水牛有腔卵泡颗粒细胞凋亡的生物形态学特征

为了解水牛有腔卵泡闭锁的起因和有腔卵泡中是否存在卵泡颗粒细胞凋亡，本研究选择青春期前水牛和成年（发情间期和发情期）水牛的卵巢，采用石蜡组织切片、HE染色技术进行光镜观察，并采用DNA原位末端标记（TUNEL）技术和透射电镜观察研究了卵泡颗粒细胞凋亡的特征。结果表明：青春期前水牛的健康有腔卵泡上无或仅有极个别凋亡的卵泡颗粒细胞（GCs），而闭锁有腔卵泡上存在有多量凋亡的GCs，凋亡的形式：GCs层内出现单个或多个凋亡细胞，卵泡腔内出现凋亡小体群。在成年水牛发情间期的闭锁大卵泡上，GCs层弯曲皱折，存在多量凋亡的GCs。发情期水牛成熟卵泡的GCs层也存在GCs凋亡，排卵前的GCs层细胞全部脱落到卵泡腔内。本研究首次在同一组织切片上先后分别应用HE染色和TUNEL检查细胞凋亡，且证实了水牛有腔卵泡GCs凋亡的存在。超微结构显示了GCs凋亡的核边集化、核浓缩及凋亡小体的形态。上述结果提示：在水牛闭锁有腔卵泡中存在着GCs凋亡，GCs凋亡是启动水牛有腔卵泡闭锁的潜在机理。     

水牛卵泡GCs体外培养发生凋亡的生物学特征

为了解水牛卵巢卵泡中颗粒细胞（GCs）和卵母细胞在体外培养过程中发生凋亡的变化特征,通过体外培养、血清撤除法诱导水牛卵泡GCs凋亡,并应用苏木精伊红（HE）染色和DNA原位末端标记的TUNEL方法观察其病理特征;通过水牛腔前卵泡体外培养、诱导或等待其中的卵母细胞及卵泡细胞发生凋亡,然后应用透射电镜技术检查其超微结构改变。结果显示：凋亡卵泡GCs的HE染色特征包括细胞体积缩小,染色较深呈紫黑色,且多成簇分布。DNA原位末端标记检查的特征是DNA发生降解,其降解碎片呈TUNEL阳性。原始卵泡体外培养中调亡卵母细胞的超微结构表现为胞核碎裂成电子密度高的大碎片,或出现形态奇特的凋亡小体;初级卵泡中的调亡GCs,细胞内外均出现凋亡小体,细胞内还见有典型的螺层状内质网结构,其中包涵部分核质。     

猪卵巢发育的组织学变化及卵泡闭锁规律研究

旨在对不同日龄猪卵巢组织结构以及卵巢发育过程中卵泡闭锁规律进行研究。取3、40、50、60、72、86、95及165日龄猪卵巢各3例,采用常规石蜡切片、HE染色和TUNEL技术检测卵巢组织结构及卵泡闭锁规律,结果表明,猪卵巢发育从组织学上可分为卵原细胞增殖期、卵泡缓慢生长期及卵泡快速生长期3个阶段。卵原细胞增殖期主要以卵原细胞的增殖分裂为特点;在卵泡缓慢生长期,原始卵泡的数量随日龄增加逐渐减少,初级卵泡数量及体积则明显增加;至卵泡快速生长期,生长卵泡的数量及体积继续增大,其数量在86日龄达到最大值;72日龄卵巢中出现三级卵泡,至95日龄卵巢中出现近成熟卵泡。在各时期的卵巢组织,均可观察到卵原细胞及卵泡的闭锁现象,以原始卵泡的退化最为显著。TUNEL检测表明,在卵原细胞增殖期,可见大量原始卵泡及初级卵泡的闭锁,其闭锁主要源自卵泡卵母细胞的凋亡。在卵泡缓慢生长期,各级生长卵泡均可出现闭锁,初级卵泡的闭锁主要由卵母细胞的凋亡引起,也有部分是同时伴有卵母细胞和卵泡细胞凋亡的;次级卵泡的闭锁则主要由于颗粒层细胞的凋亡所致。在卵泡快速生长期,随着卵泡的快速生长,各级卵泡的闭锁也变得更加明显,次级卵泡及三级卵泡的闭锁主要是由颗粒细胞凋亡引起。     

辽宁绒山羊卵泡闭锁中颗粒细胞凋亡特点的研究

本试验采用TUNEL原位法和DNA梯度电泳法等方法对卵巢上闭锁卵泡中凋亡的颗粒细胞类型和分布状况等进行研究。结果表明，辽宁绒山羊闭锁卵泡是细胞凋亡引起的。DNA梯状电泳表明，不同健康程度卵泡颗粒细胞凋亡程度不同；而颗粒细胞TUNEL原位标记表明，健康卵泡、轻度闭锁卵泡及闭锁卵泡中颗粒细胞凋亡比例分别为13.21%±1.23％、32.13%±2.13％、51.31%±3.02％；不同健康程度卵泡中类固醇激素水平不同，健康卵泡中E2水平较高，孕酮浓度较低；闭锁卵泡中E2浓度较低，P4浓度较高，健康、轻度闭锁和闭锁三类卵泡中E2/P4比值分别是2.62±0.03、0.074±0.0003、0.028±0.0004。     

二花脸猪卵巢卵泡形成与早期发育

为了研究二花脸猪卵泡的形成和发育特点,取胎儿期50、70和90(日龄)、新生1日龄及生后期20、70和120(日龄)7个阶段猪的卵巢组织样品进行组织学观察。结果表明,生殖细胞在早期卵巢中以卵母细胞巢状结构存在,原始卵泡最早出现在胎70日龄卵巢中,黄体出现在生后120日龄;以Stat3为生殖细胞的标记蛋白,进行免疫组织化学试验,统计出生前期各日龄阶段卵母细胞巢和卵泡的比例。结果表明,从胎70日龄到胎90日龄,卵母细胞巢状结构比例显著降低,单个卵泡比例显著增高,生后70日龄卵巢中出现有腔卵泡,未见黄体出现,生后120日龄卵巢中出现黄体;以PCNA作为细胞增殖的指标,进行卵巢组织的免疫组织化学定位,结果显示,在猪的卵泡形成和早期发育阶段以卵母细胞的快速增殖为主。说明二花脸猪的卵巢原始卵泡开始形成的时间在胎猪70日龄左右,初情期在生后70~120日龄,卵泡激活前以卵母细胞的生长为主。     

牛不同生长发育时期卵泡酶活性及定位

应用组织化学方法观察牛卵巢卵泡中葡萄糖-6-磷酸酶(G-6-P酶)和碱性磷酸酶(AP)在不同发育时期的活性变化及其在卵泡内的定位。旨在探讨这两种酶的活性同卵泡生长发育的关系。结果表明：不同发育时期的卵泡数量与AP和G-6-P酶活性阳性反应呈极显著正相关。其中酶活性阳性反应有腔卵泡数量多于腔前卵泡；但G-6-P酶活性阴性反应卵泡显著多于阳性卵泡，说明AP活性强于G-6-P酶。腔前卵泡酶活性位置主要在颗粒细胞和卵母细胞上，而有腔卵泡酶活性位置主要在内膜细胞上。     

哺乳动物腔前卵泡的分离与培养

哺乳动物卵巢中绝大多数卵母细胞以无腔形式存在，有腔卵泡所占比例很少。通过建立腔前卵泡的培养体系，获取大量的具有成熟和受精能力的卵母细胞，将极大地促进体外受精、核移植等胚胎工程技术的发展，并有利于研究卵泡和卵母细胞的发育规律。     

哺乳动物腔前卵泡的分离与培养

哺乳动物卵巢中绝大多数卵母细胞以无腔形式存在，有腔卵泡所占比例很少。通过建立腔前卵泡的培养体系，获取大量的具有成熟和受精能力的卵母细胞，将极大地促进体外受精、核移植等胚胎工程技术的发展，并有利于研究卵泡和卵母细胞的发育规律。     

猪卵巢颗粒细胞和黄体细胞凋亡的调控机制研究进展

母猪卵巢皮质上存在大量卵泡，卵泡由原始卵泡向初级卵泡、次级卵泡逐步发育为排卵前的成熟卵泡时，伴随着卵泡闭锁的发生。目前认为颗粒细胞凋亡是导致卵泡闭锁的主要因素，并且已经证实。卵泡成熟后会破裂排卵，如果卵子未受精，则黄体会发生萎缩并退化，形成称作白体的结缔组织，最终被组织吸收。细胞凋亡受到多基因严格控制，随着分子生物学技术的发展，已经证明存在多个信号通路。本文以猪卵巢为模型，综述了卵泡闭锁期间颗粒细胞凋亡和黄体退化过程中黄体细胞凋亡的调控机制，以期为深入研究卵巢细胞凋亡提供理论基础。     

小尾寒羊腔前卵泡体外生长发育的研究

近20年来,胚胎移植、体外受精、核移植、转基因等胚胎工程技术在理论和生产应用上已取得了很大进展.然而,所有这些技术是建立在具有完全发育能力的卵母细胞的基础之上的.大量卵母细胞的体外成熟培养是这些技术的限速步骤之一.目前采用的有腔卵泡卵母细胞的体外成熟和超数排卵,不能提供充足的卵母细胞以支撑胚胎工程技术的进一步发展.此外,卵巢中的卵母细胞绝大多数以无腔的形式存在于卵巢皮质内,有腔卵泡所占比例不到1%,屠宰场屠宰羊只后的卵巢内含有大量的腔前卵泡.如果建立腔前卵泡的体外培养体系,获得大量的具有成熟和受精能力的卵母细胞,一方面将会最大限度地挖掘保存卵巢上遗传资源;另一方面将极大的促进胚胎工程技术的研究与应用,还有利于研究卵泡和卵母细胞的生长和发育规律.     

Apoptosis inhibition by insulin-like growth factor (IGF)-I during in vitro maturation of bovine oocytes

Recently, significant progress has been achieved in improving the yield of good quality embryos in vitro. However, efforts are still required to recognize the factors and understand the mechanisms of oocyte maturation, which are essential for subsequent embryo development. The aims of the present study were to determine the frequency of apoptosis in oocytes recovered from slaughterhouse ovaries and to investigate whether insulin-like growth factor (IGF)-I action during oocyte maturation in vitro may withhold apoptosis and improve oocyte quality. Only oocytes of proper morphology with homogenous ooplasm and compact cumulus cells were selected for this study. All oocytes recovered from the slaughterhouse ovaries were divided into two groups. One group of oocytes, chosen for apoptosis detection, was examined immediately after recovery. The other group of oocytes was maturated in vitro. Oocytes were maturated with IGF-I supplementation (100 ng/ml). Oocytes without supplementation were used as a control. Apoptosis in oocytes was determined by positive results of TUNEL assay and active caspase labeling. The percentage of apoptotic oocytes detected by TUNEL fell to zero when the maturation medium was supplemented with IGF-I in comparison to the control matured oocytes (0 vs. 9.87%; P<0.05). However, active caspase labeling was only slightly decreased in the IGF-I matured oocytes compared with the control matured oocytes (1.13 vs. 2.08%; P<0.05). The results indicate that IGF-I may serve as an anti-apoptotic factor during oocyte maturation. We suggest that IGF-I may inhibit apoptosis in oocytes at the stage of caspase activation and may prevent further advancement of oocyte apoptosis.     

Detection of radiation-induced apoptosis using the comet assay

The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to stain the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis.     

Detection of Follicular Apoptosis in Water Buffalo (Bubalus bubalis) Ovary by Histology and Nick End Labelling Technique

The objective of this experiment was to assess the features and extent of follicular apoptosis in the water buffalo (Bubalus bubalis) ovary using classical histology and nick end labelling technique. Ovaries (n = 40) procured from the slaughterhouse were used for the study. The sections (5 μm) were used for detection of terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labelling (TUNEL) and classical histology (H&E). Those follicles showing ≥ 5% TUNEL positivity (TUNEL assay) and pyknotic nuclei (histology) in granulosa cells were classified as atretic. Based on histology, the atretic primary and secondary follicles (%) were 93.82 and 95.62 respectively. The histology study reveals that the rates (%) of atresia in <1, 1–3, 3–5 mm and >5 mm were 36.90, 40.50, 62.84 and 74.5 respectively. Further the atretic tertiary follicles (%) were significantly lower than the primary and secondary classes of follicles. TUNEL assay reveals that the atretic rate (%) of tertiary follicles in <1, 1–3, 3–5 and ≥ 5 mm class follicles were 50.88, 53.84, 81.81 and 36.36 respectively. The percentage of atresia in >5 mm diameter follicles were significantly lower in TUNEL than histology. Percentages of granulosa and thecal cells positive for atresia by TUNEL were 30.7 ± 0.53 and 13.82 ± 0.18 respectively per follicle. The initial structural changes in atretic follicles were seen primarily in the granulosa cells. In severely atretic follicles TUNEL positive granulosa cells along with theca cells have to be considered in assessing the rate and extent of atresia.     

Canine distemper virus causes apoptosis of Vero cells

Apoptosis of Vero cells infected with two canine distemper virus (CDV) vaccine strains was detected using TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labelling (TUNEL), flow cytometric analysis, agarose gel electrophoresis and electron microscopy (EM). By TUNEL, apoptotic cells were found in CDV-Onderstepoort (CDV-Ond)-infected cells. DNA fragments isolated from infected cells were separated by agarose gel electrophoresis and a 'ladder' pattern appeared. EM observations demonstrated that the cells undergoing cytopathic effect (CPE) possessed morphological characteristics of apoptotic cells. Flow cytometric analysis indicated that CDV could induce apoptosis of Vero cells, but the percentages of the apoptotic cells were correlated with the CPE types. The strain showing the cell-rounding type of CPE produced a much higher percentage of apoptotic cells than CDV-Ond with the syncytium type of CPE (P < 0.01). It was concluded that CDV vaccine strains could induce apoptosis of Vero cells and the apoptosis was virus strain-dependent and cell-dependent. The mechanism remains to be studied.     

Porcine circovirus induces B lymphocyte depletion in pigs with wasting disease syndrome

To disclose the mechanism of cellular injury following porcine circovirus (PCV) infection, 12 pigs were examined by the terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) method and immunohistochemistry. Histologically, the lymphoid tissues were characterized by marked apoptosis of lymphocytes, lymphocyte depletion, and macrophages and giant cells containing numerous inclusion bodies with or without apoptotic bodies. Immunohistochemically, there were many lysozyme-positive macrophages in the lymphoid follicles, while the number of CD79a-positive B lymphocytes was scanty. Apoptotic cells, which were proved to be TUNEL positive, revealed CD79a positivity. Although detectable mainly in the cytoplasm of macrophages, PCV antigens were found also in the nuclei of macrophages and apoptotic lymphocytes. Ultrastructurally, the presence of PCV virions was confirmed in apoptotic bodies phagocytosed by macrophages. These findings suggested that lymphocyte depletion with apoptotic death of B lymphocytes was caused by PCV, and that some of the inclusion bodies were phagolysosomes derived from the apoptosis. Thus, PCV may trigger the development of wasting disease syndrome by producing an immunocompromised state in pigs.     

兔脑炎原虫引起肾上皮凋亡的研究

对 16只具有兔脑炎原虫病的示病症状 ,即出现不同程度的头颈歪邪、震颤、平衡失调和转圈运动等运动障碍症状的獭兔 ,运用原位末端标记技术和基因染色技术 ,着重对病兔肾组织损伤的发生进行了研究。结果表明 :肾远曲小管和集合管是脑炎原虫主要的寄生部位 ,在此常能检出虫体或假囊 ;凋亡的细胞多见于集合小管的上皮细胞。用TUNEL染色 ,不同阶段的凋亡细胞可出现不同的病理形态学变化。初期的凋亡细胞 ,其胞核浓缩 ,胞膜增厚 ,着色较均匀 ;后期的凋亡细胞 ,其胞核的体积明显变小 ,胞浆减少 ,胞膜呈皱襞状 ,紧紧地包绕着皱缩而浓染的细胞核 ,形成典型的凋亡小体。用 P53和 Bcl- 2染色 ,由于基因表达产物的扩散 ,不仅受损细胞的核染色较深 ,而且胞浆染色也较深。研究证明 ,TUNEL 染色是一种检测由脑炎原虫引起肾细胞凋亡的灵敏、可靠的方法 ;P53和 Bcl- 2基因参与了肾细胞的凋亡过程 ;临床上病兔多尿与脱水可能主要与肾远曲小管和集合管的上皮细胞凋亡有关。     

牛有腔卵泡卵母细胞体外成熟的研究

试验从屠宰场收集了 4 8头青年黄牛、34头青年水牛的卵巢共 16 4个 ,共回收可用卵母细胞 86 4枚。水牛平均每个卵巢可回收 3.2 2枚可用卵母细胞 ,相当于黄牛 (6 .72枚 )的一半。试验结果表明 :水牛卵巢生长卵泡少 ,卵母细胞产量少、质量差 ;3种采集黄牛卵泡卵母细胞的方法 ,用抽吸加切割法平均从每个卵巢回收的可用卵数 (7.71枚 )都极显著高于抽吸法 (6 .19枚 )和切割法 (6 .4 4枚 ) (P <0 .0 1) ,但是该法手续繁杂 ,适于一次且只能收集少量卵巢时使用 ;在黄牛和水牛卵母细胞成熟培养液中用 0 .3%PVP代替 10 %FBS ,牛卵母细胞的体外成熟率无显著差异 (P >0 .0 5 )。     

Singular PCV2a or PCV2b infection results in apoptosis of hepatocytes in clinically affected gnotobiotic pigs

Porcine circovirus type 2 (PCV2) is clinically associated with respiratory disease, failure-to-thrive, hepatitis, and diarrhea; however, the precise pathogenesis of PCV2-associated disease and in particular its involvement in apoptosis is still controversial. The objectives of this study were (1) to determine whether PCV2 is associated with apoptosis by examining and comparing hepatic tissues from clinically affected or unaffected gnotobiotic pigs that were experimentally infected with PCV2, (2) to determine if there are differences between PCV2a and PCV2b in inducing hepatocyte apoptosis, and (3) to determine if there are differences between apoptosis detection systems. Forty-eight gnotobiotic pigs were separated into five groups based on inoculation status and development of clinical disease: (1) sham-inoculated, clinically-unaffected (n=4), (2) inoculated with PCV2a, clinically-unaffected (n=10), (3) inoculated with PCV2a, clinically-affected (n=6), (4) inoculated with PCV2b, clinically-unaffected, (n=13) and (5) inoculated with PCV2b, clinically-affected (n=15). Formalin-fixed, paraffin-embedded sections of liver from all pigs were analyzed for signs of apoptosis [presence of single strand DNA breaks in the nucleus by the terminal transferase dUTP nick end labeling (TUNEL) assay or presence of intra-nuclear cleaved caspase 3 (CCasp3) demonstrated by CCasp3 immunohistochemistry (IHC)]. In addition, the liver tissues were also tested for presence of cytoplasmic and intra-nuclear PCV2 antigen by an IHC assay. Specific CCasp3 and TUNEL labeling was detected in the nucleus of hepatocytes in PCV2a and PCV2b infected pigs with significantly (P<0.05) higher levels of apoptotic cells in clinically-affected pigs. Regardless of PCV2 subtype (PCV2a; PCV2b), there were higher levels of PCV2 antigen in clinically-affected pigs compared to clinically-unaffected pigs. There was no significant difference in detection rate of apoptotic cells between the TUNEL assay and CCasp3 IHC. When high amounts of PCV2 antigen were present, the incidence of CCasp3 and TUNEL staining also increased regardless of the PCV2 genotype. This suggests that PCV2-induced apoptosis of hepatocytes is important in the pathogenesis of PCV2-associated lesions and disease.