞核中也有表达。结论 1．SF-1参与调节青春期睾丸Leydig细胞中P450scc基因的转录，影响睾酮分泌；2．SF-1作为一种核受体，可能也是生精过程中重要的转录调控因子，调节B型精原细胞向初级精母细胞分化及初级精母细胞第1次减数分裂过程中特异表达的基因转录过程，从而影响青春期小鼠精子发生过程。     

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.