髭蟾属研究回顾

本文回顾了髭蟾属动物的研究历史。这些研究集中在形态、核型和蛋白质层次的系统学,以及该属动物的生物学特征方面。提出了一些尚待研究的问题。     

瑶山髭蟾的核型

髭蟾是我国特有的无尾两栖类,主要分布在川、黔、滇、桂、浙、闽等省的山区。自刘承钊于1945年建立髭蟾属(Vibrissaphora Liu)以来,已先后发表有五种,即峨嵋髭蟾(Vibrissaphora boringii Liu),崇安髭蟾(V.liui Pope),雷山髭蟾(V.leishanensis Liu et Hu),瑶山髭蟾(V.yaoshanensis Liu et Hu)和哀牢髭蟾(V.ailaomica Yang,Chen et Ma)。赵尔宓、吴贯夫(1983,1987)报道了全部髭蟾种的核型,并提出了瑶山髭     

瑶山髭蟾生态习性的研究

瑶山髭蟾主要分布在广西大瑶山海拔1000—1500米的山冲溪流附近,其生态环境是植被丰富、水源充足、气候温湿的常绿阔叶林带。全年大部分时间都栖息在陆地阴暗处的土壤及洞穴内。主要以植物和小昆虫为食。繁殖期在每年12月“大雪”之后到“冬至”前后,产卵期很短,仅在“冬至”前后10天内进行,雌雄抱对产卵一般都在黎明前完成,属一次性产卵类型,卵群粘附在石块底部的凹陷处,每次产卵298—342粒,卵径3.8—4毫米。它们除在冬季繁殖外,而且在10℃的气温条件下,仍照常活动,因此,可能没有“冬眠”习性或“冬眠”期极短。     

雷山髭蟾生态初步观察

雷山髭蟾(Vibrissphora leishanensis)主要栖息在植被类型保存较好、水源充足,海拔在800~2 100 m的山间小溪附近。每年大部分时间栖息在溪流附近陆地上比较阴暗潮湿的环境。雌雄抱对产卵从11月初开始,到月底基本结束。雌雄抱对产卵一般在午后和晚上,有集群产卵和雄蟾守窝护卵的现象。雷山髭蟾可能没有"冬眠"和"夏蛰"习性,即便有也可能很短暂。雷山髭蟾蝌蚪可能需要3年时间才发育成幼蛙。     

峨眉髭蟾精子形态结构及分类学意义

应用透射电镜、扫描电镜和光学显微镜对峨眉髭蟾(Vibrissaphoraboringii)精子的形态和超微结构研究的结果表明:峨眉髭蟾的精子具角蟾科物种精子基本的形态和结构特征,即精子头部呈螺旋状,尾部呈弯曲状;精子具锥形的顶体、纤维束构成的穿孔器、平行排列的中心粒和双轴丝;线粒体位于尾部;精子核窝不明显、无轴纤维和波动膜等特征。此外,对已有报道的角蟾科和无尾类物种精子的特征进行分析比较表明:(1)角蟾科精子细胞核呈螺旋状,中心粒平行排列,尾部具双轴丝等结构不同于无尾类其他科精子的结构,具有明显的科间差别;(2)角蟾科精子各部的量度,尾部线粒体的分布和数量,以及轴丝的排列等特征在属间和种间表现出明显的差异;(3)峨眉髭蟾和东南亚拟髭蟾指名亚种精子的形态和超微结构存在明显的差异。     

雷山髭蟾繁殖生态初步研究

2014年10月—2016年6月,采用野外定点观察的方法系统观察雷山髭蟾Vibrissaphora leishanensis的繁殖生态习性。研究表明:雷山髭蟾求偶、交配、产卵均在水体中进行;繁殖期在每年的9月底至12月底,11月中旬最集中;雄性的体长和体质量均显著大于雌性;繁殖行为包括筑"巢"、鸣叫求偶、抱对、产卵,雄蟾单个鸣叫,抱对时雄蟾主动。有共用一个"巢"穴抱对产卵的习性和集群产卵的行为。卵群直径65.13~100.47 mm,平均77.73 mm±9.47 mm(n=54);窝卵数162~394粒,平均226.79粒±7.89粒(n=54);卵径2.06~5.18 mm,平均3.82 mm±0.91 mm(n=270);孵化期为107~157 d,平均127.71 d±7.38 d(n=54)。     

雄性峨眉髭蟾“胡子”的组织结构特征

采用石蜡切片与苏木精-伊红染色及扫描电镜,对雄性峨眉髭蟾Leptobrachium boringii的角质刺及其周边皮肤进行了显微结构和亚显微结构的观察。显微结构观察发现,峨眉髭蟾的角质刺属于皮肤衍生物,突起呈倒"V"形。角质刺由表皮和真皮构成,表皮为复层扁平上皮,可分成4层;最外层细胞角质化,细胞轮廓不清,被染成深红色。真皮由疏松结缔组织构成,分辨不出致密层与疏松层,其内未见皮肤腺,但有少量色素细胞与毛细血管分布。表皮嵴伸入到真皮层,在以往的无尾两栖类研究中未见报道。角质刺基部可见皮肤褶翻起将其包裹在内,皮肤褶向上延伸形成角质刺。扫描电镜观察表明,角质刺顶端呈锥形的"小山丘"状,表面可分辨出表皮细胞轮廓,细胞为呈覆瓦状排列的角质化细胞。角质刺与皮肤交界处为多边形的角质化细胞。角质化上皮细胞的上表面与下表面均具有凹凸不平的花纹结构,细胞之间以镶嵌的方式连接。     

自然环境下雷山髭蟾卵群发育观察

2015年10月至2016年4月期间,在雷公山保护区内的雷公山工区对雷山髭蟾(Vibrissaphora leishanensis)在野外自然环境下卵群发育进行了观察。共发现产卵场14个,卵群54团。雷山髭蟾卵群从受精卵发育到鳃盖褶期的平均时间为(127.71±1.38)d(107~157 d,n=54),产卵场的气温、水温和水深对胚胎发育有一定影响。     

大巴山区锄足蟾科齿蟾属一新种——南江齿蟾的描述(两栖纲:无尾目)

对大巴山区四川南江的一种齿蟾标本作了深入研究,认为与我国已知齿蟾属     

两种髭蟾的Ag—NORs,C—带及核型的研究

本文叙述和比较了峨眉髭蟾和哀牢髭蟾的核型,C-带和Ag-NORs,结果表明两者有下列相同方面:2n=26(6+7)、除No.3为SM外,其余诸对概为M,次缢痕和Ag-NoRs位于6q,并都表现出异形现象,No.1长臂有长度异形,但所增染色体片断不呈现C-带正染,C-带正染主要是在各对染色体的着丝点区域,另外No.2短臂近着丝区域亦为正染。这些表明二者之间有很大的核带型同源性。但是二者间在核型的某些对应染色体之间,在相对长度(6对)和臂比值(2对)方面有显著性差异,故可推测其机制是相互易位和臂间倒位。另外,哀牢髭蟾未发现与性别相关的异形性染色体。     

On the stability of DNA in photoreceptor cells of rat retina

Summary DNA turnover in post-mitotic photoreceptor cells of F344 rat retina was investigated. Developing retinas of newborn rats were labelled by multiple injections of (methyl-³H)thymidine. One eye was removed on day 60 and embedded in paraffin. The groups of rats were killed 180, 365, 540 or 730 days later and the second eye was removed. Autoradiographic studies on pairs of eyes showed no detectable DNA turnover in photoreceptor cells up to the end of the experiment (near median life-span, 50% survival age). The DNA of these photoreceptor cells is not replaced through the life span of the animals; the results thus suggest that it is very stable and possibly protected in a specific manner.     

大白鼠视网膜中向同侧中枢投射的神经节细胞

在大白鼠视束和上丘注射HRP作逆行标记表明,向对侧中枢投射的神经节细胞遍布视网膜各处,而同侧投射的细胞大部分位于颞下侧边缘的新月形区域内,少数细胞分布在此区以外。与视束注射比较,上丘注射时同侧视网膜颞新月区内标记细胞数大为减少。统计测量表明,同侧投射细胞胞体直径平均比对侧投射细胞的略长。文中对上述结果的生理学意义进行了讨论。     

RETINAL CIRCADIAN RHYTHMS IN HUMANS*

Circadian rhythms in the retina may reflect intrinsic rhythms in the eye. Previous reports on circadian variability in electrophysiological human retinal measures have been scanty, and the results have been somewhat inconsistent. We studied the circadian variation of the electrooculography (EOG), electroretinography (ERG), and visual threshold (VTH) in subjects undergoing a 36h testing period. We used an ultrashort sleep-wake cycle to balance effects of sleep and light-dark across circadian cycles. Twelve healthy volunteers (10 males, 2 females; mean age 26.3 years, standard deviation [SD] 8.0 years, range 19–40 years) participated in the study. The retinal functions and oral temperature were measured every 90 min. The EOG was measured in the light, whereas the ERG and the VTH were measured in the dark. Sleep was inferred from activity detected by an Actillume monitor. The EOG peak-to-peak responses followed a circadian rhythm, with the peak occurring late in the morning (acrophase 12:22). The ERG b-wave implicit time peaked in the early morning (acrophase 06:46). No statistically significant circadian rhythms could be demonstrated in the ERG a-wave implicit time or peak-to-peak amplitude. The VTH rhythm peaked in the early morning (acrophases 07:59 for blue and 07:32 for red stimuli). All retinal rhythms showed less-consistent acrophases than the temperature and sleep rhythms. This study demonstrated several different circadian rhythms in retinal electrophysiological and psychophysical measures of healthy subjects. As the retinal rhythms had much poorer signal-to-noise ratios than the temperature rhythm, these measures cannot be recommended as circadian markers. (Chronobiology International, 18(6), 957–971, 2001)     

RETINAL CIRCADIAN RHYTHMS IN HUMANS *

Circadian rhythms in the retina may reflect intrinsic rhythms in the eye. Previous reports on circadian variability in electrophysiological human retinal measures have been scanty, and the results have been somewhat inconsistent. We studied the circadian variation of the electrooculography (EOG), electroretinography (ERG), and visual threshold (VTH) in subjects undergoing a 36h testing period. We used an ultrashort sleep-wake cycle to balance effects of sleep and light-dark across circadian cycles. Twelve healthy volunteers (10 males, 2 females; mean age 26.3 years, standard deviation [SD] 8.0 years, range 19-40 years) participated in the study. The retinal functions and oral temperature were measured every 90 min. The EOG was measured in the light, whereas the ERG and the VTH were measured in the dark. Sleep was inferred from activity detected by an Actillume monitor. The EOG peak-to-peak responses followed a circadian rhythm, with the peak occurring late in the morning (acrophase 12:22). The ERG b-wave implicit time peaked in the early morning (acrophase 06:46). No statistically significant circadian rhythms could be demonstrated in the ERG a-wave implicit time or peak-to-peak amplitude. The VTH rhythm peaked in the early morning (acrophases 07:59 for blue and 07:32 for red stimuli). All retinal rhythms showed less-consistent acrophases than the temperature and sleep rhythms. This study demonstrated several different circadian rhythms in retinal electrophysiological and psychophysical measures of healthy subjects. As the retinal rhythms had much poorer signal-to-noise ratios than the temperature rhythm, these measures cannot be recommended as circadian markers. (Chronobiology International, 18(6), 957-971, 2001)     

Melatonin modulates the neural activity in the photosensory pineal organ of the trout: Evidence for endocrine-neuronal interactions

Summary Hormonal and neural signals transmitted from the pineal organ to the brain in cold-blooded vertebrates presumably convert information about the ambient illumination into signals which may be used to mediate photoperiodic and circadian responses. The possible intrapineal function of melatonin was investigated by recording intra- and extracellularly from photoreceptors and second-order neurons in the isolated superfused pineal organ of the trout (Salmo gairdneri). Melatonin added through the perfusion bath to the explanted pineal organ caused a dose-related and reversible inhibition of ganglion cells of the luminance type whereas the hormone did not significantly affect the membrane potential of photoreceptors and their light-evoked response. The observed effects seem to be independent from photoperiod and adaptation conditions. These results suggest that melatonin provides a feedforward signal to intrapineal neurons regulating the neural output of the organ.Laboratory of Fish Biology, School of Agriculture, Nagoya University, Chikusa, Nagoya 464 Japan     

C-fos基因在大鼠缺血视网膜内的表达

实验用FOS免疫组化方法（ABC法）研究了缺血诱导的大鼠视网膜内c-fos原癌基因的表达情况。实验动物用升高眼压的方法做成视网膜缺血模型,依缺血后存活时间不同分15′、30′、1h、2h、4h、6h、12h七个组,每只大鼠右眼为缺血眼,左眼做自身对照眼,另设正常对照组。动物腹腔麻醉,4％多聚甲醛灌注固定,取双眼冰冻切片,片厚15μm。实验结果显示缺血后15′组大鼠视网膜内核层最先出现少量卵圆型浅棕色的FOS阳性神经元胞核,30分钟至1h FOS表达逐渐增强,节细胞层也出现FOS阳性胞核。缺血后2h FOS表达达最高峰,缺血后4h FOS阳性胞核逐渐减少,12h达正常组水平、自身对照眼及正常对照眼网膜节细胞层偶见FOS阳性胞核。     

真鲷视网膜结构及其视觉特性研究：Ⅱ视网膜光感受细胞超微结构研究

对真鲷光感受细胞的超微结构进行观察,结果表明：视杆外段膜盘为游离膜盘,视锥外段膜盘则为连续的膜结构,视锥和视杆均含有连接纤毛和辅助外段。花萼状突起起源于内段。椭体内充满线粒体,无球状小体。双锥椭圆体并生膜为六层,视锥内段无鳍状突起,视锥突触带,在明适应视网膜中数量增多,在暗适应视网中数量减少,视杆突触带在这两种适应网膜中数量不变,每一杆小球只有一个突触带,而锥小足有４－６个突触带。     

A Retinal Ganglion Cell Neurotrophic Factor Purified from the Superior Colliculus

Dissociated neonatal rat retinal ganglion cells can be maintained by the addition of an extract from the neonatal superior colliculus. This extract can support 95% of ganglion cells over 24 h in culture; in addition it promotes the expression of neurites from these cells. This report describes the purification of a neurotrophic factor from the superior colliculus which supports the survival of 80% of retinal ganglion cells over 24 h in vitro. The purification procedure involves a combination of dye-ligand, anion-exchange, and molecular sieve chromatography. The purified neurotrophic factor has a Stokes radius of approximately 200 A using molecular sieve chromatography in the presence of a chaotropic agent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified factor indicates that it is a glycoprotein that migrates with a molecular mass greater than 400 kDa. Further characterization of this high-molecular-mass glycoprotein by enzymatic digestion demonstrated that it is a chondroitin sulfate proteoglycan. This factor is clearly distinguishable from other neurotrophic factors that have an effect on retinal ganglion cells such as brain-derived neurotrophic factor and fibroblast growth factor. The chondroitin sulfate proteoglycan from the neonatal superior colliculus is the first proteoglycan to be identified as a neurotrophic factor.     

Retina stem cells,hopes and obstacles

Retinal degeneration is a major contributor to visual dysfunction worldwide. Although it comprises several eye diseases, loss of retinal pigment epithelial (RPE) and photoreceptor cells are the major contributors to their pathogenesis. Early therapies included diverse treatments, such as provision of anti-vascular endothelial growth factor and many survival and trophic factors that, in some cases, slow down the progression of the degeneration, but do not effectively prevent it. The finding of stem cells (SC) in the eye has led to the proposal of cell replacement strategies for retina degeneration. Therapies using different types of SC, such as retinal progenitor cells (RPCs), embryonic SC, pluripotent SCs (PSCs), induced PSCs (iPSCs), and mesenchymal stromal cells, capable of self-renewal and of differentiating into multiple cell types, have gained ample support. Numerous preclinical studies have assessed transplantation of SC in animal models, with encouraging results. The aim of this work is to revise the different preclinical and clinical approaches, analyzing the SC type used, their efficacy, safety, cell attachment and integration, absence of tumor formation and immunorejection, in order to establish which were the most relevant and successful. In addition, we examine the questions and concerns still open in the field. The data demonstrate the existence of two main approaches, aimed at replacing either RPE cells or photoreceptors. Emerging evidence suggests that RPCs and iPSC are the best candidates, presenting no ethical concerns and a low risk of immunorejection. Clinical trials have already supported the safety and efficacy of SC treatments. Serious concerns are pending, such as the risk of tumor formation, lack of attachment or integration of transplanted cells into host retinas, immunorejection, cell death, and also ethical. However, the amazing progress in the field in the last few years makes it possible to envisage safe and effective treatments to restore vision loss in a near future.